Host Cell Glycocalyx Remodeling Reveals SARS-CoV-2 Spike Protein Glycomic Binding Sites.

Yixuan Xie,Ying Sheng,Anita Vinjamuri,Mariana Barboza,Matthew Frieman,Michael Russelle S Alvarez,Carlito B Lebrilla,Marisa Mcgrath,Siyu Chen

doi:10.3389/fmolb.2022.799703

Abstract

Glycans on the host cell membrane and viral proteins play critical roles in pathogenesis. Highly glycosylated epithelial cells represent the primary boundary separating embedded host tissues from pathogens within the respiratory and intestinal tracts. SARS-CoV-2, the causative agent for the COVID-19 pandemic, reaches into the respiratory tract. We found purified human milk oligosaccharides (HMOs) inhibited the viral binding on cells. Spike (S) protein receptor binding domain (RBD) binding to host cells were partly blocked by co-incubation with exogenous HMOs, most by 2-6-sialyl-lactose (6′SL), supporting the notion that HMOs can function as decoys in defense against SARS-Cov2. To investigate the effect of host cell glycocalyx on viral adherence, we metabolically modified and confirmed with glycomic methods the cell surface glycome to enrich specific N-glycan types including those containing sialic acids, fucose, mannose, and terminal galactose. Additionally, Immunofluorescence studies demonstrated that the S protein preferentially binds to terminal sialic acids with α-(2,6)-linkages. Furthermore, site-specific glycosylation of S protein RBD and its human receptor ACE2 were characterized using LC-MS/MS. We then performed molecular dynamics calculations on the interaction complex to further explore the interactive complex between ACE2 and the S protein. The results showed that hydrogen bonds mediated the interactions between ACE2 glycans and S protein with desialylated glycans forming significantly fewer hydrogen bonds. These results supported a mechanism where the virus binds initially to glycans on host cells preferring α-(2,6)-sialic acids and finds ACE2 and with the proper orientation infects the cell.

Highlights

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19 ((Zhu et al, 2020)), encodes an extensively glycosylated spike (S) protein that protrudes from the viral surface and binds angiotensin-converting enzyme 2 (ACE2) on host cells ((Hoffmann et al, 2020; Walls et al, 2020; Wan et al, 2020; Wrapp et al, 2020; Gstöttner et al, 2021))
We first examined whether pooled samples of purified human milk oligosaccharides (HMOs) from seven different mothers could affect the binding of severe acute respiratory syndrome (SARS)-CoV-2 virus on Vero E6 cells
The HMOs 2′-fucosyllactose (2′-FL), 6′- sialyllactose (6′-SL), and lacto-N-neotetraose (LNnT) were selected for this study because they represent many of the structures and are abundant in mothers’ milk. 2′-FL and 6′-SL were produced by adding fucose or a N-acetylneuraminic acid (Neu5Ac) (Castanys-Muñoz et al, 2013) to the lactose core, respectively

Summary

Introduction

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19 ((Zhu et al, 2020)), encodes an extensively glycosylated spike (S) protein that protrudes from the viral surface and binds angiotensin-converting enzyme 2 (ACE2) on host cells ((Hoffmann et al, 2020; Walls et al, 2020; Wan et al, 2020; Wrapp et al, 2020; Gstöttner et al, 2021)). This novel SARS-SARS-Cov-2 Spike Glycomic Binding Sites. The plasma membrane protein ACE2 is abundantly expressed in humans tissues, including respiratory and intestinal epithelia, liver arteries, heart and kidney ((Hamming et al, 2004))

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Discussion

Conclusion