The d-galactose specific lectin of field bean (Dolichos lablab) seed binds sugars with extreme negative cooperativity and half-of-the-sites binding

The field bean (Dolichos lablab) lectin designated as PPO-haemagglutinin (DLL-II) is bifunctional, exhibiting both polyphenol oxidase and haemagglutinating activity. The lectin is unusual in that it binds galactose (Gal), lactose (Lac) and N-acetylgalactosamine (GalNAc) only in the presence of (NH4)2SO4 and exhibits negative cooperativity and half-of-the-sites binding. Circular dichroism, isothermal titration calorimetry and fluorescence quenching were used to assess the sugar binding in the presence of (NH4)2SO4. Comparison of the near-UV CD spectra with and without bound sugar revealed ligand induced conformational changes. The intrinsic fluorescence quenching data indicate that DLL-II exhibits weak binding to Gal in the presence of (NH4)2SO4 with a stoichiometry of one bound ligand per dimer. ITC data fitted using a two sets of sites binding model presented a similar picture. The Ka’s for Gal, Lac and GalNAc in the presence of (NH4)2SO4 were 0.16 ± 0.002, 0.21 ± 0.004 and 8.45 ± 0.78 (×10−3) M−1 respectively. The Hill plot for the binding of these sugars to DLL-II was curvilinear with a tangent slope <1.0 indicating negative cooperativity. DLL-II thus exhibits half-of-the-site binding, an extreme form of negative cooperativity in which the second ligand does not bind at all. This is the first report of a legume lectin, exhibiting half-of-the-sites binding.

Graphical abstractField bean bifunctional lectin binds galactose (Gal), lactose (Lac) and N-acetylgalactosamine (GalNAc) only in the presence of (NH4)2SO4 and exhibits negative cooperativity and half-of-the-sites binding.Figure optionsDownload full-size imageDownload high-quality image (126 K)Download as PowerPoint slideHighlights► Field bean enzymatic lectin binds galactose only in presence of (NH4)2SO4. ► Weak binding to galactose, lactose and N-galactosamine occurs with negative cooperativity. ► The stoichiometry of binding is one ligand per dimer. ► Lectin exhibits half-of-the-site binding for sugars, an extreme form of negative cooperativity. ► This is the first report of a legume lectin, exhibiting half-of-the-sites binding for sugars.