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2.
Proteomics ; 19(21-22): e1900010, 2019 11.
Article in English | MEDLINE | ID: mdl-31419058

ABSTRACT

While aberrant protein glycosylation is a recognized characteristic of human cancers, advances in glycoanalytics continue to discover new associations between glycoproteins and tumorigenesis. This glycomics-centric study investigates a possible link between protein paucimannosylation, an under-studied class of human N-glycosylation [Man1-3 GlcNAc2 Fuc0-1 ], and cancer. The paucimannosidic glycans (PMGs) of 34 cancer cell lines and 133 tissue samples spanning 11 cancer types and matching non-cancerous specimens are profiled from 467 published and unpublished PGC-LC-MS/MS N-glycome datasets collected over a decade. PMGs, particularly Man2-3 GlcNAc2 Fuc1 , are prominent features of 29 cancer cell lines, but the PMG level varies dramatically across and within the cancer types (1.0-50.2%). Analyses of paired (tumor/non-tumor) and stage-stratified tissues demonstrate that PMGs are significantly enriched in tumor tissues from several cancer types including liver cancer (p = 0.0033) and colorectal cancer (p = 0.0017) and is elevated as a result of prostate cancer and chronic lymphocytic leukaemia progression (p < 0.05). Surface expression of paucimannosidic epitopes is demonstrated on human glioblastoma cells using immunofluorescence while biosynthetic involvement of N-acetyl-ß-hexosaminidase is indicated by quantitative proteomics. This intriguing association between protein paucimannosylation and human cancers warrants further exploration to detail the biosynthesis, cellular location(s), protein carriers, and functions of paucimannosylation in tumorigenesis and metastasis.


Subject(s)
Mannose/metabolism , Neoplasms/metabolism , Cell Line, Tumor , Chromatography, Liquid , Disease Progression , Glycosylation , Humans , Tandem Mass Spectrometry
3.
Mucosal Immunol ; 12(3): 784-794, 2019 05.
Article in English | MEDLINE | ID: mdl-30846831

ABSTRACT

Helicobacter suis is the most prevalent non-Helicobacter pylori Helicobacter species in the human stomach and is associated with chronic gastritis, peptic ulcer disease, and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. H. suis colonizes the gastric mucosa of 60-95% of pigs at slaughter age, and is associated with chronic gastritis, decreased weight gain, and ulcers. Here, we show that experimental H. suis infection changes the mucin composition and glycosylation, decreasing the amount of H. suis-binding glycan structures in the pig gastric mucus niche. Similarly, the H. suis-binding ability of mucins from H. pylori-infected humans is lower than that of noninfected individuals. Furthermore, the H. suis growth-inhibiting effect of mucins from both noninfected humans and pigs is replaced by a growth-enhancing effect by mucins from infected individuals/pigs. Thus, Helicobacter spp. infections impair the mucus barrier by decreasing the H. suis-binding ability of the mucins and by decreasing the antiprolific activity that mucins can have on H. suis. Inhibition of these mucus-based defenses creates a more stable and inhabitable niche for H. suis. This is likely of importance for long-term colonization and outcome of infection, and reversing these impairments may have therapeutic benefits.


Subject(s)
Gastric Mucins/metabolism , Gastric Mucosa/physiology , Gastritis/metabolism , Helicobacter Infections/metabolism , Helicobacter heilmannii/physiology , Mucus/physiology , Ulcer/metabolism , Adult , Animals , Cell Proliferation , Chronic Disease , Female , Gastric Mucosa/microbiology , Gastritis/microbiology , Glycosylation , Helicobacter Infections/microbiology , Humans , Male , Middle Aged , Protein Binding , Swine , Ulcer/microbiology
4.
Sci Rep ; 8(1): 8655, 2018 06 05.
Article in English | MEDLINE | ID: mdl-29872119

