scholarly journals Structural O-Glycoform Heterogeneity of the SARS-CoV-2 Spike Protein Receptor-Binding Domain Revealed by Top-Down Mass Spectrometry

2021 ◽  
Author(s):  
David S. Roberts ◽  
Morgan Mann ◽  
Jake A. Melby ◽  
Eli J. Larson ◽  
Yanlong Zhu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Receptor Binding ◽  
Binding Domain ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Top Down ◽  
Protein Receptor ◽  
Top Down Mass Spectrometry

scholarly journals Online Hydrophilic Interaction Chromatography (HILIC) Enhanced Top-Down Mass Spectrometry Characterization of SARS-Cov-2 Spike Receptor Binding Domain

2022 ◽  
Author(s):  
Jesse Wilson ◽  
Aivett Bilbao ◽  
Juan Wang ◽  
Yen-Chen Liao ◽  
Dusan Velickovic ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Receptor Binding ◽  
Binding Domain ◽  
Hydrophilic Interaction Chromatography ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Hydrophilic Interaction ◽  
Top Down

SARS-CoV-2 cellular infection is mediated by the heavily glycosylated spike protein. Recombinant versions of the spike protein and the receptor binding domain (RBD) are necessary for seropositivity assays and can potentially serve as vaccines against viral infection. RBD plays key roles in the spike protein’s structure and function, and thus comprehensive characterization of recombinant RBD is critically important for biopharmaceutical applications. Liquid-chromatography coupled to mass spectrometry (LCMS) has been widely used to characterize post-translational modifications in proteins including glycosylation. Most studies of RBDs were performed at the proteolytic peptide (bottom-up proteomics) or released glycan level because of the technical challenges in resolving highly heterogenous glycans at the intact protein level. Herein, we evaluated several online separation techniques: 1. C2 reverse-phase liquid chromatography (RPLC), 2. capillary zone electrophoresis (CZE), and 3. acrylamide-based monolithic hydrophilic interaction chromatography (HILIC) to separate intact recombinant RBDs with varying combinations of glycosylations (glycoforms) for top-down MS. Within the conditions we explored, the HILIC method was superior to RPLC and CZE at separating RBD glycoforms, which differ significantly in neutral glycan groups. In addition, our top-down analysis readily captured unexpected modifications (e.g., cysteinylation, N-terminal sequence variation) and low abundance, heavily glycosylated proteoforms that may be missed by using glycopeptide data alone. The HILIC top-down MS platform holds great potential in resolving heterogenous glycoproteins for facile comparison of biosimilars in quality control applications.

scholarly journals Conjugation of Human β-Defensin 2 to Spike Protein Receptor-Binding Domain Induces Antigen-Specific Protective Immunity against Middle East Respiratory Syndrome Coronavirus Infection in Human Dipeptidyl Peptidase 4 Transgenic Mice

Vaccines ◽  
2020 ◽  
Vol 8 (4) ◽  
pp. 635
Author(s):  
Ju Kim ◽  
Ye Lin Yang ◽  
Yongsu Jeong ◽  
Yong-Suk Jang
Keyword(s):  
Middle East ◽  
Immune Responses ◽  
Receptor Binding ◽  
Dipeptidyl Peptidase ◽  
Binding Domain ◽  
Middle East Respiratory Syndrome ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Dipeptidyl Peptidase 4 ◽  
Protein Receptor

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe acute respiratory symptoms. Due to the lack of medical countermeasures, effective and safe vaccines against MERS-CoV infection are urgently required. Although different types of candidate vaccines have been developed, their immunogenicity is limited, and the dose and administration route need optimization to achieve optimal protection. We here investigated the potential use of human β-defensin 2 (HBD 2) as an adjuvant to enhance the protection provided by MERS-CoV vaccination. We found that immunization of human dipeptidyl peptidase 4 (hDPP4)-transgenic (hDPP4-Tg) mice with spike protein receptor-binding domain (S RBD) conjugated with HBD 2 (S RBD-HBD 2) induced potent antigen (Ag)-specific adaptive immune responses and protected against MERS-CoV infection. In addition, immunization with S RBD-HBD 2 alleviated progressive pulmonary fibrosis in the lungs of MERS-CoV-infected hDPP4-Tg mice and suppressed endoplasmic reticulum stress signaling activation upon viral infection. Compared to intramuscular administration, intranasal administration of S RBD-HBD 2 induced more potent mucosal IgA responses and was more effective for protecting against intranasal MERS-CoV infection. In conclusion, our findings suggest that HBD 2 potentiates Ag-specific immune responses against viral Ag and can be used as an adjuvant enhancing the immunogenicity of subunit vaccine candidates against MERS-CoV.

