CHARACTERIZATION OF SIALYLTRANSFERASE OF B16 MELANOMA-CELLS INVOLVED IN THE FORMATION OF MELANOMA-ASSOCIATED ANTIGEN GM3

Malignant melanoma cells express a melanoma-associated antigen with interspecies cross-reactivity that is recognized by monoclonal antibody M2590. This unique antigen has been shown to be a simple ganglioside, GM3 (NeuAcalpha2-3Galbeta1-4Glcbeta1-Cer), in B16 melanoma cells (Hirabayashi, Y., Hamaoka, A., Matsumoto, M., Matsubara, T., Tagawa, M., Wakabayashi, S., and Taniguchi, M. (1985): J Biol. Chem., 260, 13328-13333). In the present study we examined the enzyme activity that catalyzes the formation of the melanoma antigen from lactosylceramide (Galbeta1-4Glcbeta1-Cer) and CMP-N-acetylneuraminic acid (CMP-NcuAc) using melanoma cells derived from mice (C57BL/6) injected with B16 melanoma cells. The enzyme activity was expressed strongly in the melanoma tumor cells as compared with the activity found in normal mouse tissues. The enzyme (GM3 synthase) was successfully solubilized in the presence of 1% Triton X-100. Identification of the product was performed by TLC/enzyme-immunostaining with specific monoclonal antibodies. The optimum pH of the GM3 synthase was 6.3. For maximum enzyme activity, Triton X-100 was required as a detergent. Apparent K(m) values for CMP-NeuAc and lactosylceramide were about 0.42 and 0.057 mm, respectively. The sialyltransferase activities acting on paragloboside (Galbeta1-4GlcNAcbeta1-4Galbeta1-4Glcbeta1-Cer) and asialo-GM1 (Galbeta61-3GalNAcbeta1-4Galbeta1-4Glcbeta1-Cer) to form alpha2-3 and alpha2-6-sialylparagloboside (NeuAca2-3Galbeta1-4GlcNAcbeta1-4Galbeta1-4GIcbeta1-Cer and NeuAca2-6Galbeta1-4GlcNAcbeta1-4Galbeta1-4Glcbeta1-Cer, respectively) and GMlb(NeuAca2-3Galbeta1-3GalNAcbeta1-4Galbeta1-4Glcbeta1-Cer) were also found in the cells. A substrate competition study suggested that the GM3 synthase is different from alpha2-3sialylparagloboside and GM1b synthase.