scholarly journals The barley lectin, horcolin, binds high-mannose glycans in a multivalent fashion, enabling high-affinity, specific inhibition of cellular HIV infection

2020 ◽  
Vol 295 (34) ◽  
pp. 12111-12129
Author(s):  
Nisha Grandhi Jayaprakash ◽  
Amrita Singh ◽  
Rahul Vivek ◽  
Shivender Yadav ◽  
Sanmoy Pathak ◽  
...  
Keyword(s):  
Hiv Infection ◽  
Binding Sites ◽  
Neutralizing Antibodies ◽  
Titration Calorimetry ◽  
Carbohydrate Binding ◽  
High Affinity ◽  
Mannose Binding ◽  
Surface Envelope ◽  
High Mannose ◽  
Anti Hiv

N-Linked glycans are critical to the infection cycle of HIV, and most neutralizing antibodies target the high-mannose glycans found on the surface envelope glycoprotein-120 (gp120). Carbohydrate-binding proteins, particularly mannose-binding lectins, have also been shown to bind these glycans. Despite their therapeutic potency, their ability to cause lymphocyte proliferation limits their application. In this study, we report one such lectin named horcolin (Hordeum vulgare lectin), seen to lack mitogenicity owing to the divergence in the residues at its carbohydrate-binding sites, which makes it a promising candidate for exploration as an anti-HIV agent. Extensive isothermal titration calorimetry experiments reveal that the lectin was sensitive to the length and branching of mannooligosaccharides and thereby the total valency. Modeling and simulation studies demonstrate two distinct modes of binding, a monovalent binding to shorter saccharides and a bivalent mode for higher glycans, involving simultaneous interactions of multiple glycan arms with the primary carbohydrate-binding sites. This multivalent mode of binding was further strengthened by interactions of core mannosyl residues with a secondary conserved site on the protein, leading to an exponential increase in affinity. Finally, we confirmed the interaction of horcolin with recombinant gp120 and gp140 with high affinity and inhibition of HIV infection at nanomolar concentrations without mitogenicity.

scholarly journals Targeting the HIV-1 Spike and Coreceptor with Bi- and Trispecific Antibodies for Single-Component Broad Inhibition of Entry

2018 ◽  
Vol 92 (18) ◽  
Author(s):  
Salar N. Khan ◽  
Devin Sok ◽  
Karen Tran ◽  
Arlette Movsesyan ◽  
Viktoriya Dubrovskaya ◽  
...  
Keyword(s):  
Animal Models ◽  
Hiv Infection ◽  
Binding Sites ◽  
Neutralizing Antibodies ◽  
Single Component ◽  
Single Chain ◽  
Broadly Neutralizing Antibodies ◽  
Hiv Replication ◽  
Hiv Inhibitors ◽  
The Individual

ABSTRACTProtection against acquiring human immunodeficiency virus (HIV) infection may not require a vaccine in the conventional sense, because broadly neutralizing antibodies (bNAbs) alone prevent HIV infection in relevant animal challenge models. Additionally, bNAbs as therapeutics can effectively suppress HIV replication in infected humans and in animal models. Combinations of bNAbs are generally even more effective, and bNAb-derived multivalent antibody-like molecules also inhibit HIV replication bothin vitroandin vivo. To expand the available array of multispecific HIV inhibitors, we designed single-component molecules that incorporate two (bispecific) or three (trispecific) bNAbs that recognize HIV Env exclusively, a bispecific CrossMAb targeting two epitopes on the major HIV coreceptor, CCR5, and bi- and trispecifics that cross-target both Env and CCR5. These newly designed molecules displayed exceptional breadth, neutralizing 98 to 100% of a 109-virus panel, as well as additivity and potency compared to those of the individual parental control IgGs. The bispecific molecules, designed as tandem single-chain variable fragments (scFvs) (10E8fv-N6fv and m36.4-PRO 140fv), displayed median 50% inhibitory concentration (IC50s) of 0.0685 and 0.0131 μg/ml, respectively. A trispecific containing 10E8-PGT121-PGDM1400 Env-specific binding sites was equally potent (median IC50of 0.0135 μg/ml), while a trispecific molecule targeting Env and CCR5 simultaneously (10E8Fab-PGDM1400fv-PRO 140fv) demonstrated even greater potency, with a median IC50of 0.007 μg/ml. By design, some of these molecules lacked Fc-mediated effector function; therefore, we also constructed a trispecific prototype possessing reconstituted CH2-CH3 domains to restore Fc receptor binding capacity. The molecules developed here, along with those described previously, possess promise as prophylactic and therapeutic agents against HIV.IMPORTANCEBroadly neutralizing antibodies (bNAbs) prevent HIV infection in monkey challenge models and suppress HIV replication in infected humans. Combinations of bNAbs are more effective at suppression, and antibody-like molecules engineered to have two or three bNAb combining sites also inhibit HIV replication in monkeys and other animal models. To expand the available array of multispecific HIV inhibitors, we designed single-component molecules that incorporate two (bispecific) or three (trispecific) bNAb binding sites that recognize the HIV envelope glycoprotein (Env) or the HIV coreceptor (CCR5) or that cross-target both Env and CCR5. Several of the bi- and trispecific molecules neutralized most viruses in a diverse cross-clade panel, with greater breadth and potency than those of the individual parental bNAbs. The molecules described here provide additional options for preventing or suppressing HIV infection.

