Site-specific characterization of SARS-CoV-2 spike glycoprotein receptor-binding domain

Abstract

The novel coronavirus SARS-CoV-2, the infective agent causing COVID-19, is having a global impact both in terms of human disease as well as socially and economically. Its heavily glycosylated spike glycoprotein is fundamental for the infection process, via its receptor-binding domains interaction with the glycoprotein angiotensin-converting enzyme 2 on human cell surfaces. We therefore utilized an integrated glycomic and glycoproteomic analytical strategy to characterize both N- and O- glycan site-specific glycosylation within the receptor-binding domain. We demonstrate the presence of complex-type N-glycans with unusual fucosylated LacdiNAc at both sites N331 and N343 and a single site of O-glycosylation on T323.

Keywords: SARS-CoV-2; glycoproteomics; mass spectrometry; spike glycoprotein.

Fig. 1

Glycomic analysis of the N331 and N343 glycopeptides derived from the RBD of the S1 spike SARS-CoV-2 glycoprotein. MALDI-TOF mass spectra of permethylated N-glycans derived from (A) 329-FPNITNLCPFGE-340 glycopeptide (containing the N331 glycosylation site) and (B) 341-VFNATR-346 glycopeptide (containing the N343 glycosylation site). All signals observed are singly charged sodiated ions [M + Na]⁺ and their structural assignment is based on monosaccharide composition, MS/MS fragmentation analyses and knowledge of the biosynthetic pathways. Residues shown on top of a bracket have not had their antenna location unequivocally defined.

Fig. 2

Glycoproteomics analysis of the 341-VFNATR-346 glycopeptide derived from the RBD of the S1 spike SARS-CoV-2 glycoprotein containing the glycosylation site N343 found centered at 12.1 min in the LC elution profile. LC ES-MS (MS^e mode) containing the [M + 2H]²⁺ molecular ion found at m/z 1425.59 and corresponding to the 341-VFNATR-346 glycopeptide with a core-fucosylated bi-antennary N-glycan with fucosylated LacdiNAc antennae (permethylated N-glycan found at m/z 2674 on Figure 1B and Supplementary Table SI). The peaks in red correspond to glycopeptide fragment ions deriving from the [M + 2H]²⁺ molecular ion. “P” inside a circle corresponds to the peptide backbone of 341-VFNATR-346. Area corresponding to m/z 2120 to 2600 has a 5-fold magnification. The residues shown on top of a bracket have not had their antenna location unequivocally defined.

Fig. 3

Glycoproteomics analysis of the 320-VQPTESIVR-328 glycopeptide containing the putative glycosylation site at T325 and S325 derived from the RBD of the S1 spike SARS-CoV-2 glycoprotein. LC ES-MS (MS^e mode) data containing the [M + 2H]²⁺ molecular ion found at m/z 1171.02 and interpreted as a single Core 2 substitution at Thr 323 with the HexNAc₂Hex₂NeuAc₂ structure shown. “P” inside a circle corresponds to the peptide backbone of 320-VQPTESIVR-328. The insets correspond to zoom scan from the regions of (i) m/z 801, (ii) m/z 1004, (iii) m/z 474 and (iv) m/z 677.

Fig. 4

Glycoproteomics analysis of the 320-VQPTE-324 glycopeptide derived from the RBD of the S1 spike SARS-CoV-2 glycoprotein. LC ES-MS/MS data (CAD MS/MS mode) of a doubly charged ion species (716.8²⁺), showing clear evidence of T323 substitution with a mono-sialyl version (NeuAcHexHexNAc₂) of the assigned Core 2 structure seen in Figure 3 and described in the text. Proline cleavage y” ions at m/z 346, 549 (y₃-HexNAc) and 752 (y₃-HexNAc-HexNAc) support the interpretation made. The unlabeled higher mass signals were devoid of ¹³C isotope clusters and not assigned as of glycopeptide origin.