Altered N-/O-Glycosylation Sites On The Receptor-Binding Domain (RBD) in COVID-19 Are Related To Coronavirus Infection And Pathogenesis.

Abstract Objective: To discuss the possible significances of N-Linked and O-Linked glycosylation on the RBD of COVID-19Methods: Amino acid sequences multiple alignments of RBD used Clustal Omega (v.1.2.4) using default parameters. Prediction of potential N-linked glycosylation sites by NetNGlyc 1.0 server. Prediction of potential O-linked glycosylation sites by NetOGlyc 4.0 Server.Result: COVID-19 Spike glycoprotein has 22 potential N-linked glycosylation sites and 3 O-linked glycosylation sites. 2 of 22 N-linked glycosylation sites distributed in RBD. None of the 3 O-linked glycosylation sites distributed in RBD, which is markedly different from SARS and other bat coronavirus using ACE2 as a receptor. Comparing with its close coronavirus, but which can’t use ACE2 as a receptor, the COVID-19 has little N- and O-linked glycosylation sites.Conclusion: we show the obvious differences in glycosylation sites in RBD between COVID-19 and other coronaviruses. We speculate that altered N-/O-glycosylation sites on RBD in COVID-19 are related to its infection and pathogenesis.

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An Engineered Receptor-Binding Domain Improves the Immunogenicity of Multivalent SARS-CoV-2 Vaccines

mBio ◽

10.1128/mbio.00930-21 ◽

2021 ◽

Vol 12 (3) ◽

Author(s):

Yan Guo ◽

Wenhui He ◽

Huihui Mou ◽

Lizhou Zhang ◽

Jing Chang ◽

...

Keyword(s):

Amino Acid ◽

Receptor Binding ◽

Binding Domain ◽

Full Length ◽

Vaccine Antigen ◽

Receptor Binding Domain ◽

Protein Vaccine ◽

Wild Type ◽

S Protein ◽

Glycosylation Sites

ABSTRACT The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates viral entry into cells expressing angiotensin-converting enzyme 2 (ACE2). The S protein engages ACE2 through its receptor-binding domain (RBD), an independently folded 197-amino-acid fragment of the 1,273-amino-acid S-protein protomer. The RBD is the primary SARS-CoV-2 neutralizing epitope and a critical target of any SARS-CoV-2 vaccine. Here, we show that this RBD conjugated to each of two carrier proteins elicited more potent neutralizing responses in immunized rodents than did a similarly conjugated proline-stabilized S-protein ectodomain. Nonetheless, the native RBD is expressed inefficiently, limiting its usefulness as a vaccine antigen. However, we show that an RBD engineered with four novel glycosylation sites (gRBD) is expressed markedly more efficiently and generates a more potent neutralizing responses as a DNA vaccine antigen than the wild-type RBD or the full-length S protein, especially when fused to multivalent carriers, such as a Helicobacter pylori ferritin 24-mer. Further, gRBD is more immunogenic than the wild-type RBD when administered as a subunit protein vaccine. Our data suggest that multivalent gRBD antigens can reduce costs and doses, and improve the immunogenicity, of all major classes of SARS-CoV-2 vaccines. IMPORTANCE All available vaccines for coronavirus disease 2019 (COVID-19) express or deliver the full-length SARS-CoV-2 spike (S) protein. We show that this antigen is not optimal, consistent with observations that the vast majority of the neutralizing response to the virus is focused on the S-protein receptor-binding domain (RBD). However, this RBD is not expressed well as an independent domain, especially when expressed as a fusion protein with a multivalent scaffold. We therefore engineered a more highly expressed form of the SARS-CoV-2 RBD by introducing four glycosylation sites into a face of the RBD normally occluded in the full S protein. We show that this engineered protein, gRBD, is more immunogenic than the wild-type RBD or the full-length S protein in both genetic and protein-delivered vaccines.

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Rise and Fall of SARS-CoV-2 Lineage A.27 in Germany

Viruses ◽

10.3390/v13081491 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1491

Author(s):

Sébastien Calvignac-Spencer ◽

Matthias Budt ◽

Matthew Huska ◽

Hugues Richard ◽

Luca Leipold ◽

...

