Glycosylation and Serological Reactivity of an Expression-enhanced SARS-CoV-2 Viral Spike Mimetic

Himanshi Chawla,Sian E Jossi,Sian E Faustini,Firdaus Samsudin,Joel D Allen,Yasunori Watanabe,Maddy L Newby,Edith Marcial-Juárez,Rachel E Lamerton,Jason S Mclellan,Peter J Bond,Alex G Richter,Adam F Cunningham,Max Crispin

doi:10.1016/j.jmb.2021.167332

Abstract

Extensive glycosylation of viral glycoproteins is a key feature of the antigenic surface of viruses and yet glycan processing can also be influenced by the manner of their recombinant production. The low yields of the soluble form of the trimeric spike (S) glycoprotein from SARS-CoV-2 has prompted advances in protein engineering that have greatly enhanced the stability and yields of the glycoprotein. The latest expression-enhanced version of the spike incorporates six proline substitutions to stabilize the prefusion conformation (termed SARS-CoV-2 S HexaPro). Although the substitutions greatly enhanced expression whilst not compromising protein structure, the influence of these substitutions on glycan processing has not been explored. Here, we show that the site-specific N-linked glycosylation of the expression-enhanced HexaPro resembles that of an earlier version containing two proline substitutions (2P), and that both capture features of native viral glycosylation. However, there are site-specific differences in glycosylation of HexaPro when compared to 2P. Despite these discrepancies, analysis of the serological reactivity of clinical samples from infected individuals confirmed that both HexaPro and 2P protein are equally able to detect IgG, IgA, and IgM responses in all sera analysed. Moreover, we extend this observation to include an analysis of glycan engineered S protein, whereby all N-linked glycans were converted to oligomannose-type and conclude that serological activity is not impacted by large scale changes in glycosylation. These observations suggest that variations in glycan processing will not impact the serological assessments currently being performed across the globe.

Highlights

Recombinant viral glycoproteins are an important resource for vaccine development, diagnostics and as research reagents
Extensive glycosylation of viral glycoproteins is a key feature of the antigenic surface of viruses and yet glycan processing can be influenced by the manner of their recombinant production
We extend this observation to include an analysis of glycan engineered S protein, whereby all N-linked glycans were converted to oligomannose-type and conclude that serological activity is not impacted by large scale changes in glycosylation

Summary

Introduction

Recombinant viral glycoproteins are an important resource for vaccine development, diagnostics and as research reagents. The oligomannose-type glycan content of the detected some difference in glycosylation which glycans of the HexaPro protein (29%) could suggest differences in the conformational (Supplementary Table S1) is lower when properties between the HexaPro and 2P This moticompared to other viral glycoproteins including vated us to extend the analysis of protein conformaHIV-1 Env (60%) and LASV GPC (49%).[69,70] This tional flexibility by performing MD simulations, and is consistent with earlier observations using 2P pro- to characterize its functional behaviour by performtein which indicated that SARS-CoV-2 S is less ing serological testing. To perform the glycopeptide analysis using three protease enzymes, three 50 lg aliquots of SARSCOV-2 HexaPro were denatured for 1 h in 50 mM

Materials and Methods

Findings

Declaration of interests