PubMed | University of Edinburgh, Polyomica, Clinical Hospital Merkur, Allgemein und Viszeralchirurgie and 4 more.
Type: Journal Article | Journal: Clinical cancer research : an official journal of the American Association for Cancer Research | Year: 2016
Alternative glycosylation has significant structural and functional consequences on IgG and consequently also on cancer immunosurveillance. Because of technological limitations, the effects of highly heritable individual variations and the differences in the dynamics of changes in IgG glycosylation on colorectal cancer were never investigated before.Using recently developed high-throughput UPLC technology for IgG glycosylation analysis, we analyzed IgG glycome composition in 760 patients with colorectal cancer and 538 matching controls. Effects of surgery were evaluated in 28 patients sampled before and three times after surgery. A predictive model was built using regularized logistic regression and evaluated using a 10-cross validation procedure. Furthermore, IgG glycome composition was analyzed in 39 plasma samples collected before initial diagnosis of colorectal cancer.We have found that colorectal cancer associates with decrease in IgG galactosylation, IgG sialylation and increase in core-fucosylation of neutral glycans with concurrent decrease of core-fucosylation of sialylated glycans. Although a model based on age and sex did not show discriminative power (AUC = 0.499), the addition of glycan variables into the model considerably increased the discriminative power of the model (AUC = 0.755). However, none of these differences were significant in the small set of samples collected before the initial diagnosis.Considering the functional relevance of IgG glycosylation for both tumor immunosurveillance and clinical efficacy of therapy with mAbs, individual variation in IgG glycosylation may turn out to be important for prediction of disease course or the choice of therapy, thus warranting further, more detailed studies of IgG glycosylation in colorectal cancer. Clin Cancer Res; 22(12); 3078-86. 2016 AACR.
Stewart R.,MedImmune |
Thom G.,MedImmune |
Levens M.,MedImmune |
Guler-Gane G.,MedImmune |
And 6 more authors.
Protein Engineering, Design and Selection | Year: 2011
Ribosome display was applied to the Fc region of human immunoglobulin G (IgG1) to select for improved binding to human FcγRIIIa, the receptor expressed on human natural killer cells that mediates antibody-dependent cellular cytotoxicity (ADCC). A library of human Fcγ1 variants was generated using error-prone polymerase chain reaction, and subjected to multiple rounds of ribosome display selection against progressively decreasing concentrations of soluble human FcγRIIIa, to enrich for improved binders. Radioimmunoassay and alphascreen analyses of the aglycosylated IgG-Fc output revealed variants with improved binding to FcγRIIIa relative to wild-type IgG-Fc. Subsequent expression in human (HEK-EBNA) cells generated glycosylated IgGs with modified activity in ADCC assays. One particular variant, 125-B01 triggered enhanced ADCC (EC50 up to four-fold reduced with increased maximal lysis) relative to wild-type antibody, having more equal levels of ADCC for each allotype (V158/F158) of FcγRIIIa. Deconvolution of individual replacements within the variant showed that improved function arose from the Phe243Leu replacement within the CH2 domain, rather than the CH3 domain replacements Thr393Ala or His433Pro. Surprisingly, the oligosaccharide profiles of 125-B01 indicated more oligosaccharide chains lacking fucose, or with bisecting N-acetylglucosamine relative to wild-type IgG1, which correlates with improved function and the replacement Phe243Leu that is a carbohydrate contact residue within the CH2 domain. © The Author 2011. Published by Oxford University Press. All rights reserved.
Gotz L.,Swiss Institute of Bioinformatics |
Abrahams J.L.,Research Frontiers |
Mariethoz J.,Swiss Institute of Bioinformatics |
Rudd P.M.,National Institute for Bioprocessing Research and Training |
And 5 more authors.
Bioinformatics | Year: 2014
Sequencing oligosaccharides by exoglycosidases, either sequentially or in an array format, is a powerful tool to unambiguously determine the structure of complex N- and O-link glycans. Here, we introduce GlycoDigest, a tool that simulates exoglycosidase digestion, based on controlled rules acquired from expert knowledge and experimental evidence available in GlycoBase. The tool allows the targeted design of glycosidase enzyme mixtures by allowing researchers to model the action of exoglycosidases, thereby validating and improving the efficiency and accuracy of glycan analysis. © The Author 2014. Published by Oxford University Press.
