Molecular regulatory mechanism of cell surface garbohydrate expression and its pathology.

细胞表面碳水化合物表达的分子调控机制及其病理学。

• 批准号: 05454163
• 负责人: YAMAOKA Kazuko
• 金额: $ 4.1万
• 依托单位: Tokyo Metropolitan Institute of Medical Science
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05454163/
• 关键词: tumor growth factor; galectin; ganglioside; sialyltransferase; genome; 糖脂質; シアル酸; 糖転移酵素

(1) Intramolecular disulfide bonds are essential for the tumor growth factor activity of TGF_<gamma>2 : Amino acid sequence analysis of TGF_<gamma>2 revealed the identity 14Kda galactoside-binding protein (galectin-1) .The TGF_<gamma>2 purified from rat tumor cells, however, showed no sugar-binding activity.To analyze the difference in each activity between TGF_<gamma>2 and galectin-1, we studied the intramolecular disulfide bonds in TGF and galectin-1 by the several methods including chemical clevage of S-S bridge, MOLD-mass spectrometry and affinity chromatography.Several lines of evidences indicated that intramolecular S-S bonds in TFG_<gamma>2 is required for TGF activity and that the lectin activity is induced by thecleavage of S-S bonds in TGF_<gamma>2, possibly as a result of the conformational change.(2) Molecular cloning, expression and specificity of GD3 synthase : We obtained the cDNA encoding GD3 synthase by the expression cloning strategy.The substrate specificy of the cloned GD3 synthase showed that this enzyme synthesizes not only GD3, but GQ1b which was believed by the other sialyltransferase, SAT5.These results suggest this enzyme play a key role in the regulation of ganglioside expression in CNS.

(1)分子内二硫键是肿瘤生长因子活性所必需的。2.通过对其氨基酸序列的分析，发现了14Kda的半乳糖苷结合蛋白(Galectin-1)，而从大鼠肿瘤细胞中提纯的TGF_2则没有糖结合活性。通过S-S桥化学清除、模质谱、亲和层析等方法对转化生长因子和半乳糖凝集素-1的分子内二硫键进行了研究。一系列证据表明，转化生长因子的活性需要转化生长因子的分子内S-S键，凝集素的活性是由转化生长因子的S-S键断裂所致。(2)GD3合成酶的分子克隆、表达及特异性：通过表达克隆策略获得了GD3合成酶的编码基因，底物特异性表明该酶不仅能合成GD3，还能合成GQ1b，而另一种唾液酸基转移酶SAT5则认为该酶在中枢神经系统神经节苷脂的表达调控中起着关键作用。

项目成果

NARA,K.: ""Expression cloning of a CMP-NeuAc : NeuAcalpha2,3 Galbeta1,4Glcbeta1,1'Cer alpha2,8-Sialyltransferase (GD3 Synthase) from Human Melanoma Cells."" Proc.Natl.Acad.Sci.USA. 91. 7952-7956 (1994)

NARA,K.：“从人黑色素瘤细胞中表达克隆 CMP-NeuAc：NeuAcalpha2,3 Galbeta1,4Glcbeta1,1Cer α2,8-唾液酸转移酶（GD3 合酶）。”Proc.Natl.Acad.Sci.USA

Hidari,K.I.J.P.: "A unique biosynthetic pathway for gangliosides exist in Xenopus laevis oocytes" FEBS letters. 353. 25-28 (1994)

Hidari,K.I.J.P.：“非洲爪蟾卵母细胞中存在一种独特的神经节苷脂生物合成途径”FEBS 信件。

KAZUKO YAMAOKA: "Structural and functional characterization of a novel tumorderived rat galectin-1 having transforming growth factor(TGF)activity" J. of Biochemistry. (in press). (1996)

KAZUKO YAMAOKA：“具有转化生长因子 (TGF) 活性的新型肿瘤源性大鼠半乳糖凝集素-1 的结构和功能表征”《生物化学杂志》。

Hidari,K.I.J.P.: "In vitro synthesis of disialoganglioside(GD1alpha) from asialo-GM1 using sialyltransferase" Eur.J.Biochem.221. 603-609 (1994)

Hidari,K.I.J.P.：“使用唾液酸转移酶从 asialo-GM1 体外合成二唾液酸神经节苷脂 (GD1α)”Eur.J.Biochem.221。

Kiyomitsu Nara: "Expression Cloning of a CMP-NeuAc: NeuAcα2,3 Galβ1-4Glcβ1.1'Cer α2、8-Sialyltransferase(Go3 Synthase) from Human Melanoma Cells" Proc. Natl. Acad. Sci. USA. 91. 7952-7956 (1994)

Kiyomitsu Nara：“来自人类黑色素瘤细胞的 CMP-NeuAc：NeuAcα2,3 Galβ1-4Glcβ1.1Cer α2,8-唾液酸转移酶（Go3 合成酶）的表达克隆”，美国科学院院刊 91。 7956 (1994)