GLYCOSYLTRANSFERASES, MUCINS & MACROPHAGE RECEPTORS

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The structure-function relationships of three groups of proteins that are involved in aspects of glycoconjugate biosynthesis or function will be examined. GLYCOSYLTRANSFERASES that act in the biosynthesis of oligosaccharides in glycoconjugates will be studied in several ways. 1) cDNA expression libraries in lambda gtll will be screened with antibodies to certain transferases and clones containing transferase cDNA will be isolated and sequenced. 2) The cDNA for certain transferases will be inserted into appropriate expression vectors to obtain sufficient transferase for structure/function studies or to obtain specifically modified enzyme by site specific mutagenesis. 3) Two transferases that act in mucin oligosaccharide biosynthesis and have resisted purification will be purified and characterized. The two transferases form the core sequence GAl beta 1,3Ga1NAc-O- Ser/Thr. MUCINS that serve to lubricate the gastrointestinal, respiratory and urogenital tracts and to protect epithelial cells from injury and dehydration will be isolated and further characterized, as follows. 1) The cDNA sequence of porcine submaxillary gland cDNA will be completed and the sequence of genomic DNA for submaxillary mucin examined. In addition, the number of genes for mucin in the porcine genome will be determined. 2) Antibodies to mucin and synthetic polynucleotides based on submaxillary mucin cDNA sequence will be used to determine whether other porcine tissues contain submaxillary mucin-like protein or mRNA. It will also be determined with use of these probes whether human submaxillary apomucin is structurally related to porcine submaxillary apomucin. 3) The exact location of oligosaccharides in mucin will be determined. 4) Mucins from other porcine tissues, such as stomach, small intestine and colon will be isolated and structurally characterized. MACROPHAGE RECEPTORS for oligosaccharides will be examined, especially the receptor found only on rat and mouse Kupffer cells. 1) The sequence of the genomic DNA for rat Kupffer cells will be determined. 2) The expression of the receptor during macrophage development will be examined and a search for the exact function of the receptor will be made. 3) The structural basis for the binding of carbohydrate to the receptor will be sought.