Glycosylation is a vital post-translational modification (PTM) associated in a range of biological processes including protein folding and localization, signaling pathways, and cell-cell interactions. Glycans are found on over 50 percent of all proteins. Altered glycosylation profiles have been associated with disease progression and are a trademark of cancer. In 2011, a new type of O-linked glycosylation incorporating an N-acetylhexosamine (HexNAc) residue on the side chain oxygen of tyrosine was discovered by MS on Amyloid beta 1-X peptides and in an engineered Simple Cell line using GalNAc-binding lectins. Since its discovery, studies have identified the GalNAc-Tyr modification on over 30 human proteins and suggest that GalNAc-Tyr may be a prevalent PTM with altered expression in some disease states.<br/>While these studies provide foundational insight in to GalNAc-Tyr expression, little is known about GalNAc-Tyr biological synthesis, function, or expression in normal and disease states. We hypothesize that GalNAc-Tyr is a biologically significant but overlooked PTM due to the lack of GalNAc-Tyr selective tools and the small sampling of human tissues that have been examined. To aid in our study of GalNAc-Tyr, our lab developed G10C, a GalNAc-Tyr selective monoclonal antibody that is completely selective for GalNAc-Tyr versus other glycans with terminal GalNAc residues such as TN, Forssman, core 5 and Blood Group A.  We examined the expression levels of GalNAc-Tyr in 14 human cancer lines with G10C. Our data shows that GalNAc-Tyr is widely expressed intracellularly; however, its extracellular expression is limited. Of the cell lines tested, only LOX IMVI displayed high surface expression of GalNAc-Tyr. The high prevalence of GalNAc-Tyr across multiple cell lines suggests it may have a fundamental role in cellular biology. To determine the biological significance of GalNAc-Tyr, we first need to determine the location of this PTM. We compared immunoprecipitations of cell lysate with G10C and a control by MS proteomic analysis. Our initial investigations focused on LOX IMVI cells due to the high surface and intracellular expression levels of GalNAc-Tyr.  Our efforts have identified multiple proteins that likely contain GalNAc-Tyr. Deciphering specific glycosylation sites is challenging due to the abundance of GalNAc-Ser/Thr. By studying the fragmentation of the oxonium ion, we have identified potential GalNAc-Tyr glycosylation sites on two separate peptides.

Identifying GalNAc-Tyr glycoproteins and glycosylation sites in LOX IMVI cells

ACS Fall 2021

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