Dr. Domenico Garozzo publications
245 publications
1) Severe kidney dysfunction in sialidosis mice reveals an essential role for neuraminidase 1 in reabsorption
I.Kho, E.P.Demina, X.Pan, I.Londono, C.W.Cairo, L.Sturiale, A.Palmigiano, A.Messina, D.Garozzo, R.Ung, F.Mac-Way, E.Bonneil, P.Thibault, M.Lemaire, C.R.Morales, A.V.Pshezhetsky
JCI Insight
8(20),
166470
- 2023
DOI:
https://doi.org/10.1172/jci.insight.166470
Sialidosis is an ultra-rare multisystemic lysosomal disease caused by mutations in the neuraminidase 1 (NEU1) gene. The severe type II form of the disease manifests with a prenatal/infantile or juvenile onset, bone abnormalities, severe neuropathology, and visceromegaly. A subset of these patients present with nephrosialidosis, characterized by abrupt onset of fulminant glomerular nephropathy. We studied the pathophysiological mechanism of the disease in 2 NEU1-deficient mouse models, a constitutive Neu1-knockout, Neu1ΔEx3, and a conditional phagocyte-specific knockout, Neu1Cx3cr1ΔEx3. Mice of both strains exhibited terminal urinary retention and severe kidney damage with elevated urinary albumin levels, loss of nephrons, renal fibrosis, presence of storage vacuoles, and dysmorphic mitochondria in the intraglomerular and tubular cells. Glycoprotein sialylation in glomeruli, proximal distal tubules, and distal tubules was drastically increased, including that of an endocytic reabsorption receptor megalin. The pool of megalin bearing O-linked glycans with terminal galactose residues, essential for protein targeting and activity, was reduced to below detection levels. Megalin levels were severely reduced, and the protein was directed to lysosomes instead of the apical membrane. Together, our results demonstrated that desialylation by NEU1 plays a crucial role in processing and cellular trafficking of megalin and that NEU1 deficiency in sialidosis impairs megalin-mediated protein reabsorption.
2) N-acetylneuraminate pyruvate lyase controls sialylation of muscle glycoproteins essential for muscle regeneration and function
A.Da Silva, J.Dort, Z.Orfi, X.Pan, S.Huang, I.Kho, E.Heckel, G.Muscarnera, P.Piet van Vliet, L.Sturiale, A.Messina, D.Romeo, C.D.M.van Karnebeek, X.Wen, A.Hinek, T.Molina, G.Andelfinger, B.Ellezam, Y.Yamanaka, H.J.Olivos, C.R.Morales, J.Joyal, D.J.Lefeber, D.Garozzo, N.A.Dumont, A.V.Pshezhetsky
Science Advances
9(26)
- 2023
DOI:
https://doi.org/10.1126/sciadv.ade6308
Deleterious variants in N-acetylneuraminate pyruvate lyase (NPL) cause skeletal myopathy and cardiac edema in humans and zebrafish, but its physiological role remains unknown. We report generation of mouse models of the disease: NplR63C, carrying the human p.Arg63Cys variant, and Npldel116 with a 116-bp exonic deletion. In both strains, NPL deficiency causes drastic increase in free sialic acid levels, reduction of skeletal muscle force and endurance, slower healing and smaller size of newly formed myofibers after cardiotoxin-induced muscle injury, increased glycolysis, partially impaired mitochondrial function, and aberrant sialylation of dystroglycan and mitochondrial LRP130 protein. NPL-catalyzed degradation of sialic acid in the muscle increases after fasting and injury and in human patient and mouse models with genetic muscle dystrophy, demonstrating that NPL is essential for muscle function and regeneration and serves as a general marker of muscle damage. Oral administration of N-acetylmannosamine rescues skeletal myopathy, as well as mitochondrial and structural abnormalities in NplR63C mice, suggesting a potential treatment for human patients.
3) Soluble peptidoglycan fragments produced by Limosilactobacillus fermentum with antiproliferative activity are suitable for potential therapeutic development: A preliminary report
V.Fuochi, M.Spampinato, A.Distefano, A.Palmigiano, D.Garozzo, C.Zagni, A.Rescifina, G.Li Volti, P.M.Furneri
Frontiers in Molecular Biosciences
10,
1082526
- 2023
DOI:
https://doi.org/10.3389/fmolb.2023.1082526
Currently, the use of probiotic strains and their products represents a promising innovative approach as an antagonist treatment against many human diseases. Previous studies showed that a strain of Limosilactobacillus fermentum (LAC92), previously defined as Lactobacillus fermentum, exhibited a suitable amensalistic property. The present study aimed to purify the active components from LAC92 to evaluate the biological properties of soluble peptidoglycan fragments (SPFs). The cell-free supernatant (CFS) and bacterial cells were separated after 48 h of growth in MRS medium broth and treated for isolation of SPFs. Antimicrobial activity and proliferation analysis on the human cell line HTC116 were performed using technologies such as xCELLigence, count and viability, and clonogenic analysis. MALDI-MS investigation and docking analysis were performed to determine the molecular structure and hypothetical mode of action, respectively. Our results showed that the antimicrobial activity was mainly due to SPFs. Moreover, the results obtained when investigating the SPF effect on the cell line HCT116 showed substantial preliminary evidence, suggesting their significant cytostatic and quite antiproliferative properties. Although MALDI was unable to identify the molecular structure, it was subsequently revealed by analysis of the bacterial genome. The amino acid structure is called peptide 92. Furthermore, we confirmed by molecular docking studies the interaction of peptide 92 with MDM2 protein, the negative regulator of p53. This study showed that SPFs from the LAC92 strain exerted anticancer effects on the human colon cancer HCT116 cell line via antiproliferation and inducing apoptosis. These findings indicated that this probiotic strain might be a potential candidate for applications in functional products in the future. Further examination is needed to understand the specific advantages of this probiotic strain and improve its functional features to confirm these data. Moreover, deeper research on peptide 92 could increase our knowledge and help us understand if it will be possible to apply to specific diseases such as CRC.
4) Higher frequency of TMEM199-CDG in the southern mediterranean area is associated with c.92G>C (p.Arg31Pro) mutation
A.Fiumara, A.Sapuppo, L.Ferri, A.Arena, A.Prato, D.Garozzo, L.Sturiale, A.Morrone, R.Barone
European Journal of Medical Genetics
66(3),
104709
- 2023
DOI:
https://doi.org/10.1016/j.ejmg.2023.104709
Congenital disorders of glycosylation (CDG) are genetic multisystem diseases, characterized by defective glycoconjugate synthesis. A small number of CDG with isolated liver damage have been described, such as TMEM199-CDG, a non-encephalopathic liver disorder with Wilson disease-like phenotype. Only eight patients with TMEM199-CDG have been described including seven Europeans (originating from Greece and Italy) and one Chinese. Three patients from southern Italy (Campania) shared the same known missense mutation pathogenetic variant NM_152464.3:c. 92G > C (p.Arg31Pro), also found in a Greek patient.
Here we report a new patient from southern Italy (Sicily), with a homozygous c.92G > C p.(Arg31Pro) variant in TMEM199. The patient’s phenotype is characterized by mild non-progressive hepatopathy with a normal hepatic echo structure. A persistent increase in serum transaminases, total and low-density lipoprotein cholesterol and low serum ceruloplasmin and copper levels and normal urinary copper excretion were observed. Matrix-assisted laser desorption/ionization mass spectrometry analyses showed abnormal N- and O- protein glycosylation, indicative of a Golgi processing defect and supporting the function of TMEM199 in maintaining Golgi homeostasis. TMEM199-CDG is an ultra-rare CDG relatively frequent in the southern Mediterranean area (7 in 9 patients, 77%). It is mainly associated with the c.92G > C (p.Arg31Pro) pathogenetic allele globally reported in 4 out of 7 families (57%), including one from Greece and three unrelated families from southern Italy. The almost uniform clinical phenotype described in patients with TMEM199-CDG appears to reflect a higher prevalence of the same variant in patients from the southern Mediterranean area.
5) Phenotypic and genetic spectrum of ATP6V1A encephalopathy: a disorder of lysosomal homeostasis
R.Guerrini, D.Mei, K.Kerti-Szigeti, S.Pepe, M.Kay Koenig, G.Von Allmen, M.T Cho, K.McDonald, J.Baker, V.Bhambhani, Z.Powis, L.Rodan, R.Nabbout, G.Barcia, J.A.Rosenfeld, C.A.Bacino, C.Mignot, L.H.Power, C.J.Harris, D.Marjanovic, R.S.Møller, T.B.Hammer, The DDD Study, R.K.Filppula, P.Vieira, C.Hildebrandt, S.Sacharow, Undiagnosed Diseases Network, L.Maragliano, F.Benfenati, K.Lachlan, A.Benneche, F.Petit, J.M.De Sainte Agathe, B.Hallinan, Y.Si, I.M.Wentzensen, F.Zou, V.Narayanan, N.Matsumoto, A.Boncristiano, G.La Marca, M.Kato, K.Anderson, C.Barba, L.Sturiale, D.Garozzo, R.Bei, ATP6V1A collaborators, L.Masuelli, V.Conti, G.Novarino, A.Fassio
Brain
145(8),
2687-2703
- 2022
DOI:
https://doi.org/10.1093/brain/awac145
Vacuolar-type H+-ATPase (V-ATPase) is a multimeric complex present in a variety of cellular membranes that acts as an ATP-dependent proton pump and plays a key role in pH homeostasis and intracellular signalling pathways. In humans, 22 autosomal genes encode for a redundant set of subunits allowing the composition of diverse V-ATPase complexes with specific properties and expression. Sixteen subunits have been linked to human disease.
Here we describe 26 patients harbouring 20 distinct pathogenic de novo missense ATP6V1A variants, mainly clustering within the ATP synthase α/β
family-nucleotide-binding domain. At a mean age of 7 years (extremes: 6 weeks, youngest deceased patient to 22 years, oldest patient) clinical pictures included early lethal encephalopathies with rapidly progressive massive brain atrophy, severe developmental epileptic encephalopathies and static intellectual disability with epilepsy. The first clinical manifestation was early hypotonia, in 70%; 81% developed epilepsy, manifested as developmental epileptic encephalopathies in 58% of the cohort and with infantile spasms in 62%; 63% of developmental epileptic encephalopathies failed to achieve any developmental, communicative or motor skills. Less severe outcomes were observed in 23% of patients who, at a mean age of 10 years and 6 months, exhibited moderate intellectual disability, with independent walking and variable epilepsy. None of the patients developed communicative language. Microcephaly (38%) and amelogenesis imperfecta/enamel dysplasia (42%) were additional clinical features. Brain MRI demonstrated hypomyelination and generalized atrophy in 68%. Atrophy was progressive in all eight individuals undergoing repeated MRIs.
Fibroblasts of two patients with developmental epileptic encephalopathies showed decreased LAMP1 expression, Lysotracker staining and increased organelle pH, consistent with lysosomal impairment and loss of V-ATPase function. Fibroblasts of two patients with milder disease, exhibited a different phenotype with increased Lysotracker staining, decreased organelle pH and no significant modification in LAMP1 expression. Quantification of substrates for lysosomal enzymes in cellular extracts from four patients revealed discrete accumulation. Transmission electron microscopy of fibroblasts of four patients with variable severity and of induced pluripotent stem cell-derived neurons from two patients with developmental epileptic encephalopathies showed electron-dense inclusions, lipid droplets, osmiophilic material and lamellated membrane structures resembling phospholipids. Quantitative assessment in induced pluripotent stem cell-derived neurons identified significantly smaller lysosomes.
ATP6V1A-related encephalopathy represents a new paradigm among lysosomal disorders. It results from a dysfunctional endo-lysosomal membrane protein causing altered pH homeostasis. Its pathophysiology implies intracellular accumulation of substrates whose composition remains unclear, and a combination of developmental brain abnormalities and neurodegenerative changes established during prenatal and early postanal development, whose severity is variably determined by specific pathogenic variants.
6) CAMLG-CDG: a novel congenital disorder of glycosylation linked to defective membrane trafficking
M.P.Wilson, Z.Durin, Ö.Unal, B.G.Ng, T.Marrecau, L.Keldermans, E.Souche, D.Rymen, M.Gündüz, G.Köse, L.Sturiale, D.Garozzo, H.H.Freeze, J.Jaeken, F.Foulquier, G.Matthijs
Human molecular genetics
31(15),
2571-2581
- 2022
DOI:
https://doi.org/10.1093/hmg/ddac055
The transmembrane domain recognition complex (TRC) pathway is required for the insertion of C-terminal tail-anchored (TA) proteins into the lipid bilayer of specific intracellular organelles such as the endoplasmic reticulum (ER) membrane. In order to facilitate correct insertion, the recognition complex (consisting of BAG6, GET4 and UBL4A) must first bind to TA proteins and then to GET3 (TRC40, ASNA1), which chaperones the protein to the ER membrane. Subsequently, GET1 (WRB) and CAML form a receptor that enables integration of the TA protein within the lipid bilayer. We report an individual with the homozygous c.633 + 4A>G splice variant in CAMLG, encoding CAML. This variant leads to aberrant splicing and lack of functional protein in patient-derived fibroblasts. The patient displays a predominantly neurological phenotype with psychomotor disability, hypotonia, epilepsy and structural brain abnormalities. Biochemically, a combined O-linked and type II N-linked glycosylation defect was found. Mislocalization of syntaxin-5 in patient fibroblasts and in siCAMLG deleted Hela cells confirms this as a consistent cellular marker of TRC dysfunction. Interestingly, the level of the v-SNARE Bet1L is also drastically reduced in both of these models, indicating a fundamental role of the TRC complex in the assembly of Golgi SNARE complexes. It also points towards a possible mechanism behind the hyposialylation of N and O-glycans. This is the first reported patient with pathogenic variants in CAMLG. CAMLG-CDG is the third disorder, after GET4 and GET3 deficiencies, caused by pathogenic variants in a member of the TRC pathway, further expanding this novel group of disorders.
7) COG6-CDG: Novel variants and novel malformation
L.Cirnigliaro, P.Bianchi, L.Sturiale, D.Garozzo, G.Mangili, L.Keldermans, R.Rizzo, G.Matthijs, A.Fiumara, J.Jaeken, R.Barone
Birth Defects Research
114(5-6),
165-174
- 2022
DOI:
https://doi.org/10.1002/bdr2.1981
Background
Deficiency of Conserved Oligomeric Golgi (COG) subunits (COG1?8) is characterized by both N- and O-protein glycosylation defects associated with destabilization and mislocalization of Golgi glycosylation machinery components (COG-CDG). Patients with COG defects present with neurological and multisystem involvement and possible malformation occurrence. Eighteen patients with COG6-CDG (COG6 mutations) were reported to date. We describe a patient with COG6-CDG with novel variants and a novel clinical feature namely a congenital recto-vaginal fistula.
Methods
In-depth serum N- and O-glycosylation structural analyses were conducted by MALDI-TOF mass spectrometry. COG6 variants were identified by a gene panel and confirmed by Sanger sequencing.
Results
This female newborn presented with facial dysmorphism, distal arthrogryposis and recurrent stool discharges per vaginam. A double-contrast barium-enema X-ray study revealed a dehiscence (approximately 5 mm) at the anterior wall of the rectal ampoule communicating with the vagina consistent with a recto-vaginal fistula. She had developmental delay, corpus callosum dysgenesis, liver and gastrointestinal involvement, hyperthermia episodes and early demise. Serum N- and O-glycosylation analyses pointed to a profound Golgi disarrangement. We identified two novel variants in COG6: a deletion of 1 bp mutation c.823delA creating a shift in the reading frame and a premature stop codon and a 3 bp deletion (c.1141_1143delCTC) producing an in-frame deletion of 1 amino acid.
Conclusion
The congenital recto-vaginal fistula is a rare type of anorectal malformation that, to our knowledge, has not been reported in patients with a COG6 defect nor in patients with other COG defects. This study broadens COG6-CDG genetic landscape and spectrum of malformations.
8) SLC37A4-CDG: Second patient
M.P.Wilson, D.Quelhas, E.Leão-Teles, L.Sturiale, D.Rymen, L.Keldermans, V.Race, E.Souche, E.Rodrigues, T.Campos, E.Van Schaftingen, F.Foulquier, D.Garozzo, G.Matthijs, J.Jaeken
JIMD Reports
58,
122-128
- 2021
DOI:
https://doi.org/10.1002/jmd2.12195
Recently, a disorder caused by the heterozygous de novo c.1267C>T (p.R423*) substitution in SLC37A4 has been described. This causes mislocalization of the glucose-6-phosphate transporter to the Golgi leading to a congenital disorder of glycosylation type II (SLC37A4-CDG). Only one patient has been reported showing liver disease that improved with age and mild dysmorphism. Here we report the second patient with a type II CDG caused by the same heterozygous de novo c.1267C>T (p.R423*) mutation thereby confirming the pathogenicity of this variant and expanding the clinical picture with type 1 diabetes, severe scoliosis, and membranoproliferative glomerulonephritis. Additional clinical and biochemical data provide further insight into the mechanism and prognosis of SLC37A4-CDG.
9) HILIC-UPLC-MS for high throughput and isomeric N-glycan separation and characterization in Congenital Disorders Glycosylation and human diseases
A.Messina, A.Palmigiano, F.Esposito, A.Fiumara, A.Bordugo, R.Barone, L.Sturiale, J.Jaeken, D.Garozzo
Glycoconjugate Journal
38(2),
201-211
- 2021
DOI:
https://doi.org/10.1007/s10719-020-09947-7
N-glycan analyses may serve uncovering disease-associated biomarkers, as well as for profiling distinctive changes supporting diagnosis of genetic disorders of glycan biosynthesis named congenital disorders of glycosylation (CDG). Strategies based on liquid chromatography (LC) preferentially coupled to electrospray ionization (ESI) - mass spectrometry (MS) have emerged as powerful analytical methods for N-glycan identification and characterization. To enhance detection sensitivity, glycans are commonly labelled with a functional tag prior to LC-MS analysis. Since most derivatization techniques are notoriously time-consuming, some commercial analytical kits have been developed to speed up N-deglycosylation and N-glycan labelling of glycoproteins of pharmaceutical and biological interest such as monoclonal antibodies (mAbs). We exploited the analytical capabilities of RapiFluor-MS (RFMS) to perform, by a slightly modified protocol, a detailed N-glycan characterization of total serum and single serum glycoproteins from specific patients with CDG (MAN1B1-CDG, ALG12-CDG, MOGS-CDG, TMEM199-CDG). This strategy, accomplished by Hydrophilic Interaction Chromatography (HILIC)-UPLC-ESI-MS separation of the RFMS derivatized N-glycans, allowed us to uncover structural details of patients serum released N-glycans, thus extending the current knowledge on glycan profiles in these individual glycosylation diseases. The applied methodology enabled to differentiate in some cases either structural isomers and isomers differing in the linkage type. All the here reported applications demonstrated that RFMS method, coupled to HILIC-UPLC-ESI-MS, represents a sensitive high throughput approach for serum N-glycome analysis and a valuable option for glycan detection and separation particularly for isomeric species.
10) Clinical and radiological correlates of activities of daily living in cerebellar atrophy caused by PMM2 mutations (PMM2-CDG)
F.Pettinato, G.Mostile, R.Battini, D.Martinelli, A.Madeo, E.Biamino, D.Frattini, D.Garozzo, S.Gasperini, R.Parini, F.Sirchia, G.Sortino, L.Sturiale, G.Matthijs, A.Morrone, M.Di Rocco, R.Rizzo, J.Jaeken, A.Fiumara, R.Barone
Cerebellum
20(4),
596-605
- 2021
DOI:
https://www.doi.org/10.1007/s12311-021-01242-x
We aimed to identify clinical, molecular and radiological correlates of activities of daily living (ADL) in patients with cerebellar atrophy caused by PMM2 mutations (PMM2-CDG), the most frequent congenital disorder of glycosylation. Twenty-six PMM2-CDG patients (12 males; mean age 13 ± 11.1 years) underwent a standardized assessment to measure ADL, ataxia (brief ataxia rating scale, BARS) and phenotype severity (Nijmegen CDG rating scale, NCRS). MRI biometry of the cerebellum and the brainstem were performed in 23 patients (11 males; aged 5 months-18 years) and 19 control subjects with equal gender and age distributions. The average total ADL score was 15.3 ± 8.5 (range 3-32 out of 36 indicating severe functional disability), representing variable functional outcome in PMM2-CDG patients. Total ADL scores were significantly correlated with NCRS (r2 = 0.55, p < 0.001) and BARS scores (r2 = 0.764; p < 0.001). Severe intellectual disability, peripheral neuropathy, and severe PMM2 variants were all significantly associated with worse functional outcome. Higher ADL scores were significantly associated with decreased diameters of cerebellar vermis (r2 = 0.347; p = 0.004), hemispheres (r2 = 0.436; p = 0.005), and brainstem, particularly the mid-pons (r2 = 0.64; p < 0.001) representing the major radiological predictor of functional disability score in multivariate regression analysis. We show that cerebellar syndrome severity, cognitive level, peripheral neuropathy, and genotype correlate with ADL used to quantify disease-related deficits in PMM2-CDG. Brainstem involvement should be regarded among functional outcome predictors in patients with cerebellar atrophy caused by PMM2-CDG.
11) N-Glycomics of Human Erythrocytes
R.O.Bua, A.Messina, L.Sturiale, R.Barone, D.Garozzo, A.Palmigiano
International Journal of Molecular Sciences
22(15),
8063
- 2021
DOI:
https://doi.org/10.3390/ijms22158063
Glycosylation is a complex post-translational modification that conveys functional diversity to glycoconjugates. Cell surface glycosylation mediates several biological activities such as induction of the intracellular signaling pathway and pathogen recognition. Red blood cell (RBC) membrane N-glycans determine blood type and influence cell lifespan. Although several proteomic studies have been carried out, the glycosylation of RBC membrane proteins has not been systematically investigated. This work aims at exploring the human RBC N-glycome by high-sensitivity MALDI-MS techniques to outline a fingerprint of RBC N-glycans. To this purpose, the MALDI-TOF spectra of healthy subjects harboring different blood groups were acquired. Results showed the predominant occurrence of neutral and sialylated complex N-glycans with bisected N-acetylglucosamine and core- and/or antennary fucosylation. In the higher mass region, these species presented with multiple N-acetyllactosamine repeating units. Amongst the detected glycoforms, the presence of glycans bearing ABO(H) antigens allowed us to define a distinctive spectrum for each blood group. For the first time, advanced glycomic techniques have been applied to a comprehensive exploration of human RBC N-glycosylation, providing a new tool for the early detection of distinct glycome changes associated with disease conditions as well as for understanding the molecular recognition of pathogens.
12) Lipopolysaccharide from Gut-Associated Lymphoid-Tissue-Resident Alcaligenes faecalis: Complete Structure Determination and Chemical Synthesis of Its Lipid A
A.Shimoyama, F.Di Lorenzo, H.Yamaura, K.Mizote, A.Palmigiano, M.Pither, I.Speciale, T.Uto, S.Masui, L.Sturiale, D.Garozzo, K.Hosomi, N.Shibata, K.Kabayama, Y.Fujimoto, A.Silipo, J.Kunisawa, H.Kiyono, A.Molinaro, K.Fukase
Angewandte Chemie
60(18),
10023-10031
- 2021
DOI:
https://doi.org/10.1002/ange.202012374
Alcaligenes faecalis is the predominant Gram-negative bacterium inhabiting gut-associated lymphoid tissues, Peyer’s patches. We previously reported that an
A. faecalis lipopolysaccharide (LPS) acted as a weak agonist for Toll-like receptor 4 (TLR4)/myeloid differentiation factor-2 (MD-2) receptor as well as a potent inducer of IgA without excessive inflammation, thus suggesting that
A. faecalis LPS might be used as a safe adjuvant. In this study, we characterized the structure of both the lipooligosaccharide (LOS) and LPS from
A. faecalis. We synthesized three lipid A molecules with different degrees of acylation by an efficient route involving the simultaneous introduction of 1- and 4’-phosphates. Hexaacylated
A. faecalis lipid A showed moderate agonistic activity towards TLR4-mediated signaling and the ability to elicit a discrete interleukin-6 release in human cell lines and mice. It was thus found to be the active principle of the LOS/LPS and a promising vaccine adjuvant candidate.
13) Aberrant sialylation in a patient with a HNF1α variant and liver adenomatosis
L.Sturiale, M.C.Nassogne, A.Palmigiano, A.Messina, I.Speciale, R.Artuso, G.Bertino, N.Revencu, X.Stephénne, C.De Castro, G.Matthijs, R.Barone, J.Jaeken, D.Garozzo
iScience
24(4),
102323
- 2021
DOI:
https://doi.org/10.1016/j.isci.2021.102323
Glycosylation is a fundamental post-translational modification of proteins that boosts their structural diversity providing subtle and specialized biological properties and functions. All those genetic diseases due to a defective glycan biosynthesis and attachment to the nascent glycoproteins fall within the wide area of congenital disorders of glycosylation (CDG), mostly causing multisystem involvement. In the present paper, we detailed the unique serum N-glycosylation of a CDG-candidate patient with an unexplained neurological phenotype and liver adenomatosis harboring a recurrent pathogenic
HNF1α variant. Serum transferrin isoelectric focusing showed a surprising N-glycosylation pattern consisting on hyposialylation, as well as remarkable hypersialylation. Mass spectrometry-based glycomic analyses of individual serum glycoproteins enabled to unveil hypersialylated complex N-glycans comprising up to two sialic acids per antenna. Further advanced MS analysis showed the additional sialic acid is bonded through an α2-6 linkage to the peripheral N-acetylglucosamine residue.
14) Tear N-glycomics in vernal and atopic keratoconjunctivitis
A.Messina, A.Palmigiano, C.Tosto, D.Romeo, L.Sturiale, D.Garozzo, A.Leonardi
Allergy
76(8),
2500-2509
- 2021
DOI:
https://doi.org/10.1111/all.14775
Purpose: Tear fluid
N-Glycome from patients affected with vernal (VKC) and atopic keratoconjunctivitis (AKC) was investigated to identify specific changes in tears and to recognize possible glyco-biomarkers.
Methods: The analysis of the
N-glycans was performed using matrix-assisted laser desorption ionization mass spectrometry on single tear samples. Tears from control normal subjects (CTRL), VKC and AKC patients were processed and treated with peptide
N-glycosidase F (PNGase F) to deglycosylate
N-glycoproteins. Released
N-glycans were purified, permethylated, and analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and tandem mass spectrometry (MALDI-TOF MS and MALDI-TOF MS/MS).
Results: More than 150 complex
N-glycans, including highly fucosylated biantennary, triantennary, tetra-antennary, and bisecting species, were observed in our spectra. Three distinct patterns for CTRL, VKC, and AKC patients were identified in terms of relative intensities for some
N-glycans structures. Major variations involved bisecting and hyperfucosylated glycoforms.
The most intense ions were associated with species at
m/z 1907.0 (asialo, agalacto, bisected, biantennary structure-NGA2B) in CTRL MS profiles, at
m/z 2605.3 and 2966.5 in VKC, and at
m/z 2792.4 in AKC corresponding to a well-known biantennary, disialylated
N-glycan. Several peaks were associated with structures bearing one or two Lewis X epitopes. Structures were confirmed by MS/MS analysis. Quantitative differences among the three groups were statistically significant.
Conclusions: Tear MS profiles are rich in specific glycoforms, particularly those with a high fucosylation degree, indicating both core and peripheral decoration. Tear
N-glycome analysis provided important information for a better comprehension of VKC and AKC alterations at the molecular level.
15) MALDI-MS cerebrospinal fluid (CSF) N-glycan profiles in neurodegenerative diseases
A.Palmigiano, A.Messina, F.Esposito, R.Barone, G.Mostile, A.Nicoletti, L.Sturiale, D.Romeo, C.Sanfilippo, M.Zappia, D.Garozzo
Workshop IPCB 27 - 29 October 2020
,
47
- 2020
Glycosylation is a key post-translational protein modification in different biological functions such as cellular adhesion, recognition and signalling, and changes in protein glycosylation have been recognized in different neurodegeneration disorders [1]. Alzheimer’s Disease (AD) and Parkinson’s Disease (PD) are the most common neurodegenerative diseases. Both pathologies are multifactorial diseases presenting clinically heterogeneous symptoms and prognosis and ultimately resulting in neurons loss of functions and death. AD and PD are diagnosed by clinical and neuropsychological criteria, only proved by post-mortem autopsy. Therefore, there is a great need of diagnostic tools able of detecting the diseases in their early stages when preventative therapies could ameliorate patients’ conditions before irreversible neuronal damages. We performed Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) CSF N-glycosylation analysis of released and permethylated N-glycans from a cohort including 21 AD, 11 mild cognitive impairment (MCI), 19 PD patients and 19 control subjects (age- and gender-matched). PD-CSF spectra denoted a significant increase of high-mannose 5 (M5, m/z 1579.8), agalactosylated biantennary (G0, m/z 1661.8), agalactosylated bisected biantennary (G0B, m/z 1906.9) bisected, agalactosylated core fucosylated N-glycans (G0BF, m/z 2081.0). Although no unique profile emerged for AD and MCI, principal component analysis (PCA) allows to separate AD and MCI in two categories, according to bisecting-N-glycans relative intensities. AD1 and MCI1 showed significant increase of bisecting structures and an overall decrease of sialylated species compared to healthy controls, while AD2 and MCI2 showed a slightly reduction of those species. Interestingly, the observed divergences in MCI1 and MCI2 glycosylation profiles reflected the different clinical follow-up of the respective class of patients: 5 MCI1 patients out 5 converted to AD within 36 months from diagnosis, while all the MCI2 subjects (6 out 6) remained stable over the time, suggesting that increasing amount of N-glycans with bisected GlcNAc is a biochemical hallmark of AD in the pre-dementia phase [2]. MALDI-MS CSF N-glycome profiling enabled detection of peculiar changes in subject affected by neurodegenerative diseases, helpful in monitoring diseases development and progression, thus representing a source of potential biomarkers and therapeutic targets.
16) Chlorovirus PBCV-1 protein A064R has three of the transferase activities necessary to synthesize its capsid protein N-linked glycans
I.Speciale, M.Laugieri, E.Noel, S.Lin, T.Lowary, A.Molinaro, G.Duncan, I.Agarkova, D.Garozzo, M.Tonetti, J.Van Etten, C.De Castro
Proceedings of the National Academy of Sciences of The United States of America
- 2020
DOI:
https://doi.org/10.1073/pnas.2016626117
The chloroviruses are unusual because they are predicted to encode most, if not all, of the machinery to synthesize the glycans attached to their major capsid proteins. Here we show that two of the virus-encoded proteins A064R and A061L are functionally active. A064R has three domains: The first two are GTs and the third domain is a methyltransferase. A061L has a methyltransferase activity. The action of these two enzymes produce the fragment 2,3-di-O-methyl-α-L-Rha-(1→ 2)-β-L-Rha, which is part of the complex N-linked glycan attached to the virus capsid protein. A064R domain 2 is a member of a new GT family. This provides direct evidence that the synthesis of PBCV-1 glycans are accomplished with virus-encoded enzymes.
17) Structure of the unusual Sinorhizobium fredii HH103 lipopolysaccharide and its role in symbiosis
F.Di Lorenzo, I.Speciale, A.Silipo, C.Alías-Villegas, S.Acosta-Jurado, M.Rodríguez-Carvajal, M.Dardanelli, A.Palmigiano, D.Garozzo, J.Ruiz-Sainz, A.Molinaro, J.Vinardell
Journal of Biological Chemistry
295 (32),
10969-10987
- 2020
DOI:
https://doi.org/10.1074/jbc.RA120.013393
Rhizobia are soil bacteria that form important symbiotic associations with legumes, and rhizobial surface polysaccharides, such as K-antigen polysaccharide (KPS) and lipopolysaccharide (LPS), might be important for symbiosis. Previously, we obtained a mutant of Sinorhizobium fredii HH103, rkpA, that does not produce KPS, a homopolysaccharide of a pseudaminic acid derivative, but whose LPS electrophoretic profile was indistinguishable from that of the WT strain. We also previously demonstrated that the HH103 rkpLMNOPQ operon is responsible for 5-acetamido-3,5,7,9-tetradeoxy-7-(3-hydroxybutyramido)-L-glycero-L-manno-nonulosonic acid [Pse5NAc7(3OHBu)] production and is involved in HH103 KPS and LPS biosynthesis and that an HH103 rkpM mutant cannot produce KPS and displays an altered LPS structure. Here, we analyzed the LPS structure of HH103 rkpA, focusing on the carbohydrate portion, and found that it contains a highly heterogeneous lipid A and a peculiar core oligosaccharide composed of an unusually high number of hexuronic acids containing β-configured Pse5NAc7(3OHBu). This pseudaminic acid derivative, in its α-configuration, was the only structural component of the S. fredii HH103 KPS and, to the best of our knowledge, has never been reported from any other rhizobial LPS. We also show that Pse5NAc7(3OHBu) is the complete or partial epitope for a mAb, NB6-228.22, that can recognize the HH103 LPS, but not those of most of the S. fredii strains tested here. We also show that the LPS from HH103 rkpM is identical to that of HH103 rkpA but devoid of any Pse5NAc7(3OHBu) residues. Notably, this rkpM mutant was severely impaired in symbiosis with its host, Macroptilium atropurpureum.
18) ALG12-CDG N-glycome discovers unusual hybrid and high mannose glycans
F.Esposito, S.Bianca, D.Garozzo, A.Messina, A.Palmigiano, L.Sturiale, C.Barone, A.Novelli, J.Jaeken, R.Barone
EurocabXX, June 30 - July 4, 2019 Leiden (Netherland)
- 2019
19) ALG12-CDG: novel glycophenotype insights endorse the molecular defect
L.Sturiale, S.Bianca, D.Garozzo, A.Terracciano, E.Agolini, A.Messina, A.Palmigiano, F.Esposito, Ch.Barone, A.Novelli, A.Fiumara, J.Jaeken, R.Barone
Glycoconjugate journal
36,
461-472
- 2019
DOI:
https://doi.org/10.1007/s10719-019-09890-2
Congenital disorders of glycosylation (CDG) are genetic diseases characterized by deficient synthesis (CDG type I) and/or abnormal processing (CDG type II) of glycan moieties linked to protein and lipids. The impact of the molecular defects on protein glycosylation and in turn on the clinical phenotypes of patients with CDG is not yet understood. ALG12-CDG is due to deficiency of ALG12 α1,6-mannosyltransferase that adds the eighth mannose residue on the dolichol-PP-oligosaccharide precursor in the endoplasmic reticulum. ALG12-CDG is a severe multisystem disease associated with low to deficient serum immunoglobulins and recurrent infections. We thoroughly investigated the glycophenotype in a patient with novel ALG12 variants and immunodeficiency. We analyzed serum native transferrin, as first line test for CDG and we profiled serum IgG and total serum N-glycans by a combination of consolidated (N-glycan analysis by MALDI MS) and innovative mass spectrometry-based protocols, such as GlycoWorks RapiFluor N-glycan analysis coupled with LC-ESI MS. Intact serum transferrin showed, as expected for a CDG type I defect, underoccupancy of N-glycosylation sites. Surprisingly, total serum proteins and IgG N-glycans showed some specific changes, consisting in accumulating amounts of definite high-mannose and hybrid structures. As a whole, ALG12-CDG behaves as a dual CDG (CDG-I and II defects) and it is associated with distinct, abnormal glycosylation of total serum and IgG N-glycans. Glycan profiling of target glycoproteins may endorse the molecular defect unraveling the complex clinical phenotype of CDG patients.
20) The cerebrospinal fluid (CSF) N-glycome as a novel biomarker of Parkinson’s disease. A mass spectrometry-based CSF n-glycosylation study of patients affected by Parkinson’s disease
A.Palmigiano, A.Messina, F.Esposito, R.Barone, G.Mostile, A.Nicoletti, L.Sturiale, D.Romeo, D.Garozzo, M.Zappia
Glycoconjugate journal
- 2019
Parkinson’s Disease (PD) is a clinically heterogeneous, multifactorial, age-related neurodegenerative disorder. PD is characterized by some pathological features such as cytoplasmic Lewy bodies accumulation in substantia nigra pars compacta, loss of dopaminergic neurons, inflammation, mitochondrial dysfunctions, that lead to neuronal degeneration and death. Glycosylation is a common post-translational protein modification with multiple biological functions. Glycosylation changes have been recently found in serum of patients with PD. However, N-glycosylation profiling in PD cerebrospinal fluid (CSF glycome) is still almost unexplored. We aimed to study CSF glycome in PD in order to identify potential glycosylation changes associated with PD. We performed Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI MS) CSF N-glycosylation analysis of released and permethylated N-glycans from a cohort including 19 PD patients and 19 control subjects (age- and gender-matched). Control CSF spectra were characterized by a base peak at 2792.4 Da, corresponding to a biantennary A2 complex N-glycan. Spectra from PD-CSF denote a significant increase of high-mannose 5 (M5, m/z 1579.8), agalactosylated biantennary (G0, m/z 1661.8), agalactosylated bisected biantennary (G0B, m/z 1906.9) bisected, agalactosylated core fucosylated N-glycans (G0BF, m/z 2081.0). CSF N-glycome could shed new light on the pathological mechanisms that lead to the disease development and progression in PD.