scholarly journals Combining a Fusion Inhibitory Peptide Targeting the MERS-CoV S2 Protein HR1 Domain and a Neutralizing Antibody Specific for the S1 Protein Receptor-Binding Domain (RBD) Showed Potent Synergism against Pseudotyped MERS-CoV with or without Mutations in RBD

Viruses ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 31 ◽  
Author(s):  
Cong Wang ◽  
Chen Hua ◽  
Shuai Xia ◽  
Weihua Li ◽  
Lu Lu ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Neutralizing Antibody ◽  
Binding Domain ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Inhibitory Peptide ◽  
Protein Receptor ◽  
Neutralizing Monoclonal Antibody ◽  
Peptide Targeting ◽  
Combinatorial Strategy

Middle East respiratory syndrome coronavirus (MERS-CoV) has continuously posed a threat to public health worldwide, yet no therapeutics or vaccines are currently available to prevent or treat MERS-CoV infection. We previously identified a fusion inhibitory peptide (HR2P-M2) targeting the MERS-CoV S2 protein HR1 domain and a highly potent neutralizing monoclonal antibody (m336) specific to the S1 spike protein receptor-binding domain (RBD). However, m336 was found to have reduced efficacy against MERS-CoV strains with mutations in RBD, and HR2P-M2 showed low potency, thus limiting the clinical application of each when administered separately. However, we herein report that the combination of m336 and HR2P-M2 exhibited potent synergism in inhibiting MERS-CoV S protein-mediated cell–cell fusion and infection by MERS-CoV pseudoviruses with or without mutations in the RBD, resulting in the enhancement of antiviral activity in contrast to either one administered alone. Thus, this combinatorial strategy could be used in clinics for the urgent treatment of MERS-CoV-infected patients.

scholarly journals Can the SARS-CoV-2 Spike Protein Bind Integrins Independent of the RGD Sequence?

2021 ◽  
Vol 11 ◽  
Author(s):  
Christopher A. Beaudoin ◽  
Samir W. Hamaia ◽  
Christopher L.-H. Huang ◽  
Tom L. Blundell ◽  
Antony P. Jackson
Keyword(s):  
Receptor Binding ◽  
Conformational Changes ◽  
Sequence Similarity ◽  
Binding Domain ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Binding Motifs ◽  
Rgd Motif ◽  
Rgd Sequence ◽  
Protein Receptor

The RGD motif in the Severe Acute Syndrome Coronavirus 2 (SARS-CoV-2) spike protein has been predicted to bind RGD-recognizing integrins. Recent studies have shown that the spike protein does, indeed, interact with αVβ3 and α5β1 integrins, both of which bind to RGD-containing ligands. However, computational studies have suggested that binding between the spike RGD motif and integrins is not favourable, even when unfolding occurs after conformational changes induced by binding to the canonical host entry receptor, angiotensin-converting enzyme 2 (ACE2). Furthermore, non-RGD-binding integrins, such as αx, have been suggested to interact with the SARS-CoV-2 spike protein. Other viral pathogens, such as rotaviruses, have been recorded to bind integrins in an RGD-independent manner to initiate host cell entry. Thus, in order to consider the potential for the SARS-CoV-2 spike protein to bind integrins independent of the RGD sequence, we investigate several factors related to the involvement of integrins in SARS-CoV-2 infection. First, we review changes in integrin expression during SARS-CoV-2 infection to identify which integrins might be of interest. Then, all known non-RGD integrin-binding motifs are collected and mapped to the spike protein receptor-binding domain and analyzed for their 3D availability. Several integrin-binding motifs are shown to exhibit high sequence similarity with solvent accessible regions of the spike receptor-binding domain. Comparisons of these motifs with other betacoronavirus spike proteins, such as SARS-CoV and RaTG13, reveal that some have recently evolved while others are more conserved throughout phylogenetically similar betacoronaviruses. Interestingly, all of the potential integrin-binding motifs, including the RGD sequence, are conserved in one of the known pangolin coronavirus strains. Of note, the most recently recorded mutations in the spike protein receptor-binding domain were found outside of the putative integrin-binding sequences, although several mutations formed inside and close to one motif, in particular, may potentially enhance binding. These data suggest that the SARS-CoV-2 spike protein may interact with integrins independent of the RGD sequence and may help further explain how SARS-CoV-2 and other viruses can evolve to bind to integrins.

