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

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

Xu Z1, Li X1, Zhou S1, Xie W1, Wang J1,2, Cheng L1,3,4, Wang S1, Guo S1, Xu Z1, Cao X5, Zhang M2, Yu B5, Narimatsu H6,7, Tao SC1,3,4, Zhang Y1,2,7.

Author information

1
Key Laboratory of Systems Biomedicine (Ministry of Education) and Collaborative Innovation Center of Systems Biomedicine, Shanghai Center for Systems Biomedicine (SCSB), Shanghai Jiao Tong University, Shanghai, P. R. China.
2
State Key Laboratory of Microbial metabolism, School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai, P. R. China.
3
State Key Laboratory of Oncogenes and Related Genes, Shanghai, P. R. China.
4
School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, P. R. China.
5
Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, P. R. China.
6
Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.
7
SCSB (China) - AIST (Japan) Joint Medical Glycomics Laboratory, Shanghai Jiao Tong University, Shanghai, P. R. China.

Abstract

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‚ÄČ368 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

PMID:
28394504
DOI:
10.1002/pmic.201600485
[Indexed for MEDLINE]

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