Structural Characterization of N‐Linked Glycans in the Receptor Binding Domain of the SARS‐CoV‐2 Spike Protein and their Interactions with Human Lectins

Abstract

The glycan structures of the receptor binding domain of the SARS‐CoV2 spike glycoprotein expressed in human HEK293F cells have been studied by using NMR. The different possible interacting epitopes have been deeply analysed and characterized, providing evidence of the presence of glycan structures not found in previous MS‐based analyses. The interaction of the RBD 13C‐labelled glycans with different human lectins, which are expressed in different organs and tissues that may be affected during the infection process, has also been evaluated by NMR. In particular, 15N‐labelled galectins (galectins‐3, ‐7 and ‐8 N‐terminal), Siglecs (Siglec‐8, Siglec‐10), and C‐type lectins (DC‐SIGN, MGL) have been employed. Complementary experiments from the glycoprotein perspective or from the lectin's point of view have permitted to disentangle the specific interacting epitopes in each case. Based on these findings, 3D models of the interacting complexes have been proposed.

Structural- and Site-Specific N-Glycosylation Characterization of COVID-19 Virus Spike with StrucGP

“…In previous studies, the glycan compositions at each glycosite of the S protein have been systematically analyzed. ,,− Some structural motifs on the S protein such as LacdiNAc and Mannose 6-phosphate (M6P) have also reported previously. , The structural information on N- glycan released from the receptor binding domain (RBD) without glycosite information has also been investigated. , In this study, we further characterized additional structural features of N- glycans at each glycosite of the COVID-19 virus S protein (both trimer and protomer expressed in HEK293 cells) by using our recently developed glycopeptide interpretation software, StrucGP …”

“… 12 , 16 The structural information on N- glycan released from the receptor binding domain (RBD) without glycosite information has also been investigated. 19 , 20 In this study, we further characterized additional structural features of N- glycans at each glycosite of the COVID-19 virus S protein (both trimer and protomer expressed in HEK293 cells) by using our recently developed glycopeptide interpretation software, StrucGP. 21 …”

“…12,16 The structural information on N-glycan released from the receptor binding domain (RBD) without glycosite information has also been investigated. 19,20 In this study, we further characterized additional structural features of N-glycans at each glycosite of the COVID-19 virus S protein (both trimer and protomer expressed in HEK293 cells) by using our recently developed glycopeptide interpretation software, StrucGP. 21 The protein samples were digested into peptides using three individual enzymes followed by triplicate LC-MS/MS analyses of each peptide sample with two fragmentation energies (low and high HCD energies) on each glycopeptide peak.…”

The two domains of human galectin-8 bind sialyl- and fucose-containing oligosaccharides in an independent manner. A 3D view by using NMR

“…Indeed, the interaction of Gal8 N-term with the glycans of the receptor binding domain of SARS CoV2 has been shown to involve the sialyl epitope. 28 These results, together with the ligand-based NMR experiments 29 described above, highlight the direct participation of the Neu5Ac moiety in lectin binding. Noteworthy, binding specificity completely switched in the case of the histo-blood group antigen A type II 6.…”

Structural Insights into the Molecular Recognition Mechanism of the Cancer and Pathogenic Epitope, LacdiNAc by Immune‐Related Lectins

“…In this context, LacdiNAc (GalNAcβ1‐4GlcNAc, LDN) is a terminal epitope found on the chains of N ‐ and O ‐glycoproteins in both mammals and pathogens [6,7] . LDN is highly expressed in parasitic helminths such as Schistosoma mansoni [7] and was recently identified as a part of the N ‐linked glycans in the receptor binding domain of the SARS‐CoV‐2 spike protein [8] . Additionally, LDN is a well‐established cancer biomarker overexpressed on glycoproteins associated with several human cancers, including colon, pancreas, ovarian and prostate cancers [9–13] .…”