scholarly journals Bacterial expression and purification of functional recombinant SARS-CoV-2 spike receptor binding domain

2021 ◽  
Author(s):  
Janani Prahlad ◽  
Lucas R. Struble ◽  
William E. Lutz ◽  
Savanna A. Wallin ◽  
Surender Khurana ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Disulfide Bonds ◽  
Rapid Diagnosis ◽  
Binding Domain ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Expression And Purification ◽  
Protein Receptor ◽  
The World ◽  
Active Antigen

AbstractThe COVID-19 pandemic caused by SARS-CoV-2 has applied significant pressure on overtaxed healthcare around the world, underscoring the urgent need for rapid diagnosis and treatment. We have developed a bacterial strategy for the expression and purification of the SARS-CoV-2 spike protein receptor binding domain using the CyDisCo system to create and maintain the correct disulfide bonds for protein integrity and functionality. We show that it is possible to quickly and inexpensively produce functional, active antigen in bacteria capable of recognizing and binding to the ACE2 (angiotensin-converting enzyme) receptor as well as antibodies in COVID-19 patient sera.

scholarly journals Protein Footprinting, Conformational Dynamics and Interface-Adjacent Neutralization ‘Hotspots’ in the SARS-CoV-2 Spike Protein Receptor Binding Domain / Human ACE2 Interaction

2020 ◽  
Author(s):  
Dominic Narang ◽  
Matthew Balmer ◽  
D. Andrew James ◽  
Derek Wilson
Keyword(s):  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Conformational Dynamics ◽  
Binding Domain ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Angiotensin Converting Enzyme 2 ◽  
Additional Information ◽  
Protein Receptor ◽  
Hdx Ms

This study provides an HDX-MS based analysis of the interaction between the SARS-CoV-2 spike protein and the human Angiotensin Converting Enzyme 2. <div><br></div><div>- The data agree exactly with the X-ray co-crystal structure of this complex, but provide additional information based on shifts in dynamics that are observed just outside the interface. </div><div><br></div><div>- These dynamic changes occur specifically in regions that are the primary targets of neutralizing antibodies that target spike protein, suggesting that the neutralization mechanism may result from suppression of dynamic shifts in the spike Receptor Binding Domain (RBD) that are necessary for favorable binding thermodynamics in the spike / ACE2 interaction.</div>

scholarly journals Polymersomes decorated with SARS-CoV-2 spike protein receptor binding domain elicit robust humoral and cellular immunity

2021 ◽  
Author(s):  
Lisa R Volpatti ◽  
Rachel P Wallace ◽  
Shijie Cao ◽  
Michal Raczy ◽  
Ruyi Wang ◽  
...  
Keyword(s):  
T Cell ◽  
Antibody Response ◽  
Receptor Binding ◽  
Neutralizing Antibody ◽  
Binding Domain ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Vaccination Site ◽  
Monophosphoryl Lipid A ◽  
Protein Receptor

A diverse portfolio of SARS-CoV-2 vaccine candidates is needed to combat the evolving COVID-19 pandemic. Here, we developed a subunit nanovaccine by conjugating SARS-CoV-2 Spike protein receptor binding domain (RBD) to the surface of oxidation-sensitive polymersomes. We evaluated the humoral and cellular responses of mice immunized with these surface-decorated polymersomes (RBDsurf) compared to RBD-encapsulated polymersomes (RBDencap) and unformulated RBD (RBDfree), using monophosphoryl lipid A-encapsulated polymersomes (MPLA PS) as an adjuvant. While all three groups produced high titers of RBD-specific IgG, only RBDsurf elicited a neutralizing antibody response to SARS-CoV-2 comparable to that of human convalescent plasma. Moreover, RBDsurf was the only group to significantly increase the proportion of RBD-specific germinal center B cells in the vaccination-site draining lymph nodes. Both RBDsurf and RBDencap drove similarly robust CD4+ and CD8+ T cell responses that produced multiple Th1-type cytokines. We conclude that multivalent surface display of Spike RBD on polymersomes promotes a potent neutralizing antibody response to SARS-CoV-2, while both antigen formulations promote robust T cell immunity.

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