21) HILIC-MS based glycomics in the diagnosis of Congenital Disorders of Glycosylation
F.Esposito, A.Palmigiano, A.Messina, R.Barone, L.Sturiale, D.Garozzo
Glycoconjugate journal
- 2019
N-glycans are often studied to discover disease-associated biomarkers, as well as to identify changing in glycomic profiles supporting diagnosis of those disorders directly connected to impaired glycan biosynthesis of glycoconjugates, such as congenital disorders of glycosylation (CDG). Strategies based on liquid chromatography (LC) coupled preferentially to electrospray ionization (ESI) - mass spectrometry (MS) have emerged as powerful analytical methods for N-glycan identification and characterization. To enhance detection sensitivity, glycans are commonly functionalized with a tag prior to LC-MS analysis. Since the majority of the techniques used for glycan derivatization are notoriously time-consuming, some commercial analytical kits were developed to perform a rapid deglycosylation and labelling of N-glycans linked specifically to monoclonal antibodies (mAbs). Adopting one of these commercial kits, RapiFluor-MS (RFMS), through a slightly modified protocol, we performed the enzymatic release and the N-glycan labelling of total serum and single serum glycoproteins from selected CDG patients (MAN1B1-CDG, ALG12-CDG, MOGS-CDG, TMEM199-CDG). Hydrophilic Interaction Chromatography (HILIC)-UPLC-ESI-MS separation of derivatized N-glycans allowed us to differentiate either structural isomers and isomers differing for the linkage type, as hybrid glycan structures accumulating in N-glycan profile of MAN1B1-CDG and ALG12-CDG. According to patient immunological phenotype, serum IgG analysis of the Glucosidase 1-deficient patient (MOGS-CDG) showed significant N-glycosylation differences compared to control as the occurrence of the accumulating Glc3-Man7-GlcNAc2 glycoform (in accordance with the molecular defect) and, moreover, a generalized increase of sialylation. Serum N-glycan profiling of TMEM199-CDG showed, aside an increased amount of undersialylated biantennary structures, also an overall increase of the triantennary N-glycan component. All these applications demonstrated that RFMS method coupled to HILIC-UPLC-ESI-MS, represents a sensitive high throughput approach for serum N-glycome analysis and a valuable option for glycan detection and separation particularly for isomeric species.
22) Combined mass spectrometry methods for serum N-glycoprotein profiling enhance the awareness of the molecular patho-mechanism in ALG12-CDG
L.Sturiale, S.Bianca, A.Terracciano, E.Agolini, A.Messina, A.Palmigiano, F.Esposito, C.Barone, A.Novelli, A.Fiumara, D.Garozzo, J.Jaeken, R.Barone
Glycoconjugate journal
- 2019
Congenital disorders of glycosylation (CDG) are inherited metabolic diseases affecting the glycan biosynthesis of glycoconjugates. They represent an expanding group of multisystemic diseases with variable phenotypes and prevalent neurological involvement. More than 120 genetic disorders have been associated to defective glycosylation, mainly in the protein N-glycosylation pathway. Among these, CDG type I (CDG-I) occur in the cytosol or in the endoplasmic reticulum (ER) affecting dolichol-linked oligosaccharide synthesis, whereas CDG type II (CDG-II) involve the N-linked oligosaccharide processing in the Golgi. ALG12-CDG (MIM: 607143) is caused by mutations of the human orthologue of the yeast asparagine-linked glycosylation (ALG)12 gene, encoding the mannosyltransferase which adds in the ER an α-mannosyl residue to the core Man(α1-6) of the dolichol-PP-oligosaccharide precursor, thus ensuring its correct shape and branching before the oligosyltransferase (OST) complex action. Nine patients with ALG12-CDG have been reported so far. We investigated by MALDI MS and UHPLC-ESI MS the glyco-phenotype of a novel patient with unreported variants, sharing most of the clinical signs of patients with ALG12 deficiency, including severe recurrent infections with hypogammaglobulinemia and B cell dysfunction. MALDI MS analysis of native transferrin showed underoccupancy of N-glycosylation sites, a typical feature of CDG-I. N-glycome analysis by MALDI MS, either on total serum N-glycan pool and on IgG released N-glycans, revealed the abnormal occurrence of high-mannose and hybrid N-glycan species. These accumulating glycoforms, further analyzed by UHPLC-ESI MS, corresponded to unbranched structures with α1,2-terminal mannose residues, previously identified in serum of patients with MAN1B1-CDG (CDG-II defect). Glycosylation analyses on the observed ALG12-CDG patient revealed a combination of CDG-I and CDG-II defects, these last associated with abnormal IgG N-glycan profile, consistent with the immunophenotype. Glycan characterization of target glycoproteins may endorse the molecular defect unraveling the complex clinical phenotype of CDG patients.
23) CSF N-Glycoproteomics Using MALDI MS Techniques in Neurodegenerative Diseases
A.Messina, A.Palmigiano, R.O.Bua, D.Romeo, R.Barone, L.Sturiale, M.Zappia, D.Garozzo
Cerebrospinal Fluid (CSF) Proteomics
2044,
255-272
- 2019
CSF diagnostics has proved to be a formidable testing ground for N-glycoproteomic analysis of neurological diseases.
To characterize specific N-glycan profiles of CSF in early and advanced phases of Alzheimer’s disease, as well as in lysosomal storage disorders such as Tay-Sachs disease, we set up in our lab a robust and feasible protocol by coupling bioanalytical methods and mass spectrometry analysis.
Starting from a few microliters of CSF, after protein denaturation, reduction, and alkylation, N-glycans are released from glycoproteins using the peptide-N-glycosidase F (PNGase F) and purified. The analysis of permethylated N-glycans by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and MALDI-TOF MS/MS allowed us to identify specific glyco-structures and also to distinguish between isobaric N-glycans.
24) SLC35A2-CDG: Functional characterization, expanded molecular, clinical, and biochemical phenotypes of 30 unreported Individuals
B.G.Ng, P.Sosicka, S.Agadi, M.Almannai, C.A.Bacino, R.Barone, L.D.Botto, J.E.Burton, C.Carlston, B.Hon-Yin Chung, J.S.Cohen, D.Coman, K.M.Dipple, N.Dorrani, W.B.Dobyns, A.F.Elias, L.Epstein, W.A.Gahl, D.Garozzo, T.B.Hammer, J.Haven, D.Héron, M.Herzog, G.E.Hoganson, J.M.Hunter, M.Jain, J.Juusola, S.Lakhani, H.Lee, J.Lee, K.Lewis, N.Longo, C.Marques Lourenço, C.C.Y.Mak, D.McKnight, B.A.Mendelsohn, C.Mignot, G.Mirzaa, W.Mitchell, H.Muhle, S.F.Nelson, M.Olczak, C.G.S.Palmer, A.Partikian, M.C.Patterson, T.M.Pierson, S.C.Quinonez, B.M.Regan, M.E.Ross, M.J.Guillen Sacoto, F.Scaglia, I.E.Scheffer, D.Segal, N.Shah Singhal, P.Striano, L.Sturiale, J.D.Symonds, S.Tang, E.Vilain, M.Willis, L.A.Wolfe, H.Yang, S.Yano, Z.Powis, S.F.Suchy, J.A.Rosenfeld, A.C.Edmondson, S.Grunewald, H.H.Freeze
Human Mutation
40(7),
908-925
- 2019
DOI:
https://doi.org/10.1002/humu.23731
Pathogenic de novo variants in the X-linked gene SLC35A2 encoding the major Golgi-localized UDP-galactose transporter required for proper protein and lipid glycosylation cause a rare type of congenital disorder of glycosylation known as SLC35A2-congenital disorders of glycosylation (CDG; formerly CDG-IIm). To date, 29 unique de novo variants from 32 unrelated individuals have been described in the literature. The majority of affected individuals are primarily characterized by varying degrees of neurological impairments with or without skeletal abnormalities. Surprisingly, most affected individuals do not show abnormalities in serum transferrin N-glycosylation, a common biomarker for most types of CDG. Here we present data characterizing 30 individuals and add 26 new variants, the single largest study involving SLC35A2-CDG. The great majority of these individuals had normal transferrin glycosylation. In addition, expanding the molecular and clinical spectrum of this rare disorder, we developed a robust and reliable biochemical assay to assess SLC35A2-dependent UDP-galactose transport activity in primary fibroblasts. Finally, we show that transport activity is directly correlated to the ratio of wild-type to mutant alleles in fibroblasts from affected individuals.
25) Characterization of the Salmonella Typhimurium core oligosaccharide and its reducing end 3-deoxy-D-manno-oct-2-ulosonic acid used for conjugate vaccine production
G.De Benedetto, F.Micoli, S.Londero, L.Salvini, L.Sturiale, D.Garozzo, N.Ravenscroft, C.Giannelli, P.Cescutti
Carbohydrate Research
481,
43-51
- 2019
DOI:
https://doi.org/10.1016/j.carres.2019.05.014
One of the strategies adopted for the development of a bivalent conjugate vaccine against invasive nontyphoidal
Salmonella consists of linking the O-antigen component of
S.Typhimurium and
S.Entertidis lipopolysaccharides to the carrier protein CRM
197, a non-toxic variant of diphtheria toxin. The conjugation reaction uses the reducing end residue 3-deoxy-D-
manno-oct-2-ulosonic acid (Kdo) of the core to which the O-antigen chain is bound (OAg-core). OAg-core chains are cleaved from the lipid A directly in the fermentation broth by mild acid treatment. Kdo has been reported to undergo structural changes under these conditions and therefore the Kdo at the reducing end was thoroughly analysed to verify its structural integrity. For this purpose, low molecular mass OAg-core chains extracted from
S.Typhimurium wild type bacteria and core oligosaccharides extracted from
S.Typhimurium bacteria mutated not to produce O-antigen repeats were characterized by GLC-MS, MALDI-TOF-MS and NMR spectroscopy. Moreover, a combination of
1H-
1H and
1H-
13C experiments confirmed the linkage positions, sequence and structure of the octasaccharide core with 5-linked Kdo present at the reducing end in its native structure: α-Glc
pNAc-(1→2)-α-Glc
p-(1→2)-α-Gal
p-(1→3)-[α-Gal
p-(1→6)]-α-Glc
p-(1→3)-[α-Hep
p-(1→7)]-α-Hep
p-(1→3)-α-Hep
p-(1→5)-Kdo.
26) The N-glycan structures of the antigenic variants of chlorovirus PBCV-1 major capsid protein help to identify the virus-encoded glycosyltransferases
I.Speciale, G.A.Duncan, L.Unione, I.V.Agarkova, D.Garozzo, J.Jiménez-Barbero, S.Lin, T.L.Lowary, A.Molinaro, E.Noel, M.E.Laugieri, M.G.Tonetti, J.L.Van Etten, C.De Castro
The Journal of Biological Chemistry
294(14),
5688-5699
- 2019
DOI:
https://doi.org/10.1074/jbc.RA118.007182
The chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1) is a large dsDNA virus that infects the microalga Chlorella variabilis NC64A. Unlike most other viruses, PBCV-1 encodes most, if not all, of the machinery required to glycosylate its major capsid protein (MCP). The structures of the four N-linked glycans from the PBCV-1 MCP consist of nonasaccharides, and similar glycans are not found elsewhere in the three domains of life. Here, we identified the roles of three virus-encoded glycosyltransferases (GTs) that have four distinct GT activities in glycan synthesis. Two of the three GTs were previously annotated as GTs but the third GT was identified in this study. We determined the GT functions by comparing the wild-type glycan structures from PBCV-1 with those from a set of PBCV-1 spontaneous GT genes mutants resulting in antigenic variants having truncated glycan structures. According to our working model, the virus gene a064r encodes a GT with three domains: domain 1 has a β-L-rhamnosyltransferase activity, domain 2 has an α-L-rhamnosyltransferase activity and domain 3 is a methyltransferase that decorates two positions in the terminal α-L-rhamnose (Rha) unit. The a075l gene encodes a β-xylosyltransferase that attaches the distal D-xylose (Xyl) unit to the L-fucose (Fuc) that is part of the conserved N-glycan core region. Lastly, gene a071r encodes a GT that is involved in the attachment of a semiconserved element, α-D-Rha, to the same L-Fuc in the core region. Our results uncover GT activities that assemble four of the nine residues of the PBCV-1 MCP N-glycans.
27) Profilo glicomico delle sieroproteine in un paziente con deficit dell'enzima Glucosidasi I (MOGS-CDG)
F.Esposito, A.Palmigiano, A.Messina, R.Barone, L.Sturiale, G.Cantalupo, T.Lo Barco, G.Rodella, A.Fiumara, A.Bordugo, D.Garozzo
IX Congresso Nazionale SIMMESN - "Malattie Metaboliche Ereditarie: tra presente e futuro", 21-23 novembre 2018, Catania
- 2018
28) Biophysical Approaches to Solve the Structures of the Complex Glycan Shield of Chloroviruses
C.De Castro, G.A. Duncan, D.Garozzo, A.Molinaro, L.Sturiale, M.Tonetti, J.L. Van Etten
Glycobiophysics
1104,
237-257
- 2018
The capsid of
Paramecium bursaria chlorella virus (PBCV
-1) contains a heavily glycosylated major capsid protein, Vp54. The capsid protein contains four glycans, each N-linked to Asn.
The glycan structures are unusual in many aspects: (1) they are attached by a Β-glucose linkage, which is rare in nature; (2) they are highly branched and consist of 8-10 neutral monosaccharides; (3) all four glycoforms contain a dimethylated rhamnose as the capping residue of the main chain, a hyper-branched fucose residue and two rhamnose residues with opposite absolute configurations; (4) the four glycoforms differ by the nonstoichiometric presence of two monosaccharides, L-arabinose and D-mannose; (5) the N-glycans from all of the chloroviruses have a strictly conserved core structure; and (6) these glycans do not resemble any structures previously reported in the three domains of life. The structures of these N-glycoforms remained elusive for years because initial attempts to solve their structures used tools developed for eukaryotic-like systems, which we now know are not suitable for this noncanonical glycosylation pattern. This chapter summarizes the methods used to solve the chlorovirus complex glycan structures with the hope that these methodologies can be used by scientists facing similar problems.
29) Lipid A Structure and Immunoinhibitory Effect of the Marine Bacterium Cobetia pacifica KMM 3879T
F.Di Lorenzo, A.Palmigiano, S.Albitar-Nehme, M.Pallach, M.Kokoulin, N.Komandrova, L.Romanenko, M.L.Bernardini, D.Garozzo, A.Molinaro, A.Silipo
European Journal of Organic Chemistry
2018 (20-21),
2707-2716
- 2018
The structural elucidation of lipopolysaccharides (LPSs) from Gram-negative marine bacteria, along with the assessment of their immunological properties, is a fascinating and active research field. Such studies can aid understanding of adaptation phenomena that occur in the marine environment, but they can also open up new perspectives on the design and development of new immunoregulatory drugs. In this paper, we report the structural characterization of the lipid A component of the LPS isolated from the marine bacterium Cobetia pacifica KMM 3879T, which is characterized by a family of structures differing in their acylation patterns. The structural assignment was achieved through extensive chemical analysis and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Moreover, cellular immunology studies on the LPS highlighted its low immunostimulatory impact, as well as a very interesting and promising inhibitory activity of the toxic effects of Escherichia coli LPS.
30) Mutation and Suppressor Analysis of the Essential Lipopolysaccharide Transport Protein LptA Reveals Strategies To Overcome Severe Outer Membrane Permeability Defects in Escherichia coli
F.A.Falchi, E.A.Maccagni, S.Puccio, C.Peanoe, C.De Castro, A.Palmigiano, D.Garozzo, A.M.Martorana, A.Polissi, G.Dehò, P.Sperandeo
Journal of Bacteriology
200(7),
e00487-17
- 2018
DOI:
https://doi.org/10.1128/JB.00487-17
In Gram-negative bacteria, lipopolysaccharide (LPS) contributes to the robust permeability barrier of the outer membrane (OM), preventing the entry of toxic molecules, such as detergents and antibiotics. LPS is transported from the inner membrane (IM) to the OM by the Lpt multiprotein machinery. Defects in LPS transport compromise LPS assembly at the OM and result in increased antibiotic sensitivity. LptA is a key component of the Lpt machine that interacts with the IM protein LptC and chaperones LPS through the periplasm. We report here the construction of lptA41, a quadruple mutant in four conserved amino acids potentially involved in LPS or LptC binding. Although viable, the mutant displays increased sensitivity to several antibiotics (bacitracin, rifampin, and novobiocin) and the detergent SDS, suggesting that lptA41 affects LPS transport. Indeed, lptA41 is defective in Lpt complex assembly, and its lipid A carries modifications diagnostic of LPS transport defects. We also selected and characterized two phenotypic bacitracin-resistant suppressors of lptA41. One mutant, in which only bacitracin sensitivity is suppressed, harbors a small in-frame deletion in mlaA, which codes for an OM lipoprotein involved in maintaining OM asymmetry by reducing accumulation of phospholipids in the outer leaflet. The other mutant, in which bacitracin, rifampin, and SDS sensitivity is suppressed, harbors an additional amino acid substitution in LptA41 and a nonsense mutation in opgH, encoding a glycosyltransferase involved in periplasmic membrane-derived oligosaccharide synthesis. Characterization of the suppressor mutants highlights different strategies adopted by the cell to overcome OM defects caused by impaired LPS transport.
31) CSF N-Glycomics Using MALDI MS Techniques in Alzheimer’s Disease
A.Palmigiano, A.Messina, R.O.Bua, R.Barone, L.Sturiale, M.Zappia, D.Garozzo
Biomarkers for Alzheimer’s Disease Drug Development
,
75-91
- 2018
In this chapter, we present the methodology currently applied in our laboratory for the structural elucidation of the cerebrospinal fluid (CSF) N-glycome.
N-glycans are released from denatured carboxymethylated glycoproteins by digestion with peptide-N-glycosidase F (PNGase F) and purified using both C
18 Sep-Pak® and porous graphitized carbon (PGC) HyperSep
TM Hypercarb
TM solid-phase extraction (SPE) cartridges. The glycan pool is subsequently permethylated to increase mass spectrometry sensitivity. Molecular assignments are performed through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) analysis considering either the protein N-inked glycosylation pathway or MALDI TOF MS/MS data. Each stage has been optimized to obtain high-quality mass spectra in reflector mode with an optimal signal-to-noise ratio up to
m/z 4800. This method has been successfully adopted to associate specific N-glycome profiles to the early and the advanced phases of Alzheimer’s disease.
32) Hypoacylated LPS from Foodborne Pathogen Campylobacter jejuni
Induces Moderate TLR4-Mediated Inflammatory Response in Murine Macrophages
K.V.Korneev, A.N.Kondakova, E.N.Sviriaeva, N.A.Mitkin, A.Palmigiano, A.A.Kruglov, G.B.Telegin, M.S.Drutskaya, L.Sturiale, D.Garozzo, S.A.Nedospasov, Y.A.Knirel, D.V.Kuprash
Frontiers in Cellular and Infection Microbiology
8,
Art. 58
- 2018
Toll-like receptor 4 (TLR4) initiates immune response against Gram-negative bacteria upon specific recognition of lipid A moiety of lipopolysaccharide (LPS), the major component of their cell wall. Some natural differences between LPS variants in their ability to interact with TLR4 may lead to either insufficient activation that may not prevent bacterial growth, or excessive activation which may lead to septic shock. In this study we evaluated the biological activity of LPS isolated from pathogenic strain of Campylobacter jejuni, the most widespread bacterial cause of foodborne diarrhea in humans. With the help of hydrophobic chromatography and MALDI-TOF mass spectrometry we showed that LPS from a C. jejuni strain O2A consists of both hexaacyl and tetraacyl forms. Since such hypoacylation can result in a reduced immune response in humans, we assessed the activity of LPS from C. jejuni in mouse macrophages by measuring its capacity to activate TLR4-mediated proinflammatory cytokine and chemokine production, as well as NFκB-dependent reporter gene transcription. Our data support the hypothesis that LPS acylation correlates with its bioactivity.
33) Advanced LC-MS Methods for N-Glycan Characterization
A.Palmigiano, A.Messina, L.Sturiale, D.Garozzo
Comprehensive Analytical Chemistry . Chapter 6 (ISSN 0166-526X)
79,
147-172
- 2018
N-glycosylation is one of the main posttranslational modifications involving proteins that belong to all the three domains of life and plays a pivotal role in many biological functions, including intercellular signalling and recognition.
Glycosylation analysis in humans is an added value and a fundamental tool to characterize those genetic defects concerning glycan biosynthesis, such as congenital disorders of glycosylation. In addition, glycan alterations are associated with pathophysiology of major diseases as cancer and neurodegenerative dysfunctions. Concurrently, there is a growing interest of biopharmaceutical industries for the development of high-throughput methods for glycan analysis of therapeutic glycoproteins.N-linked glycans are commonly analyzed by liquid chromatography (LC) interfaced with fluorescence detection, but in the recent years a growing number of research laboratories extended the application by coupling LC with mass spectrometry (MS) techniques. Furthermore, LC-MS has become the primary device for smart high-throughput analyses.Here we report an outline of the prevailing LC-MS techniques set up for N-glycan analysis, paying special attention to N-glycomic characterization of human serum, as the most available and informative biological source for diagnosis and disease investigations.
34) An Unexplained Congenital Disorder of Glycosylation-II in a Child with Neurohepatic Involvement, Hypercholesterolemia and Hypoceruloplasminemia
P.L.Calvo, M.Spada, I.Rabbone, M.Pino, F.Porta, F.Cisarò, S.Reggiani, A.B.Cefalù, L.Sturiale, D.Garozzo, D.J.Lefeber, J.Jaeken
JIMD Reports
35,
1-7
- 2017
We report on a 12-year-old adopted boy with psychomotor disability, absence seizures, and normal brain MRI.
He showed increased (but initially, at 5 months, normal) serum cholesterol, increased alkaline phosphatases, transiently increased transaminases and hypoceruloplasminemia with normal serum and urinary copper. Blood levels of immunoglobulins, haptoglobin, antithrombin, and factor XI were normal. A type 2 serum transferrin isoelectrofocusing and hypoglycosylation of apoCIII pointed to a combined N- and O-glycosylation defect. Neither CDG panel analysis with 79 CDG-related genes, nor whole exome sequencing revealed the cause of this CDG. Whole genome sequencing was not performed since the biological parents of this adopted child were not available.
35) Neuraminidases 3 and 4 regulate neuronal function by catabolizing brain gangliosides
X.Pan, C.B.P.De Aragão, J.P.Velasco-Martin, D.A.Priestman, H.Y.Wu, K.Takahashi, K.Yamaguchi, L.Sturiale, D.Garozzo, F.M.Platt, N.Lamarche-Vane, C.R.Morales, T.Miyagi, A.V.Pshezhetsky
FASEB J.
31,
3467-3483
- 2017
Gangliosides (sialylated glycolipids) play an essential role in the CNS by regulating recognition and signaling in neurons. Metabolic blocks in processing and catabolism of gangliosides result in the development of severe neurologic disorders, including gangliosidoses manifesting with neurodegeneration and neuroinflammation. We demonstrate that 2 mammalian enzymes, neuraminidases 3 and 4, play important roles in catabolic processing of brain gangliosides by cleaving terminal sialic acid residues in their glycan chains. In neuraminidase 3 and 4 double-knockout mice, GM3 ganglioside is stored in microglia, vascular pericytes, and neurons, causing micro- and astrogliosis, neuroinflammation, accumulation of lipofuscin bodies, and memory loss, whereas their cortical and hippocampal neurons have lower rate of neuritogenesis in vitro Double-knockout mice also have reduced levels of GM1 ganglioside and myelin in neuronal axons. Furthermore, neuraminidase 3 deficiency drastically increased storage of GM2 in the brain tissues of an asymptomatic mouse model of Tay-Sachs disease, a severe human gangliosidosis, indicating that this enzyme is responsible for the metabolic bypass of β-hexosaminidase A deficiency. Together, our results provide the first in vivo evidence that neuraminidases 3 and 4 have important roles in CNS function by catabolizing gangliosides and preventing their storage in lipofuscin bodies
36) Structure of the Lipopolysaccharide from the Bradyrhizobium sp. ORS285 rfaL Mutant Strain
F.Di Lorenzo, A.Palmigiano, K.A.Duda, M.Pallach, N.Busset, L.Sturiale, E.Giraud, D.Garozzo, A.Molinaro, A.Silipo
ChemistryOpen
6,
541-553
- 2017
The importance of the outer membrane and of its main constituent, lipopolysaccharide, in the symbiosis between rhizobia and leguminous host plants has been well studied. Here, the first complete structural characterization of the entire lipopolysaccharide from an O-chain-deficient
Bradyrhizobium ORS285
rfaL mutant is achieved by a combination of chemical analysis, NMR spectroscopy, MALDI MS and MS/MS. The lipid A structure is shown to be consistent with previously reported
Bradyrhizobium lipid A, that is, a heterogeneous blend of penta- to hepta-acylated species carrying a nonstoichiometric hopanoid unit and possessing very-long-chain fatty acids ranging from 26:0(25-OH) to 32:0(31-OH). The structure of the core oligosaccharide region, fully characterized for the first time here, is revealed to be a nonphosphorylated linear chain with methylated sugar residues, with a heptose residue exclusively present in the outer core region, and with the presence of two singly substituted 3-deoxy-D-
manno-oct-2-ulosonic acid (Kdo) residues, one of which is located in the outer core region. The lipid A moiety is linked to the core moiety through an uncommon 4-substituted Kdo unit.
37) The Deep-Sea Polyextremophile Halobacteroides lacunaris TB21 Rough-Type LPS: Structure and Inhibitory Activity towards Toxic LPS
F.D.Lorenzo, A.Palmigiano, I.Paciello, M.Pallach, D.Garozzo, M.L.Bernardini, V.Cono, M.M.Yakimov, A.Molinaro, A.Silipo
Marine Drugs
15(7),
201
- 2017
The structural characterization of the lipopolysaccharide (LPS) from extremophiles has important implications in several biomedical and therapeutic applications. The polyextremophile Gram-negative bacterium Halobacteroides lacunaris TB21, isolated from one of the most extreme habitats on our planet, the deep-sea hypersaline anoxic basin Thetis, represents a fascinating microorganism to investigate in terms of its LPS component. Here we report the elucidation of the full structure of the R-type LPS isolated from H. lacunaris TB21 that was attained through a multi-technique approach comprising chemical analyses, NMR spectroscopy, and Matrix-Assisted Laser Desorption Ionization (MALDI) mass spectrometry. Furthermore, cellular immunology studies were executed on the pure R-LPS revealing a very interesting effect on human innate immunity as an inhibitor of the toxic Escherichia coli LPS.
38) MALDI-MS profiling of serum O-glycosylation and N-glycosylation in COG5-CDG
A.Palmigiano, R.O.Bua, R.Barone, D.Rymen, L. Régal, N.Deconinck, C.Dionisi-Vici, C.W.Fung, D.Garozzo, J.Jaeken, L.Sturiale
Journal of Mass Spectrometry
52(6),
372-377
- 2017
DOI:
https://doi.org/10.1002/jms.3936
Congenital disorders of glycosylation (CDG) are due to defective glycosylation of glycoconjugates. Conserved oligomeric Golgi (COG)-CDG are genetic diseases due to defects of the COG complex subunits 1-8 causing
N-glycan and
O-glycan processing abnormalities. In COG-CDG, isoelectric focusing separation of undersialylated glycoforms of serum transferrin and apolipoprotein C-III (apoC-III) allows to detect
N-glycosylation and
O-glycosylation defects, respectively. COG5-CDG (COG5 subunit deficiency) is a multisystem disease with dysmorphic features, intellectual disability of variable degree, seizures, acquired microcephaly, sensory defects and autistic behavior. We applied matrix-assisted laser desorption/ionization-MS for a high-throughput screening of differential serum
O-glycoform and
N-glycoform in five patients with COG5-CDG. When compared with age-matched controls, COG5-CDG showed a significant increase of apoC-III
0a (aglycosylated glycoform), whereas apoC-III
1 (mono-sialylated glycoform) decreased significantly. Serum
N-glycome of COG5-CDG patients was characterized by the relative abundance of undersialylated and undergalactosylated biantennary and triantennary glycans as well as slight increase of high-mannose structures and hybrid glycans. Using advanced and well-established MS-based approaches, the present findings reveal novel aspects on
O-glycan and
N-glycan profiling in COG5-CDG patients, thus providing an increase of current knowledge on glycosylation defects caused by impairment of COG subunits, in support of clinical diagnosis.
39) Xanthomonas citri pv. citri Pathotypes: LPS Structure and Function as Microbe-Associated Molecular Patterns
F.Di Lorenzo, A.Silipo, L.B. Andersen Gersby, A.Palmigiano, R.Lanzetta, D.Garozzo, C.Boyer, O.Pruvost, M.Newman, A.Molinaro
ChemBioChem
18(8),
772-781
- 2017
Xanthomonas citri pv. citri is the pathogen responsible for Asiatic citrus canker, one of the most serious citrus diseases worldwide. The lipopolysaccharide (LPS) molecule has been demonstrated to be involved in X. citri pv. citri virulence. Despite enormous progress in investigations of the molecular mechanisms for bacterial pathogenicity, determination of the detailed LPS structure-activity relationship is limited, as the current knowledge is mainly based on structural determination of one X. citri pv. citri strain. As X. citri pv. citri strains are distinguished into three main pathogenicity groups, we characterized the full structure of the LPS from two pathotypes that differ in their host-range specificity. This revealed an intriguing difference in LPS O-chain structure. We also tested the LPSs and isolated lipid A moieties for their ability to act as microbe-associated molecular patterns in Arabidopsis thaliana. Both LPS/lipid As induced ROS accumulation, but no difference was observed between the two pathotypes.
40) Recessive mutations in SLC35A3 cause early onset epileptic encephalopathy with skeletal defects
C.Marini, K.Hardies, T.Pisano, P.May, S.Weckhuysen, E.Cellini, A.Suls, D.Mei, R.Balling, P.D.Jonghe, I.Helbig, D.Garozzo, EuroEpiNOMICS, R.Guerrini
American Journal of Medical Genetics Part A
173(4),
1119-1123
- 2017
We describe the clinical and whole genome sequencing (WGS) study of a non-consanguineous Italian family in which two siblings, a boy and a girl, manifesting a severe epileptic encephalopathy (EE) with skeletal abnormalities, carried novel SLC35A3 compound heterozygous mutations. Both siblings exhibited infantile spasms, associated with focal, and tonic vibratory seizures from early infancy. EEG recordings showed a suppression-burst (SB) pattern and multifocal paroxysmal activity in both. In addition both had quadriplegia, acquired microcephaly, and severe intellectual disability. General examination showed distal arthrogryposis predominant in the hands in both siblings and severe left dorso-lumbar convex scoliosis in one. WGS of the siblings-parents quartet identified novel compound heterozygous mutations in SLC35A3 in both children. SLC35A3 encodes the major Golgi uridine diphosphate N-acetylglucosamine transporter. With this study, we add SLC35A3 to the gene list of epilepsies. Neurological symptoms and skeletal abnormalities might result from impaired glycosylation of proteins involved in normal development and function of the central nervous system and skeletal apparatus.
41) The Lipid A from Rhodopseudomonas palustris Strain BisA53 LPS Possesses a Unique Structure and Low Immunostimulant Properties
F.Di Lorenzo, A.Palmigiano, S.Al Bitar-Nehme, L.Sturiale, K.A.Duda, D.Gully, R.Lanzetta, E.Giraud, D.Garozzo, M.L.Bernardini, A.Molinaro, A.Silipo
Chemistry A European Journal
23(15),
3637-3647
- 2017
The search for novel lipid A analogues from any biological source that can act as antagonists, displaying inhibitory activity towards the production of pro-inflammatory cytokines, or as immunomodulators in mammals, is a very topical issue. To this aim, the structure and immunological properties of the lipopolysaccharide lipid A from the purple nonsulfur bacterium
Rhodopseudomonas palustris strain BisA53 have been determined. This lipid A displays a unique structural feature, with a non-phosphorylated skeleton made up of the tetrasaccharide Manρ-α-(1→4)-GlcρN3N-β-1→6-GlcρN3N-α-(1→1)-α-GalρA, and four primary amide-linked 14:0(3-OH) and, as secondary O-acyl substituents, a 16:0 and the very long-chain fatty acid 26:0(25-OAc), appended on the GlcρN3N units. This lipid A architecture is definitely rare, so far identified only in the genus
Bradyrhizobium. Immunological tests on both murine bone-marrow-derived and human monocyte-derived macrophages revealed an extremely low immunostimulant capability of this LPS lipid A.
42) Identification of N-glycan profiles in tears of vernal and atopic keratoconjunctivitis patients
A.Leonardi, A.Messina, A.Ruaro, A.La Gloria Valerio, D.Garozzo
Allergy
71(S102),
140
- 2016
43) Prevotella denticola Lipopolysaccharide from a Cystic Fibrosis Isolate Possesses a Unique Chemical Structure
F.Di Lorenzo, A.Silipo, Th.Matier, A.Hanuszkiewicz, J.S.Elborn, R.Lanzetta, L.Sturiale, A.Scamporrino, D.Garozzo, M.A.Valvano, M.M.Tunney, A.Molinaro
European Journal of Organic Chemistry
2016(9),
1732-1738
- 2016
We report the first complete structural characterization of the lipopolysaccharide (LPS) from a cystic fibrosis (CF) clinical isolate of Prevotella denticola (B003V1S1X). Chemical, spectroscopic, and spectrometric analyses revealed a unique rough-type LPS (LOS) structure. The structure has a highly negatively charged heptasaccharide core region containing hexoses, with the first two sugars, 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and mannose, highly phosphorylated. Furthermore, the lipid A moiety has the typical structure for the genus Prevotella, and was also highly phosphorylated.
44) CSF N-Glycan Profile Reveals Sialylation Deficiency in a Patient with
GM2 Gangliosidosis Presenting as Childhood Disintegrative Disorder
R.Barone, L.Sturiale, A.Fiumara, A.Palmigiano, R.O.Bua, R.Rizzo, M.Zappia, D.Garozzo
Autism Research
9,
423-428
- 2016
Protein N-glycosylation consists in the synthesis and processing of the oligosaccharide moiety (N-glycan) linked to a protein and it serves several functions for the proper central nervous system (CNS) development and function. Previous experimental and clinical studies have shown the importance of proper glycoprotein sialylation for the synaptic function and the occurrence of autism spectrum disorders (ASD) in the presence of sialylation deficiency in the CNS. Late-onset Tay Sachs disease (LOTSD) is a lysosomal disorder caused by mutations in the HEXA gene resulting in GM2-ganglioside storage in the CNS. It is characterized by progressive neurological impairment and high co-occurrence of psychiatric disturbances. We studied the N-glycome profile of the cerebrospinal fluid (CSF) in a 14 year-old patient with GM2- gangliosidosis (LOTSD). At the age of 4, the patient presented regressive autism fulfilling criteria for childhood disintegrative disorder (CDD). A CSF sample was obtained in the course of diagnostic work-up for the suspicion of an underlying neurodegenerative disorder. We found definite changes of CSF N-glycans due to a dramatic decrease of sialylated biantennary and triantennary structures and an increase of asialo-core fucosylated bisected N-glycans. No changes of total plasma N-glycans were found. Herein findings highlight possible relationships between the early onset psychiatric disturbance featuring CDD in the patient and defective protein sialylation in the CNS. In conclusion, the study first shows aberrant N-glycan structures of CSF proteins in LOTSD; unveils possible pathomechanisms of GM2-gangliosidosis; supports existing relationships between neuropsychiatric disorders and unproper protein glycosylation in the CNS.
45) The structure of the lipooligosaccharide from Xanthomonas oryzae pv. Oryzae: the causal agent of the bacterial leaf blight in rice
F.Di Lorenzo, A.Palmigiano, A.Silipo, Y.Desakic, D.Garozzo, R.Lanzetta, N.Shibuyac, A.Molinaro
Carbohydrate Research
427,
38-43
- 2016
The structure of the lipooligosaccharide (LOS) from the rice pathogen
Xanthomonas oryzae pv.
oryzae has been elucidated. The characterization of the core oligosaccharide structure was obtained by the employment of two chemical degradation protocols and by analysis of the products via NMR spectroscopy. The structure of the lipid A portion was achieved by MALDI mass spectrometry analysis on purified lipid A. The LOS from
Xanthomonas oryzae pv.
oryzae revealed to possess the same core structure of
Xanthomonas campestris pv.
campestris and interesting novel features on its lipid A domain. The evaluation of the biological activity of both LOS and isolated lipid A was also executed.
46) N-Linked Glycans of Chloroviruses Sharing a Core Architecture without Precedent
C.De Castro, I.Speciale, G.Duncan, D.D.Dunigan, I.Agarkova, R.Lanzetta, L.Sturiale, A.Palmigiano, D.Garozzo, A.Molinaro, M.Tonetti, J.L.Van Etten
Angewandte Chemie International Edition
55,
654-658
- 2016
DOI:
https://doi.org/10.1002/ange.201509150
N-glycosylation is a fundamental modification of proteins and exists in the three domains of life and in some viruses, including the chloroviruses, for which a new type of core N-glycan is herein described. This N-glycan core structure, common to all chloroviruses, is a pentasaccharide with a β-glucose linked to an asparagine residue which is not located in the typical sequon N-X-T/S. The glucose is linked to a terminal xylose unit and a hyperbranched fucose, which is in turn substituted with a terminal galactose and a second xylose residue. The third position of the fucose unit is always linked to a rhamnose, which is a semiconserved element because its absolute configuration is virus-dependent. Additional decorations occur on this core N-glycan and represent a molecular signature for each chlorovirus.
