Antibodies Directed Against GalNAc- and GlcNAc-O-Tyrosine Posttranslational Modifications - a New Tool for Glycoproteomic Detection

Inn 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 alpha-GalNAc-O-Tyr modifications, and studies propose that beta-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 alpha-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 alpha-GlcNAc-O-Tyr modified RhoA GTPase.

Automated summary

In the last decade, protein mucin-type O-glycosylation and O-GlcNAcylation have been found to modify Tyr residues in addition to Thr and Ser residues. These modifications, known as GlcNAc-O-Tyr and GalNAc-O-Tyr, have been identified on various proteins and have been implicated in bacterial virulence. However, their biological functions and biosynthesis are not well understood due to a lack of detection tools. In this study, antibodies were raised to selectively detect proteins carrying these modifications, and synthetic O-glycopeptides were prepared for microarray analysis. The antibodies showed specific detection of GlcNAc- and GalNAc-O-Tyr modified proteins, providing a valuable tool for further research.