ABSTRACT

Aberrant glycosylation has been associated with a number of diseases including cancer. Our aim was to elucidate changes in whole plasma N-glycosylation between colorectal cancer (CRC) cases and controls in one of the largest cohorts of its kind. A set of 633 CRC patients and 478 age and gender matched controls was analysed. Additionally, patients were stratified into four CRC stages. Moreover, N-glycan analysis was carried out in plasma of 40 patients collected prior to the initial diagnosis of CRC. Statistically significant differences were observed in the plasma N-glycome at all stages of CRC, this included a highly significant decrease in relation to the core fucosylated bi-antennary glycans F(6)A2G2 and F(6)A2G2S(6)1 (P < 0.0009). Stage 1 showed a unique biomarker signature compared to stages 2, 3 and 4. There were indications that at risk groups could be identified from the glycome (retrospective AUC = 0.77 and prospective AUC = 0.65). N-glycome biomarkers related to the pathogenic progress of the disease would be a considerable asset in a clinical setting and it could enable novel therapeutics to be developed to target the disease in patients at risk of progression.


Subject(s)
Blood Proteins/chemistry , Colorectal Neoplasms/epidemiology , Colorectal Neoplasms/pathology , Polysaccharides/blood , Case-Control Studies , Female , Humans , Male , Middle Aged , Neoplasm Staging , Risk Assessment
5.
Virulence ; 9(1): 898-918, 2018 12 31.
Article in English | MEDLINE | ID: mdl-29638186

ABSTRACT

Helicobacter suis colonizes the stomach of most pigs and is the most prevalent non-Helicobacter pylori Helicobacter species found in the human stomach. In the human host, H. suis contributes to the development of chronic gastritis, peptic ulcer disease and MALT lymphoma, whereas in pigs it is associated with gastritis, decreased growth and ulcers. Here, we demonstrate that the level of H. pylori and H. suis binding to human and pig gastric mucins varies between individuals with species dependent specificity. The binding optimum of H. pylori is at neutral pH whereas that of H. suis has an acidic pH optimum, and the mucins that H. pylori bind to are different than those that H. suis bind to. Mass spectrometric analysis of mucin O-glycans from the porcine mucin showed that individual variation in binding is reflected by a difference in glycosylation; of 109 oligosaccharide structures identified, only 14 were present in all examined samples. H. suis binding to mucins correlated with glycans containing sulfate, sialic acid and terminal galactose. Among the glycolipids present in pig stomach, binding to lactotetraosylceramide (Galß3GlcNAcß3Galß4Glcß1Cer) was identified, and adhesion to Galß3GlcNAcß3Galß4Glc at both acidic and neutral pH was confirmed using other glycoconjugates. Together with that H. suis bound to DNA (used as a proxy for acidic charge), we conclude that H. suis has two binding modes: one to glycans terminating with Galß3GlcNAc, and one to negatively charged structures. Identification of the glycan structures H. suis interacts with can contribute to development of therapeutic strategies alternative to antibiotics.


Subject(s)
Gastric Mucins/metabolism , Glycolipids/metabolism , Helicobacter Infections/microbiology , Helicobacter Infections/veterinary , Helicobacter heilmannii/metabolism , Polysaccharides/metabolism , Swine Diseases/metabolism , Animals , Gastric Mucosa/metabolism , Glycosylation , Helicobacter Infections/metabolism , Helicobacter heilmannii/genetics , Humans , Stomach/microbiology , Swine , Swine Diseases/microbiology
6.
Sci Rep ; 8(1): 242, 2018 01 10.
Article in English | MEDLINE | ID: mdl-29321476

