MOLECULAR MECHANISMS OF COMPLEX CARBOHYDRATE RECOGNITION

Complex carbohydrates serve diverse functions in biological systems. One of their roles is to act as recognition markers on the surface of cells and on extracellular glycoproteins. These oligosaccharides are selectively recognized by endogenous sugar-binding proteins that are designated animal lectins. Recognition of endogenous and exogenous carbohydrates by animal lectins is an important aspect of cell-cell adhesion, serum glycoprotein turnover and antibody-independent innate immunity. Many animal lectins contain discrete, Ca2+-dependent carbohydrate-recognition domains (C-type CRDs) of approximately 130 amino acids. These modules share an underlying sequence motif, suggesting that they are folded in similar ways and that, although each binds to a unique spectrum of sugars, the underlying mechanism for sugar recognition by all of these domains is likely to be related. It is proposed to study certain key C-type CRDs which serve as models for the way in which all CRDs in this class function. The goals are: (1) To develop a detailed molecular description of how individual CRDs interact with monosaccharides. (2) To define the arrangement of multiple CRDs within animal lectins, in order to understand how the intact lectin molecules interact with multivalent oligosaccharides. (3) To determine the changes in CRDs that are responsible for their loss of ligand-binding activity at low pH, which allows release of ligands in endosomes following endocytosis. A combination of x-ray crystallography, site-directed mutagenesis, limited proteolysis, expression of protein fragments, spectroscopic analysis of protein-sugar interactions, nuclear magnetic resonance, and computational modeling will be employed.