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

在脊椎动物横纹肌中，巨大的丝状蛋白连接素(3000-3700 kDa)从粗丝上的Z线延伸到肌节中心的M线，有助于组织收缩的肌节。在静止的肌节中，连接蛋白分子非常灵活，大约1μm长，可以被动拉伸，导致张力生成。在小龙虾的近爪肌中，巨大的肌节在静止时长8.3μm，是脊椎动物横纹肌的3.5倍，拉伸时可伸展到13μm。为了阐明是什么样的一级结构使得这种显著的延伸成为可能，我们对小龙虾爪部肌肉中编码无脊椎动物连接蛋白(I-Connectin)的cDNA进行了测序。从53,424个碱基(17,352个残基；1960 kDa)的cDNA序列中预测了小龙虾I-连接蛋白的整个序列。小龙虾I-Connectin包含一个新的SEK重复序列，还含有两个PEVK结构域和一个kettin区域。Kettin是I-连接蛋白的一种小亚型。利用SEK重复序列的抗体进行免疫印迹试验，发现I-连接蛋白也存在于昆虫腿部肌肉和藤壶腹肌的长肌节中。用改进的原子力显微镜系统(IFM)对单个重组SEK多肽的作用力进行了测量。力-伸长曲线符合蠕虫链(WLC)模型，得到的持续长度为0.37 nm，表明SEK区域是一个全长的无规卷曲。免疫荧光显微镜显示，这两个重复序列、长间隔区和两个PEVK结构域有助于肌节的延伸。这些区域可以允许3.5μm的跨度作为一种新的复合弹簧。

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.