scholarly journals Insights into Hydrophobicity and the Chaperone-like Function of αA- and αB-crystallins

2005 ◽  
Vol 280 (23) ◽  
pp. 21726-21730 ◽  
Author(s):  
M. Satish Kumar ◽  
Mili Kapoor ◽  
Sharmistha Sinha ◽  
G. Bhanuprakash Reddy
Keyword(s):  
Binding Sites ◽  
Chaperone Activity ◽  
Titration Calorimetry ◽  
Affinity Binding ◽  
Hydrophobic Surfaces ◽  
Ans Binding ◽  
High Affinity Binding ◽  
Chaperone Function ◽  
High Affinity ◽  
Αb Crystallin

α-Crystallin, composed of two subunits, αA and αB, has been shown to function as a molecular chaperone that prevents aggregation of other proteins under stress conditions. The exposed hydrophobic surfaces of α-crystallins have been implicated in this process, but their exact role has not been elucidated. In this study, we quantify the hydrophobic surfaces of αA- and αB-crystallins by isothermal titration calorimetry using 8-anilino-1-napthalenesulfonic acid (ANS) as a hydrophobic probe and analyze its correlation to the chaperone potential of αA- and αB-crystallins under various conditions. Two ANS binding sites, one with low and another with high affinity, were clearly detected, with αB showing a higher number of sites than αA at 30 °C. In agreement with the higher number of hydrophobic sites, αB-crystallin demonstrated higher chaperone activity than αA at this temperature. Thermodynamic analysis of ANS binding to αA- and αB-crystallins indicates that high affinity binding is driven by both enthalpy and entropy changes, with entropy dominating the low affinity binding. Interestingly, although the number of ANS binding sites was similar for αA and αB at 15 °C, αA was more potent than αB in preventing aggregation of the insulin B-chain. Although there was no change in the number of high affinity binding sites of αA and αB for ANS upon preheating, there was an increase in the number of low affinity sites of αA and αB. Preheated αA, in contrast to αB, exhibited remarkably enhanced chaperone activity. Our results indicate that although hydrophobicity appears to be a factor in determining the chaperone-like activity of α-crystallins, it does not quantitatively correlate with the chaperone function of α-crystallins.

scholarly journals Correction: The barley lectin, horcolin, binds high-mannose glycans in a multivalent fashion, enabling high-affinity, specific inhibition of cellular HIV infection

2021 ◽  
Vol 297 (3) ◽  
pp. 101158
Author(s):  
Nisha Grandhi Jayaprakash ◽  
Amrita Singh ◽  
Rahul Vivek ◽  
Shivender Yadav ◽  
Sanmoy Pathak ◽  
...  
Keyword(s):  
Hiv Infection ◽  
Specific Inhibition ◽  
High Affinity ◽  
High Mannose

scholarly journals Phylogenetic and specificity studies of two-domain GNA-related lectins: generation of multispecificity through domain duplication and divergent evolution

