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. 2021 Nov 25;74:103712. doi: 10.1016/j.ebiom.2021.103712

Figure 1.

Figure 1

N-glycosylation in spike is highly conserved in SARS-CoV-2 variants and essential for ACE2 binding. (a) Western blot analysis of S protein expression in HEK293T-spike cells that transfected with spike for 24h, followed by treatment with the indicated inhibitor for an additional 24 h. N-linked inhibitors [2.5 μg/mL Tunicamycin (TM), 10 μM NGI-1, 20 mg/mL 2-DG, 10 μM Swainsonine (SW), and 50 μM Castanospermine (CSA)]; 10 μM O-GlcNAc-β-N-acetylglucosaminidase inhibitor (PUGNAc); 10 μM O-GlcNAcase inhibitor (Thiamet G), and 10 μM O-linked glycosyltransferase inhibitor [Benzyl 2-acetamido-2-deoxy-α-D-galactopyranoside (BADG)]. The non-glycosylated form in lane 2 represents a spike with TM treatment. Circle, glycosylated spike (g-spike); arrow, non-glycosylated spike (ng-spike). (b) The glycosylation pattern of the S protein. S protein was treated with PNGase F or Endo H, and analyzed by glycoprotein staining (left panel) and Coomassie blue staining (right panel). Black circle, g-spike; arrowhead, ng-spike. Negative control (-), Trypsin inhibitor soybean (20 kDa); positive control (+), horseradish peroxidase (44 kDa). (c) 3D structure diagrams of the ACE2 and spike in SARS-CoV-2 (7A95) structure rendered as a surface with NXT/S motif glycans resolved in the cryo-EM map rendered as red spheres. (d) Sequence identity analysis of three SARS-CoV-2 distinct surface residues (N17, N149, N657), RBD residues (N331, N343), and the susceptible residues (E484, N501, and D614) of the variants in 57311 S proteins retrieved from NCBI-Virus-database. (e) Flow cytometry analysis of ACE2 binding to HEK293T-spike cells with NGI-1 and Thiamet G treatment (right panel). Glycosylated and de-glycosylated S protein molecular weight were assessed by Western blot (left panel). (f) ELISA detection of the indicated concentration of ACE2 binding to precoated SARS-CoV-2 ECD after DMSO or PNGase F treatment. Data shown are means ± SD from representative triplicates from three independent experiments. ***p < 0.001 (Two-way ANOVA). (g) Binding affinity of g-spike or ng-spike with immobilized ACE2 measured by BioLayer interferometry.