Enzymatic Sulfation of Gangliotriaosylceramide in Human Renal Cancer Cells

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Biosynthesis of sulfoglycolipid is markedly increased in a human renal cancer cell line, SMKT-R3. We investigated the sulfotransferase catalyzing the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to gangliotriaosylceramide (Gg3Cer) in SMKT-R3 cells. On thin-layer chromatography, the reaction product comigrated with Gg3Cer III³-sulfate (SM2b), not with Gg3Cer II³-sulfate (SM2a). To examine which monosaccharide of Gg3Cer was sulfated, the product was treated with β-hexosaminidase A. Unlike authentic SM2a, of which the non-reducing terminal N-acetylgalactosamine was cleaved off, the product was not hydrolyzed. These results suggest that sulfate was transferred to the non-reducing terminal N-acetylgalactosamine. Gg3Cer sulfotransferase activity was independent of divalent cations but stimulated by Fe²⁺, and the optimal pH was approximately 6.5. The apparent K_m values were 102 μM for PAPS and 348 umM for Gg3Cer. Gg3Cer II³, III³-bis-sulfate (SB2) and a sulfotransferase activity synthesizing SB2 from SM2a were also detected in SMKT-R3 cells.