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

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

JOURNAL=Frontiers in Chemistry

VOLUME=9

YEAR=2021

URL=https://www.frontiersin.org/journals/chemistry/articles/10.3389/fchem.2021.689521

DOI=10.3389/fchem.2021.689521

ISSN=2296-2646

ABSTRACT=<p>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 <italic>N</italic>-glycosites and site-specific <italic>N</italic>-glycans in the S protein protomer. Herein, we report the <italic>O-</italic>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-<italic>N-</italic>glycosylation using PNGase F, we determined the GalNAc-type <italic>O-</italic>glycosylation pattern of S proteins, including <italic>O</italic>-glycosites and the six most common <italic>O</italic>-glycans occupying them, <italic>via</italic> Byonic identification and manual validation. Finally, 255 intact <italic>O</italic>-glycopeptides composed of 50 peptides sequences and 43 <italic>O</italic>-glycosites were discovered by higher energy collision-induced dissociation (HCD), and three <italic>O</italic>-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 <italic>O</italic>-glycans such as HexNAc(1) and HexNAc(1)Hex (1). In contrast, in the human cell-expressed S protein S1 subunit, 407 intact <italic>O</italic>-glycopeptides composed of 34 peptides sequences and 30 <italic>O</italic>-glycosites were discovered by HCD, and 11 <italic>O</italic>-glycosites were unambiguously assigned by EThcD. However, the measurement of O-glycosylation occupancy hasn’t been made. Most glycosites were modified by sialylated <italic>O</italic>-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 <italic>O</italic>-glycoprotein; the <italic>O</italic>-glycosites and <italic>O</italic>-glycan compositions vary with the host cell type. These comprehensive <italic>O</italic>-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.</p>