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Antibodies directed against GalNAc- and GlcNAc-O-Tyrosine posttranslational modifications – a new tool for glycoproteomic detection
Umeå University, Faculty of Science and Technology, Department of Chemistry. Leibniz-Institut für Analytische Wissenschaften, Dortmund, Germany. (Westerlind)ORCID iD: 0000-0002-9835-7045
Umeå University, Faculty of Science and Technology, Department of Chemistry. Leibniz-Institut für Analytische Wissenschaften, Dortmund, Germany. (Westerlind)ORCID iD: 0000-0003-0672-6921
Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Universität Freiburg, Freiburg, Germany.ORCID iD: 0000-0002-2876-0189
Umeå University, Faculty of Science and Technology, Department of Chemistry. Leibniz-Institut für Analytische Wissenschaften, Dortmund, Germany. (Westerlind)ORCID iD: 0000-0002-4841-6238
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2023 (English)In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 29, no 29, article id e202300392Article in journal (Refereed) Published
Abstract [en]

In the last decade, it was discovered that protein mucin-type O-glycosylation and O-GlcNAcylation modify Tyr residues besides the well explored Thr and Ser amino acids. Several glycoproteomic studies have identified α-GalNAc-O-Tyr modifications, and studies propose that β-GlcNAc-O-Tyr also exists as a new group of posttranslational modifications (PTMs). Specific bacterial toxins have further been identified to modify host GTPases with α-GlcNAc-O-Tyr to promote bacterial virulence. Despite being identified on numerous proteins, the biological roles, biosynthesis and expression of GalNAc- and GlcNAc-O-Tyr modifications are poorly understood. A major obstacle is the lack of tools to specifically detect and identify proteins containing these modifications. With this in mind, we prepared vaccine constructs and raised antibodies to enable selective detection of proteins carrying these new PTMs. The obtained polyclonal antibody sera were evaluated using ELISA and glycopeptide microarrays and were found to be highly selective for GlcNAc- and GalNAc-O-Tyr glycopeptides over the corresponding Ser- and Thr-modifications. For microarray analysis, synthetic GlcNAc- and GalNAc-O-Tyr Fmoc-amino acids were prepared and applied in Fmoc-SPPS to obtain an extensive O-glycopeptide library. After affinity purification, the antibodies were applied in western blot analysis and showed specific detection of α-GlcNAc-O-Tyr modified RhoA GTPase.

Place, publisher, year, edition, pages
Wiley-VCH Verlagsgesellschaft, 2023. Vol. 29, no 29, article id e202300392
Keywords [en]
antibodies, vaccine, glycoproteomics, glycopeptides, glycosylation, PTM
National Category
Organic Chemistry
Research subject
Biorganic Chemistry; biological chemistry
Identifiers
URN: urn:nbn:se:umu:diva-187279DOI: 10.1002/chem.202300392ISI: 000970554600001PubMedID: 37052513Scopus ID: 2-s2.0-85152586008OAI: oai:DiVA.org:umu-187279DiVA, id: diva2:1591692
Note

Originally included in thesis in manuscript form. 

Available from: 2021-09-07 Created: 2021-09-07 Last updated: 2023-06-19Bibliographically approved
In thesis
1. Development and Evaluation of Tools to Explore Posttranslational HexNAc-Tyrosine and Mucin-Type O-Glycosylation
Open this publication in new window or tab >>Development and Evaluation of Tools to Explore Posttranslational HexNAc-Tyrosine and Mucin-Type O-Glycosylation
2021 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Glycosylation is the most abundant form of post-translational modification (PTM). Recently, O-glycosylation attracted much attention in the glycoproteomic field due to its association with various diseases, such as pathogenic infections and cancer. However, glycoproteomic analysis of O-linked glycosylation is highly challenging due its structural diversity and complexity. New and efficient methods need to be developed to obtain a better understanding of the biological functions of O-glycans. In the presented thesis, glycopeptide microarrays were used as tools to explore the role of mucin type O-glycosylation in cancer, bacterial adhesion processes and galectin recognition on a molecular level, and to get insights into a new group of tyrosine O-glycosylation. A better understanding of these carbohydrate-protein interactions on a molecular level could facilitate the development of glycomimetic inhibitors to fight bacterial infections or block glycan binding proteins involved in cancer progression, or improve the design of novel carbohydrate-based cancer vaccines.

In the first part of this work, tools were developed to elucidate the role of a novel group of PTMs, where N-acetylhexosamine (HexNAc = α-GalNAc, α- or β-GlcNAc) was found to modify the hydroxyl group of tyrosine. Synthetic glycopeptides carrying this new modification, as well as glycopeptide microarray libraries were prepared to evaluate the abilities of plant lectins (carbohydrate-binding proteins) to detect HexNAc-O-Tyr modifications. These lectins are commonly used in glycoproteomic work flows to detect and enrich glycopeptides and -proteins. Additionally, HexNAc-O-Tyr-specific rabbit antibodies were raised and immunologically analyzed by enzyme-linked immunosorbent assays, western blot and microarray binding studies.

In the second part of the presented thesis, synthetic mucin glycopeptide microarray libraries were prepared and employed to explore carbohydrate-protein interactions of galectins, bacterial lectins and tumor specific antibodies. Mucin glycoproteins are part of the mucus barrier that protects the host against invading pathogens. However, bacteria and viruses have co-evolved with the human host and have developed strategies to promote virulence, for example by adhering to glycans on the host cell-surface. To combat bacterial infections, their virulence and pathogenicity must be understood on a molecular level. In this work, mucin glycopeptides were enzymatically modified with different fucose motifs and used to determine the fine binding specificities of fucose-recognizing lectins LecB from Pseudomonas aeruginosa and the Clostridium difficile toxin A. Furthermore, a synthesis strategy was developed to generate simplified mucin core glycopeptides that could be used as scaffolds to enzymatically generate LacdiNAc modified glycopeptides. They could be used in microarray binding studies to evaluate the glycan binding preferences of various proteins, including the Helicobacter pylori lectin LabA and human galectins, which play roles in cancer development and progression. Aberrant glycosylation of mucin glycoproteins has been associated with various types of cancer. Tumor specific carbohydrate antigens on mucins represent attractive antigenic targets for the development of effective anti-cancer vaccines. In this work, antibodies induced by tumor-associated MUC1 glycopeptide-bacteriophage Qβ vaccine conjugates were immunologically analyzed using MUC1 glycopeptide microarray libraries.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2021. p. 171
Keywords
Glycopeptides, mucins, mucin type-O-glycosylation, microarrays, tyrosine-O-HexNAcylation, lectins, MUC1 cancer vaccines, TcdA, LecB, galectins, LacdiNAc, antibodies
National Category
Organic Chemistry
Research subject
biological chemistry; Biorganic Chemistry; Organic Chemistry
Identifiers
urn:nbn:se:umu:diva-187282 (URN)978-91-7855-646-5 (ISBN)978-91-7855-647-2 (ISBN)
Public defence
2021-10-01, Glasburen, Kemiskt Biologiskt Centrum, Linnaeus väg 6, Umeå, 09:00 (English)
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Supervisors
Available from: 2021-09-10 Created: 2021-09-07 Last updated: 2021-09-09Bibliographically approved

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Behren, SandraSchorlemer, ManuelWesterlind, Ulrika

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