Expression Cloning and Functional Characterization of Human cDNA for Ganglioside GM3 Synthase

Abstract

Ganglioside GM3 is a major glycosphingolipid in the plasma membrane and is widely distributed in vertebrates. We describe here the isolation of a human cDNA whose protein product is responsible for the synthesis of GM3. The cloned cDNA spanned 2,359 base pairs, with an open reading frame encoding a protein of 362 amino acids with a predicted molecular mass of 41.7 kDa. The deduced primary structure shows features characteristic of the sialyltransferase family, including a type II transmembrane topology and the sialylmotifs L at the center and S at the C-terminal region. An amino acid substitution from aspartic acid to histidine was demonstrated at a position invariant in sialylmotif L of all the other sialyltransferases so far cloned. The best acceptor substrate for the gene product was lactosylceramide, and cells transfected with the cloned cDNA clearly exhibited de novo synthesis of GM3, with a measurable decrease in the precursor lactosylceramide. Despite the ubiquitous distribution of ganglioside GM3 in human tissues, a major 2.4-kilobase transcript of the gene was found in a tissue-specific manner, with predominant expression in brain, skeletal muscle, and testis, and very low expression in liver. Ganglioside GM3 is a major glycosphingolipid in the plasma membrane and is widely distributed in vertebrates. We describe here the isolation of a human cDNA whose protein product is responsible for the synthesis of GM3. The cloned cDNA spanned 2,359 base pairs, with an open reading frame encoding a protein of 362 amino acids with a predicted molecular mass of 41.7 kDa. The deduced primary structure shows features characteristic of the sialyltransferase family, including a type II transmembrane topology and the sialylmotifs L at the center and S at the C-terminal region. An amino acid substitution from aspartic acid to histidine was demonstrated at a position invariant in sialylmotif L of all the other sialyltransferases so far cloned. The best acceptor substrate for the gene product was lactosylceramide, and cells transfected with the cloned cDNA clearly exhibited de novo synthesis of GM3, with a measurable decrease in the precursor lactosylceramide. Despite the ubiquitous distribution of ganglioside GM3 in human tissues, a major 2.4-kilobase transcript of the gene was found in a tissue-specific manner, with predominant expression in brain, skeletal muscle, and testis, and very low expression in liver. NeuAcα2–3Galβ1–4Glcβ1–1′Cer GalNAcβ1–4(NeuAcα2–3)Galβ1–4Glcβ1–1′Cer Galβ1–3GalNAcβ1–4 (NeuAcα2–3)Galβ1–4Glcβ1–1′Cer (ganglioside nomenclature is based on that of Svennerholm (35Svennerholm L. J. Neurochem. 1963; 10: 613-623Crossref PubMed Scopus (1313) Google Scholar)) 12-O-tetradecanoylphorbol-13 acetate monoclonal antibody fluorescein isothiocyanate fluorescence-activated cell sorter base pairs phosphate-buffered saline. It is known that sialic acid-containing glycosphingolipids, gangliosides, have various important biological functions (1Hakomori S. Annu. Rev. Immunol. 1984; 2: 103-126Crossref PubMed Scopus (541) Google Scholar, 2Hakomori S. J. Biol. Chem. 1990; 265: 18713-18716Abstract Full Text PDF PubMed Google Scholar), and their functions as well as their biosynthesis are currently clarified. In vertebrates, almost all the ganglio-series gangliosides are synthesized from a common precursor, ganglioside GM3,1 which has the simplest structure among the major gangliosides. GM3itself is known to participate in induction of differentiation (3Nojiri H. Takaku F. Tetsuka T. Motoyoshi K. Miura Y. Saito M. Blood. 1984; 64: 534-541Crossref PubMed Google Scholar, 4Nojiri H. Takaku F. Terui Y. Miura Y. Saito M. