title: Systematic identification of the protein substrates of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase-T1/T2/T3 using a human proteome microarray.
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 16 368 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.
authors.affiliation: 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.
authors.forename: Zhijue
authors.initials: Z
authors.lastname: Xu
authors.affiliation: 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.
authors.forename: Xing
authors.initials: X
authors.lastname: Li
authors.affiliation: 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.
authors.forename: Shumin
authors.initials: S
authors.lastname: Zhou
authors.affiliation: 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.
authors.forename: Wenxian
authors.initials: W
authors.lastname: Xie
authors.affiliation: 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.|State Key Laboratory of Microbial metabolism, School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai, P. R. China.
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.affiliation: 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.|State Key Laboratory of Oncogenes and Related Genes, Shanghai, P. R. China.|School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, P. R. China.
authors.forename: Li
authors.initials: L
authors.lastname: Cheng
authors.affiliation: 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.
authors.forename: Sheng
authors.initials: S
authors.lastname: Wang
authors.affiliation: 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.
authors.forename: Shujuan
authors.initials: S
authors.lastname: Guo
authors.affiliation: 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.
authors.forename: Zhaowei
authors.initials: Z
authors.lastname: Xu
authors.affiliation: Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, P. R. China.
authors.forename: Xin
authors.initials: X
authors.lastname: Cao
authors.affiliation: State Key Laboratory of Microbial metabolism, School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai, P. R. China.
authors.forename: Menghui
authors.initials: M
authors.lastname: Zhang
authors.affiliation: Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, P. R. China.
authors.forename: Biao
authors.initials: B
authors.lastname: Yu
authors.affiliation: Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.|SCSB (China) - AIST (Japan) Joint Medical Glycomics Laboratory, Shanghai Jiao Tong University, Shanghai, P. R. China.
authors.forename: Hisashi
authors.initials: H
authors.lastname: Narimatsu
authors.affiliation: 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.|State Key Laboratory of Oncogenes and Related Genes, Shanghai, P. R. China.|School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, P. R. China.
authors.forename: Sheng-Ce
authors.initials: SC
authors.lastname: Tao
authors.affiliation: 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.|State Key Laboratory of Microbial metabolism, School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai, P. R. China.|SCSB (China) - AIST (Japan) Joint Medical Glycomics Laboratory, Shanghai Jiao Tong University, Shanghai, P. R. China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D001386:Azides / Q000032:analysis* / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D017350:N-Acetylgalactosaminyltransferases / Q000032:analysis* / Q000378:metabolism; D040081:Protein Array Analysis / Q000379:methods*; D020033:Protein Isoforms; D020543:Proteome / Q000032:analysis*; D013379:Substrate Specificity; D014536:Uridine Diphosphate N-Acetylgalactosamine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D000097763:Polypeptide N-acetylgalactosaminyltransferase
chemical_list: D001386:Azides; D017350:N-Acetylgalactosaminyltransferases; D020033:Protein Isoforms; D020543:Proteome; D014536:Uridine Diphosphate N-Acetylgalactosamine; D000097763:Polypeptide N-acetylgalactosaminyltransferase; C000622611:UDP-N-azidoacetylgalactosamine
keywords: Click chemistry; Glycoproteomics; Glycosyltransferase; Human proteome microarray