47) CSF N-glycoproteomics for early diagnosis in Alzheimer’s disease
A.Palmigiano, R.Barone, L.Sturiale, C.Sanfilippo, R.O.Bua, D.Romeo, A.Messina, M.L.Capuana, T.Maci, F.Le Pira, M.Zappia, D.Garozzo
Journal of Proteomics
131,
29-37
- 2016
DOI:
https://doi.org/10.1016/j.jprot.2015.10.006
This work aims at exploring the human CSF (Cerebrospinal fluid) N-glycome by MALDI MS techniques, in order to assess specific glycosylation pattern(s) in patients with Alzheimer’s disease (n:24) and in subjects with mild cognitive impairment (MCI) (n:11), these last as potential AD patients at a pre-dementia stage. For comparison, 21 healthy controls were studied. We identified a group of AD and MCI subjects (about 40-50% of the studied sample) showing significant alteration of CSF N-glycome profiling, consisting of a decrease in the overall sialylation degree and an increase in species bearing bisecting GlcNAc. Noteworthy, all the MCI patients that converted to AD within the clinical follow-up, had an abnormal CSF glycosylation profile. Based on the studied cohort, CSF glycosylation changes may occur before an AD clinical onset. Previous studies specifically focused on the key role of glycosyltransferase GnT-III on AD-pathogenesis, addressing the patho-mechanism to specific sugar modification of BACE-1 glycoprotein with bisecting GlcNAc. Our patients addressed protein N-glycosylation changes at an early phase of the whole biomolecular misregulation on AD, pointing to CSF N-glycome analyses as promising tool to enhance early detection of AD and also suggesting alternative therapeutics target molecules, such as specific glyco-enzymes.
48) Structural Relationship of the Lipid A Acyl Groups to Activation of Murine Toll-Like Receptor 4 by Lipopolysaccharides from Pathogenic Strains of Burkholderia mallei, Acinetobacter baumannii, and Pseudomonas aeruginosa
K.V.Korneev, N.P.Arbatsky, A.Molinaro, A.Palmigiano, R.Z.Shaikhutdinova, M.M.Shneider, G.B.Pier, A.N.Kondakova, E.N.Sviriaeva, L.Sturiale, D.Garozzo, A.A.Kruglov, S.A.Nedospasov, M.S.Drutskaya, Y.A.Knirel, D.V.Kuprash
Frontiers in Immunology
6
- 2015
Toll-like receptor 4 (TLR4) is required for activation of innate immunity upon recognition of lipopolysaccharide (LPS) of Gram-negative bacteria. The ability of TLR4 to respond to a particular LPS species is important since insufficient activation may not prevent bacterial growth while excessive immune reaction may lead to immunopathology associated with sepsis. Here, we investigated the biological activity of LPS from Burkholderia mallei that causes glanders, and from the two well-known opportunistic pathogens Acinetobacter baumannii and Pseudomonas aeruginosa (causative agents of nosocomial infections). For each bacterial strain, R-form LPS preparations were purified by hydrophobic chromatography and the chemical structure of lipid A, an LPS structural component, was elucidated by HR-MALDI-TOF mass spectrometry. The biological activity of LPS samples was evaluated by their ability to induce production of proinflammatory cytokines, such as IL-6 and TNF, by bone marrow-derived macrophages. Our results demonstrate direct correlation between the biological activity of LPS from these pathogenic bacteria and the extent of their lipid A acylation.
49) TEAR FLUID N-GLYCAN PROFILING TO INVESTIGATE BIOMARKERS IN VERNAL AND ATOPIC KERATOCONJUNCTIVITIS
A.Leonardi, C.Tosto, A.Messina, A.Palmigiano, R.O.Bua, D.Romeo, L.Sturiale, D.Garozzo
Investigative Ophthalmology & Visual Science
56(7),
5875
- 2015
Proteomics and glycogene-chips studies revealed that several tear proteins are glycosylated. Glycoproteins play a role in different function of the ocular surface including protection against pathogens, immunoregulation and prevention of desiccation. In the present study, we analyze the N-linked glycome of tear fluid from patients affected by vernal (VKC) and atopic keratoconjunctivitis (AKC) in order to identify potential biomarkers for these diseases. To date, no specific laboratory test are suitable for VKC and AKC diagnosis and monitoring.
Tear samples from 23 VKC patients, 7 AKC patients and 11 control subjects were diluted 1/10 in a denaturant solution, reduced, alkylated and treated with N-glycosidase F (PNGase F), an enzyme which specifically deglycosylates N-glycoproteins. Released N-glycans were purified, chemically derivate by permethylation and analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS and MALDI TOF/TOF MS/MS). The eligibility of mass spectrometry for the study of glycosylation is due to its high sensibility and ability to analyze complex mixtures of glycans from biological samples.
Our approach allowed to identify more than 150 complex N-glycans, including biantennary, triantennary, tetraantennary and bisecting species. Highly fucosylated structures were also found. Data analysis showed changes in terms of relative intensities for some structures. In VKC, the most significant peaks were related to mass to charge ratios 1906.7 and 2592.3, corresponding to a bisecting and to a N-glycan structure bearing two Lewis-X epitope, respectively.In AKC the most intense peak is found at m/z 2792.4, and corresponds to the well-known biantennary, disialylated N-glycan. Each structure reveals significant differences between healthy and pathological conditions, with major variations involving bisecting and hyperfucosylated glycoforms. Structures were confirmed in detail by MS/MS analysis.
MALDI-TOF-MS is a valuable technique for biomarker detection and characterization of defective glycan structures. Identified peaks could be used as potential biomarkers for VKC and AKC. Further structural characterization of the disease-associated N-glycans may confirm these preliminary results and allow to discover additional biomarkers.
50) Determination of the structure of the O-antigen and the lipid A from the entomopathogenic bacterium Pseudomonas entomophila lipopolysaccharide along with its immunological properties
I.Speciale, I.Paciello, L.Lembo Fazio, L.Sturiale, A.Palmigiano, R.Lanzetta, M.Parrilli, D.Garozzo, B.Lemaitre, M.L.Bernardini, A.Molinaro, C.De Castro
Carbohydrate Research
412,
20-27
- 2015
DOI:
https://doi.org/10.1016/j.carres.2015.04.017
The structure and the immunology of the lipopolysaccharide (LPS) of
Pseudomonas entomophila, an entomopathogenic bacterium isolated from the fruit fly
Drosophila melanogaster, was characterized. The O-antigen portion was established and resulted to be built up of a repetitive unit constituted by four monosaccharide residues, all L configured, all deoxy at C-6 and with an acetamido function at C-2: →3)-α-L-FucNAc-(1→4)-α-L-FucNAc-(1→3)-α-L-FucNAc-(1→3)-β-L-QuiNAc-(1→ The structural analysis of lipid A, showed a mixture of different species. The diphosphorylated glucosamine backbone carries six fatty acids consistent with the composition C10:0 3(OH), C12:0 2(OH) and C12:0 3(OH), whereas other species differs by the number of phosphates and/or of fatty acids. The immunology experiments demonstrated that the LPS structure of
P. entomophila displayed a low ability to engage the TLR4-mediated signaling correlated to a significant antagonistic activity toward hexaacylated LPS structures.
51) N-glycosylation of Chloroviruses: the end of a paradigm
C.De Castro, G.Duncan, I.Agarkova, I.Speciale, L.Sturiale, A.Palmigiano, R.Lanzetta, M.Parrilli, D.Garozzo, A.Molinaro, M.Tonetti, J.L.Van Etten
11th Jenner glycobiology and medicine Symposium, National Centre for Scientific Research (CNRS), Paris, France, 19-21 April 2015
- 2015
52) CSF N-glycan MS profiling may enable early diagnosis in some patients with Alzheimer Disease
L.Sturiale, R.Barone, A.Palmigiano, C.Sanfilippo, F.Le Pira, R.O.Bua, D.Romeo, M.Zappia, D.Garozzo
11th Jenner glycobiology and medicine Symposium, National Centre for Scientific Research (CNRS), Paris, France, 19-21 April 2015
- 2015
53) N-glycomics of tear fluid in patients with Vernal and Atopic Keratoconjunctivitis
A.Messina, C.L.Tosto, A.Palmigiano, L.Sturiale, A.Leonardi, D.Garozzo
11th Jenner glycobiology and medicine Symposium, National Centre for Scientific Research (CNRS), Paris, France, 19-21 April 2015
- 2015
54) A nationwide survey of PMM2-CDG in Italy: high frequency
of a mild neurological variant associated with the L32R mutation
R.Barone, M.Carrozzi, R.Parini, R.Battini, D.Martinelli, M.Elia, M.Spada, F.Lilliu, G.Ciana, A.Burlina, V.Leuzzi, M.Leoni, L.Sturiale, G. Matthijs, J.Jaeken, M.Di Rocco, D.Garozzo, A.Fiumara
Journal of Neurology
,
154-164
- 2015
PMM2-CDG (PMM2 gene mutations) is the most common congenital disorder of N-glycosylation. We conducted a nationwide survey to characterize the frequency, clinical features, glycosylation and genetic correlates in Italian patients with PMM2-CDG. Clinical information was obtained through a questionnaire filled in by the referral physicians including demographics, neurological and systemic features, neuroimaging data and genotype. Glycosylation analyses of serum transferrin were complemented by MALDI-Mass Spectrometry (MALDI-MS). Between 1996 and 2012, data on 37 Italian patients with PMM2-CDG were collected. All the patients with a severe phenotype were unable to walk unaided, 84 % had severe intellectual disability and 81 % microcephaly. Conversely, among 17 mildly affected patients 82 % had independent ambulation, 64 % had borderline to mild intellectual disability and 35 % microcephaly. Epilepsy and stroke-like events did not occur among patients with the mild phenotype. The rate and extent of systemic involvement were more pronounced in severely affected patients. The L32R misfolding mutation of the PMM2 gene occurred in 70 % of the patients with the mild phenotype and was associated with a less severe underglycosylation of serum Tf at MALDI-MS analyses. Despite their different disease severity, all patients had progressive (olivo)ponto-cerebellar atrophy that was the hallmark clinical feature for the diagnosis. A mild neurological phenotype of PMM2-CDG marked by preserved ambulatory ability and autonomy and associated with L32R mutation is particularly frequent in Italy. PMM2-CDG should be considered in patients with even mild developmental disability and/or unexplained progressive cerebellar atrophy.
55) Persistent cystic fibrosis isolate Pseudomonas aeruginosa strain RP73 exhibits an under-acylated LPS structure responsible of its low inflammatory activity
F.Di Lorenzo, A.Silipo, I.Bianconi, N.I.Loré, A. Scamporrino, L.Sturiale, D.Garozzo, R.Lanzetta, M.Parrilli, A.Bragonzi, A.Molinaro
Molecular Immunology
63,
166-175
- 2015
Pseudomonas aeruginosa, the major pathogen involved in lethal infections in cystic fibrosis (CF) population, is able to cause permanent chronic infections that can persist over the years. This ability to chronic colonize CF airways is related to a series of adaptive bacterial changes involving the immunostimulant lipopolysaccharide (LPS) molecule. The structure of LPSs isolated from several P. aeruginosa strains showed conserved features that can undergo chemical changes during the establishment of the chronic infection. In the present paper, we report the elucidation of the structure and the biological activity of the R-LPS (lipooligosaccharide, LOS) isolated from the persistent CF isolate P. aeruginosa strain RP73, in order to give further insights in the adaptation mechanism of the pathogen in the CF environment. The complete structural analysis of P. aeruginosa RP73 LOS was achieved by chemical analyses, NMR spectroscopy and MALDI MS spectrometry, while the assessment of the biological activity was attained testing the in vivo pro-inflammatory capacity of the isolated LOS molecule. While a typical CF LPS is able to trigger a high immune response and production of pro-inflammatory molecules, this P. aeruginosa RP73 LOS showed to possess a low pro-inflammatory capacity. This was possible due to a singular chemical structure possessing an under-acylated lipid A very similar to the LPS of P. aeruginosa found in chronic lung diseases such as bronchiectstasis.
56) Covalently linked hopanoid-lipid A improves outer-membrane resistance of a Bradyrhizobium symbiont of legumes
A.Silipo, G.Vitiello, D.Gully, L.Sturiale, C.Chaintreuil, J.Fardoux, D.Gargani, H.I.Lee, G.Kulkarni, N.Busset, R.Marchetti, A.Palmigiano, H.Moll, R.Engel, R.Lanzetta, L.Paduano, M.Parrilli, W.S.Chang, O.Holst, D.K.Newman, D.Garozzo, G.D’Errico, E.Giraud, A.Molinaro
Nature Communications
5,
5106
- 2014
DOI:
https://doi.org/10.1038/ncomms6106
Lipopolysaccharides (LPSs) are major components of the outer membrane of Gram-negative bacteria and are essential for their growth and survival. They act as a structural barrier and play an important role in the interaction with eukaryotic hosts. Here we demonstrate that a photosynthetic
Bradyrhizobium strain, symbiont of
Aeschynomene legumes, synthesizes a unique LPS bearing a hopanoid covalently attached to lipid A. Biophysical analyses of reconstituted liposomes indicate that this hopanoid-lipid A structure reinforces the stability and rigidity of the outer membrane. In addition, the bacterium produces other hopanoid molecules not linked to LPS. A hopanoid-deficient strain, lacking a squalene hopene cyclase, displays increased sensitivity to stressful conditions and reduced ability to survive intracellularly in the host plant. This unusual combination of hopanoid and LPS molecules may represent an adaptation to optimize bacterial survival in both free-living and symbiotic states.
57) Thermophiles as Potential Source of Novel Endotoxin Antagonists: the Full Structure and Bioactivity of theLipo-oligosaccharide from Thermomonas hydrothermalis
F.Di Lorenzo, I.Paciello, L.L.Fazio, L.Albuquerque, L.Sturiale, M.S.Da Costa, R.Lanzetta, M.Parrilli, D.Garozzo, M.L.Bernardini, A.Silipo, A.Molinaro
Chembiochem
15(14),
2146-2155
- 2014
Thermomonas hydrothermalis is a Gram-negative thermophilic bacterium that is able to live at 50 °C. This ability is attributed to chemical modifications, involving those to bacterial cell-wall components, such as proteins and (glyco)lipids. As the main component of the outer membrane of Gram-negative bacteria, lipopolysaccharides (LPSs) are exposed to the environment, thus they can undergo structural chemical changes to allow thermophilic bacteria to live at their optimal growth temperature. Furthermore, as one of the major target of the eukaryotic innate immune system, LPS elicits host immune response in a structure-dependent mode; thus the uncommon chemical features of thermophilic bacterial LPSs might exert a different biological action on the innate immune system-an antagonistic effect, as shown in studies of LPS structure-activity relationship in the ongoing research into antagonist LPS candidates. Here, we report the complete structural and biological activity analysis of the lipo-oligosaccharide isolated from Thermomonas hydrothermalis, achieved by a multidisciplinary approach (chemical analysis, NMR, MALDI MS and cellular immunology). We demonstrate a tricky and interesting structure combined with a very interesting effect on human innate immunity.
58) Tear Fluid N-Glycan Profiling To Investigate Biomarkers In Vernal Keratoconjunctivitis
C.L.Tosto, A.Messina, A.Palmigiano, R.O.Bua, A.Scamporrino, L.Sturiale, A.Leonardi, D.Garozzo
XIV Convegno-Scuola sulla Chimica dei Carboidrati, Pontignano (Si), 22-25 Giugno 2014
- 2014
The tears covering the ocular surface are an important component of the extracellular environment of the superficial epithelial cell layer. Secretory and plasma contributions result in a fluid containing lipids, carbohydrates, proteins, and electrolytes which can affect the activity of the cells of the ocular surface
1. Different studies, based on proteomics and glycogene-chips, revealed several tear proteins are glycosylated, such as lacritin and proline rich protein 1. Mucins and carbohydrate-binding proteins were found to be among the most highly expressed glycogenes in human conjunctiva. These glycoproteins play a role in protection against pathogens and prevention of ocular surface desiccation
2,3.
Figure 1. MALDI-TOF analysis of permethylated N-glycans from control human tear sample
Figure 2. MALDI-TOF analysis of permethylated N-glycans from pathological human tear sample
In the present study, we analyze the N-linked glycome of tear fluid from patients affected by vernal keratoconjunctivitis (VKC). VKC is a bilateral, chronic sight-threatening and severe inflammatory ocular disease mainly occurring in children and young male adults. Its predominant symptom is intense ocular itching, associated with tearing, mucous stringy discharge, severe photophobia, blepharospasm and foreign body sensation. Severe and inappropriately treated VKC can lead to severe ocular complications such as corneal ulcers and scarring, corticosteroids-induced glaucoma and visual impairment. To date, no specific laboratory test is suitable for VKC diagnosis and monitoring. Tear samples of VKC patients and control subjects were treated with N-glycosidase F (PNGase F), an enzyme which specifically deglycosylates N-glycoprotein. Released N-glycans are purified and chemically derivatized by permethylation to improve the sensitivity of detection of molecular ions. Samples are analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). It is a valuable technique for biomarker detection and characterization of defective glycan structures. The eligibility of mass spectrometry for the study of glycosylation is due to its high sensibility and ability to analyze complex mixtures of glycans from biological samples. This approach allowed to distinguish tear fluid glycosylation pattern of VKC patients from control subjects (
Figures 1 and
2).
Spectra show tear fluid is rich in N-glycan structures. Data analysis evidences changes in terms of relative intensities for some structures. Preliminary study suggests the most significant peaks are related to mass to charge ratios 1906.7, 2592.3 and 2765.9. Each one of these three structures to 2966.5 ratios reveal meaningful changes between healthy and pathological conditions, as evidenced by the following histogram: These peaks could be used as potential biomarkers for VKC disease. Further structural characterization of the disease-associated N-glycans could confirm these preliminary results and allow to discover additional biomarkers.
- 1) Zhou L., Beuerman R. W., Foo Y., Liu S., Ang L. P., Tan D. T. "Characterisation of human tear proteins using high-resolution mass spectrometry". Ann. Acad. Med. Singapore 2006, 35 (6), 400-407
- 2) Li N., Wang N., Zheng J., Liu X. M., Lever O. W., Erickson P. M., Li L. A. "Characterization of human tear proteome using multiple proteomic analysis techniques". J. Proteome Res. 2005, 4 (6), 2052-2061
- 3) Argüeso P. "Glycobiology of the ocular surface: mucins and lectins". Jpn J Ophthalmol. 2013 Mar, 57(2), 150-155
59) Tay-Sachs CSF N-Glycan Profile by MALDI MS Reveals Incomplete Glycosylation
A.Scamporrino, A.Palmigiano, R.O.Bua, A.Messina, C.L.Tosto, A.Fiumara, R.Barone, L.Sturiale, D.Garozzo
XIV Convegno-Scuola sulla Chimica dei Carboidrati, Pontignano (Si), 22-25 Giugno 2014
- 2014
60) Red blood cell N-glycan profiling by MALDI-MS
R.O.Bua, C.L.Tosto, A.Palmigiano, A.Scamporrino, A.Messina, R.Barone, L.Sturiale, D.Garozzo
XIV Convegno-Scuola sulla Chimica dei Carboidrati, Pontignano (Si), 22-25 Giugno 2014
- 2014
Protein glycosylation is one of the most common post-translational modifications in mammalian cells, estimated to be found on over 50% of all human proteins. Changes in N-linked glycosylation have long been associated with disease development, and acquired glycan modifications have been described in multifactor diseases such as cancer and inflammatory disorders. The characterization of protein glycosylation is often a challenge due to the heterogeneity of glycoforms. MS-based methods are capable of structural characterization of unknown glycans and high-throughput analysis of known glycan structures. Due to the advancement in MS techniques, MALDI-TOF mass spectrometry has become an eligible technique for the study of glycan populations derived from biological samples (1). As red blood cell (RBC) glycosylated membrane proteins can be directly isolated from blood, purified and readily available, glycosylation analysis of erythrocyte membrane glycoproteins might be the key for detection of glyco-biomarkers for diagnostic and therapeutic purposes. There are few studies that investigated N-glycosylation of erythrocyte membrane glycoproteins using MS techniques, all referred to congenital dyserythropoietic anemia type II (CDA II), also called hereditary erythroblastic multinuclearity with positive acidified-serum test (HEMPAS). Fukunda et al. (2) in 1987 developed a method based on fast-atom bombardment (FAB) MS, whereas Denecke et al. (3) in 2008 compared erythrocyte band 3 mass mapping from HEMPAS with that from control by MALDI-TOF MS following SDS-PAGE and lectin-binding strategies. Both of these studies found accordingly unprocessed oligosaccharides (high mannose, hybrid, and truncated complex species) in the red blood cell membrane glycoproteins from HEMPAS patients suggesting the disease is caused by a defect disturbing Golgi processing in erythroblasts. In the present study, we reported the application of high-sensitivity MALDI MS-based glycomic methodologies to the analysis of N-linked glycans derived from human erythrocyte membrane proteins in order to produce a fingerprint of erythrocyte N-glycans. The obtained spectrum (see figure) contains a full complement of high-mannose type structures and a series of complex glycans comprising a mixture of bi- tri- and tetra-antennary structures. The main peaks correspond to fucosylated mono and disialo- biantennary oligosaccharides with bisecting N-acetylglucosamine (GlcNAc). Moreover, due to the extension of lactosamine repeating units, glycans larger than 8 kDa were detected. Our developed MS strategy led to a considerable improvement in upper mass range sensitivity and in signal-to-noise ratio, in addition to a substantive increases in resolution of MALDI-TOF mass spectra, allowing the detailed mapping of N-glycans derived from human erythrocyte membrane proteins. These results could provide a new tool to detect potential biomarkers, or allow the recognition of distinct glycosylation changes associated with disease conditions.
- 1) Barone R, Sturiale L, Garozzo D. 2009. Mass spectrometry in the characterization of human genetic N- glycosylation defects. Mass Spectrometry Reviews 28:517-542.
- 2) Fukuda MN, Dell A, Scartezzini P. 1987. Primary Defect of Congenital Dyserythropoietic Anemia Type II. Failure in glycosylation of erythrocyte lactosaminoglycan protein caused by lowered N-acetyl-glycosaminyltrasferase II. JBC 262:7195-7206
- 3) Denecke J, Kranz C, Nimtz M, Conradt HS, Brune T, Heimpel H, Marquardt T. 2008. Characterization of N- glycosylation phenotype of erythrocyte membrane proteins in congenital dyserythropoietic anemia type II (CDA II/HEMPAS). Glycoconj J. 25:375-382
61) Cerebrospinal Fluid N-Glycomics May Enable Early Diagnosis in Alzheimer Disease
A.Palmigiano, R.O.Bua, A.Messina, C.L.Tosto, A.Scamporrino, L.Sturiale, R.Barone, F.Le Pira, M.Zappia, D.Garozzo
XIV Convegno-Scuola sulla Chimica dei Carboidrati, Pontignano (Si), 22-25 Giugno 2014
- 2014
The ability to predict the conversion from MCI to AD is one of the most important research topic in the field of neurodegenerative diseases; in particular it may represent an useful method for the diagnosis of early stages of AD. Several studies have been made in this regard by exploiting the diagnostic power of various predictors as: functional and neuropsychological measures, CSF biomarkers (ρTau, Aβ 42), magnetic resonance imaging data (MRI).
In our study we compare MALDI MS CSF N-glycan profiles from eight control subjects, 11 MCI and 25 AD patients.
Control spectra were characterized by a base peak at 2792 m/z corresponding to a biantennary A2 complex N-glycan.
AD glycol-profiles were classified according to the ratio of NGA2FB/A2 glycans: we named AD1 profiles those characterized by a NGA2FB to A2 quotient over 50% (11 AD patients). AD2 profiles (14 AD patients) exhibited glycosylation patterns with a NGA2FB relative intensity below 50%. Of particular note, these last patients gave, in the majority of cases, a glycosylation ratio below the mean value from controls (36.06 ± 5.47).
Interestingly, MCI patients exhibiting AD1 spectra profile conversed to AD (5 patients out of 6, the 6th patient refused monitoring) while MCI patients showing AD2 spectra profile don’t converse to AD (no conversion for 5 patients out of 5).
CSF N-glycome profile could shed new light on the different mechanisms that lead to the development of AD even if those mechanism are still unclear.
62) N-Linked Glycans From PBCV-1 and Related Viruses Have A New Type Of Core Region
I.Speciale, D.Garozzo, J.R.Gurnon, R.Lanzetta, A.Molinaro, A.Palmigiano, M.Parrilli, F.Piacente, L.Sturiale, M.G.Tonetti, J.L.Van Etten, C.De Castro
XIV Convegno-Scuola sulla Chimica dei Carboidrati, Pontignano (Si), 22-25 Giugno 2014
- 2014
63) Coffee enhances the expression of chaperones and antioxidant proteins in rats with nonalcoholic fatty liver disease
F.Salomone, G.Li Volti, P.Vitaglione, F.Morisco, V.Fogliano, A.Zappalà, A.Palmigiano, D.Garozzo, N.Caporaso, G.D’Argenio, F.Galvano
Translational Research
163,
593-602
- 2014
Coffee consumption is inversely related to the degree of liver injury in patients with nonalcoholic fatty liver disease (NAFLD). Molecular mediators contributing to coffee’s beneficial effects in NAFLD remain to be elucidated. In this study, we administrated decaffeinated espresso coffee or vehicle to rats fed an high-fat diet (HFD) for 12 weeks and examined the effects of coffee on liver injury by using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) proteomic analysis combined with mass spectrometry. Rats fed an HFD and water developed panacinar steatosis, lobular inflammation, and mild fibrosis, whereas rats fed an HFD and coffee exhibited only mild steatosis. Coffee consumption increased liver expression of the endoplasmic reticulum chaperones glucose-related protein 78 and protein disulfide-isomerase A3; similarly, coffee drinking enhanced the expression of the mitochondrial chaperones heat stress protein 70 and DJ-1. Furthermore, in agreement with reduced hepatic levels of 8-isoprostanes and 8-hydroxy-2’-deoxyguanosine, proteomic analysis showed that coffee consumption induces the expression of master regulators of redox status (i.e., peroxiredoxin 1, glutathione S-transferase α2, and D-dopachrome tautomerase). Last, proteomics revealed an association of coffee intake with decreased expression of electron transfer flavoprotein subunit α, a component of the mitochondrial respiratory chain, involved in de novo lipogenesis. In this study, we were able to identify by proteomic analysis the stress proteins mediating the antioxidant effects of coffee; moreover, we establish for the first time the contribution of specific coffee-induced endoplasmic reticulum and mitochondrial chaperones ensuring correct protein folding and degradation in the liver.
64) Identification of human tear fluid biomarkers in vernal keratoconjunctivitis using iTRAQ quantitative proteomics
A.Leonardi, A.Palmigiano, E.Mazzola, A.Messina, E.Milazzo, M.Bortolotti, D.Garozzo
Allergy
69(2),
254-260
- 2014
DOI:
https://doi.org/10.1111/all.12331
BACKGROUND: Understanding and treating vernal keratoconjunctivitis (VKC) has been a challenge because the pathogenesis is unclear and antiallergic therapy often unsuccessful. The aim of the study was to analyze peptide profiles in human tears using mass spectrometry to elucidate compositional differences between healthy subjects and patients affected by VKC.
METHODS: Tears were collected from healthy subjects and VKC patients. Digested samples were treated with iTRAQ (isobaric tag for relative and absolute quantitation). Separation of tryptic peptides was realized using a MicroHPLC interfaced with a microfraction collector. MS and MS/MS mass spectra were performed using a MALDI TOF/TOF 4800 Applied Biosystem spectrometer. Protein Pilot
TM software with Paragon
TM algorithm v4.1.46 or GPS
TM with Mascot engine was used as search engines with SwissProt or IPI human as the databases.
RESULTS: A significant number of peptides were examined, and 78 proteins were successfully identified. In all VKC samples, levels of serum albumin, transferrin, and hemopexin were found up to 100 times higher than control tear levels and correlated to the severity of disease. Hemopexin, transferrin, mammaglobin B, and secretoglobin 1D were found significantly over-expressed in VKC samples compared with the control samples. Tear samples from patients treated with topical cyclosporine or corticosteroids showed a dramatic reduction in these protein levels.
CONCLUSIONS:LC MALDI MS and isobaric tag for relative and absolute quantitation technique may be useful in the quantitative and qualitative characterization of the peptidoma of human tears. These techniques may identify target proteins to be used in the diagnosis and management of VKC and other inflammatory ocular surface conditions.
65) CSF N-GLYCANS PROFILES TO INVESTIGATE BIOMARKERS IN ALZHEIMER’S DISEASE
A.Palmigiano, R.Barone, C.Tosto, R.O.Bua, L.Sturiale, F.Le Pira, M.Zappia, D.Garozzo
17th European Carbohydrate Symposium (July 7 -11 2013 Tel-Aviv, Israel)
- 2013
Alzheimer’s disease (AD) is the most common cause of dementia in the elderly, it’s a progressive, irreversible, degenerative brain disorder, resulting in memory, thinking and behavior impairments; the causes of AD are currently being researched but no definite answer exists yet, moreover there’s a lack of accurate diagnostic method for AD [1].
The composition of Cerebrospinal fluid (CSF) is rapidly and directly influenced by the brain, so CSF represents an appealing source for diagnostic biomarkers potentially able to describe neuropathological state and trajectory [2].
CSF N-glycans are characterized by large portions of bisecting N-acetylglucosammine while in blood such glycoforms cannot persist due to the existence of specific hepatic clearance mechanisms. Bisecting N-glycans are so called "brain type"[3]. Elevated expression levels of N-acetylglucosaminyl transferase III (GnT-III), the glycosyltransferase responsible for synthesizing a bisecting GlcNAc residue, may have a protective effect on β-amyloid production in AD [4].
We have analyzed a total of 35 individual CSF samples: 10 controls (from patients who underwent to minor orthopedic surgery), 13 AD samples and 12 from other neurodegenerative diseases such as mild cognitive impairment (MCI), Parkinson’s disease (PD) and Tay-Sachs disease. The number of analyzed samples is still limited but there are important differences between pathological and control spectra with a dramatic enhancement of bisecting structures in AD patients’ spectra expecially for the fucosylated ones. In particular NGA2FB glycan (asyalo, agalacto, bisected biantennary fucosylated) is over-expressed in AD patients. In order to differentiate between isobaric serum and brain type N-glycan structures we perform a target MALDI MSMS analysis.
[1] R. Barone et al. Journal of Proteomics 75, 5123-5139, 2012
[2] R.J. Perrin et al. PLoS ONE 6, 1, e16032
[3] A. Hoffmann et al. FEBS Letters 359, 164-168, 1995
[4] K. Akasaka-Manya et al. Glycobiology 20, 1, 99-106, 2010
66) CSF N-GLYCANS PROFILES TO INVESTIGATE BIOMARKERS IN ALZHEIMER’S DISEASE
A.Palmigiano, R.Barone, C.Tosto, R.O.Bua, L.Sturiale, F.Le Pira, M.Zappia, D.Garozzo
International symphosium on chemical glycobiology (June 29-July 1 2013 Shanghai, China)
- 2013
Alzheimer’s disease (AD) is the most common cause of dementia in the elderly, it’s a progressive, irreversible, degenerative brain disorder, resulting in memory, thinking and behavior impairments; the causes of AD are currently being researched but no definite answer exists yet, moreover there’s a lack of accurate diagnostic method for AD [1]. The composition of Cerebrospinal fluid (CSF) is rapidly and directly influenced by the brain, so CSF represents an appealing source for diagnostic biomarkers potentially able to describe neuropathological state and trajectory [2]. CSF N-glycans are characterized by large portions of bisecting N-acetylglucosammine while in blood such glycoforms cannot persist due to the existence of specific hepatic clearance mechanisms. Bisecting N-glycans are so called "brain type"[3]. Elevated expression levels of N-acetylglucosaminyl transferase III (GnT-III), the glycosyltransferase responsible for synthesizing a bisecting GlcNAc residue, may have a protective effect on β-amyloid production in AD [4]. We have analyzed a total of 35 individual CSF samples: 10 controls (from patients who underwent to minor orthopedic surgery), 13 AD samples and 12 from other neurodegenerative diseases such as mild cognitive impairment (MCI), Parkinson’s disease (PD) and Tay-Sachs disease. The number of analyzed samples is still limited but there are important differences between pathological and control spectra with a dramatic enhancement of bisecting structures in AD patients’ spectra expecially for the fucosylated ones. In particular NGA2FB glycan (asyalo, agalacto, bisected biantennary fucosylated) is over-expressed in AD patients. In order to differentiate between isobaric serum and brain type N-glycan structures we perform a target MALDI MSMS analysis.
67) Structure of very unusual N-Glycans Associated with the Major Capsid Protein of Chlorovirus PBCV-1: Nature can synthesize a different class of complex N-Glycans
C.De Castro, A.Molinaro, M.Tonetti, J.Gurnonc, L.Sturiale, A.Palmigiano, D.Garozzo, J. L. Van Etten
International symphosium on chemical glycobiology (June 29-July 1 2013 Shanghai, China)
- 2013
The envelope protein Vp54 from the prototype chlorovirus, Paramecium bursaria chlorella virus (PBCV-1) comprises ~40% of total virus protein and presents four N-glycosylated sites.
In depth chemical, spectroscopical, and spectrometrical analysis has disclosed the structure of four different N-linked oligosaccharides and the type of substitution at each glycosylation site. Vp54 glycosylation is unusual in many respects: i) a β-glucose, and not an aminosugar, is connected to the protein, ii) N-linked glycans are not located in a typical N-X-(T/S) consensus site, iii) four glycoforms are present. The structure of four glycoforms share a common core region and differences are related to the non-stoichiometric presence of two monosaccharides. The more abundant species comprises nine neutral monosaccharide residues, organized in a highly branched fashion; among the most relevant features, a dimethylated rhamnose as capping residue of the main chain, a hyperbranched fucose unit, and two rhamnose residues with opposite absolute configuration deserve to be mentioned.
This protein glycosylation is new and different from what so far reported in the three domains of life. Considering that Chloroviruses and other members of the family Phycodnaviridae have a long evolutionary history possibly dating back to the time when eukaryotes arose from prokaryotes, we suggest that the chlorovirus glycosylation pathway is ancient, possibly existing prior to the development of the ER and Golgi, and involves new and still unexplored mechanisms.
68) MAN1B1 Deficiency: An Unexpected CDG-II
D.Rymen, R.Peanne, M.B.Millón, V.Race, L.Sturiale, D.Garozzo, P.Mills, P.Clayton, C.G.Asteggiano, D.Quelhas, A.Cansu, E.Martins, M.C.Nassogne, M.Gonçalves-Rocha, H.Topaloglu, J.Jaeken, F.Foulquier, G.Matthijs
Plos Genetics
9,
1-13
- 2013
Congenital disorders of glycosylation (CDG) are a group of rare metabolic diseases, due to impaired protein and lipid glycosylation. In the present study, exome sequencing was used to identify MAN1B1 as the culprit gene in an unsolved CDGII patient. Subsequently, 6 additional cases with MAN1B1-CDG were found. All individuals presented slight facial dysmorphism, psychomotor retardation and truncal obesity. Generally, MAN1B1 is believed to be an ER resident alpha-1,2-mannosidase acting as a key factor in glycoprotein quality control by targeting misfolded proteins for ER-associated degradation (ERAD). However, recent studies indicated a Golgi localization of the endogenous MAN1B1, suggesting a more complex role for MAN1B1 in quality control. We were able to confirm that MAN1B1 is indeed localized to the Golgi complex instead of the ER. Furthermore, we observed an altered Golgi morphology in all patients’ cells, with marked dilatation and fragmentation. We hypothesize that part of the phenotype is associated to this Golgi disruption. In conclusion, we linked mutations in MAN1B1 to a Golgi glycosylation disorder. Additionally, our results support the recent findings on MAN1B1 localization. However, more work is needed to pinpoint the exact function of MAN1B1 in glycoprotein quality control, and to understand the pathophysiology of its deficiency.
69) Mutations in SLC35A3 cause autism spectrum disorder, epilepsy and arthrogryposis
S.Edvardson, A.Ashikov, C.Jalas, L.Sturiale, A.Shaag, A.Fedick, N.R.Treff, D.Garozzo, R.Gerardy-Schahn, O.Elpeleg
Journal of Medical Genetics
50(11),
733-739
- 2013
Background The heritability of autism spectrum disorder is currently estimated at 55%. Identification of the molecular basis of patients with syndromic autism extends our understanding of the pathogenesis of autism in general. The objective of this study was to find the gene mutated in eight patients from a large kindred, who suffered from autism spectrum disorder, arthrogryposis and epilepsy.
Methods and results By linkage analysis and exome sequencing, we identified deleterious mutations in SLC35A3 in these patients. SLC35A3 encodes the major Golgi uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) transporter. In Golgi vesicles isolated from patient fibroblasts the transport of the respective nucleotide sugar was significantly reduced causing a massive decrease in the content of cell surface expressed highly branched N-glycans and a concomitant sharp increase of lower branched glycoforms.
Conclusions Spontaneous mutation in SLC35A3 has been discovered in cattle worldwide, recapitulating the human phenotype with arthrogryposis and additional skeletal defects known as Complex Vertebral Malformation syndrome. The skeletal anomalies in the mutant cattle and in our patients, and perhaps even the neurological symptoms are likely the consequence of the lack of high-branched N-glycans and the concomitant abundance of lower-branched glycoforms at the cell surface. This pattern has previously been associated with growth arrest and induction of differentiation. With this study, we add SLC35A3 to the gene list of autism spectrum disorders, and underscore the crucial importance of UDP-GlcNAc in the regulation of the N-glycan branching pathway in the Golgi apparatus.
