Site-specific N-glycosylation Characterization of Recombinant SARS-CoV-2 Spike Proteins using High-Resolution Mass Spectrometry

Yong Zhang; Wanjun Zhao; Yonghong Mao; Shisheng Wang; Yi Zhong; Tao Su; Meng Gong; Xiaofeng Lu; Jingqiu Cheng; Hao Yang

For the recombinant protein S1 subunit expressed in human cells, all 13 N-glycosites were assigned unambiguously (Table S2 ). Finally, we profiled all 22 potential N-glycosites of S protein (Table S3 and S4). These sites were preferentially . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.28.013276 doi: bioRxiv preprint distributed in the S1 subunit of the N-terminus and the S2 subunit of the C-terminus, including two sites in the RBD ( Fig. 2A and 2B ). To visualize the N-glycosylation on the protein structure, all of the experimentally determined N-glycosites were hand-marked on the surface of the trimeric S protein following refinement of the recently reported SARS-CoV-2 S protein Cryo-EM structure (PDB: 6VSB) ( Based on these findings, we further analyzed the conservation of glycosites among 753 SARS-CoV-2 genome sequences from the Global Initiative on Sharing All Influenza Data (GISAID) database. After the removal of redundant sequences of the S protein at the amino acid residue level, we refined 145 S protein variants. A very low frequency of alterations in 38 residue sites was found, uniformly spanning the full length of the S protein among all S variants, except for the substitution G614D, which was identified at a relatively high frequency in 47 variants (Table S5) . However, nearly all of the 22 N-glycosylated sequons were conserved in the S protein, except for the loss of the N717 glycosite due to the T719A substitution in only one S variant.