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Lee, Young-Choon,Kojima, Naoya,Tsuji, Shuich,Inoue, Mio,Liu, Hong,Kono, Mari,Hamamoto, Toshiro,Takashima, Shou 東亞大學校附設遺傳工學硏究所 1999 遺傳工學硏究 Vol.- No.6
The cDNA encoding a new type of α2,3-sialyl-transferase (mST3Gal V) was cloned from mouse brain cDNA library by PCR-based cloning approach using a pair of degenerate primers deduced from the nucleotide sequence information of mouse ST3Gal III and IV. The predicted amino acid sequence of mST3Gal V showed 27.3% and 26.4% identity to mST3Gal III and IV, respectively. The recombinant soluble mST3Gal V fused with protein-A, which expressed in the culture media of COS-7 cells, showed activity toward lactosyl-ceramide (LacCer), and synthesized GM3. The apparent Km value for LacCer was 9.3 uM.mST3Gal V did not exhibit any activity toward other substrates we tested in this study, including glycolipids, glycoproteins and disaccharides. The mST3Gal V cDNA transfected F28-7 cells, which express large amount of lactosylceramide and very small amount of GM3 at native stage, expressed a large amount of GM3. The ST3Gal V gene was strongly expressed in mouse brain and liver, which contained a large amount of ganglioside. The gene expression seemed to be coincident with ganglioside expression in mouse. Thus, we conclude that mST3Gal V is the fifth-type α2,3-sialyltransferase carrying GM3 synthetic activity.
Lee, Young-Choon,Kono, Mari,Ohyama, Yuji,Hamamoto, Toshiro,Kojima, Naoya,Tsuji, Shuichi 東亞大學校附設遺傳工學硏究所 1998 遺傳工學硏究 Vol.- No.5
Four types of B-galactoside α2,3-sialyltransferase(ST3GalI-IV) have been cloned from several animals, but some contradictory observations regarding their substrate specificities and expression have been reported. Therefore, it is necessary to concurrently analyze the substrate specificities of the four enzymes, of which the source should be one animal. Accordingly, the acceptor substrate specificities and gene expression of mST3Gal I-IV were analyzed. Since we had already cloned ST3Gal I and II, as previously reported(Lee, Y.-C.et al., Eur.J.Biochem.,216,377-385(1993);J.Biol. Chem., 269,10028-10033(1994), the cDNAs of ST3Gal III and IV were cloned from mouse cDNA libraries. Each of the four enzymes was expressed in COS-7 cells as a recombinant enzyme fused with protein A, and applied on an IgG-Sepharose gel to eliminate endogenous sialytransferase activity. ST3Gal I and II showed the highest activity toward GalB1,3GalNAc(typeIII), very low activity toward GalB1,3GalNAc(typeIII), very low activity toward GalB1,3GlcNAc(typeI), but none toward GalB1,4GlcNAc(typeII). ST3Gal III and IV exhibited high activity toward the type I and II disaccharides, but very low activity toward the type I and II disaccharides, but very low activity toward the type I and III one. On the other hand, asialo-GM1(Gg4Cer) was as good a substrate for ST3Gal I and II as the type III disaccharide, though ST3Gal III and IV hardly utilized glycolipids as substrates, as indicated by in vitro experiments. Northern blot analysis revealed that enzymes of the ST3Gal-family are expressed mainly in a tissue-specific manner. The ST3Gal I gene was strongly expressed in spleen and salivary gland, and weakly in brain, liver, heart, kidney, and thymus. The ST3Gal II gene was strongly expressed in brain, and weakly in colon, thymus, salivary gland, and testis, and developmentally expressed in liver, heart, kidney, and spleen. The ST3Gal III and IV genes were expressed in a wide variety of tissues. These differences in tissue specific expression suggest the expression of each ST3Gal influences the distribution of sialyl-glycoconjugates in vivo.