Campbell M.P.,Research Frontiers |
Hayes C.A.,Gothenburg University |
Struwe W.B.,National Institute for Bioprocessing Research and Training |
Wilkins M.R.,University of Sydney |
And 7 more authors.
Proteomics | Year: 2012
Despite the success of several international initiatives the glycosciences still lack a managed infrastructure that contributes to the advancement of research through the provision of comprehensive structural and experimental glycan data collections. UniCarbKB is an initiative that aims to promote the creation of an online information storage and search platform for glycomics and glycobiology research. The knowledgebase will offer a freely accessible and information-rich resource supported by querying interfaces, annotation technologies and the adoption of common standards to integrate structural, experimental and functional data. The UniCarbKB framework endeavors to support the growth of glycobioinformatics and the dissemination of knowledge through the provision of an open and unified portal to encourage the sharing of data. In order to achieve this, the framework is committed to the development of tools and procedures that support data annotation, and expanding interoperability through cross-referencing of existing databases. Database URL: http://www.unicarbkb.org. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Duffy F.J.,National Institute for Bioprocessing Research and Training |
Rudd P.M.,National Institute for Bioprocessing Research and Training
Bioinformatics | Year: 2015
Motivation: Sequencing glycan structures is a difficult problem that requires the use of multiple experimental approaches. One powerful approach to glycan sequencing is the combination of liquid chromatography with sequential exoglycosidase digestions; however, interpreting this can be difficult and time-consuming. To aid this process, we introduce GlycoProfileAssigner, software for automated structural assignment of glycan profile data from liquid chromatography experiments. Results: GlycoProfileAssigner has been tested on human IgG data, and can retrieve the correct structure in 14 out of 16 peaks tested. Availability and Implementation: The programme and its source code is available at https://bitbucket.org/fergaljd/glycoprofileassigner. © 2015 The Author 2015. Published by Oxford University Press. All rights reserved.
Doherty M.,National Institute for Bioprocessing Research and Training |
McManus C.A.,National Institute for Bioprocessing Research and Training |
McManus C.A.,University of Western Australia |
McManus C.A.,Phylogica Ltd |
And 2 more authors.
Methods in Molecular Biology | Year: 2012
N-linked oligosaccharides are complex non-template-derived structures that are attached to the side chains of asparagine, via the nitrogen atom. Specific changes in the N-glycans of serum glycoproteins have been associated with the pathogenesis of many diseases. The oligosaccharides present on the C H2 domain of immunoglobulins are known to modulate the effector functions of the molecule. These glycans provoke various biological effects, necessitating the development of robust high-throughput technology in order to fully characterize the N-glycosylation profile. This chapter describes in detail four methods to release N-glycans from the glycoprotein of interest. Two of these protocols, referred to as the "In-Gel Block" and "1D sodium dodecyl sulfate-polyacrylamide gel electrophoresis" methods, require immobilization of the glycoprotein prior to analysis. An automated method is also described, involving the purification of immunoglobulins directly from fermentation media, and, finally, an "In-solution method" is detailed, which directly releases the N-glycans into solution. HILIC and WAX-HPLC are used to analyze the N-glycan profile. Exoglycosidase enzymes digestion arrays, in combination with computer-assisted data analysis, are used to determine both the sequence and linkage of the N-glycans present. © 2012 Springer Science+Business Media, LLC.
