Mannosylated hemagglutinin peptides bind cyanovirin-N independent of disulfide-bonds in complementary binding sites

Schilling, P.; Kontaxis, Georg; Dragosits, Martin; Schiestl, Robert H.; Becker, Christian F. W.; Maier, Irene

doi:10.1039/d0ra01128b

Cited by 3 publications

(18 citation statements)

References 57 publications

(68 reference statements)

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“…Disulfide bond variants were created by substitution of Cys and insertion of polar residue pairs Glu -Arg with slightly decreasing thermal stability and helical structures in V4 and V5. As the number of disulfide bonds in vicinity to the glycantargeting pocket in CVN2 decreased from 4 (CVN2L0) to 2 (variants V3-5), binding affinity to HA protein decreased (Schilling et al, 2020).…”

Section: Mechanisms Of Cyanovirin-n Binding To Viral Spikesmentioning

confidence: 99%

“…Whereas the glycan shield on the membrane-distal HA top part induced high-affinity binding to CV-N, CVN2 binding to HA close to a stabilizing disulfide bridge of HA top [4 N-linked glycans at N54, N97, N181, N301 (Bizebard et al, 1995)] has further been observed at its low-affinity sites. HA binding to at least one high-affinity carbohydrate binding site (H) of CVN2 has been examined at K D of 275 nM (A/Wisconsin/67/05) (Schilling et al, 2020), and Ebola GP1,2 bound to 2H of CVN2 with affinities in the lower nanomolar range (K D =26 nM) as measured via surface plasmon resonance (SPR) (Maier et al, 2021). Higher density of glycans on HA protein achieved binding with polar Glu-Arg residues instead of cystines in CVN2L0, and an association between respective mannosylated peptides (K D =10 µM for dimannosylated HA peptide, DM) and CVN2L0 or mutated Glu-Arg pairing near the HA binding pockets (Schilling et al, 2020).…”

Section: Mechanisms Of Cyanovirin-n Binding To Viral Spikesmentioning

confidence: 99%

“…HA binding to at least one high-affinity carbohydrate binding site (H) of CVN2 has been examined at K D of 275 nM (A/Wisconsin/67/05) (Schilling et al, 2020), and Ebola GP1,2 bound to 2H of CVN2 with affinities in the lower nanomolar range (K D =26 nM) as measured via surface plasmon resonance (SPR) (Maier et al, 2021). Higher density of glycans on HA protein achieved binding with polar Glu-Arg residues instead of cystines in CVN2L0, and an association between respective mannosylated peptides (K D =10 µM for dimannosylated HA peptide, DM) and CVN2L0 or mutated Glu-Arg pairing near the HA binding pockets (Schilling et al, 2020). Testing H on domains B, which was impacted by replacing a disulfide bond into ionic residues, and domain A forming the low-affinity carbohydrate binding site (L), the dimeric domain-swapped CVN2L0 molecule showed binding to HA top region and the whole molecule.…”

Section: Mechanisms Of Cyanovirin-n Binding To Viral Spikesmentioning

confidence: 99%

“…Testing H on domains B, which was impacted by replacing a disulfide bond into ionic residues, and domain A forming the low-affinity carbohydrate binding site (L), the dimeric domain-swapped CVN2L0 molecule showed binding to HA top region and the whole molecule. Binding affinity was presented as SPR K D values: A variant with altered binding-affinity was expressed in E. coli, purified, and positively tested for binding to HA-protein (H3N2) and DM, and showed a conformational change upon binding HA with either H or L carbohydrate binding sites and K D1 = 49 nM and K D2 = 8 µM (Schilling et al, 2020;Maier et al, 2021). Disulfide bond variants were created by substitution of Cys and insertion of polar residue pairs Glu -Arg with slightly decreasing thermal stability and helical structures in V4 and V5.…”

