STT3A基因敲除HEK293细胞

STT3A and STT3B are the main catalytic subunits of the oligosaccharyltransferase complex (OST-A and OST-B in mammalian cells), which primarily mediate cotranslational and post-translocational N-linked glycosylation, respectively. To determine the specificity of STT3A and STT3B, we performed proteomic and glycoproteomic analyses in the gene knock-out (KO) and wild-type HEK293 cells. In total, 3961 proteins, 4265 unique N-linked intact glycopeptides and 629 glycosites representing 349 glycoproteins were identified from all these cells. Deletion of the STT3A gene had a greater impact on the protein expression than deletion of STT3B, especially on glycoproteins. In addition, total mannosylated N-glycans were reduced and fucosylated N-glycans were increased in STT3A-KO cells, which were caused by the differential expression of glycan-related enzymes. Interestingly, hyperglycosylated proteins were identified in KO cells, and the hyperglycosylation of ENPL was caused by the endoplasmic reticulum (ER) stress due to the STT3A deletion. Furthermore, the increased expression of the ATF6 and PERK indicated that the unfolded protein response also happened in STT3A-KO cells. Overall, the specificity of STT3A and STT3B revealed that defects in the OST subunit not only broadly affect N-linked glycosylation of the protein but also affect protein expression.

Viral fusion proteins decorate their antigenic surface with N-linked glycans which support processes such as protein folding, cell-specific interactions and shielding of vulnerable antibody epitopes. Asparagine-linked glycosylation is catalyzed by the oligosaccharyltransferase (OST) complexes containing the catalytic subunits STT3A or STT3B, which act predominantly co- and post-translationally, respectively. Here, we investigated the contributions of STT3A and STT3B to glycan attachment to recombinant SARS-CoV-2 Spike (S) and influenza A (H3N2) virus hemagglutinin (HA). Soluble proteins and pseudotyped viruses were produced in wild-type and STT3A or STT3B-knockout (KO) 293T cells. Site-specific glycan analysis of the recombinant proteins revealed significant changes at only a limited number of glycosylation sites upon deletion of either STT3A or STT3B, indicating partial redundancy in maintaining overall site occupancy. However, STT3A-KO reduced glycosylation at N717 and N1074 on the SARS-CoV-2 S protein, while STT3B KO reduced glycosylation at N483 on influenza HA, suggesting isoform-specific preferences for distinct glycosylation sequons. Infectivity assays further suggested that the glycosylation of both SARS-CoV-2 and influenza A (H3N2) viral glycoproteins are more dependent on STT3A, with STT3B contributing to a lesser but detectable extent. These findings highlight distinct, context-dependent and site-specific contributions of STT3A and STT3B to viral glycoprotein glycosylation, with implications for vaccine antigen design.