O-GalNAc glycosylation is the initial step of the mucin-type O-glycosylation. In humans, it is catalyzed by a family of 20 homologous UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts). So far, there is very limited information on their protein substrate specificities. In this study, we developed an on-chip ppGalNAc-Ts assay that could rapidly and systematically identify the protein substrates of each ppGalNAc-T. In detail, we utilized a human proteome microarray as the protein substrates and UDP-GalNAz as the nucleotide sugar donor for click chemistry detection. From a total of 16 368 human proteins, we identified 570 potential substrates of ppGalNAc-T1, T2, and T3. Among them, 128 substrates were overlapped, while the rest were isoform specific. Further cluster analysis of these substrates showed that the substrates of ppGalNAc-T1 had a closer phylogenetic relationship with that of ppGalNAc-T3 compared with ppGalNAc-T2, which was consistent with the topology of the phylogenetic tree of these ppGalNAc-Ts. Taken together, our microarray-based enzymatic assay comprehensively reveals the substrate profile of the ppGalNAc-T1, T2, and T3, which not only provides a plausible explanation for their partial functional redundancy as reported, but clearly implies some specialized roles of each enzyme in different biological processes.
Keywords: Click chemistry; Glycoproteomics; Glycosyltransferase; Human proteome microarray.
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