@article {Sanda2020.07.05.187344, author = {Miloslav Sanda and Lindsay Morrison and Radoslav Goldman}, title = {N and O glycosylation of the SARS-CoV-2 spike protein}, elocation-id = {2020.07.05.187344}, year = {2020}, doi = {10.1101/2020.07.05.187344}, publisher = {Cold Spring Harbor Laboratory}, abstract = {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 the 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 sites that differs from the SARS-CoV spike protein of 2002. We observe that the spike protein 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 we confirmed that the spike protein is heavily N-glycosylated. Our recently developed LC-MS/MS 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{\textquoteright}s glycosylation and enables the investigation of the influence of the O-glycosylation on its proteolytic activation.Competing Interest StatementThe authors have declared no competing interest.}, URL = {https://www.biorxiv.org/content/early/2020/07/06/2020.07.05.187344}, eprint = {https://www.biorxiv.org/content/early/2020/07/06/2020.07.05.187344.full.pdf}, journal = {bioRxiv} }