Keyword(s):

Amino Acid ◽

Receptor Binding ◽

Relative Abundance ◽

Immune Escape ◽

Amino Acid Substitutions ◽

Receptor Binding Domain ◽

Spike Glycoprotein ◽

German State ◽

Mutational Pattern ◽

B Lineage

Here, we report on the increasing frequency of the SARS-CoV-2 lineage A.27 in Germany during the first months of 2021. Genomic surveillance identified 710 A.27 genomes in Germany as of 2 May 2021, with a vast majority identified in laboratories from a single German state (Baden-Wuerttemberg, n = 572; 80.5%). Baden-Wuerttemberg is located near the border with France, from where most A.27 sequences were entered into public databases until May 2021. The first appearance of this lineage based on sequencing in a laboratory in Baden-Wuerttemberg can be dated to early January ’21. From then on, the relative abundance of A.27 increased until the end of February but has since declined—meanwhile, the abundance of B.1.1.7 increased in the region. The A.27 lineage shows a mutational pattern typical of VOIs/VOCs, including an accumulation of amino acid substitutions in the Spike glycoprotein. Among those, L18F, L452R and N501Y are located in the epitope regions of the N-terminal- (NTD) or receptor binding domain (RBD) and have been suggested to result in immune escape and higher transmissibility. In addition, A.27 does not show the D614G mutation typical for all VOIs/VOCs from the B lineage. Overall, A.27 should continue to be monitored nationally and internationally, even though the observed trend in Germany was initially displaced by B.1.1.7 (Alpha), while now B.1.617.2 (Delta) is on the rise.

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Murine Coronavirus Evolution In Vivo: Functional Compensation of a Detrimental Amino Acid Substitution in the Receptor Binding Domain of the Spike Glycoprotein

Journal of Virology ◽

10.1128/jvi.79.12.7629-7640.2005 ◽

2005 ◽

Vol 79 (12) ◽

pp. 7629-7640 ◽

Cited By ~ 18

Author(s):

Sonia Navas-Martin ◽

Susan T. Hingley ◽

Susan R. Weiss

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Receptor Binding ◽

Amino Acid Substitution ◽

Binding Domain ◽

Receptor Binding Domain ◽

Spike Glycoprotein ◽

Functional Compensation

ABSTRACT Murine coronavirus A59 strain causes mild to moderate hepatitis in mice. We have previously shown that mutants of A59, unable to induce hepatitis, may be selected by persistent infection of primary glial cells in vitro. These in vitro isolated mutants encoded two amino acids substitutions in the spike (S) gene: Q159L lies in the putative receptor binding domain of S, and H716D, within the cleavage signal of S. Here, we show that hepatotropic revertant variants may be selected from these in vitro isolated mutants (Q159L-H716D) by multiple passages in the mouse liver. One of these mutants, hr2, was chosen for more in-depth study based on a more hepatovirulent phenotype. The S gene of hr2 (Q159L- R654H -H716D- E1035D ) differed from the in vitro isolates (Q159L-H716D) in only 2 amino acids (R654H and E1035D). Using targeted RNA recombination, we have constructed isogenic recombinant MHV-A59 viruses differing only in these specific amino acids in S (Q159L-R654H-H716D-E1035D). We demonstrate that specific amino acid substitutions within the spike gene of the hr2 isolate determine the ability of the virus to cause lethal hepatitis and replicate to significantly higher titers in the liver compared to wild-type A59. Our results provide compelling evidence of the ability of coronaviruses to rapidly evolve in vivo to highly virulent phenotypes by functional compensation of a detrimental amino acid substitution in the receptor binding domain of the spike glycoprotein.