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 782-786Crossref PubMed Scopus (218) Google Scholar), modulation of proliferation (2Hakomori S. J. Biol. Chem. 1990; 265: 18713-18716Abstract Full Text PDF PubMed Google Scholar, 5Bremer E. Hakomori S. Bowen-Pope D.F. Raines E. Ross R. J. Biol. Chem. 1984; 259: 6818-6825Abstract Full Text PDF PubMed Google Scholar), maintenance of fibroblast morphology (6Meivar-Levy I. Sabanay H. Bershadsky A.D. Futerman A.H. J. Biol. Chem. 1997; 272: 1558-1564Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), signal transduction (7Hakomori S. Yamamura S. Handa K. Ann. N. Y. Acad. Sci. 1998; 845: 1-10Crossref PubMed Scopus (123) Google Scholar), and integrin-mediated cell adhesion (8Kojima N. Hakomori S.-I. Glycobiology. 1991; 1: 623-630Crossref PubMed Scopus (58) Google Scholar). Molecular cloning of genes whose protein products catalyze the transfer of sialic acid through an α-2,3 linkage has been reported (14Sasaki K. Watanabe E. Kawashima K. Sekine S. Dohi T. Oshima M. Hanai N. Nishi T. Hasegawa M. J. Biol. Chem. 1993; 268: 22782-22787Abstract Full Text PDF PubMed Google Scholar, 15Kitagawa H. Paulson J.C. Biochem. Biophys. Res. Commun. 1993; 194: 375-382Crossref PubMed Scopus (124) Google Scholar, 16Kitagawa H. Paulson J.C. J. Biol. Chem. 1994; 269: 17872-17878Abstract Full Text PDF PubMed Google Scholar, 17Giordanengo V. Bannwarth S. Laffont C. Van Miegem V. HarduinLepers A. Delannoy P. Lefebvre J.-C. Eur. J. Biochem. 1997; 247: 558-566Crossref PubMed Scopus (44) Google Scholar), and these protein products had the same acceptor substrates,i.e. the oligosaccharides of O- and N-linked glycoproteins and glycolipids. However, none of the gene products so far reported is known to be involved in the synthesis of ganglioside GM3. We demonstrated previously that the level of GM3 synthase activity was dramatically enhanced during the monocytic differentiation of HL-60 cells (21Nakamura M. Tsunoda A. Sakoe K. Gu J. Nishikawa A. Taniguchi N. Saito M. J. Biol. Chem. 1992; 267: 23507-23514Abstract Full Text PDF PubMed Google Scholar). Using a cDNA library prepared from the differentiated HL-60 cells, we have isolated a cDNA encoding the GM3 synthase by a modified expression cloning method. In the present study, we demonstrate that the GM3 synthase shows some features clearly distinct from those of all the other sialyltransferases so far cloned, although it possesses several features common to members of the sialyltransferase family. The human myelogenous leukemia cell line HL-60, and the mouse lung carcinoma cell line 3LL-J5 (22Inokuchi J. Jimbo M. Kumamoto Y. Shimeno H. Nagamatsu A. Clin. Exp. Metastasis. 1993; 11: 27-36Crossref PubMed Scopus (13) Google Scholar), which completely lacks acidic glycosphingolipids, and its derivative cell lines, 3LL-HK46, which stably expresses the polyoma virus large tumor antigen, and 3LL-ST28, which is a line permanently transfected with human GD3 synthase cDNA, were cultured in RPMI 1640 medium containing 10% fetal calf serum. HL-60 cells were inoculated at 2–3 × 105 cells/ml and treated with 24 nm TPA for 48 h, and poly(A)+ RNA was isolated using a Fast Track mRNA isolation kit (Invitrogen). After reverse transcription, followed by double-strand synthesis and blunt-ending of the termini, cDNA was ligated with BstXI adapter (Invitrogen) and cloned into pCEV18, which is a derivative of pCEV7 (24Ito N. Yonehara S. Schreurs J. Gorman D.M. Maruyama K. Ishii A. Yahara I. Arai K. Miyajima A. Science. 