70) Structure of N-linked oligosaccharides attached to chlorovirus PBCV-1 major capsid protein reveals unusual class of complex N-glycans
C.De Castro, A.Molinaro, F.Piacente, J.R.Gurnon, L.Sturiale, A.Palmigiano, R.Lanzetta, M.Parrilli, D.Garozzo, M.G.Tonetti, J.L.Van Etten
Proceedings of the National Academy of Sciences of The United States of America
110,
13956-13960
- 2013
DOI:
https://doi.org/10.1073/pnas.1313005110
The major capsid protein Vp54 from the prototype chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1) contains four Asnlinked glycans. The structure of the four N-linked oligosaccharides and the type of substitution at each glycosylation site was determined by chemical, spectroscopic, and spectrometric analyses. Vp54 glycosylation is unusual in many ways, including: (i) unlike most viruses, PBCV-1 encodes most, if not all, of the machinery to glycosylate its major capsid protein; (ii) the glycans are attached to the protein by a β-glucose linkage; (iii) the Asn-linked glycans are not located in a typical N-X-(T/S) consensus site; and (iv) the process probably occurs in the cytoplasm. The four glycoforms share a common core structure, and the differences are related to the nonstoichiometric presence of two monosaccharides. The most abundant glycoform consists of nine neutral monosaccharide residues, organized in a highly branched fashion. Among the most distinctive features of the glycoforms are (i) a dimethylated rhamnose as the capping residue of the main chain, (ii) a yperbranched fucose unit, and (iii) two rhamnose residues with opposite absolute configurations. These glycoforms differ from what has been reported so far in the three domains of life. Considering that chloroviruses and other members of the family Phycodnaviridae may have a long evolutionary history, we suggest that the chlorovirus glycosylation pathway is ancient, possibly existing before the development of the endoplasmic reticulum and Golgi pathway, and involves still unexplored mechanisms.
71) Mannosidase I deficiency: An unexpected CDG-II with intellectual disability and dysmorphic features
G.Matthijs, D.Rymen, R.Péanne, V.Race, L.Sturiale, D.Garozzo, P.Mills, P.Clayton, J.Jaeken, F.Foulquier
European Human Genetics Conference 2013 location:Paris, France (June 8-11, 2013)
21,
29-29
- 2013
72) Chemistry and Biology of the Potent Endotoxin from a Burkholderia dolosa Clinical Isolate from a Cystic Fibrosis
Patient
F.Di Lorenzo, L.Sturiale, A.Palmigiano, L.Lembo-Fazio, I.Paciello, C.P.Coutinho, I.S -Correia, M.L.Bernardini, R.Lanzetta, D.Garozzo, A.Silipo, A.Molinaro
ChemBioChem
14,
1105-1115
- 2013
This is the first report of the chemical and biological properties of the lipooligosaccharide (LOS) endotoxin isolated from Burkholderia dolosa IST4208, an isolate recovered from a cystic fibrosis (CF) patient in a Portuguese CF center. B. dolosa is a member of the Burkholderia cepacia complex, a group of closely related species that are highly problematic and opportunistic pathogens in CF. B. dolosa infection leads to accelerated loss of lung function and decreased survival. The structural determination of its endotoxin was achieved using a combination of chemistry and spectroscopy, and has revealed a novel endotoxin structure. The purified LOS was tested for its immunostimulatory activity on human HEK 293 cells expressing TLR-4, MD-2, and CD-14. In these assays, the LOS showed strong proinflammatory activity.
73) Structure and Immunological Activity of the Lipopolysaccharide Isolated from
the Species Alkalimonas delamerensis
A.Silipo, F.Di Lorenzo, L.Lembo Fazio, I.Paciello, L.Sturiale, A.Palmigiano, M.Parrilli, W.D.Grant, D.Garozzo, R.Lanzetta, M.L.Bernardini, A.Molinaro
European Journal of Organic Chemistry
2013(13),
2653-2665
- 2013
We have defined the complete structure and assessed the biological activity of the lipopolysaccharide (LPS) from Alkalimonas delamerensis, an alkaliphilic bacterium isolated from soda lakes in China and East Africa. The structural determination, which was achieved by the use of chemical, spectroscopic and spectrometric approaches, indicates a novel Rough type lipopolysaccharide that is rich in negatively charged groups in the lipid A inner core region. The proinflammatory activity of the pure lipopolysaccharide has also been defined by testing the LPS as a stimulant in human HEK293 cells, which express the CD14/MD2/TLR4 complex (HEK293-hTLR4), and in bone marrow-derived macrophages.
74) Bone Dysplasia as a Key Feature in Three Patients with a Novel Congenital Disorder of Glycosylation (CDG)
Type II Due to a Deep Intronic Splice Mutation in TMEM165
R.Zeevaert,F.De Zegher, L.Sturiale, D.Garozzo, M.Smet, M.Moens, G.Matthijs, J. Jaeken
JIMD Reports
8,
145-152
- 2013
Three patients belonging to two families presented with a psychomotor-dysmorphism syndrome including postnatal growth deficiency and major spondylo-, epi-, and metaphyseal skeletal involvement.
Other features were muscular hypotrophy, fat excess, partial growth hormone deficiency, and, in two of the three patients, episodes of unexplained fever. Additional investigations showed mild to moderate increases of serum transaminases (particularly of aspartate transaminase (AST)), creatine kinase (CK), and lactate dehydrogenase (LDH), as well as decreased coagulation factors VIII, IX, XI, and protein C. Diagnostic work-up revealed a type 2 serum transferrin isoelectrofocusing (IEF) pattern and a cathodal shift on apolipoprotein C-III IEF pointing to a combined N- and O-glycosylation defect. Known glycosylation disorders with similar N-glycan structures lacking galactose and sialic acid were excluded. Through a combination of homozygosity mapping and expression profiling, a deep intronic homozygous mutation (c.792 + 182G>A) was found in TMEM165 (TPARL) in the three patients. TMEM165 is a gene of unknown function, possibly involved in Golgi proton/calcium transport. Here we present a detailed clinical description of the three patients with this mutation. The TMEM165 deficiency represents a novel type of CDG (TMEM165-CDG). This disorder enlarges the group of CDG caused by deficiencies in proteins that are not specifically involved in glycosylation but that have functions in the organization and homeostasis of the intracellular compartments and the secretory pathway, like COG-CDG and ATP6V0A2-CDG.
75) Maldi mass spectrometry for the structural investigation of intact lipopolysaccharides
A.Silipo, A.Molinaro, R.Lanzetta, M.Parrilli, L.Sturiale, A.Messina, A.Palmigiano, D.Garozzo, A.Scamporrino
Atti XIII Convegno-Scuola sulla Chimica dei Carboidrati 24-27 giugno 2012 Certosa di Pontignano (Siena)
,
PC-19
- 2012
Lipopoligosaccharides (LPS) are powerful Gram-negative glycolipids that evade the immune system and invade host animal and vegetal cells. The structural elucidation of LPS is pivotal to understanding the mechanisms of infection at the molecular level. The structural nature of LOS has been the main obstacle for structural analysis by matrix-assisted laser desorption/ ionization (MALDI) mass spectrometry (MS). Recently1,2, we have published strategies able to obtain MALDI mass spectra of Lipooligosaccharides (LOS) to reveal the fine chemical structure with minimal structural variations. The high-quality MALDI mass spectra show LOS species characteristic of molecular ions and defined fragments due to decay in the ion source. The in-source decay yields B-type ions, which correspond to core oligosaccharide(s), and Y-type ions, which are related to lipid A unit(s). MALDI tandem time-of-flight (TOF/TOF) MS of lipid A allowed for the elucidation of its structure directly from purified intact LOS without the need for any chemical manipulations. These findings constitute a significant advancement in the analysis of such an important biomolecule by MALDI MS. Here, we?ll show our preliminary results on MALDI MS of intact LPS. From these spectra (figure 1) it is possible to obtain the mw of the lipid a moiety, of the core oligosaccharides and of the o-chain repeating unit.
76) Identification of N-Glycosylation in the CSF in Patients with Neurodegenerative diseases
A.Palmigiano, R.Barone, M.Zappia, L.Sturiale, D.Garozzo
Atti XIII Convegno-Scuola sulla Chimica dei Carboidrati 24-27 giugno 2012 Certosa di Pontignano (Siena)
,
OC-15
- 2012
Neurodegenerative diseases are defined as hereditary and sporadic conditions which are characterized by progressive nervous system dysfunction. These disorders are often associated with atrophy of the affected central or peripheral structures of the nervous system. They include diseases such as Alzheimer’s Disease and other dementias, Brain Cancer, Degenerative Nerve Diseases, Encephalitis, Epilepsy, Genetic Brain Disorders, Head and Brain Malformations, Hydrocephalus, Stroke, Parkinson’s Disease, Multiple Sclerosis, Amyotrophic Lateral Sclerosis (ALS or Lou Gehrig’s Disease), Huntington’s Disease, Prion Diseases, Creutzfeld-Jakob disease (CJD) and others. The prevalence of neurodegenerative disorders is increasing, but diagnostic biomarkers and effective treatments are lacking. Cerebrospinal fluid (CSF) is produced from the choroid plexus within the ventricles of the brain and circulates through the ventricular system and around the outside of brain; because its composition is rapidly and directly influenced by the brain, CSF represents an appealing source for diagnostic biomarkers. Glycosylation is one of the most common and most complex post-translational modifications (PTMs) of secreted and cell surface proteins and plays a critical role in protein-protein, protein-cell, and cell-cell interactions including antibody binding, protein degradation, cellular endocytosis, and protease protection. Glycosylation is cell-type specific and reproducible for a given physiological state. Glycoproteins occur usually as a collection of glycoforms that differ in the structure of the component oligosaccharides. Glycoform abundance and glycan structures can be altered significantly in disease, resulting in a characteristic signature as a means of investigating complex pathophysiological processes and revealing novel biomarkers and drug targets. Here we describe a promising strategy for discovering N-glycan biomarkers in individual CSF in patients with neurodegenerative diseases based on analyzing total glycan profiles rather than the sugars on particular glycoproteins.
77) Identification of N-Glycosylation in the CSF in Patients with Alzheimer’s disease by MALDI Mass Spectrometry
A.Palmigiano, R.Barone, M.Zappia, L.Sturiale, D.Garozzo
Massa 2012 Congresso della Divisione di Spettrometria di Massa della Società Chimica Italiana (SCI) 1-5 Luglio 2012, Mondello (PA)
- 2012
Neurodegenerative diseases are defined as hereditary and sporadic conditions which are characterized by progressive nervous system dysfunction. These disorders are often associated with atrophy of the affected central or peripheral structures of the nervous system. They include diseases such as Alzheimer’s Disease and other dementias, Brain Cancer, Degenerative Nerve Diseases, Encephalitis, Epilepsy, Genetic Brain Disorders, Head and Brain Malformations, Hydrocephalus, Stroke, Parkinson's Disease, Multiple Sclerosis, Amyotrophic Lateral Sclerosis (ALS or Lou Gehrig's Disease), Huntington’s Disease, Prion Diseases, Creutzfeld-Jakob disease (CJD) and others. The prevalence of neurodegenerative disorders is increasing, but diagnostic biomarkers and effective treatments are lacking. Cerebrospinal fluid (CSF) is produced from the choroid plexus within the ventricles of the brain and circulates through the ventricular system and around the outside of brain; because its composition is rapidly and directly influenced by the brain, CSF represents an appealing source for diagnostic biomarkers. Glycosylation is one of the most common and most complex post-translational modifications (PTMs) of secreted and cell surface proteins and plays a critical role in protein-protein, protein-cell, and cell-cell interactions including antibody binding, protein degradation, cellular endocytosis, and protease protection. Glycosylation is cell-type specific and reproducible for a given physiological state. Glycoproteins occur usually as a collection of glycoforms that differ in the structure of the component oligosaccharides. Glycoform abundance and glycan structures can be altered significantly in disease, resulting in a characteristic signature as a means of investigating complex pathophysiological processes and revealing novel biomarkers and drug targets. Here we describe a promising strategy for discovering N-glycan biomarkers in individual CSF in patients with neurodegenerative diseases based on analyzing total glycan profiles rather than the sugars on particular glycoproteins.
78) Deficiency of subunit 6 of the conserved oligomeric golgi complex (COG6-CDG): second patient, different phenotype
P.J.Goyens, S.Huybrechts , C.De Laet, P.Bontems, S.Rooze, H.Souayah , Y.Sznajer, L.Sturiale, D.Garozzo, G.Matthijs, A.Ferster, J.Jaeken
Journal of Inherited metabolic disease
35,
S114
- 2012
CDG-II due to defects in one of the eight subunits of the COG-complex represent a novel group of protein glycosylation disorders. We describe a 42-month-old girl with COG6 deficiency. First child of healthy consanguineous Moroccan parents, she presented at birth with dysmorphic features: microcephaly, polydactyly, broad palpebral fissures, retrognathia and anal anteposition. The clinical phenotype was further characterised by multiorgan involvement including mild psychomotor retardation, inflammatory bowel disease, micronodular liver cirrhosis, proximal tubulopathy, associated with life-threatening and recurrent infections due to combined Tand B-cell dysfunction and neutrophil dysfunction. No bleeding tendency was observed. Mutation analysis showed the patient to be homozygous for the c.G1646T mutation in the COG6 gene. She is the second reported patient with COG6 deficiency and carrier of the same mutation as the index patient (Lübbehusen et al, 2010). Although both patients are homozygous for the same mutation, they present a markedly different clinical picture. Indeed immunodeficiency as well as inflammatory bowel disease has not been described previously in patients with any COG-CDG. Mutation c.G1646T produces instability of mRNA rather than a degradation of mutated protein; individual factors might therefore interfere with the degradation speed of mRNA and contribute to the inter-individual phenotypical variation.
79) DPM2-CDG: A Muscular Dystrophy-Dystroglycanopathy Syndrome with Severe Epilepsy
R.Barone, Ch.Aiello, V.Race, E.Morava, F.Foulquier, M.Riemersma, Ch.Passarelli, D.Concolino, M.Carella, F.Santorelli, W.Vleugels, E.Mercuri, D.Garozzo, L.Sturiale, S.Messina, J.Jaeken, A.Fiumara, R.A.Wevers, E.Bertini, G.Matthijs, D. J.Lefeber
Annals of Neurology
72,
550-558
- 2012
Objective:
Congenital disorders of glycosylation (CDG) are a group of metabolic diseases due to defects in protein and lipid glycosylation. We searched for the primary defect in 3 children from 2 families with a severe neurological phenotype, including profound developmental delay, intractable epilepsy, progressive microcephaly, severe hypotonia with elevated blood creatine kinase levels, and early fatal outcome. There was clinical evidence of a muscular dystrophy-dystroglycanopathy syndrome, supported by deficient O-mannosylation by muscle immunohistochemistry.
Methods:
Biochemical and molecular methods were combined to pinpoint the defect in the glycosylation pathway in the endoplasmic reticulum.
Results:
Metabolic investigations revealed CDG-I, pointing to a defect in protein N-glycosylation in the endoplasmic reticulum. Analysis of lipid-linked oligosaccharides in fibroblasts showed accumulation of Dol-PP-GlcNAc2-Man5. DNA analysis revealed mutations in DPM2, 1 of the subunits of the dolichol-phosphate-mannose (DPM) synthase; the patient in the first family is compound heterozygous for 2 mutations (c.68A>G, predicting a missense mutation p.Y23C and c.4-1G>C, a splice mutation), whereas the patients in the second family are homozygous for the same missense mutation (c.68A>G, p.Y23C).
Interpretation:
We describe a new CDG, due to a deficiency of DPM2. Hence, mutations have now been described in the genes for the 3 subunits of DPM: DPM1, DPM2, and DPM3, whereby DPM2-CDG links the congenital disorders of glycosylation to the congenital muscular dystrophies.
80) Glycomics of pediatric and adulthood diseases of the central nervous system
R.Barone , L.Sturiale, A.Palmigiano, M. Zappia, D.Garozzo
Journal of Proteomics
75,
5123-5139
- 2012
Glycosylation consists in the covalent linkage of a carbohydrate structure to membrane bound and secreted glycoconjugates. It is a common post-translational modification that serves multiple functions in cell differentiation, signaling and intercellular communication. Unlike DNA/RNA/protein, the addition of complex carbohydrates is not-template driven and it is conceivable that both genetics and environmental factors might interact to influence glycosylation machinery in several pathological processes. Over the last few decades, the recognition of Congenital Disorders of Glycosylation (CDG) as an increasing number of genetic diseases of glycosylation with almost constant nervous system involvement, dramatically illustrated the consequences of abnormal glycosylation as improper CNS development and function. In addition, CDG recognition contributed to postulate that aberrant glycosylation processes might play a role in multifactorial, complex CNS diseases. On this context, CNS glycomics explores the effects of possible aberrant glycosylation to identify potential glyco-biomarkers useful for the diagnosis and ultimately for potential intervention strategies in neurological diseases. Up to date, CNS glycomics is an emerging, still uncharted area because of the specificity of CNS glycosylation, the complexity of the neurological disorders and for the inaccessibility and invasiveness of disease relevant samples. Here we review current knowledge on clinical glycomics of nervous system diseases, starting with CDG to include those pediatric and adulthood neuropsychiatric diseases where some evidences suggest that multifactor determinants converge to dysregulate glycosylation. Conventional and mass spectrometrybased high throughput technology for glyco-biomarker detection in CNS diseases is reported.
81) Investigation of bacterial resistance to the immune system response: Cepacian depolymerisation by reactive oxygen species
B.Cuzzi, P.Cescutti, L.Furlanis, C.Lagatolla, L.Sturiale, D.Garozzo, R.Rizzo
Innate Immunity
18(4),
661-671
- 2012
Reactive oxygen species (ROS) are part of the weapons used by the immune system to kill and degrade infecting microorganisms. Bacteria can produce macromolecules, such as polysaccharides, that are able to scavenge ROS. Species belonging to the
Burkholderia cepacia complex are involved in serious lung infection in cystic fibrosis patients and produce a characteristic polysaccharide, cepacian. The interaction between ROS and bacterial polysaccharides was first investigated by killing experiments, where bacteria cells were incubated with sodium hypochlorite (NaClO) with and without prior incubation with cepacian. The results showed that the polysaccharide had a protective effect towards bacterial cells. Cepacian was then treated with different concentrations of NaClO and the course of reactions was followed by means of capillary viscometry. The degradation products were characterised by size-exclusion chromatography, NMR and mass spectrometry. The results showed that hypochlorite depolymerised cepacian, removed side chains and O-acetyl groups, but did not cleave the glycosidic bond between glucuronic acid and rhamnose. The structure of some oligomers produced by NaClO oxidation is reported.
82) Structure of the lipopolysaccharide isolated from the novel species Uruburuella sius
A.Silipo, L.Sturiale, C.De Castro, R.Lanzetta , M.Parrilli, D.Garozzo, A.Molinaro
Carbohydrate Research
357 (2012),
75-82
- 2012
Uruburuella suis is a novel species isolated from lungs and heart of pigs with pneumonia and pericarditis. Phenotypic and phylogenetic evidences showed that it represented a hitherto unknown subline within the family
Neisseriaceae. In the present work we defined the whole structure of the LPS isolated from
Uruburuella suis. The structural determination, which was achieved by chemical, spectroscopic and spectrometric approaches, indicates a novel rough type lipopolysaccharide rich in negatively charged groups in the lipid A-inner core region. The elucidation of the structural features of the LPS from
Uruburuella suis is a first step toward the comprehension of the characteristics of the cell envelope in such new and interesting microorganisms.
83) Structural Study of the Lipopolysaccharide O-Antigen Produced by the
Emerging Cystic Fibrosis Pathogen Pandoraea pulmonicola
F.Di Lorenzo, A.Silipo, A.Costello, L.Sturiale, D.Garozzo, M.Callaghan, R.Lanzetta, M.Parrilli, S.McClean, A.Molinaro
European Journal of Organic Chemistry
2012(11),
2243-2249
- 2012
This is the first report on the molecular structure of the Ochain of the lipopolysaccharide produced by the emerging and highly virulent clinical isolate
Pandoraea pulmonicola strain LMG 18108, an opportunistic human pathogen in cystic fibrosis patients. Monosaccharide analysis and 2D NMR spectroscopy revealed a novel polysaccharide, the structure of which consists of the trisaccharide repeating unit [→2-β-D-Quip3NAcyl-(1→4)-α-D-GalpNAc-(1→3)-α-D-GlcpNAc-1→]
n. The quinovosamine residue was found to be linked by a very unusual acyl substituent, a five-membered-ring acyl residue, the 3-hydroxy-2,3-dimethyl-5-oxoprolyl group.
84) Deficiency of Subunit 6 of the Conserved Oligomeric
Golgi Complex (COG6-CDG): Second Patient, Different
Phenotype
S.Huybrechts, C.De Laet, P.Bontems, S.Rooze, H.Souayah, Y.Sznajer, L.Sturiale, D.Garozzo, G.Matthijs, A.Ferster, J.Jaeken, P.Goyens
JIMD Reports
4,
103-108
- 2012
We describe a 27-month-old girl with COG6 deficiency. She is the first child of healthy consanguineous Moroccan parents.
She presented at birth with dysmorphic features including microcephaly, post-axial polydactyly, broad palpebral fissures, retrognathia, and anal anteposition. The clinical phenotype was further characterised by multiorgan involvement including mild psychomotor retardation, and microcephaly, chronic inflammatory bowel disease, micronodular liver cirrhosis, associated with lifethreatening and recurrent infections due to combined T- and B-cell dysfunction and neutrophil dysfunction. Mutation analysis showed the patient to be homozygous for the c.G1646T mutation in the COG6 gene. She is the second reported patient with a deficiency of subunit 6 of the COG complex. Although both patients are homozygous for the same mutation, they present a markedly different clinical picture. Indeed immunodeficiency as well as inflammatory bowel disease has not been described previously in patients with any COG-CDG.
85) ENCEFALOPATIA EPILETTICA PRECOCE ASSOCIATA A DIFETTI CONGENITI DELLA GLICOSILAZIONE (CDG).
M.Falchi, M.A.Donati, E.Procopio, R.Barone, L.Sturiale, D.Garozzo, C.Barba, R.Guerrini
SINPIA 2011: la NEUROPSICHIATRIA dell’infanzia e dell’adolescenza: dalla ricerca alla clinica (Pisa 11-14 Maggio 2011)
,
22
- 2011
I difetti congeniti della glicosilazione, (Congenital Disorder of Glycosylation CDG), sono patologie neurogenetiche dovute al difetto di enzimi implicati nella glicosilazione delle proteine. Il quadro clinico è eterogeneo con uno spettro di sintomi e segni variamente rappresentati nei singoli difetti. Scopo: descriviamo due pazienti con CDG-I, in cui la manifestazione principale è una encefalopatia epilettica precoce. La diagnosi di CDG-I posta in due pazienti affetti da encefalopatia epilettica, microcefalia acquisita, dismorfismi facciali, artrogriposi, scoliosi e tetraparesi spastica, sottolinea l’importanza di effettuare gli accertamenti specifici per questo gruppo di malattie genetiche rare. Metodologie e soggetti: Paziente 1: 2 anni e tre mesi; ritardo di crescita intrauterina e ipocinesia fetale, parto alla 34a settimana. Alla nascita parametri auxologici nella norma, severa ipotonia artrogriposi , distress respiratorio. Dai due mesi crisi epilettiche farmaco resistenti, microcefalia progressiva, grave ritardo psicomotorio, tetraparesi spastica, infezioni respiratorie frequenti con distress. Esame clinico: microcefalia, dismorfismi facciali, collo corto e grave scoliosi. Artrogriposi arti superiori e inferiori. Tetraparesi spastica. Esami strumentali RM: semplificazione della girazione con atrofia diffusa. EMG/ VCN: nella norma, BAEP e PEV: modesta alterazione della trasmissione centrale. Eco addome: fegato di dimensioni ed ecostruttura incrementate. Fondo oculare: iride stellata e atrofia ottica. EEG: scarsa organizzazione, anomalie parossistiche multifocali; crisi ad esordio emisferico destro o sinistro, con arresto psicomotorio, versione capo, manifestazioni toniche asimmetriche, talora clonie. Paziente 2: 16 mesi. Gravidanza normodecorsa, parto eutocico. Parametri auxologici nella norma. Alla nascita motricità povera. Ad un mese crisi parziali, pluriquotidiane farmacoresistenti, grave ritardo psicomotorio, infezioni respiratorie ricorrenti. Esame obiettivo: microcefalia, aplasia cutis dello scalpo. Dismorfismi facciali. Artrogriposi degli arti superiori e inferiori. Scoliosi. Tetraparesi spastica.Esami strumentali. RM encefalo: semplificazione della girazione con atrofia diffusa. EMG/ENG e PEV: nella norma, BAEP: bassa ampiezza con latenza conservata. Eco addome e fondo oculare nella norma. EEG: rallentato, anomalie parossistiche multifocali; numerose crisi ad esordio emisferico destro o sinistro, versione del capo, manifestazioni toniche asimmetriche, incostantemente clonie. Esami di laboratorio dei due pazienti: Isoelettrofocusing della transferrina sierica (IEF) : aumento di sialo-tranferrina con assenza di asialo-transferrina in entrambi. Analisi MALDI-TOF della transferrina sierica: in entrambi i pazienti difetto di glicosilazione in accordo con analisi IEF . Oligosaccaridi legati ai lipidi (LLO) su fibroblasti: profilo alterato nel primo paziente, in corso nel secondo. Analisi di mutazione del gene ALG3 (CDG-Id) in corso. Risultati e discussione: entrambi i pazienti presentano encefalopatia epilettica ad esordio precoce associata a microcefalia acquisita, dismorfismi facciali, artrogriposi e tetraparesi spastica. La RM encefalo in entrambi i casi mostra una semplificazione della girazione con progressiva e diffusa atrofia. Le crisi epilettiche ad esordio nei primi 3 mesi e farmacoresistenti, mostrano un pattern elettroclinico caratterizzato da coinvolgimento sincrono o asincrono dei due emisferi con manifestazioni cliniche prevalentemente di tipo motorio. L’IEF eseguita nel primo anno di vita ha mostrato in entrambi i pazienti, un pattern di CDG di tipo I. Le sindromi CDG sono un gruppo di difetti clinicamente e biochimicamente eterogeneo, probabilmente sotto diagnosticato. Si sottolinea l’importanza di eseguire l’analisi IEF su siero in pazienti affetti da encefalopatia epilettica da causa non nota specie in presenza di dimorfismi facciali, epatopatia e artrogriposi.
86) DISORDINI CONGENITI DELLA GLICOSILAZIONE: LA NOSTRA ESPERIENZA IN EUROGLYCANET (2005-2010)
R.Barone, L.Sturiale, D.Garozzo, D.Cocuzza, G.Sorge, A.Fiumara
SINPIA 2011: la NEUROPSICHIATRIA dell’infanzia e dell’adolescenza: dalla ricerca alla clinica (Pisa 11-14 Maggio 2011)
,
19
- 2011
I disordini congeniti della glicosilazione (Congenital Disorders of Glycosylation - CDG) sono patologie neurometaboliche dovute a difetti di sintesi delle glicoproteine e altri glicoconiugati. Ad oggi, sono state identificate 25 forme di CDG dovute a difetti della N-glicosilazione. I disordini della glicosilazione sono caratterizzati da ritardo dello sviluppo di grado variabile e possibile presenza di anomalie malformative del sistema nervoso centrale (atrofia cerebellare), dismorfie, microcefalia, deficit sensoriali, epilessia, neuropatia periferica e patologia multisistemica. Euroglycanet è un network europeo finalizzato alla diffusione di strumenti adeguati per la diagnosi clinica dei pazienti affetti da CDG, per la identificazione dei difetti molecolari e per lo sviluppo di procedure terapeutiche. Scopo: riportiamo la nostra esperienza nella diagnosi dei disordini della glicosilazione e descriviamo i fenotipi clinici identificati attraverso un lavoro di screening per queste patologie svolto nel contesto di Euroglycanet. Pazienti e Metodi: durante gli ultimi 5 anni (2005-2010), presso il Dipartimento di Pediatria, Università di Catania, oltre 3000 campioni di pazienti Italiani con sospetto clinico per CDG, sono stati analizzati con esame isoelettrofocusing della transferrina sierica (test di primo livello per i difetti della N-glicosilazione). Sui campioni diagnosticati veniva ulteriormente effettuata caratterizzazione dello stato di glicosilazione tramite tecniche di spettrometria di massa, attraverso metodiche appositamente sviluppate presso l’ICTP-CNR di Catania. Successivamente tali campioni venivano inviati in altri laboratori Euroglycanet per analisi biochimica e molecolare. Risultati: sono stati identificati 21 pazienti con difetti molecolari della N-glicosilazione dovuti a deficit della sintesi del precursore glicoproteico dolicol-oligosaccaride nel citosol e reticolo-endoplasmico (CDG tipo I). Quattordici pazienti erano affetti da PMM2-CDG (CDG-Ia), che rappresenta la forma più frequente tra queste patologie. Rispetto alla forma classica, la presenza di pazienti con PMM2-CDG e fenotipo ’mild’ veniva definita in relazione al pattern di sviluppo, assenza di microcefalia e assenza di malformazione cerebellare tipo Dandy-Walker. In pazienti con fenotipo ’mild’ veniva osservata associazione genotipo-fenotipo per la ricorrenza in tutti questi soggetti di eterozigosi composta in presenza della mutazione p.L32R del gene PMM2. I sette pazienti non PMM2-CDG (38%), presentavano dismorfie (7/7), microcefalia (6/7), ritardo dello sviluppo di grado severo (6/7), epilessia farmaco-resistente con esordio nel primo anno di vita (6/7), deficit visivo (5/7) e assenza di atrofia cerebellare (6/7). In questi soggetti, i profili di glicosilazione della Tf osservati tramite spettrometria di massa MALDI-TOF erano significativamente differenti rispetto a quelli osservati in pazienti con PMM2-CDG. Conclusioni: la presenza di epilessia farmaco-resistente con esordio precoce, microcefalia e deficit visivo in pazienti identificati con CDG-I, definiscono nella popolazione studiata un fenotipo frequente e differente dalla forma classica (PMM2-CDG/CDG-Ia). Questi risultati indicano che la diagnosi di CDG dovrebbe essere considerata tra le malattie metaboliche causa di encefalopatia epilettica precoce.
87) Clinical and biochemical characterization of a CDG-I patient with fetal akinesia
L.Sturiale, R.Barone, L.Keldermans, A.Fiumara, M.Falchi, R.Guerrini, M.A.Donati, G.Matthijs, D.Garozzo
4th International Meeting on Congenital Disorders of Glycosylation (Leuven, Belgium, 13-14 January 2011)
,
49
- 2011
This work reports on clinical and biochemical characterization of a CDG-I patient with arthrogryposis and developmental anomalies (fetal akinesia).
Clinical data: the patient was born pre-term (34 weeks) to healthy unrelated parents, after a pregnancy complicated with intrauterine growth restriction between 20 and 32 weeks and decreased intrauterine fetal movement. At birth she had severe hypotonia, distinct facial features and multiple congenital contractures (arthrogryposis). Since the age 2 months she manifested drug-resistant epilepsy. At age 8 months microcephaly (OFC: 41 cm), severe and progressive kyphoscoliosis with thoracal deformity were recorded. There were absent psychomotor development, no visual fixation, axial hypotonia and reduction of active limb movements with normoactive deep tendon reflexes. Moderate hepatomegaly. Developmental anomalies included simplified gyral pattern and cerebellar hypoplasia.
Laboratory investigations (age 8 months): increased serum transaminases levels (SGOT 106 IU/l, SGPT 103 IU/l). Blood CK levels, clotting times, plasma FVIII levels, thyroid hormones, karyotype, array CGH: normal.
88) Mass spectrometry contribution to the diagnosis and characterization of Congenital Disorders of Glycosylation (CDG):
our experience in the EUROGLYCANET European network
L.Sturiale, R.Barone, A.Fiumara, G.Sorge, J.Jaeken, D.Garozzo
16th European Carbohydrate Symposium (July 3-7, Sorrento -Naples, Italy)
,
538
- 2011
Congenital Disorders of Glycosylation (CDG) comprise an ever increasing group of inborn errors in the proteins glycosylation pathway, representing a paradigm as they disclose the direct connection between glycosylation changes and human diseases. Since the first description in 1980 [1], about 40 CDG forms were discovered, each with a variable clinical spectrum, from multisystem diseases to single organ involvements, causing high morbidity and even mortality. Sharing information and competences is an essential step in order to identify new CDG types and to promote awareness and early diagnosis. Mass spectrometry contributed significantly to this research field to identify possible underglycosylation profiles of intact individual glycoproteins and/or to map glycan population of the sample under study, typically serum or plasma. Here we report on serum glycosylation analyses by MALDI-MS in patients with CDG recruited in the context of Euroglycanet, an European network focused on diagnosis and understanding CDG [2]. During the last five years (2005-2010), almost 3000 samples of Italian patients with clinical suspicion of CDG were analyzed by serum Transferrin (Tf) isoelectric focusing (IEF) in a clinical setting at the University Paediatric Hospital in Catania. Patient with abnormal IEF pattern underwent MALDI MS glycosylation analyses at the ICTP-CNR in Catania, where a specific method for intact glycoprotein investigation and N-glycan analyses was set up [3]. Eighteen patients showed by MS the Tf abnormal glycosylation profile characteristic of type I CDG, encompassing defects in the assembly of dolichol-linked oligosaccharide in the Endoplasmic reticulum. Among these, PMM2-CDG (or CDGIa) was most frequently found (11 cases). Of particular importance, one patient affected with epileptic encephalopathy, had a newly identified CDG type due to DPM2 gene mutation [4]. Moreover, MS glycosylation analysis was performed on seventy-one serum samples with type II IEF pattern, collected from the Euroglycanet partners. Two of these samples showed recognizable glycan processing defects, respectively caused by gene mutations of subunit 5 and subunit 7 of the Conserved Oligomeric Golgi (COG) complex (COG5-CDG and COG7-CDG) [5-6]. Four more samples with combined Type I and Type II Tf MS profiles were also found; this significant information makes disease comprehension at molecular basis and diagnosis particularly challenging in these patients.
89) The MALDI TOF/TOF Mass Spectrometer as a Chemical Lab for the structural characterization of lipooligosaccharides
L.Sturiale, A.Palmigiano, A.Silipo, R.Lanzetta, M.Parrilli, A.Molinaro, D.Garozzo
16th European Carbohydrate Symposium (July 3-7, Sorrento -Naples, Italy)
,
45
- 2011
Lipooligosaccharide (LOS) is a powerful Gram negative glycolipid involved in immune system
elusion and invasion of host animal and vegetal cells. Its structural elucidation is pivotal to
understand at molecular levels mechanisms of infections. Due to its amphiphilic nature, it has
resisted structural matrix assisted laser desorption/ionization (MALDI) analysis. Our approach
has now resolved this important issue, permitting us to obtain high resolution MALDI mass
spectra that are rich of information. In fact, they show both LOS quasi-molecular ions, and
also well defined fragments due to a in-source decay which yields B-type ions corresponding to
core oligosaccharide(s), and Y-type ions relating to lipid A unit(s). Moreover, MALDI tandem
Time of Flight (TOF/TOF) mass spectrometry (MS) of lipid A allows its complete structural
elucidation straight from the purified intact LOS, without any chemical manipulation. These
findings constitute a significant improvement in the analysis of such important biomolecule by
MS.
90) O-Acetyl location on Cepacian, the principal exopolysaccharide of Burkholderia cepacia complex bacteria
P.Cescutti, G.Impallomeni, D.Garozzo, R.Rizzo
Carbohydrate Research
346 (18),
2905-2912
- 2011
Cepacian is an exopolysaccharide produced by the majority of the isolates belonging to the
Burkholderia cepacia complex bacteria, a group of 17 species, some of which infect cystic fibrosis patients, sometime with fatal outcome. The repeating unit of cepacian consists of a backbone having a trisaccharidic repeating unit with three side chains, as reported in the formula below. The exopolysaccharide is also acetylated, carrying from one to three acetyl esters per repeating unit, depending on the strain examined. The consequences of
O-acetyl substitution in a polysaccharide are important both for its biological functions and for industrial applications, including the preparation of conjugated vaccines, since
O-acetyl groups are important immunogenic determinants. The location of acetyl groups was achieved by NMR spectroscopy and ESI mass spectrometry and revealed that these substituents are scattered in non-stoichiometric ratio on many sugar residues in different positions, a feature which adds to the already unique carbohydrate structure of the polysaccharide.
91) Reflectron MALDI TOF and MALDI TOF/TOF mass spectrometry reveal novel structural details of native lipooligosaccharides
L.Sturiale, A.Palmigiano, A.Silipo, Y.A.Knirel, A.Anisimov, R.Lanzetta, M.Parrilli, A.Molinaro, D.Garozzo
Journal of Mass Spectrometry
46,
1135-1142
- 2011
Lipooligosaccharides (LOS) are powerful Gram-negative glycolipids that evade the immune system and invade host animal and vegetal cells. The structural elucidation of LOS is pivotal to understanding the mechanisms of infection at the molecular level. The amphiphilic nature of LOS has been the main obstacle for structural analysis by matrix-assisted laser desorption/ ionization (MALDI) mass spectrometry (MS). Our approach has resolved this important issue and has permitted us to obtain reflectron MALDI mass spectra of LOS to reveal the fine chemical structure with minimal structural variations. The high-quality MALDI mass spectra show LOS species characteristic of molecular ions and defined fragments due to decay in the ion source. The in-source decay yields B-type ions, which correspond to core oligosaccharide(s), and Y-type ions, which are related to lipid A unit(s). MALDI tandem time-of-flight (TOF/TOF) MS of lipid A allowed for the elucidation of its structure directly from purified intact LOS without the need for any chemical manipulations. These findings constitute a significant advancement in the analysis of such an important biomolecule by MALDI MS.
