UDP-galactosyltransferase (GT) is primarily a Golgi membrane- bound enzyme that participates in the coordinate biosynthesis of the carbohydrate moieties of glycoproteins and glycolipids. In mammary tissue, GT can interact with the hormonally regulated protein-lactalbumin. This complex (lactose synthetase) is responsible for the biosynthesis of the unique mammalian disaccharide, lactose. Galactosyltransferase has also been localized to the plasma membrane of a diverse variety of cells and tissues by immunohistochemical procedures. This cell surface distribution supports the hypothesis that, in addition to its biosynthetic role, this transferase also has a functional role in intercellular recognition/adhesion. Our long range goals are to determine the structure, function and organization of GT in both intracellular and plasma membranes and to understand the mechanism responsible for compartmentalization of this membrane bound enzyme. The proposed experiments outlined in this proposal lay the foundation for these goals. This proposal takes advantage of: (1) a partial cDNA clone isolated from a Lambdal gt11 expression library that encodes bovine GT and (2) a series of monoclonal antibodies that identify four unique structural/functional domains within the bovine GT polypeptide including the alpha-lactalbumin binding site. Additionally, several of these antibodies are able to selectively recognize either the cell surface GT or the intracellular Golgi-associated GT as analyzed by immunohistochemical staining. These observations suggest a structural difference between the intracellular and cell surface form of GT. Using these highly specific and complementary probes for GT, we plan: (1) to isolate a full- length cDNA clone for bovine GT. (2) To characterize the mRNA structure including any alternatively spliced intermediates. (3) To construct an expression vector containing the complete protein coding sequence, in order to determine after transfection, if GT is synthesized and inserted into both the intracellular Golgi membrane and the plasma membrane. (4) To localize the four structure/function domains within the GT polypeptide as identified by our monoclonal antibodies by utilization the Lambdal gt11 expression system in conjunction with Western blot analysis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM038310-03
Application #
3294642
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1988-04-01
Project End
1992-03-31
Budget Start
1990-04-01
Budget End
1992-03-31
Support Year
3
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Type
Schools of Medicine
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Shaper, N L; Meurer, J A; Joziasse, D H et al. (1997) The chicken genome contains two functional nonallelic beta1,4-galactosyltransferase genes. Chromosomal assignment to syntenic regions tracks fate of the two gene lineages in the human genome. J Biol Chem 272:31389-99
Rajput, B; Shaper, N L; Shaper, J H (1996) Transcriptional regulation of murine beta1,4-galactosyltransferase in somatic cells. Analysis of a gene that serves both a housekeeping and a mammary gland-specific function. J Biol Chem 271:5131-42
Russo, R N; Shaper, N L; Taatjes, D J et al. (1992) Beta 1,4-galactosyltransferase: a short NH2-terminal fragment that includes the cytoplasmic and transmembrane domain is sufficient for Golgi retention. J Biol Chem 267:9241-7
Taatjes, D J; Roth, J; Shaper, N L et al. (1992) Immunocytochemical localization of beta 1,4 galactosyltransferase in epithelial cells from bovine tissues using monoclonal antibodies. Glycobiology 2:579-89
Joziasse, D H; Shaper, N L; Kim, D et al. (1992) Murine alpha 1,3-galactosyltransferase. A single gene locus specifies four isoforms of the enzyme by alternative splicing. J Biol Chem 267:5534-41
Shaper, N L; Lin, S P; Joziasse, D H et al. (1992) Assignment of two human alpha-1,3-galactosyltransferase gene sequences (GGTA1 and GGTA1P) to chromosomes 9q33-q34 and 12q14-q15. Genomics 12:613-5
Joziasse, D H; Shaper, N L; Shaper, J H et al. (1991) Gene for murine alpha 1----3-galactosyltransferase is located in the centromeric region of chromosome 2. Somat Cell Mol Genet 17:201-5
Joziasse, D H; Shaper, J H; Jabs, E W et al. (1991) Characterization of an alpha 1----3-galactosyltransferase homologue on human chromosome 12 that is organized as a processed pseudogene. J Biol Chem 266:6991-8
Russo, R N; Shaper, N L; Shaper, J H (1990) Bovine beta 1----4-galactosyltransferase: two sets of mRNA transcripts encode two forms of the protein with different amino-terminal domains. In vitro translation experiments demonstrate that both the short and the long forms of the enzyme are type II mem J Biol Chem 265:3324-31
Joziasse, D H; Shaper, N L; Salyer, L S et al. (1990) Alpha 1----3-galactosyltransferase: the use of recombinant enzyme for the synthesis of alpha-galactosylated glycoconjugates. Eur J Biochem 191:75-83

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