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      Glycosylation of extracellular vesicles: current knowledge, tools and clinical perspectives

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          ABSTRACT

          It is now acknowledged that extracellular vesicles (EVs) are important effectors in a vast number of biological processes through intercellular transfer of biomolecules. Increasing research efforts in the EV field have yielded an appreciation for the potential role of glycans in EV function. Indeed, recent reports show that the presence of glycoconjugates is involved in EV biogenesis, in cellular recognition and in the efficient uptake of EVs by recipient cells. It is clear that a full understanding of EV biology will require researchers to focus also on EV glycosylation through glycomics approaches. This review outlines the major glycomics techniques that have been applied to EVs in the context of the recent findings. Beyond understanding the mechanisms by which EVs mediate their physiological functions, glycosylation also provides opportunities by which to engineer EVs for therapeutic and diagnostic purposes. Studies characterising the glycan composition of EVs have highlighted glycome changes in various disease states, thus indicating potential for EV glycans as diagnostic markers. Meanwhile, glycans have been targeted as molecular handles for affinity-based isolation in both research and clinical contexts. An overview of current strategies to exploit EV glycosylation and a discussion of the implications of recent findings for the burgeoning EV industry follows the below review of glycomics and its application to EV biology.

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          Most cited references60

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          GlycoWorkbench: a tool for the computer-assisted annotation of mass spectra of glycans.

          Mass spectrometry is the main analytical technique currently used to address the challenges of glycomics as it offers unrivalled levels of sensitivity and the ability to handle complex mixtures of different glycan variations. Determination of glycan structures from analysis of MS data is a major bottleneck in high-throughput glycomics projects, and robust solutions to this problem are of critical importance. However, all the approaches currently available have inherent restrictions to the type of glycans they can identify, and none of them have proved to be a definitive tool for glycomics. GlycoWorkbench is a software tool developed by the EUROCarbDB initiative to assist the manual interpretation of MS data. The main task of GlycoWorkbench is to evaluate a set of structures proposed by the user by matching the corresponding theoretical list of fragment masses against the list of peaks derived from the spectrum. The tool provides an easy to use graphical interface, a comprehensive and increasing set of structural constituents, an exhaustive collection of fragmentation types, and a broad list of annotation options. The aim of GlycoWorkbench is to offer complete support for the routine interpretation of MS data. The software is available for download from: http://www.eurocarbdb.org/applications/ms-tools.
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            On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database.

            The SWISS-PROT protein sequence data bank contains at present nearly 75,000 entries, almost two thirds of which include the potential N-glycosylation consensus sequence, or sequon, NXS/T (where X can be any amino acid but proline) and thus may be glycoproteins. The number of proteins filed as glycoproteins is however considerably smaller, 7942, of which 749 have been characterized with respect to the total number of their carbohydrate units and sites of attachment of the latter to the protein, as well as the nature of the carbohydrate-peptide linking group. Of these well characterized glycoproteins, about 90% carry either N-linked carbohydrate units alone or both N- and O-linked ones, attached at 1297 N-glycosylation sites (1.9 per glycoprotein molecule) and the rest are O-glycosylated only. Since the total number of sequons in the well characterized glycoproteins is 1968, their rate of occupancy is 2/3. Assuming that the same number of N-linked units and rate of sequon occupancy occur in all sequon containing proteins and that the proportion of solely O-glycosylated proteins (ca. 10%) will also be the same as among the well characterized ones, we conclude that the majority of sequon containing proteins will be found to be glycosylated and that more than half of all proteins are glycoproteins.
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              Molecular lipidomics of exosomes released by PC-3 prostate cancer cells.

              The molecular lipid composition of exosomes is largely unknown. In this study, sophisticated shotgun and targeted molecular lipidomic assays were performed for in-depth analysis of the lipidomes of the metastatic prostate cancer cell line, PC-3, and their released exosomes. This study, based in the quantification of approximately 280 molecular lipid species, provides the most extensive lipid analysis of cells and exosomes to date. Interestingly, major differences were found in the lipid composition of exosomes compared to parent cells. Exosomes show a remarkable enrichment of distinct lipids, demonstrating an extraordinary discrimination of lipids sorted into these microvesicles. In particular, exosomes are highly enriched in glycosphingolipids, sphingomyelin, cholesterol, and phosphatidylserine (mol% of total lipids). Furthermore, lipid species, even of classes not enriched in exosomes, were selectively included in exosomes. Finally, it was found that there is an 8.4-fold enrichment of lipids per mg of protein in exosomes. The detailed lipid composition provided in this study may be useful to understand the mechanism of exosome formation, release and function. Several of the lipids enriched in exosomes could potentially be used as cancer biomarkers.
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                Author and article information

                Journal
                J Extracell Vesicles
                J Extracell Vesicles
                ZJEV
                zjev20
                Journal of Extracellular Vesicles
                Taylor & Francis
                2001-3078
                2018
                04 March 2018
                : 7
                : 1
                : 1442985
                Affiliations
                [ a ] Exosomes Laboratory. CIC bioGUNE, CIBER , Bizkaia, Spain
                [ b ] Glycotechnology Laboratory, CIC BiomaGUNE , San Sebastian, Spain
                [ c ] Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences, and Bijvoet Center for Biomolecular Research, Utrecht University , Utrecht, The Netherlands
                [ d ] CIBER-BBN , San Sebastian, Spain
                [ e ] IKERBASQUE Basque Foundation for science , Bilbao, Spain
                Author notes
                CONTACT Charles Williams cwilliams@ 123456cicbiogune.es ; Juan M. Falcon-Perez jfalcon@ 123456cicbiogune.es Exosomes Laboratory. CIC bioGUNE, CIBER , Bizkaia, 48160, Spain
                Niels-Christian Reichardt nreichardt@ 123456cicbiomagune.es Glycotechnology Laboratory, CIC BiomaGUNE , Paseo Miramon 182, San Sebastian 20009, Spain
                Author information
                http://orcid.org/0000-0001-7781-8693
                http://orcid.org/0000-0003-2321-2113
                http://orcid.org/0000-0003-3111-5954
                http://orcid.org/0000-0002-9092-7023
                Article
                1442985
                10.1080/20013078.2018.1442985
                5844028
                29535851
                6a591b9f-be56-49c6-b52c-4523a8dd9248
                © 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of The International Society for Extracellular Vesicles.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License ( http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 21 September 2017
                : 14 February 2018
                Page count
                Figures: 2, Tables: 1, References: 112, Pages: 15
                Funding
                Funded by: Ramón Areces Foundation 10.13039/100008054
                Funded by: JMF 10.13039/100001245
                Funded by: CIC bioGUNE
                Funded by: CIC biomaGUNE
                This work has been funded by the Ramón Areces Foundation to JMF and is co-supported by CIC bioGUNE and CIC biomaGUNE.
                Categories
                Review Article
                Review Article

                extracellular vesicles,exosomes,microvesicles,glycans,glycomics,glycosylation,lectins,glycoengineering

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