Abstract

The immediate objectives of the proposed research are sixfold: 1. Separation, characterization, and purification of GalNAcT-2(UDP-GalNAc:GbOse3Cer(Betal-3)N-acetylgalactosaminyltransferase) and GalNAcT-3(UDP-GalNAc:GbOse4Cer(Alphal-3)N-acetyl-galactosaminyltransferase) present in the detergent solubilized supernatants from guinea pig tumor cell lines 104Cl (tumorigenic) and 106B(nontumorigenic), respectively. 2. Determination of the exact chemical linkages in the Forssman biosynthetic products of the 104Cl and 106B GalNAcT-3-catalyzed reactions. 3. Purification and kinetic studies of GalNAcT-1(UDP-GalNAc:GM3 or Lac-Cer(Betal-4)N-acetyl-galactosaminyltransferase) from guinea pig bone marrow. 4. Determination of the effects of tunicamycin and insulin on GalNAcT-2 and GalNAcT-3 activities in 104Cl and 106B guinea pig tumor cells. 5. Biosynthesis in vitro of the tumor-specific antigens sialosylAlpha2-3Le-x, sialosylAlpha2-3Le-a in neuroblastoma IMR-32 cells and GD3 and Fuc-GM1 in rat prostate PA-I/II cells. 6. Characterization of GalT-5(UDP-Gal:nLcOse4Cer(Alphal-3)galactosyltransferase) from neuroblastoma IMR-32 cells and study of its activity under various growth conditions during the cell cycle and in the presence of differentiating agent ((But)2cAMP and HMBA). Our overall goal is to develop an understanding of the specificities of glycolipid:glycosyltransferases in three tumor cell lines of guinea pig and human origin. Tunicamycin inhibits glycoconjugate biosynthesis. However, inhibition of glycosylation of glycosyltransferase is a novel observation. At the end of this project an attempt will be made to establish a correlation between inhibition of a glycosyltransferase (GalNAcT-2) by tunicamycin and the change of a tumor-specific antigen (Forssman GSL) on the surface of 104Cl and 106B cells using 125I-labeled Helix pomatia and Dolichos biflorus lectins and Siga toxin. Screening of the mouse genomic library, prepared from a partial EcoRI digestion of AJ mouse genomic DNA (Shotgun in Charon 4a) with a 32P-labeled cDNA insert of GalNAcT-2 will be performed. Using 125I-mono and polyclonal antibodies the translated products for particular 104Cl GalNAcT-2 DNA sequences will also be screened.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA014764-11
Application #
3164008
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1979-04-01
Project End
1991-06-30
Budget Start
1987-07-01
Budget End
1988-06-30
Support Year
11
Fiscal Year
1987
Total Cost
Indirect Cost

  Institution

Name
University of Notre Dame
Department
Type
Schools of Arts and Sciences
DUNS #
824910376
City
Notre Dame
State
IN
Country
United States
Zip Code
46556

  Publications

Basu, Subhash; Ma, Rui; Moskal, Joseph R et al. (2012) Ganglioside biosynthesis in developing brains and apoptotic cancer cells: X. regulation of glyco-genes involved in GD3 and Sialyl-Lex/a syntheses. Neurochem Res 37:1245-55
Ma, Rui; Hopp, Elizabeth A; Decker, N Matthew et al. (2011) Regulation of glycosyltransferase genes in apoptotic breast cancer cells induced by L: -PPMP and cisplatin. Adv Exp Med Biol 705:621-42
Ma, Rui; Matthew Decker, N; Anilus, Vesta et al. (2009) Post-translational and transcriptional regulation of glycolipid glycosyltransferase genes in apoptotic breast carcinoma cells: VII. Studied by DNA-microarray after treatment with L-PPMP. Glycoconj J 26:647-61
Ray, S; Kelley, T J; Fan, L et al. (1994) Characterization of DNA polymerase-alpha/primase complex from developing embryonic chicken brains. Indian J Biochem Biophys 31:226-35
Kelley, T J; Moghaddas, S; Bose, R et al. (1993) Inhibition of immunopurified DNA polymerase-alpha from PA-3 prostate tumor cells by platinum (II) antitumor drugs. Cancer Biochem Biophys 13:135-46
Basu, M; Basu, S S; Li, Z et al. (1993) Biosynthesis and regulation of Le(x) and SA-Le(x) glycolipids in metastatic human colon carcinoma cells. Indian J Biochem Biophys 30:324-32
Schaeper, R J; Das, K K; Li, Z et al. (1992) In vitro biosynthesis of GbOse4Cer (globoside) and GM2 ganglioside by the (1-->3) and (1-->4)-N-acetyl beta-D-galactosaminyltransferases from embryonic chicken brain. Solubilization, purification, and characterization of the transferases. Carbohydr Res 236:227-44
Ray, S; Kelley, T J; Campion, S et al. (1991) Developmental expression of the embryonic chicken brain DNA polymerase alpha and its binding with monoclonal antibodies against human KB cell DNA polymerase alpha. Cell Growth Differ 2:567-73
Ghosh, S; Lee, S; Brown, T A et al. (1991) Use of exoglycosidases from Mercenaria mercenaria (hard shelled clam) as a tool for structural studies of glycosphingolipids and glycoproteins. Anal Biochem 196:252-61
Das, K K; Basu, M; Basu, S et al. (1991) Biosynthesis in vitro of GlcA beta 1-3nLcOse4Cer by a novel glucuronyltransferase (GlcAT-1) from embryonic chicken brain. J Biol Chem 266:5238-43

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