1990; 247: 324-327Crossref PubMed Scopus (291) Google Scholar). The cDNA library was divided into eight parts, and each part was amplified separately in Escherichia coli DH10B (Life Technologies, Inc.). After purification with Qiagen Tip (Qiagen), 100 μg of the plasmid DNA was introduced into 5 × 106 3LL-HK46 cells by electroporation (180 V, 600 μF). Thirty-six to 48 h after transfection, the cells were harvested, washed with PBS(−), and reacted with anti-GM3 mAb M2590 for 30 min on ice and stained with FITC-conjugated rabbit anti-mouse IgG/A/M (Zymed Laboratories Inc.) for 30 min on ice. The immunologically stained cells were isolated using an EPICS Elite ESP cell sorter (Coulter), and the plasmid DNA was rescued from the 5% of the cells sorted into the strongest fluorescent fields. After two rounds of the transfection and the sorting, 5 × 1063LL-HK46 cells were co-transfected with both 100 μg of the plasmid DNA rescued from the strongly fluorescent cells and 20 μg of pBKCMVh2,8ST, harboring human GD3 synthase cDNA (19Nara K. Watanabe Y. Maruyama K. Kasahara K. Nagai Y. Sanai Y. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7952-7956Crossref PubMed Scopus (123) Google Scholar), and GD3 expression on the host cell surface was detected using anti-GD3 mAb R24 as a specific marker for enrichment of the targeted cDNA. The 0.6% of the total cells applied that were distributed into the strongest fluorescent fields were sorted using a FACS Vantage cell sorter (Becton Dickinson). E. coliDH10B cells were electroporated with the plasmid DNA recovered after this sorting and seeded into nine 96-well microplates to yield 100 colonies/well, and the subpool showing the strongest positive signals in the flow cytometric profile was chosen using sibling selection. Finally, using 3LL-ST28 as well as 3LL-HK46 cell lines as host cells, a single candidate cDNA, pCEV4C7, was cloned by seeding 24 96-well microplates with one colony per well. 3LL-HK46 and 3LL-ST28 cells were transfected with 100 μg of either pCEV18 or pCEV4C7 by electroporation. After 36 h, the cells were harvested and used as an enzyme source. The enzymatic activity of GM3 synthase was measured as described previously (19Nara K. Watanabe Y. Maruyama K. Kasahara K. Nagai Y. Sanai Y. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7952-7956Crossref PubMed Scopus (123) Google Scholar). Protein concentrations were estimated by the Pierce protein assay procedure with bovine serum albumin as a standard. MTN blots (CLONTECH) were used to determine the tissue distribution of GM3 synthase mRNA. A 2,066-bpEcoRI-EcoRI fragment of the pCEV4C7 cDNA was gel-purified, labeled with [α-32P]dCTP, and used as a probe. The blots were also hybridized with a radiolabeled human glyceraldehyde-3-phosphate dehydrogenase gene probe to control for the amount and quality of RNA in each sample. We first found that newly synthesized ganglioside GM3 was barely detectable on the surface of recipient 3LL cells using anti-GM3 monoclonal antibody M2590. We therefore devised a strategy for cloning the GM3 synthase gene on the basis of the facts that anti-GD3 monoclonal antibody R24 is an IgG protein, whereas M2590 mAb is an IgM protein, and that the GD3 synthetic sialyltransferase (sialyltransferase-II, SAT-II) cDNA had been successfully cloned and was available for our use (21Nakamura M. Tsunoda A. Sakoe K. Gu J. Nishikawa A. Taniguchi N. Saito M. J. Biol. Chem. 1992; 267: 23507-23514Abstract Full Text PDF PubMed Google Scholar); the polyoma virus large T antigen was first introduced into the mouse lung carcinoma cell line 3LL-J5 (22Inokuchi J. Jimbo M. Kumamoto Y. Shimeno H. Nagamatsu A. Clin. Exp. Metastasis. 1993; 11: 27-36Crossref PubMed Scopus (13) Google Scholar) to produce the 3LL-HK46 cell line, both of which were completely deficient in gangliosides but rich in lactosylceramide (the precursor of ganglioside GM3). Thereafter, transfecting human GD3 synthase cDNA into 3LL-HK46 cells, we successfully established the 3LL-ST28 cell line, which stably expressed ganglioside GD3 synthase activity. 