In this project the structures and biosynthesis of glycoprotein and glycolipid antigens of malignant melanoma, originally detected by human serum antibodies and mouse monoclonal antibodies, will be analyzed. We have previously shown that the sera of some patients contain antibodies reacting only with autologous tumor cells. One of these antibodies (FD) is immunoprecipitating and detects a glycoprotein of 90,000 kD. The glycoprotein is also present on other cells but these forms lack the determinant detected by serum FD. This glycoprotein (gp90) will be purified and its biochemical characteristics, including partial amino acid sequence and glycosylation features, will be analyzed. Attempts will be made to clone the gene coding for gp90 using lambda gt11 expression libraries, oligonucleotide probes, and transfection approaches. Comparison of protein and nucleotide data for gp90 from the autologous melanoma with that from other cell types should demonstrate the basis for the FD restricted epitope.
The aim i s to understand the significance of restricted antigens such as FD and the immune response to them in patients. We will also continue our studies of gangliosides characteristic of malignant melanoma, particularly GD3. A key step resulting in the expression of this ganglioside is the activity of the enzyme (GD synthetase; CMP-N- acetylneuraminic acid:GM3 sialyltransferase) that converts GM3 to GD3 since normal melanocytes have extremely low levels of GD3. The enzymatic properties of this enzyme including detergent requirements, pH optimum, ion requirements, Km for acceptor and donor, etc., will be studied. Also, the activity of the transferase in different cell types will be assayed to determine whether GD3 levels are controlled mainly by enzyme levels. The sialyltransferase will be purified using biochemical and affinity chromatography methods and its biochemical characteristics, including partial amino acid sequence and glycosylation, will be analyzed. Monoclonal antibodies to the purified enzyme will be produced and used to further analyze its properties and distribution. Experiments will also be initiated to clone the gene encoding GD3 synthetase using transfection and other strategies. Overall goals of this project are to understand the control of restricted determinant expression and of characteristic glycolipid synthesis and also to provide reagents for the immunodiagnosis and therapy of melanoma.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA021445-15
Application #
3165560
Study Section
Experimental Immunology Study Section (EI)
Project Start
1977-04-01
Project End
1993-05-31
Budget Start
1991-06-01
Budget End
1992-05-31
Support Year
15
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Sloan-Kettering Institute for Cancer Research
Department
Type
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10065
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Ruan, S; Lloyd, K O (1992) Glycosylation pathways in the biosynthesis of gangliosides in melanoma and neuroblastoma cells: relative glycosyltransferase levels determine ganglioside patterns. Cancer Res 52:5725-31
Nagata, Y; Yamashiro, S; Yodoi, J et al. (1992) Expression cloning of beta 1,4 N-acetylgalactosaminyltransferase cDNAs that determine the expression of GM2 and GD2 gangliosides. J Biol Chem 267:12082-9
Lloyd, K O (1991) Humoral immune responses to tumor-associated carbohydrate antigens. Semin Cancer Biol 2:421-31
Furukawa, K; Arita, Y; Satomi, N et al. (1990) Tumor necrosis factor enhances GD3 ganglioside expression in cultured human melanocytes. Arch Biochem Biophys 281:70-5
Feickert, H J; Anger, B R; Cordon-Cardo, C et al. (1990) Cell-surface antigens of human lung tumors detected by mouse monoclonal antibodies: definition of blood-group- and non-blood-group-related antigenic systems. Int J Cancer 46:1007-13
Lloyd, K O; Old, L J (1989) Human monoclonal antibodies to glycolipids and other carbohydrate antigens: dissection of the humoral immune response in cancer patients. Cancer Res 49:3445-51
Thampoe, I J; Furukawa, K; Vellve, E et al. (1989) Sialyltransferase levels and ganglioside expression in melanoma and other cultured human cancer cells. Cancer Res 49:6258-64
Furukawa, K S; Furukawa, K; Real, F X et al. (1989) A unique antigenic epitope of human melanoma is carried on the common melanoma glycoprotein gp95/p97. J Exp Med 169:585-90
Tai, T; Kawashima, I; Furukawa, K et al. (1988) Monoclonal antibody R24 distinguishes between different N-acetyl- and N-glycolylneuraminic acid derivatives of ganglioside GD3. Arch Biochem Biophys 260:51-5

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