Section: Mechanisms Of Cyanovirin-n Binding To Viral Spikesmentioning

confidence: 99%

“…Cyanobacterial lectin CV-N displayed antiviral activity, which is mediated by nanomolar binding to high-mannose oligosaccharide modifications on envelope spike proteins (Mazur-Marzec et al, 2021), against human immunodeficiency virus 1 (HIV-1) (Boyd, 1997;Bolmstedt et al, 2001), influenza (O'Keefe et al, 2003), herpes virus, hepatitis C, severe acute respiratory syndrome coronavirus (SARS-CoV)-2 (Naidoo et al, 2021;Li et al, 2022), among others, and binds Ebola virus (Jensen et al, 2014). Structural analyses and binding affinity assays indicated cross-linking of two high-or low-affinity carbohydrate binding sites in a domain-swapped CVN2 dimer (Boyd, 1997;Bewley et al, 2002;Barrientos et al, 2006) by bivalent binding in the micromolar range to enhance avidity to viral envelope glycoproteins (Schilling et al, 2020) and to inhibit viral entry (Boyd, 1997;O'Keefe et al, 2003;Barrientos et al, 2006). Selective binding of the terminal-accessible disaccharide Mana1-2Mana on oligomannose-8 D1D3 arms and oligomannose-9 was comprised by two binding sites of differing affinities located on opposite protein protomers, thereby reaching nanomolar binding affinities (Bewley, 2001;Bewley and Otero-Quintero, 2001).…”

Section: Introductionmentioning

confidence: 99%

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Engineering recombinantly expressed lectin-based antiviral agents

Maier

2022

Front. Cell. Infect. Microbiol.

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Cyanovirin-N (CV-N), a lectin from Nostoc ellipsosporum was found an infusion inhibitory protein for human immunodeficiency virus (HIV)-1. A tandem-repeat of the engineered domain-swapped dimer bound specific sites at hemagglutinin (HA), Ebola and HIV spike glycoproteins as well as dimannosylated HA peptide, N-acetyl-D-glucosamine and high-mannose containing oligosaccharides. Among these, CV-N bound the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein at a dissociation constant (KD) of 18.6 µM (and KD=260 µM to RBD), which was low-affinity carbohydrate-binding as compared with the recognition of the other viral spikes. Binding of dimannosylated peptide to homo-dimeric CVN2 and variants of CVN2 that were pairing Glu-Arg residues sterically located close to its high-affinity carbohydrate binding sites, was measured using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Binding affinity increased with polar interactions, when the mutated residues were used to substitute a single, or two disulfide bonds, in CVN2. Site-specific N-linked glycans on spikes were mediating the infection with influenza virus by broadly neutralizing antibodies to HA and lectin binding to HA was further investigated via modes of saturation transfer difference (STD)-NMR. Our findings showed that stoichiometry and the lectin’s binding affinity were revealed by an interaction of CVN2 with dimannose units and either the high- or low-affinity binding site. To understand how these binding mechanisms add to viral membrane fusion we compare our tested HA-derived peptides in affinity with SARS-CoV-2 glycoprotein and review lectins and their mechanisms of binding to enveloped viruses for a potential use to simulate neutralization ability.

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Section: Mechanisms Of Cyanovirin-n Binding To Viral Spikesmentioning

confidence: 99%

Section: Mechanisms Of Cyanovirin-n Binding To Viral Spikesmentioning

confidence: 99%

Section: Mechanisms Of Cyanovirin-n Binding To Viral Spikesmentioning

confidence: 99%

Section: Mechanisms Of Cyanovirin-n Binding To Viral Spikesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Engineering recombinantly expressed lectin-based antiviral agents

Maier

2022

Front. Cell. Infect. Microbiol.