3LL-HK46 cells were used as host cells for the first stage of our expression cloning, in which co-transfection was performed with GD3 synthase cDNA to detect the GD3expression on the cell surface. Then, in the second stage, 3LL-ST28 cells served as recipient cells to efficiently isolate the GM3 synthase gene. The cDNA library was prepared in the vector pCEV18 from poly(A)+ RNA of HL-60 cells that had been differentiated along the monocytic lineage with phorbol ester and was introduced into the host cells by electroporation. For the first two cycles of cell sorting, the transfected cells were stained with M2590 followed by a FITC-conjugated second antibody and subjected to cell sorting. The 5% of the cells sorted into the strongest fluorescent fields was recovered, and the plasmid DNA was rescued by the method of Hirt (25Hirt B. J. Mol. Biol. 1967; 26: 365-369Crossref PubMed Scopus (3352) Google Scholar). Thus enriched plasmid DNA was co-transfected into the host cells with the plasmid pBKCMVh2,8ST (19Nara K. Watanabe Y. Maruyama K. Kasahara K. Nagai Y. Sanai Y. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7952-7956Crossref PubMed Scopus (123) Google Scholar), which harbors human GD3 synthase cDNA. After sorting the cells that were stained with anti-GD3 monoclonal antibody R24 and FITC-conjugated anti-mouse IgG antibody, we obtained a single candidate clone (termed 4C7) via two sequential sibling selections using 3LL-ST28 cells as the hosts. Fig. 1 shows the FACS profile of 3LL cells transiently transfected with clone 4C7. Strongly positive cells were found only when ST28 cells, and not HK46 cells, were used as the hosts. This result of the difficulty in detecting GM3 molecule by FACS suggests different localization or organization of the GM3 on the cell surface in different cell lines (23Ichikawa S. Sakiyama H. Suzuki G. Hidari K.I.P. Hirabayashi Y. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4638-4643Crossref PubMed Scopus (221) Google Scholar). The insert DNA of clone 4C7 spans 2,359 bp with a single open reading frame of 1,089 bp starting at nucleotide 278, which is compatible with Kozak’s rule (26Kozak M. J. Cell Biol. 1989; 108: 229-241Crossref PubMed Scopus (2810) Google Scholar) (Fig. 2 A). The predicted primary sequence contains 362 amino acids with a molecular mass of 41.7 kDa, which is very close to that of the deglycosylated form (43 kDa) of the purified GM3 synthase from rat liver microsomes (27Melkerson-Watson L.J. Sweeley C.C. J. Biol. Chem. 1991; 266: 4448-4457Abstract Full Text PDF PubMed Google Scholar). As shown by the hydropathy analysis (Fig. 2 B), there exists a single anchor helix led by cationic amino acid residues in the N-terminal portion, suggesting that the 4C7 product might be a type II transmembrane protein (28Klein P. Kanehisa M. Delisi C. Biochim. Biophys. Acta. 1985; 815: 468-471Crossref PubMed Scopus (629) Google Scholar). The characteristic sequences found in all the sialyltransferases so far cloned, sialylmotifs L and S (29Drickamer K. Glycobiology. 1993; 3: 2-3Crossref PubMed Scopus (91) Google Scholar), were also found in the center and the C-terminal region of the 4C7 product, respectively, and it should be noted that there was one drastic and nonconservative substitution, of His for Asp (at position 177 in the peptide, in sialylmotif L), which is invariant in all the other known sialyltransferases (11Kim Y.-J. Kim K.-S. Do S.-I. Kim C.-H. Kim S.-K. Lee Y.-C. Biochem. Biophys. Res. Commun. 1997; 235: 327-330Crossref PubMed Scopus (50) Google Scholar, 12Scheidegger E.P. Sternberg L.R. Roth J. Lowe J.B. J. Biol. Chem. 1995; 270: 22685-22688Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 13Nakayama J. Fukuda M.N. Fredette B. Ranscht B. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7031-7035Crossref PubMed Scopus (218) Google Scholar, 14Sasaki K. Watanabe E. Kawashima K. Sekine S. Dohi T. Oshima M. Hanai N. Nishi T. Hasegawa M. J. Biol. Chem. 1993; 268: 22782-22787Abstract Full Text PDF PubMed Google Scholar, 15Kitagawa H. Paulson J.C. Biochem. Biophys. Res. Commun. 