EXoO-Tn: Tag-n-Map the Tn Antigen in the Human Proteome

Tn antigen (Tn), a single N-acetylgalactosamine (GalNAc) monosaccharide attached to protein Ser and Thr residues, is found on most solid tumors yet rarely detected in adult tissues: featuring it one of the most distinctive signatures of cancers. Although it is prevalent in cancers, Tn-glycosylation sites are not entirely clear owing to the lack of suitable technology. Knowing the Tn-glycosylation sites will spur the development of the new vaccines, diagnostics, and therapeutics of cancers. Here, we report a novel technology named EXoO-Tn for large-scale mapping of Tn-glycosylation sites. EXoO-Tn utilizes glycosyltransferase C1GalT1 and isotopically-labeled UDP-Gal(13C6) to tag and convert Tn to Gal(13C6)-Tn. This exquisite Gal(13C6)-Tn structure is recognized by a human-gut-bacterial enzyme, called OpeRATOR, that specifically cleaves N-termini of the Gal(13C6)-Tn-occupied Ser and Thr residues to yield site-containing glycopeptides. The enzymes C1GalT1 and OpeRATOR could be used concurrently in one-pot. The effectiveness of EXoO-Tn was evaluated by analyzing Jurkat cells, where 947 Tn-glycosylation sites from 480 glycoproteins were mapped. Bioinformatic analysis of the identified site-specific Tn-glycoproteins revealed conserved motif, cellular localization, relative position in proteins, and mapped site-specific Tn-glycoproteome in different studies. Given the significance of Tn in cancers, EXoO-Tn is anticipated to have broad utilities in clinical study of cancers.