ABSTRACT

Sample collection, handling and storage are the most critical steps for ensuring the highest preservation of specimens. Pre-analytical variability can influence the results as protein signatures alter rapidly after tissue excision or during long-term storage. Hence, we evaluated current state-of-the-art biobank preservation methods from a glycomics perspective and analyzed O-glycan alterations occurring in the gastric cancer tissues. Paired tumor and adjacent normal tissue samples were obtained from six patients undergoing gastric cancer surgery. Collected samples (n = 24) were either snap-frozen or heat stabilized and then homogenized. Glycans were released from extracted glycoproteins and analyzed by LC-MS/MS. In total, the relative abundance of 83 O-glycans and 17 derived structural features were used for comparison. There was no statistically significant difference found in variables between snap frozen and heat-stabilized samples, which indicated the two preservation methods were comparable. The data also showed significant changes between normal and cancerous tissue. In addition to a shift from high sialylation in the cancer area towards blood group ABO in the normal area, we also detected that the LacdiNAc epitope (N,N'-diacetyllactosamine) was significantly decreased in cancer samples. The O-glycan alterations that are presented here may provide predictive power for the detection and prognosis of gastric cancer.


Subject(s)
Gastric Mucosa/metabolism , Polysaccharides/metabolism , Stomach Neoplasms/metabolism , Aged , Aged, 80 and over , Chromatography, Liquid , Female , Glycoproteins/metabolism , Glycosylation , Humans , Male , Metabolomics/methods , Neoplasm Staging , Stomach Neoplasms/pathology , Tandem Mass Spectrometry
7.
Glycobiology ; 27(12): 1099-1108, 2017 12 01.
Article in English | MEDLINE | ID: mdl-28973482

ABSTRACT

Juvenile idiopathic arthritis (JIA) encompasses all forms of chronic idiopathic arthritis that arise before age 16. Previous studies have found JIA to be associated with lower Fc galactosylation of circulating IgG, but the overall spectrum of glycan changes and the net impact on IgG function are unknown. Using ultra performance liquid chromatography (UPLC), we compared IgG glycosylation in 54 subjects with recent-onset untreated JIA with 98 healthy pediatric controls, paired to biophysical profiling of affinity for 20 IgG receptors using a high-throughput multiplexed microsphere assay. Patients with JIA exhibited an increase in hypogalactosylated and hyposialylated IgG glycans, but no change in fucosylation or bisection, together with alteration in the spectrum of IgG ligand binding. Supervised machine learning demonstrated a robust capacity to discriminate JIA subjects from controls using either glycosylation or binding data. The binding signature was driven predominantly by enhanced affinity for Fc receptor like protein 5 (FcRL5), a noncanonical Fc receptor expressed on B cells. Affinity for FcRL5 correlated inversely with galactosylation and sialylation, a relationship confirmed through enzymatic manipulation. These results demonstrate the capacity of combined structural and biophysical IgG phenotyping to define the overall functional impact of IgG glycan changes and implicate FcRL5 as a potential cellular sensor of IgG glycosylation.


Subject(s)
Arthritis, Juvenile , Binding Sites, Antibody , Immunoglobulin Fc Fragments , Immunoglobulin G , Receptors, Fc , Adolescent , Arthritis, Juvenile/blood , Arthritis, Juvenile/immunology , Child , Child, Preschool , Female , Glycosylation , Humans , Immunoglobulin Fc Fragments/blood , Immunoglobulin Fc Fragments/immunology , Immunoglobulin G/blood , Immunoglobulin G/immunology , Infant , Male , Receptors, Fc/blood , Receptors, Fc/immunology
8.
Mol Cell Proteomics ; 16(5): 743-758, 2017 05.
Article in English | MEDLINE | ID: mdl-28461410

ABSTRACT

The mucin O-glycosylation of 10 individuals with and without gastric disease was examined in depth in order to generate a structural map of human gastric glycosylation. In the stomach, these mucins and their O-glycosylation protect the epithelial surface from the acidic gastric juice and provide the first point of interaction for pathogens such as Helicobacter pylori, reported to cause gastritis, gastric and duodenal ulcers and gastric cancer. The rational of the present study was to map the O-glycosylation that the pathogen may come in contact with. An enormous diversity in glycosylation was found, which varied both between individuals and within mucins from a single individual: mucin glycan chain length ranged from 2-13 residues, each individual carried 34-103 O-glycan structures and in total over 258 structures were identified. The majority of gastric O-glycans were neutral and fucosylated. Blood group I antigens, as well as terminal α1,4-GlcNAc-like and GalNAcß1-4GlcNAc-like (LacdiNAc-like), were common modifications of human gastric O-glycans. Furthemore, each individual carried 1-14 glycan structures that were unique for that individual. The diversity and alterations in gastric O-glycosylation broaden our understanding of the human gastric O-glycome and its implications for gastric cancer research and emphasize that the high individual variation makes it difficult to identify gastric cancer specific structures. However, despite the low number of individuals, we could verify a higher level of sialylation and sulfation on gastric O-glycans from cancerous tissue than from healthy stomachs.


