The C-type (Ca2+-dependent) animal lectins are large and diverse family of cell-surface carbohydrate-binding proteins that includes serum mannose-binding proteins (MBPs), which mediate immunoglobulin-independent recognition and killing of pathogens; hepatic lectins, membrane receptors that regulate the levels of serum glycoproteins; and P-selectin, which mediates adhesion of leukocytes to endothelium at regions of tissue injury, and platelet-leukocyte interactions at sites of inflammation and hemmorhage. Each contains a carbohydrate-recognition domain (CRD) that confers Ca2+-dependent carbohydrate binding, attached to effector domains responsible for the function of the molecule. The different contexts in which the C-type lectins function require a variety of carbohydrate specificities.
The aim of this proposal is to understand the biophysical basis of the specificity of the C-type lectins, using x-ray crystallography in conjunction with available thermodynamic and site- directed mutagenesis data. Lectins display extremely weak affinity for monovalent ligands, but multivalent interactions amplify energetically small differences in affinity for preferred and non-preferred ligands to produce tight binding to, and discrimination among, carbohydrates. Thus, two structural questions will be addressed: 1) What are the determinants of weak, specific binding to monovalent ligands? 2) How are these proteins and their ligands spatially organized to achieve high-avidity multivalent interactions at the cell surface? The following structures, alone and complexed with carbohydrate ligands, will be determined: 1) A trimeric fragment of rat MBP-A that contains the CRD, alone and complexed with multivalent ligands, to visualize directly multivalent interactions between this protein and a pathogenic cell surface. Crystals of the unliganded protein that diffract to at least 2.5 A resolution have been prepared. 2) The CRD from rat MBP-C, to compare its specificity for the internal trimannosyl core of N-linked oligosaccharides with that of MBP-A for terminal sugars. Crystals that diffract past 2.0 A resolution have been prepared. 3) MBP-A CRDs whose specificities have been switched from mannose to galactose by site- directed mutagenesis, with varying degrees of discrimination between these two ligands, to assess the contribution of individual residues to specificity. 4) CRDs from the rat and chicken hepatic lectins, which respectively recognize galactose- or N-acetylglucosamine-terminated oligosaccharides that serve as tags for removal of glycoproteins from circulation in these organisms. 5) The P-selectin CRD, which recognizes cell surfaces bearing a specific sialic acid- and fucose-containing carbohydrate structure.
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