Conserved role of spike S2 domain N-glycosylation across betacoronaviruses

npj Viruses, Journal Year: 2025, Volume and Issue: 3(1)

Published: Jan. 25, 2025

Abstract Besides acting as an immunological shield, the N-glycans of SARS-CoV-2 are also critical for viral life cycle. As S2 subunit spike is highly conserved across betacoronaviruses, we determined functional significance five ‘stem N-glycans’ located in between N1098-N1194. Studies were performed with 31 Asn-to-Gln mutants, betacoronavirus virus-like particles and single-cycle replicons. Deletions stem enhanced S1 shedding from trimeric spike, reduced ACE2 binding abolished syncytia formation. When three or more deleted, expression on cell surface incorporation into virions was both reduced. Viral entry function progressively lost upon deleting N1098 glycan combination additional glycosite modifications. In addition to SARS-CoV-2, SARS-CoV MERS-CoV prevented target cells. These data suggest multiple roles N-glycans, evolutionarily properties these complex carbohydrates human betacoronaviruses.

Language: Английский

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N-Glycome Profile of the Spike Protein S1: Systemic and Comparative Analysis from Eleven Variants of SARS-CoV-2

Biomolecules, Journal Year: 2023, Volume and Issue: 13(9), P. 1421 - 1421

Published: Sept. 20, 2023

The SARS-CoV-2 virus rapidly spread worldwide, threatening public health. Since it emerged, the scientific community has been engaged in development of effective therapeutics and vaccines. subunit S1 spike protein mediates viral entry into host is therefore one major research targets. extensively glycosylated, there compelling evidence that glycans protect virus’ active site from human defense system. Therefore, investigation glycome alterations different variants will provide a view glycan evolution its relationship with pathogenesis. In this study, we explored N-glycosylation expression for eleven variants: five concern (VOC), including alpha, beta, gamma, delta, omicron, six interest (VOI), epsilon, eta, iota, lambda, kappa, mu. results showed significant differences N-glycome abundance all variants. VOC large increase sialofucosylated glycans, greatest omicron variant. contrast, fucosylated most VOI. Two compositions, GlcNAc4,Hex5,Fuc,NeuAc (4-5-1-1) GlcNAc6,Hex8,Fuc,NeuAc (6-8-1-1), were abundant structures across We believe our data contribute to understanding protein’s structural between mutations.

Language: Английский

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Near-Infrared Light Exposure Triggers ROS to Downregulate Inflammatory Cytokines Induced by SARS-CoV-2 Spike Protein in Human Cell Culture

Antioxidants, Journal Year: 2023, Volume and Issue: 12(10), P. 1824 - 1824

Published: Oct. 2, 2023

The leading cause of mortality from SARS-CoV-2 is an exaggerated host immune response, triggering cytokine storms, multiple organ failure and death. Current drug- vaccine-based therapies are limited efficacy against novel viral variants. Infrared therapy a non-invasive safe method that has proven effective inflammatory conditions for over 100 years. However, its mechanism action poorly understood not received widespread acceptance. We herein investigate whether near-infrared (NIR) light exposure in human primary alveolar macrophage cells could downregulate cytokines triggered by the spike (S) protein or lipopolysaccharide (LPS), via what underlying mechanism. Our results showed dramatic reduction pro-inflammatory within days NIR treatment, while anti-inflammatory were upregulated. Mechanistically, stimulated mitochondrial metabolism, induced transient bursts reactive oxygen species (ROS) activated antioxidant gene transcription. These, turn, downregulated ROS cytokines. A causal relationship was shown between induction cellular downregulation S. If confirmed clinical trials, this would provide immediate defense variants other infectious diseases.