Subject(s)
Gastric Mucins/chemistry , Polysaccharides/chemistry , Blood Group Antigens/chemistry , Chromatography, Liquid , Epitopes/metabolism , Gastric Mucins/metabolism , Humans , Mucin 5AC/chemistry , Mucin 5AC/metabolism , Polysaccharides/metabolism , Tandem Mass Spectrometry
9.
Mol Cell Proteomics ; 2017 Mar 13.
Article in English | MEDLINE | ID: mdl-28289177

ABSTRACT

The mucin O-glycosylation of 10 individuals with and without gastric disease was examined in depth in order to generate a structural map of human gastric glycosylation. In the stomach, these mucins and their O-glycosylation protect the epithelial surface from the acidic gastric juice and provide the first point of interaction for pathogens such as Helicobacter pylori, reported to cause gastritis, gastric and duodenal ulcers and gastric cancer. The rational of the present study was to map the O-glycosylation that the pathogen may come in contact with. An enormous diversity in glycosylation was found, which varied both between individuals and within mucins from a single individual: mucin glycan chain length ranged from 2-13 residues, each individual carried 34-103 O-glycan structures and in total over 258 structures were identified. The majority of gastric O-glycans were neutral and fucosylated. Blood group I antigens, as well as terminal α1,4-GlcNAc-like and GalNAcß1-4GlcNAc-like (LacdiNAc-like), were common modifications of human gastric O-glycans. Furthemore, each individual carried 1-14 glycan structures that were unique for that individual. The diversity and alterations in gastric O-glycosylation broaden our understanding of the human gastric O-glycome and its implications for gastric cancer research and emphasize that the high individual variation makes it difficult to identify gastric cancer specific structures. However, despite the low number of individuals, we could verify a higher level of sialylation and sulfation on gastric O-glycans from cancerous tissue than from healthy stomachs.

10.
Methods Mol Biol ; 1503: 97-108, 2017.
Article in English | MEDLINE | ID: mdl-27743361

ABSTRACT

The understanding of glycosylation alterations in health and disease has evolved significantly and glycans are considered to be relevant biomarker candidates. High-throughput analytical technologies capable of generating high-quality, large-scale glycoprofiling data are in high demand. Here, we describe an automated sample preparation workflow and analysis of N-linked glycans from plasma samples using hydrophilic interaction liquid chromatography with fluorescence detection on an ultrahigh-performance liquid chromatography (UHPLC) instrument. Samples are prepared in 96-well plates and the workflow features rapid glycoprotein denaturation, enzymatic glycan release, glycan purification on solid-supported hydrazide, fluorescent labeling, and post-labeling cleanup with solid-phase extraction. The development of a novel approach for plasma N-glycan analysis and its implementation on a robotic platform significantly reduces the time required for sample preparation and minimizes technical variation. It is anticipated that the developed method will contribute to expanding high-throughput capabilities to analyze protein glycosylation.


