tetano
Editor, Senior Moderator
Front Chem
. 2021 Sep 6;9:689521.
doi: 10.3389/fchem.2021.689521. eCollection 2021.
O-Glycosylation Landscapes of SARS-CoV-2 Spike Proteins
Yong Zhang[SUP] 1 [/SUP], Wanjun Zhao[SUP] 2 [/SUP], Yonghong Mao[SUP] 3 [/SUP], Yaohui Chen[SUP] 3 [/SUP], Shanshan Zheng[SUP] 1 [/SUP], Wei Cao[SUP] 1 [/SUP], Jingqiang Zhu[SUP] 2 [/SUP], Liqiang Hu[SUP] 1 [/SUP], Meng Gong[SUP] 1 [/SUP], Jingqiu Cheng[SUP] 1 [/SUP], Hao Yang[SUP] 1 [/SUP]
Affiliations
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.
. 2021 Sep 6;9:689521.
doi: 10.3389/fchem.2021.689521. eCollection 2021.
O-Glycosylation Landscapes of SARS-CoV-2 Spike Proteins
Yong Zhang[SUP] 1 [/SUP], Wanjun Zhao[SUP] 2 [/SUP], Yonghong Mao[SUP] 3 [/SUP], Yaohui Chen[SUP] 3 [/SUP], Shanshan Zheng[SUP] 1 [/SUP], Wei Cao[SUP] 1 [/SUP], Jingqiang Zhu[SUP] 2 [/SUP], Liqiang Hu[SUP] 1 [/SUP], Meng Gong[SUP] 1 [/SUP], Jingqiu Cheng[SUP] 1 [/SUP], Hao Yang[SUP] 1 [/SUP]
Affiliations
- PMID: 34552909
- PMCID: PMC8450404
- DOI: 10.3389/fchem.2021.689521
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.