Artemenko N.V.,National Institute for Bioprocessing Research and Training |
McDonald A.G.,Trinity College Dublin |
Davey G.P.,Trinity College Dublin |
Rudd P.M.,National Institute for Bioprocessing Research and Training
Methods in Molecular Biology | Year: 2012
Glycans are crucial to the functioning of multicellular organisms. They may also play a role as mediators between host and parasite or symbiont. As many proteins (>50%) are posttranslationally modified by glycosylation, this mechanism is considered to be the most widespread posttranslational modification in eukaryotes. These surface modifications alter and regulate structure and biological activities/functions of proteins/biomolecules as they are largely involved in the recognition process of the appropriate structure in order to bind to the target cells. Consequently, the recognition of glycans on cellular surfaces plays a crucial role in the promotion or inhibition of various diseases and, therefore, glycosylation itself is considered to be a critical protein quality control attribute for commercial therapeutics, which is one of the fastest growing segments in the pharmaceutical industry. With the development of glycobiology as a separate discipline, a number of databases and tools became available in a similar way to other well-established "omics." Alleviating the recognized shortcomings of the available tools for data storage and retrieval is one of the highest priorities of the international glycoinformatics community. In the last decade, major efforts have been made, by leading scientific groups, towards the integration of a number of major databases and tools into a single portal, which would act as a centralized data repository for glycomics, equipped with a number of comprehensive analytical tools for data systematization, analysis, and comparison. This chapter provides an overview of the most important carbohydrate-related databases and glycoinformatic tools. © 2012 Springer Science+Business Media, LLC.
PubMed | National Institute for Bioprocessing Research and Training and University of Girona
Type: Journal Article | Journal: Theranostics | Year: 2016
New markers based on PSA isoforms have recently been developed to improve prostate cancer (PCa) diagnosis. However, novel approaches are still required to differentiate aggressive from non-aggressive PCa to improve decision making for patients. PSA glycoforms have been shown to be differentially expressed in PCa. In particular, changes in the extent of core fucosylation and sialylation of PSA N-glycans in PCa patients compared to healthy controls or BPH patients have been reported. The objective of this study was to determine these specific glycan structures in serum PSA to analyze their potential value as markers for discriminating between BPH and PCa of different aggressiveness. In the present work, we have established two methodologies to analyze the core fucosylation and the sialic acid linkage of PSA N-glycans in serum samples from BPH (29) and PCa (44) patients with different degrees of aggressiveness. We detected a significant decrease in the core fucose and an increase in the 2,3-sialic acid percentage of PSA in high-risk PCa that differentiated BPH and low-risk PCa from high-risk PCa patients. In particular, a cut-off value of 0.86 of the PSA core fucose ratio, could distinguish high-risk PCa patients from BPH with 90% sensitivity and 95% specificity, with an AUC of 0.94. In the case of the 2,3-sialic acid percentage of PSA, the cut-off value of 30% discriminated between high-risk PCa and the group of BPH, low-, and intermediate-risk PCa with a sensitivity and specificity of 85.7% and 95.5%, respectively, with an AUC of 0.97. The latter marker exhibited high performance in differentiating between aggressive and non-aggressive PCa and has the potential for translational application in the clinic.
Knezevic A.,Genos Ltd |
Bones J.,National Institute for Bioprocessing Research and Training |
Kracun S.K.,Copenhagen University |
Gornik O.,University of Zagreb |
And 3 more authors.
Analyst | Year: 2011
A rapid glycomic profiling method is described wherein N-glycans from plasma samples individually labelled with aniline, 2-aminobenzamide and 2-aminoacridone are mixed, co-injected and separated in the same HILIC-fluorescence run. Transfer of the multiplexed method to UPLC-fluorescence permits an increase in sample throughput from 24 to 864 plasma samples per day. © 2011 The Royal Society of Chemistry.
PubMed | National Institute for Bioprocessing Research and Training
Type: Journal Article | Journal: Bioinformatics (Oxford, England) | Year: 2015
Sequencing glycan structures is a difficult problem that requires the use of multiple experimental approaches. One powerful approach to glycan sequencing is the combination of liquid chromatography with sequential exoglycosidase digestions; however, interpreting this can be difficult and time-consuming. To aid this process, we introduce GlycoProfileAssigner, software for automated structural assignment of glycan profile data from liquid chromatography experiments.GlycoProfileAssigner has been tested on human IgG data, and can retrieve the correct structure in 14 out of 16 peaks tested.The programme and its source code is available at https://bitbucket.org/fergaljd/glycoprofileassigner: firstname.lastname@example.orgSupplementary data are available at Bioinformatics online.