N-Glycan Modification in Covid-19 Pathophysiology: In vitro Structural Changes with Limited Functional Effects

J Clin Immunol. 2021 Feb;41(2):335-344. doi: 10.1007/s10875-020-00905-4. Epub 2020 Nov 27.

Abstract

In 2014, we reported two siblings with a rare congenital disorder of glycosylation due to mutations in mannosyl-oligosaccharide glucosidase (MOGS). The glycan alteration derived from this disease resulted in an in vitro infection resistance to particular enveloped, N-glycosylation-dependent viruses as influenza and HIV. As part of the global effort to find safe and effective antiviral therapies for Covid-19, we assessed the in vitro activity of the FDA-approved α-glucosidase inhibitor miglustat against SARS-CoV-2. Expression plasmids encoding SARS-CoV-2 spike (S) and human ACE2 glycoproteins (GP) were tested to evaluate N-glycan modifications induced by α-glucosidase inhibition. Immunoprecipitation was used to assess binding between these two GP. Cell-to-cell fusion was assessed by immunofluorescence of cocultures of SARS-CoV-2 S and ACE2-expressing cells. Miglustat effect on immune response was tested by measuring cytokine release from PBMC exposed to purified SARS-CoV-2 S. In our overexpression system, miglustat successfully and specifically modified N-glycans in both SARS-CoV-2 S and its main receptor ACE2. Binding between these two GP was not affected by glycan modifications. A surrogate marker for viral cytopathic effect, measured as receptor-dependent SARS-CoV-2 S-driven cell-to-cell fusion, was not disrupted by miglustat treatment. This observation was further confirmed in MOGS-null transfected cells. Miglustat produced no statistically significant effects on cytokine production following SARS-CoV-2 S glycoprotein stimulation of PBMC. Our work shows that despite clear N-glycan alteration in the presence of miglustat, the functions of the Covid-19-related glycoproteins studied were not affected, making it unlikely that miglustat can change the natural course of the disease.

Keywords: ACE2; Glycosylation; SARS-CoV-2; miglustat; spike; α-glucosidase.

Publication types

  • Research Support, N.I.H., Intramural

MeSH terms

  • Angiotensin-Converting Enzyme 2 / genetics
  • Angiotensin-Converting Enzyme 2 / metabolism
  • Animals
  • COVID-19 / metabolism*
  • COVID-19 / virology*
  • Cytokines / metabolism
  • Estrogen Receptor alpha / metabolism
  • Glycosylation
  • HEK293 Cells
  • Host-Pathogen Interactions*
  • Humans
  • Mice
  • NIH 3T3 Cells
  • Peptidyl-Dipeptidase A / genetics
  • Peptidyl-Dipeptidase A / metabolism
  • Polysaccharides / metabolism*
  • SARS-CoV-2*

Substances

  • Cytokines
  • ESR1 protein, human
  • Estrogen Receptor alpha
  • Polysaccharides
  • Peptidyl-Dipeptidase A
  • ACE2 protein, human
  • Angiotensin-Converting Enzyme 2