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 S1 subunit expressed in human cells, the occupancy of N-glycans on each N-glycosite was quite nonuniform. Surprisingly, six N-glycosites (N122, N165, N282, N331, N343, and N657) were decorated with markedly heterogeneous N-glycans of up to 139 types. The average occupancies of all glycosites presented as an overwhelming proportion (~75%) of complex N-glycans and a small proportion of hybrid (~13%) or high-mannose (~12%) N-glycans (Fig. 3F ). The glycan occupancy on two N-glycosites (N331 and N343) of RBD were identified ( Fig. 3E and 3F ). The high occupancy of RBD glycosites by various N-glycan compositions implies that N-glycosylation might be associated with the recognition of RBD to ACE2 receptor, since the interaction between RBD and ACE2 mainly depends on polar residue interactions(11). Our results suggest that S proteins expressed in different cells display distinct N-glycosylation patterns. In particular, the glycosylation of the S protein in human cells exhibits remarkable heterogeneity on N-glycosites. However, the N-glycan types on each glycosite is primarily determined by the host cells rather than the location of different glycosites ( Fig. 3E-3F, Fig. S8 ).