Comprehensive characterization of N- and O- glycosylation of SARS-CoV-2 human receptor angiotensin converting enzyme 2

Abstract

The emergence of the COVID-19 pandemic caused by SARS-CoV-2 has created the need for development of new therapeutic strategies. Understanding the mode of viral attachment, entry and replication has become a key aspect of such interventions. The coronavirus surface features a trimeric spike (S) protein that is essential for viral attachment, entry and membrane fusion. The S protein of SARS-CoV-2 binds to human angiotensin converting enzyme 2 (hACE2) for entry. Herein, we describe glycomic and glycoproteomic analysis of hACE2 expressed in HEK293 cells. We observed high glycan occupancy (73.2 to 100%) at all seven possible N-glycosylation sites and surprisingly detected one novel O-glycosylation site. To deduce the detailed structure of glycan epitopes on hACE2 that may be involved in viral binding, we have characterized the terminal sialic acid linkages, the presence of bisecting GlcNAc, and the pattern of N-glycan fucosylation. We have conducted extensive manual interpretation of each glycopeptide and glycan spectrum, in addition to using bioinformatics tools to validate the hACE2 glycosylation. Our elucidation of the site-specific glycosylation and its terminal orientations on the hACE2 receptor, along with the modeling of hACE2 glycosylation sites can aid in understanding the intriguing virus-receptor interactions and assist in the development of novel therapeutics to prevent viral entry. The relevance of studying the role of ACE2 is further increased due to some recent reports about the varying ACE2 dependent complications with regard to age, sex, race, and pre-existing conditions of COVID-19 patients.

Data, Reagents, Assays and Merits of Proteomics for SARS-CoV-2 Research and Testing

“…Although not covered in this work, it is well established that virus-host interactions are heavily reliant on post-translational modifications (PTMs) on virus and host proteins. Both the virus spike protein as well as the host receptors ACE2 and TMPRSS2 are glycoproteins and several studies are emerging that characterize the glycosylation pattern of individual proteins ( 60 , 61 , 62 , 63 ). Unfortunately, global and protein-specific surface glycosylation profiling is still challenging but could become an important future avenue to understand better how complex glycan patterns both of the virus as well as host cells determine the tropism of SARS-CoV-2.…”

Structural analysis of COVID-19 spike protein in recognizing the ACE2 receptor of different mammalian species and its susceptibility to viral infection

“…Besides, the absence of a glycosylation site at position Asn103 in all other mammals also plays an essential role in spike recognition of the ACE2 receptor. In human, the ACE2 receptor contains seven N-linked glycosylation sites (Asn53, Asn90, Asn103, Asn322, Asn432, Asn546 and Asn690) and three O-linked glycosylation sites (S155, S496 and S730) (Shajahan et al 2020a ). Out of seven N-linked glycosylation sites, five (Asn53, Asn90, Asn103, Asn322 and Asn546) are aligned close to the spike 2 recognition site and might have positive role in binding with spike 2 protein thereby increasing the binding affinity.…”

“…By LC–MS/MS, a particular pattern of sugar moieties were reported for both N-linked and O-linked glycosylation sites. In N-linked sites, a bisecting N-acetyl Glucosamine, an N-glycan fucosylation with the sialic acid as terminal sugar moiety was observed (Shajahan et al 2020a ; Zhao et al 2020a ). While, in the crystal structures of the human ACE2 receptor (PDB ID: 6M0J and 6M18) sugar moieties were observed at the N-linked glycosylation sites Asn53, Asn90, Asn103, Asn322, Asn432, Asn546 and Asn690.…”

“…2 ). Based on the previously reported study (Shajahan et al 2020a ) the sugar molecules were modeled at these N-linked glycosylation sites as shown in Fig. 2 b.…”

“…Although there is the loss of a crucial network of interactions, individual interactions were observed between SARS-CoV-2 spike protein and the ACE2 receptor. To form a stable complex the glycan moieties of SARS-CoV-2 spike protein interact with the glycans of the ACE2 receptor (Han et al 2007b; Shajahan et al 2020b ; Zhao et al 2020a ). Hence, the glycan-glycan interaction between ACE2 receptor and SARS-CoV-2 spike protein could contribute to the susceptibility for infections.…”