92) From micro- to femto-moles, from small to giant molecules: the route of modern Mass Sectrometry
A.Palmigiano, D.Garozzo
Seminars in organic synthesis
SCI June 13-17, 2011 Gargnano (BS),
264-279
- 2011
The definition of a mass spectrometer from the America Society for Mass Spectrometry (ASMS) is:
"A mass spectrometer is an instrument that measures the masses of individual molecules thathave been converted to ions; i. e. molecules that have been electrically charged"; using simply words: a machine used to weigh molecules. This definition is obviously correct, but the mass spectrometer is much more than an instrument. It is a complete laboratory for the investigation of molecules, clusters, and other species under the environment-free conditions of the gas phase. For this reason Mass Spectrometry (MS) is widely used today by almost all chemists and many researchers from neighbouring disciplines such as physics, medicine, or biology as a powerful analytical tool. Today the mass spectrometer is more and more present in the pharmaceutical industry, in the biological labs, in the hospitals and in many other bio-labs, while until few years ago it was present only in specialized chemical lab. This revolution starts at the end of 1980s with the introduction of two different methods able to desorbs and ionize molecules with molecular weights in a range from few hundreds daltons to millions. Until then, a few techniques, fast atom bombardment (FAB), plasma desorption (PD) and desorption chemical ionization (DCI) were able to carry out in the gas phase and to ionize molecules with a molecular weight greater than one thousand, but they all required high concentrations of sample and they did not work at all for larger molecules such as proteins or polymers. Then, in 1988, Electrospray ionization (ESI) invented by John B. Fenn and Matrix assisted laser desorption (MALDI) introduced by Franz Hillenkamp and Michael Karas appeared almost simultaneously. These desorption-ionization methods revolutionized MS and are the main forms of ionization to this day. In the beginning the two techniques were not believed robust enough, but at the end of 1990s almost all MS instruments on the market were MALDI or ESI.
93) Two Argentinean Siblings with CDG-Ix: A Novel Type of Congenital Disorder of Glycosylation?
M.B.Bistuè Millòn, M.A.Delgado, N.B.Azar, N.Guelbert, L.Sturiale, D.Garozzo, G.Matthijs, J.Jaeken, R.Dodelson de Kremer, C.G.Asteggiano
JIMD Reports
1/2011,
65-72
- 2011
Congenital disorders of glycosylation (CDG) are genetic diseases caused by abnormal protein and lipid glycosylation.
In this chapter, we report the clinical, biochemical, and molecular findings in two siblings with an unidentified CDG (CDG-Ix). They are the first and the third child of healthy consanguineous Argentinean parents. Patient 1 is now a 11-year-old girl, and patient 2 died at the age of 4 months. Their clinical picture involved liver dysfunction in the neonatal period, psychomotor retardation, microcephaly, seizures, axial hypotonia, feeding difficulties, and hepatomegaly. Patient 1 also developed strabismus and cataract. They showed a type 1 pattern of serum sialotransferrin. Enzymatic analysis for phosphomannomutase and phosphomannose isomerase in leukocytes and fibroblasts excluded PMM2-CDG and MPI-CDG. Lipid-linked oligosaccharide (LLO) analysis showed a normal profile. Therefore, this result could point to a deficiency in the dolichol metabolism. In this context, ALG8-CDG, DPAGT1-CDG, and SRD5A3-CDG were analyzed and no defects were identified. In conclusion, we could not identify the genetic deficiency in these patients yet. Further studies are underway to identify the basic defect in them, taking into account the new CDG types that have been recently described.
94) The impact of mass spectrometry in the diagnosis of congenital disorders of glycosylation
L.Sturiale, R.Barone, D.Garozzo
Journal of Inherited Metabolic Disease
34(4),
891-899
- 2011
Contribution of mass spectrometry (MS) in the diagnosis and characterization of congenital disorders of glycosylation (CDG) has long been known. CDG type I diseases are characterized by the under-occupancy of protein N-glycosylation sites. Electrospray (ESI) MS and matrix assisted laser desorption ionization (MALDI) MS are effective for underglycosylation analyses of intact serum Transferrin (Tf) in CDG-I patients by mass determination of individual component glycoforms. Thus, highthroughput methods developed to speed-up analytical times found increasing application in clinical testing for CDG detection. ESI MS recognizable glycoform profiles of serum Tf have been reported in CDG-I different from PMM2-CDG and in individual CDG-II defects. MALDI MS analysis of acidic and neutral N-linked glycans released from total plasma or targeted glycoproteins, is the mainstream tool to explore abnormal oligosaccharide structure and changes in the relative amount of individual oligosaccharides in CDG-II patients. Here we briefly review stateof-the-art and updates of MS-based applications for the diagnosis of CDG with special emphasis to detectable glycosylation profiles reported in different CDG types.
95) COG5-CDG with a Mild Neurohepatic Presentation
C.W.Fung. G.Matthijs, L.Sturiale, D.Garozzo, K.Y.Wong, R.Wong, V.Wong, J.Jaeken
JIMD Reports
DOI 10.1007/8904_2011_61
- 2011
The conserved oligomeric Golgi (COG) complex is an eight subunit protein involved in the retrograde transport of Golgi components.
It affects the localization of several Golgi glycosyltransferases and hence is involved in N- and O-glycosylation. Genetic defects in this complex belong to the rapidly expanding family of congenital disorders of glycosylation (CDG). Patients have been reported with defects of subunit 1 (CDG1-CDG), subunit 4 (CDG4-CDG), subunit 5 (CDG5-CDG), subunit 6 (CDG6-CDG), subunit 7 (CDG7-CDG), and subunit 8 (CDG8-CDG). This paper is on the second reported patient with COG5-CDG. She showed a mild neurohepatic disease with central as well as peripheral neurological involvement while in the first reported patient (with a different mutation) only mild central neurological involvement was reported.
96) Identification of tear fluid biomarkers in vernal keratoconjunctivitis syndrome using iTRAQ quantitive proteomics
A.Palmigiano, E.Mazzola, A.Messina, S.Milazzo, A.Leonardi, D.Garozzo
58th ASMS Conference on Mass Spectrometry and Allied Topics - May 23-27 2010 Salt Lake City, Utah
- 2010
97) Identification of tear fluid biomarkers in vernal keratoconjunctivitis using iTRAQ quantitive proteomics
M.Bortolotti, A.Palmigiano, A.Messina, E.Mazzola, S.Milazzo, D.Garozzo, A.Leonardi
Annual meeting ARVO 2010 - May 2-6 2010 Fort Lauderdale, Florida
- 2010
98) Clinical characterization of a novel congenital disorder of glycosylation (DPM2 mutation)
R.Barone, V.Race, L.Sturiale, R.Bammens, W.Vleugels, L.Keldermans, D.Garozzo, F.Foulquier, J.Jaeken, G.Sorge, A.Fiumara, G.Matthijs
Journal of Inherited Metabolic Disease
33,
S74
- 2010
Background:The human dolichol-phosphate-mannose (DPM) synthase is a heterotrimeric complex composed of DPM1, DPM2 and DPM3. Only mutations in DPM1 (the catalytic subunit) and more recently in DPM3 have been described.
Case Report: The girl was born to unrelated parents at the 32th week of gestation complicated by polyhydramnios. At birth she had normal growth parameters, severe hypotonia and dysmorphism. At six months, microcephaly (OFC: 33.5 cm), keel thorax,widely spaced nipples and joint flexion contractures were seen. Repeated laboratory investigations showed elevation of serum transaminases and CPK levels and coagulopathy. At age 20 months there were severe hypotonia, no head control, reduced active limb movements with normal osteotendineous reflexes. She experienced pharmacoresistant generalized tonic seizures and myoclonic jerks. Funduscopy showed optic atrophy. Brain MRI documented loss of cerebral white matter, without cerebellar atrophy. She died at age 36 months from pneumonia. Serum Transferrin (Tf) IEF showed overt increase of disialo-Tf and a very faint asialo-Tf band (type I pattern). PMM and PMI enzymes were normal. MALDI-MS of serum Tf showed di-glycosylated Tf and an additional glycoform owing to mono-glycosylated Tf. Unlike CDG-Ia (PMM deficiency), no a-glycosylated Tf species were detectable. Lipid-linked oligosaccharides analyses showed an accumulation of dol-PP-GlcNAc2-Man5. No mutations in DPM1, ALG3 or MPDU1 were detected. The patient was compound heterozygote for a splice mutation (c.4-1G>C) and a missense mutation (c.68A>G, p.Y23C) in the DPM2 gene.
Conclusions: Prominent neurological involvement, dysmorphism and fatal outcome were hallmark clinical features in the first CDG patient with DPM2 mutation.
99) The structure of the carbohydrate backbone of the lipooligosaccharide from an alkaliphilic Halomonas sp.
A.Silipo, V.Gargiulo, L.Sturiale, R.Marchetti, P.Prizeman, W.D.Grant, C.De Castro, D.Garozzo, R.Lanzetta, M.Parrilli, A.Molinaro
Carbohydrate Research
345(13),
1971-1975
- 2010
The structure of the carbohydrate backbone of the core-lipid A region of the lipooligosaccharide (LOS) of the alkaliphilic and slightly halophilic bacterium Halomonas sp. has been elucidated. The LOS was fully deacylated, dephosphorylated, and reduced at the free reducing end. The structure, obtained by means of compositional analysis, 2D NMR spectroscopy, and MALDI mass spectrometry, was determined as the following:
All sugars are D-pyranoses; Hep is L-glycero-D-manno-heptose and Kdo is 3-deoxy-D-manno-oct- 2-ulosonic acid.
100) Against the rules: A marine bacterium, Loktanella rosea, possesses a unique lipopolysaccharide
T.Ieranò, A.Silipo, E.L.Nazarenko, R.P.Gorshkova, E.P.Ivanova, D.Garozzo, L.Sturiale, R.Lanzetta, M.Parrilli, A.Molinaro
Glycobiology
20(5),
586-593
- 2010
Bacteria are an inimitable source of new glyco-structures potentially useful in medicinal and environmental chemistry. Lipopolysaccharides (LPS; endotoxins) are the major components of the outer membrane of Gram-negative bacteria; being exposed toward the external environment they can undergo structural changes and thus, they often possess peculiar chemical features that allow them to thrive in harsh chemical and physical environments. Marine bacteria have evolved and adapted over millions of years in order to succeed in different environments, finding a niche for their survival characterized by severe physical or chemical parameters. The present work focuses on the structural investigation of the LPS from Loktanella rosea, a marine Gram-negative bacterium. Through chemical analysis, 2D nuclear magnetic resonance and matrix-assisted laser desorption ionization mass spectrometry investigations, a unique LPS carbohydrate backbone has been defined. The lipid Askeleton consists of a trisaccharide backbone lacking the typical phosphate groups and is characterized by two β-glucosamines and an α-galacturonic acid. The core region is built up of three ulosonic acids, with two 3-deoxy-D-manno-oct-2-ulopyranosonic acid residues, one of which is carrying a neuraminic acid. This carbohydrate structure is an exceptional variation from the typical architectural skeleton of endotoxins which consequently implies a very different biosynthesis.
101) The structure of the carbohydrate backbone of the lipooligosaccharide from the halophilic bacterium Arcobacter halophilus
A.Silipo, L.Sturiale, V.Perino, D.Garozzo, R.Lanzetta,.M.Parrilli, A.Molinaro
Carbohydrate Research
345(6),
850-853
- 2010
A novel oligosaccharide was isolated and identified from the lipooligosaccharide fraction of the halophilic marine bacterium Arcobacter halophilus. The complete structure was achieved by chemical analysis, 2D NMR spectroscopy, and MALDI mass spectrometry as the following:
α-Glc-(1 → 7)-α-Hep-(1 → 5)-α-Kdo4P-(2 → 6)-β-GlcN4P-(1 → 6)-α-GlcN1P.
102) Structural Elucidation of a Novel B. cenocepacia ET-12 Lipooligosaccharide Isolated from a Cystic Fibrosis Patient after Lung Transplantation
T.Ieranò, A.Silipo, L.Sturiale, D.Garozzo, P.Corris, J.Perry, R.Lanzetta, M.Parrilli, A.De Soyza, A.Molinaro
European Journal of Organic Chemistry
2010(7),
1299-1306
- 2010
In this work, we carried out the elucidation of a novel lipooligosaccharide (LOS) from a clinical strain of the most virulent strain of the Burkholderia cepacia complex, which was of B. cenocepacia ET-12 epidemic strain lineage. The strain was isolated from a cystic fibrosis patient who had successfully undergone lung transplantation. This molecule presents some specific chemical differences when compared to the LOS reported so far from other B. cenocepacia ET-12 clones. The new endotoxin was also tested as a cytokine enhanceron human myelomonocytic U937 cells. Lipopolysaccharides are the main virulence factors of Gram-negative bacteria, and since they are exposed to the extracellular space, they are deeply involved in bacterial adaptation to the host environment. The characterisation of structural motifs present in these molecules is essential to the comprehension of the persistence/ survival mechanisms and inflammatory potential of these bacteria.
103) Quantitative proteomic analysis of human tear fluids using differentially labelled tags iTRAQ with LC-MALDI MS and MS/MS in VKC Patients
D.Garozzo
Quantitative Techniques in Mass Spectometry-Based Proteomics 12th 13th October 2009 Vienna: IMP/IMBA
- 2009
104) LC MALDI MS nell’analisi del fluido lacrimale
D.Garozzo
Cromatografia Nano Capillare e Spettrometria di Massa Applicazioni di Proteomica - Pisa 22 Aprile 2009 (Area della Ricerca del CNR)
- 2009
105) β-Amyloid Monomers Are Neuroprotective
M.L.Giuffrida, F.Caraci, B.Pignataro, S.Cataldo, P.De Bona, V.Bruno, G.Molinaro, G.Pappalardo, A.Messina, A.Palmigiano, D.Garozzo, F.Nicoletti, E.Rizzarelli, A.Copani
Journal of Neuroscience
29(34),
10582-10587
- 2009
The 42-aa-long β-amyloid protein--Aβ1-42--is thought to play a central role in the pathogenesis of Alzheimer’s disease (AD) (Walsh and Selkoe, 2007). Data from AD brain (Shankar et al., 2008), transgenic APP (amyloid precursor protein)-overexpressing mice (Lesné et al., 2006), and neuronal cultures treated with synthetic Aβ peptides (Lambert et al., 1998) indicate that self-association of Aβ1-42 monomers into soluble oligomers is required for neurotoxicity. The function of monomeric Aβ1-42 is unknown. The evidence that Aβ1-42 is present in the brain and CSF of normal individuals suggests that the peptide is physiologically active (Shoji, 2002). Here we show that synthetic Aβ1-42 monomers support the survival of developing neurons under conditions of trophic deprivation and protect mature neurons against excitotoxic death, a process that contributes to the overall neurodegeneration associated with AD. The neuroprotective action of Aβ1-42 monomers was mediated by the activation of the PI-3-K (phosphatidylinositol-3-kinase) pathway, and involved the stimulation of IGF-1 (insulin-like growth factor-1) receptors and/or other receptors of the insulin superfamily. Interestingly, monomers of Aβ1-42 carrying the Arctic mutation (E22G) associated with familiar AD (Nilsberth et al., 2001) were not neuroprotective. We suggest that pathological aggregation of Aβ1-42 may also cause neurodegeneration by depriving neurons of the protective activity of Aβ1-42 monomers. This "loss-of-function" hypothesis of neuronal death should be taken into consideration when designing therapies aimed at reducing Aβ burden.
106) First structural characterization of Burkholderia vietnamiensis lipooligosaccharide from
cystic fibrosis-associated lung transplantation strains
T.Ieranò, A.Silipo, L.Sturiale, D.Garozzo, C.Bryant, R.Lanzetta, M.Parrilli, C.Aldridge, F.K.Gould, P.A.Corris, C.M.Anjam Khan, A.De Soyza, A.Molinaro
Glycobiology
19,
1214-1223
- 2009
This is the first structural elucidation of the lipooligosaccharide (LOS) endotoxin isolated from Burkholderia vietnamiensis, a clinically important member of Burkholderia cepacia complex, a group of over 10 opportunistic species that are highly problematic in cystic fibrosis.We have characterized a novel LOS structure extracted from two clonal strains of B. vietnamiensis isolated from a cystic fibrosis patient who underwent lung transplantation. Strains were selected from the pretransplantation and post-transplantation periods and endotoxin was extracted. Subsequent analysis interestingly revealed identical oligosaccharidic sequences, but variation in lipid A moieties. Further, both LOS fractions were tested for their immunostimulatory activity on human myelomonocytic U937 cells and for signaling on an HEK293 cell line stably expressing both TLR 4 and MD-2. We observed an increase in lipid A acylation and a resultant increase in biological activity in bio-reporter assays of TNF-α secretion in the post-transplantation strain.
107) A new mutation in COG7 extends the spectrum of COG subunit deficiencies
R.Zeevaert, F.Foulquier, D.Cheillan, I.Cloix, N.Guffon, L.Sturiale, D.Garozzo, G.Matthijs, J.Jaeken
European Journal of Medical Genetics
52,
303-305
- 2009
We describe a patient homozygous for a novel mutation in COG7, coding for one of the subunits of the Conserved Oligomeric Golgi complex, involved in retrograde vesicular trafficking. His brother showed a similar clinical syndrome and glycosylation defect but no DNA could be obtained from this patient. This mutation, c.170-7A > G, activates a cryptic splice acceptor and leads to the insertion of 2 amino acids at protein level (p.56-57insAT). The insertion disturbs the structure and function of the Conserved Oligomeric Golgi complex. In comparison to the previously described patients with a different COG7 mutation, intrauterine growth retardation and dysmorphic features were absent and there was a longer survival.
108) Mass spectrometry in the characterization of human genetic N-glycosylation defects
R.Barone, L.Sturiale, D.Garozzo
Mass Spectrometry Reviews
28(3),
517-542
- 2009
Human genetic diseases that affect N-glycosylation result from the defective synthesis of the N-linked sugar moiety (glycan) of glycoproteins. The role of glycans for proper protein folding and biological functions is illustrated in the variety and severity of clinical manifestations shared by congenital disorders of glycosylation (CDG). This family of inherited metabolic disorders includes defects in the assembly of the oligosaccharide precursor that lead to an under-occupancy of N-glycosylation sites (CDG-I), and defects of glycan remodeling (CDG-II). Mass spectrometry constitutes a key tool for characterization of CDG-I defects by mass resolution of native protein glycoforms that differ for glycosylation-site occupancy. Glycan MS analyses in CDG-II is mandatory to detect whenever possible a repertoire of structures to pinpoint candidate enzymes and genes responsible for the abnormal N-glycan synthesis. In this manuscript, we review the MS applications in the area of CDG and related disorders with a special emphasis on those techniques that have been already applied or might become functional for diagnosis, characterization, and treatment monitoring in some specific conditions.
109) Chemically modified tetranitro-oxacalix[4]arenes: synthesis and conformational preferences of tetra-N-(1-octyl)ureido-oxacalix[4]arenes
C.Capici, D.Garozzo, G.Gattuso, A.Messina, A.Notti, M.F.Parisi, I.Pisagattia, S.Pappalardo
ARKIVOC
VIII,
199-211
- 2009
Tetranitro-oxacalix[4]arenes 1–5, prepared by direct SNAr reaction of 1,5-difluoro-2,4-dinitrobenzene with the appropriate aromatic diol (pyrocatechol, resorcinol, hydroquinone, 2,7-dihydroxynaphthalene, and 4,4´-dihydroxybiphenyl), were subjected to Raney-nickel reduction to provide the corresponding tetraamino-oxacalix[4]arenes 6–10, which upon treatment with an excess of 1-octyl isocyanate were converted into the title compounds 11–15, featuring a pair of 1,3-bis-[N-(1-octyl)ureido]phenylene moieties doubly connected at their 4,6-positions by rigid spacers of varied geometry. All new oxacalix[4]arenes were characterized by MALDI-TOF spectrometry and NMR spectroscopy. 1H NMR data and ab initio calculations support saddle-shaped conformations for oxacalix[4]arenes incorporating pyrocatechol, resorcinol and 2,7-dihydroxynaphthalene nucleophilic components, and boat-shaped conformations for derivatives possessing hydroquinone and 4,4´-dihydroxybiphenyl spacers.
110) Long-standing mild hypertransaminasaemia caused by congenital disorder of glycosylation (CDG) type IIx
P.L.Calvo, S.Pagliardini, M.Baldi, A.Pucci, L.Sturiale, D.Garozzo, T.Vinciguerra, C.Barbera, J.Jaeken
Journal of inherited metabolic disease
31 (Suppl 2),
S437-S440
- 2008
A 32 year-old asymptomatic male came to our attention with a 21-year history, documented elsewhere, of puzzling increases in his serum transaminase level. At first, very low serum ceruloplasmin level suggested Wilson disease. Two liver biopsies showed mild portal inflammation, steatosis and mild fibrosis. Further investigation revealed low levels of the glycoproteins AT III and clotting factor XI, leading to a diagnosis of congenital disorder of glycosylation (CDG) type II. Further studies as to the cause of this "apparently new" CDG, are ongoing. On the basis of our data and a literature review, we suggest that subjects with asymptomatic hypertransaminasaemia be screened for CDG
111) Multiplexed glycoproteomic analysis of glycosylation disorders by sequential IgY immunoseparation and MALDI TOF MS
L.Sturiale, R.Barone, A.Messina, A.Palmigiano, D.Garozzo
XI Convegno-Scuola sulla Chimica dei Carboidrati, Pontignano (Si), 22-26 Giugno 2008
- 2008
The physiological roles of glycan structures in the fine-tuning of multiple biological processes is depicted in the congenital disorders pf glycosylation (CDG) clinical spectrum which is broad and virtually involves all organ systems. A major challenge in CDG research concerns the possibility to screen different native intact glycoproteins for their possible macroheterogeneity due to site glycosylation underoccupancy which is mandatory for characterization of CDG-I defects. In addition, the investigation of glycoprotein microheterogeneity (structural abnormalities of N-glycan structures at glycosylation sites) is required for detecting CDG-II disorders. Glycoproteomics of CDG and secondary glycosylation disorders such as galactosemia (GALT deficiency) and hereditary fructose intolerance or HFI, is aimed to investigate glycosylation changes of proteins with the following purposes: 1) biomarker detection for diagnosis and treatment monitoring (whenever possible); 2) characterization of defective glycan structures to pinpoint possible enzyme/gene defects in patients with unknown basic defect; 3) insight into the pathogenesis by elucidating structure-function relationships. We applied IgY immunoaffinity separation and MALDI MS for clinical proteomics of post-translational modification (glycosylation) as schematised in figure, with the aim to set up a high-throughput method to analyze macroheterogeneity and microheterogeneity of protein glycoforms. The method was validated by the analyses of human underglycosylated serum glycoproteins in CDG-I, CDG-II and secondary glycosylation disorders and applied for biomarker detection in the diagnostic procedure (CDG-I and II) and treatment monitoring (galactosemia and HFI).
112) Clinical characteristics and mass spectrometry glycosylation profile in two CDG-I patients
R.Barone, L.Sturiale, M.D.Cocuzza, G.Rapicavoli, G.Sorge, A.Fiumara, D.Garozzo
IV Euroglycanet Meeting, Worms, 1-4 ottobre 2008
- 2008
We report on two unrelated Sicilian patients which share some clinical features and have a similar and peculiar mass spectrometry (MS) glycosylation profile of native Transferrin (Tf) and alpha1-antitrypsin (AAT).
Clinical data
Two unrelated girls aged 9 month (patient 1) and 20 month (patient 2) presented with almost absent psychomotor development and marked hypotonia. They both were born pre-term from healthy non-consanguineous parents, after a pregnancy complicated with polyhydramnios. The two patients had congenital microcephaly, craniofacial dysmorphy, limb anomalies with joint restrictions. Malformation anomalies were more severe in patient 1. Brain MRI documented a decrease of cerebral white matter and thinning of the corpus callosum. They presented increased serum transaminases levels (<110 UI/l) and coagulopathy. Serum Transferrin (Tf) isoelectric focusing (IEF) showed a type I pattern with a very faint asialo band in the two patients, while it was normal in their respective parents. CDG-Ia and CDG-Ib were ruled out in as PMM and PMI enzyme activities were found in the normal ranges.
Mass Spectrometry analyses
MALDI spectra of native Tf showed in these patients in addition to the main normal peak at ˜79,5 kDa (di-glycosylated Tf), a significant component at ˜77,4 kDa (mono-glycosylated Tf). Hypoglycosylation profiles were observed in the AAT mass spectra from both the patients. MALDI analyses of the acidic N-glycans from Tf did not reveal truncated and/or abnormal species.
Conclusions
The reported patients have common clinical features. MS glycosylation analyses of serum Tf is also similar between these patients and it suggests the increase of mono-glycosylated but not a-glycosylated Tf species.
113) Multiplexed glycoproteomic analysis of glycosylation disorders by sequential IgY immunoseparation and MALDI TOF MS
L.Sturiale, R.Barone, A.Messina, A.Palmigiano, D.Garozzo
XXI Congresso Nazionale di Chimica Analitica, Arcavacata di Rende (CS), 21 - 25 Settembre 2008
- 2008
The physiological roles of glycan structures in the fine-tuning of multiple biological processes is depicted in the congenital disorders pf glycosylation (CDG) clinical spectrum which is broad and virtually involves all organ systems. A major challenge in CDG research concerns the possibility to screen different native intact glycoproteins for their possible macroheterogeneity due to site glycosylation underoccupancy which is mandatory for characterization of CDG-I defects. In addition, the investigation of glycoprotein microheterogeneity (structural abnormalities of N-glycan structures at glycosylation sites) is required for detecting CDG-II disorders. Glycoproteomics of CDG and secondary glycosylation disorders such as galactosemia (GALT deficiency) and hereditary fructose intolerance or HFI, is aimed to investigate glycosylation changes of proteins with the following purposes: 1) biomarker detection for diagnosis and treatment monitoring (whenever possible); 2) characterization of defective glycan structures to pinpoint possible enzyme/gene defects in patients with unknown basic defect; 3) insight into the pathogenesis by elucidating structure-function relationships. We applied IgY immunoaffinity separation and MALDI MS for clinical proteomics of post-translational modification (glycosylation) as schematised in figure, with the aim to set up a high-throughput method to analyze macroheterogeneity and microheterogeneity of protein glycoforms. The method was validated by the analyses of human underglycosylated serum glycoproteins in CDG-I, CDG-II and secondary glycosylation disorders and applied for biomarker detection in the diagnostic procedure (CDG-I and II) and treatment monitoring (galactosemia and HFI).
114) The structure and proinflammatory activity of the lipopolysaccharide from Burkholderia multivorans and the differences between clonal strains colonizing pre and posttransplanted lungs
T.Ieranò, A.Silipo, L.Sturiale, D.Garozzo, H.Brookes, C.M.Anjam Khan, C.Bryant, F.K.Gould, P.A.Corris, R.Lanzetta, M.Parrilli, A.De Soyza, A.Molinaro
Glycobiology
18(11),
871-881
- 2008
The Burkholderia cepacia complex is a group of Gramnegative bacteria that are opportunistic pathogens for humans especially in cystic fibrosis patients. Lipopolysaccharide (LPS) molecules are potent virulence factors of Gram-negative bacteria organisms essential for bacterial survival. A complete analysis of the bacterial lipopolysaccharide structure to function relationship is required to understand the chemical basis of the inflammatory process. We have therefore investigated the structures of lipopolysaccharides from clonally identical Burkholderia multivorans strains (genomovar II) isolated pre- and post-lung transplantation through compositional analysis, mass spectrometry, and 2D NMR spectroscopy. We tested the LPS proinflammatory activity as a stimulant of human myelomonocytic U937 cell cytokine induction and assessed TLR4/MD2 signaling. Marked changes between the paired strains were found in the lipid A-inner core region. Such structural variations can contribute to the bacterial survival and persistence of infections despite the loss of a CF milieu following lung transplantation.
115) Self-Assembly Dynamics of Modular Homoditopic Bis-calix[5]arenes and Long-Chain á,ù-Alkanediyldiammonium Components
G.Gattuso, A.Notti, A.Pappalardo, M.F.Parisi, I.Pisagatti, S.Pappalardo, D.Garozzo, A.Messina, Y.Cohen, S.Slovak
Journal of Organic Chemistry
73(18),
7280-7289
- 2008
Homoditopic building blocks
1, featuring two π-rich cone-like calix[5]arene moieties connected at their narrow rims by a rigid
o-, m-, or
p-xylyl spacer in a centrosymmetric divergent arrangement, show a remarkable tendency to spontaneously and reversibly self-assemble with the complementary homoditopic α,ω-alkanediyldiammonium dipicrate guest salts
C8−C12·2Pic through iterative intermolecular inclusion events, forming supramolecular assemblies whose composition and dynamics strongly depend upon the length of the connector, the geometry of the spacer, as well as the concentration and/or molar ratios between the two components.
1H NMR spectroscopy and ESI-MS studies of
1/Cn·2Pic modular homoditopic pairs support the formation of discrete (bis)-
endo-cavity assemblies with the shorter
C8 and
C9 connectors, and/or (poly)capsular assemblies with the longer
C10−C12 components under appropriate concentrations and molar ratios (50 mM equimolar solutions).
1H NMR titration experiments and diffusion NMR studies provide clear evidence for the self-assembly dynamics of the complementary pairs here investigated.
116) Multiplexed glycoproteomic analysis of glycosylation disorders by sequential yolk immunoglobulins immunoseparation and MALDI-TOF MS
L.Sturiale, R.Barone, A.Palmigiano, C.N.Ndosimao, P.Briones, M.Adamowicz, J.Jaeken, D.Garozzo
Proteomics
8(18),
3822-3832
- 2008
This study applied yolk immunoglobulins immunoaffinity separation and MALDI-TOF MS for clinical proteomics of congenital disorders of glycosylation (CDG) and secondary glycosylation disorders [galactosemia and hereditary fructose intolerance (HFI)]. Serum transferrin (Tf) and α1-antitrypsin (AAT) that are markers for CDG, were purified sequentially to obtain high-quality MALDI mass spectra to differentiate single glycoforms of the native intact glycoproteins. The procedure was found feasible for the investigation of protein macroheterogeneity due to glycosylation site underoccupancy then ensuing the characterization of patients with CDG group I (N-glycan assembly disorders). Following PNGase F digestion of the purified glycoprotein, the characterization of protein microheterogeneity by N-glycan MS analysis was performed in a patient with CDG group II (processing disorders). CDG-Ia patients showed a typical profile of underglycosylation where the fully glycosylated glycoforms are always the most abundant present in plasma with lesser amounts of partially and unglycosylated glycoforms in this order. Galactosemia and HFI are potentially fatal diseases, which benefit from early diagnosis and prompt therapeutic intervention. In symptomatic patients with galactosemia and in those with HFI, MALDI MS of Tf and AAT depicts a hypoglycosylation profile with a significant increase of underglycosylated glycoforms that reverses by dietary treatment, representing a clue for diagnosis and treatment monitoring.
117) Clinical phenotype correlates to glycoprotein phenotype in a sib pair with CDG-Ia
R.Barone, L.Sturiale, V.Sofia, A.Ignoto, A.Fiumara, G.Sorge, D.Garozzo, M.Zappia
American Journal of Medical Genetics Part A
146A(16),
2103-2108
- 2008
Congenital disorder of glycosylation (CDG) type Ia (PMM2 mutations) is the most common genetic disorder of protein N-glycosylation. The wide clinical spectrum with mild to severe impairment of neurological function and extensive allelic heterogeneity hamper phenotype-genotype comparison. We report on two male adult siblings with the PMM2 mutations c. 385G > A (p.V129M) and c. 422G > A (p.R141H) and partially different clinical phenotype. Patient 2 has a more severe degree of neurological and systemic involvement and a more pronounced decrease in levels of serum glycoproteins. MALDI-TOF mass spectrometry of serum transferrin and alpha-1-antitrypsin shows more pronounced glycosylation defects in the more severely affected patient. Glycoproteomic analysis may reveal differences in CDG-Ia patients with different disease severity and might endorse clinical characterization of CDG-Ia patients.
118) Rhizobium rubiT: A Gram-Negative Phytopathogenic Bacterium Expressing the Lewis B Epitope on the Outer Core of its Lipooligosaccharide Fraction
V.Gargiulo, D.Garozzo, R.Lanzetta, A.Molinaro, L.Sturiale, C.De Castro, M.Parrilli
ChemBioChem
9(11),
1830-1835
- 2008
The structure of the core oligosaccharide from the phytopathogenic bacterium Rhizobium rubi was deduced by combining information from complementary chemical approaches (alkaline and acid hydrolysis), similar to the "overlap peptide" strategy. This structure is new and it contains two main oligosaccharide backbones that differ in the substitution degree of the external Kdo unit. The relevant feature shared by both oligosaccharides is the presence of a tetrasaccharide motif that is similar to the blood group Lewis B antigen (LeB). This epitope differs from LeB in the glycosidic configuration of the glucosamine unit (α and not β) and in the occurrence of acetyls substituents at O3 and/or O4 of the galactose moiety. Other notable structural features are the location of the Dha residue, the presence of a α-glucose unit that is linked to the inner Kdo unit, the high number of acid sugars and the highly branched core structure.
119) Clinical and biochemical features in a Congolese infant with congenital disorder of glycosylation (CDG)-IIx
N.C.Nsibu, J.Jaeken, H.Carchon, M.Mampunza, L.Sturiale, D.Garozzo, M.N.L.Mashako, M.P.Tshibassu
European Journal of Paediatric Neurology
12,
257-261
- 2008
We describe an infant girl with psychomotor retardation, growth retardation, mild facial dysmorphy, evidence of liver involvement and a type 2 pattern of serum sialotransferrins. Serum transferrin glycan analysis with MALDI-TOF showed an extremely altered N-glycan pattern with a large number of truncated asialoglycans pointing to a severely defective Nglycan processing. The basic defect in this patient with CDG-IIx has not yet been identified.
120) The Acylation and Phosphorylation Pattern of Lipid A from Xanthomonas Campestris Strongly Influence its Ability to Trigger the Innate Immune Response in Arabidopsis
A.Silipo, L.Sturiale, D.Garozzo, G.Erbs, T.Tandrup Jensen, R.Lanzetta, J.Maxwell Dow, M.Parrilli, M.A.Newman, A.Molinaro
ChemBioChem
9(6),
896-904
- 2008
Lipopolysaccharides (LPSs) are major components of the cell surface of Gram-negative bacteria. LPSs comprise a hydrophilic heteropolysaccharide (formed by the core oligosaccharide and the O-specific polysaccharide) that is covalently linked to the glycolipid moiety lipid A, which anchors these macromolecules to the external membrane. LPSs are one of a group of molecules called pathogen-associated molecular patterns (PAMPs) that are indispensable for bacterial growth and viability, and act to trigger innate defense responses in eukaryotes. We have previously shown that LPS from the plant pathogen Xanthomonas campestris pv. campestris (Xcc) can elicit defense responses in the model plant Arabidopsis thaliana. Here we have extended these studies by analysis of the structure and biological activity of LPS from a nonpathogenic Xcc mutant, strain 8530. We show that this Xcc strain is defective in core completion and introduces significant modification in the lipid A region, which involves the degree of acylation and nonstoichiometric substitution of the phosphate groups with phosphoethanolamine. Lipid A that was isolated from Xcc strain 8530 did not have the ability to induce the defense-related gene PR1 in Arabidopsis, or to prevent the hypersensitive response (HR) that is caused by avirulent bacteria as the lipid A from the wild-type could. This suggests that Xcc has the capacity to modify the structure of the lipid A to reduce its activity as a PAMP. We speculate that such effects might occur in wild-type bacteria that are exposed to stresses such as those that might be encountered during plant colonization and disease.
121) Structural characterizations of lipids A by MS/MS of doubly charged ions on a hybrid linear ion trap/orbitrap mass spectrometer
A.Silipo, C.De Castro, R.Lanzetta, A.Molinaro, M.Parrilli, G.Vago, L.Sturiale, A.Messina, D.Garozzo
Journal of Mass Spectrometry
43,
478-484
- 2008
DOI:
https://doi.org/10.1002/jms.1333
Here, a new "one pot" and fast approach is described, based on electrospray ionization (ESI) of negative ions by using a hybrid linear ion trap/orbitrap mass spectrometer (LTQ/orbitrap) for MS and MS/MS analysis. By this method the distribution of the primary and secondary acyl residues of the intact lipid A is inferred by analysis of the ESI spectra measured in positive and negative mode. The analysis of these data allows an unequivocal assignment of the fatty acid distribution. This methodology was successfully tested on two different lipid A with known structures, deriving from the Agrobacterium tumefaciens and Escherichia coli lipopolysaccharides.
122) Full structural characterization of Shigella flexneri M90T serotype 5 wild-type R-LPS and its ΔgalU mutant: glycine residue location in the inner core of the lipopolysaccharide
A.Molinaro, A.Silipo, C.De Castro, L.Sturiale, G.Nigro, D.Garozzo, M.L.Bernardini, R.Lanzetta, M.Parrilli
Glycobiology
18,
260-269
- 2008
Shigella flexneri is a Gram-negative bacterium responsible for serious enteric infections that occur mainly in the terminal ileum and colon. High interest in Shigella, as a human pathogen, is driven by its antibiotic resistance and the necessity to develop a vaccine against its infections.Vaccines of the last generation use carbohydrate moieties of the lipopolysaccharide as probable candidates. For this reason, the primary structure of the core oligosaccharide from the R-LPS produced by S. flexneri M90T serotype 5 using chemical analysis, nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MALDI), is herein reported. This is the first time that the core oligosaccharide primary structure by S. flexneri M90T is established in an unambiguous multidisciplinary approach. Chemical and spectroscopical investigation of the de-acetylated LPS showed that the inner core structure is characterized by a L,D-Hep-(1 →7)-L,D-Hep-(1 →3)-L,D-Hep-(1 →5)-[Kdo-(2 →4)]-Kdo sequence that is the common structural theme identified in Enterobacteriaceae. In particular, in S. flexneri M90T serotype 5 LPS, a glucosamine residue is additionally sitting at O-7 of the last heptose whereas the outer core is characterized by glucose and galactose residues. Also, in order to exactly define the position of glycine that is an integral constituent of the core region of the LPS, we created a S. flexneri M90T ΔgalU mutant and studied its LOS. In this way it was possible to establish that glycine is sitting at O-6 of the second heptose in the inner core.