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Identification of a SARS-like bat coronavirus that shares structural features with the spike glycoprotein receptor-binding domain of SARS-CoV-2

Access Microbiology ◽

10.1099/acmi.0.000166 ◽

2020 ◽

Vol 2 (11) ◽

Author(s):

Conchita Fraguas Bringas ◽

David Booth

Keyword(s):

Receptor Binding ◽

Structural Features ◽

Binding Domain ◽

Receptor Binding Domain ◽

Spike Glycoprotein ◽

Angiotensin Converting Enzyme 2 ◽

Reliable Model ◽

High Conservation ◽

Host Range Determination ◽

Bat Coronavirus

SARS-CoV-2 is a recently emerged coronavirus that binds angiotensin-converting enzyme 2 (ACE2) for cell entry via its receptor-binding domain (RBD) on a surface-expressed spike glycoprotein. Studies show that despite its similarities to severe acute respiratory syndrome (SARS) coronavirus, there are critical differences in key RBD residues when compared to SARS-CoV-2. Here we present a short in silico study, showing that SARS-like bat coronavirus Rs3367 shares a high conservation with SARS-CoV-2 in important RBD residues for ACE2 binding: SARS-CoV-2’s Phe486, Thr500, Asn501 and Tyr505; implicated in receptor-binding strength and host-range determination. These features were not shared with other studied bat coronaviruses belonging to the betacoronavirus genus, including RaTG13, the closest reported bat coronavirus to SARS-CoV-2’s spike protein. Sequence and phylogeny analyses were followed by the computation of a reliable model of the RBD of SARS-like bat coronavirus Rs3367, which allowed structural insight of the conserved residues. Superimposition of this model on the SARS-CoV-2 ACE2-RBD complex revealed critical ACE2 contacts are also maintained. In addition, residue Asn488Rs3367 interacted with a previously defined pocket on ACE2 composed of Tyr41, Lys353 and Asp355. When compared to available SARS-CoV-2 crystal structure data, Asn501SARS-CoV-2 showed a different interaction with the ACE2 pocket. Taken together, this study offers molecular insights on RBD-receptor interactions with implications for vaccine design.

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Peptide inhibitors of the interaction of the SARS-CoV-2 receptor-binding domain with the ACE2 cell receptor

Biomeditsinskaya Khimiya ◽

10.18097/pbmc20216703244 ◽

2021 ◽

Vol 67 (3) ◽

pp. 244-250

Author(s):

R.Sh. Bibilashvili ◽

M.V. Sidorova ◽

U.S. Dudkina ◽

M.E. Palkeeva ◽

A.S. Molokoedov ◽

...

Keyword(s):

Amino Acid ◽

Receptor Binding ◽

Disulfide Bonds ◽

Protein S ◽

Cell Receptor ◽

Binding Domain ◽

Amino Acid Sequences ◽

Receptor Binding Domain ◽

Amino Acid Residues ◽

Chimeric Molecules

Computer simulation has been used to identify peptides that mimic the natural target of the SARS-CoV-2 coronavirus spike (S) protein, the angiotensin converting enzyme type 2 (ACE2) cell receptor. Based on the structure of the complex of the protein S receptor-binding domain (RBD) and ACE2, the design of chimeric molecules consisting of two 22-23-mer peptides linked to each other by disulfide bonds was carried out. The chimeric molecule X1 was a disulfide dimer, in which edge cysteine residues in the precursor molecules h1 and h2 were connected by the S-S bond. In the chimeric molecule X2, the disulfide bond was located in the middle of the molecule of each of the precursor peptides. The precursors h1 and h2 modelled amino acid sequences of α1- and α2-helices of the extracellular peptidase domain of ACE2, respectively, keeping intact most of the amino acid residues involved in the interaction with RBD. The aim of the work was to evaluate the binding efficiency of chimeric molecules and their RBD-peptides (particularly in dependence of the middle and edge methods of fixing the initial peptides h1 and h2). The proposed polypeptides and chimeric molecules were synthesized by chemical methods, purified (to 95-97% purity), and characterized by HPLC and MALDI-TOF mass spectrometry. The binding of the peptides to the SARS-CoV-2 RBD was evaluated by microthermophoresis with recombinant domains corresponding in sequence to the original Chinese (GenBank ID NC_045512.2) and the British (B. 1.1.7, GISAID EPI_ISL_683466) variants. Binding to the original RBD of the Chinese variant was detected in three synthesized peptides: linear h2 and both chimeric variants. Chimeric peptides were also bound to the RBD of the British variant with micromolar constants. The antiviral activity of the proposed peptides in Vero cell culture was also evaluated.