Language: Английский

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Enhanced Surface Accessibility of SARS-CoV-2 Omicron Spike Protein Due to an Altered Glycosylation Profile

ACS Infectious Diseases, Journal Year: 2024, Volume and Issue: 10(6), P. 2032 - 2046

Published: May 10, 2024

SARS-CoV-2 spike (S) proteins undergo extensive glycosylation, aiding in proper folding, enhancing stability, and evading host immune surveillance. In this study, we used mass spectrometric analysis to elucidate the N-glycosylation characteristics disulfide bonding of recombinant derived from Omicron variant (B.1.1.529) comparison with D614G variant. Furthermore, conducted microsecond-long molecular dynamics simulations on resolve how different N-glycans impact conformational sampling two variants. Our findings reveal that protein maintains an overall resemblance terms site-specific glycan processing bond formation. Nonetheless, alterations glycans were observed at certain sites. These changes, synergy mutations within protein, result increased surface accessibility macromolecule, including ectodomain, receptor-binding domain, N-terminal domain. Additionally, mutagenesis pull-down assays role glycosylation a specific sequon (N149); furthermore, correlation MD simulation HDX-MS identified several high-dynamic areas proteins. insights contribute our understanding interplay between structure function, thereby advancing effective vaccination therapeutic strategies.

Language: Английский

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Glycoprofile Comparison of the SARS-CoV-2 Spike Proteins Expressed in CHO and HEK Cell Lines

Molecular Biotechnology, Journal Year: 2024, Volume and Issue: unknown

Published: Oct. 1, 2024

Abstract Coronavirus SARS-CoV-2 spike protein remains a key focus of research due to continued need for diagnostic and therapeutic tools monitor respond new variants. Glycosylation the is critical protein’s functions in viral attachment host cell entry. For scalable cost-effective production protein, expression system-driven divergence glycosylation patterns on recombinant proteins needs be fully understood. This study assessed N -glycosylation profiles full-length trimeric expressed either Human Embryonic Kidney (HEK Expi293F) or Chinese Hamster Ovary (CHO-S) cells. Glycopeptide analysis was performed using tandem mass spectrometry workflow BioPharma $${\hbox {Finder}}^{\textrm{TM}}$$ Finder TM incorporating HEK CHO glycan databases characterisation. The results outline important differences variety types -glycan generated by two lines across 22 known sites protein. A notable increase terminal sialylation, as well presence potentially immunogenic -glycolylneuraminic acid at functionally site, observed CHO-S derived With potential relatively vast more complex repertoire (182 glycans relative 39 human glycans) produce functional implications with this adds valuable knowledge aid Quality Design approaches enable Multi Attribute Monitoring specific proteoform analyses. can further inform antigen development future variants order devise updated tests vaccine designs.

Language: Английский

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Structural and Functional Insights into the Evolution of SARS-CoV-2 KP.3.1.1 Spike Protein

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 10, 2024

Summary The JN.1-sublineage KP.3.1.1 recently emerged as the globally prevalent SARS-CoV-2 variant, demonstrating increased infectivity and antibody escape. We investigated how mutations a deletion in spike protein (S) affect ACE2 binding Mass spectrometry revealed new glycan site at residue N30 altered glycoforms neighboring N61. Cryo-EM structures showed that rearrangement of adjacent residues did not significantly change overall structure, up-down ratio receptor-binding domains (RBDs), or binding. Furthermore, S structure with hACE2 further confirmed an epistatic effect between F456L Q493E on Our analysis shows variants after late 2023 are now incorporating reversions to found other sarbecoviruses, including glycan, Q493E, others. Overall, these results inform structural functional consequences mutations, current evolutionary trajectory, immune evasion.

Language: Английский

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Conserved role of spike S2 domain N-glycosylation across beta-coronavirus family

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Sept. 5, 2024

Abstract Besides acting as an immunological shield, the N-glycans of SARS-CoV-2 are also critical for viral life cycle. As S2 subunit spike is highly conserved across beta-coronaviruses, we determined functional significance five ‘stem N-glycans’ located in between N1098-N1194. Studies were performed with 31 Asn-to-Gln mutants, beta-coronavirus virus-like particles and single-cycle replicons. Deletions stem enhanced S1 shedding from trimeric spike, reduced ACE2 binding abolished syncytia formation. When three or more deleted, expression on cell surface incorporation into virions was both reduced. Viral entry function progressively lost upon deleting N1098 glycan combination additional glycosite modifications. In addition to SARS-CoV-2, SARS-CoV MERS-CoV prevented target cells. These data suggest multiple roles N-glycans, evolutionarily properties these complex carbohydrates human beta-coronaviruses. Author Summary Previous work shows that N-linked glycans essential Few natural mutations have been observed S2-subunit glycoprotein GISAID data, absent post-fusion structure lie equidistant, ~4 nm apart, suggesting significance. Upon testing hypothesis function, noted including regulation S1-subunit shedding, cells virions, syncytial formation/cell-cell fusion entry. other Thus, represent targets development countermeasures against future outbreaks.