2007 ◽  
Vol 404 (1) ◽  
pp. 51-61 ◽  
Author(s):  
Els J. M. Van Damme ◽  
Sachiko Nakamura-Tsuruta ◽  
David F. Smith ◽  
Maté Ongenaert ◽  
Harry C. Winter ◽  
...  
Keyword(s):  
Binding Sites ◽  
Sequence Divergence ◽  
Structural Data ◽  
Divergent Evolution ◽  
Carbohydrate Binding ◽  
Dual Specificity ◽  
Domain Duplication ◽  
Galanthus Nivalis ◽  
High Mannose ◽  
Microarray Analyses

A re-investigation of the occurrence and taxonomic distribution of proteins built up of protomers consisting of two tandem arrayed domains equivalent to the GNA [Galanthus nivalis (snowdrop) agglutinin] revealed that these are widespread among monotyledonous plants. Phylogenetic analysis of the available sequences indicated that these proteins do not represent a monophylogenetic group but most probably result from multiple independent domain duplication/in tandem insertion events. To corroborate the relationship between inter-domain sequence divergence and the widening of specificity range, a detailed comparative analysis was made of the sequences and specificity of a set of two-domain GNA-related lectins. Glycan microarray analyses, frontal affinity chromatography and surface plasmon resonance measurements demonstrated that the two-domain GNA-related lectins acquired a marked diversity in carbohydrate-binding specificity that strikingly contrasts the canonical exclusive specificity of their single domain counterparts towards mannose. Moreover, it appears that most two-domain GNA-related lectins interact with both high mannose and complex N-glycans and that this dual specificity relies on the simultaneous presence of at least two different independently acting binding sites. The combined phylogenetic, specificity and structural data strongly suggest that plants used domain duplication followed by divergent evolution as a mechanism to generate multispecific lectins from a single mannose-binding domain. Taking into account that the shift in specificity of some binding sites from high mannose to complex type N-glycans implies that the two-domain GNA-related lectins are primarily directed against typical animal glycans, it is tempting to speculate that plants developed two-domain GNA-related lectins for defence purposes.

scholarly journals GalNAc-Specific Soybean Lectin Inhibits HIV Infection of Macrophages through Induction of Antiviral Factors

2017 ◽  
Vol 92 (6) ◽  
Author(s):  
Runhong Zhou ◽  
Xu Wang ◽  
Hang Liu ◽  
Le Guo ◽  
Qijian Su ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Surface ◽  
Hiv Infection ◽  
Binding Capacity ◽  
Target Cells ◽  
Significant Activity ◽  
Mitogenic Effect ◽  
Mannose Binding ◽  
Soybean Lectin ◽  
Anti Hiv

ABSTRACTAlthough it has been shown that some mannose-binding lectins (MBLs) exhibit significant activity against HIV infection, little is known about whetherN-acetylgalactosamine (GalNAc)-binding lectins have the ability to inhibit HIV infection. Here, we demonstrate that a soybean-derived lectin (SBL) with GalNAc-binding affinity could potently suppress HIV infection of macrophages in a dose-dependent fashion. Unlike the MBLs, which block HIV only through binding to the glycosylated envelope proteins (gp120 and gp41) of the virus, SBL inhibited HIV at multiple steps of the virus infection/replication cycle. SBL could activate the beta interferon (IFN-β)–STAT signaling pathway, resulting in the upregulation of a number of antiviral interferon-stimulated genes (ISGs) in macrophages. In addition, SBL treatment of macrophages induced the production of C-C chemokines, which bind to HIV entry coreceptor CCR5. Deglycosylation of cell surface galactosyl moieties or presaturation of GalNAc-binding capacity could compromise SBL-mediated induction of the antiviral factors. Furthermore, SBL exerted its anti-HIV activity in the low nanomolar range with no mitogenic effect on CD4+T cells, a major advantage in the development of SBL as a potential anti-HIV agent compared with MBLs. These data indicate a necessity to further investigate SBL as an alternative and cost-effective anti-HIV natural product.IMPORTANCEMannose-binding lectins (MBLs) can block the attachment of HIV to target cells and have been suggested as anti-HIV microbicides. However, the mitogenic effect of MBLs on CD4+T cells limits this potential in clinical settings. Lectins with galactose (Gal)- orN-acetylgalactosamine (GalNAc)-binding specificity are another important category of carbohydrate-binding proteins (CBP). Compared to high-mannose N-linked glycans, GalNAc-type glycans present much less in HIV gp120 or gp41 glycosylation. Here, we demonstrate that GalNAc-specific soybean lectin (SBL) triggers antiviral signaling via recognition of the cell surface galactosyl group of macrophages, which results in the suppression of HIV at multiple steps. More importantly, SBL has no mitogenic effect on the activation of CD4+T cells, a major advantage in the development of Gal/GalNAc-specific lectins as naturopathic anti-HIV agents.

