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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM025766-29
Application #
3484574
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1978-09-01
Project End
1993-08-31
Budget Start
1989-09-01
Budget End
1990-08-31
Support Year
29
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Duke University
Department
Type
Schools of Medicine
DUNS #
071723621
City
Durham
State
NC
Country
United States
Zip Code
27705
Perez-Vilar, J; Eckhardt, A E; DeLuca, A et al. (1998) Porcine submaxillary mucin forms disulfide-linked multimers through its amino-terminal D-domains. J Biol Chem 273:14442-9
Perez-Vilar, J; Eckhardt, A E; Hill, R L (1996) Porcine submaxillary mucin forms disulfide-bonded dimers between its carboxyl-terminal domains. J Biol Chem 271:9845-50
Roth, J; Wang, Y; Eckhardt, A E et al. (1994) Subcellular localization of the UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-mediated O-glycosylation reaction in the submaxillary gland. Proc Natl Acad Sci U S A 91:8935-9
Wang, Y; Agrwal, N; Eckhardt, A E et al. (1993) The acceptor substrate specificity of porcine submaxillary UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase is dependent on the amino acid sequences adjacent to serine and threonine residues. J Biol Chem 268:22979-83
Wang, Y; Abernethy, J L; Eckhardt, A E et al. (1992) Purification and characterization of a UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase specific for glycosylation of threonine residues. J Biol Chem 267:12709-16
Tiemeyer, M; Brandley, B K; Ishihara, M et al. (1992) The binding specificity of normal and variant rat Kupffer cell (lectin) receptors expressed in COS cells. J Biol Chem 267:12252-7
Eckhardt, A E; Timpte, C S; Abernethy, J L et al. (1991) Porcine submaxillary mucin contains a cystine-rich, carboxyl-terminal domain in addition to a highly repetitive, glycosylated domain. J Biol Chem 266:9678-86
Hoyle, G W; Hill, R L (1991) Structure of the gene for a carbohydrate-binding receptor unique to rat kupffer cells. J Biol Chem 266:1850-7
Timpte, C S; Eckhardt, A E; Abernethy, J L et al. (1988) Porcine submaxillary gland apomucin contains tandemly repeated, identical sequences of 81 residues. J Biol Chem 263:1081-8
Hoyle, G W; Hill, R L (1988) Molecular cloning and sequencing of a cDNA for a carbohydrate binding receptor unique to rat Kupffer cells. J Biol Chem 263:7487-92

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