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O-linked sialoglycans modulate the proteolysis of SARS-CoV-2 spike and likely contribute to the mutational trajectory in variants of concern

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journal contribution
posted on 2023-03-22, 11:15 authored by 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, Benjamin Schumann
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.


Crick (Grant ID: CC1283, Grant title: Crick legacy study CC1283) Crick (Grant ID: CC2112, Grant title: Wilkinson CC2112) Crick (Grant ID: CC2127, Grant title: Schumann CC2127) Crick (Grant ID: CC1063, Grant title: STP Proteomics) Crick (Grant ID: CC1065, Grant title: STP Chemical Biology) Crick (Grant ID: CC1068, Grant title: STP Structural Biology) Rosetrees Trust (Grant ID: M926, Grant title: RT M926) Crick (Grant ID: CC2060, Grant title: Gamblin CC2060)