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Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells
Published on: April 28, 2010
Systematic identification of the protein substrates of UDP-GalNAc:polypeptide
Zhijue Xu1, Xing Li1, Shumin Zhou1
1Key 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.
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.
Insights
Researchers developed a novel on-chip assay to identify protein substrates for UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts). This study reveals 570 potential substrates for ppGalNAc-T1, T2, and T3, clarifying their roles in O-glycosylation.
Area of Science:
- Biochemistry
- Glycobiology
- Proteomics
Background:
- O-GalNAc glycosylation initiates mucin-type O-glycosylation and is catalyzed by UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts).
- Limited information exists regarding the specific protein substrates recognized by individual ppGalNAc-T enzymes.
Purpose of the Study:
- To develop a high-throughput method for systematically identifying protein substrates of ppGalNAc-Ts.
- To characterize the substrate specificities of ppGalNAc-T1, T2, and T3.
Main Methods:
- Development of an on-chip enzymatic assay using a human proteome microarray.
- Utilized UDP-GalNAz as the nucleotide sugar donor for click chemistry detection.
- Screened 16,368 human proteins to identify ppGalNAc-T substrates.
Main Results:
- Identified 570 potential protein substrates for ppGalNAc-T1, T2, and T3.
- Discovered 128 overlapping substrates and numerous isoform-specific substrates.
- Cluster analysis revealed phylogenetic relationships between enzyme substrates mirroring enzyme phylogeny.
Conclusions:
- The developed assay comprehensively maps the substrate profiles of ppGalNAc-T1, T2, and T3.
- Findings explain the partial functional redundancy observed among these enzymes.
- Results suggest specialized biological roles for each ppGalNAc-T isoform.
