O-Linked Sialoglycans Modulate the Proteolysis of SARS-CoV-2 Spike and Likely Contribute to the Mutational Trajectory in Variants of Concern
O-Linked Sialoglycans Modulate the Proteolysis of SARS-CoV-2 Spike and Likely Contribute to the Mutational Trajectory in Variants of Concern.
Edgar Gonzalez-Rodriguez, Mia Zol-Hanlon, Ganka Bineva-Todd, Andrea Marchesi, Mark Skehel, Keira E. Mahoney, Chloë Roustan, Annabel Borg, Lucia Di Vagno, Svend Kjær, Antoni G. Wrobel, Donald J. Benton, Philipp Nawrath, Sabine L. Flitsch, Dhira Joshi, Andrés Manuel González-Ramírez, Katalin A. Wilkinson, Robert J. Wilkinson, Emma C. Wall, Ramón Hurtado-Guerrero, Stacy A. Malaker, and Benjamin Schumann*. ACS Cent. Sci. 2023, XXXX, XXX, XXX-XXX
Publication Date:February 16, 2023
https://doi.org/10.1021/acscentsci.2c01349
The emergence of a polybasic cleavage motif for the protease furin in SARS-CoV-2 spike has been established as a major factor for human viral transmission. The region N-terminal to that motif is extensively mutated in variants of concern (VOCs). Besides furin, spikes from these variants appear to rely on other proteases for maturation, including TMPRSS2. Glycans near the cleavage site have raised questions about proteolytic processing and the consequences of variant-borne mutations. Here, we identify that sialic acid-containing O-linked glycans on Thr678 of SARS-CoV-2 spike influence furin and TMPRSS2 cleavage and posit O-linked glycosylation as a likely driving force for the emergence of VOC mutations. We provide direct evidence that the glycosyltransferase GalNAc-T1 primes glycosylation at Thr678 in the living cell, an event that is suppressed by mutations in the VOCs Alpha, Delta, and Omicron. We found that the sole incorporation of N-acetylgalactosamine did not impact furin activity in synthetic O-glycopeptides, but the presence of sialic acid reduced the furin rate by up to 65%. Similarly, O-glycosylation with a sialylated trisaccharide had a negative impact on TMPRSS2 cleavage. With a chemistry-centered approach, we substantiate O-glycosylation as a major determinant of spike maturation and propose disruption of O-glycosylation as a substantial driving force for VOC evolution.
The emergence of a polybasic cleavage motif for the protease furin in SARS-CoV-2 spike has been established as a major factor for human viral transmission. The region N-terminal to that motif is extensively mutated in variants of concern (VOCs). Besides furin, spikes from these variants appear to rely on other proteases for maturation, including TMPRSS2. Glycans near the cleavage site have raised questions about proteolytic processing and the consequences of variant-borne mutations. Here, we identify that sialic acid-containing O-linked glycans on Thr678 of SARS-CoV-2 spike influence furin and TMPRSS2 cleavage and posit O-linked glycosylation as a likely driving force for the emergence of VOC mutations. We provide direct evidence that the glycosyltransferase GalNAc-T1 primes glycosylation at Thr678 in the living cell, an event that is suppressed by mutations in the VOCs Alpha, Delta, and Omicron. We found that the sole incorporation of N-acetylgalactosamine did not impact furin activity in synthetic O-glycopeptides, but the presence of sialic acid reduced the furin rate by up to 65%. Similarly, O-glycosylation with a sialylated trisaccharide had a negative impact on TMPRSS2 cleavage. With a chemistry-centered approach, we substantiate O-glycosylation as a major determinant of spike maturation and propose disruption of O-glycosylation as a substantial driving force for VOC evolution.