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Thanabalasingham G.,University of Oxford | Thanabalasingham G.,Churchill Hospital | Huffman J.E.,University of Edinburgh | Kattla J.J.,National Institute for Bioprocessing Research and Training NIBRT | And 49 more authors.
Diabetes | Year: 2013

A recent genome-wide association study identified hepatocyte nuclear factor 1-a (HNF1A) as a key regulator of fucosylation. We hypothesized that loss-of-function HNF1A mutations causal for maturity-onset diabetes of the young (MODY) would display altered fucosylation of N-linked glycans on plasma proteins and that glycan biomarkers could improve the efficiency of a diagnosis of HNF1A-MODY. In a pilot comparison of 33 subjects with HNF1A-MODY and 41 subjects with type 2 diabetes, 15 of 29 glycan measurements differed between the two groups. The DG9-glycan index, which is the ratio of fucosylated to nonfucosylated triantennary glycans, provided optimum discrimination in the pilot study and was examined further among additional subjects with HNF1A-MODY (n = 188), glucokinase (GCK)-MODY (n = 118), hepatocyte nuclear factor 4-α (HNF4A)-MODY (n = 40), type 1 diabetes (n = 98), type 2 diabetes (n = 167), and nondiabetic controls (n = 98). The DG9-glycan index was markedly lower in HNF1A-MODY than in controls or other diabetes subtypes, offered good discrimination between HNF1AMODY and both type 1 and type 2 diabetes (C statistic ≥0.90), and enabled us to detect three previously undetected HNF1A mutations in patients with diabetes. In conclusion, glycan profiles are altered substantially in HNF1A-MODY, and the DG9-glycan index has potential clinical value as a diagnostic biomarker of HNF1A dysfunction. © 2013 by the American Diabetes Association.


Struwe W.B.,National Institute for Bioprocessing Research and Training NIBRT | Struwe W.B.,University of Oxford | Gough R.,University College Dublin | Gallagher M.E.,University College Dublin | And 5 more authors.
Molecular and Cellular Proteomics | Year: 2015

The Gram-negative bacteria Campylobactor jejuni is the primary bacteria responsible for food poisoning in industrialized countries, and acute diarrheal illness is a leading cause of mortality among children in developing countries. C. jejuni are commensal in chickens. They are particularly abundant in the caecal crypts, and poultry products are commonly infected as a result of crosscontamination during processing. The interactions between C. jejuni and chicken intestinal tissues as well as the pathogenic molecular mechanisms of colonization in humans are unknown, but identifying these factors could provide potential targets to reduce the incidence of campylobacteriosis. Recently, purified chicken intestinal mucin was shown to attenuate adherence and invasion of C. jejuni in the human colorectal adenocarcinoma cell line HCT-8 in vitro, and this effect was attributed to mucin O-glycosylation. Mucins from different regions of the chicken intestine inhibited C. jejuni binding and internalization differentially, with large intestine small intestine caecum. Here, we use LC-MS to perform a detailed structural analysis of O-glycans released from mucins purified from chicken large intestine, small intestine, and caecum. The O-glycans identified were abundantly sulfated compared with the human intestines, and sulfate moieties were present throughout the chicken intestinal tract. Interestingly, alpha 1-2 linked fucose residues, which have a high binding affinity to C. jejuni, were identified in the small and large intestines. Additionally, N-glycolylneuraminic/N-acetylneuraminic acid containing structures present as Sda-like epitopes were identified in large intestine samples but not small intestine or caecum. O-glycan structural characterization of chicken intestinal mucins provides insights into adherence and invasion properties of C. jejuni, and may offer prospective candidate molecules aimed at reducing the incidence of infection. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.


Mehta N.,University of Georgia | Porterfield M.,University of Georgia | Struwe W.B.,National Institute for Bioprocessing Research and Training NIBRT | Struwe W.B.,University of Oxford | And 6 more authors.
Journal of Proteome Research | Year: 2016

Environmental and metabolic processes shape the profile of glycoprotein glycans expressed by cells, whether in culture, developing tissues, or mature organisms. Quantitative characterization of glycomic changes associated with these conditions has been achieved historically by reductive coupling of oligosaccharides to various fluorophores following release from glycoprotein and subsequent HPLC or capillary electrophoretic separation. Such labeling-based approaches provide a robust means of quantifying glycan amount based on fluorescence yield. Mass spectrometry, on the other hand, has generally been limited to relative quantification in which the contribution of the signal intensity for an individual glycan is expressed as a percent of the signal intensity summed over the total profile. Relative quantification has been valuable for highlighting changes in glycan expression between samples; sensitivity is high, and structural information can be derived by fragmentation. We have investigated whether MS-based glycomics is amenable to absolute quantification by referencing signal intensities to well-characterized oligosaccharide standards. We report the qualification of a set of N-linked oligosaccharide standards by NMR, HPLC, and MS. We also demonstrate the dynamic range, sensitivity, and recovery from complex biological matrices for these standards in their permethylated form. Our results indicate that absolute quantification for MS-based glycomic analysis is reproducible and robust utilizing currently available glycan standards. © 2016 American Chemical Society.


