TTLL7 is a mammalian β-tubulin polyglutamylase required for growth of MAP2-positive neurites
TTLL7 is a mammalian β-tubulin polyglutamylase required for growth of MAP2-positive neurites
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DOI：
10.1074/jbc.m603984200
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发表时间：
2006-10-13
影响因子：
4.8
通讯作者：
Setou, Mitsutoshi
中科院分区：
文献类型：
--
作者：
Ikegami, Koji;Mukai, Masahiro;Setou, Mitsutoshi
微管形成影响细胞形状并为细胞提供结构支持的细胞骨架框架。神经系统中的微管经历独特的翻译后修饰，其微管蛋白亚基的C末端的多聚谷氨酰化。哺乳动物的酶，执行β-微管蛋白多聚谷氨酰化，以及它们的生理功能，在神经元组织仍然难以捉摸。我们报告鉴定的哺乳动物多聚谷氨酰胺酶与β-微管蛋白的特异性，以及它的分布和神经突生长的功能。为了确定推定的微管蛋白多聚谷氨酰化酶，我们搜索微管蛋白酪氨酸连接酶样（TTLL）蛋白的主要在神经系统中表达。在13种TTLL蛋白中，TTLL 7在神经系统中转录水平最高。重组TTLL 7在体外催化微管蛋白多聚谷氨酰化，对β-微管蛋白具有高度偏好。当在HEK 293 T细胞中表达时，TTLL 7表现出对β-微管蛋白的特异性，而不是对β-微管蛋白或核小体组装蛋白1的特异性。与这些发现一致，在上级颈神经节神经元的原代培养物中TTLL 7的敲低引起多聚谷氨酰化β-微管蛋白的损失。在用神经生长因子刺激PC 12细胞分化后，TTLL 7的水平伴随β-微管蛋白的多聚谷氨酰化而增加。短干扰RNA介导的TTLL 7敲低抑制了神经生长因子刺激的MAP（微管相关蛋白）2阳性神经突在PC 12细胞中的生长。TTLL 7在上级颈神经节的富含MAP 2的体树突部分内积累，这与其在MAP 2阳性神经突生长中的作用一致，多聚谷氨酰化β-微管蛋白也是如此。抗TTLL 7抗体显示TTLL 7在小鼠脑中分布于体树突隔室中。这些发现表明TTLL 7是β-微管蛋白聚谷氨酰胺酶，并且是PC 12细胞中MAP 2阳性神经突生长所需的。
Microtubules form a cytoskeletal framework that influences cell shape and provides structural support for the cell. Microtubules in the nervous system undergo a unique post-translational modification, polyglutamylation of the C termini of their tubulin subunits. The mammalian enzymes that perform beta-tubulin polyglutamylation as well as their physiological functions in the neuronal tissue remain elusive. We report identification of a mammalian polyglutamylase with specificity for beta-tubulin as well as its distribution and function in neurite growth. To identify putative tubulin polyglutamylases, we searched tubulin tyrosine ligase-like (TTLL) proteins for those predominantly expressed in the nervous system. Of 13 TTLL proteins, TTLL7 was transcribed at the highest level in the nervous system. Recombinant TTLL7 catalyzed tubulin polyglutamylation with high preference to beta-tubulin in vitro. When expressed in HEK293T cells, TTLL7 demonstrated specificity for beta-tubulin and not for beta-tubulin or nucleosome assembly protein 1. Consistent with these findings, knockdown of TTLL7 in a primary culture of superior cervical ganglion neurons caused a loss of polyglutamylated beta-tubulin. Following stimulation of PC12 cells with nerve growth factor to differentiate, the level of TTLL7 increased concomitantly with polyglutamylation of beta-tubulin. Short interference RNA-mediated knockdown of TTLL7 repressed nerve growth factor-stimulated MAP ( microtubule-associated protein) 2- positive neurite growth in PC12 cells. Consistent with having a role in the growth of MAP2-positive neurites, TTLL7 accumulated within a MAP2-enriched somatodendritic portion of superior cervical ganglion, as did polyglutamylated beta-tubulin. Anti-TTLL7 antibody revealed that TTLL7 was distributed in a somatodendritic compartment in the mouse brain. These findings indicate that TTLL7 is a beta-tubulin polyglutamylase and is required for the growth of MAP2- positive neurites in PC12 cells.