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Lauc G.,University of Zagreb | Lauc G.,Genos Glycoscience Research Laboratory | Pezer M.,Genos Glycoscience Research Laboratory | Rudan I.,University of Edinburgh | Campbell H.,University of Edinburgh
Biochimica et Biophysica Acta - General Subjects | Year: 2016

Background: The majority of human proteins are being modified by covalent attachment of complex oligosaccharides-glycans. Both glycans and polypeptide parts of a protein contribute to its structure and function, but contrary to polypeptide that is defined by the sequence of nucleotides in the corresponding gene, glycans are shaped by complex dynamic interactions between hundreds of enzymes, transcription factors, ion channels and other proteins. Scope of review: An overviewof current knowledge about the importance of N-glycans in normal human physiology and disease mechanisms, exemplified by IgG N-glycans. Major conclusions: Recent technological development enabled systematic analysis of glycome composition in large epidemiological cohorts and clinical studies. However, the majority of these studies is still missing any glycomic component, and consequently also lacks this layer of biological information. Individual variation in glycosylation is potentially important for individualized disease risk, disease course and response to therapy. Evidence in support of this hypothesis is accumulating, but further studies are needed to enable understanding of the role of changes in protein glycosylation in disease. General significance: Glycans are involved in virtually all physiological processes. Inter-individual variation in glycome composition is large, and these differences associate with disease risk, disease course and the response to therapy. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc. © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license.

Trbojevic Akmacic I.,Genos Glycoscience Research Laboratory | Ugrina I.,Genos Glycoscience Research Laboratory | Stambuk J.,Genos Glycoscience Research Laboratory | Gudelj I.,Genos Glycoscience Research Laboratory | And 4 more authors.
Biochemistry (Moscow) | Year: 2015

Glycosylation affects structure, folding, and function of numerous proteins. Aberrant glycosylation has been shown to be associated with different diseases. A wide range of analytical methods is available for glycan analysis of antibodies (mainly IgG), but analysis of plasma glycans is less established due to additional challenges encountered with higher complexity of the sample. Here we describe development and optimization of a high-throughput sample preparation method for hydrophilic interaction liquid chromatography and ultra-performance liquid chromatography analysis of plasma N-glycans. Clean-up of labeled glycans was found to be the largest source of variation, and we tested cellulose, silica gel, Bio-Gel, and hydrophilic GHP filter as stationary phases for solid-phase extraction. All stationary phases were shown to be suitable for purification of labeled glycans, but GHP filter plate in combination with cold 96% acetonitrile had the highest reproducibility and was easiest to work with. The method was further optimized with Plackett - Burman screening design and validated in terms of analysis of major step variation and between-day and between-person variation. The developed method is fast, cost-effective, and easy to perform, and it has very good reproducibility during long period of time, enabling the detection of biological variability of the plasma N-glycome. © 2015 Pleiades Publishing, Ltd.

Kao D.,Friedrich - Alexander - University, Erlangen - Nuremberg | Danzer H.,Friedrich - Alexander - University, Erlangen - Nuremberg | Collin M.,Lund University | Gross A.,Friedrich - Alexander - University, Erlangen - Nuremberg | And 5 more authors.
Cell Reports | Year: 2015

Immunoglobulin G (IgG) glycosylation modulates antibody activity and represents a major source of heterogeneity within antibody preparations. Depending on their glycosylation pattern, individual IgG glycovariants present in recombinant antibody preparations may trigger effects ranging from enhanced pro-inflammatory activity to increased anti-inflammatory activity. In contrast, reduction of IgG glycosylation beyond the central mannose core is generally believed to result in impaired IgG activity. However, this study reveals that a mono- or disaccharide structure consisting of one N-acetylglucosamine with or without a branching fucose residue is sufficient to retain the activity of the most active human and mouse IgG subclasses in vivo and further directs antibody activity to cellular Fcγ receptors. Notably, the activity of minimally glycosylated antibodies is not predicted by in vitro assays based on a monomeric antibody-Fcγ-receptor interaction analysis, whereas in vitro assay systems using immune complexes are more suitable to predict IgG activity in vivo. © 2015 The Authors.

Trbojevic-Akmacic I.,Genos Glycoscience Research Laboratory | Vilaj M.,University of Zagreb | Lauc G.,Genos Glycoscience Research Laboratory | Lauc G.,University of Zagreb
Expert Review of Proteomics | Year: 2016

ABSTRACT: Introduction: Glycosylation of immunoglobulin G (IgG) is important for its effector functions and was shown to be related to age, sex and disease status of an individual. Adding glycomic information to genome-wide association studies (GWAS) and large clinical trials is enabling insight into the functional relevance of changes in glycosylation, as well as molecular mechanisms behind these changes. Large-scale studies require sensitive, robust and affordable high-throughput methodologies for glycosylation analysis, which are currently available in only a limited number of laboratories. Areas covered: This review focuses on currently used high-throughput approaches for N-glycosylation analysis of IgG, as well as some recent advances in the areas of deglycosylation, trypsin digestion, labeling, purification, derivatization and automation of current workflows. Relevant literature was searched using the PubMed database. Expert commentary: Development, optimization and validation of robust, affordable and simple high-throughput glycosylation analysis methods is essential for discovery and validation of diagnostic and prognostic glycan biomarkers. Although significant advances in glycosylation analysis have been made in recent years, currently used protocols will have to be further optimized to enable subsequent analysis of glycosylation on all levels with the limited initial sample and in the minimal amount of time, which is still a challenging task. © 2016 Informa UK Limited, trading as Taylor & Francis Group.

Prenc E.,Croatian Cooperative Group for Hematologic Diseases | Pulanic D.,University of Zagreb | Pulanic D.,Josip Juraj Strossmayer University of Osijek | Pucic-Bakovic M.,Genos Glycoscience Research Laboratory | And 6 more authors.
Biochimica et Biophysica Acta - General Subjects | Year: 2016

Background: Glycans, complex oligosaccharides, are directly involved in almost every biological process, have a fundamental role in the immune system, and are probably involved in nearly every human disease. However, glycosylation has been greatly ignored in the area of allogeneic hematopoietic stem cell transplantation (alloHSCT) and graft versus host disease (GVHD). Both acute and chronic GVHD are multisystemic debilitating immunological disturbances arising after alloHSCT. Scope of review: In this paper, we review the glycosylation research already done in the field of alloHSCT and GVHD and evaluate further potential of glycan analysis in GVHD by looking into resembling inflammatory and autoimmune conditions. Major Conclusions: Glycan research could bring significant improvement in alloHSCT procedure with reduction in following complications, such as GVHD. Identifying glycan patterns that induce self-tolerance and the ones that cause the auto- and allo-immune response could lead to innovative and tissue-specific immunomodulative therapy instead of the current immunosuppressive treatment, enabling preservation of the graft-versus-tumor effect. Moreover, improved glycan pattern analyses could offer a more complete assessment and greatly needed dynamic biomarkers for GVHD. General significance: This review is written with a goal to encourage glycan research in the field of alloHSCT and GVHD as a perspective tool leading to improved engraftment, discovery of much needed biomarkers for GVHD, enabling an appropriate therapy and improved monitoring of therapeutic response.This article is part of a special issue titled "Glycans in personalised medicine"; guest editor: Professor Gordan Lauc. © 2016 Elsevier B.V.

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