Institute of General and Molecular Pathology, University of Tartu, Tartu, Estonia.
Cell Div. 2012 Sep 17;7(1):21. doi: 10.1186/1747-1028-7-21.
Previously we have reported on the development of a new mouse anti-titin monoclonal antibody, named MAb Titl 5 H1.1, using the synthetic peptide N-AVNKYGIGEPLESDSVVAK-C which corresponds to an amino acid sequence in the A-region of the titin molecule as immunogen. In the human skeletal muscles, MAb Titl 5 H1.1 reacts specifically with titin in the A-band of the sarcomere and in different non-muscle cell types with nucleus and cytoplasm, including centrioles. In this report we have studied the evolutionary aspects of the binding of MAb Tit1 5 H1.1 with its target antigen (titin).
We have specified the epitope area of MAb Tit1 5 H1.1 by subpeptide mapping to the hexapeptide N-AVNKYG-C. According to protein databases this amino acid sequence is located in the COOH-terminus of several different Fn3 domains of the A-region of titin molecule in many organisms, such as human being, mouse, rabbit, zebrafish (Danio rerio), and even in sea squirt (Ciona intestinalis). Our immunohisto- and cytochemical studies with MAb Tit1 5 H1.1 in human, mouse and zebrafish tissues and cell cultures showed a striated staining pattern in muscle cells and also staining of centrioles, cytoplasm and nuclei in non-muscle cells.
The data confirm that titin can play, in addition to the known roles in striated muscle cells also an important role in non-muscle cells as a centriole associated protein. This phenomenon is highly conserved in the evolution and is related to Fn3 domains of the titin molecule. Using titin A-band-specific monoclonal antibody MAb Tit1 5 H1.1 it was possible to locate titin in the sarcomeres of skeletal muscle cells and in the centrioles, cytoplasm and nuclei of non-muscle cells in phylogenetically so distant organisms as Homo sapiens, Mus musculus and zebrafish (Danio rerio).
此前，我们曾报道过一种新型的肌联蛋白单克隆抗体 MAb Titl 5 H1.1 的开发，该抗体使用合成肽 N-AVNKYGIGEPLESDSVVAK-C 作为免疫原，该肽对应肌联蛋白分子 A 区的一个氨基酸序列。在人类骨骼肌中，MAb Titl 5 H1.1 特异性地与肌联蛋白在肌节的 A 带反应，并且在不同的非肌肉细胞类型中与细胞核和细胞质反应，包括中心粒。在本报告中，我们研究了 MAb Tit1 5 H1.1 与其靶抗原（肌联蛋白）结合的进化方面。
我们通过亚肽作图将 MAb Tit1 5 H1.1 的表位区域指定为六肽 N-AVNKYG-C。根据蛋白质数据库，该氨基酸序列位于人类、小鼠、兔子、斑马鱼（Danio rerio）甚至海鞘（Ciona intestinalis）等多种生物体的肌联蛋白分子 A 区的几个不同 Fn3 结构域的羧基末端。我们用 MAb Tit1 5 H1.1 在人、鼠和斑马鱼组织和细胞培养中的免疫组织化学和细胞化学研究显示，肌肉细胞呈条纹状染色模式，非肌肉细胞的中心粒、细胞质和细胞核也呈染色。
这些数据证实，除了在横纹肌细胞中的已知作用外，肌联蛋白还可以作为一种中心粒相关蛋白在非肌肉细胞中发挥重要作用。这种现象在进化中高度保守，与肌联蛋白分子的 Fn3 结构域有关。使用肌联蛋白 A 带特异性单克隆抗体 MAb Tit1 5 H1.1，我们能够在进化上如此遥远的生物，如智人、小家鼠和斑马鱼（Danio rerio）的骨骼肌细胞的肌节和非肌肉细胞的中心粒、细胞质和细胞核中定位肌联蛋白。
