N- and O‑Glycosylation of the SARS-CoV‑2 Spike Protein

Covid-19
pandemic outbreak is the reason of the current world health
crisis. The development of effective antiviral compounds and vaccines
requires detailed descriptive studies of SARS-CoV-2 proteins. The
SARS-CoV-2 spike (S) protein mediates virion binding to the human
cells through its interaction with the ACE2 cell surface receptor
and is one of the prime immunization targets. A functional virion
is composed of three S1 and three S2 subunits created by furin cleavage
of the spike protein at R682, a polybasic cleavage site that differs
from the SARS-CoV spike protein of 2002. By analysis of the protein
produced in HEK293 cells, we observe that the spike is O-glycosylated
on a threonine (T678) near the furin cleavage site occupied by core-1
and core-2 structures. In addition, we have identified eight additional
O-glycopeptides on the spike glycoprotein and confirmed that the spike
protein is heavily N-glycosylated. Our recently developed liquid chromatography–mass
spectrometry methodology allowed us to identify LacdiNAc structural
motifs on all occupied N-glycopeptides and polyLacNAc structures on
six glycopeptides of the spike protein. In conclusion, our study substantially
expands the current knowledge of the spike protein’s glycosylation
and enables the investigation of the influence of O-glycosylation
on its proteolytic activation.