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Structure and binding properties of Pangolin-CoV spike glycoprotein inform the evolution of SARS-CoV-2

Nature Communications ◽

10.1038/s41467-021-21006-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Antoni G. Wrobel ◽

Donald J. Benton ◽

Pengqi Xu ◽

Lesley J. Calder ◽

Annabel Borg ◽

...

Keyword(s):

Receptor Binding ◽

Binding Domain ◽

Spike Protein ◽

Receptor Binding Domain ◽

Spike Glycoprotein ◽

Binding Properties ◽

Origin And Evolution ◽

Closed Conformation ◽

Bat Coronavirus

AbstractCoronaviruses of bats and pangolins have been implicated in the origin and evolution of the pandemic SARS-CoV-2. We show that spikes from Guangdong Pangolin-CoVs, closely related to SARS-CoV-2, bind strongly to human and pangolin ACE2 receptors. We also report the cryo-EM structure of a Pangolin-CoV spike protein and show it adopts a fully-closed conformation and that, aside from the Receptor-Binding Domain, it resembles the spike of a bat coronavirus RaTG13 more than that of SARS-CoV-2.

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Structure and binding properties of Pangolin-CoV Spike glycoprotein inform the evolution of SARS-CoV-2.

10.21203/rs.3.rs-83072/v1 ◽

2020 ◽

Author(s):

Antoni G. Wrobel ◽

Donald J. Benton ◽

Pengqi Xu ◽

Annabel Borg ◽

Chloë Roustan ◽

...

Keyword(s):

Receptor Binding ◽

Binding Domain ◽

Receptor Binding Domain ◽

Spike Glycoprotein ◽

Binding Properties ◽

Origin And Evolution ◽

Closed Conformation ◽

Bat Coronavirus

Abstract Coronaviruses of bats and pangolins are implicated in the origin and evolution of the pandemic SARS-CoV-2. We show that Spikes from Pangolin-CoVs, closely related to SARS-CoV-2, bind strongly to human and pangolin ACE2 receptors. We also report Cryo-EM structure of Pangolin-CoV S and show it adopts a fully-closed conformation and that, aside from the Receptor-Binding Domain, it resembles the spike of a bat coronavirus RaTG13 more than that of SARS-CoV-2.

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Role of key point Mutations in Receptor Binding Domain of SARS-CoV-2 Spike Glycoprotein

Infectious Disorders - Drug Targets ◽

10.2174/1871526520666200804161650 ◽

2020 ◽

Vol 20 ◽

Author(s):

Bipin Singh

Keyword(s):

Receptor Binding ◽

Point Mutations ◽

Binding Domain ◽

Receptor Binding Domain ◽

Spike Glycoprotein ◽

Angiotensin Converting Enzyme 2 ◽

Recent Outbreak ◽

Novel Coronavirus ◽

Genomic Similarity

: The recent outbreak of novel coronavirus (SARS-CoV-2 or 2019-nCoV) and its worldwide spread is posing one of the major threats to human health and the world economy. It has been suggested that SARS-CoV-2 is similar to SARSCoV based on the comparison of the genome sequence. Despite the genomic similarity between SARS-CoV-2 and SARSCoV, the spike glycoprotein and receptor binding domain in SARS-CoV-2 shows the considerable difference compared to SARS-CoV, due to the presence of several point mutations. The analysis of receptor binding domain (RBD) from recently published 3D structures of spike glycoprotein of SARS-CoV-2 (Yan, R., et al. (2020); Wrapp, D., et al. (2020); Walls, A. C., et al. (2020)) highlights the contribution of a few key point mutations in RBD of spike glycoprotein and molecular basis of its efficient binding with human angiotensin-converting enzyme 2 (ACE2).

