Turnacioglu K K, Mittal B, Dabiri G A, Sanger J M, Sanger J W
Department of Cell and Developmental Biology, Pennsylvania Muscle Institute, University of Pennsylvania, Philadelphia 19104-6058, USA.
Cell Struct Funct. 1997 Feb;22(1):73-82. doi: 10.1247/csf.22.73.
Originally, zeugmatin was identified as a 600-800 kD muscle specific protein in Z-bands of cardiac and skeletal muscles by Maher et al. (1985). In this presentation we review our work on myofibrillogenesis and present evidence that zeugmatin is actually part of the Z-Band region of titin and that this region of titin plays an important role in the assembly of the Z-bands and myofibrils. Rhee et al. (1994) reported that during myofibrillogenesis, zeugmatin antibody localization is detected in fully formed Z-bands in the mature myofibrils, in the Z-bodies of the nascent myofibrils, but not in the Z-bodies of the premyofibrils. These observations lead to the suggestion that zeugmatin might be responsible for the fusion of the Z-bodies to form the solid Z-bands of the mature myofibrils (Rhee et al. 1994). As part of a study to test aspects of this model of myofibrillogenesis, we isolated a 1.8 kb cDNA from a chicken cardiac expression library using an anti-zeugmatin antibody (Turnacioglu et al., 1996). We found this chicken cDNA to be 60% identical at the amino acid level to a segment of the Z-band region of human cardiac titin (connectin) sequenced by Labeit and Kolmerer (1995). This homology along with Western blot analysis with purified titin, suggested that zeugmatin is in fact part of the N-terminal region of chicken titin. When expressed in non-muscle cells, Z1.1 product colocalized with the alpha-actinin in stress fiber dense bodies and focal adhesions. Cultures of non-muscle cells, skeletal myotubes and cardiomyocytes were also transfected with a fusion construct (Z1.1GFP) consisting of the Z1.1 kb cDNA linked to the cDNA for green fluorescent protein (GFP). The Z1.1 kb cDNA encodes only 362 of the approximately 2,000 amino acids which comprise the Z-band region of titin; nevertheless, the Z1.1GFP fusion protein targets in vivo to the alpha-actinin rich Z-bands of contracting myofibrils. A dominant negative phenotype was observed in living cells expressing high levels of this Z1.1GFP fusion protein with inhibition of myofibrillogenesis as well as the disassembly of preexisting myofibrils in these cells. These data indicate that the Z-band region of titin (connectin) plays an important role in organizing and maintaining the structure of the myofibril.
最初，马赫等人（1985年）在心肌和骨骼肌的Z线中，将接合素鉴定为一种600 - 800千道尔顿的肌肉特异性蛋白。在本次报告中，我们回顾了我们在肌原纤维形成方面的工作，并提供证据表明接合素实际上是肌联蛋白Z带区域的一部分，并且肌联蛋白的这一区域在Z带和肌原纤维的组装中起重要作用。李等人（1994年）报道，在肌原纤维形成过程中，在成熟肌原纤维中完全形成的Z带、新生肌原纤维的Z体中可检测到接合素抗体定位，但在原肌原纤维的Z体中未检测到。这些观察结果表明，接合素可能负责Z体融合以形成成熟肌原纤维的坚实Z带（李等人，1994年）。作为测试该肌原纤维形成模型各方面的一项研究的一部分，我们使用抗接合素抗体从鸡心脏表达文库中分离出一个1.8 kb的cDNA（图尔纳乔格鲁等人，1996年）。我们发现该鸡cDNA在氨基酸水平上与拉贝特和科尔梅勒（1995年）测序的人心肌肌联蛋白（连接蛋白）Z带区域的一段序列有60%的同一性。这种同源性以及用纯化的肌联蛋白进行的蛋白质印迹分析表明，接合素实际上是鸡肌联蛋白N端区域的一部分。当在非肌肉细胞中表达时，Z1.1产物与α - 辅肌动蛋白在应力纤维致密体和粘着斑中共定位。非肌肉细胞、骨骼肌肌管和心肌细胞的培养物也用由与绿色荧光蛋白（GFP）的cDNA连接的1.1 kb cDNA组成的融合构建体（Z1.1GFP）进行了转染。1. |1 kb cDNA仅编码构成肌联蛋白Z带区域的约2000个氨基酸中的362个；然而，Z1.1GFP融合蛋白在体内靶向收缩肌原纤维中富含α - 辅肌动蛋白的Z带。在表达高水平这种Z1.1GFP融合蛋白的活细胞中观察到显性负性表型，这些细胞中的肌原纤维形成受到抑制，并且预先存在的肌原纤维发生解体。这些数据表明，肌联蛋白（连接蛋白）的Z带区域在组织和维持肌原纤维的结构中起重要作用。