Subject(s)
Chromatography, High Pressure Liquid/methods , Glycomics/methods , Glycoproteins/chemistry , Polysaccharides/analysis , Chromatography, High Pressure Liquid/instrumentation , Equipment Design , Glycomics/instrumentation , Glycoproteins/blood , Glycoproteins/isolation & purification , Glycosylation , High-Throughput Screening Assays/instrumentation , High-Throughput Screening Assays/methods , Humans , Plasma/chemistry , Polysaccharides/blood , Polysaccharides/isolation & purification , Protein Denaturation , Software , Solid Phase Extraction/instrumentation , Solid Phase Extraction/methods
11.
Methods Mol Biol ; 1503: 235-264, 2017.
Article in English | MEDLINE | ID: mdl-27743371

ABSTRACT

The access to biodatabases for glycomics and glycoproteomics has proven to be essential for current glycobiological research. This chapter presents available databases that are devoted to different aspects of glycobioinformatics. This includes oligosaccharide sequence databases, experimental databases, 3D structure databases (of both glycans and glycorelated proteins) and association of glycans with tissue, disease, and proteins. Specific search protocols are also provided using tools associated with experimental databases for converting primary glycoanalytical data to glycan structural information. In particular, researchers using glycoanalysis methods by U/HPLC (GlycoBase), MS (GlycoWorkbench, UniCarb-DB, GlycoDigest), and NMR (CASPER) will benefit from this chapter. In addition we also include information on how to utilize glycan structural information to query databases that associate glycans with proteins (UniCarbKB) and with interactions with pathogens (SugarBind).


Subject(s)
Glycomics/methods , Glycoproteins/chemistry , Polysaccharides/chemistry , Proteomics/methods , Animals , Carbohydrate Conformation , Chromatography, High Pressure Liquid/methods , Databases, Chemical , Databases, Protein , Humans , Mass Spectrometry/methods , Nuclear Magnetic Resonance, Biomolecular/methods , Protein Conformation , Software
12.
J Proteome Res ; 15(9): 3255-65, 2016 09 02.
Article in English | MEDLINE | ID: mdl-27428249

ABSTRACT

The dromedary camel (Camelus dromedarius) is an agriculturally important species of high economic value but of low reproductive efficiency. Serum and IgG N-glycosylation are affected by physiological and pathogenic changes and might therefore be a useful diagnostic tool in camel livestock management. This study presents the first comprehensive annotation of the N-glycome from dromedary camel serum as well as their single-domain and conventional antibodies and its subsequent application for camel pregnancy diagnostics. N-glycans were released by PNGaseF, labeled with 2-aminobenzamide (2-AB), and analyzed by hydrophilic interaction liquid chromatography with fluorescent detection (HILIC-UPLC-FLD), enzymatic sequencing and mass spectrometry (MS). The use of a high-throughput robotic platform for sample preparation allowed the rapid generation of glycomics data from pregnant (n = 8) and nonpregnant (n = 8) camels of the Majaheem and Wadha breed. IgG N-glycans dominate the glycan profile of camel serum and present a mixture of core-fucosylated and noncore-fucosylated N-glycans which can contain N-glycolylneuraminic and N-acetylneuraminic acid. Significant pregnancy-associated but breed-independent increases in galactosylation, core-fucosylation, sialylation, and decreases in serum O-acetylation were observed. The monitoring of IgG and serum N-glycosylation presents an attractive complementary test for camel pregnancy diagnostics and presents an interesting tool for biomarker discovery in camel health and breeding.


Subject(s)
Glycomics/methods , Immunoglobulin G/metabolism , Polysaccharides/analysis , Serum/metabolism , Animals , Biomarkers/analysis , Camelus , Chromatography, Liquid , Diagnosis , Female , Glycosylation , Mass Spectrometry , Polysaccharides/metabolism , Pregnancy
13.
Future Microbiol ; 11: 721-36, 2016 06.
Article in English | MEDLINE | ID: mdl-27200457

ABSTRACT

AIM: The aim of this study was to identify and characterize EndoS-like enzymes in Streptococcus dysgalactiae subspecies dysgalactiae (SDSD). MATERIALS & METHODS: PCR, DNA sequencing, recombinant protein expression, lectin blot, ultra high performance liquid chromatography analysis and a chitinase assay were used to identify ndoS-like genes and characterize EndoSd. RESULTS: EndoSd were found in four SDSD strains. EndoSd hydrolyzes the chitobiose core of the glycan on IgG. The amino acid sequence of EndoSd is 70% identical to EndoS in S. pyogenes, but it has a unique C-terminal sequence. EndoSd secretion is influenced by the carbohydrate composition of the growth medium. CONCLUSION: Our findings indicate that IgG glycan hydrolyzing activity is present in SDSD, and that the activity can be attributed to the here identified enzyme EndoSd.


