Publications

• Accumulation of unusual gangliosides G(Q3) and G(P3) in breast cancer cells expressing the G(D3) synthase.

  Steenackers A., Vanbeselaere J., Cazet A., Bobowski M., Rombouts Y., Colomb F., Le Bourhis X., Guerardel Y., Delannoy P.

  Glycosphingolipids from the ganglio-series are usually classified in four series according to the presence of 0 to 3 sialic acid residues linked to lactosylceramide. The transfer of sialic acid is catalyzed in the Golgi apparatus by specific sialyltransferases that show high specificity toward gly ... >> More

  Glycosphingolipids from the ganglio-series are usually classified in four series according to the presence of 0 to 3 sialic acid residues linked to lactosylceramide. The transfer of sialic acid is catalyzed in the Golgi apparatus by specific sialyltransferases that show high specificity toward glycolipid substrates. ST8Sia I (EC 2.4.99.8, SAT-II, SIAT 8a) is the key enzyme controlling the biosynthesis of b- and c-series gangliosides. ST8Sia I is expressed at early developmental stages whereas in adult human tissues, ST8Sia I transcripts are essentially detected in brain. ST8Sia I together with b- and c-series gangliosides are also over-expressed in neuroectoderm-derived malignant tumors such as melanoma, glioblastoma, neuroblastoma and in estrogen receptor (ER) negative breast cancer, where they play a role in cell proliferation, migration, adhesion and angiogenesis. We have stably expressed ST8Sia I in MCF-7 breast cancer cells and analyzed the glycosphingolipid composition of wild type (WT) and GD3S+ clones. As shown by mass spectrometry, MCF-7 expressed a complex pattern of neutral and sialylated glycosphingolipids from globo- and ganglio-series. WT MCF-7 cells exhibited classical monosialylated gangliosides including G(M3), G(M2), and G(M1a). In parallel, the expression of ST8Sia I in MCF-7 GD3S+ clones resulted in a dramatic change in ganglioside composition, with the expression of b- and c-series gangliosides as well as unusual tetra- and pentasialylated lactosylceramide derivatives G(Q3) (II(3)Neu5Ac(4)-Gg(2)Cer) and G(P3) (II(3)Neu5Ac(5)-Gg(2)Cer). This indicates that ST8Sia I is able to act as an oligosialyltransferase in a cellular context. << Less

  Molecules 17:9559-9572(2012) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Expression cloning of a CMP-NeuAc:NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1'Cer alpha 2,8-sialyltransferase (GD3 synthase) from human melanoma cells.

  Nara K., Watanabe Y., Maruyama K., Kasahara K., Nagai Y., Sanai Y.

  Using an expression cloning approach, we have isolated a cDNA encoding GD3 synthase (CMP-NeuAc:NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1'Cer alpha 2,8-sialyltransferase, EC 2.4.99.8), which is a key regulatory enzyme determining the prominence of the ganglioside biosynthesis pathway. The cloned cDNA ... >> More

  Using an expression cloning approach, we have isolated a cDNA encoding GD3 synthase (CMP-NeuAc:NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1'Cer alpha 2,8-sialyltransferase, EC 2.4.99.8), which is a key regulatory enzyme determining the prominence of the ganglioside biosynthesis pathway. The cloned cDNA encodes a 341-amino acid protein containing a single transmembrane domain at its N-terminal region, suggesting that the protein has a type II transmembrane topology. The sequence of alpha 2,8-sialyltransferase showed a high level of similarity with other sialyltransferases at two conserved regions typical in the sialyltransferase family. Transfected cells containing the cloned cDNA expressed GD3 ganglioside on the cell surface, which was detectable with specific anti-GD3 antibody by immunofluorescence and immunostaining after separation of isolated glycolipids on thin-layer chromatography. The cDNA hybridized to a single mRNA species of 2.4 kb in melanoma cells. This sialyltransferase is distinctive in catalyzing the formation of the alpha 2-8 linkage of sialic acids. << Less

  Proc. Natl. Acad. Sci. U.S.A. 91:7952-7956(1994) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Isolation of GD3 synthase gene by expression cloning of GM3 alpha-2,8-sialyltransferase cDNA using anti-GD2 monoclonal antibody.

  Haraguchi M., Yamashiro S., Yamamoto A., Furukawa K., Takamiya K., Lloyd K.O., Shiku H., Furukawa K.

