Abstract
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.
Highlights
N-Linked glycans on glycoprotein-120 are critical to the biology of HIV for its infectious cycle
We describe the characterization of a mannosebinding lectin from the coleoptile of Hordeum vulgare, detailing the thermodynamic fingerprint of its interaction with mannose and mannooligosaccharides by isothermal titration calorimetry (ITC), which reveals subtle features of its recognition of bi- and triantennary glycans
The 146-residue protein sequence retrieved from Uniprot (ID: Q5U9T2) was annotated to have the jacalin-like lectin domain by the Pfam database
Summary
The 146-residue protein sequence retrieved from Uniprot (ID: Q5U9T2) was annotated to have the jacalin-like lectin domain by the Pfam database. This was not observed for smaller sugars, where the role of the secondary site is absent or negligible Engagement of both CBS I and II pockets in bivalent binding mode by manno-oligosaccharides Man5/7, by their nonreducing terminal mannosyl residues at their D1 and D3 arms, respectively, potentiated the avidity of the interaction (Supporting information, Fig. S5–S8). This, was not maintained across the simulation time as can be observed in RMSF analysis of the glycones and the residence time of the hydrogen bond interaction between the D2 arm and the protein (2–5% of the simulation time) (Fig. S9) This is consistent with the observation of relatively poor enthalpy for binding in the ITC data, implying a reduced number of hydrogen bonds and an increased entropy owing to the high flexibility of the arms of Man. The in silico studies indicated a possibility of one pocket offering better interaction than the other
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