Subject(s)
Acetylglucosaminidase/metabolism , Bacterial Proteins/metabolism , Immunoglobulin G/metabolism , Polysaccharides/metabolism , Streptococcus/enzymology , Acetylglucosaminidase/chemistry , Acetylglucosaminidase/genetics , Animals , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Disaccharides/metabolism , Humans , Hydrolysis , Immunoglobulin G/chemistry , Phylogeny , Polysaccharides/chemistry , Streptococcus/chemistry , Streptococcus/classification , Streptococcus/genetics , Substrate Specificity
14.
Data Brief ; 7: 814-33, 2016 Jun.
Article in English | MEDLINE | ID: mdl-27077082

ABSTRACT

Gastric carcinoma MKN45 cells stably transfected with the full-length ST3GAL4 gene were characterised by glycomic and sialoproteomic analysis. Complementary strategies were applied to assess the glycomic alterations induced by ST3GAL4 overexpression. The N- and O-glycome data were generated in two parallel structural analyzes, based on PGC-ESI-MS/MS. Data on glycan structure identification and relative abundance in ST3GAL4 overexpressing cells and respective mock control are presented. The sialoproteomic analysis based on titanium-dioxide enrichment of sialopeptides with subsequent LC-MS/MS identification was performed. This analysis identified 47 proteins with significantly increased sialylation. The data in this article is associated with the research article published in Biochim Biophys Acta "Glycomic analysis of gastric carcinoma cells discloses glycans as modulators of RON receptor tyrosine kinase activation in cancer" [1].

15.
Biochim Biophys Acta ; 1860(8): 1795-808, 2016 Aug.
Article in English | MEDLINE | ID: mdl-26721331

ABSTRACT

BACKGROUND: Terminal α2-3 and α2-6 sialylation of glycans precludes further chain elongation, leading to the biosynthesis of cancer relevant epitopes such as sialyl-Lewis X (SLe(X)). SLe(X) overexpression is associated with tumor aggressive phenotype and patients' poor prognosis. METHODS: MKN45 gastric carcinoma cells transfected with the sialyltransferase ST3GAL4 were established as a model overexpressing sialylated terminal glycans. We have evaluated at the structural level the glycome and the sialoproteome of this gastric cancer cell line applying liquid chromatography and mass spectrometry. We further validated an identified target expression by proximity ligation assay in gastric tumors. RESULTS: Our results showed that ST3GAL4 overexpression leads to several glycosylation alterations, including reduced O-glycan extension and decreased bisected and increased branched N-glycans. A shift from α2-6 towards α2-3 linked sialylated N-glycans was also observed. Sialoproteomic analysis further identified 47 proteins with significantly increased sialylated N-glycans. These included integrins, insulin receptor, carcinoembryonic antigens and RON receptor tyrosine kinase, which are proteins known to be key players in malignancy. Further analysis of RON confirmed its modification with SLe(X) and the concomitant activation. SLe(X) and RON co-expression was validated in gastric tumors. CONCLUSION: The overexpression of ST3GAL4 interferes with the overall glycophenotype of cancer cells affecting a multitude of key proteins involved in malignancy. Aberrant glycosylation of the RON receptor was shown as an alternative mechanism of oncogenic activation. GENERAL SIGNIFICANCE: This study provides novel targets and points to an integrative tumor glycomic/proteomic-profiling for gastric cancer patients' stratification. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.


