Systematic identification of the protein substrates of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase-T1/T2/T3 using a human proteome microarray

Proteomics. 2017 Jun;17(11). doi: 10.1002/pmic.201600485.


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.

MeSH terms

  • Azides / analysis*
  • Azides / metabolism
  • Enzyme Assays / methods*
  • HEK293 Cells
  • Humans
  • N-Acetylgalactosaminyltransferases / analysis*
  • N-Acetylgalactosaminyltransferases / metabolism
  • Protein Array Analysis / methods*
  • Protein Isoforms
  • Proteome / analysis*
  • Substrate Specificity
  • Uridine Diphosphate N-Acetylgalactosamine / analogs & derivatives*
  • Uridine Diphosphate N-Acetylgalactosamine / analysis
  • Uridine Diphosphate N-Acetylgalactosamine / metabolism


  • Azides
  • Protein Isoforms
  • Proteome
  • UDP-N-azidoacetylgalactosamine
  • Uridine Diphosphate N-Acetylgalactosamine
  • N-Acetylgalactosaminyltransferases
  • polypeptide N-acetylgalactosaminyltransferase