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Studies on molecular structure of C-protein and its role in myofibrillogensis

C蛋白分子结构及其在肌原纤维形成中的作用研究

基本信息

批准号：
06454024
负责人：
OBINATA Takashi
金额：
$ 4.35万
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：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

项目摘要

C-protein is a major myosin-binding protein involved in myosin assembly and registration of thick filaments in the sarcomeres during myofibrillogenesis. Distinct C-protein isoforms, fast (F), slow (S), and cardiac (C), are present in both chicken and mammals, and C-isoform is known to be first expressed during chicken muscle development. The present studies were carried out aiming to clarify the molecular structure of C-protein and its role in myofibrillogensis, and the following results were obtained.1) The complete cDNA sequence encoding chicken cardiac (C) C-protein and the deduced amino acid sequence were determined. Seven IgC2 domains, 3 fibronectin type III domains and a phosphorylation site were specified in the molecule and the C-terminal IgC2 was designated to be essential for myosin-binding. In addition, two variants of C isoform which differ only in the presence or absence of the phosphorylation site were detected. Both were detected in cardiac muscle through developmental s … More tages, while only the latter was in embryonic skeletal muscle.2) In order to clarify expression pattern of C-protein isoforms and their roles in mammalian muscle development, the cDNAs encoding mouse skeletal muscle (F and S) C-protein were cloned and determined the entire sequences. In adult mouse tissues, the messages for C-, F-, S- isoforms were detected in a tissue-specific fasion by Northern blotting ; interestingly, however, both F- and S-isoforms were detectable in EDL (extensor digitorum longus) muscle which has been categorized as a typical fast muscle. Although C-isoform is known to be expressed first and transiently during development of chicken skeletal muscles, C-isoform was not detected at all in mouse skeletal muscle through developmental stages, but S-isoform became detectable first, followed by appearance of F-isoform. Basically the same results were obtained at a protein level by means of Western blot and indirect immunofluorescence methods with isoform-specific antibodies. Less

c蛋白是一种主要的肌球蛋白结合蛋白，参与肌原纤维形成过程中肌节中肌球蛋白的组装和粗丝的登记。不同的C蛋白异构体，快速(F)，慢速(S)和心脏(C)，在鸡和哺乳动物中都存在，已知C-异构体在鸡肌肉发育期间首次表达。本研究旨在阐明c蛋白的分子结构及其在肌原纤维病中的作用，得到以下结果：1)确定了鸡心脏(C) C蛋白的完整cDNA序列及推导出的氨基酸序列。在分子中指定了7个IgC2结构域，3个纤维连接蛋白III型结构域和一个磷酸化位点，c端IgC2被指定为肌球蛋白结合所必需的。此外，还检测到C异构体的两种变体，其差异仅在于磷酸化位点的存在或不存在。两者均在发育阶段的心肌中检测到，而只有后者在胚胎骨骼肌中检测到。2)为了阐明c蛋白同工型的表达模式及其在哺乳动物肌肉发育中的作用，克隆了编码小鼠骨骼肌（F和S） c蛋白的cdna，并确定了其全序列。在成年小鼠组织中，通过Northern blotting以组织特异性的方式检测C-， F-， S-亚型的信息；然而，有趣的是，在EDL（指长伸肌）肌肉中都可以检测到F-和s -异构体，EDL被归类为典型的快速肌肉。虽然已知c -异构体在鸡骨骼肌发育过程中首先短暂表达，但在小鼠骨骼肌发育阶段均未检测到c -异构体，但s -异构体首先被检测到，然后出现f -异构体。在蛋白水平上，采用同种异型特异性抗体的Western blot和间接免疫荧光方法获得了基本相同的结果。少