1993; 194: 375-382Crossref PubMed Scopus (124) Google Scholar, 16Kitagawa H. Paulson J.C. J. Biol. Chem. 1994; 269: 17872-17878Abstract Full Text PDF PubMed Google Scholar, 17Giordanengo V. Bannwarth S. Laffont C. Van Miegem V. HarduinLepers A. Delannoy P. Lefebvre J.-C. Eur. J. Biochem. 1997; 247: 558-566Crossref PubMed Scopus (44) Google Scholar, 18Stamenkovic I. Asheim H.C. Deggerdal A. Blomhoff H.K. Smeland E.B. Funderud S. J. Exp. Med. 1990; 172: 641-643Crossref PubMed Scopus (49) Google Scholar, 19Nara K. Watanabe Y. Maruyama K. Kasahara K. Nagai Y. Sanai Y. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7952-7956Crossref PubMed Scopus (123) Google Scholar, M. S. A. K. K. H. K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar) (Fig. A and base that the 4C7 protein sequence with other sialyltransferases that catalyze of the α-2,3 and of sialic acid to the acceptor but not with that catalyze of the linkage to the sialic acid of and strongly that the 4C7 protein should be into the sialyltransferase nucleotide and its deduced amino acid sequences of clone 4C7. The nucleotide sequence is on the with sequences of the The amino acid sequence is on the The predicted transmembrane and the are with the L and S are and the characteristic His in the sialylmotif L is the sequence of clone 4C7. hydropathy of the 4C7 product by the method of and Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). the the of sialylmotifs L and S. of amino acid sequence of sialylmotif L in human sialyltransferases so far clone 4C7 H. Paulson J.C. Biochem. Biophys. Res. Commun. 1993; 194: 375-382Crossref PubMed Scopus (124) Google Scholar); (14Sasaki K. Watanabe E. Kawashima K. Sekine S. Dohi T. Oshima M. Hanai N. Nishi T. Hasegawa M. J. Biol. Chem. 1993; 268: 22782-22787Abstract Full Text PDF PubMed Google Scholar); H. Paulson J.C. J. Biol. Chem. 1994; 269: 17872-17878Abstract Full Text PDF PubMed Google Scholar); V. Bannwarth S. Laffont C. Van Miegem V. HarduinLepers A. Delannoy P. Lefebvre J.-C. Eur. J. Biochem. 1997; 247: 558-566Crossref PubMed Scopus (44) Google Scholar); gene I. Asheim H.C. Deggerdal A. Blomhoff H.K. Smeland E.B. Funderud S. J. Exp. Med. 1990; 172: 641-643Crossref PubMed Scopus (49) Google Scholar); GD3 synthase (19Nara K. Watanabe Y. Maruyama K. Kasahara K. Nagai Y. Sanai Y. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7952-7956Crossref PubMed Scopus (123) Google Scholar, M. S. A. K. K. H. K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar); E.P. Sternberg L.R. Roth J. Lowe J.B. J. Biol. Chem. 1995; 270: 22685-22688Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar); J. Fukuda M.N. Fredette B. Ranscht B. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7031-7035Crossref PubMed Scopus (218) Google Scholar); (11Kim Y.-J. Kim K.-S. Do S.-I. Kim C.-H. Kim S.-K. Lee Y.-C. Biochem. Biophys. Res. Commun. 1997; 235: 327-330Crossref PubMed Scopus (50) Google that the 4C7 product the synthesis of ganglioside GM3, we the enzymatic activity in 3LL cells transiently transfected with As shown in Fig. both 3LL-HK46 and 3LL-ST28 cells exhibited GM3 synthase activity only when the cells were transfected with An with the same as ganglioside was when transfected 3LL-ST28 cells were used as the enzyme (Fig. which suggests that GD3 synthase might catalyze the synthesis of ganglioside It is or not GD3 synthase is with synthase K. Watanabe Y. Kawashima I. T. Nagai Y. Sanai Y. Eur. J. Biochem. 1996; PubMed Scopus Google Scholar, J. Fukuda M.N. Hirabayashi Y. A. K. Nishi T. Fukuda M. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, Y.-J. Kim K.-S. Do S.-I. Kim C.-H. Kim S.-K. Lee Y.