9728447 Hedrick The long term goal of this research is to determine the macromolecular composition, the biosynthesis, and the fertilization function of egg cortical granule components. The macromolecular components of the cortical granules modify the molecular structure and the supramolecular architecture of the egg envelope. This modification maintains the diploid state of the fertilized egg chromosomes by preventing additional sperm from fusing with the egg. The enzyme beta-N-acetylglucosaminidase was identified in the cortical granules of eggs from the frog Xenopus laevis. N-acetylglucosamine residues of the egg envelope glycoproteins may function in sperm binding to the egg. Their enzymatic removal may prevent additional sperm from fusing with egg. The specific objectives of the research are to provide evidence for this proposal by: 1) determining the protein structure of the egg enzyme using recombinant DNA cloning methods, 2) identifying egg envelope glycoproteins that are substrates for the enzyme, 3) determining the monosaccharide structure(s) of the carbohydrate moiety of these glycoproteins, and 4) relating this structural information to its biological role by: a) causing the glycosidic hydrolysis of envelope glycoproteins with exogenously added enzyme, and b) preventing the glycosidic hydrolysis of envelope glycoprotein by endogenously released enzyme using specific inhibitors of beta-N-acetylglucosaminidase activity. This research will identify the molecules involved in sperm-egg binding and how modification of the envelope glycoproteins by an enzyme released from the egg alters the structure of the molecules outside the egg. Accomplishing this research will provide a molecular understanding as to how an egg enzyme's protein structure determines its specific action on its egg envelope substrate, and how glycosidic modification of the envelope prevents sperm-egg interactions. From this understanding of the structure-function relations of the molecules involved in fertilization may come information relevant to regulating fertilization and early developmental events.
