Roles of Host Cell Glycans in SARS-CoV-2 Infection
Creators: Nguyen, Linh Le Hoai
Publisher: name: University of Alberta Library
Year: 2024

<p>Many human viruses utilize carbohydrates (glycans) present on the surface of host cells during the infection process. Uncovering the glycans recognized and their functional roles is essential to a comprehensive understanding of viral infections. This thesis focuses on the development and application of native mass spectrometry (nMS) approaches to identify the host glycan ligands of SARS-CoV-2 and to determine the strength and location of these interactions.<br>
    In Chapter 2, a suite of nMS methods was used to survey the glycan binding properties of SARS-CoV-2 Wuhan-Hu-1 (WT) spike (S) protein and receptor binding domain (RBD). Catch-and-release (CaR)-nMS screening of a defined glycan library against the RBD identified the oligosaccharides of the gangliosides GM1 and GM2 as the top binders. Other sialoglycans and neutral glycans, including ABH blood group antigens, also bind. Screening of natural N-glycan libraries from human lung and intestinal tissue revealed a preference for mono- and disialylated complex N-glycans. The affinities of a subset of ligands identified from screening were quantified using the direct nMS assay. Notably, the affinities of GM1 and GM2 oligosaccharides are comparable in magnitude to those measured for heparan sulfate (HS) oligosaccharides, which serve SARS-CoV-2 co-receptors. According to the results of CaR-nMS and nMS measurements, GM1 and HS oligosaccharides bind competitively to RBD. Guided by these findings, cell binding (by RBD) and entry (pseudotyped lentivirus) experiments were carried out. The results show that host sialoglycans, including sialylated glycolipids, influence binding and entry. Finally, the functional role of acidic glycolipids in SARS-CoV-2 infection was confirmed using viral assays.<br>
    ABO blood group is associated with susceptibility to SARS-CoV-2 infection, with group A individuals exhibiting an increased risk. However, the mechanism whereby ABO blood group status influences infection remains unclear. In Chapter 3, the affinities of the ABH blood group antigens (subtypes I-VI) for the RBD of SARS-CoV-2 WT and variants of concern (VOCs - Alpha, Beta, Delta and Omicron) were quantified by nMS. RBD is found to bind to subset of the ABH antigens. However, the detected interactions are weak (Kd 0.5–5 mM). Moreover, high concentrations of free ABH antigen failed to inhibit cell infection. These findings argue against ABH antigens playing a functional role in SARS-CoV-2 cell entry.<br>
     In Chapter 4, the influence of SARS-CoV-2 RBD mutations on glycan binding was investigated. Comparison of CaR-nMS screening data acquired for WT, Alpha, Beta, Delta and Omicron RBD revealed comparable glycan binding (relative to WT) for Alpha and Omicron, diminished binding to Beta, and more promiscuous binding to Delta. The changes in affinity associated with the specific mutations suggested amino acid residues that are involved in the glycan binding site(s). Importantly, it was found that the RBD glycan binding properties are influenced by glycosylation, with truncation of N-glycans weakening binding. According to the results of molecular dynamics MD simulation, decrease of glycan binding affinity is related to the critical role of the glycosylation at N343 in RBD folding.<br>
    In Chapter 5, the glycan binding sites of WT SARS-CoV-2 RBD were localized using a newly developed antibody masking CaR-nMS assay and competitive nMS and mass photometry (MP) assays and insights gained in Chapter 4. Together, these data suggest that that HS binding occurs at shallow bowl-shaped cavity (aa 420-430, aa 441-452 and 490-512) of receptor binding motif (RBM), which is the binding site of primary receptor (angiotensin-converting enzyme 2, ACE2), while sialylated glycans bind at the distal end of RBM (aa 470-486 with parts of aa 441-452 and 490-512). Neutral glycans were predicted to bind in region (aa 490-512) between ACE2 binding site and hydrophobic beta sheet core, which is distinct from sialylated glycans.<br>
    Chapter 6 describes the results of a fundamental study of protein binding to glycosphingolipids (GSLs) in soluble model membranes (nanodiscs), performed using MP and nMS. Key findings are that GSLs are distributed non-statistically across the lipid bilayer and engage in clustering that is sensitive to protein binding. Specifically, the affinities and stoichiometries measured for multivalent proteins are strongly modulated by GSL clustering, which can overwhelm affinity gains from multivalent binding. These results serve as a foundation for understanding GSL binding in natural cellular environments. </p>