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

Glycosylation processing in insect and human cells can yield a common intermediate N-glycan, which will further elongate in human cells but is only trimmed in insect cells to form end products(19). Consequently, the S protein expressed in human cells displayed a larger size and a much higher proportion of complex N-glycans than that expressed in insect cells, owing to the additional elongation of the glycan backbone with multiple oligosaccharides (Fig. 3C-3F ). Our findings are in line with those of previous studies, that demonstrated the predominant complex of N-glycans attached to MERS-CoV and SARS-CoV proteins (4, 5, 28) . Moreover, two recent preprint studies have revealed that the complex N-glycans dominate the glycosites on human cell-expressed SARS-CoV-2 S protein(29, 30). In contrast, S protein expression in insect cells led to a high ratio of high-mannose N-glycans ( Fig. 3C and 3E ), which has also been found in the insect cell-produced HCoV-NL63 S protein, despite expression in a different insect cell, the Drosophila S2 cell, in the previous study(15).