Kono M, Yoshida Y, Kojima N, Tsuji S
Molecular Glycobiology, Frontier Research Program, The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-01, Japan.
J Biol Chem. 1996 Nov 15;271(46):29366-71. doi: 10.1074/jbc.271.46.29366.
The cDNAs encoding a new alpha2,8-sialyltransferase (ST8Sia V) were cloned from a mouse brain cDNA library by means of a polymerase chain reaction-based method using the nucleotide sequence information on mouse ST8Sia I (GD3 synthase) and mouse ST8Sia III (Siaalpha2,3Galbeta1,4GlcNAcalpha2,8-sialyltransferase ), both of which exhibit activity toward glycolipids. The predicted amino acid sequence of ST8Sia V shows 36.1% and 15.0% identity to those of mouse ST8Sia I and III, respectively. The recombinant protein A-fused ST8Sia V expressed in COS-7 cells exhibited an alpha2, 8-sialyltransferase activity toward GM1b, GD1a, GT1b, and GD3, and synthesized GD1c, GT1a, GQ1b, and GT3, respectively. The apparent Km values for GM1b, GD1a, GT1b and GD3 were 1.1, 0.082, 0.070, and 0.28 mM, respectively. However, ST8Sia V did not exhibit activity toward GM3. Thus, the substrate specificity of ST8Sia V is different from those of ST8Sia I and III, both of which exhibit activity toward GM3. Transfection of the ST8Sia V gene into COS-7 cells, which express GD1a as a major glycolipid, led to the expression of determinants for monoclonal antibody 4F10, which recognizes GT1a and GQ1b, suggesting that ST8Sia V exhibits activity toward gangliosides GD1a and/or GT1b in vivo. The expression of the ST8Sia V gene was tissue- and developmental stage-specific, and was clearly different from those of other alpha2,8-sialyltransferase genes. The ST8Sia V gene was strongly expressed in the brain and weakly in other tissues such as the liver. In addition, its expression was greater in the adult than fetal brain. These results strongly indicate that ST8Sia V is a candidate for SAT-V, the alpha2,8-sialyltransferase involved in GD1c, GT1a, GQ1b, and GT3 synthesis.
利用基于聚合酶链反应的方法，借助小鼠ST8Sia I（GD3合酶）和小鼠ST8Sia III（Siaα2,3Galβ1,4GlcNAcα2,8-唾液酸转移酶）的核苷酸序列信息，从小鼠脑cDNA文库中克隆出编码一种新型α2,8-唾液酸转移酶（ST8Sia V）的cDNA。这两种酶均对糖脂具有活性。ST8Sia V的预测氨基酸序列与小鼠ST8Sia I和III的预测氨基酸序列分别具有36.1%和15.0%的同一性。在COS-7细胞中表达的重组蛋白A融合型ST8Sia V对GM1b、GD1a、GT1b和GD3表现出α2,8-唾液酸转移酶活性，并分别合成了GD1c、GT1a、GQ1b和GT3。GM1b、GD1a、GT1b和GD3的表观Km值分别为1.1、0.082、0.070和0.28 mM。然而，ST8Sia V对GM3没有活性。因此，ST8Sia V的底物特异性不同于ST8Sia I和III，后两者均对GM3具有活性。将ST8Sia V基因转染到以GD1a作为主要糖脂的COS-7细胞中，导致了单克隆抗体4F10识别的决定簇的表达，该抗体识别GT1a和GQ1b，这表明ST8Sia V在体内对神经节苷脂GD1a和/或GT1b具有活性。ST8Sia V基因的表达具有组织和发育阶段特异性，且明显不同于其他α2,8-唾液酸转移酶基因。ST8Sia V基因在脑中强烈表达，在肝脏等其他组织中表达较弱。此外，其在成脑中的表达高于胎儿脑。这些结果有力地表明，ST8Sia V是参与GD1c、GT1a、GQ1b和GT3合成的α2,8-唾液酸转移酶SAT-V的候选者。
