Angata Kiyohiko, Chan Dominic, Thibault Joseph, Fukuda Minoru
Glycobiology Program, Cancer Research Center, The Burnham Institute, La Jolla, California 92037, USA.
J Biol Chem. 2004 Jun 11;279(24):25883-90. doi: 10.1074/jbc.M401562200. Epub 2004 Apr 2.
Polysialic acid, a homopolymer of alpha2,8-linked sialic acid expressed on the neural cell adhesion molecule (NCAM), is thought to play critical roles in neural development. Two highly homologous polysialyltransferases, ST8Sia II and ST8Sia IV, which belong to the sialyltransferase gene family, synthesize polysialic acid on NCAM. By contrast, ST8Sia III, which is moderately homologous to ST8Sia II and ST8Sia IV, adds oligosialic acid to itself but very inefficiently to NCAM. Here, we report domains of polysialyltransferases required for NCAM recognition and polysialylation by generating chimeric enzymes between ST8Sia IV and ST8Sia III or ST8Sia II. We first determined the catalytic domain of ST8Sia IV by deletion mutants. To identify domains responsible for NCAM polysialylation, different segments of the ST8Sia IV catalytic domain, identified by the deletion experiments, were replaced with corresponding segments of ST8Sia II and ST8Sia III. We found that larger polysialic acid was formed on the enzymes themselves (autopolysialylation) when chimeric enzymes contained the carboxyl-terminal region of ST8Sia IV. However, chimeric enzymes that contain only the carboxyl-terminal segment of ST8Sia IV and the amino-terminal segment of ST8Sia III showed very weak activity toward NCAM, even though they had strong activity in polysialylating themselves. In fact, chimeric enzymes containing the amino-terminal portion of ST8Sia IV fused to downstream sequences of ST8Sia III inhibited NCAM polysialylation in vitro, although they did not polysialylate NCAM. These results suggest that in polysialyltransferases the NCAM recognition domain is distinct from the polysialylation domain and that some chimeric enzymes may act as a dominant negative enzyme for NCAM polysialylation.
多唾液酸是一种在神经细胞黏附分子（NCAM）上表达的α2,8-连接唾液酸的同聚物，被认为在神经发育中起关键作用。两种高度同源的多唾液酸转移酶ST8Sia II和ST8Sia IV属于唾液酸转移酶基因家族，它们在NCAM上合成多唾液酸。相比之下，与ST8Sia II和ST8Sia IV中度同源的ST8Sia III会将寡唾液酸添加到自身，但对NCAM的添加效率非常低。在此，我们通过在ST8Sia IV与ST8Sia III或ST8Sia II之间生成嵌合酶，报告了NCAM识别和多唾液酸化所需的多唾液酸转移酶结构域。我们首先通过缺失突变体确定了ST8Sia IV的催化结构域。为了鉴定负责NCAM多唾液酸化的结构域，将缺失实验确定的ST8Sia IV催化结构域的不同片段替换为ST8Sia II和ST8Sia III的相应片段。我们发现，当嵌合酶包含ST8Sia IV的羧基末端区域时，酶自身会形成更大的多唾液酸（自身多唾液酸化）。然而，仅包含ST8Sia IV羧基末端片段和ST8Sia III氨基末端片段的嵌合酶对NCAM的活性非常弱，尽管它们在自身多唾液酸化方面具有很强的活性。事实上，包含ST8Sia IV氨基末端部分与ST8Sia III下游序列融合的嵌合酶在体外抑制了NCAM的多唾液酸化，尽管它们不会对NCAM进行多唾液酸化。这些结果表明，在多唾液酸转移酶中，NCAM识别结构域与多唾液酸化结构域不同，并且一些嵌合酶可能作为NCAM多唾液酸化的显性负性酶起作用。