PubMed | Macquarie University, Max Planck Institute of Colloids and Interfaces, Fudan University, National Institute for Bioprocessing Research and Training NIBRT and 9 more.
Type: Journal Article | Journal: Glycoconjugate journal | Year: 2016

The Human Disease Glycomics/Proteome Initiative (HGPI) is an activity in the Human Proteome Organization (HUPO) supported by leading researchers from international institutes and aims at development of disease-related glycomics/glycoproteomics analysis techniques. Since 2004, the initiative has conducted three pilot studies. The first two were N- and O-glycan analyses of purified transferrin and immunoglobulin-G and assessed the most appropriate analytical approach employed at the time. This paper describes the third study, which was conducted to compare different approaches for quantitation of N- and O-linked glycans attached to proteins in crude biological samples. The preliminary analysis on cell pellets resulted in wildly varied glycan profiles, which was probably the consequence of variations in the pre-processing sample preparation methodologies. However, the reproducibility of the data was not improved dramatically in the subsequent analysis on cell lysate fractions prepared in a specified method by one lab. The study demonstrated the difficulty of carrying out a complete analysis of the glycome in crude samples by any single technology and the importance of rigorous optimization of the course of analysis from preprocessing to data interpretation. It suggests that another collaborative study employing the latest technologies in this rapidly evolving field will help to realize the requirements of carrying out the large-scale analysis of glycoproteins in complex cell samples.


PubMed | National Institute for Bioprocessing Research and Training NIBRT, University College Dublin and Gothenburg University
Type: Journal Article | Journal: Molecular & cellular proteomics : MCP | Year: 2015

The Gram-negative bacteria Campylobactor jejuni is the primary bacteria responsible for food poisoning in industrialized countries, and acute diarrheal illness is a leading cause of mortality among children in developing countries. C. jejuni are commensal in chickens. They are particularly abundant in the caecal crypts, and poultry products are commonly infected as a result of cross-contamination during processing. The interactions between C. jejuni and chicken intestinal tissues as well as the pathogenic molecular mechanisms of colonization in humans are unknown, but identifying these factors could provide potential targets to reduce the incidence of campylobacteriosis. Recently, purified chicken intestinal mucin was shown to attenuate adherence and invasion of C. jejuni in the human colorectal adenocarcinoma cell line HCT-8 in vitro, and this effect was attributed to mucin O-glycosylation. Mucins from different regions of the chicken intestine inhibited C. jejuni binding and internalization differentially, with large intestine>small intestine>caecum. Here, we use LC-MS to perform a detailed structural analysis of O-glycans released from mucins purified from chicken large intestine, small intestine, and caecum. The O-glycans identified were abundantly sulfated compared with the human intestines, and sulfate moieties were present throughout the chicken intestinal tract. Interestingly, alpha 1-2 linked fucose residues, which have a high binding affinity to C. jejuni, were identified in the small and large intestines. Additionally, N-glycolylneuraminic/N-acetylneuraminic acid containing structures present as Sd(a)-like epitopes were identified in large intestine samples but not small intestine or caecum. O-glycan structural characterization of chicken intestinal mucins provides insights into adherence and invasion properties of C. jejuni, and may offer prospective candidate molecules aimed at reducing the incidence of infection.


McDonald A.G.,Trinity College Dublin | McDonald A.G.,National Institute for Bioprocessing Research and Training NIBRT | Hayes J.M.,Trinity College Dublin | Hayes J.M.,National Institute for Bioprocessing Research and Training NIBRT | And 11 more authors.
Journal of Cell Science | Year: 2014

Protein N-glycosylation is a common post-translational modification that produces a complex array of branched glycan structures. The levels of branching, or antennarity, give rise to differential biological activities for single glycoproteins. However, the precise mechanism controlling the glycan branching and glycosylation network is unknown. Here, we constructed quantitative mathematical models of N-linked glycosylation that predicted new control points for glycan branching. Galactosyltransferase, which acts on Nacetylglucosamine residues, was unexpectedly found to control metabolic flux through the glycosylation pathway and the level of final antennarity of nascent protein produced in the Golgi network. To further investigate the biological consequences of glycan branching in nascent proteins, we glycoengineered a series of mammalian cells overexpressing human chorionic gonadotropin (hCG). We identified a mechanism in which galactosyltransferase 4 isoform regulated N-glycan branching on the nascent protein, subsequently controlling biological activity in an in vivo model of hCG activity. We found that galactosyltransferase 4 is a major control point for glycan branching decisions taken in the Golgi of the cell, which might ultimately control the biological activity of nascent glycoprotein. © 2014. Published by The Company of Biologists Ltd.

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