O‑Linked Sialoglycans Modulate the Proteolysis of SARS-CoV‑2 Spike and Likely Contribute to the Mutational Trajectory in Variants of Concern

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.