We have isolated a cDNA clone encoding human 3-phosphoadenylylsulfate:galactosylceramide 3-sulfotransferase (EC 2.8.2.11). Degenerate oligonucleotides, based on amino acid sequence data for the purified enzyme, were used as primers to amplify fragments of the gene from human renal cancer cell cDNA by the polymerase chain reaction method. The amplified cDNA fragment was then used as probe to screen a human renal cancer cell cDNA library. The isolated cDNA clone contained an open reading frame encoding 423 amino acids including all of the peptides that were sequenced. The deduced amino acid sequence predicts a type II transmembrane topology and contains two potential N-glycosylation sites. There is no significant homology between this sequence and either the sulfotransferases cloned to date or other known proteins. Northern blot analysis demonstrated that a 1.9-kilobase mRNA was unique to renal cancer cells. When the cDNA was inserted into the expression vector pSVK3 and transfected into COS-1 cells, galactosylceramide sulfotransferase activity in the transfected cells increased from 8-to 16-fold over that of controls, and the enzyme product, sulfatide, was expressed on the transformed cells.Sulfoglycolipids are a class of acidic glycolipids containing sulfate esters on their oligosaccharide chains which were originally found in the human brain by Thudichum (1). Sulfoglycolipids are abundant in myelin, spermatozoa, kidney, and small intestine (for review, see Ref.2) and have been implicated in a variety of physiological functions through their interactions with extracellular matrix proteins, cellular adhesive receptors, blood coagulation systems, complement activation systems, cation transport systems, and microorganisms (for a review, see Ref.3). The addition of sulfate ester is catalyzed by a sulfotransferase with PAPS 1 serving as the sulfate donor.

We have purified 3'-phosphoadenosine-5'-phosphosulfate:GalCer sulfotransferase [EC 2.8.2.11] from a human renal cancer cell line SMKT-R3 through a combination of affinity chromatographies using galactosylsphingosine, 3',5'-bisphosphoadenosine and heparin as ligands. The purified sulfotransferase showed a specific activity of 1.2 mumol/min/mg, which is 300 times more than the highest activity among the enzyme preparations purified so far from other sources. Homogeneity of the purified sulfotransferase was supported by the facts that the enzyme preparation showed a single protein band with an apparent molecular mass of 54 kDa on reducing SDS-PAGE and that protein bands coincided with the enzyme activity on both native PAGE and nonreducing SDS-PAGE. GalCer was the best acceptor for the purified enzyme. LacCer, GalAAG, and GalDG were also good acceptors. GlcCer, Gg3Cer, Gg4Cer, Gb4Cer, and nLc4Cer did serve as acceptors although the relative activities were low. On the other hand, the enzyme could not act on Gb3Cer, which possesses alpha-galactoside at the nonreducing terminus. Neither galactose nor lactose served as an acceptor. These observations suggest that the sulfotransferase prefers beta-glycoside, especially beta-galactoside, at the nonreducing termini of sugar chains attached to a lipid moiety.

Summary A cell line (SMKT-R3) established from human renal cell carcinoma was characterised for the presence of sulfolipids and glycolipid sulfotransferases. Sulfolipids were found to constitute a large part of the acidic glycolipid fraction in SMKT-R3 cells. These findings were confirmed by metabolic labelling with IIS-sulfate. These sulfolipids were expressed at the surface of SMKT-R3 cells as ascertained by cytofluorometry using a monoclonal antibody directed to sulfolipids. Furthermore, markedly high activity levels of glycolipid sulfotransferases were observed in SMKT-R3 cells compared with other cell lines. These results suggest that the increased synthesis of sulfolipids in renal cell carcinoma tissue (Sakakibara et al., 1989. Cancer Res., 49, 335-339) is due to the elevation of the sulfotransferase activities of renal carcinoma cells themselves.Glycolipids have been known to undergo marked cancerassociated changes (Hakomori, 1985). In particular, acidic glycolipids with sialic acid residues, called gangliosides, have been well studied. On the other hand, reports on cancerassociated changes of the other acidic glycolipids, sulfolipids, which contain sulfate residues, are relatively rare (Siddiqui et al., 1978;Gasa et al., 1979;Yoda et al., 1979;Hattori et al., 1981;Mitsuyama et al., 1983;Hiraiwa et al., 1988;Hiraiwa et al., 1990). The synthesis of sulfolipids is catalysed by PAPS:GalCer sulfotransferase (EC 2.8.2.11) (Balasubramanian & Bachhawat, 1965). Although the sulfotransferase from rat kidney (Tennekoon et al., 1985) and testis (Sakac et al., 1992) has been recently purified, the human enzyme has not.In our previous studies, sulfolipids were found to increase markedly in human renal cell carcinoma (Sakakibara et al., 1989), but not in Wilms' tumour (Sakakibara et al., 1991). The increment of the sulfolipid contents in renal cell carcinoma was associated with enhanced activity of glycolipid sulfotransferase in the cancer tissues (Sakakibara et al., 1989). Furthermore, the level of the sulfotransferase appeared to be elevated in sera from patients with renal cell carcimona , and hepatocellular carcinoma DEAE-Sephadex A-25 and Sephadex G-25 were obtained from Pharmacia-LKB. GalCer and LacCer were purified in this laboratory from bovine brain and horse red cell membranes, respectively. Other reagents were of analytical grade.Several human cell lines, A-431 (epidermoid carcinoma), PC-3 (lung adenocarcinoma), HL-60 (acute promyelocyte leukaemia), K-562 (chronic myelogenous leukaemia), were a gift from the Japanese Cancer Research Resources Bank.Cell culture SMKT-R3 cells were established from human renal cell carcinoma as described previously (Miyao et al., 1989), and cultured in Dulbecco's modified minimal essential medium supplemented with 10% foetal bovine serum. Preparation ofglycolipidsCell monolayers were washed with Tris-buffered saline and harvested by scraping with a rubber policeman. Then the cell suspensions were centrifuged and washed three times with Tris-buffered saline. The cell pellets ...