Role of myosin-binding poteins in myofibrillogenesis

C蛋白是横纹肌的主要肌球蛋白结合蛋白。为了阐明C-蛋白在肌纤维形成中的作用，我们进行了以下实验，获得了如下结果：1)小鼠C-蛋白基因序列及C-蛋白异构体的表达克隆了小鼠快骨骼(F)和慢骨骼(S)C-蛋白的cDNA，并测定了它们的全序列。已知心脏(C)-异构体在鸡骨骼肌发育过程中最先和短暂地表达，而在小鼠骨骼肌中，C-异构体在发育过程中没有检测到。首先检测到S异构体，然后出现F异构体，在退化的小鼠骨骼肌中，S异构体的表达增加。2)鸡心脏C蛋白中的两种异构体及其表达我们在鸡心脏C蛋白中检测到两种异构体(类型I和类型II)，并测定了它们的全序列；它们的区别仅在于包含或不包含…更多的15个氨基酸残基的序列(P-seq)，其中包括A-激酶的磷酸化位点。我们制备了P-Seq的特异性抗体。用这两种异构体和另一种抗心肌C蛋白的单抗(C-315)免疫细胞化学方法，我们发现在发育过程中，两种异构体在心房肌和心室肌中共表达和共定位，而在胚胎骨骼肌中几乎不表达。3)心肌细胞内C-蛋白(MyBP-C)和H-蛋白(MyBP-H)与肌球蛋白结合的竞争。C-蛋白和H-蛋白是肌球蛋白结合蛋白，定位于肌瘤的A带区域。为了阐明这两种肌球蛋白结合蛋白的功能关系，我们将编码这两种蛋白的cDNA导入原代培养的鸡肌细胞，并检测它们组装成肌原纤维的情况。结果表明，在培养的心肌细胞中过表达MyBP-C或MyBP-H可分别降低MyBP-H和MyBP-C的含量。这些数据表明，MyBP-H可以与MyBP-C竞争结合细胞内的肌球蛋白。较少

C-protein is a major myosin-binding protein characteristic of striated muscles. In order to clarity the roles of C-protein in myofibrillogenesis, we carried out the following experiments and obtained the results as described.1) Mouse C-protein cDNA sequences and C-protein isoform expressionThe cDNAs encoding mouse fast skeletal (F) and slow skeletal (S) C-proteins were cloned and their entire sequences were determined. Cardiac (C)-isoform is known to be expressed first and transiently during development of chicken skeletal muscles, but in mouse skeletal muscle, C-isoform was not detected through developmental stages. S-isoform became detectable first, followed by appearance of F-isoform, In the degenerating mouse skeletal muscles, expression of S-isoform is increased.2) Two isoforms in chicken cardiac C-protein and their expressionWe detected two variants (type I and type II) in chicken cardiac C-protein and determined their entire sequences ; they differ only by the inclusion or exclu … More sion of a sequence of 15 amino acid residues (P-seq) which includes a phosphorylation site for A-kinase. We prepared the antibody specific for P-seq. By immunocytochemical methods with this and the other monoclonal antibody to cardiac C-protein (C-315), we found that two isoforms were coexpressed and colocalized in both atrial and ventricular muscles through development, but type I was scarcely expressed in embryonic skeletal muscle. These observations were confirmed by a RT-PCR method.3) Competition of C-protein (MyBP-C) with H-protein (MyBP-H) for binding to myosin in cardiac muscle cellsC-Protein and H-protein are myosin-binding proteins which localize in A-band regions of sarcomeres. To clarify functional relationship of these two myosin binding proteins, we introduced cDNAs encoding these proteins into primary cultures of chicken myocytes and examined their assembly into myofibrils. The results obtained indicate that overexpression of MyBP-C or MyBP-H in cultured cardiac myocytes can reduce the content of MyBP-H or MyBP-C, respectively. These data indicate that MyBP-H can compete with MyBP-C for binding to myosin in the cells. Less