Subject(s)
Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Lewis X Antigen/biosynthesis , Neoplasm Proteins/biosynthesis , Polysaccharides/biosynthesis , Receptor Protein-Tyrosine Kinases/metabolism , Stomach Neoplasms/metabolism , Glycomics , Humans , Lewis X Antigen/genetics , Neoplasm Proteins/genetics , Polysaccharides/genetics , Receptor Protein-Tyrosine Kinases/genetics , Sialyl Lewis X Antigen , Sialyltransferases/biosynthesis , Sialyltransferases/genetics , Stomach Neoplasms/genetics , beta-Galactoside alpha-2,3-Sialyltransferase
16.
J Biol Chem ; 290(38): 22991-3008, 2015 Sep 18.
Article in English | MEDLINE | ID: mdl-26203194

ABSTRACT

Glypicans are multifunctional cell surface proteoglycans involved in several important cellular signaling pathways. Glypican-1 (Gpc1) is the predominant heparan sulfate proteoglycan in the developing and adult human brain. The two N-linked glycans and the C-terminal domain that attach the core protein to the cell membrane are not resolved in the Gpc1 crystal structure. Therefore, we have studied Gpc1 using crystallography, small angle x-ray scattering, and chromatographic approaches to elucidate the composition, structure, and function of the N-glycans and the C terminus and also the topology of Gpc1 with respect to the membrane. The C terminus is shown to be highly flexible in solution, but it orients the core protein transverse to the membrane, directing a surface evolutionarily conserved in Gpc1 orthologs toward the membrane, where it may interact with signaling molecules and/or membrane receptors on the cell surface, or even the enzymes involved in heparan sulfate substitution in the Golgi apparatus. Furthermore, the N-glycans are shown to extend the protein stability and lifetime by protection against proteolysis and aggregation.


Subject(s)
Glypicans/chemistry , Adult , Crystallography, X-Ray , Glycosylation , Glypicans/genetics , Glypicans/metabolism , HEK293 Cells , Humans , Protein Structure, Tertiary
17.
Carbohydr Res ; 389: 174-85, 2014 May 07.
Article in English | MEDLINE | ID: mdl-24680513

ABSTRACT

The IgG N-glycome provides sufficient complexity and information content to serve as an excellent source for biomarker discovery in mammalian health. Since oligosaccharides play a significant role in many biological processes it is very important to understand their structure. The glycosylation is cell type specific as well as highly variable depending on the species producing the IgG. We evaluated the variation of N-linked glycosylation of human, bovine, ovine, equine, canine and feline IgG using three orthogonal glycan separation techniques: hydrophilic interaction liquid chromatography (HILIC)-UPLC, reversed phase (RP)-UPLC and capillary electrophoresis with laser induced fluorescence detection (CE-LIF). The separation of the glycans by these high resolution methods yielded different profiles due to diverse chemistries. However, the % abundance of structures obtained by CE-LIF and HILIC-UPLC were similar, whereas the analysis by RP-UPLC was difficult to compare as the structures were separated by classes of glycans (highly mannosylated, fucosylated, bisected, fucosylated and bisected) resulting in the co-elution of many structures. The IgGs from various species were selected due to the complexity and variation in their N-glycan composition thereby highlighting the complementarity of these separation techniques.


Subject(s)
Chemical Fractionation/methods , Health , Immunoglobulin G/isolation & purification , Immunoglobulin G/metabolism , Polysaccharides/metabolism , Animals , Cats , Cattle , Dogs , Glycosylation , Horses , Humans , Oligosaccharides/metabolism
18.
J Proteome Res ; 13(2): 385-94, 2014 Feb 07.
Article in English | MEDLINE | ID: mdl-24359113