  For the isolation of ganglioside GD3 synthase (EC 2.4.99.8) cDNA, we developed an expression cloning approach that used an anti-GD2 monoclonal antibody for selection. A host recipient cell line that we have named KF3027-Hyg5 was also utilized. This cell line expresses high levels of GM2 as well as ... >> More

  For the isolation of ganglioside GD3 synthase (EC 2.4.99.8) cDNA, we developed an expression cloning approach that used an anti-GD2 monoclonal antibody for selection. A host recipient cell line that we have named KF3027-Hyg5 was also utilized. This cell line expresses high levels of GM2 as well as GM3 but no GD3 or GD2 and was constructed from mouse B16 melanoma cells transfected with the polyoma large tumor antigen gene (KF3027) and the previously cloned beta-1,4-N-acetylgalactosaminyltransferase (EC 2.4.1.92) cDNA. Four rounds of transfection, monoclonal antibody 3F8 panning, and Hirt extraction resulted in the isolation of two cDNA clones, transfection of which directed the expression of GD3 in KF3027 and B16 melanoma cells and GD3 and GD2 in KF3027-Hyg5 cells. The cDNA contained a 1650-bp insert and a single open reading frame. The deduced amino acid predicted a type II membrane topology consisting of cytoplasmic (14 aa), transmembrane (18 aa), and catalytic (309 aa) domains. The sequence also predicted the presence of a sialyl motif similar to that found in the other sialyltransferases cloned so far. As expected, mRNA of this gene (2.6 kb) was strongly expressed in human melanoma lines. << Less

  Proc. Natl. Acad. Sci. U.S.A. 91:10455-10459(1994) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Acceptor substrate specificity of a cloned GD3 synthase that catalyzes the biosynthesis of both GD3 and GD1c/GT1a/GQ1b.

  Nara K., Watanabe Y., Kawashima I., Tai T., Nagai Y., Sanai Y.

  To address the role of alpha2,8-sialyltransferase (GD3 synthase) in the biosynthesis of gangliosides, we examined the substrate specificity of the enzyme. In the ganglioside synthesis pathway, it has been generally accepted that sialyltransferase II (SAT II) catalyzes the production of GD3 from GM ... >> More

  To address the role of alpha2,8-sialyltransferase (GD3 synthase) in the biosynthesis of gangliosides, we examined the substrate specificity of the enzyme. In the ganglioside synthesis pathway, it has been generally accepted that sialyltransferase II (SAT II) catalyzes the production of GD3 from GM3, and sialyltransferase V (SAT V) catalyzes the production of GD1c/GT1a/GQ1b from GM1h/GD1a/GT1b. However, acceptor specificity of the clones GD3 synthase that was isolated from human melanoma cells [Nara, K., Watanabe, Y., Maruyama, K., Kasahara, K., Nagai. Y. & Sanai, Y. (1994) Proc. Natl Acad. Sci. USA 91, 7952-7956] has revealed that this enzyme utilized the gangliosides containing the terminal Sia(alpha2-3)Gas structure of the carbohydrate moiety, which includes GM3, GM1b, GD1a and GT1B as exogenous substrates. Kinetic data also showed that the enzyme was able to utilize both GM3 and GM1b/GD1a/GT1b as acceptor substrates. These data indicate that the enzyme catalyzes the formation of not only GD3 but also GD1c, GT1a, and GQ1B in vitro. Furthermore, by transfection of the cloned human alpha2,8-sialyltransferase cDNA, transient and stable expression of GT1a and GQ1b wa also observed in COS-7 cells and Swiss 3T3 cells that originally lacked SAT II and SAT V activities. These observations indicate that the enzyme has both SAT II and SAT V activities in vivo. << Less

  Eur. J. Biochem. 238:647-652(1996) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Expression cloning of a GM3-specific alpha-2,8-sialyltransferase (GD3 synthase).

  Sasaki K., Kurata K., Kojima N., Kurosawa N., Ohta S., Hanai N., Tsuji S., Nishi T.

  A cDNA encoding a GM3-specific alpha-2,8-sialyltransferase (GD3 synthase) was obtained from an expression cDNA library of human melanoma cell line WM266-4 by enrichment of Namalwa KJM-1 cells highly expressing GD3 using an anti-GD3 antibody and a fluorescence-activated cell sorter. Selection of B- ... >> More

  A cDNA encoding a GM3-specific alpha-2,8-sialyltransferase (GD3 synthase) was obtained from an expression cDNA library of human melanoma cell line WM266-4 by enrichment of Namalwa KJM-1 cells highly expressing GD3 using an anti-GD3 antibody and a fluorescence-activated cell sorter. Selection of B-cell line Namalwa cells expressing transfected cDNAs in the presence of anti-GD3 monoclonal antibody KM641 gave a cDNA (pAMo-GD3) encoding a protein with a type II transmembrane topology as found for mammalian glycosyltransferases. The following evidence confirms that the cDNA encodes an alpha-2,8-sialyltransferase, which specifically converts GM3 to GD3. (i) Transfection of pAMo-GD3 into Namalwa KJM-1 cells leads to the appearance of GD3 and a GD3 synthase activity. (ii) Northern blot analysis revealed a correlation between the expression of this gene and GD3 in several cell lines. (iii) The putative COOH-terminal active domain of this cloned enzyme fused with protein A has been purified with IgG-Sepharose beads and has been shown to possess GD3-synthesizing activity, excluding the possibility that the cloned cDNA encodes a transacting factor inducing a GD3 synthase. The deduced primary sequence also contains the "sialyl motif" conserved among all the sialyltransferases cloned to date. The polymerase chain reaction analysis reveals that this gene is located on chromosome 12. << Less

  J. Biol. Chem. 269:15950-15956(1994) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Expression cloning of a human GT3 synthase. GD3 and GT3 are synthesized by a single enzyme.