scholarly journals Peptide Scanning of SARS-CoV and SARS-CoV-2 Spike Protein Subunit 1 Reveals Potential Additional Receptor Binding Sites

2021 ◽  
Author(s):  
Weilin Lin ◽  
Jannatul Rafeya ◽  
Vanessa Roschewitz ◽  
David Smith ◽  
Adrian Keller ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Binding Sites ◽  
Neutralizing Antibodies ◽  
Solid Phase ◽  
Solid Phase Peptide Synthesis ◽  
Spike Protein ◽  
Loss Of Function ◽  
Protein Subunit ◽  
Viral Attachment ◽  
High Affinity

The binding of SARS-CoV and SARS-CoV-2 to the ACE2 receptor on human cells is mediated by the spike protein subunit 1 (S1) on the virus surfaces, while the receptor binding domains (RBDs) of S1 are the major determinants for the interaction with ACE2 and dominant targets of neutralizing antibodies. However, at the virus-host interface, additional biomolecular interactions, although being relatively weak in affinity and low in specificity, could also contribute to viral attachment and play important roles in gain- or loss-of-function mutations. In this work, we performed a peptide scanning of the S1 domains of SARS-CoV and SARS-CoV-2 by synthesizing 972 16-mer native and mutated peptide fragments using a high throughput in situ array synthesis technology. By probing the array using fluorescently labelled ACE2, a number of previously unknown potential receptor binding sites of S1 have been revealed. 20 peptides were synthesized using solid phase peptide synthesis, in order to validate and quantify their binding to ACE2. Four ACE2-binding peptides were selected, to investigate whether they can be assembled through a biotinylated peptide/neutravidin system to achieve high affinity to ACE2. A number of constructs exhibited high affinity to ACE2 with Kd values of pM to low nM.

scholarly journals Overview of the Structure–Function Relationships of Mannose-Specific Lectins from Plants, Algae and Fungi

2019 ◽  
Vol 20 (2) ◽  
pp. 254 ◽  
Author(s):  
Annick Barre ◽  
Yves Bourne ◽  
Els Van Damme ◽  
Pierre Rougé
Keyword(s):  
Structure Function ◽  
Binding Site ◽  
High Specificity ◽  
Binding Specificity ◽  
Binding Pocket ◽  
Carbohydrate Binding ◽  
Specific Recognition ◽  
Mannose Binding ◽  
High Mannose ◽  
Function Relationship

To date, a number of mannose-binding lectins have been isolated and characterized from plants and fungi. These proteins are composed of different structural scaffold structures which harbor a single or multiple carbohydrate-binding sites involved in the specific recognition of mannose-containing glycans. Generally, the mannose-binding site consists of a small, central, carbohydrate-binding pocket responsible for the “broad sugar-binding specificity” toward a single mannose molecule, surrounded by a more extended binding area responsible for the specific recognition of larger mannose-containing N-glycan chains. Accordingly, the mannose-binding specificity of the so-called mannose-binding lectins towards complex mannose-containing N-glycans depends largely on the topography of their mannose-binding site(s). This structure–function relationship introduces a high degree of specificity in the apparently homogeneous group of mannose-binding lectins, with respect to the specific recognition of high-mannose and complex N-glycans. Because of the high specificity towards mannose these lectins are valuable tools for deciphering and characterizing the complex mannose-containing glycans that decorate both normal and transformed cells, e.g., the altered high-mannose N-glycans that often occur at the surface of various cancer cells.

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

Molecules ◽  
2021 ◽  
Vol 26 (12) ◽  
pp. 3621
Author(s):  
Irene Maier ◽  
Robert H. Schiestl ◽  
Georg Kontaxis
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Affinity Maturation ◽  
Viral Envelope ◽  
Biologically Active ◽  
Carbohydrate Binding ◽  
Affinity Binding ◽  
Protein Patterns ◽  
High Affinity Binding ◽  
High Affinity

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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