Subtle Influence of ACE2 Glycan Processing on SARS-CoV-2 Recognition

Joel D Allen,Yasunori Watanabe,Himanshi Chawla,Maddy L Newby,Max Crispin

doi:10.1016/j.jmb.2020.166762

Joel D Allen, Yasunori Watanabe + Show 3 more

Open Access

https://doi.org/10.1016/j.jmb.2020.166762

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Abstract

The severity of SARS-CoV-2 infection is highly variable and yet the molecular basis for this effect remains elusive. One potential contribution are differences in the glycosylation of target human cells, particularly as SARS-CoV-2 has the capacity to bind sialic acid which is a common, and highly variable, terminal modification of glycans. The viral spike glycoprotein (S) of SARS-CoV-2 and the human cellular receptor, angiotensin-converting enzyme 2 (ACE2) are both densely glycosylated. We therefore sought to investigate whether the glycosylation state of ACE2 impacts the interaction with SARS-CoV-2 viral spike. We generated a panel of engineered ACE2 glycoforms which were analyzed by mass spectrometry to reveal the site-specific glycan modifications. We then probed the impact of ACE2 glycosylation on S binding and revealed a subtle sensitivity with hypersialylated or oligomannose-type glycans slightly impeding the interaction. In contrast, deglycosylation of ACE2 did not influence SARS-CoV-2 binding. Overall, ACE2 glycosylation does not significantly influence viral spike binding. We suggest that any role of glycosylation in the pathobiology of SARS-CoV-2 will lie beyond its immediate impact of receptor glycosylation on virus binding.

Highlights

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19, encodes an extensively glycosylated spike (S) protein that protrudes from the viral surface.[1,2] The S protein is a trimeric class I fusion protein, composed of two functional subunits, S1 and S2, which are responsible for receptor binding and membrane fusion, respectively.[1,3] The S protein binds to cell surface angiotensin-converting enzyme 2 (ACE2) which initiates host-cell entry and results in membrane fusion.[4]
We suggest that any role of glycosylation in the pathobiology of SARS-CoV-2 will lie beyond its immediate impact of receptor glycosylation on virus binding
To enable the determination of the glycosylation and binding capabilities of ACE2, a soluble recombinant ACE2 ectodomain that possesses all bar the last N-linked glycan site was expressed in Human Embryonic Kidney (HEK) 293F cells (Figure 1(A))

Summary

Introduction

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19, encodes an extensively glycosylated spike (S) protein that protrudes from the viral surface.[1,2] The S protein is a trimeric class I fusion protein, composed of two functional subunits, S1 and S2, which are responsible for receptor binding and membrane fusion, respectively.[1,3] The S protein binds to cell surface angiotensin-converting enzyme 2 (ACE2) which initiates host-cell entry and results in membrane fusion.[4]. To understand the impact of the ACE2 glycosylation upon SARS-CoV-2 binding we first defined the glycans present on the soluble variant used in this study (Figure 1(A)).

Results

Conclusion