1. O-glycosylation of SARS-CoV-2 spike protein by host O-glycosyltransferase strengthens its trimeric structure
- Author
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Xu Zhijue, Zhang Han, Tian Jiaqi, Ku Xin, Wei Rumeng, Hou Jingli, Zhang Can, Yang Fang, Zou Xia, Li Yang, Kaji Hiroyuki, Tao Sheng-Ce, Kuno Atsushi, Yan Wei, Da Lin-Tai, and Zhang Yan
- Subjects
O-glycosylation ,SARS-CoV-2 ,spike protein ,glycosyltransferase ,ppGalNAc-T ,Biochemistry ,QD415-436 ,Genetics ,QH426-470 - Abstract
Protein O-glycosylation, also known as mucin-type O-glycosylation, is one of the most abundant glycosylation in mammalian cells. It is initially catalyzed by a family of polypeptide GalNAc transferases (ppGalNAc-Ts). The trimeric spike protein (S) of SARS-CoV-2 is highly glycosylated and facilitates the virus’s entry into host cells and membrane fusion of the virus. However, the functions and relationship between host ppGalNAc-Ts and O-glycosylation on the S protein remain unclear. Herein, we identify 15 O-glycosites and 10 distinct O-glycan structures on the S protein using an HCD-product-dependent triggered ETD mass spectrometric analysis. We observe that the isoenzyme T6 of ppGalNAc-Ts (ppGalNAc-T6) exhibits high O-glycosylation activity for the S protein, as demonstrated by an on-chip catalytic assay. Overexpression of ppGalNAc-T6 in HEK293 cells significantly enhances the O-glycosylation level of the S protein, not only by adding new O-glycosites but also by increasing O-glycan heterogeneity. Molecular dynamics simulations reveal that O-glycosylation on the protomer-interface regions, modified by ppGalNAc-T6, potentially stabilizes the trimeric S protein structure by establishing hydrogen bonds and non-polar interactions between adjacent protomers. Furthermore, mutation frequency analysis indicates that most O-glycosites of the S protein are conserved during the evolution of SARS-CoV-2 variants. Taken together, our finding demonstrate that host O-glycosyltransferases dynamically regulate the O-glycosylation of the S protein, which may influence the trimeric structural stability of the protein. This work provides structural insights into the functional role of specific host O-glycosyltransferases in regulating the O-glycosylation of viral envelope proteins.
- Published
- 2024
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