Enzymatic sulfation of gangliotriaosylceramide in human renal cancer cells.
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 beta-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 Km values were 102 microM for PAPS and 348 microM for Gg3Cer. Gg3Cer II3,III3-bis-sulfate (SB2) and a sulfotransferase activity synthesizing SB2 from SM2a were also detected in SMKT-R3 cells.
Kobayashi
T
T
Biochemistry Laboratory, Hokkaido University School of Medicine, Sapporo.
Honke
K
K
Gasa
S
S
Saga
Y
Y
Miyazaki
T
T
Tadano-Aritomi
K
K
Ishizuka
I
I
Makita
A
A
D000116:Acetylgalactosamine / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002292:Carcinoma, Renal Cell / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D002417:Cattle; D002855:Chromatography, Thin Layer; D005732:Gangliosides; D006017:Glycolipids / Q000378:metabolism; D006028:Glycosphingolipids / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007680:Kidney Neoplasms / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D051381:Rats; D013431:Sulfates / Q000378:metabolism*; D015238:Sulfotransferases / Q000737:chemistry / Q000378:metabolism*; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
D005732:Gangliosides; D006017:Glycolipids; D006028:Glycosphingolipids; D013431:Sulfates; C038244:ganglio-N-triaosylceramide; D015238:Sulfotransferases; D001619:beta-N-Acetylhexosaminidases; D000116:Acetylgalactosamine