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Authors: Sun, Z. et al

Link to paper: https://www.biorxiv.org/content/10.1101/2020.04.29.068098v1

Journal/ Pre-Print: bioRxiv

Tags: Molecular biology; Modelling; Proteomics; Structural biology

Research Highlights 

1. In vitro investigation of post-translational modifications (mainly glycans, also methyl and hydroxyproline groups) of recombinant SARS-CoV-2 S and human ACE2 proteins followed by structural modelling of glycosylation

2. Glycans camouflage >65% of the SARS-CoV-2 S protein

3. Binding affinity between SARS-CoV-2 S protein and ACE2 is not affected by the presence of absence of glycans.

Summary 

The authors performed analysis of purified recombinant SARS-CoV-2 S and human ACE2 proteins expressed by insect-derived Sf9 cells, in order to determine the position and compositions of post-translational modifications (PTMs), including glycans, methylation, and hydroxyproline. The authors identified multiple glycosylation sites (20/21 possible glycosites in S protein, 7/7 in ACE2), which suggest camouflage by glycans of the S protein when the PTMs are added to the cryo-EM structures of the S and ACE2 proteins. Additionally, the authors suggest that the binding affinity of the S protein to ACE2 is not affected by glycosylation.

Impact for SARS-CoV2/COVID19 research efforts

Understand the virology and/or cell biology of SARS-CoV2/COVID19

Binding affinity between SARS-CoV-2 S protein and ACE2 is not affected by the presence of absence of glycans.

Understand the immune response to SARS-CoV2/COVID19

Develop a vaccine for SARS-CoV2/COVID19

The presence of a large amount of glycans could affect the development of vaccines through modulation of protein immunogenicity.

Study Type

In vitro study

Strengths and limitations of the paper

Novelty: Determination of glycosylation on SARS-CoV-2 S protein and human ACE2. The authors found that glycans conceal the vast majority of the S protein, which could help immune evasion. Similarly to SARS-CoV1, binding affinity between the S protein and ACE2 is not affected by glycosylation of the S protein and ACE2. The authors also found methylation and hydroxyproline in the S protein and ACE2.

Standing in the field: The large coverage by glycans of the SARS-CoV-2 S protein and the binding affinity between the S protein and ACE2 are in agreement with the literature. Several other aspects of SARS-CoV-2 S protein, mostly oligomannose glycans, no or few sialic acid

incorporation, no phosphorylation and acetylation, are in line with what have been observed for SARS-CoV1. Similarly, deglycosylation of ACE2 does not affect the binding affinity of the SARS-CoV1 spike protein.

Appropriate statistics: Mention of duplicates, but actually only one sample was tested for some of the in vitro work. Should really have been triplicates of each condition. Statistics described for the proteomics analysis.

Viral model used: Wuhan Hu-1; GenBank number QHD43416.1) 115 was synthesized (Genscript) with codons optimized for insect cell expression.

Translatability: The presence of a large amount of glycans could affect development of an immune response, as well as development of vaccines through modulation of protein immunogenicity.Main limitations: No discussion/ reference of other PTM studies (e.g. https://www.biorxiv.org/content/10.1101/2020.04.01.020966v1; https://www.biorxiv.org/content/10.1101/2020.04.07.030445v1; https://science.sciencemag.org/content/early/2020/05/01/science.abb9983). Glycosylation status of ACE2 and S protein has been obtained following purification from insect cell and HEK 293 cells, respectively, which well might not represent the glycosylation outcome in humans or with live viruses. In line with other research in the field, it is likely that the glycan shield is not as extensive and likely to be less polymannose than seen here in a real virus and trimer. For the ACE2-SARS-CoV-2 S protein-protein binding assay, the authors did not test deglycosylated ACE2 with deglycosylated SARS-CoV-2 S, which is an important control to determine whether glycosylation affects protein-protein interaction. It remains unknown whether the presence of two other PTMs, methylation and hydroxyproline, in the S protein and ACE2 have any biological function.