Morioka Tetsuo, Yao Jian, Suzuki Yasuhito, Oite Takashi
Department of Cellular Physiology, Institute of Nephrology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.
Kidney Int. 2004 Dec;66(6):2214-23. doi: 10.1111/j.1523-1755.2004.66011.x.
An Experimental model of proliferative glomerulonephritis induced by an antibody against Thy-1 antigen has been established. However, the pathophysiologic role and the critical epitope of Thy-1 molecule for induction of mesangial cell dysfunction remain unknown. We have reported that monoclonal antibody 1-22-3 recognizes specific epitope which could transduce highly effective activation in mesangial cells. Identification of functional domains on cell surfaces is indispensable for understanding the molecular mechanisms of mesangial cell function. This study was undertaken to determine the functional domain containing the specific epitope recognized by monoclonal antibody 1-22-3.
A series of glutathione-S-transferase (GST)-truncated-Thy-1 proteins were generated using pGEX 4T-1 vector. COS cells were transiently transfected with plasmid vectors which could express the rat Thy-1 and mutant-Thy-1.
Western blot analysis using recombinant GST-truncated-Thy-1 revealed that 1-22-3 bound to epitope at amino acids 15-23 (LRLDCRHEN). Enzyme-linked immunosorbent assay (ELISA) revealed that synthetic LRLDCRHEN peptides could inhibit the binding of 1-22-3 to rat mesangial cells and GST-Thy-1 protein. Using peptides as antigens, ELISA showed that 1-22-3 bound to the LRLDCRHEN but not to the RVNLFSDRF, which was corresponding to at amino acids 59-67 of rat Thy-1. 1-22-3 could bind the COS cells which express rat Thy-1 proteins, but could not bind rat truncated-Thy-1 which lacks residues 15-23.
Critical epitope detected by 1-22-3 in this study may play an important role in mesangial function and injury.
已经建立了一种由抗Thy-1抗原抗体诱导的增殖性肾小球肾炎实验模型。然而，Thy-1分子在诱导系膜细胞功能障碍中的病理生理作用和关键表位仍不清楚。我们已经报道单克隆抗体1-22-3识别特定表位，该表位可在系膜细胞中传导高效激活。鉴定细胞表面的功能域对于理解系膜细胞功能的分子机制是必不可少的。本研究旨在确定包含单克隆抗体1-22-3识别的特定表位的功能域。
使用pGEX 4T-1载体产生一系列谷胱甘肽-S-转移酶（GST）截短的Thy-1蛋白。用可表达大鼠Thy-1和突变型Thy-1的质粒载体瞬时转染COS细胞。
使用重组GST截短的Thy-1进行的蛋白质印迹分析显示，1-22-3与氨基酸15-23（LRLDCRHEN）处的表位结合。酶联免疫吸附测定（ELISA）显示合成的LRLDCRHEN肽可抑制1-22-3与大鼠系膜细胞和GST-Thy-1蛋白的结合。以肽为抗原，ELISA显示1-22-3与LRLDCRHEN结合，但不与对应于大鼠Thy-1氨基酸59-67的RVNLFSDRF结合。1-22-3可与表达大鼠Thy-1蛋白的COS细胞结合，但不能与缺乏残基15-23的大鼠截短型Thy-1结合。
本研究中1-22-3检测到的关键表位可能在系膜功能和损伤中起重要作用。
