The long range goal of this project is to elucidate the molecular basis for the species specific interaction of the sperm and egg surfaces during fertilization in sea urchins. We hope to establish the structural basis for the specific recognition and binding of cell surface sulfated polysaccharides by the sperm adhesive protein, bindin, at a molecular level of resolution. We also hope to gain some insight into the process of sperm and egg plasma membrane fusion during fertilization. We propose a molecular biological and protein engineering approach to understanding the structure and function relationships of bindin. We will isolate and sequence additional cDNAs for other species of bindin in order to understand the sequence homologies and primary species differences between bindins. We have recently succeeded in overproducing bindin in E. coli and purifying it from bacteria in a biologically-active and species-specific form. This provides us with a source of large amounts of bindin and allows us to manipulate the structure of bindin using recombinant DNA technology. We propose to attempt to crystallize this recombinant bindin in order to facilitate the elucidation of its three dimensional structure. We will examine the molecular basis for the species specificity of bindin by constructing hybrid bindins from species-specific domains of two different species of bindins. We will analyze the active sites of bindin for sulfated fucan binding and gel-phase selective membrane binding. To accomplish this goal, we propose to examine the biological properties of synthetic peptide analogs of bindin and a series of altered bindin polypeptides produced by site-directed mutagenesis and expressed in E. coli. We will examine the structure and composition of the complementary egg surface receptor for bindin and attempt to identify any additional egg surface receptor components using multiple independent approaches. We also propose to attempt to isolate the acrosomal plamsa membrane of sperm and develope an in vitro assay for membrane fusion using purified sperm and egg plasma membrane components. Bindin is the first eukaryotic adhesive protein to be overproduced in microorganisms in a full-sized, biologically active form. These investigations should advance our understanding of the molecular basis of gamete interaction which could be useful for understanding infertility. Knowledge gained from these studies may also be applicable to other types of adhesion molecules and useful for the targeting of drugs and macromolecules.
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