O-Glycosylation Landscapes of SARS-CoV-2 Spike Proteins

Yong Zhang; Wanjun Zhao; Yonghong Mao; Yaohui Chen; Shanshan Zheng; Wei Cao; Jingqiang Zhu; Liqiang Hu; Meng Gong; Jingqiu Cheng; Hao Yang

doi:10.3389/fchem.2021.689521

O-Glycosylation Landscapes of SARS-CoV-2 Spike Proteins

Front Chem. 2021 Sep 6:9:689521. doi: 10.3389/fchem.2021.689521. eCollection 2021.

Authors

Affiliations

¹ Key Laboratory of Transplant Engineering and Immunology, MOH, Frontiers Science Center for Disease-related Molecular Network, Institutes for Systems Genetics, West China Hospital, Sichuan University, Chengdu, China.
² Department of Thyroid Surgery, West China Hospital, Sichuan University, Chengdu, China.
³ Institute of Thoracic Oncology, West China Hospital, Sichuan University, Chengdu, China.

Abstract

The densely glycosylated spike (S) proteins that are highly exposed on the surface of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) facilitate viral attachment, entry, and membrane fusion. We have previously reported all the 22 N-glycosites and site-specific N-glycans in the S protein protomer. Herein, we report the O-glycosylation landscapes of SARS-CoV-2 S proteins, which were characterized through high-resolution mass spectrometry. Following digestion with trypsin and trypsin/Glu-C, and de-N-glycosylation using PNGase F, we determined the GalNAc-type O-glycosylation pattern of S proteins, including O-glycosites and the six most common O-glycans occupying them, via Byonic identification and manual validation. Finally, 255 intact O-glycopeptides composed of 50 peptides sequences and 43 O-glycosites were discovered by higher energy collision-induced dissociation (HCD), and three O-glycosites were confidently identified by electron transfer/higher energy collision-induced dissociation (EThcD) in the insect cell-expressed S protein. Most glycosites were modified by non-sialylated O-glycans such as HexNAc(1) and HexNAc(1)Hex (1). In contrast, in the human cell-expressed S protein S1 subunit, 407 intact O-glycopeptides composed of 34 peptides sequences and 30 O-glycosites were discovered by HCD, and 11 O-glycosites were unambiguously assigned by EThcD. However, the measurement of O-glycosylation occupancy hasn't been made. Most glycosites were modified by sialylated O-glycans such as HexNAc(1)Hex (1)NeuAc (1) and HexNAc(1)Hex (1)NeuAc (2). Our results reveal that the SARS-CoV-2 S protein is an O-glycoprotein; the O-glycosites and O-glycan compositions vary with the host cell type. These comprehensive O-glycosylation landscapes of the S protein are expected to provide novel insights into the viral binding mechanism and present a strategy for the development of vaccines and targeted drugs.

Keywords: EThcD fragmentation; O-glycosylation; SARS-CoV-2; mass spectrometry; spike protein.