-C. Biochem. Biophys. Res. Commun. 1997; 235: 327-330Crossref PubMed Scopus (50) Google Scholar). In to the on purified GM3 synthase from rat liver (27Melkerson-Watson L.J. Sweeley C.C. J. Biol. Chem. 1991; 266: 4448-4457Abstract Full Text PDF PubMed Google Scholar) and rat U. Gu Gu T. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar), the substrate of the 4C7 product was found to be to lactosylceramide as the K. C. and M. in It has not been the 4C7 product is or and oligosaccharides of glycoproteins as However, this acceptor for lactosylceramide, and not for the of other with on the substrate of purified the of substrate the 4C7 gene The glycosphingolipid in clone 3LL-HK46 cells was by and it was clearly demonstrated that the de of GM3 in the transfected cells, along with an decrease in lactosylceramide. K. C. and M. in The hydropathy (Fig. 2 or strongly in and sialylmotif S the of the that the enzyme might be to the of the The expression of GM3 synthase in human was by A major transcript in was detected at various level in all human it was expressed in brain, skeletal muscle, and testis, whereas it was very expressed in and In some tissues, a of was detected (Fig. A). in the gene expression of in brain, analysis of various of the human was performed with the same probe. As shown in Fig. GM3 synthase mRNA was widely distributed in human brain, but expression was in the and This the molecular cloning of a human sialyltransferase involved in the biosynthesis of ganglioside GM3. In our expression cloning, it was found that newly synthesized ganglioside GM3 was barely detectable with anti-GM3 antibody M2590 on the surface of transfected 3LL-HK46 cells in FACS analysis (Fig. This was by or in the GD3 synthase the product ganglioside GD3 was detected using its specific antibody R24 (Fig. of expression cloning are and use of an has also been reported M. S. A. K. K. H. K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar). The human GM3 synthase cDNA cloned in the present and its product some different from those of the other sialyltransferases so far cloned. An invariant aspartic acid was found in the C-terminal region of the sialylmotif L of all the other sialyltransferases (Fig. 2 This acidic amino acid was by the amino acid in GM3 synthetic this amino acid is in the GM3 of and A. and M. in this be to the GM3 synthetic activity. It this substitution the the enzyme and its and or not the substitution is to GM3 of all the only lactosylceramide as an acceptor for the by the cloned human GM3 In it has been reported that the purified GM3 synthase from rat liver exhibited a substrate (27Melkerson-Watson L.J. Sweeley C.C. J. Biol. Chem. 1991; 266: 4448-4457Abstract Full Text PDF PubMed Google Scholar) and that the enzyme from rat both and as as well as lactosylceramide U. Gu Gu T. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). we rule in the of the enzyme these the of with different substrate for analysis that the human GM3 synthase gene was expressed in a tissue-specific (Fig. A). The expression of GM3 synthase RNA in (Fig. be responsible for the in of GM3 and of other gangliosides with which might be to A. E. K. Futerman A.H. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, R. Futerman A.H. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar) and S. K. Hirabayashi Y. J. Neurochem. 1995; PubMed Scopus Google Scholar). The expression of GM3 synthase was enhanced by the of HL-60 and cells with the differentiation TPA H. Takaku F. Terui Y. Miura Y. Saito M. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 782-786Crossref PubMed Scopus (218) Google Scholar). of GM3 synthase has not been of the expression of the GM3 synthase gene should yield into the by which cells be to into We Taniguchi and Maruyama and for the anti-GM3 mAb and the plasmid pCEV18,