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Cyanovirin-N Binding to N-Acetyl-d-glucosamine Requires Carbohydrate-Binding Sites on Two Different Protomers

Maier,

Kontaxis,

Zimmermann

et al. 2024

Biochemistry

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Cyanovirin-N (CV-N) binds high-mannose oligosaccharides on enveloped viruses with two carbohydrate-binding sites, one bearing high affinity and one low affinity to Manα(1−2)Man moieties. A tandem repeat of two CV-N molecules (CVN2) was tested for antiviral activity against human immunodeficiency virus type I (HIV-1) by using a domain-swapped dimer. CV-N was shown to bind N-acetylmannosamine (ManNAc) and N-acetyl-D-glucosamine (GlcNAc) when the carbohydrate-binding sites in CV-N were free to interact with these monosaccharides independently. CVN2 recognized ManNAc at a K d of 1.4 μM and bound this sugar in solution, regardless of the lectin making amino acid side chain contacts on the targeted viral glycoproteins. An interdomain cross-contacting residue Glu41, which has been shown to be hydrogen bonding with dimannose, was substituted in the monomeric CV-N. The amide derivative of glucose, GlcNAc, achieved similar high affinity to the new variant CVN-E41T as high-mannose Nglycans, but binding to CVN2 in the nanomolar range with four binding sites involved or binding to the monomeric CVN-E41A. A stable dimer was engineered and expressed from the alanine-to-threoninesubstituted monomer to confirm binding to GlcNAc. In summary, low-affinity binding was achieved by CVN2 to dimannosylated peptide or GlcNAc with two carbohydrate-binding sites of differing affinities, mimicking biological interactions with the respective N-linked glycans of interest and cross-linking of carbohydrates on human T cells for lymphocyte activation.

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Cyanovirin-N Binds Viral Envelope Proteins at the Low-Affinity Carbohydrate Binding Site without Direct Virus Neutralization Ability

2021

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Glycan-targeting antibodies and pseudo-antibodies have been extensively studied for their stoichiometry, avidity, and their interactions with the rapidly modifying glycan shield of influenza A. Broadly neutralizing antiviral agents bind in the same order when they neutralize enveloped viruses regardless of the location of epitopes to the host receptor binding site. Herein, we investigated the binding of cyanovirin-N (CV–N) to surface-expressed glycoproteins such as those of human immunodeficiency virus (HIV) gp120, hemagglutinin (HA), and Ebola (GP)1,2 and compared their binding affinities with the binding response to the trimer-folded gp140 using surface plasmon resonance (SPR). Binding-site knockout variants of an engineered dimeric CV–N molecule (CVN2) revealed a binding affinity that correlated with the number of (high-) affinity binding sites. Binding curves were specific for the interaction with N-linked glycans upon binding with two low-affinity carbohydrate binding sites. This biologically active assembly of a domain-swapped CVN2, or monomeric CV–N, bound to HA with a maximum KD of 2.7 nM. All three envelope spike proteins were recognized at a nanomolar KD, whereas binding to HIV neutralizing 2G12 by targeting HA and Ebola GP1,2 was measured in the µM range and specific for the bivalent binding scheme in SPR. In conclusion, invariant structural protein patterns provide a substrate for affinity maturation in the membrane-anchored HA regions, as well as the glycan shield on the membrane-distal HA top part. They can also induce high-affinity binding in antiviral CV–N to HA at two sites, and CVN2 binding is achieved at low-affinity binding sites.

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Mannosylated hemagglutinin peptides bind cyanovirin-N independent of disulfide-bonds in complementary binding sites

Abstract: Di-mannosylated peptides reveal mannose binding to cyanovirin-N (CV-N) low-affinity binding sites.

Cited by 3 publications

References 57 publications

Engineering recombinantly expressed lectin-based antiviral agents

Engineering recombinantly expressed lectin-based antiviral agents

Cyanovirin-N Binding to N-Acetyl-d-glucosamine Requires Carbohydrate-Binding Sites on Two Different Protomers

Cyanovirin-N Binds Viral Envelope Proteins at the Low-Affinity Carbohydrate Binding Site without Direct Virus Neutralization Ability

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