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INVERTEBRATE CONNECTIN, AN ELASTIC PROTEIN IN THE GIANT SARCOMERE OF INVERTEBRATE STRIATED MUSCLE

无脊椎动物连接蛋白，无脊椎动物横纹肌巨肌节中的一种弹性蛋白

基本信息

批准号：
13440248
负责人：
KIMURA Sumiko
金额：
$ 9.6万
依托单位：
CHIBA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13440248/
关键词：
I-Connectin Giant sarcomere Elastic protein PEVK obliquely striated muscle Titin striated muscle invertebrate 1分子計測ザリガニ

项目摘要

The giant filamentous protein, connectin (3000-3700 kDa) contributes to organize a contractile sarcomere by extending from the Z line over the thick filament up to the M line at the center of the sarcomere in vertebrate striated muscle. A molecule of connectin is very flexible and approximately 1 μm long in resting sarcomeres, and can be passively stretched resulting in tension generation.In crayfish claw closer muscle, the giant sarcomeres are 8.3 μm long at rest, 3.5 times longer than vertebrate striated muscle sarcomeres and they are extensible up to 13 μm upon stretch. In order to clarify what kind of primary structure makes such a remarkable extension possible, we sequenced the cDNA encoding the Invertebrate-connectin (I-connectin) in crayfish claw closer muscle. The entire sequence of crayfish I-connectin was predicted from cDNA sequences of 53,424 bp (17,352 residues ; 1960 kDa). Crayfish I-connectin contains a novel SEK repeats, and also two PEVK domains and one kettin region. Kettin is a small isoform of I-connectin. Immunoblot tests using antibody to the SEK repeats, revealed the presence of I-connectin also in long sarcomeres of insect leg muscle and barnacle ventral muscle. The force measurements of the single recombinant SEK polypeptide were performed by intemolecular force microscopy(IFM), a refined AFM system. The force versus extension curves were well fit to the wormlike chain(WLC) model and the obtained persistence length of 0.37 nm indicates that the SEK region is a random coil for full length. Immunofluorescence microscopy demonstrated that the two repeats, the long spacer and the two PEVK domains contribute to sarcomere extension. These regions may allow a span of a 3.5 μm distance as a new class of composite spring.

在脊椎动物横纹肌中，巨大的丝状蛋白连接蛋白（3000-3700 kDa）通过从粗丝上的Z线延伸到肌节中心的M线，有助于组织可收缩的肌节。连接蛋白分子非常柔韧，在静止的肌节中长度约为1 μm，可以被动拉伸从而产生张力。在小龙虾的爪状肌中，静止时的巨型肌节长度为8.3 μm，是脊椎动物横纹肌肌节长度的3.5倍，拉伸时可伸展至13 μm。为了弄清是什么样的初级结构使这种显著的延伸成为可能，我们对小龙虾爪近肌中编码无脊椎连接蛋白（I-connectin）的cDNA进行了测序。从全长53,424 bp（17,352个残基，1960 kDa）的cDNA序列中预测了小龙虾I-connectin的全序列。小龙虾I-connectin包含一个新的SEK重复序列，以及两个PEVK结构域和一个kettin区域。Kettin是I-connectin的一个小同工异构体。利用SEK重复序列抗体的免疫印迹检测显示，昆虫腿肌和藤壶腹肌的长肌节中也存在I-connectin。单重组SEK多肽的力测量采用改良的分子力显微镜（IFM）系统进行。力-伸曲线与蠕虫状链（WLC）模型拟合良好，得到的持续长度为0.37 nm，表明SEK区域是全长随机线圈。免疫荧光显微镜显示，两个重复序列，长间隔和两个PEVK结构域有助于肌节延伸。这些区域的间距可达3.5 μm，是一种新型的复合弹簧。