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- Title
Antibodies Directed Against GalNAc- and GlcNAc-OTyrosine Posttranslational Modifications -- a New Tool for Glycoproteomic Detection.
- Authors
Behren, Sandra; Schorlemer, Manuel; Schmidt, Gudula; Aktories, Klaus; Westerlind, Ulrika
- Abstract
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-OTyr 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 Thrmodifications. 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.
- Subjects
IMMUNOGLOBULINS; POST-translational modification; WESTERN immunoblotting; BIOSYNTHESIS; BACTERIAL toxins
- Publication
Chemistry - A European Journal, 2023, Vol 29, Issue 29, p1
- ISSN
0947-6539
- Publication type
Article
- DOI
10.1002/chem.202300392