123) Self-assembly of a nucleotide-calixarene hybrid in a triangular supramolecule
G.M.L.Consoli, G.Granata, D.Garozzo, T.Mecca, C.Geraci
Tetrahedron Letters
48,
7974-7977
- 2007
The self-assembly of a thymine nucleotide-calixarene hybrid (1) in CDCl3 as a solvent was investigated. FT-IR, ESI-MS, 1H and DOSY-NMR spectra evidenced that compound 1 (ammonium or sodium salt) self-assembles in a triangular trimeric supramolecule by thymine-thymine hydrogen bonding. The saline form is crucial for the arrangement in the cyclic trimer as the protonation of the nucleotide phosphate groups leads the assembly toward a dimeric species.
124) Detailed characterization of the lipid A fraction from the nonpathogen Acinetobacter radioresistens strain S13
S.Leone, L.Sturiale, E.Pessione, R.Mazzoli, C.Giunta, R.Lanzetta, D.Garozzo, A.Molinaro, M.Parrilli
Journal of Lipid Research
48,
1045-1051
- 2007
The genus Acinetobacter is composed of ubiquitous, generally nonpathogen environmental bacteria. Interest concerning these microorganisms has increased during the last 30 years, because some strains, belonging to the so-called A. baumannii-A. calcoaceticus complex, have been implicated in some severe pathological states in debilitated and hospitalized patients. The involvement of lipopolysaccharides (LPSs) as virulence factors in infections by Acinetobacter has been proven, and ongoing studies are aimed toward the complete serological characterization of the O-polysaccharides from LPSs isolated in clinical samples. Conversely, no characterization of the lipid A fraction from Acinetobacter strains has been performed. Here, the detailed structure of the lipid A fraction from A. radioresistens S13 is reported for the first time. A. radioresistens strains have never been isolated in cases of infectious disease. Nevertheless, it is known that the lipid A structure, with minor variations, is highly conserved across the genus; thus, structural details acquired from studies of this nonpathogen strain represent a useful basis for further studies of pathogen species.
125) The Complete Structure and Pro-inflammatory Activity of the Lipooligosaccharide of the Highly Epidemic and Virulent Gram-Negative Bacterium Burkholderia cenocepacia ET-12 (Strain J2315)
A.Silipo, A.Molinaro, T.Ieranò, A.De Soyza, L.Sturiale, D.Garozzo, C.Aldridge, P.A.Corris, C.M.Anjam Khan, R.Lanzetta, M.Parrilli
Chemistry-A European Journal
13,
3501-3511
- 2007
Members of genus Burkholderia include opportunistic Gram-negative bacteria that are responsible for serious infections in immunocompromised and cystic fibrosis (CF) patients. The Burkholderia cepacia complex is a group of microorganisms composed of at least nine closely related genomovars. Among these, B. cenocepacia is widely recognized to cause epidemics associated with excessive mortality. Species that belong to this strain are problematic CF pathogens because of their high resistance to antibiotics, which makes respiratory infections difficult to treat and impossible to eradicate. Infection by these bacteria is associated with higher mortality in CF and poor outcomes following lung transplantation. One virulence factor contributing to this is the pro-inflammatory lipopolysaccharide (LPS) molecules. Thus, the knowledge of the lipopolysaccharide structure is an essential prerequisite to the understanding of the molecular mechanisms involved in the inflammatory process. Such data are instrumental in aiding the design of antimicrobial compounds and for developing therapeutic strategies against the inflammatory cascade. In particular, defining the structure of the LPS from B. cenocepacia ET-12 clone LMG 16656 (also known as J2315) is extremely important given the recent completion of the sequencing project at the Sanger Centre using this specific strain. In this paper we address this issue by defining the pro-inflammatory activity of the pure lipopolysaccharide, and by describing its full primary structure. The activity of the lipopolysaccharide was tested as a stimulant in human myelomonocytic U937 cells. The structural analysis was carried out by compositional analysis, mass spectrometry and 2D NMR spectroscopy on the intact lipooligosacchride (LOS) and its fragments, which were obtained by selective chemical degradations.
126) Syntheses, Structures, and Anion-Binding Properties of Two Novel Calix[2]benzo[4]pyrroles
G.Cafeo, F.H.Kohnke, A.J.P.White, D.Garozzo, A.Messina
Chemistry-A European Journal
13,
649-656
- 2007
Calix[2]benzo[4]pyrrole m-6 and p-6, each containing two dipyrromethane moieties and two m-phenylene or p-phenylene units, respectively, were readily synthesised from pyrrole, 1,3- and 1,4-bis(1,1’-dimethylhydroxymethyl)benzene, (m-4 and p-4, respectively) and acetone. Macrocycles m-6 and p-6 were tested as receptors for a selection of anions, such as acetate, dihydrogenphosphate and fluoride. The X-ray structures of m-6 and p-6 and those of the complexes m-6-F-, m-6-Cl- and m-6-CH3COO- (with an nBu4N+ counterion) were also determined.
127) The Outer Membrane of the Marine Gram-Negative Bacterium Alteromonas addita is Composed of a Very Short-Chain Lipopolysaccharide with a High Negative Charge Density
S.Leone, A.Molinaro, L.Sturiale, D.Garozzo, E.L.Nazarenko, R.P.Gorshkova3, E.P.Ivanova, L.S.Shevchenko, R.Lanzetta, M.Parrilli
European Journal of Organic Chemistry
2007(7),
1113-1122
- 2007
The complete structure of the lipopolysaccharide isolated from the Gram-negative marine bacterium Alteromonas addita, type strain KMM 3600T = R10SW13T, has been elucidated by means of a combined chemical approach and state-of-the-art NMR and MS analyses. Isolation and characterisation of the lipid A moiety and the core oligosaccharide were pursued separately after either acid or alkaline treatment of the lipopolysaccharide. The structure detected was identified as a novel, highly negatively charged, deep-rough lipopolysaccharide in which a trisaccharide subunit is connected to a typical lipid A glucosamine backbone. Within the core oligosaccharide, a phosphodiester bridge connects a glucose unit to a heptose residue.
128) Borderline mental development in a congenital disorder of glycosylation (CDG) type Ia patient with multisystemic involvement (intermediate phenotype)
R.Barone, L.Sturiale, A.Fiumara, G.Uziel, D.Garozzo, J.Jaeken
Journal of Inherited Metabolic Disease
30,
107
- 2007
CDG Ia (phosphomannomutase deficiency) has a wide clinical spectrum with the most severe affected patients having multisystemic disease in addition to severe nervous system involvement. We report a patient with CDG Ia and an intermediate phenotype due to mild neurological impairment and borderline cognitive abilities despite the occurrence of typical extraneurological symptoms. These included liver involvement, coagulopathy and failure to thrive with enteropathy. Genotype analyses showed that he was compound heterozygous for T237R/C241S mutations. This observation underlines that the CDG Ia clinical spectrum may include intraindividual variability that might reflect different degrees of glycosylation abnormalities among distinct body compartments. CDG Ia should be considered in cases of unexplained liver involvement and/or enteropathy in patients with mild developmental delay and subtle neurological signs.
129) Differences in disease severity reflect diversity of glycoproteome in two sibs with CDG-Ia
R.Barone, L.Sturiale, V.Sofia, A.Ignoto, A.Fiumara, D.Garozzo, M.Zappia
3rd International Meeting on Congenital Disorders of Glycosylation. 18-19 October, 2007, Paris, France
- 2007
130) Multiplexed glycoproteomic analysis of glycosylation disorders by sequential IgY immunoseparation and MALDI TOF MS
L.Sturiale, R.Barone, A.Messina, A.Palmigiano, D.Garozzo
3rd International Meeting on Congenital Disorders of Glycosylation. 18-19 October, 2007, Paris, France
- 2007
131) Proteomic analysis of human tear fluids using LC-MALDI MS and MS/MS
A.Messina, L.Sturiale, R.Barone, A.Palmigiano, MG.Mazzone, A.Leonardi, D.Garozzo
Italian Proteomic Association. 2nd Annual National Conference. 26-29 Giugno 2007, Acitrezza (CT). Italy
- 2007
132) Differences in severity of clinical phenotype reflect diversity of glycoproteome in two sibs with CDG-Ia
L.Sturiale, R.Barone, V.Sofia, A.Messina, A.Palmigiano, A.Fiumara, M.Zappia, D.Garozzo
Italian Proteomic Association. 2nd Annual National Conference. 26-29 Giugno 2007, Acitrezza (CT). Italy
- 2007
133) Glycoproteomics of congenital disorders of glycosylation and secondary glycosylation disorders
R.Barone, L.Sturiale, A.Messina, A.Palmigiano, D.Garozzo
Italian Proteomic Association. 2nd Annual National Conference, 26-29 Giugno 2007, Acitrezza (CT), Italy
- 2007
134) Exopolysaccharides as Bacterial Defence Tools: the Case of Burkholderia cepacia complex
P.Cescutti, Y.Herasimenka, G.Impallomeni, L.Sturiale, A.Palmigiano, D.Garozzo, R.Rizzo
14th European Carbohydrate Symposium (EUROCARB 2007), Lubeck (Germania), 2-7 Settembre, 2007
- 2007
135) Structural Analysis of the Polysaccharides from Echinacea angustifolia Radix
R.Cozzolino, P.Malvagna, E.Spina, A.Giori, N.Fuzzati, A.Anelli, D.Garozzo, G.Impallomeni
XVIII Convegno Italiano di Scienza e Tecnologia delle Macromolecole, Catania, 16-20 Settembre, 2007
- 2007
136) Dall’analisi dei monosaccaridi all’analisi del glicoma; dalle micromoli alle femtomoli: i progressi della spettrometria di massa nell’analisi dei carboidrati
D.Garozzo
X Convegno-scuola sulla chimica dei carboidrati, 25-29 Giugno 2006, Certosa di Pontignano - Siena
- 2006
137) Silver-Russell syndrome-like features in a patient with abnormal transferrin glycosylation
R.Barone, A.Fiumara, E.Pasquini, MA.Donati, L.Sturiale, D.Garozzo, G.Sorge
EUROGLYCANET meeting, 28-29 April, 2006, Porto, Portugal.
- 2006
138) Advances in purification methods of serum glycoproteins for MALDI-MS analysis of N-glycome in CDG patients and in galactosemia
L.Sturiale, R.Barone, D.Garozzo
EUROGLYCANET meeting, 28-29 April, 2006, Porto, Portugal
- 2006
139) Differences in severity of clinical phenotype reflect diversity of glycosylation status in two sibs with phosphomannomutase deficiency (CDG-Ia) due to V129M/R141H mutations
R.Barone L.Sturiale, V.Sofia, A.Ignoto, L.Pavone, D.Garozzo, M.Zappia
XXXVII Congresso Società Italiana di Neurologia. 14-18 Ottobre 2006. Bari
- 2006
140) Analisi del glicoma del fluido lacrimale in pazienti affetti da disordini congeniti della glicosilazione (CDG)
L.Sturiale, R.Barone, A.Messina, A.Scuderi, A.Leonardi, M.Santocono, MG.Mazzone, D.Garozzo
X Convegno-scuola sulla chimica dei carboidrati. 25-29 Giugno 2006. Certosa di Pontignano - Siena
- 2006
141) Advances in Purification Methods of Serum Glycoproteins for
MALDI-MS Analysis of N- Glycome in Patients with Glycosylation
Disorders
L.Sturiale, R.Barone, D.Garozzo
Glycobiology
16(11),
1135
- 2006
Genetic defects of the N-glycosylation pathway, named Congenital Disorders of Glycosylation (CDGs), result in abnormalities of N-glycome with aberrant glycan structures and changes in the relative levels of normal glycan moiety. Understanding N-glycan profile may be useful for characterization of known CDG types and to identify glycosylation processing defects in unsolved patients. CDG are heterogeneous disorders with variable clinical findings and multisystem involvement. As glycosylation defects are usually associated with abnormal glycoprotein folding and activity, it is plausible that the variety of clinical signs in CDG underlies abnormalities in a plethora of glycosylated molecules. Serum Transferrin was widely used so far to characterize N-glycan profile in patients with CDG; an alternative approach was based on the analysis of N-linked glycan released from total plasma. Our present work on N-glycome analyses in patients with CDG and related disorders is based on the systematic characterization, in addition to Transferrin, of multiple abundant serum glycoproteins, including acute-phase proteins. On this regard, we are working for creation of N-glycan profiling panel of each patient by the following steps: 1) purification of target glycoproteins by using sequentially, selective immunoaffinity columns on a few amount of unique serum sample. 2) characterization of the intact glycoprotein by MALDI mass spectrometry: this fundamental step allows us to analyze the rate and extent of deglycosylation (N-glycosylation site underoccupancy). 3) MALDI analyses of N-glycan structures. The observed occurrence of underglycosylation and abnormal glycan structure of AAT in CDG-Ia may link to possible unbalance of protease/antiprotease system in these patients.
142) Lower rim arylation of calix[n]arenes with extended perfluorinated domains
S.Buscemi, A.Pace, A.Palumbo Piccionello, S.Pappalardo, D.Garozzo, T.Pilati, G.Gattuso, A.Pappalardo, I.Pisagatti, M.F.Parisi
Tetrahedron Letters
47,
9049-9052
- 2006
Exhaustive O-arylation of p-tert-butylcalix[n]arenes 2 (n = 4-8) with an excess of 3-pentadecafluoroheptyl-5-pentafluorophenyl- 1,2,4-oxadiazole 3 and K2CO3 in refluxing acetonitrile provides an easy entry to a new family of perfluorinated calix[n]arenes 1. The cyclic tetramer furnishes a mixture of cone, partial cone, and 1,2-alternate conformers, while the larger macrocycles afford single products. The structures of all new compounds are substantiated by NMR techniques and MALDI-TOF mass spectral data. Single-crystal X-ray diffraction studies on the pentamer derivative 1b reveal a distorted cone-in conformation of the calixarene cup.
143) Structural Analysis of the Deep Rough Lipopolysaccharide from Gram Negative Bacterium Alteromonas macleodii ATCC 27126T: The First Finding of β-Kdo in the Inner Core of Lipopolysaccharides
V.Liparoti, A.Molinaro, L.Sturiale, D.Garozzo, E.L.Nazarenko, R.P.Gorshkova, E.P.Ivanova, L.S.Shevcenko, R.Lanzetta, M.Parrilli
European Journal of Organic Chemistry
2006(20),
4710-4716
- 2006
Alteromonas macleodii ATCC 27126T is a Gram-negative marine bacterium isolated from a sea water sample collected from around the Hawaiian Islands. The structure of the lipooligosaccharide derived from its outer membrane has been fully determined using either alkaline or acid hydrolysis. Alkaline treatment, aimed at recovering the complete carbohydrate backbone, was carried out by mild hydrazinolysis (de-O-acylation) followed by de-N-acylation using hot KOH and furnished a single core glycoform. Mild acid hydrolysis was employed to obtain the lipid A moiety which was selectively de-O-acylated and analysed to determine its primary structure. The structural elucidation of both fractions was carried out by chemical analyses, 2D NMR spectroscopy and MALDI-TOF mass spectrometry and revealed a novel lipooligosaccharide with an unusual structure.
144) Full Structural Characterisation of the Lipooligosaccharide of a Burkholderia pyrrocinia Clinical Isolate
A.Silipo, A.Molinaro, D.Comegna, L.Sturiale, P.Cescutti, D.Garozzo, R.Lanzetta, M.Parrilli
European Journal of Organic Chemistry
2006(21),
4874-4883
- 2006
This paper deals with the full structural elucidation of the lipooligosaccharide from the Gram-negative bacterium Burkholderia pyrrocinia. B. pyrrocinia is one of the nine species included in the Burkholderia cepacia complex (BCC), a group of important opportunistic pathogens in patients with cystic fibrosis and chronic granulomatous disease. B. pyrrocinia strain BTS7, isolated from a patient with cystic fibrosis, was found to exclusively produce a lipooligosaccharide (LOS). This component of the external membrane plays a key role in the virulence of BCC bacteria and is required for resistance to antimicrobial compounds and for bacterial survival. Here we present, for the first time, a detailed study of the structure of the lipooligosaccharide (LOS) elucidated by means of compositional analysis, MALDI and ESI mass spectrometry and 2D NMR spectroscopy. The LOS of B. pyrrocinia was degraded by complete deacylation, dephosphorylation and reduction. The major oligosaccharide representing the carbohydrate backbone was isolated by size-exclusion chromatography and identified. The structural determination of molecules (such as the LOS of B. pyrrocinia) involved in the activation of the pro-inflammatory processes is the first step in the adoption of new therapeutic strategies against lung infections.
145) Structural analysis of the polysaccharides from Echinacea angustifolia radix
R.Cozzolino, P.Malvagna, E.Spina, A.Giori, N.Fuzzati, A.Anelli, D.Garozzo, G.Impallomeni
Carbohydrate Polymers
65,
263-272
- 2006
In this paper we report the characterization by monosaccharide and linkage analyses and by NMR spectroscopy and size exclusion chromatography of the carbohydrate fraction extracted from Echinacea angustifolia radix. In addition, the products of endo-pectin lyase, endo-pectate lyase, endo-polygalacturonase, endo-galactanase, and endo-arabinase digestion were characterized by MALDI-TOF mass spectrometry. The data obtained during this study showed that the carbohydrate fraction extracted from E. angustifolia radix is constituted by two polysaccharides with molecular weight of about 128,000 and 4500 Da. The low molecular weight polysaccharide corresponds to inulin while the high molecular weight component is a high metoxy pectin in which the backbone structure of the smooth region is constituted by α-(1-4)-polygalacturonan partially methyl esterified (60%) and acetylated (9%) and with the hairy regions containing 2-O- and 2,4-O-rhamnopyranose, 5-O- and 3,5-O-arabinofuranose, 3,6-galactopyranose, and terminal rhamnopyranose, arabinofuranose, arabinopyranose, galactopyranose, and galacturonopyranose. Mass spectrometry data of a galactanase treated sample showed evidence of a novel structure in the pectin hairy region, namely a galactose-galacturonic acid alternating sequence.
146) Structural elucidation of the core-lipid A backbone from the lipopolysaccharide of Acinetobacter radioresistens S13, an organic solvent tolerant Gram-negative bacterium
S.Leone, A.Molinaro, E.Pessione, R.Mazzoli, C.Giunta, L.Sturiale, D.Garozzo, R.Lanzetta, M.Parrilli
Carbohydrate Research
341,
582-590
- 2006
The structure of the core oligosaccharide of the lipopolysaccharide from an organic solvent tolerant Gram-negative bacterium,
Acinetobacter radioresistens S13, was investigated by chemical analysis, NMR spectroscopy and MALDI-TOF mass spectrometry. All the experiments were performed on the oligosaccharides obtained either by alkaline degradation or mild acid hydrolysis. The data showed the presence of two novel oligosaccharide molecules containing a trisaccharide of 3-deoxy-D-
manno- octulopyranosonic acid in the inner core region and a glucose rich outer core whose structure is the following:
R = H in the main oligosaccharide and β-Glc in the minor product. The bacterium was grown on aromatic (phenol and benzoic acid) and nonaromatic carbon sources and the core oligosaccharide resulted to occur always with this novel structure.
147) Advances in purification methods of serum glycoproteins for MALDI_MS analysis of N-Glycome in patients with glycosylation disorders.
L.Sturiale, R.Barone, D.Garozzo
2005 Annual Meeting of the Society for Glycobiology. 9-12 November, 2005. Boston
- 2005
148) Mass spectrometric approaches for the N-glycosylation analysis in congenital disordrs of glycosylation (CDG) and galactosemia
L.Sturiale, R.Barone, M.Perez, G.Sorge, J.Jaaken, D.Garozzo
Italian Human Proteomic Organization (IHUPO). Terzo Congresso Nazionale. 29-Settembre- 1 Ottobre 2005, Lodi-Milano
- 2005
149) Mass spectrometry strategies for the N-glycosylation analysis of serum glycoproteins in patients with congenital disorders of glycosylation (CDG) and galactosemia
L.Sturiale, R.Barone, G.Sorge, M.Perez, A.Messina, S.Tortorelli, J.F.O&rsquoBrien, J.Jaeken, D.Garozzo
Glycoproteomics - Protein Modifications for Versatile Functions. June 28 - 30, 2005, Dubrovnik, Croatia
- 2005
150) Hypoglycosylation with increate fucosylation and branching of serum transferrin N-glycans in untreated galactosemia
L.Sturiale, R.Barone, A.Fiumara, M.Perez, M.Zaffanello, G.Sorge, L.Pavone, J.Jaeken, D.Garozzo
1st Euroglycanet meeting, March, 28-30, 2005, Athens, Greece
- 2005
151) Proteomics of gluten: mapping of the 1Bx7 glutenin subunit in Chinese Spring cultivar by matrix-assisted laser desorption/ionization
G.Alberghina, R.Cozzolino, S.Fisichella, D.Garozzo, A.Savarino
Rapid Communications in Mass Spectrometry
19(14),
2069
- 2005
The verification of the cDNA-deduced sequence of the high molecular weight glutenin subunit 1Bx7 in Chinese Spring cultivar was achieved by direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis of the tryptic fragments. The published sequence of the 1Bx7 subunit contains 5 Lys and 15 Arg residues but, due to the presence of three Arg-Pro bonds, which are generally resistant to cleavage by trypsin, or cleaved to a very limited extent by trypsin, 19 peptides can be predicted. The identification of the tryptic fragments was achieved by direct MALDI-MS analysis by using three different matrices (DHB, SA and HCCA) in combination with the most compatible sample preparation procedures in order to obtain the maximum sequence coverage. MALDI analysis of the 1Bx7 tryptic digest resulted in the identification of the expected peptides and additional fragments arising from non-specific cleavages; the fragments that were not detected are peptides with low mass (from 147.2 to 317.4), so we obtained a sequence coverage of 98.8%. The results reported here also indicated that the sequence of the 1Bx7 subunit from cv. Chinese Spring is different from the cDNA-deduced sequence reported in the literature; in particular, a possible insertion of the hexapeptide QPGQGQ within the sequence Gln630-Tyr725 was suggested. Finally, it is possible to rule out glycosylation of the 1Bx7 subunit, or any other post-translational modification, to within the detection limits of the method.
152) New conditions for matrix-assisted laser desorption/ionization mass spectrometry of native bacterial R-type lipopolysaccharides
L.Sturiale, D.Garozzo, A.Silipo, R.Lanzetta, M.Parrilli, A.Molinaro
Rapid Communications in Mass Spectrometry
19(13),
1829
- 2005
A new sample preparation method for matrix-assisted laser desorption/ionization (MALDI) analysis of native rough-type lipopolysaccharides (R-type LPSs) is presented. In our MALDI mass spectra, besides the [M-H]- ions, abundant ions originating from the cleavage between the 3- deoxy-D-manno-oct-2-ulosonic acid (Kdo) unit and the lipid A moiety are always present, giving important pieces of information about the structure of the molecules analyzed. Remarkably, in most cases, the comparison of the MALDI mass spectra of the intact R-type LPS with the Odeacylated one allowed us to obtain the structure of the lipid A moiety.
153) Complete Structural Elucidation of a Novel Lipooligosaccharide from the Outer Membrane of the Marine Bacterium Shewanella pacifica
A.Silipo, S.Leone, A.Molinaro, L.Sturiale, D.Garozzo, E.L.Nazarenko, R.P.Gorshkova, E.P.Ivanova, R.Lanzetta, M.Parrilli
European Journal of Organic Chemistry
2005(11),
2281
- 2005
Shewanella pacifica is a Gram-negative microrganism that is able to grow in sea water. A novel lipooligosaccharide (LOS) has been isolated from the outer membrane of this bacterium and its primary structure fully characterised. For the first time, the presence of a 2,3-dihydroxypropanoic acid residue (glyceric acid) has been identified in the core region. The complete structure of the LOS was determined by compositional and methylation analyses, by MALDI mass spectrometry, and by 1H, 13C and 31P NMR spectroscopy on the oligosaccharides formed by selective degradation of the LOS. Strong alkaline treatment, aimed at recovering and identifying the complete carbohydrate backbone, was carried out by hydrazinolysis followed by de-N-acylation with hot KOH, whereas mild hydrazinolysis (de-O-acylation) allowed us to gain information about the nature of the phosphate and other non-carbohydrate substituents on the core oligosaccharide. Mild acid hydrolysis was employed to obtain a lipid A moiety with which further degradation and mass spectrometry experiments were carried out in order to determine its primary structure.
154) The complete structure of the core carbohydrate backbone from the LPS of marine halophilic bacterium Pseudoalteromonas carrageenovora type strain IAM 12662T
A.Silipo, R.Lanzetta, M.Parrilli, L.Sturiale, D.Garozzo, E.L.Nazarenko, R.P.Gorshkova, E.P.Ivanovac, A.Molinaro
Carbohydrate Research
340(8),
1475
- 2005
The complete novel structure of the components of the core oligosaccharide fraction from the LOS of the halophilic marine bacterium
Pseudoalteromonas carrageenovora was characterized. The fully de-acylated lipooligosaccharide was studied by means of compositional analysis, matrix-assisted laser desorption/ionization mass spectrometry and complete
1H and
13C and
31P NMR spectroscopy. The core oligosaccharide is composed by a mixture of species differing for the length of the sugar chain and the phosphorylation pattern:
All sugars are D-pyranoses. Hep is L-glycero-D-manno-heptose, Kdo is 3-deoxy-D-manno-oct-2-ulosonic acid, P is phosphate, residues and substituents in italic are not stoichiometrically linked.
155) Synthesis and characterization of poly(amidoamine)-platinum(II) complexes. Detailed speciation by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry
A.Mazzaglia, L.Monsù Scolaro, D.Garozzo, P.Malvagna, R.Romeo
Journal of Organometallic Chemistry
690(8),
1978
- 2005
MALDI and ESI-MS have been applied to the characterization of the reaction products between the labile cis-[Pt(DMSO)2Cl2] (1) and trans-[Pt(DMSO)2Cl(CH3)] (2) complexes with the simplest poly(amidoamine) ligand (PAMAM, G = 0, 1,2-diaminoethane as core). The comparison of the mass spectra of the starting G0 and those of the metallo-dendrimers formed upon mixing of the reagents in an equimolecular ratio, and the analysis of the isotopic distribution in the ESI spectra, have revealed the formation of cationic and neutral mononuclear complexes with PAMAM as ligand, e.g., cis-[Pt(DMSO)(PAMAM)Cl]Cl or trans- (C,N)[Pt(DMSO)(PAMAM)Cl(CH3)], together with various minor components, which have been identified as derivatives from defective structures of PAMAM. The geometry of the main products has been deduced from the values of the protons coupling constants with the isotopically abundant 195Pt. The metal-to-ligand bond is restricted to the peripheral amino groups of PAMAM which shows sufficient flexibility to involve either one or two branches in the coordination bonding.
156) Identificazione rapida di batteri mediante spettrometria di massa MALDI-TOF: dallo spettro del lipooligosaccaride all’individuazione di batteri gram-negativi
L.Sturiale, R.Cozzolino, M.Perez, A.Messina, D.Garozzo
Convegno finale del progetto Trattamento di prodotti freschi altamente deperibili per garantirne qualità, sicurezza e salubrità . 16-19 Marzo, 2005, Ariano Irpino (AV)
- 2005
157) Mass spectrometric strategies for the N-glycosylation analysis of serum glycoproteins in patients with congenital disorders of glycosylation (CDG) and galactosemia
L.Sturiale, R.Barone, M.Perez, G.Sorge, J.Jaeken, D.Garozzo
GLYCO XVIII. XVIII International Symposium on Glycoconjugates, September 4-9, 2005, Firenze, Italy
- 2005
158) Hypoglycosylation with increased fucosylation and branching of serum transferrin
N-glycans in untreated galactosemia
L.Sturiale, R.Barone, A.Fiumara, M.Perez, M.Zaffanello, G.Sorge, L.Pavone, S.Tortorelli, J.F.O’Brien, J.Jaeken, D.Garozzo
Glycobiology
15,
1268
- 2005
Untreated classic galactosemia (galactose-1-phosphate uridyltransferase [GALT] deficiency) is known as a secondary congenital disorders of glycosylation (CDG) characterized by galactose deficiency of glycoproteins and glycolipids (processing defect or CDG-II). The mechanism of this undergalactosylation has not been established. Here we show that in untreated galactosemia, there is also a partial deficiency of whole glycans of serum transferrin associated with increased fucosylation and branching as seen in genetic glycosylation assembly defects (CDG-I). Thus galactosemia seems to be a secondary "dual" CDG causing a processing as well as an assembly N-glycosylation defect. We also demonstrated that in galactosemia patients, transferrin N-glycan biosynthesis is restored upon dietary treatment.
159) Proteomic Analysis of Human Tear Fluids in Healthy and Pathological Conditions
R.Cozzolino, M. Perez, A.Messina, L.Sturiale, D.Garozzo, M.G.Mazzone, M.Santocono
53rd ASMS Conference (June 5-9 2005)
- 2005
The ocular tear film is a complex mixture of ions, small molecules, glycoproteins and proteins, which form a thin film over the surface of the cornea and conjunctiva. Since directly in contact with the environment, it plays a critical role in protecting against microbial challenge and preserving visual acuity. Changes in tear film composition are usually related to several eye diseases like dry eye, Sjögren syndrome, conjunctivitis, blepharitis and so on. To elucidate compositional differences between healthy subjects and patients affected by different eye diseases, we thoroughly studied their tear proteomic profiles by MALDI-TOF MS, focusing in particular the low mass range region, in which defensins and bioactive peptides are present in order to find biomarkers for disease diagnosis.
160) Structural characterization of the carbohydrate backbone of the lipooligosaccharide of the marine bacterium Arenibacter certesii strain KMM 3941T
A.Silipo, A.Molinaro, E.L.Nazarenko, L.Sturiale, D.Garozzo, R.P.Gorshkova, O.I.Nedashkovskaya, R.Lanzetta, M.Parrilli
Carbohydrate Research
340(16),
2540
- 2005
The structure of the carbohydrate backbone of the lipooligosaccharide (LOS) of the marine bacterium
Arenibacter certesii strain KMM 3941
T has been elucidated. The structure was obtained by means of compositional analysis, matrix-assisted laser desorption/ionization mass spectrometry and complete
1H and
13C and
31P NMR spectroscopy. It shows novel and interesting aspects and is the first description of
Arenibacter lipopolysaccharides. Strong and mild alkaline treatments, to fully deacylate and only to O-deacylate the LOS were performed in order to determine the core structure. The core consists of a mixture of species differing by the presence of a non-stoichiometric α-D-rhamnose residue. The Kdo unit is substituted at O-5 by α-mannose and at O-4 by a α-galactosyluronic acid phosphate. The lipid A is constituted by a bis-phosphorylated disaccharide unit composed by a 2,3- diamino-2,3-dideoxy-β-D-glucopyranose (DAG) residue as non-reducing end and a GlcN as reducing end.
161) A calix[5]arene-based heterotetratopic host for molecular recognition of long-chain, ion-paired á,ù-alkanediyldiammonium salts
D.Garozzo, G.Gattuso, A.Notti, A.Pappalardo, S.Pappalardo, M.F.Parisi, M.Perez, I.Pisagatti
Angewandte Chemie-International Edition
44(31),
4892
- 2005
The anions went in two by two: Sep. ion-pair binding of nano-sized α,ω-alkanediyldiammonium dichloride salts is achieved in org. media with high efficiency and selectivity by a heterotetratopic receptor that comprises two converging calix[5]arene units to tightly encapsulate the linear dication, as well as two ureido side functions to simultaneously bind the chloride counteranions through hydrogen bonding.
162) Mass spectrometric strategies for the N-glycosylation analisys of serum glycoproteins in patients with congenital disorders of glycosylation (CDG) and galactosemia
L.Sturiale, R.Barone, M.Perez, G.Sorge, J.Jaeken, D.Garozzo
Glycoconjugate Journal
22 (4/5/6) ,
202
- 2005
Congenital disorders of glycosylation (CDG) and galactosemia are inherited metabolic diseases which have in common the occurrence of an abnormal glycosylation pattern of some serum glycoproteins. CDG are due to defects in the synthesis of N-glycans and less frequently affect the O-glycosylation pathway. As glycoproteins are ubiquitous molecules, CDG present as multisystemic disorders with a prominent central nervous system involvement and a variable clinical spectrum. CDG type I defines defects owing to impaired synthesis of the lipid-linked oligosaccharide and/or its transfer to the nascent glycoprotein in the cytosol and in the endoplasmic reticulum, CDG type II refers to abnormalities in the glycoprotein processing at the Golgi level. The most common form of galactosemia, due to a deficiency of galactose-1-phosphate uridyltransferase (GalT), causes accumulation of galactose and galactose-1-phosphate in blood and tissues, and if untreated, produces severe symptoms as mental retardation, cirrhosis of the liver, and cataracts. In both CDG and galactosemia an abnormal glycosylation pattern involving multiple serum glycoproteins may be promptly detectable by the isoelectric focusing (IEF) of serum proteins following by immunodetection of transferrin isoforms which show a cathodal shift due to the absence of terminal negatively charged sialic acid residues. Although IEF allows the measurements of the sialylation degree in serum transferrin, it is uninformative about the presence of isoforms due to the partial occupancy of one or both transferrin glycosylation sites and the detailed analysis of the N-glycan structures. In order to identify glycosylation abnormalities both in CDG and in galactosemia patients, additional techniques such as matrix assisted laser-desorption ionisation (MALDI) and Electrospray mass spectrometry may be essential. We used mass spectrometry based strategy to define the glycosylation degree of intact glycoproteins and to detect changes in serum protein N-glycan profiles. These findings open the way to a better understanding of the biochemical mechanisms of defective glycosylation.
163) Increased fucosylation and branching of serum transferrin N-glycans in long-term untreated galactosemic patients
L.Sturiale R.Barone, G.Sorge, M.Zaffanello, A.Fiumara, G.Impallomeni, D.Garozzo
US/Japan Glyco 2004 - Joint meeting of the society for glycobiology and the japanese society of carbohydrate research. November 17-20, 2004, Honolulu, Hawaii
- 2004
164) Glycomics: a frontier in proteomics
D.Garozzo
Primiero discussion group on mass spectrometry. New frontiers in proteomics . November 7-10, 2004. Fiera di Primiero (TN)
- 2004
165) New Fragmentation Mechanisms in MALDI-TOF/TOF MS/MS of Carbohydrates
D.Garozzo
International Symposium on Mass Spectrometry in Proteomics and Molecular Profiles. 29th January 2004. Università della Calabria
- 2004
166) From calixfurans to heterocyclophanes containing isopyrazole units
G.Cafeo, D.Garozzo, F.H.Kohnke, S.Pappalardo, M.F.Parisi, R.Pistone Nascone, D.J.Williams
Tetrahedron
60(8),
1895
- 2004
Cyclic poly-1,4-diketones 2, obtained by the oxidn. of the furan units present in calix[4]furan 1a and calix[6]furan 1c have been converted into the novel heterocyclophanes 4a and 4c contg. four and six isopyrazole units, resp. Soln. studies have demonstrated the ability of 4a and 4c to act as ligands for transition metals. The crystal structures of 4a and the coordination compd. formed by 4c with 2 equiv. of cis-PtCl2(DMSO)2 have been detd. In the solid state 4c is shown to bind arom. substrates within its cavity.
167) New fragmentation mechanisms in matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry of carbohydrates
E.Spina, L.Sturiale, D.Romeo, G.Impallomeni, D.Garozzo, D.Waidelich, M.Glueckmann
Rapid Communications in Mass Spectrometry
18(4),
392
- 2004
The spectra recorded by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS/MS) of complex carbohydrates from human milk are presented. Besides ions originating from glycosidic cleavages and from sugar ring fragmentations, these spectra show intense peaks that may be assigned to ions produced by three new fragmentation pathways involving a six-atom rearrangement. These ions, together with the A fragments from sugar ring fragmentations, open the possibility of obtaining a complete mapping of the linkage positions present in the carbohydrates investigated by MALDI-TOF/TOF.
168) Multivalent binding of galactosylated cyclodextrin vesicles to lectin
A.Mazzaglia, D.Forde, D.Garozzo, P.Malvagna, B.J.Ravoo, R.Darcy
Organic & Biomolecular Chemistry
2(7),
957
- 2004
Amphiphilic β-cyclodextrins with alkylthio chains at the primary-hydroxyl side and galactosylthio-oligo-(ethylene glycol) units at the secondary-hydroxyl side, which form nanoparticles and vesicles, show multivalent effects in their binding to lectin.
