N-Glycan Modification in Covid-19 Pathophysiology: In vitro Structural Changes with Limited Functional Effects
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ORIGINAL ARTICLE
N-Glycan Modification in Covid-19 Pathophysiology: In vitro Structural Changes with Limited Functional Effects Cristiane J. Nunes-Santos 1 & Hye Sun Kuehn 1 & Sergio D. Rosenzweig 1 Received: 20 August 2020 / Accepted: 28 October 2020 # This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2020
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 SARSCoV-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 Glycosylation . α-glucosidase . miglustat . SARS-CoV-2 . spike . ACE2
Introduction In 2014, we reported two siblings with a rare congenital disorder of glycosylation (CDG) due to mutations in mannosyloligosaccharide glucosidase (MOGS) [1]. These patients were resistant to infections by particular, enveloped viruses with Nglycosylated glycoproteins. Lessons learned from these siblings, and previous attempts to manipulate MOGS, highlight the reliance of particular viruses on the host N-glycosylation machinery and a vulnerability that can be exploited in favor of Cristiane J. Nunes-Santos and Hye Sun Kuehn contributed equally to this work. * Sergio D. Rosenzweig [email protected] 1
Immunology Service, Department of Laboratory Medicine, National Institutes of Health (NIH) Clinical Center, 10 Center Dr., Bldg 10, Rm. 2C410F, Bethesda, MD 20892, USA
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