  Nakayama J., Fukuda M.N., Hirabayashi Y., Kanamori A., Sasaki K., Nishi T., Fukuda M.

  Gangliosides of the C series such as GT3 are polysialylated glycosphingolipids whose synthesis is developmentally regulated. Here we report the expression cDNA cloning and characterization of GT3 synthase that adds the second alpha-2,8-sialic acid to GD3, NeuNAcalpha2-->8NeuNAcalpha2-->3Galbeta1-- ... >> More

  Gangliosides of the C series such as GT3 are polysialylated glycosphingolipids whose synthesis is developmentally regulated. Here we report the expression cDNA cloning and characterization of GT3 synthase that adds the second alpha-2,8-sialic acid to GD3, NeuNAcalpha2-->8NeuNAcalpha2-->3Galbeta1-->4Glc-->Cer, thus forming GT3, NeuNAcalpha2-->8NeuNAcalpha2-->8NeuNAc alpha2-->3Galbeta1--> 4Glc-->Cer. Unexpectedly, the cloned cDNA was found to be identical to the cDNA that encodes GD3 synthase. The newly identified enzyme was therefore named GD3/GT3 synthase (GD3/GT3ST). GD3/GT3ST synthesized GT3 most efficiently when GM3, NeuNAcalpha2-->3Galbeta1-->4Glc-->Cer, was incubated as an acceptor, indicating that GD3/GT3ST is a polysialyltransferase that can transfer more than one sialic acid residue via alpha-2,8 linkage to gangliosides. Moreover, a longer period of incubation of GD3 with GD3/GT3ST produced a significant amount of GT3 and higher polysialogangliosides. Among various cell lines expressing GD3/GT3ST, higher polysialogangliosides including GT3 were detected only in cell lines where the amount of GD3/GT3 mRNA is sufficiently high. The expression of GD3/GT3ST mRNA among human tissues is highly restricted to fetal and adult brains. The GD3/GT3ST gene was found to be located at chromosome 12, region p12. Taken together, these results indicate that C series polysialogangliosides are synthesized by a ganglioside-specific polysialyltransferase, GD3/GT3ST, that is specifically expressed in neural tissues. << Less

  J. Biol. Chem. 271:3684-3691(1996) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Molecular cloning and expression of a sixth type of alpha 2,8-sialyltransferase (ST8Sia VI) that sialylates O-glycans.

  Takashima S., Ishida H.K., Inazu T., Ando T., Ishida H., Kiso M., Tsuji S., Tsujimoto M.

  A novel member of the mouse alpha2,8-sialyltransferase (ST8Sia) family, designated ST8Sia VI, was identified by BLAST analysis of expressed sequence tags. The sequence of ST8Sia VI encodes a protein of 398 amino acids and shows 42.0 and 38.3% amino acid sequence identities to mouse alpha2,8-sialyl ... >> More

  A novel member of the mouse alpha2,8-sialyltransferase (ST8Sia) family, designated ST8Sia VI, was identified by BLAST analysis of expressed sequence tags. The sequence of ST8Sia VI encodes a protein of 398 amino acids and shows 42.0 and 38.3% amino acid sequence identities to mouse alpha2,8-sialyltransferases ST8Sia I (GD3 synthase) and ST8Sia V (GD1c, GT1a, GQ1b, and GT3 synthases), respectively. The recombinant soluble form of ST8Sia VI expressed in COS-7 cells exhibited alpha2,8-sialyltransferase activity toward both glycolipids and glycoproteins that have the NeuAcalpha2,3(6)Gal sequence at the nonreducing end of their carbohydrate groups. This enzyme formed NeuAcalpha2,8NeuAc structures, but not oligosialic or polysialic acid structures. Analysis of the fetuin sialylated by ST8Sia VI indicated that ST8Sia VI prefers O-glycans to N-glycans as acceptor substrates. Substrate specificities and kinetic properties also showed that ST8Sia VI prefers O-glycans to glycolipids as acceptor substrates. ST8Sia VI also exhibited activity toward oligosaccharides such as sialyllactose and sialyllactosamine, and the structure of the minimal acceptor substrate for ST8Sia VI was determined as the NeuAcalpha2,3(6)Gal sequence. The expression of the ST8Sia VI gene was ubiquitous, and the highest expression was observed in kidney, with three major transcripts of 8.2, 3.8, and 2.7 kb. This is the first report of a mammalian alpha2,8-sialyltransferase that sialylates O-glycans preferentially. << Less

  J. Biol. Chem. 277:24030-24038(2002) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.