169) Caratterizzazione strutturale di polisaccaridi mediante spettrometria di massa
D.Garozzo, G.Impallomeni, D.Romeo, E.Spina, L.Sturiale
XXVI Convegno Scuola dell’Associazione Italiana di Scienza e Tecnologia delle Macromolecole, Gargnano (BS) 24-28 Maggio, 2004
- 2004
170) Analisi del Glicoma del Siero di Pazienti Affetti da Disordini Congeniti della Glicosilazione (CDG) e da Galattosemia
L.Sturiale, R.Barone, A.Fiumara, G.Sorge, G.Impallomeni, D.Garozzo
IX Convegno sulla Chimica dei Carboidrati, Certosa di Pontignano (Siena), 21-23 Giugno, 2004
- 2004
171) Glycomics of human serum: mass spectrometry investigations of serum glycoproteins in patients with congenital disorders of glycosylation (CDG) and glactosemia
L.Sturiale, R.Barone, A.Fiumara, G.Sorge, G.Impallomeni, D.Garozzo
2nd Italian Human Proteome Organization National Meeting. Proteomics impact in basic and clinical research. 16-18 September, 2004 - Chieti, Italy
- 2004
172) Preliminary results on proteomics of human tear fluids : Lack of bioactive peptides and defensins in disease subjects
M.Perez, A.Messina, R.Cozzolino, L.Sturiale, MG.Mazzone, D.Garozzo
2nd Italian Human Proteome Organization National Meeting. Proteomics impact in basic and clinical research. 16-18 September, 2004 - Chieti, Italy
- 2004
173) Proteomic of human tear fluids in healthy and pathological conditions
MG.Mazzone, M.Santocono, M.Perez, A.Messina, R.Cozzolino, L.Suriale, D.Garozzo
4th International Conference on the lacrimal gland, tear film, ocular surface and dry eye syndromes: basic science and clinical relevance. November17-20, 2004. Fajardo, Puerto Rico
- 2004
174) Increased fucosylation and branching of serum transferrin N-glycans in long-term untreated Galactosemic patients
L.Sturiale, R.Barone, G.Sorge, M.Zaffanello, A.Fiumara, G.Impallomeni, D.Garozzo
Glycobiology
14(11),
1154
- 2004
Galactosemia is an autosomal recessive disorder caused, in the most common form, by mutation in the galactose-1-phosphate uridyltransferase (GALT) gene (ch. 9p13). The resulting enzyme deficiency leads to anomalous accumulation of galactose and gatactose-1-phosphate in blood and tissues and produces severe symptoms as mental retardation, cirrhosis of the liver and cataracts, prevented by a galactose-free diet. In untreated galactosemic patients, isoform patterns of serum transferrin, lysosomal enzymes b-hexosaminidase and a-fucosidase and follicle stimulating hormone are abnormal, due to the increase of relatively neutral isoforms corresponding to less sialylated carbohydrate structures [1-2]. These evidences are similar to those observed in the Congenital Disorders of Glycosylation (CDG) which are inherited disorders characterized by a defective synthesis of the carbohydrate moiety of multiple serum glycoproteins. In order to investigate the glycosylation abnormalities in galactosemic patients, we used MALDI mass spectrometry to individuate the glycosylation degree of intact glycoproteins and to achieve the fully characterization of the N-linked oligosaccharide structures. Particularly, we focused on the glycosylation pattern of rivanol purified serum transferrin (the classical biochemical marker of CDGs) in two galactosemia patients before treatment and during follow-up on galactose-free diet. The results were compared with those obtained in subjects with CDG-Ia (PMM deficiency) and healthy controls. The galactosemic patients were both overexposed to dietary galactose (11 and 5 weeks respectively) because of an initial false negative newborn screening following red blood cell transfusion. In long-term untreated galactosemia we found a severe underoccupancy of both transferrin N-glycosylation sites according to the isoelectric focusing pattern and the profile of MALDI-TOF mass spectra of the intact glycoprotein. Release of N-glycans after PNGase F digestion of the same samples, followed by MALDI-TOF analysis both in negative and in positive polarity, revealed a great heterogeneity of glycoforms ranging from truncated biantennary species deprived of sialic acid and/or galactose, to triantennary and tetraantennary species at higher molecular weight. Moreover, we found a significant increase of the fucosylation degree of all the glycoforms, already reported for CDG-I [3], but never observed before in galactosemia. Such abnormal findings were not observed upon dietary treatment. These evidences suggest that in long-term untreated galactosemia, defects in both the assembly as well as in the processing pathways may occur. These findings open the way to a better comprehension of the biochemical mechanisms regarding the defective glycosylation pathway in galactosemia.
175) Structure of minor oligosaccharides from the lipopolysaccharide fraction from Pseudomonas stutzeri OX1
S.Leone, V.Izzo, L.Sturiale, D.Garozzo, R.Lanzetta, M.Parrilli, A.Molinaro, A.Di Donato
Carbohydrate Research
339(16),
2657
- 2004
A minor oligosaccharide fraction was isolated after complete de-acylation of the lipooligosaccharide extracted from
Pseudomonas stutzeri OX1. The full structure of this oligosaccharide was obtained by chemical degradation, NMR spectroscopy and MALDI-TOF MS spectrometry. These experiments showed the presence of two novel oligosaccharides (OS1 and OS2):
where R = (S)-Pyr(-->4,6) in OS1 and α-Rha-(1-->3) in OS2. All sugars are D-pyranoses, except Rha, which is L-pyranose. Hep is L-
glycero-D-
manno-heptose, Kdo is 3-deoxy-D-
manno-oct-2-ulosonic acid, Pyr is pyruvic acid, P is phosphate.
176) Structural Determination of the O-Chain Moieties of the Lipopolysaccharide Fraction from Agrobacterium radiobacter DSM 30147
C.De Castro, E.Bedini, D.Garozzo, L.Sturiale, M.Parrilli
European Journal of Organic Chemistry
18,
3842
- 2004
Two O-chain structures were identified after acid hydrolysis of the lipopolysaccharide fraction of Agrobacterium radiobacter (type strain). The first is constituted by the linear tetrasaccharide repeating unit [2)-α-L-Rhap-(1 3)-α-L-Rhap- (1 3)-α-L-Rhap-(1 2)-α-L-Rhap-(1 ]n and the second by the (1 2)-branched repeating unit α-D-Manp-(1 2)-[3)-α-DFucp-( 1 3)-α-D-Fucp-(1 ]n.The two structures were determined mainly by 1D- and 2D NMR spectroscopy together with chemical-degradation methods. A detailed analysis of the NOESY spectrum, supported by molecular dynamics calculations, suggested that some unexpected NOEs were due to the sum of many small dipolar effects, whose identification was possible only by considering the 3D structure of an Ochain oligosaccharide bigger than the repeating unit.
177) The complete structure of the lipooligosaccharide from the halophilic bacterium Pseudoalteromonas issachenkonii KMM 3549T
A.Silipo, S.Leone, R.Lanzetta, M.Parrilli, L.Sturiale, D.Garozzo, E.L.Nazarenko, R.P.Gorshkova, E.P.Ivanova, N.M.Gorshkovac, A.Molinaro
Carbohydrate Research
339,
1985
- 2004
Novel lipooligosaccharide components were isolated and identified from the lipooligosaccharide fraction of the halophilic marine bacterium Pseudoalteromonas issachenkonii type strain KMM 3549T. The complete structure was achieved by chemical analysis, 2D NMR spectroscopy and MALDI mass spectrometry as the following: β-Gal-(1->7)-α-Hep3P-(1->5)-α-Kdo4P-(2->6)-LipidAα-Glc-(1->4)-β-Gal-(1->4) All sugars are D-pyranoses. Hep is L-glycero-D-manno-heptose, Kdo is 3-deoxy-D-manno-oct-2-ulosonic acid, P is phosphate, residues and substituents in italic are not stoichiometrically linked. In addition, by MALDI mass spectrometry of the intact LOS, the lipid A moiety was also identified as a mixture of penta-, tetra- and triacylated species.
178) A novel type of highly negatively charged lipooligosaccharide from Pseudomonas stutzeriOX1 possessing two 4,6- O-(1-carboxy)-ethylidene residues in the outer core region 4,6- O-(1-carboxy)-ethylidene residues in the outer core region
S.Leone, V.Izzo, A.Silipo, L.Sturiale, D.Garozzo, R.Lanzetta, M.Parrilli, A.Molinaro, A.Di Donato
European Journal of Biochemistry
271(13),
2691
- 2004
Pseudomonas stutzeri OXI is a Gram-negative microorganism able to grow in media containing aromatic hydrocarbons. A novel lipo-oligosaccharide from P. stutzeri OX1 was isolated and characterized. For the first time, the presence of two moieties of 4,6-O-(1-carboxy)-ethylidene residues (pyruvic acid) was identified in a core region; these two residues were found to possess different absolute configuration. The structure of the oligosaccharide backbone was determined using either alkaline or acid hydrolysis. Alkaline treatment, aimed at recovering the complete carbohydrate backbone, was carried out by mild hydrazinolysis (de-Oacylation) followed by de-N-acylation using hot KOH. The lipo-oligosaccharide was also analyzed after acid treatment, attained by mild hydrolysis with acetic acid, to obtain information on the nature of the phosphate and acyl groups. The two resulting oligosaccharides were isolated by gel permeation chromatography, and investigated by compositional and methylation analyses, by MALDI mass spectrometry, and by 1H-, 31P- and 13C-NMR spectroscopy. These experiments led to the identification of the major oligosaccharide structure representative of core region-lipid A. All sugars are D-pyranoses and a-linked, if not stated otherwise. Based on the structure found, the hypothesis can be advanced that pyruvate residues are used to block elongation of the oligosaccharide chain. This would lead to a less hydrophilic cellular surface, indicating an adaptive response of P. sutzeriOX1 to a hydrocarbon-containing environment. A novel lipo-oligosaccharide from P. stutzeri OX1 was isolated and characterized. For the first time, the presence of two moieties of 4,6-O-(1-carboxy)-ethylidene residues (pyruvic acid) was identified in a core region; these two residues were found to possess different absolute configuration. The structure of the oligosaccharide backbone was determined using either alkaline or acid hydrolysis. Alkaline treatment, aimed at recovering the complete carbohydrate backbone, was carried out by mild hydrazinolysis (de-Oacylation) followed by de-N-acylation using hot KOH. The lipo-oligosaccharide was also analyzed after acid treatment, attained by mild hydrolysis with acetic acid, to obtain information on the nature of the phosphate and acyl groups. The two resulting oligosaccharides were isolated by gel permeation chromatography, and investigated by compositional and methylation analyses, by MALDI mass spectrometry, and by 1H-, 31P- and 13C-NMR spectroscopy. These experiments led to the identification of the major oligosaccharide structure representative of core region-lipid A. All sugars are D-pyranoses and a-linked, if not stated otherwise. Based on the structure found, the hypothesis can be advanced that pyruvate residues are used to block elongation of the oligosaccharide chain. This would lead to a less hydrophilic cellular surface, indicating an adaptive response of P. sutzeriOX1to a hydrocarbon-containing environment.
179) Structure Elucidation of the Highly Heterogeneous Lipid A from the Lipopolysaccharide of the Gram-Negative Extremophile Bacterium Halomonas Magadiensis Strain 21 M1 Lipopolysaccharide of the Gram-Negative Extremophile Bacterium Halomonas Magadiensis Strain 21 M1
A.Silipo, L.Sturiale, D.Garozzo, C.De Castro, R.Lanzetta, M.Parrilli, W.D.Grant, A.Molinaro
European Journal of Organic Chemistry
10,
2263
- 2004
Halomonas magadiensis (formerly named Halomonas magadii) is a Gram-negative extremophilic and alkaliphilic bacterium isolated from Lake Magadi, which is located in the East African Rift Valley. Several members of the halomonad group of bacteria have been shown to inhabit the alkaline brines, including a new member, Halomonas magadiensis strain 21 M1 (NCIMB 13595), an organism that grows at high pH and relatively high salt concentration. The unusual structure of the lipid A family derived from the lipopolysaccharide of Halomonas magadiensis is reported herein. The structure was determined using chemical analysis, NMR spectroscopy and MALDI-TOF mass spectrometry. Lipid A was also analysed after either de-O-acylation or dephosphorylation. The resultant mixture was very heterogeneous in fatty acid substitution, from heptaacyl to triacyl species. The various lipid A molecules obtained by the removal of one or more acyl substituents from the heptaacyl species are described below.
180) Determination of linkage positions and sequence of complex carbohydrates by MALDI TOF-TOF mass spectrometry.
E.Spina, L.Sturiale, G.Impallomeni, D.Garozzo, D.Waidelich, M.Glueckmann
Primo congresso Nazionale IHUPO, 26-27 Settembre 2003. Napoli, Italy
- 2003
181) Analysis of human tear proteins by capillary LC/ESI-MS
R.Cozzolino, V.Enea, MG.Mazzone, D.Garozzo
Primo congresso Nazionale IHUPO, 26-27 Settembre 2003. Napoli, Italy
- 2003
182) Structural characterization of the O-specific lipopolisaccharide chains by MALDI-MS
L.Sturiale, A.Molinaro R.Lanzetta, D.Garozzo
12th European Carbohydrate Symposium, July 6-11, 2003, Grenoble, France
- 2003
183) Proprietà Strutturali di Polisaccaridi Prodotti dal Batterio Patogeno Opportunista Burkholderia cepacia
P.Cescutti, Y. Herasimenka, R.Urbani, P.Sist, R.Rizzo, G.Impallomeni, L.Sturiale, D.Garozzo
XVI Convegno Italiano di Scienza e Tecnologia delle Macromolecole, Pisa, 22-25 Settembre, 2003
- 2003
184) Exopolysaccharides produced by a clinical strain of Burkholderia cepacia isolated from a cystic fibrosis patient
P.Cescutti, G.Impallomeni, D.Garozzo, L.Sturiale, Y.Herasimenka, C.Lagatolla, R.Rizzo
Carbohydrate Research
338(23),
2687
- 2003
Burkholderia cepacia is an opportunistic pathogen involved in pulmonary infections related to cystic fibrosis. A clinical strain, BTS13, was isolated and the production of exopolysaccharides was tested growing the bacteria on two different media, one of which was rich in mannitol as carbon source. The primary structure of the polysaccharides was determined using mostly mass spectrometry and NMR spectroscopy. On both media an exopolysaccharide having the following repeating unit was produced: → 5) -β-KDop-(2→3)-β-D-Galp2Ac-(1→4)-α-D-Galp-(1→3)-β-D-Galp-(1→. This polysaccharide has already been described as the biosynthetic product of another Burkholderia species, B. pseudomallei, the microbial agent causing melioidosis. In addition to this, when grown on the mannitol-rich medium, B. cepacia strain BTS13 produced another polysaccharide that was established to be levan: →6)-β-D-Fruf-(2→. The content of levan was about 20% (w/w) of the total amount of polymers. The ability of B. cepacia to produce these two exopolysaccharides opens new perspectives in the investigation of the role of polysaccharides in lung infections.
185) Identification of some adulteration in milk and in water buffalo mozzarella cheese by MALDI TOF MS
R.Cozzolino, S.Passalacqua, S.Salemi, D.Garozzo
Abstracts of the 51st Conference on Mass Spectrometry and Allied Topics, Montreal, June 2003
,
1704
- 2003
Water buffalo mozzarella cheese and "Pecorino" cheese are two of the most typical Italian products. Because water buffalo milk and ewe milk are more expensive than bovine milk, this is some times fraudulently added to raw ovine and water buffalo milk samples leading to products that are sold to the same prize of the authentic cheese. Several analytical methodologies have been developed in order to detect this kind of adulteration but the slowness of these methods makes them of limited value in the diary industry routine screening of milk. Recently, matrix-assisted laser desorption/ionisation mass spectrometry has shown to be a powerful analytical tool in providing a valid fingerprint of milk protein profiles. The method, in fact, has been successful employed for monitoring structural damages caused by thermal industrial processes, like pasteurisation and sterilisation, as well as to reveal differences in the protein pattern of milk samples coming from various mammal species.
186) Sequencing underivatized oligosaccharides using a MALDI-TOF/TOF tandem mass spectrometer
D.Garozzo, E.Spina, F.J.Mayer-Posner, V.Sauerland
Abstracts of the 51st Conference on Mass Spectrometry and Allied Topics, Montreal, June 2003
,
1300
- 2003
Analysis of carbohydrates is an important problem in biochemical analysis but the structural elucidation of a complex carbohydrate molecule is analytically difficult because it requires the knowledge of several parameters such as sugar sequence, reducing end, linkage type between the monosaccharides and the anomeric configuration. Because of its large mass range and high sensitivity, matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry is now widely used in the molecular mass determination of underivatized oligo- and polysaccharides (1-5) and since the introduction of the postsource
decay (PSD) technique, providing fragmentation spectra, some papers dealing with the PSD of oligosaccharides have been published
187) Advances in MALDI-MS methods to investigate the structure of bacterial Lipopolysaccharides
L.Sturiale, A.Molinaro, R.Lanzetta, D.Garozzo
Abstracts of the 51st Conference on Mass Spectrometry and Allied Topics, Montreal, June 2003
,
1293
- 2003
Lipopolysacchrides (LPSs) are the main components of the outer membrane of bacterial cells and contribute significantly to patho-physiological effects and virulence of these organisms. Such glycoconjugates, also known as endotoxin of gram-negative bacteria, are comprised of three distinct regions covalently bound: a lipid A moiety (which acts as hydrophobic anchor of LPSs in the bacterial membrane) an acidic oligosaccharide core, and an O-antigen, a long chain of oligosaccharide repeating units extending out into the external environment. The LPSs structures vary greatly among different bacterial species, particularly, O-antigen changes also between diverse strains and exhibits moreover a remarkable polydispersity due to the presence of molecules differing in the number of repeating units. This work presents some improved MALDI-MS approaches to elucidate the structure of these high complex species.
188) Inclusion Networks of a Calix[5]arene-Based Exoditopic Receptor and Long-Chain Alkyldiammonium Ions
D.Garozzo, G.Gattuso, F.H.Kohnke, A.Notti, S.Pappalardo, M.F.Parisi, I.Pisagatti, A.J.P.White, D.J.Williams
Organic Letters
5(22),
4025
- 2003
Tail-to-tail connection of two cone calix[5]arene moieties by a rigid p-xylyl spacer affords the new exoditopic receptor 3 featuring two ð-rich cavities (assembling cores) in a centrosymmetric divergent rrangement, as established by a single-crystal X-ray analysis. 1H NMR complexation studies of 3 with alkyldiammonium ions support the formation of discrete bis-endo-cavity complexes and/or capsular assemblies along a polymer chain (polycaps), according to the length of the connector.
189) Identification of High Molecular Mass Proteins in Tear Fluid by MALDI MS
R.Cozzolino, V.Enea, M.G.Mazzone, D.Garozzo
50th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, Florida, June 2-6, 2002
- 2002
Tear fluid, a complex mixture of ions, small molecules, glycoproteins and proteins, refers to fluid present as the precorneal film and in the conjunctival sac. As it is directly in contact with the environment, it mainly serves to protect the eye from exogenous antigenic substances and to maintain the structural integrity of the cornea and the conjunctival sac [1]. Regarding to this, tear proteins play an important role in the maintenance of the eye surface integrity, in fact in patients with external eye diseases, the level of certain tear proteins may be altered [2-4]. Consequently, many attempts have been made to correlate any qualitative and/or quantitative change in tear protein with eye disorders, but at the moment problems are habitually encountered finding appropriate, sensitive and yet rapid and simple techniques for the analysis of tear proteins. SDS-PAGE has been demonstrated to be a useful methods for the analysis of tear protein composition, as the major tear proteins sIgA, lactoferrin, TSPA and lysozyme can be separated with high resolution [5]. Furthermore, up to now with the two-dimensional electrophoretic technique at least 60 components have been detected in tears [6]. Recently, MALDI-TOF MS has been proposed in the ocular environment as a diagnostic tool for evaluating the pattern of low molecular weight peptide species (<20kDa) in tears [8-9]. On the contrary, there are no published data on the detection of the high molecular mass tear proteins. In view of these results, the aim of this study has been to build a biochemical profile of high molecular mass proteins in tears and to determine its repeatability by MALDI-TOF MS. A total of 90 tear samples were collected from 20 subjects. In particular, figure 1 presents the MALDI TOF spectrum of a tear sample using sinapinic acid as matrix. In figure 1, besides the peaks due to peptide species [8-9], it is possible to clearly detect two intense mass signals at m/z 14690 Da and 17443 Da which, according to the data reported in literature and in particular to some study obtained by SDS-PAGE [10-11], can be due to Lysozyme and Tear Specific Prealbumin (TSPA), respectively, while at m/z around 35kDa there are some peaks which can be attributed to the so called Protein G (TSPA dimmer). Comparable data were obtained with other MALDI matrices, as DHB, THAP, DHAP, a-ciano etc. and for all the samples studied. These data have demonstrated that tear samples, opportunely diluted and analysed by MALDI TOF using sinapinic acid as a matrix, give rise to spectra showing peaks only up to about 35 KDa. On the contrary, the same studies have shown the difficulty in obtaining molecular ion signals corresponding to the high molecular mass tear proteins such as Albumin and Lactoferrin. In view of the fact that Lactoferrin is soluble at lightly basic pH, it was thought to dilute tear samples with 0.05M NH3 and analyse the so treated samples using different matrices. As shown in figure 2 this way of proceeding allowed to observe a peak at about m/z 82000, if DHB was employed as matrix, the MW of which corresponds to Lactoferrin. Unfortunately, the dilution with 0.05M NH3 did not permit to reveal the albumin and other high molecular weight tear proteins mass signals. In order to overcome this problem, tear samples were treated with Centri/Por Centrifughe Concentrators which allow to concentrate, fractionate and desalt the high molecular mass proteins. Following this kind of procedure it can be obtained two fractions called filtrate (which contains compounds that can pass the membrane) and retentate (which is the aliquot containing the high MW species). Figure 3 presents the MALDI spectrum of the retentate of a tear sample, gained with Centri/Por Centrifughe. This analysis shows that treating the sample in that way, other two peaks at m/z about 67kDA and 92KDa can be detected, using sinapinic acid as a matrix. According to the data reported in literature [10-11], these two mass signals can be attributed to Albumin and Progelatinase B, respectively. The approach for a rapid, simple and accurate detection of human tear protein profile proposed here has demonstrated that the majority of high molecular mass proteins in tears are repeatedly present. Thanks to the successful data obtained, this method can be considered a first attempt in order to characterize tear high molecular protein species and to identify qualitative changes in tear protein patterns coming from healthy people and subject affected by eye diseases.
190) New Results on MALDI MS of Widely Polydisperse Hydrosoluble Polymers
P.Malvagna, G.Impallomeni, R.Cozzolino, E.Spina, D.Garozzo
50th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, Florida, June 2-6, 2002
- 2002
Since its introduction in 1988 (1), Matrix Assisted Laser Desorption Mass Spectrometry (MALDI MS) has become an important analytical tool in the analysis of macromolecules. However, while it was found that average molecular mass (MM) and molecular mass dispersion (MMD) estimates by MALDI MS for narrowly dispersed polymers were in agreement with those obtained by conventional methods of MM determination (size exclusion chromatography (SEC), end group titration, light scattering), on the contrary, several authors reported that for polymers with a broad MMD (synthetic polymers and polysaccharides) MALDI yielded erroneous results. Specifically, when the polydispersity index reached values around 1.10 the MM obtained by SEC and the MM calculated by MALDI might differ up to about 20%. With broader MMDs, which are quite common, MALDI spectra failed to give reliable MM values, these being much lower than those obtained by classical methods (2-4). To overcome these difficulties, it was introduced the so-called SEC-MALDI technique (7-10), wherein the samples are fractionated by SEC, in order to reduce the polydispersity of the original sample to values compatible with MALDI analysis (i.e., < 1.1) (2-12). The collected SEC fractions are then analyzed by MALDI MS, and the mass spectra of these nearly monodisperse samples allow the computation of reliable values of the number-average and weight-average molecular weight (Mn and Mw) corresponding to each fraction. When the MM of each fraction is plotted vs. the elution volume, an accurate calibration of the chromatogram may be obtained. The SEC trace and the calibration constants are fed into the SEC software, which outputs the Mn and the Mw of the polymer. On the other hand, it must be remembered that the quality of a MALDI mass spectrum, even of simple molecules, is highly dependent from the morphology of the irradiated matrix/sample crystals, with the consequence that significant variations in peaks presence, intensity, resolution and mass accuracy are often obtained by focusing the laser on different regions of the same sample (13-14). To alleviate this problem, considerable efforts have been made toward the development of new sample preparation techniques able to conduct to the formation of a homogeneous "solid solution" following the matrix/sample mixture. However, these procedures can improve the quality of MALDI mass spectra of monodisperse samples, but fail in the case of polydisperse polymers. The main problem in the co-crystallization of a polydisperse polymer with the solid matrix is, in fact, the phase separation due to the difference in the interactions between the species in solution, which occurs during solvent evaporation. These considerations may explain why it is not possible to obtain the correct MM and MMD of polydisperse polymers and the suppression of desorption and ionization of heavier molecules by the lighter, which, in general, can better mix with the matrix. On this subject, it is known that polyvynilpyrrolidone (PVP) is a material which can form complexes with many small molecules, including DHB, increasing the solubility of these in water. The formation of complexes between the polymer and DHB assures the intimate sample and matrix mixing, that is essential in order to obtain good MALDI spectra. Furthermore, it guarantees that all the polymer molecules, independently from their dimension, are deeply mixed with the matrix. For these reasons, it represented a good candidate to obtain a MALDI mass spectrum of a polydisperse sample, and we analyzed PVP with Mw of 40000 and 160000 using DHB as the solid matrix. The MALDI mass spectra of PVP with Mw of 40000 is reported in figure 1. This spectrum shows a broad molecular weight distribution from which it is possible to calculate the values of Mn and Mw, which are 26000 and 48000, in good agreement with those obtained by SEC in our lab and those reported by the manufacturer.Similar results have been achieved for the analysis of PVP 160000. In fact, also in this second case the Mn and Mw obtained are the ones expected for this polymer (data not shown). These results are absolutely different from those gained for all other synthetic polymers and shown by many authors (2-12), who, on the contrary, observed a relevant difference between the real MM and MMD and those measured by MALDI MS. A possible explanation of this failure is the polymer and/or matrix phase separation during the drying process, with the consequence that the spectrum obtained is representative only of the lower molecular weight species present in the polymer sample. The exception discussed in the present paper relative to PVP, a molecule which interacts with the matrix, seems to confirm this hypothesis. In view of these first and encouraging results, we decided to test a completely different method of sample preparation of polydisperse polymers. Instead of the "dried drop" technique, in which the slow crystallization of the matrix/sample mixture produces phase separation and fractionation of the polymer sample, as recently demonstrated (15), we used freeze drying of the matrix/sample solution where it may be expected that the original dispersed state of the polymer is maintained, provided that the cooling process by which the solution is solidified is fast enough. The first sample we checked was dextran, a polysaccharide constituted by glucose units linked alpha 1-6 . The MALDI mass spectrum of a dextran with a MW of 150000 Daltons prepared by freeze drying the solution containing the sample and the matrix (DHB) is reported in figure 2. From the MMD here presented the Perseptive computer software calculated Mn and Mw values of 100000 and 150000, which are in good agreement with the values obtained by conventional techniques and reported by the manufacturer. After this experiment, we studied other two polysaccharides: Lichenan and Nigeran which, because of their low solubility in water, are difficult to analyze by SEC. The MALDI mass spectra obtained by the 'freezedrying preparation' give, also in this case, the correct molecular weight distribution for these polymers. In particular, figure 3 presents the MALDI analysis of Nigeran The last sample we analyzed was a synthetic hydrosoluble polymer, polyethyleneoxide (PEO) Mw 100000. The MALDI mass spectrum obtained by the 'freeze drying preparation' (data not shown), shows a MMD from mass 10000 to above mass 250000. The Mn and Mw calculated are in good agreement with those reported by the manufacturer. Our results demonstrate that this method allows to obtain, for the first time, reliable spectra of the molecular weight distribution of polydisperse samples, extending the usefulness of MALDI MS. Including DHB, increasing the solubility of these in water. The formation of complexes between the polymer and DHB assures the intimate sample and matrix mixing, that is essential in order to obtain good MALDI spectra. Furthermore, it guarantees that all the polymer molecules, independently from their dimension, are deeply mixed with the matrix. For these reasons, it represented a good candidate to obtain a MALDI mass spectrum of a polydisperse sample, and we analyzed PVP with Mw of 40000 and 160000 using DHB as the solid matrix. The MALDI mass spectra of PVP with Mw of 40000 is reported in figure 1. This spectrum shows a broad molecular weight distribution from which it is possible to calculate the values of Mn and Mw, which are 26000 and 48000, in good agreement with those obtained by SEC in our lab and those reported by the manufacturer.Similar results have been achieved for the analysis of PVP 160000. In fact, also in this second case the Mn and Mw obtained are the ones expected for this polymer (data not shown). These results are absolutely different from those gained for all other synthetic polymers and shown by many authors (2-12), who, on the contrary, observed a relevant difference between the real MM and MMD and those measured by MALDI MS. A possible explanation of this failure is the polymer and/or matrix phase separation during the drying process, with the consequence that the spectrum obtained is representative only of the lower molecular weight species present in the polymer sample. The exception discussed in the present paper relative to PVP, a molecule which interacts with the matrix, seems to confirm this hypothesis. In view of these first and encouraging results, we decided to test a completely different method of sample preparation of polydisperse polymers. Instead of the "dried drop" technique, in which the slow crystallization of the matrix/sample mixture produces phase separation and fractionation of the polymer sample, as recently demonstrated (15), we used freeze drying of the matrix/sample solution where it may be expected that the original dispersed state of the polymer is maintained, provided that the cooling process by which the solution is solidified is fast enough. The first sample we checked was dextran, a polysaccharide constituted by glucose units linked alpha 1-6 . The MALDI mass spectrum of a dextran with a MW of 150000 Daltons prepared by freeze drying the solution containing the sample and the matrix (DHB) is reported in figure 2. From the MMD here presented the Perseptive computer software calculated Mn and Mw values of 100000 and 150000, which are in good agreement with the values obtained by conventional techniques and reported by the manufacturer. After this experiment, we studied other two polysaccharides: Lichenan and Nigeran which, because of their low solubility in water, are difficult to analyze by SEC. The MALDI mass spectra obtained by the 'freezedrying preparation' give, also in this case, the correct molecular weight distribution for these polymers. In particular, figure 3 presents the MALDI analysis of Nigeran The last sample we analyzed was a synthetic hydrosoluble polymer, polyethyleneoxide (PEO) Mw 100000. The MALDI mass spectrum obtained by the 'freeze drying preparation' (data not shown), shows a MMD from mass 10000 to above mass 250000. The Mn and Mw calculated are in good agreement with those reported by the manufacturer. Our results demonstrate that this method allows to obtain, for the first time, reliable spectra of the molecular weight distribution of polydisperse samples, extending the usefulness of MALDI MS.
191) Guest-induced capsular assembly of calix[5]arenes
D.Garozzo, G.Gattuso, F.H.Kohnke, P.Malvagna, A.Notti, S.Occhipinti, S.Pappalardo, M.F.Parisi, I.Pisagatti
Tetrahedron Letters
43(43),
7663
- 2002
Novel dimeric capsules are generated from the non-covalent assembly of cone calix[5]arenes and alkyldiammonium ions[H3N-(CH2)n-NH3]² featuring an appropriate length of the spacer joining the two charged end groups. ¹H NMR spectroscopy and electrospray ionisation mass spectrometry provide evidence that in the capsule a single ditopic guest holds together a pair of calix[5]arene units, which are oriented rim-to-rim to form a closed cavity encapsulating the shape-complementary dication.
192) Identification of adulteration in water buffalo mozzarella and in ewe cheese by using whey proteins as biomarkers and matrix-assisted laser desorption/ionization mass spectrometry
R.Cozzolino, S.Passalacqua, S.Salemi, D.Garozzo
Journal of Mass Spectrometry
37(9),
985
- 2002
A rapid and accurate method to identify bovine and ewe milk adulteration of fresh water buffalo mozzarella cheese by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is described. The differentiation among mozzarella made from water buffalo milk and from mixtures of less expensive bovine and, more recently, ewe milk with water buffalo milk is achieved using whey proteins, -lactalbumin and -lactoglobulins as molecular markers. It is worth noting that the method proposed here is, to our knowledge, the first strategy able to characterize possible fraudulent additions of ewe milk in samples of water buffalo milk devoted to the production of water buffalo mozzarella cheese. In addition, a linear relationship was found between the relative response of the molecular ion and the abundance of the analysed whey proteins. This demonstrates that this approach can be used to determine the amount of bovine or ovine milk added to water buffalo milk employed for mozzarella cheese production. Furthermore, this method also appears suitable for the analysis of ewe cheese. Hence these findings open the way to a new field for mass spectrometry in the evaluation of possible fraudulence in dairy industry production.
193) New results on matrix-assisted laser desorption/ionization mass spectrometry of widely polydisperse hydrosoluble polymers
P.Malvagna, G.Impallomeni, R.Cozzolino, E.Spina, D.Garozzo
Rapid Communications in Mass Spectrometry
16(16),
1599
- 2002
Polyvinylpyrrolidone (PVP) of molecular weight (Mw) 40 000 and 160 000 and polydispersity of 1.8-2.2 was shown to yield matrix-assisted laser desorption/ionization (MALDI) mass spectra with the correct molar mass distribution of the polymer. This is a unique behavior among widely polydisperse samples and was ascribed to the capacity of PVP to complex the MALDI matrix. This result prompted us to test a new sample preparation technique consisting of flash-freezing followed by freeze-drying of the solution of the polymer with the matrix. In this paper we describe our preliminary results on MALDI-MS of polydisperse polymers obtained by this technique. This sample preparation method allowed us to obtain, for the first time, spectra yielding the true molecular weight distribution of polydisperse samples such as dextran, lichenan, nigeran, and poly(ethylene oxide).
194) Structural determination of lipid A of the lipopolysaccharide from Pseudomonas reactans
A.Silipo, R.Lanzetta, D.Garozzo, P.Lo Cantore, N.S.Iacobellis, A.Molinaro, M.Parrilli, A.Evidente
European Journal of Biochemistry
269(10),
2498
- 2002
The chemical structure of lipid A from the lipopolysaccharide of the mushroom-associated bacterium Pseudomonas reactans, a pathogen of cultivated mushroom, was elucidated by compositional analysis and spectroscopic methods (MALDI-TOF and two-dimensional NMR). The sugar backbone was composed of the beta-(1'-->6)-linked d-glucosamine disaccharide 1-phosphate. The lipid A fraction showed remarkable heterogeneity with respect to the fatty acid and phosphate composition. The major species are hexacylated and pentacylated lipid A, bearing the (R)-3-hydroxydodecanoic acid [C12:0 (3OH)] in amide linkage and a (R)-3-hydroxydecanoic [C10:0 (3OH)] in ester linkage while the secondary fatty acids are present as C12:0 and/or C12:0 (2-OH). A nonstoichiometric phosphate substitution at position C-4' of the distal 2-deoxy-2-amino-glucose was detected. Interestingly, the pentacyl lipid A is lacking a primary fatty acid, namely the C10:0 (3-OH) at position C-3'. The potential biological meaning of this peculiar lipid A is also discussed.
