Viral envelope proteins fused to multiple distinct fluorescent reporters to probe receptor binding

Protein Science, Год журнала: 2024, Номер 33(4)

Опубликована: Март 27, 2024

Abstract Enveloped viruses carry one or multiple proteins with receptor‐binding functionalities. Functional receptors can be glycans, proteinaceous, both; therefore, recombinant protein approaches are instrumental in attaining new insights regarding viral envelope properties. Visualizing and measuring receptor binding typically entails antibody detection direct labeling, whereas fluorescent fusions attractive tools molecular biology. Here, we report a suite of distinct fusions, both N‐ C‐terminal, for influenza A virus hemagglutinins SARS‐CoV‐2 spike RBD. The contained three six barrels were applied directly to cells assess

Язык: Английский

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The HCoV-HKU1 N-Terminal Domain Binds a Wide Range of 9-O-Acetylated Sialic Acids Presented on Different Glycan Cores

ACS Infectious Diseases, Год журнала: 2024, Номер 10(11), С. 3880 - 3890

Опубликована: Окт. 12, 2024

Coronaviruses (CoVs) recognize a wide array of protein and glycan receptors by using the S1 subunit spike (S) glycoprotein. The contains two functional domains: N-terminal domain (S1-NTD) C-terminal (S1-CTD). S1-NTD SARS-CoV-2, MERS-CoV, HCoV-HKU1 possesses an evolutionarily conserved binding cleft that facilitates weak interactions with sialic acids on cell surfaces. employs 9-O-acetylated α2-8-linked disialylated structures for initial binding, followed TMPRSS2 receptor virus–cell fusion. Here, we demonstrate NTD has broader repertoire than previously recognized. We presented Fc chimeras nanoparticle system to mimic densely decorated surface HCoV-HKU1. These proteins were expressed HEK293S GnTI– cells, generating species carrying Man-5 structures, often observed near site CoVs. This multivalent presentation high mannose-containing revealed much profile compared its fully glycosylated counterpart. Using microarrays, α2-3-linked sialylated LacNAc are also bound, comparable OC43 NTD, suggesting glycan-binding modality. Further characterization specificity indicated promiscuous toward sialoglycans, independent core (glycolipids, N- or O-glycans). may employ additional sialoglycan trigger conformational changes in glycoprotein expose S1-CTD proteinaceous binding.

Язык: Английский

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Comparative Analysis of Host Cell Entry Efficiency and Neutralization Sensitivity of Emerging SARS-CoV-2 Lineages KP.2, KP.2.3, KP.3, and LB.1

Vaccines, Год журнала: 2024, Номер 12(11), С. 1236 - 1236

Опубликована: Окт. 30, 2024

New SARS-CoV-2 lineages continue to evolve and may exhibit new characteristics regarding host cell entry efficiency potential for antibody evasion. Here, employing pseudotyped particles, we compared the efficiency, ACE2 receptor usage, sensitivity antibody-mediated neutralization of four emerging lineages, KP.2, KP.2.3, KP.3, LB.1. The XBB.1.5 JN.1 served as controls. Our findings reveal that LB.1 enter cells efficiently in an ACE2-dependent manner, KP.3 is more adept at entering Calu-3 lung than JN.1. However, variants differed their capacity employ orthologues from animal species entry, suggesting differences interactions. Moreover, demonstrate only two out seven therapeutic monoclonal (mAbs) preclinical development retain robust neutralizing activity against sublineages tested, while three mAbs displayed strongly reduced lacked any tested. Furthermore, our results show evade by antibodies induced infection or vaccination with greater JN.1, particularly individuals without hybrid immunity. This study indicates differ interactions suggest evasion drove emergence these variants.

Язык: Английский

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Characterization of Site-Specific N- and O-Glycopeptides from Recombinant Spike and ACE2 Glycoproteins Using LC-MS/MS Analysis

International Journal of Molecular Sciences, Год журнала: 2024, Номер 25(24), С. 13649 - 13649

Опубликована: Дек. 20, 2024

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in hundreds of millions infections and deaths globally. Although vaccination campaigns are mitigating the emerging viral variants continue to pose challenges. spike (S) protein SARS-CoV-2 plays a critical role entry binding angiotensin-converting enzyme (ACE2) receptor, making both proteins essential targets for therapeutic vaccine development. glycosylation these influences their structure function. This underscores need detailed site-specific glycoproteomic analysis. In this study, we characterized N- or O-glycosylation profiles recombinant receptor-binding domain (RBD) ACE2 expressed from Expi293F cells, as well S2 subunit plant (N. benthamiana) cells. Using high-resolution Orbitrap Eclipse Tribrid mass spectrometer equipped with Ultimate 3000 RSLCnano I-GPA (Integrated GlycoProteome Analyzer) developed previous 148 28 O-glycopeptides RBD, 71 N-glycopeptides subunit, 139 were characterized. addition, report post-translational modifications (PTMs) glycan, including mannose-6-phosphate (M6P) GlcNAc-1-phosphate-6-O-mannose N-glycan RBD ACE2, O-acetylation O-glycan identified first time proteins. relative abundance distribution according glycosites glycan types analyzed quantified O (only RBD)-glycopeptides S2, using I-GPA. Asn331 Asn1098 Asn103 majorly N-glycosylated, dominant glycan-type was complex high-mannose S2. These findings will provide valuable insights into patterns that influence function immunogenicity offer new perspectives development vaccines antibody-based therapies against COVID-19.

