Glycomics-Informed Glycoproteomics Reveals Complex N-linked Glycosylation of ACE2
We also analyzed ACE2 glycosylation utilizing the same glycomic and glycoproteomic approaches described for S protein. Glycomic analyses of released N-linked glycans (Figure 5 A; Table S3) revealed that the majority of glycans on ACE2 are complex with limited high-mannose and hybrid glycans, and we were unable to detect sulfated N-linked glycans. Glycoproteomic analyses revealed that occupancy was high (> 75%) at all six sites, and significant microheterogeneity dominated by complex N-glycans was observed for each site (Figures 5B–5G; Tables S5–S8). We also observed, consistent with the O-glycomics (Figure S5; Table S4), that Ser 155 and several S/T residues at the C terminus of ACE2 outside of the peptidase domain were O-glycosylated, but stoichiometry was extremely low (less than 2%; Tables S9 and S10).
Figure 5 Glycomics-Informed Glycoproteomics of Soluble Human ACE2 Reveals High Occupancy, Complex N-linked Glycosylation
(A) Glycans released from soluble, purified ACE2 were permethylated and analyzed by MSn. Structures were assigned, grouped by type and structural features, and prevalence was determined based on ion current. The pie chart shows basic division by broad N-glycan type. The bar graph provides additional detail about the glycans detected. The most abundant structure with a unique categorization by glycomics for each N-glycan type in the pie chart, or above each feature category in the bar graph, is indicated.
(B–G) Glycopeptides were prepared from soluble human ACE2 by using multiple combinations of proteases, analyzed by LC-MSn, and the resulting data were searched by using several different software packages. All six sites of N-linked glycosylation are presented here. Displayed in the bar graphs are the individual compositions observed graphed in terms of assigned spectral counts. Representative glycans (as determined by glycomics analysis) for several abundant compositions are shown in SNFG format. The pie chart (analogous to Figure 3 for SARS-CoV-2 S) for each site is displayed in the upper corner of each panel.
(B) N053.
(C) N090.
(D) N103.
(E) N322.
(F) N432.
(G) N546, a site that does not exist in three in 10,000 people.