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

J Mol Biol. 2022 Jan 30;434(2):167332. doi: 10.1016/j.jmb.2021.167332. Epub 2021 Oct 27.

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.

Keywords: SARS-CoV-2; antibody; glycoprotein; glycosylation; serology.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies, Viral / blood
  • Antibodies, Viral / immunology*
  • Binding Sites / genetics
  • COVID-19 / immunology*
  • COVID-19 / virology
  • Glycosylation
  • Humans
  • Immunoglobulin A / blood
  • Immunoglobulin A / immunology
  • Immunoglobulin G / blood
  • Immunoglobulin G / immunology
  • Immunoglobulin M / blood
  • Immunoglobulin M / immunology
  • Mannose / metabolism
  • Mutation, Missense / genetics
  • Mutation, Missense / immunology*
  • Oligosaccharides / metabolism
  • Polysaccharides / metabolism
  • Proline / genetics
  • Proline / immunology
  • Proline / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / immunology
  • Recombinant Proteins / metabolism
  • SARS-CoV-2 / immunology*
  • SARS-CoV-2 / metabolism
  • SARS-CoV-2 / physiology
  • Spike Glycoprotein, Coronavirus / genetics
  • Spike Glycoprotein, Coronavirus / immunology*
  • Spike Glycoprotein, Coronavirus / metabolism

Substances

  • Antibodies, Viral
  • Immunoglobulin A
  • Immunoglobulin G
  • Immunoglobulin M
  • Oligosaccharides
  • Polysaccharides
  • Recombinant Proteins
  • Spike Glycoprotein, Coronavirus
  • spike protein, SARS-CoV-2
  • Proline
  • Mannose