Our research for the next year involves the completion of our analysis of the asparagine-linked oligosaccharides of BHK and polyoma-transformed BHK cells by sequential lectin-affinity chromatography (SLAC) and methylation analyses. Two lectins, E-phytohemagglutinin and one from Datura stamonium, will be added to the analysis of the glycopeptides. Next, each of the 29 L-PHA-resistant PyBHK clones will be screened for its ability not to bind labeled L-PHA using the procedure we have developed for the parental PyBHK and the BHK cells. Any clone which can be isolated that is L-PHA?-? will be radiolabeled with [2-?3?H]-mannose, and its Asnlinked glycopeptides analyzed by SLAC and compared to those from the parental PyBHK cells. The next series of experiments involves the production of tumors in hamsters by injection of PyBHK cells and their lectin-resistant variants. We will be able to compare the Asn-linked glycopeptides of the tumors by radiolabeling in organ culture and then SLAC analysis. We wish to know if a particular oligosaccharide phenotype is selected for in vivo. We will finish the analysis of the glycosyltransferases that participate in the synthesis of the Asn-linked oligosaccharides. We will determine if the specific activities of these enzymes change upon transformation and will attempt to explain the differences in the oligosaccharide structures between the two cell types in terms of enzyme activity differences.
The final aim will be to finish the examination of the various glycoproteins on the surfaces of the BHK, PyBHK, and variant cells which express the highly branched structures and determine if the increase in branched structures occurs on all or only a few of these glycoproteins. This analysis, coupled with the results of the glycosyltransferase activity comparisons, should allow us to focus on the immediate cause in the changes in oligosaccharide branching patterns. (A)

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
3R01CA035377-06S1
Application #
3172985
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1983-08-01
Project End
1989-12-31
Budget Start
1988-08-01
Budget End
1989-12-31
Support Year
6
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Miami School of Medicine
Department
Type
Schools of Medicine
DUNS #
City
Miami
State
FL
Country
United States
Zip Code
33101
Ripka, J; Pierce, M (1992) Novel glycosylation-defective baby hamster kidney cells. Biochem Biophys Res Commun 186:102-13
Shoreibah, M G; Hindsgaul, O; Pierce, M (1992) Purification and characterization of rat kidney UDP-N-acetylglucosamine: alpha-6-D-mannoside beta-1,6-N-acetylglucosaminyltransferase. J Biol Chem 267:2920-7
Palcic, M M; Ripka, J; Kaur, K J et al. (1990) Regulation of N-acetylglucosaminyltransferase V activity. Kinetic comparisons of parental, Rous sarcoma virus-transformed BHK, and L-phytohemagglutinin-resistant BHK cells using synthetic substrates and an inhibitory substrate analog. J Biol Chem 265:6759-69
Crawley, S C; Hindsgaul, O; Alton, G et al. (1990) An enzyme-linked immunosorbent assay for N-acetylglucosaminyltransferase-V. Anal Biochem 185:112-7
Ripka, J; Pierce, M; Fregien, N (1990) Co-transformation of Lec 1 CHO cells with N-acetylglucosaminyltransferase 1 activity and a selectable marker. J Cell Biochem 42:117-22
Ripka, J; Pierce, M; Fregien, N (1989) DNA-mediated transformation of N-acetylglucosaminyltransferase I activity into an enzyme deficient cell line. Biochem Biophys Res Commun 159:554-60
Arango, J; Pierce, M (1988) Comparison of N-acetylglucosaminyltransferase V activities in Rous sarcoma-transformed baby hamster kidney (RS-BHK) and BHK cells. J Cell Biochem 37:225-31
Srivastava, O P; Hindsgaul, O; Shoreibah, M et al. (1988) Recognition of oligosaccharide substrates by N-acetyl-glucosaminyltransferase-V. Carbohydr Res 179:137-61
Hindsgaul, O; Tahir, S H; Srivastava, O P et al. (1988) The trisaccharide beta-D-GlcpNAc-(1----2)-alpha-D-Manp-(1----6)-beta-D-Manp, as its 8-methoxycarbonyloctyl glycoside, is an acceptor selective for N-acetylglucosaminyltransferase V. Carbohydr Res 173:263-72
Pierce, M; Arango, J; Tahir, S H et al. (1987) Activity of UDP-GlcNAc:alpha-mannoside beta(1,6)N-acetylglucosaminyltransferase (GnT V) in cultured cells using a synthetic trisaccharide acceptor. Biochem Biophys Res Commun 146:679-84

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