Altered O-glycosylation Level of SARS-CoV-2 Spike Protein by Host O-glycosyltransferase Strengthens Its Trimeric Structure

Xu, Z.; Ku, X.; Tian, J.; Zhang, H.; Hou, J.; Zhang, C.; Shi, J.; Li, Y.; Kaji, H.; Tao, S.-c.; Kuno, A.; Yan, W.; Da, L.-T.; Zhang, Y.

doi:10.1101/2021.04.06.438614

bioRxiv · 10.1101/2021.04.06.438614

Altered O-glycosylation Level of SARS-CoV-2 Spike Protein by Host O-glycosyltransferase Strengthens Its Trimeric Structure

Xu, Z.·Ku, X.·Tian, J.·Zhang, H.·Hou, J.·Zhang, C.·Shi, J.·Li, Y.·Kaji, H.·Tao, S.-c.·Kuno, A.·Yan, W.·Da, L.-T.·Zhang, Y.

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Abstract

The trimeric spike protein (S) mediates host-cell entry and membrane fusion of SARS-CoV-2. S protein is highly glycosylated, whereas its O-glycosylation is still poorly understood. Herein, we site-specifically examine the O-glycosylation of S protein through a mass spectrometric approach with HCD-triggered-ETD model. We identify 15 high-confidence O-glycosites and at least 10 distinct O-glycan structures on S protein. Peptide microarray assays prove that human ppGalNAc-T6 actively participates in O-glycosylation of S protein. Importantly, the upregulation of ppGalNAc-T6 expression can profoundly enhance the O-glycosylation level by generating new O-glycosites and increasing both O-glycan heterogeneity and intensities. Further molecular dynamics simulations reveal that the O-glycosylation on the protomer-interface regions, which are mainly modified by ppGalNAc-T6, can potentially stabilize the trimeric S protein structure. Our work provides deep molecular insights of how viral infection harnesses the host O-glycosyltransferases to dynamically regulate the O-glycosylation level of the viral envelope protein responsible for membrane fusion.

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Xu, Z., Ku, X., Tian, J., Zhang, H., Hou, J., Zhang, C., Shi, J., Li, Y., Kaji, H., Tao, S.-c., Kuno, A., Yan, W., Da, L.-T., Zhang, Y.. 2021-04-06. Altered O-glycosylation Level of SARS-CoV-2 Spike Protein by Host O-glycosyltransferase Strengthens Its Trimeric Structure. https://doi.org/10.1101/2021.04.06.438614

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