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Site-specific N-glycosylation Characterization of Recombinant SARS-CoV-2 Spike Proteins using High-Resolution Mass Spectrometry

View ORCID ProfileYong Zhang, Wanjun Zhao, View ORCID ProfileYonghong Mao, Shisheng Wang, Yi Zhong, Tao Su, Meng Gong, Xiaofeng Lu, Jingqiu Cheng, Hao Yang
doi: https://doi.org/10.1101/2020.03.28.013276
Yong Zhang
aKey Laboratory of Transplant Engineering and Immunology, MOH, West China-Washington Mitochondria and Metabolism Research Center, West China Hospital, Sichuan University, Chengdu 610041, China
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Wanjun Zhao
bDepartment of Thyroid Surgery, West China Hospital, Sichuan University, Chengdu 610041, China
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Yonghong Mao
cThoracic Surgery Research Laboratory, West China Hospital, Sichuan University, Chengdu 610041, China
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Shisheng Wang
aKey Laboratory of Transplant Engineering and Immunology, MOH, West China-Washington Mitochondria and Metabolism Research Center, West China Hospital, Sichuan University, Chengdu 610041, China
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Yi Zhong
aKey Laboratory of Transplant Engineering and Immunology, MOH, West China-Washington Mitochondria and Metabolism Research Center, West China Hospital, Sichuan University, Chengdu 610041, China
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Tao Su
aKey Laboratory of Transplant Engineering and Immunology, MOH, West China-Washington Mitochondria and Metabolism Research Center, West China Hospital, Sichuan University, Chengdu 610041, China
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Meng Gong
aKey Laboratory of Transplant Engineering and Immunology, MOH, West China-Washington Mitochondria and Metabolism Research Center, West China Hospital, Sichuan University, Chengdu 610041, China
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Xiaofeng Lu
aKey Laboratory of Transplant Engineering and Immunology, MOH, West China-Washington Mitochondria and Metabolism Research Center, West China Hospital, Sichuan University, Chengdu 610041, China
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Jingqiu Cheng
aKey Laboratory of Transplant Engineering and Immunology, MOH, West China-Washington Mitochondria and Metabolism Research Center, West China Hospital, Sichuan University, Chengdu 610041, China
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Hao Yang
aKey Laboratory of Transplant Engineering and Immunology, MOH, West China-Washington Mitochondria and Metabolism Research Center, West China Hospital, Sichuan University, Chengdu 610041, China
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  • For correspondence: yanghao@scu.edu.cn
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ABSTRACT

The global pandemic of severe acute pneumonia syndrome (COVID-19) caused by SARS-CoV-2 urgently calls for prevention and intervention strategies. The densely glycosylated spike (S) protein highly exposed on the viral surface is a determinant for virus binding and invasion into host cells as well as elicitation of a protective host immune response. Herein, we characterized the site-specific N-glycosylation of SARS-CoV-2 S protein using stepped collision energy (SCE) mass spectrometry (MS). Following digestion with two complementary proteases to cover all potential N-glycosylation sequons and integrated N-glycoproteomics analysis, we revealed the N-glycosylation profile of SARS-CoV-2 S proteins at the levels of intact N-glycopeptides and glycosites, along with the glycan composition and site-specific number of glycans. All 22 potential canonical N-glycosites were identified in S protein protomer. Of those, 18 N-glycosites were conserved between SARS-CoV and SARS-CoV-2 S proteins. Nearly all glycosites were preserved among the 753 SARS-CoV-2 genome sequences available in the public influenza database Global Initiative on Sharing All Influenza Data. By comparison, insect cell-expressed SARS-CoV-2 S protein contained 38 N-glycans, which were primarily assigned to the high-mannose type N-glycans, whereas the human cell-produced protein possessed up to 140 N-glycans largely belonging to the complex type. In particular, two N-glycosites located in the structurally exposed receptor-binding domain of S protein exhibited a relatively conserved N-glycan composition in human cells. This N-glycosylation profiling and determination of differences between distinct expression systems could shed light on the infection mechanism and promote development of vaccines and targeted drugs.

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Posted March 29, 2020.
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Site-specific N-glycosylation Characterization of Recombinant SARS-CoV-2 Spike Proteins using High-Resolution Mass Spectrometry
Yong Zhang, Wanjun Zhao, Yonghong Mao, Shisheng Wang, Yi Zhong, Tao Su, Meng Gong, Xiaofeng Lu, Jingqiu Cheng, Hao Yang
bioRxiv 2020.03.28.013276; doi: https://doi.org/10.1101/2020.03.28.013276
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Site-specific N-glycosylation Characterization of Recombinant SARS-CoV-2 Spike Proteins using High-Resolution Mass Spectrometry
Yong Zhang, Wanjun Zhao, Yonghong Mao, Shisheng Wang, Yi Zhong, Tao Su, Meng Gong, Xiaofeng Lu, Jingqiu Cheng, Hao Yang
bioRxiv 2020.03.28.013276; doi: https://doi.org/10.1101/2020.03.28.013276

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