Language: Английский

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Genome sequencing of SARS-CoV-2 omicron variants in Delhi reveals alterations in immunogenic regions in spike glycoprotein

Frontiers in Immunology, Journal Year: 2023, Volume and Issue: 14

Published: Oct. 2, 2023

The SARS-CoV-2 omicron variants keep accumulating a large number of mutations in the spike (S) protein, which contributes to greater transmissibility and rapid rise dominance within populations. identification their affinity cellular angiotensin-converting enzyme-2 (ACE-2) receptor immune evasion Delhi NCR region was under-acknowledged. study identifies some (Y505 reversion, G339H, R346T/N) genomes from Delhi, India, probable implications for altering response binding ACE-2. have influenced neutralizing activity antibodies against variant, shows partial escape. However, researchers are currently exploring various mitigation strategies tackle potential decline efficacy or effectiveness existing future SARS-CoV-2. These include modifying vaccines target specific variants, such as developing multivalent vaccine formulations, alternative delivery methods. To address this, it is also necessary understand impact these different perspective, especially terms alterations antigenic determinants. In this study, we done whole genome sequencing (WGS) COVID-19 samples NCR, analyzed spike's mutation with an emphasis on alterations. epitope formation, loss/gain efficiency, interaction epitopes has been studied. Some variant seem be progenitors upcoming India. Our analyses suggested that weakening interactions may lead resistance circulating genomes.

Language: Английский

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Impact of mutations on the plant-based production of recombinant SARS-CoV-2 RBDs

Frontiers in Plant Science, Journal Year: 2023, Volume and Issue: 14

Published: Oct. 6, 2023

Subunit vaccines based on recombinant viral antigens are valuable interventions to fight existing and evolving viruses can be produced at large-scale in plant-based expression systems. The often derived from glycosylated envelope proteins of the virus glycosylation plays an important role for immunogenicity by shielding protein epitopes. receptor-binding domain (RBD) SARS-CoV-2 spike is a principal target vaccine development has been plants, but yields RBD variants were low N-glycosylation different concern less studied. Here, we investigated six transiently expressed leaves Nicotiana benthamiana. All purified functional terms receptor binding displayed almost full N-glycan occupancy both sites with predominately complex N-glycans. Despite high structural sequence conservation variants, detected variation yield which attributed lower differences unintentional proteolytic processing C-terminal polyhistidine tag used purification. Glycoengineering towards human-type profile core α1,6-fucose, showed that reactivity neutralizing antibody S309 differs depending variant.

Language: Английский

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Enhanced surface accessibility of SARS-CoV-2 Omicron spike protein due to an altered glycosylation profile

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: Nov. 23, 2023

Abstract SARS-CoV-2 spike (S) proteins undergo extensive glycosylation, aiding proper folding, enhancing stability, and evading host immune surveillance. In this study, we used mass spectrometric analysis to elucidate the N-glycosylation characteristics disulfide bonding of recombinant derived from Omicron variant (B.1.1.529) in comparison with D614G variant. Furthermore, conducted microsecond-long molecular dynamics simulations on resolve how different N-glycans impact conformational sampling two variants. Our findings reveal that protein maintains an overall resemblance terms site-specific glycan processing bond formation. Nonetheless, alterations glycans were observed at certain sites. These changes, synergy mutations within protein, result increased surface accessibility macromolecule, including ectodomain, receptor-binding domain, N-terminal domain. insights contribute our understanding interplay between structure function, thereby advancing effective vaccination therapeutic strategies. Teaser Through spectrometry simulations, is found be less covered by when compared

Language: Английский

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