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Cloning and sequence analysis of cDNAs encoding the α1, α2 and α3 chains of mouse collagen VI

Biochemical Journal ◽

10.1042/bj2910787 ◽

1993 ◽

Vol 291 (3) ◽

pp. 787-792 ◽

Cited By ~ 8

Author(s):

R Z Zhang ◽

T C Pan ◽

R Timpl ◽

M L Chu

Keyword(s):

Amino Acid ◽

Amino Acid Sequences ◽

Untranslated Regions ◽

Cdna Libraries ◽

Cdna Clones ◽

Collagen Vi ◽

Central Segment ◽

Glycosylation Sites ◽

Key Features ◽

Triple Helical Domain

cDNA clones encoding the alpha 1, alpha 2 and alpha 3 chains of mouse collagen VI have been isolated by screening cDNA libraries with the corresponding human probes. The composite cDNAs for the alpha 1, alpha 2, and alpha 3 chains are 2.5, 1.6 and 2.9 kb in size respectively. The alpha 1 and alpha 2 cDNAs encode the C-terminal portions of the chains as well as the entire 3′-untranslated regions, while the alpha 3 cDNAs encode a central segment of 959 amino acids flanking the triple-helical domain. The deduced amino acid sequences share 86-88% identity with the human counterparts and 67-73% identity with the chicken equivalents. Alignment of the deduced amino acid sequences of mouse, human and chicken collagens reveal that the key features of the protein, including the cysteine residues, imperfections in the Gly-Xaa-Xaa regions, Arg-Gly-Asp sequences and potential N-glycosylation sites, are mostly conserved.

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ReaxFF-based molecular dynamics simulation of the interaction between plasma ROS and the receptor-binding domain of the spike protein in the capsid protein of SARS-CoV-2

Journal of Physics D Applied Physics ◽

10.1088/1361-6463/ac360e ◽

2021 ◽

Author(s):

Huichao Wang ◽

Tong Zhao ◽

Shuhui Yang ◽

Liang Zou ◽

Xiaolong Wang ◽

...

Keyword(s):

Molecular Dynamics ◽

Amino Acid ◽

Capsid Protein ◽

Receptor Binding ◽

Hydrogen Abstraction ◽

Binding Domain ◽

Spike Protein ◽

Receptor Binding Domain ◽

Amino Acid Residues ◽

Global Epidemic

Abstract Under the severe situation of the current global epidemic, researchers have been working hard to find a reliable way to suppress the infection of the virus and prevent the spread of the epidemic. Studies have shown that the recognition and binding of human angiotensin-converting enzyme 2 (ACE2) by the receptor-binding domain (BRD) of spike protein on the surface of SARS-CoV-2 is a crucial step for SARS-CoV-2 to invade human receptor cells, and blocking this process can inhibit the virus from invading human normal cells. Plasma treatment can disrupt the structure of the RBD and effectively block the binding process. However, the mechanism by which plasma blocks the recognition and binding between the two is not clear. In this study, reaction process between reactive oxygen species (ROS) in plasma and the molecular model of RBD was simulated using a reactive molecular dynamics method. The results showed that the destruction of RBD molecule by ROS was triggered by hydrogen abstraction reactions. O and OH abstracted H atoms from RBD, while the H atoms of H2O2 and HO2 were abstracted by RBD. The hydrogen abstraction resulted in the breakage of C-H, N-H, O-H and C=O bonds and the formation of C=C, C=N bonds. The addition reaction of OH increased the number of O-H bonds and caused the formation of C-O, N-O and O-H bonds. The dissociation of N-H bonds led to the destruction of the original structure of peptide bonds and amino acid residues, change the type of amino acid residues, and caused the conversion of N-C and N=C, C=O and C-O. The simulation partially elucidated the microscopic mechanism of the interaction between ROS in plasma and the capsid protein of SARS-CoV-2, providing theoretical support for the control of SARS-CoV-2 infection by plasma, a contribution to overcoming the global epidemic problem.

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