195) Identification of adulteration in milk by matrix-flight mass spectrometry
R.Cozzolino, S.Passalacqua, S.Salemi, P.Malvagna, E.Spina, D.Garozzo
Journal of Mass Spectrometry
36(9),
1031
- 2001
The development is described of a rapid, simply and accurate analytical method aimed at evaluating both the presence of cow milk in either raw ewe and water buffalo milk samples employed in industrial processes and the addition of powdered milk to samples of fresh raw milk, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The presence of adulteration is defined by evaluating the protein patterns coming from the most abundant whey proteins, alpha -lactalbumin and beta -lactoglobulin, used as molecular markers. As no pretreatment of the milk samples is required and owing to the speed and ease of use of MALDI-MS the proposed analytical protocol can be used as a routine strategy for the identification of possible adulteration of the raw fresh milk samples that the dairy industry receives from producers every day. Copyright © 2001 John Wiley & Sons, Ltd
196) Matrix-assisted laser desorpition/ionization mass spectrometric peptide mapping of high molecular weight glutenin subunits 1Bx7 and 1Dy7 in Cheyenne cultivar
R.Cozzolino, S.Di Giorgi, S.Fisichella, D.Garozzo, D.Lafiandra, A.Palermo
Rapid Communications in Mass Spectrometry
15(10),
778
- 2001
"This study describes the verification of the cDNA-deduced amino acid sequences of high molecular weight glutenin subunits 1Dy10 and 1Bx7 in Cheyenne cultivar by direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis of their tryptic fragments omitting chromatographic pre-separation. These polypeptides have a conserved structure consisting of a long central repetitive domain that prevents the application of conventional sequencing procedures such as Edman degradation. The published sequence of subunit 1Dy10 contains 7 Lys and 13 Arg residues; thus the production of 21 tryptic peptides is expected. The cDNA-deduced sequence for 1Bx7 subunit includes 5 Lys and 15 Arg residues, but the presence of three Arg-Pro bonds, which are normally not cleaved by trypsin, predicts only 19 tryptic peptides. Three different matrices (DHB, SA and HCCA) in combination with the most compatible sample preparation procedures were used in order to obtain the maximum 1Dy10 and 1Bx7 sequence coverage. MALDI analysis of the 1Dy10 tryptic digest resulted in the identification of all 21 expected peptides. In the case of 1Bx7 MALDI analysis resulted in the identification of 17 of the 19 expected peptides, giving a sequence coverage of 99.3%. These results were sufficient to rule out glycosylation of the 1Dy10 and 1Bx7 proteins and to assess the absence of any other post-translational modification, to within the detection limits of the method. Copyright © 2001 John Wiley & Sons, Ltd"
197) Proteomics of gluten: mapping of subunit 1 Ax2* in Cheyenne cultivar by matrix-assisted laser desorption/ionization
R.Cozzolino, S.Di Giorgi, S.Fisichella, D.Garozzo, D.Lafiandra, A.Palermo
Rapid Communications in Mass Spectrometry
15(14),
1129
- 2001
This paper reports results on the verification of the 1Ax2* high molecular weight glutenin subunit sequence in Cheyenne cultivar. The gene sequence of the protein is known but recently some text changes have been made, and furthermore until now no characterization of post-translational modifications has been reported. The two published sequences, named I and II, differ in four residues at positions 23, 208, 475, and 611. The first sequence contains 20 Arg and 6 Lys residues, producing 26 tryptic fragments, since the Arg(109)-Pro(110) bond is generally not cleaved by trypsin. The second sequence contains 19 Arg and 6 Lys residues, producing 25 tryptic peptides, again because of the Arg(109)-Pro(110) bond. Both sequences generate two cyanogen bromide fragments. Matrix-assisted laser desorption/ionization analysis of the tryptic digest of the high-MW glutenin subunit 1Ax2* resulted in the identification of 24 out of the 26 expected peptides for sequence I, a sequence coverage of 99.5%. These results were sufficient to rule out sequence II and any protein glycosylation and any other post-translational modifications to within the detection limits of the method. It was found that the choice of matrix considerably influenced the sequence coverage in peptide mapping. Copyright © 2001 John Wiley & Sons, Ltd
198) Spettrometria di Massa MALDI TOF di Macromolecole
D.Garozzo, E.Spina, R.Cozzolino
AIM Magazine
54(1),
33
- 2000
199) Sequencing of oligosaccharides by collision-induced dissociation matrix-assisted laser desorption/ionization mass spectrometry
E.Spina, R.Cozzolino, E.Ryan, D.Garozzo
Journal of Mass Spectrometry
35(8),
1042
- 2000
A study of the collision-induced dissociation post-source decay (PSD) spectra of free oligosaccharides is presented. These spectra, when obtained with helium as collision gas, show X-1,X-5 fragments containing the reducing end sugar. The presence of these fragments permits Y ions and, consequently, B and C peaks to be identified, This is a common behaviour from which it has been possible to delineate a general method for the easy assignment of the peaks in PSD spectra of underivatized neutral sugars, allowing the sequence of a real unknown to be obtained. Copyright © 2000 John Wiley & Sons, Ltd
200) Studies on the primary structure of short polysaccharides using SEC MALDI mass spectroscopy
D.Garozzo, E.Spina, R.Cozzolino, P.Cescutti, W.F.Fett
Carbohydrate Research
323(1-4),
139
- 2000
The introduction of size-exclusion chromatography (SEC) analysis of polysaccharides prior to MALDI mass spectroscopy accounts for the determination of the molecular mass of the repeating unit when neutral homopolymers are investigated. In the case of natural polysaccharides characterised by more complicated structural features (presence of non-carbohydrate substituents, charged groups, etc.), this mass value usually is in agreement with more than one sugar composition. Therefore, it is not sufficient to give the correct monosaccharidic composition of the polysaccharide investigated. To solve this problem, MALDI spectra were recorded on the permethylated sample and post-source decay experiments were performed on precursor ions. In this way, the composition (in terms of Hex, HexNAc, etc.), size and sequence of the repeating unit were determined. (C) 2000 Elsevier Science Ltd. All rights reserved
201) Structure analysis of underivatized oligosaccharides using postsource decay with collision-induced fragmentation, in matrix-assisted
laser desorption ionization mass spectrometry
D.Garozzo, E.Spina, R.Cozzolino
47th ASMS Conference on Mass Spectrometry and Allied Topics, Dallas, Texas, June 13-17, 1999
- 1999
In this paper we show the PSD spectra of some underivatized oligosaccharides obtained using collision induced decomposition (CID) by using helium as the collision gas. We observe some relevant differences with the spectra of the same oligosaccharides obtained without a collision gas or by using argon and recently reported. The most important difference is the presence of X1,5 fragments which are highly diagnostic allowing to obtain the sequence of the oligosaccharides analyzed. We have observed that CID spectra of underivatized oligosaccharides, unlike other compounds as peptides are quite different from that observed without CID. The most important difference is the presence of intense X1,5 ions containing the reducing end. By the presence of these ions in the CID PSD spectra we can now distinguish the Y ions from all others and in this way, sequence the oligosaccharide (in terms of Hex, HexNAc, etc.) by the CID PSD spectrum alone. This constitutes a big improvement respect the PSD spectra obtained without CID where, although a lot of information on the sequence of the oligosaccharides analyzed are obtained, the presence of "internal" fragments and the difficulty of discriminate B and C fragments from Y and Z can originate some problems in unequivocal assign the sequence from a PSD spectrum alone
202) Synthesis of 5,5 '-bicalix[6]arene and 5,5 '-bicalix[8]arene systems
A.Bottino, F.Cunsolo, M.Piattelli, D.Garozzo, P.Neri
Journal of Organic Chemistry
64(21),
8018
- 1999
203) Use of Hydroxyacetophenones as Matrices for the Analysis of High Molecular Weight Glutenin Mixtures by Matrix-assisted Laser Desorption/Ionization Mass Spectrometry
D.Garozzo, R.Cozzolino, S.Di Giorgi, S.Fisichella, D.Lafiandra
Rapid Communications in Mass Spectrometry
13(21),
2084
- 1999
A selection of hydroxyacetophenones has been investigated as matrices for the analysis, by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), of high molecular weight (HMW) glutenin mixtures from three wheat varieties. Mass spectra were obtained directly from the HMW glutenin extracts without any preliminary purification and separation steps. According to the quality of the mass spectra, obtained using different hydroxyacetophenones, it has been possible to classify the matrices on the basis of their suitability for the analyte properties. Only two of the matrices considered showed to be compatible with the HMW glutenin mixtures analysis, although a large amount of other highly complex protein mixtures are present, This study indicates that MALDI-MS, as a stand-alone technique, Is particularly useful for the direct determination of the complete HMW subunits profile and their molecular weights, Copyright © 1999 John Wiley & Sons, Ltd
204) The structure of exocellular polysaccharide from the Cyanobacterium Cyanospira capsulata
D.Garozzo, G.Impallomeni, E.Spina, L.Sturiale
Carbohydrate Research
307(1-2),
113
- 1998
The exocellular polysaccharide produced by the cyanobacterium Cyanospira capsulata has been subjected to partial acid hydrolysis and N-deacetylation-nitrous acid deamination. The oligosaccharides released have been isolated by weak anion exchange and aqueous size exclusion chromatography, and characterized by a combination of 1D and 2D nuclear magnetic resonance spectroscopy, mass spectrometry, sugar compostion and linkage analyses. The polysaccharide has an octasaccharide repeating unit with the following structure: [GRAPHICS] (C) 1998 Elsevier Science Ltd. All rights reserved. [References: 29]
205) Maldi Ms of oligo and polysaccharides in Mass Spectrometry in the Biomolecular Sciences, R. M. Caprioli et al (eds)
D.Garozzo
Kluwer Academic Publishers
1997,
477
- 1997
206) Effect of methylation of beta-cyclodextrin on the formation of inclusion complexes with aromatic compounds - an ionspray mass spectrometry investigation
P.Cescutti, D.Garozzo, R.Rizzo
Carbohydrate Research
302(1-2),
1
- 1997
The investigation of the inclusion complexes obtained with heptakis-(2,6-di-O-methyl)-beta-cyclodextrin (DM-beta-Cdx) or heptakis-(2,3,6-tri-O-methyl)-beta-cyclodextrin (PM-beta-Cdx) and either 1-anilinonaphthalene-8-sulfonate (ANS) or 2-p-toluidinylnaphthalene-6-sulfonate (TNS) was carried out by means of ionspray mass spectrometry, both in the positive and in the negative ion mode. All the data collected showed that the heptakis-(2,3,6-tri-O-methyl)-beta-cyclodextrin interacted to a very little extent with TNS and not at all with ANS. On the contrary, heptakis-(2,6-di-O-methyl)-beta-cyclodextrin formed complexes with both aromatic molecules showing a more effective interaction with TNS. Small variations in the number of methoxy substituents in the DM-beta-Cdx molecule did not affect the complexation behaviour significantly. The spectra recorded at different orifice potentials indicated that the complex of heptakis-(2,6-di-O-methyl)-beta-cyclodextrin with TNS is more stable than the one formed with ANS. These results agreed on one hand with the conformations of both the heptakis-(2,6-di-O-methyl)-beta-cyclodextrin and heptakis-(2,3,6-tri-O-methyl)-beta-cyclodextrin, established by X-ray diffraction studies, and, on the other hand, with the different complexation behaviour of the guest aromatic molecules due to their own geometry. (C) 1997 Elsevier Science Ltd. [References: 17]
207) Discrimination of isomeric oligosaccharides and sequencing of unknowns by post source decay matrix-assisted laser desorption ionization time-of-flight mass spectrometry
D.Garozzo, V.Nasello, E.Spina, L.Sturiale
Rapid Communications in Mass Spectrometry
11(14),
1561
- 1997
The PSD spectra of some isomeric oligosaccharides are investigated in order to study the fragmentation mechanisms. Results show that intense peaks are formed from one or more glycosidic linkage cleavages, while ring fragmentation produces very weak signals. Two analytical applications of post source decay (PSD) to the structural analysis of oligosaccharides are also illustrated: the discrimination of isomeric structures and the identification of a real unknown by combining PSD data with the linkage analysis results.
208) Study of the inclusion complexes of aromatic molecules with cyclodextrins usin ionspray mass spectrometry
P.Cescutti, D.Garozzo, R.Rizzo
Carbohydrate Research
290(2),
105
- 1996
"The formation of inclusion complexes between cyclodextrins (cyclohexa-, cyclohepta-, and cyclooctamylose) and either 1-anilinonaphthalene-8-sulfonate or 2-p-toluidinylnaphthalene-6-sulfonate was investigated by ionspray mass spectrometry operated both in the positive and in the negative ion mode. This soft ionisation technique allowed the detection of the inclusion complexes; the presence of false positives was excluded by increasing the voltage at the orifice which caused breakage of the electrostatic adducts and some fragmentation of the free cyclodextrin molecules, but left the inclusion complexes intact. The spectra recorded in the negative mode showed the presence of complexes formed by two cyclodextrin molecules and one aromatic molecule; such stoichiometry was not detected in the positive mode.
209) Matrix Assisted Laser Desorption/Ionization Mass Spectrometry of Polysaccharides
D.Garozzo, G.Impallomeni, E.Spina, L.Sturiale, F.Zanetti
Rapid Communications in Mass Spectrometry
9(10),
937
- 1995
In this paper we report matrix-assisted laser desorption/ionization (MALDI) mass spectra of dextrans up to 100 000 Da molecular weight and compare these measurements with those obtained by conventional size-exclusion chromatography with low-angle laser light scattering (SEC/LALLS). These results extend the usefulness of MALDI-MS to higher molecular weight polysaccharides and give some insights into the MALDI process
210) Identification of N-acetylglucosamine and 4-O(1carboxyethyl)mannose in the exopolysaccharide from Cyanospyra capsulata
D.Garozzo, G.Impallomeni, E.Spina, L.Sturiale, A.Cesaro, P.Cescutti
Carbohydrate Research
270(1),
97
- 1995
211) Molecular and Structural Characterization of Polydisperse Polymers and Copolymers by Combining Maldi-Tof Mass Spectrometry with GPC Fractionation
G.Montaudo, D.Garozzo, M.S.Montaudo, C.Puglisi, F.Samperi
Macromolecules
28,
7983
- 1995
Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) allows detection of large molecules such as those present in synthetic and natural macromolecules. Until recently, it was reported that MALDI-TOF measurements can provide correct molecular weight (MW) averages only for samples with a narrow MW distribution (M(w)/M(n) less than or equal to 1.20). We have now developed a methodology for polydisperse samples. We recorded the GPC trace of two polydisperse polymeric samples, namely, poly(butylene adipate) (PEA) and poly(butylene adipate-co-butylene succinate) (PBAS), collecting about 40-50 fractions per run. Selected fractions were analyzed by MALDI-TOF, and the average MW of each fraction was determined, allowing calibration of the GPC curves against absolute MW. The two calibrated GPC traces were then used to compute average MW and molecular weight distributions (MWD) of the unfractionated samples. End group analysis from MALDI-TOF spectra revealed that the PEA sample is composed of seven different types of chains. For the copolymer sample, PBAS, analysis of the MALDI spectra established a random sequence distribution of comonomer units. The succinate/adipate molar ratio calculated from the MALDI spectra is in good agreement with the molar ratio found by NMR. [References: 25]
212) Quantitative Determination of B(1-2) Cyclic Glucans by MALDI-MS
D.Garozzo, E.Spina, L.Sturiale, G.Montaudo, R.Rizzo
Rapid Communications in Mass Spectrometry
8(5),
358
- 1994
The use of matrix-assisted laser desorption/ionization mass spectrometry for the determination of the molecular weight distribution of cyclic beta (1-2) glucans is reported here. Quantitative response is demonstrated by the use of different instruments and by comparison with high-performance liquid chromatography/refractive index detection analysis.
213) Quantitative Applications of Matrix-Assisted Laser Desorption Ionization with Time-of-Flight Mass Spectrometry - Determination of Copolymer Composition in Bacterial Copolyesters
R.Abate, A.Ballistreri, G.Montaudo, D.Garozzo, G.Impallomeni, G.Critchley, K.Tanaka
Rapid Communications in Mass Spectrometry
7(11),
1033
- 1993
Poly(beta-hydroxybutyrate-co-beta-hydroxyvalerate) of microbial origin has been analyzed by matrix-assisted laser desorption/ionization (MALDI) in a time-of-flight mass spectrometer, after partial methanolysis, using two different instruments. Our results show that the mass spectral intensities recorded in the MALDI mode correlate with previously reported fast-atom bombardment and direct chemical ionization data. MALDI compares favourably with these other two techniques in that it is more suitable to the analysis of complex mixtures and of higher molecular masses.
214) Negative-Ion Fab Ms of 3-O-Branched Oligosaccharides
D.Garozzo, G.Impallomeni, G.Montaudo, F.Spina
Abstracts of Papers of the American Chemical Society
204,
78
- 1992
215) Structure of Underivatized Branched Oligosaccharides by Negative-Ion Fast-Atom-Bombardment Mass-Spectrometry
D.Garozzo, G.Impallomeni, G.Montaudo, E.Spina
Rapid Communications in Mass Spectrometry
6(9),
550
- 1992
We report here a study by negative-ion fast-atom bombardment (FAB) mass spectrometry of branched oligosaccharides containing a 3-O glycosidic linkage. Our results show that discrimination of the monosaccharide unit linked in the 3-O position is made possible by the analysis of the negative-ion FAB mass spectra. In the mass spectrum of A-trisaccharide (alpha-Fuc1-2-(alpha-GalNAc1-3)-Gal) the peak at m/z 307 can be explained as elimination of a neutral molecule of GaINAc (221 u) from the molecular ion. Also in the mass spectrum of 3-fucosyllactose (alpha-Fuc1-3-(beta-Gal1-4)-Glc) an intense peak at m/z 323, corresponding to the elimination of the fucose linked at the 3 position of glucose is present. To investigate further the fragmentations of the 3-O-linked sugars, the negative-ion FAB mass spectra of disaccharides have also been studied
216) Sequence Distribution of Beta-Hydroxyalkanoate Units in Bacterial Copolyesters Determined by Desorption Chemical Ionization Mass-Spectrometry
R.Abate, D.Garozzo, R.Rapisardi, A.Ballistreri, G.Montaudo
Rapid Communications in Mass Spectrometry
6(11),
702
- 1992
The sequence distributions of poly(beta-hydroxyalkanoate) copolymers were determined by analyzing oligomers obtained by the pyrolysis direct-chemical-ionization (DCI) mass spectrometry technique. Oligomers up to nonamers were identified and the comparison between the experimental and calculated peak intensities makes it possible to calculate repeating unit compositions and sequence distributions. Comparison with other earlier methods are given
217) On the Mechanism of Thermal-Degradation of Polypivalolactone
D.Garozzo, G.Montaudo
Macromolecules
24(6),
1416
- 1991
Evidence is presented for the formation of cyclic oligomers, obsd. at relatively large intensities by direct pyrolysis-CI mass spectrometry. This suggests that they are primary thermal degrdn. products generated by a chain-end initiated, intramol. exchange mechanism. This conclusion is offered as an alternative to the proposal of L. E. Manring et al. (1990), which states that the thermal degrdn. of polypivalolactone proceeds by a stepwise living process.
218) Microstructure of Bacterial Poly(Beta-Hydroxybutyrate-Co-Beta-Hydroxyvalerate) by Fast-Atom-Bombardment Mass-Spectrometry Analysis of the Partial Pyrolysis Products
A.Ballistreri, G.Montaudo, D.Garozzo, M.Giuffrida, M.S.Montaudo
Macromolecules
24(6),
1231
- 1991
The microstructure of poly(beta-hydroxybutyrate-co-beta-hydroxyvalerate) (P(HB-co-HV)) has been studied by mass spectrometry. We describe a novel method for the structural analysis of P(HB-co-HV) copolymers, based on the direct FAB-MS analysis of the partial pyrolysis products. The FAB spectra obtained allow the identification of the copolymer microstructure up to the hexads level, and a comparison between the experimental and calculated peak intensities makes it possible to discern clearly if the sample is made of a single random copolymer or of a mixture of two random copolymers. In addition, an algorithm has been developed that also allows the determination of both the amount of the random copolymers in the binary P(HB-co-HV) mixtures, as well as the copolymer composition of the individual copolymers. The highly discriminating power of the method developed here is due to the possibility of fitting the calculated statistical abundances with the MS experimental values corresponding to higher oligomers, i.e., hexamers as compared to diad and triad monomer sequences seen by C-13 NMR
219) Linkage Analysis in Disaccharides by Electrospray Mass-Spectrometry
D.Garozzo, G.Impallomeni, E.Spina, B.N.Green, T.Hutton
Carbohydrate Research
221(1),
253
- 1991
The electrospray mass spectrometry of (1.fwdarw.2)-sophorose, (1.fwdarw.3)-laminaribiose, (1.fwdarw.4)-cellobiose, (1.fwdarw.6)-gentiobiose, and maltose are reported. Each mass spectrum contains peaks assocd. with fragmentations which involve the glycosidic linkages and reflect the positions of the linkages. Electrospray ionization thus permits linkage anal. without resorting to tandem mass spectrometry.
220) Linkage Analysis in Disaccharides by Electrospray Mass-Spectrometry (Es Ms)
D.Garozzo, G.Impallomeni, E.Spina
Abstracts of Papers of the American Chemical Society
202,
48
- 1991
221) Microstructure of Bacterial Poly(B-hydroxybutirate-co-B-hydroxyvalerate) by Fast Atom Bombardment Mass SpectrometryAnalysis of their Partial Degradation Products
A.Ballistreri, D.Garozzo, M.Giuffrida, G.Montaudo
Novel Biodegradable Microbial Polymers ,E.A.Dawes Ed., Kluwer
1990,
49
- 1990
The partial methanolysis and the partial pyrolysis of bacterially synthesized .beta.-hydroxybutyric acid-.beta.-hydroxyvaleric acid copolymers were performed and the oligomers produced were analyzed by fast-atom-bombardment mass spectrometry. An algorithm was developed to distinguish pure random copolymers from mixts. of random copolymers, using the relative abundances of the oligomers which could be deduced from the mass spectra.
222) Determination of Linkage Position and Identification of the Reducing End in Linear Oligosaccharides by Negative-Ion Fast Atom Bombardment Mass-Spectrometry
D.Garozzo, M.Giuffrida, G.Impallomeni, A.Ballistreri, G.Montaudo
Analytical Chemistry
62(3),
279
- 1990
Neg. ion fast-atom-bombardment (FAB) mass spectra allowed the detn. of the linkage position and sugar sequences in a series of (underivatized) disaccharides and of linear oligosaccharides. Discrimination of 1-4, 1-6, 1-3, and 1-2 linkage type and detn. of the reducing end and of the monosaccharide sequence is made possible by the anal. of the neg. metastable ions produced in linked scans FAB MS. The peculiarity of neg. ionization is believed to consist in a selective deprotonation of the anomeric hydroxyl (reducing end of the oligosaccharide chain), which is more acidic with respect to the remaining OH groups. Once the neg. charge is localized at the oligosaccharide reducing end, the ion fragmentation of this ring occurs rapidly and the mass losses obsd. are found to be diagnostic of the glycoside linkage type between adjacent sugar units. The overall neg. ions fragmentation process outlined above allows the simultaneous identification of the reducing end of the chain, of the monosaccharide units sequence, and of the linkage type between adjacent units.
223) Odd-Electron Molecular Ion and Loss of Toluene in Fast Atom Bombardment Mass-Spectra of Some Carotenoids
S.Caccamese, D.Garozzo
Organic Mass Spectrometry
25(3),
137
- 1990
Pos.-ion fast atom bombardment (FAB) and B/E linked scan FAB mass spectra of seven carotenoids are reported. In all cases the M+.bul. ions are obsd., and the [M + H]+ ions are absent in the hydrocarbons and weak in the oxygenated compds. The usefulness of B/E linked scan FAB mass spectra to distinguish isomers and to attribute the loss of toluene from the M+.bul. to an ionic fragmentation and not to a thermal process is discussed.
224) Primary thermal decomposition processes in aliphatic polyamides
A.Ballistreri, D.Garozzo, M.Giuffrida, G.Impallomeni, G.Montaudo
Polymer Degradation and Stability
23(1),
25
- 1989
The primary thermal decompn. processes of a series of aliph. polyamides were investigated by direct chem. ionization and tandem mass spectrometry. Intramol. exchange process was the preferred thermal decompn. mechanism for many of the polyamides. Decompn. through a C-H H transfer reaction occurred as a secondary thermal process, except in the case of nylon 3 because of its particular structure. A quite different decompn. pathway was followed by nylons from adipic acid (nylons 6.6 and 11.6), where specific structural factors made the formation of cyclopentanone preferred. These polymers decompd. via a C-H H transfer to N with the formation of compds. having amine and/or ketoamide end groups.
225) Sequencing bacterial poly(β-hydroxybutyrate-co-β-hydroxyvalerate) by partial methanolysis, HPLC fractionation, and fast-atom-bombardment mass spectrometry analysis
A.Ballistreri, D.Garozzo, M.Giuffrida, G.Impallomeni, G.Montaudo
Macromolecules
22(5),
2107
- 1989
The partial methanolysis of poly(β.-hydroxybutyrate) (I) and of poly(β-hydroxybutyrate-co-β-hydroxyvalerate) (II) was performed, and reaction kinetics was optimized to produce oligomers with mol. wts. below the detection limit (.apprx.2000) of the mass spectrometer used for the subsequent anal. The fractionation of the methanolysis products was achieved by HPLC, and the fractions collected were analyzed by fast-atom-bombardment mass spectrometry (FAB-MS) in the presence of NaCl. FAB spectra of oligomers from I and II consisted only of pseudomol. ions MH+ and MNa+, allowing for identification of the methanolysis products, estn. of the copolymer compn., and the detn. of the sequence distribution.
226) Analytical degradation: an approach to the structural analysis of microbial polyesters by different methods
A.Ballistreri, D.Garozzo, M.Giuffrida, G.Impallomeni, G.Montaudo
Journal of Analytical and Applied Pyrolysis
16(3),
239
- 1989
"A sample of pure poly(.beta.-hydroxybutyrate) (I) and a sample of 87:13 .beta.-hydroxybutyrate-.beta.-hydroxyvalerate copolymer (II) were investigated by different degrdn. Methods: (a) direct pyrolysis-mass spectrometry; (b) fast-atom-bombardment mass spectrometry (FABMS) anal. Of the partial pyrolysis products; and © FABMS anal. Of the partial methanolysis products. The pyrolysis products originating from I and II are thermally labile, so that oligomers higher than the trimer cannot distill undecomposed and remain undetected by electron impact or chem. Ionization MS. However, the pyrolysis products can be desorbed by FABMS up to the decamer level. With the partial pyrolysis method it is not possible to obtain reliable compn. And sequence distribution data, which may, however, be obtained by the FABMS anal. Of the partial methanolysis products of I and II."
227) Determination of linkage position in disaccharides by negative-ion fast-atom bombardment mass spectrometry
A.Ballistreri, G.Montaudo, D.Garozzo, M.Giuffrida, G.Impallomeni
Rapid Communications in Mass Spectrometry
3(9),
302
- 1989
Neg.-ion fast-atom bombardment linked-scan spectra at const. B/E allow differentiation of the linkage position in hexose-hexose disaccharides. Sequence information can also be obtained when there is a mol. wt. difference between the monosaccharide units in the carbohydrate mol.
228) Primary thermal fragmentation processes in poly(ethylene oxalate) investigated by mass spectrometry
A.Ballistreri, D.Garozzo, M.Giuffrida, G.Impallomeni, G.Montaudo
Polymer Degradation and Stability
21(4),
311
- 1988
The primary thermal decompn. mechanism of poly(ethylene oxalate) (I) was investigated by pyrolysis-mass spectrometry. Several mass spectrometric techniques were used to identify compds. present in the pyrolysis mixt.: comparison of electron impact and chem. ionization spectra, high-resoln. accurate mass measurements, and tandem mass spectrometry (daughter- and parent-ion spectra). The results obtained indicate that intramol. exchange reactions predominate in the primary thermal fragmentation processes yielding cyclic oligomers up to tetramer. I, prepd. by condensation polymn., was shown by 1H-NMR to contain 7% of diethylene glycol (II) units along the polymer chain. A small amt. of cyclic oligomers contg. II units was detected among the pyrolysis products from I.
229) Thermal decomposition processes in polyhydrazides and polyoxamides investigated by mass spectrometry
A.Ballistreri, D.Garozzo, G.Montaudo, A.Pollicino, M.Giuffrida
Polymer
28(1),
139
- 1987
The thermal decompn. processes of some totally arom. or totally aliph. polyhydrazides and polyoxamides were studied by direct pyrolysis mass spectrometry, using both chem. ionization and electron impact modes. The results indicate that the primary thermal decompn. processes of these polymers (contg. -CO-CO- and -NR-NR- linkages) are strongly influenced by structural factors.
230) Mass spectrometric characterization of poly(ethylene terephthalate-co-p-oxybenzoate)
D.Garozzo, M.Giuffrida, G.Montaudo, R.W.Lenz
Journal of Polymer Science part A: Polymer Chemistry
25,
271
- 1987
p-Acetoxybenzoic acid-ethylene glycol-terephthalic acid copolymer [52237-98-6] samples having various compns. were examd. by direct pyrolysis-mass spectrometry to det. the thermal stabilities and the sequence distributions of the oxybenzoate and ethylene terephthalate units as a function of compn. Thermal stability as measured by the rate of volatilization increased with increasing oxybenzoate content, as did the amt. of char residue formed. The electron-impact mass spectra of volatile fragments showed the formation of predominantly linear fragments with COOH and vinyl end groups. Dimer, trimer, and tetramer fragments contg. either or both types of units were identified and their relative amts. were estd. from peak intensities. The results were consistent with those expected for random distributions of the 2 units, i.e., for statistical copolymers.
231) Fast atom bombardment mass spectrometry identification of oligomers contained in poly(.epsilon.-caprolactam) and poly(butylene isophthalate)
A.Ballistreri, D.Garozzo, M.Giuffrida, G.Montaudo, A.Filippi, C.Guaita, P.Manaresi, F.Pilati
Macromolecules
20(5),
1029
- 1987
Cyclic and linear oligomers with mol. wts. .ltoreq.1450 of poly(.epsilon.-caprolactam) [25038-54-4] and poly(butylene isophthalate) [28087-45-8] were identified by fast-atom-bombardment mass spectrometry employing collision-activated decompn. ion scanning. Anal. of the oligomers by high-performance liq. chromatog. provided information only on compds. with mol. wts. .ltoreq.800.
232) Thermal decomposition processes in aromatic-aliphatic polyamides investigated by mass spectrometry
A.Ballistreri, D.Garozzo, P.Maravigna, G.Montaudo, M.Giuffrida
Journal of Polymer Science part A: Polymer Chemistry
25,
1049
- 1987
The thermal decompn. processes of some arom.-aliph. polyamides, derived from terephthalic acid and aliph. diamines, were studied by flash pyrolysis gas chromatog.-mass spectrometry and by direct pyrolysis-mass spectrometry, using both chem. ionization and electron impact modes. The primary thermal decompn. proceeded via a .beta.-CH hydrogen transfer process, with formation of pyrolysis products contg. amide and olefin end groups. Nitrile end groups were also formed by dehydration of the amide groups formed in the primary decompn. process.
233) Analysis of polymers by mass spectrometry. Metastable mapping of transitions from pyrolysis products of an aromatic polyamide
A.Ballistreri, D.Garozzo, M.Giuffrida, G.Montaudo
Journal of Analytical and Applied Pyrolysis
12(1),
3
- 1987
Metastable mapping was applied to the anal. of the mixt. of the pyrolysis products obtained from an arom. polyamide, pyrolyzed directly in a mass spectrometer. This technique permitted a rapid anal. of the components and provided a method for assigning structures through the study of mol. ions, characteristic fragmentations, and comparisons of daughter and parent ions of authentic samples. Collisionally activated decompn. spectra were obtained in order to induce a high intensity in the metastable transition. The thermal decompn. mechanism occurred via an intramol. exchange and a concomitant NH hydrogen transfer process with the formation of compds. with amine and/or succinimide end-groups.
234) Identification of the ions produced by fast atom bombardment mass spectrometry in some polyesters and polyamides
A.Ballistreri, D.Garozzo, M.Giuffrida, G.Montaudo
Analytical Chemistry
59(17),
2024
- 1987
"Fast atom bombardment (FAB) mass spectrometry was applied to the anal. Of poly(ethylene adipate) poly(.epsilon.-caprolactone), nylon 66 and nylon 6. The peaks present in the spectra of the crude polymers were identified as corresponding to protonated mol. Ions of preformed cyclic oligomers and of low-mol.-wt. Compds. Contained in the polymer samples; these species were desorbed intact from the glycerol matrix under FAB conditions. When the polymer investigated were accurately purified from low-mol.-wt. Compds., no significant peaks were obsd. In their FAB mass spectra. Instead, the FAB mass spectra of the mixts. Extd. From the polymers were very similar to those obtained for crude polymers."
235) Mechanism of thermal decomposition of nylon 66
A.Ballistreri, D.Garozzo, M.Giuffrida, G.Montaudo
Macromolecules
20(12),
2991
- 1987
Thermal degrdn. of nylon 66 as studied by direct pyrolysis into the mass spectrometer proceeded via a C-H hydrogen-transfer reaction to nitrogen with formation of compds. contg. amine and ketoamide end groups. These primary degrdn. products further reacted or decompd. to form cyclopentanone, aminohexamethylene isocyanate, and compds. contg. amine and/or Schiff base groups.
236) Effect of N-methyl substitution on the thermal decomposition processes in aliphatic-aromatic polyamides
A.Ballistreri, D.Garozzo, G.Montaudo, M.Giuffrida
Journal of Polymer Science part A: Polymer Chemistry
25,
2351
- 1987
The thermal decompn. processes of two polyamides, derived from succinic acid and two arom. diamines, were studied by direct pyrolysis mass spectrometry. Fast-atom-bombardment mass spectrometry was also used to provide addnl. information for the elucidation of the thermal degrdn. mechanism of the polymers. The thermal stability of the N-methyl-substituted polyamide was higher than that of the unsubstituted polyamide. The difference in the thermal degrdn. mechanism accounted for the difference in the thermal stability of the two polyamides. The unsubstituted polyamide decompd. via an intramol. exchange and a concomitant N-H hydrogen transfer process with formation of compds. with amine and/or succinimide end groups. The N-methyl-substituted polyamide decompd. via an .alpha. C-H hydrogen transfer process from the Me group to the N atom with formation of compds. with amine and/or 2,5-piperidinedione end groups.
237) Direct mass spectrometry of polymers. XIV. Thermal fragmentation processes in poly-Schiff bases
A.Ballistreri, D.Garozzo, M.Giuffrida, P.Maravigna, G.Montaudo
Journal of Polymer Science part A: Polymer Chemistry
24,
331
- 1986
The thermal fragmentation occurring in the title polymers is characterized by H transfer reactions. In the case of a totally arom. poly-Schiff base, the thermal fragmentation process involves H transfer from the methyne group with formation of fragments bearing nitrile and/or Ph end groups. In the case of arom.-aliph. poly-Schiff bases, H transfer occurs from the aliph. methylene groups. The latter process involves a lower energy and therefore occurs at lower temps. compared to the totally arom. polymer, with formation of thermal fragments bearing olefin and/or imine end groups. Beside these fragments, several thermal fragmentation compds. are also evolved by multiple H transfer reactions.
238) Primary thermal decomposition processes in aliphatic polyesters investigated by chemical ionization mass spectrometry
D.Garozzo, M.Giuffrida, G.Montaudo
Macromolecules
19(6),
1643
- 1986
"The thermal decompn. Mechanisms of several polyesters (derived from aliph. Diols and dicarboxylic acids) and polylactones were studied by direct pyrolysis-mass spectrometry, using both pos. Chem. Ionization and neg. Chem. Ionization. The thermally formed compds. Were not stable under electron impact conditions. Instead, the results obtained by pos. And neg. Chem. Ionization indicated that intramol. Exchange reactions predominated in the primary thermal fragmentation processes, causing the formation of cyclic oligomers, which were particularly stable under chem. Ionization conditions. The only exception was poly-.beta.-propiolactone [24938-43-0]; in this case the thermal decompn. Mechanism involved .beta.-hydrogen transfer reactions."
239) Mixtures of cyclic oligomers of poly(lactic acid) analyzed by negative chemical ionization and thermospray mass spectrometry
D.Garozzo, M.Giuffrida, G.Montaudo
Polymer Bulletin
15(4),
353
- 1986
The distribution of the cyclic oligomers of poly(lactic acid) (I) formed both by pyrolysis of I and by equilibration with a catalyst was studied by the title method. The relative amts. of the oligomers present in the mixt. were also detd. by these methods.
240) Primary thermal fragmentation processes in poly(lactic acid) investigated by positive and negative chemical ionization mass spectrometry
D.Garozzo, G.Montaudo, M.Giuffrida
Polymer Degradation and Stability
15(2),
143
- 1986
The mechanism of thermal decompn. of poly(lactic acid) [104585-16-2] was studied by direct pyrolysis-mass spectrometry using electron impact (EI), pos. chem. ionization (CI) and neg. chem. ionization (NCI) methods. The thermally formed cyclic oligomers of lactic acid are not stable under EI conditions. The results obtained by CI and NCI indicate that intramol. exchange reactions predominate in the primary thermal fragmentation processes.
241) Thermal decomposition processes in aliphatic-aromatic polyamides investigated by mass spectrometry
A.Ballistreri, D.Garozzo, M.Giuffrida, P.Maravigna, G.Montaudo
Macromolecules
19(11),
2693
- 1986
The thermal decompn. processes of some polyamides derived from aliph. dicarboxylic acids and arom. diamines were studied by flash pyrolysis chromatog.-mass spectrometry and by direct pyrolysis-mass spectrometry, using both chem. ionization and electron impact modes. The thermal decompn. of these polyamides is strongly influenced by the structure of the aliph. dicarboxylic acid units. The formation of compds. with succinimide and amine end groups is obsd. in the pyrolysis of the polyamides contg. succinic units. In the pyrolysis of polyamides contg. adipic units, the primary thermal degrdn. products are compds. with amine and ketoamide end-groups. In the pyrolysis of the latter polyamides, secondary thermal fragments are also formed: cyclopentanone, CO2, and compds. with azomethine and isocyanate groups.
242) Thermal degradation processes of polyamides investigated by collision-activated decomposition mass spectrometry/mass spectrometry
A.Ballistreri, D.Garozzo, M.Giuffrida, G.Montaudo
Polymer Degradation and Stability
16(4),
337
- 1986
Linked scanning mass spectrometry was applied to the anal. of the mixt. of pyrolysis products obtained from some polyamides, pyrolyzed directly in a mass spectrometer. Collision-activated decompn. spectra were obtained in order to induce a higher intensity in the metastable transition. Linked scanning mass spectrometry provided a fast method for unambiguously assigning chem. structure through the study of mol. ions, characteristic fragmentations, and comparisons of daughter and parent ions of authentic samples.
243) Identification of polymers by library search of pyrolysis mass spectra and pattern recognition analysis
D.Garozzo, G.Montaudo
Journal of Analytical and Applied Pyrolysis
9(1),
1
- 1985
A library of mass spectra of polymers contg. .apprx.200 entries was described. These spectra were obtained by direct pyrolysis-electron-impact mass spectrometry, i.e., by heating the polymers in the direct insertion probe for solid samples at a const. heating rate and recording repetitive mass spectra during the temp. rise. The library could be used both as a data base for library searches and as a training set for a pattern recognition anal. The algorithm used to generate and search the files and a few applications of the library search and pattern recognition anal. were presented.
244) Mass spectral characterization and thermal decomposition mechanism of poly(dimethylsiloxane)
A.Ballistreri, D.Garozzo, G.Montaudo
Macromolecules
17(7),
1312
- 1984
The pyrolysis of di-Me siloxane was shown by mass spectrometry to form cyclic oligomers by intramol. exchange. The distribution of pyrolysis products was calcd. This distribution was not changed by the addn. of NaOH catalyst, although NaOH did lower the max. polymer decompn. temp. Cyclic oligomers present in di-Me siloxane from its prepn. could also be detected and identified by mass spectrometry.
245) Identification of polymers by library search of mass spectra
S.Foti, D.Garozzo, G.Montaudo
La Chimica e L’industria
65,
641
- 1983
The generation of a data bank of mass spectra of polymers obtained by direct pyrolysis-mass spectrometry and a computer retrieval procedure based on this data bank were described. The use of the retrieval system allowed the identification of unknown polymers.
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