ABSTRACT

Galactose intoxication and over-restriction in galactosemia may affect glycosylation pathways and cause multisystem effects. In this study, we describe an applied hydrophilic interaction chromatography ultra-performance liquid chromatography high-throughput method to analyze whole serum and extracted IgG N-glycans with measurement of agalactosylated (G0), monogalactosylated (G1), and digalactosylated (G2) structures as a quantitative measure of galactose incorporation. This was applied to nine children with severe galactosemia (genotype Q188R/Q188R) and one child with a milder variant (genotype S135L/S135L). The profiles were also compared with those obtained from three age-matched children with PMM2-CDG (congenital disorder of glycosylation type Ia) and nine pediatric control samples. We have observed that severe N-glycan assembly defects correct in the neonate following dietary restriction of galactose. However, treated adult galactosemia patients continue to exhibit ongoing N-glycan processing defects. We have now applied informative galactose incorporation ratios as a method of studying the presence of N-glycan processing defects in children with galactosemia. We identified N-glycan processing defects present in galactosemia children from an early age. For G0/G1, G0/G2, and (G0/G1)/G2 ratios, the difference noted between galactosemia patients and controls was found to be statistically significant (p = 0.002, 0.01, and 0.006, respectively).


Subject(s)
Galactosemias/metabolism , Polysaccharides/metabolism , Adolescent , Case-Control Studies , Child , Child, Preschool , Chromatography, Liquid/methods , Female , Humans , Infant , Male
19.
Anal Chem ; 85(18): 8841-9, 2013 Sep 17.
Article in English | MEDLINE | ID: mdl-23919734

ABSTRACT

One of today's key challenges is the ability to decode the functions of complex carbohydrates in various biological contexts. To generate high-quality glycomics data in a high-throughput fashion, we developed a robotized and low-cost N-glycan analysis platform for glycoprofiling of immunoglobulin G antibodies (IgG), which are central players of the immune system and of vital importance in the biopharmaceutical industry. The key features include (a) rapid IgG affinity purification and sample concentration, (b) protein denaturation and glycan release on a multiwell filtration device, (c) glycan purification on solid-supported hydrazide, and (d) glycan quantification by ultra performance liquid chromatography. The sample preparation workflow was automated using a robotic liquid-handling workstation, allowing the preparation of 96 samples (or multiples thereof) in 22 h with excellent reproducibility and, thus, should greatly facilitate biomarker discovery and glycosylation monitoring of therapeutic IgGs.


Subject(s)
Antibodies, Anti-Idiotypic/analysis , Automation, Laboratory/methods , High-Throughput Screening Assays/methods , Immunoglobulin G/analysis , Glycosylation
20.
Rheumatology (Oxford) ; 52(9): 1572-82, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23681398

ABSTRACT

OBJECTIVE: Glycosylation is the most common post-translational modification and is altered in disease. The typical glycosylation change in patients with inflammatory arthritis (IA) is a decrease in galactosylation levels on IgG. The aim of this study is to evaluate the effect of anti-TNF therapy on whole serum glycosylation from IA patients and determine whether these alterations in the glycome change upon treatment of the disease. METHODS: Serum samples were collected from 54 IA patients before treatment and at 1 and 12 months after commencing anti-TNF therapy. N-linked glycans from whole serum samples were analysed using a high-throughput hydrophilic interaction liquid chromatography-based method. RESULTS: Glycosylation on the serum proteins of IA patients changed significantly with anti-TNF treatment. We observed an increase in galactosylated glycans from IgG, also an increase in core-fucosylated biantennary galactosylated glycans and a decrease in sialylated triantennary glycans with and without outer arm fucose. This increase in galactosylated IgG glycans suggests a reversing of the N-glycome towards normal healthy profiles. These changes are strongly correlated with decreasing CRP, suggesting a link between glycosylation changes and decreases in inflammatory processes. CONCLUSION: Glycosylation changes in the serum of IA patients on anti-TNF therapy are strongly associated with a decrease in inflammatory processes and reflect the effect of anti-TNF on the immune system.


Subject(s)
Antirheumatic Agents/therapeutic use , Arthritis, Psoriatic/drug therapy , Arthritis, Rheumatoid/drug therapy , Blood Proteins/metabolism , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Adult , Aged , Antirheumatic Agents/pharmacology , Arthritis, Psoriatic/blood , Arthritis, Rheumatoid/blood , Female , Glycosylation/drug effects , Humans , Immunoglobulin G/metabolism , Male , Middle Aged
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