Язык: Английский

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Bioluminescent detection of viral surface proteins using branched multivalent protein switches

RSC Chemical Biology, Год журнала: 2023, Номер 5(2), С. 148 - 157

Опубликована: Ноя. 23, 2023

Fast and reliable virus diagnostics is key to prevent the spread of viruses in populations. A hallmark presence multivalent surface proteins, a property that can be harnessed control conformational switching sensor proteins. Here, we introduce new platform (dark-LUX) for detection viral proteins consisting general bioluminescent framework post-translationally functionalized with separately expressed binding domains. The relies on (1) plug-and-play bioconjugation different

Язык: Английский

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The HCoV-HKU1 N-terminal domain binds a wide range of 9-O-acetylated sialic acids presented on different glycan cores

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Май 24, 2024

Coronaviruses recognize a wide array of protein and glycan receptors using the S1 subunit spike (S) glycoprotein. The contains two functional domains: N-terminal (S1-NTD) C-terminal (S1-CTD). S1-NTD SARS-CoV-2, MERS-CoV, HCoV-HKU1 possess an evolutionarily conserved binding cleft that facilitates weak interactions with sialic acids on cell surfaces. employs 9-

Язык: Английский

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Elusive Protein–Glycosphingolipid Interactions Revealed by Membrane Anchor-Assisted Native Mass Spectrometry

Journal of the American Chemical Society, Год журнала: 2024, Номер 146(31), С. 21700 - 21709

Опубликована: Июль 25, 2024

Interactions between glycan-binding proteins (GBPs) and glycosphingolipids (GSLs) present in cell membranes are implicated a wide range of biological processes. However, studying GSL binding is hindered by the paucity purified GSLs weak affinities typical monovalent GBP–GSL interactions. Native mass spectrometry (nMS) performed using soluble model promising approach for discovery GBP ligands, but detection interactions remains challenging. The work introduces MEmbrane ANchor-assisted nMS (MEAN-nMS) low-affinity complexes. assay utilizes membrane anchor, produced covalent cross-linking lipid membrane, to localize on surface promote binding. Ligands identified intact complexes or catch-and-release (CaR) strategy, wherein released from upon collisional activation detected (MEAN-CaR-nMS). To establish reliability, library gangliosides incorporated into nanodiscs was screened against human immune lectins, results compared with corresponding ganglioside oligosaccharides. Without analysis yielded predominantly false negatives. In contrast, all ligands were MEAN-(CaR)-nMS, no positives. highlight potential MEAN-CaR-nMS ligand discovery, natural viral uncover elusive ligands. Finally, nMS-based directly cells demonstrated. This breakthrough paves way shotgun glycomics screening cells.

Язык: Английский

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Bioluminescent detection of viral surface proteins using branched multivalent protein switches

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2023, Номер unknown

Опубликована: Сен. 22, 2023

Abstract Fast and reliable virus diagnostics is key to prevent the spread of viruses in populations. A hallmark presence multivalent surface proteins, a property that can be harnessed control conformational switching sensor proteins. Here, we introduce new platform (dark-LUX) for detection viral proteins consisting general bioluminescent framework post-translationally functionalized with separately expressed binding domains. The relies on 1) plug-and-play bioconjugation different via SpyTag/SpyCatcher technology create branched protein structures, 2) an optimized turn-on switch based complementation split-luciferase NanoBiT upon target 3) straightforward exploration linker space. influenza (IAV) hemagglutinin (HA) neuraminidase (NA) were used as relevant targets establish proof principle optimize parameters such properties, choice domains optimal combination competing components SmBiT DarkBiT. allows rapid conjugation exchange various including scFvs, nanobodies de novo designed binders variety targets, construction heterobivalent head stem region hemagglutinin. modularity thus optimization scaffold properties existing well suited quickly adapt effectively detect newly evolving epitopes.

Язык: Английский

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Viral envelope proteins fused to multiple distinct fluorescent reporters to probe receptor binding

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2023, Номер unknown

Опубликована: Окт. 23, 2023

Abstract Enveloped viruses carry one or multiple proteins with receptor binding functionalities. Functional receptors can either be glycans, proteinaceous both, recombinant protein approaches are instrumental to gain more insight into these properties. Visualizing and measuring normally entails antibody detection direct labelling, whereas fluorescent fusions attractive tools in molecular biology. Here we report a suite of different fusions, both N- and/or C-terminal, for influenza A virus hemagglutinins SARS-CoV-2 spike RBD. The contained total three six barrels were applied directly cells determine

Язык: Английский

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Sialic acid analog as inhibitor for human coronavirus OC43-a study by molecular dynamics simulations

Journal of Carbohydrate Chemistry, Год журнала: 2023, Номер 42(4-6), С. 197 - 215

Опубликована: Июль 24, 2023

Human coronavirus OC43 (HCoV-OC43) causes severe respiratory tract illness in humans. In the present study, we carried out 100 ns molecular dynamics (MD) simulations to investigate conformational of HCoV-OC43 spike (S) glycoprotein complexes with sialic acid (SIA) analogs. The binding affinity between S and SIA analogs is analyzed using NAMD MM-PBSA free energy calculation. It concluded from results MM/PBSA that order specificity toward 9-O-Ac-Sia > Neu5Gc zanamivir Neu5Ac2en.

Язык: Английский

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