Tetsuo Morioka1, Jian Yao, Yasuhito Suzuki, Takashi Oite. 1. Department of Cellular Physiology, Institute of Nephrology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan. morioka@med.niigata-u.ac.jp
Abstract
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: Critical epitope detected by 1-22-3 in this study may play an important role in mesangial function and injury.
BACKGROUND: 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. METHODS: 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 ratThy-1 and mutant-Thy-1. RESULTS: 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 ratThy-1. 1-22-3 could bind the COS cells which express ratThy-1 proteins, but could not bind rat truncated-Thy-1 which lacks residues 15-23. CONCLUSION: Critical epitope detected by 1-22-3 in this study may play an important role in mesangial function and injury.