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{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7443692","sourcedb":"PMC","sourceid":"7443692","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7443692","text":"The highly glycosylated SARS-CoV-2 Spike protein, unlike several other viral proteins including HIV-1 (Watanabe et al., 2019) but in agreement with another recent report (Watanabe et al., 2020a), presents significantly more processing of N-glycans toward complex glycosylation, suggesting that steric hindrance to processing enzymes is not a major factor at most sites (Figures 2 and 3). However, the N-glycans still provide considerable shielding of the peptide backbone (Figure 4). Our glycomics-guided glycoproteomic data are generally in strong agreement with the trimer immunogen data recently published by Crispin (Watanabe et al., 2020a), although we also observed sulfated N-linked glycans; were able to differentiate branching, bisected, and diLacNAc containing structures by glycomics; and observed less occupancy on the two most C-terminal N-linked sites by using a different approach. Our detection of sulfated N-linked glycans at multiple sites on S is in agreement with a recent manuscript re-analyzing the Crispin data (https://www.biorxiv.org/content/10.1101/2020.05.31.125302v1). Sulfated N-linked glycans could potentially play key roles in immune regulation and receptor binding as in other viruses (Wang et al., 2009). This result is especially significant in that sulfated N-glycans were not observed when we performed glycomics on ACE2. At each individual site, the glycans we observed on our immunogen appear to be slightly more processed, but the overlap between our analysis and the Crispin’s group results (Watanabe et al., 2020a) at each site in terms of major features are nearly superimposable. This agreement differs substantially when comparing our and Crispin’s data (Watanabe et al., 2020a) to that of the Azadi group (Shajahan et al., 2020), which analyzed S1 and S2 that had been expressed individually. When expressed as two separate polypeptides and not purified for trimers, several unoccupied sites of N-linked glycosylation were observed and processing at several sites was significantly different (Shajahan et al., 2020) than we and others (Watanabe et al., 2020a) observed. Although O-glycosylation has recently been reported for individually expressed S1 and S2 domains of the Spike glycoprotein (Shajahan et al., 2020), in trimeric form the level of O-glycosylation is extremely low, with the highest level of occupancy we observed being 11% at T0323 (Figure 4E). The low level of O-linked occupancy we observed is in agreement with the Crispin group’s analysis of a Spike Trimer immunogen (Watanabe et al., 2020a) but differs significantly from the Azadi group’s analyses of individually expressed S1 and S2 (Shajahan et al., 2020). Thus, the context in which the Spike protein is expressed and purified before analysis significantly alters the glycosylation of the protomer that is reminiscent of previous studies looking at expression of the HIV-1 envelope Spike (Behrens et al., 2017; Watanabe et al., 2019). The soluble ACE2 protein examined here contains six highly utilized sites of N-linked glycosylation dominated by complex type N-linked glycans (Figure 5). O-glycans were also present on this glycoprotein but at very low levels of occupancy at all sites 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