Characterization of amphioxus nebulin and its similarity to human nebulin
Characterization of amphioxus nebulin and its similarity to human nebulin
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DOI：
10.1242/jeb.022681
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发表时间：
2009-03-01
影响因子：
2.8
通讯作者：
Kimura, Sumiko
中科院分区：
文献类型：
--
作者：
Hanashima, Akira;Kubokawa, Kaoru;Kimura, Sumiko
肌肉中大分子的鉴定很重要，但很难通过蛋白质化学来实现。在这项研究中，我们从文昌鱼的横纹肌中分离出了星云蛋白 cDNA，并对其他脊索动物的星云蛋白 C 末端区域进行了表征。虽然与人类的序列同源性只有26%，但文昌鱼星云蛋白的C端区域具有相似的35个氨基酸星云蛋白重复序列和SH3结构域的结构基序。使用针对细菌产生的重组肽产生的特异性抗体进行原位间接免疫荧光分析，我们确定该星云蛋白片段位于肌节的 Z 线，类似于人星云蛋白。体外 Pull-down 和共沉降测定表明，C 末端区域与肌动蛋白、α-肌动蛋白和连接蛋白（肌联蛋白）结合。这些结果表明，文昌鱼星云蛋白的 C 末端区域在维持横纹肌结构方面发挥着与人类星云蛋白相似的作用。这是首次报道文昌鱼肌肉中星云蛋白的确切位置。
Identification of a large molecule in muscle is important but difficult to approach by protein chemistry. In this study we isolated nebulin cDNA from the striated muscle of amphioxus, and characterized the C-terminal regions of nebulins from other chordates. Although the sequence homology with that of human is only 26%, the C-terminal region of amphioxus nebulin has similar structural motifs of 35 amino acid nebulin repeats and an SH3 domain. Using in situ indirect immunofluorescence analysis with a specific antibody raised to the bacterially produced recombinant peptide, we identified that this nebulin fragment is located in the Z-line of the sarcomere, similar to human nebulin. Pull-down and co-sedimentation assays in vitro showed that the C-terminal region binds to actin, alpha-actinin and connectin (titin). These results suggest that the C-terminal region of amphioxus nebulin plays a similar role in maintaining striated muscle structure to that of human nebulin. This is the first report of the exact location of nebulin in amphioxus muscle.