Structure–Function Analysis of C-Type Animal Lectins

Abstract

C-Type carbohydrate recognition domains (CRDs) are common modules in Ca 2+ -dependent animal lectins. Different C-type CRDs bind a wide variety of sugar ligands, reflecting the diverse biological processes that they mediate. The CRDs are often clustered in oligomers of lectin polypeptides, so they bind to complex, multivalent ligands with high affinity. Proteins containing C-type lectin-like domains (CTLDs) can be identified by sequence analysis, which can lead to predictions of possible sugar-binding activity. This chapter describes procedures that provide empirical methods for demonstrating sugar-binding activity in CTLDs and provides a means of preparing quantities sufficient for biochemical characterization and structural analysis. Approaches to determining the specificity of sugar binding and screening for high-affinity ligands are discussed. The chapter also discusses the expression system of C-type CRDs, production of native CRDs in Escherichia coli (E. coli), production as inclusion bodies in E. coli followed by renaturation, affinity chromatography of CRDs, solid-phase-binding assays of C-type CRDs, radiolabeled CRDs as probes for identification of high-affinity ligands, and identification of ligands by affinity chromatography on immobilized CRDs.