Fig. 1: The multibasic cleavage site of SARS-CoV-2 spike protein is glycosylated sequentially by GalNAc-T3 and T7 in vitro.
a The domain structure of SARS-CoV-2 spike protein and the sequence alignment of listed coronaviruses around SARS-CoV-2 furin site and TMPRSS2 site. b MALDI-TOF analysis of GalNAcylation reactions catalyzed by purified GalNAc-Ts on the synthetic multibasic peptide of SARS-CoV-2 spike protein (674-693). Reactions were performed and analyzed as described in the Methods. An increase of 203 Da corresponds to the addition of one GalNAc residue. c ETD-MS2 spectrum of the O-GalNAcylated peptide from the GalNAc-T3 reaction. The mass of c- and z- fragment ions (e.g. the delta mass between c4 and c5) unambiguously assigned the GalNAc modification to T678. d MALDI-TOF analysis of GalNAcylation reactions catalyzed by purified GalNAc-Ts on T678-O-GalNAcylated peptide. An increase of 203 Da, corresponding to the modification with an additional GalNAc residue, was only observed with GalNAc-T7. e ETD-MS2 spectrum of the doubly O-GalNAcylated peptide from the GalNAc-T7 reaction. The mass of c- and z- fragment ions (e.g. the delta mass between c11 and c13) indicated the second GalNAc residue is located at S686. The GalNAc residues are denoted as yellow squares according to Consortium for Functional Glycomics (CFG) standard. Source data are provided as a Source Data file.
