NEW ELASTIC STRUCTURE OF GIANT SARCOMERE

在脊椎动物的横纹肌中，连接素(也称为肌联蛋白)(MM3000 KDa)从粗丝上的Z线延伸到肌节的M线。在静止状态下，一个分子跨越半个肌节，大约1.2μ米长。另一方面，在小龙虾的巨大肌节中，长度为8.3μm的小龙虾爪子更近的肌肉在静止时具有类似于连接蛋白的弹性蛋白的功能。3000 kDa的蛋白质是如何覆盖这么远的距离的？从从小龙虾Clow Closer cDNA文库中克隆的cDNA序列中预测了3000 kDa的蛋白质全序列。发现了与脊椎动物连接素明显不同的序列。首先，存在68个和71个氨基酸残基的新重复基序。其次，富含脊椎动物连接素的免疫球蛋白C2基序或纤维连接蛋白III型的数量非常少。第三，有两个大的弹性PEVK结构域，而在脊椎动物连接中只有一个结构域。第四，连接蛋白、投射蛋白和抽动蛋白共有的肌球蛋白轻链激酶结构域缺失。免疫荧光显微镜显示，当肌节伸展到肌节长度~13μm时，68个残基重复序列和与免疫球蛋白结构域之间的长间距显著延长。圆二色谱表明，新的68个重复序列的融合蛋白由53%的β-Sheet和22%的β-Turn组成。

In vertebrate striated muscle, connectin (also called titin ) (MM 3000 kDa) extends from the Z line over the thick filament up to the M line of the sarcomere. One molecule spans a half sarcomere approximately 1.2μm long at rest. On the other hand, in giant sarcomeres of crayfish claw closer muscle the length of which is 8.3μm long at rest, a similar size of an elastic protein functions as connetin. How does the 3000 kDa protein cover such a long distance?The entire sequence of the 3000 kDa protein was predicted from the cDNA sequences cloned from crayfish claw closer cDNA library. Markedly different sequence from that of vertebrate connectin has been found. First of all, there are novel repeating motifs of 68 and 71 amino acid residues. Second, the number of immunogloblin C2 motif or fibronectin Type III, abundant in vertebrate connectin, is very small. Third, there are two large elastic PEVK domains, as compared to only one domain in vertebrate connectin. Fourth, the myosin light chain kinase domain common to connectin, projectin and twitchin is missing. Immunofluorescence microscopy using antibodies raised to three recombinant peptides demonstrated that the 68 residue repeats and a long spacer between the repeats and immunoglobulin domains were remarkably extensible when the sarcomere was stretched to a sarcomere length of 〜13μm. Furthermore, circular dichroism spectra suggested that a fusion protein of novel 68 repeat consists of 53% β-sheet and 22% β-turn.