Biochemical and biophysical characterization of human recombinant IgE-binding protein, an S-type animal lectin

IgE-binding protein (εBP) was originally identified by virtue of its affinity for IgE. It is now known to be a β-galactoside-binding lectin with the characteristic of an S-type carbohydrate recognition domain. The protein is composed of two domains: the amino-terminal domain consisting of tandem repeats and the carboxyl-terminal domain containing sequences shared by other S-type carbohydrate recognition domains. The amino-terminal domain also contains a number of potential recognition sites for collagenase cleavage. In this study, human εBP was first expressed in Escherichia coli, and the carboxyl-terminal domain (εBP-C) was then generated by collagenase digestion of εBP. By equilibrium dialysis, the association constants of εBP and εBP-C for lactose were found to be similar (6.0 ± 0.70) x 10⁴ M^-1 and (4.7 ± 0.27) x 10⁴ M^-1, respectively. Both polypeptides contain only one lactose-binding site/molecule. By an assay involving binding of ¹²⁵I-labeled eBP or εBP-C to solid phase IgE, and inhibition of this binding by saccharides, it was determined that εBP-C retains the saccharide specificity of εBP. Importantly, although unlabeled εBP-C inhibited the binding of the radiolabeled εBP to IgE, unlabeled εBP caused increased binding to IgE, suggesting self-association among εBP molecules. Oligomeric structures resulting from self-association of εBP were confirmed by chemical cross-linking studies. Furthermore, εBP possesses hemagglutination activity on rabbit erythrocytes, whereas εBP-C lacks such activity. Based on these results, we propose a structural model for multivalency of εBP: dimerization or oligomerization of εBP occurs through intermolecular interaction involving the amino-terminal domain.