Characterisation of an Autoantigenic Epitope on Cartilage Oligomeric Matrix Protein Recognised by mAb 1D10

【作者】 韩东； 韩魏巍； 熊丽； 刘德立； 耿辉；

【机构】 华中师范大学生命科学院；

【摘要】 Introduction:Cartilage oligomeric matrix protein(COMP)is a joint-specific arthritogenic antigen associated with rheumatoid arthritis and experimental arthritis animal model.We recently developed a mAb 1D10 recognizes the C-terminal globular domain of COMP and proved that mAb 1D10 is pathogenic.The present study was aimed to identify the antigenic epitope of monoclonal anti-cartilage oligomeric matrix protein antibody 1D10.Methods:A filamentous phage library that displays random linear dodecapeptides was employed to identify epitope of mAb 1D10,and the selected phages binding specificity were confirmed by ELISA.Confirmation relevantion of interaction between mAb 1D10 and COMP molecule was tested by calcium chelation analysis and by western blot.Synthetic peptides derived from COMP sequence were designed and analyzed for inhibitory activity of selected phages and COMP molecule with mAb ID10.Results:We selected two similar peptide-presenting phages(HSFKWLDSPRLR-phage and HSFQWLDSPRLR-phage)which interact specifically with mAb 1D10.Homology search and structure analysis identified the peptide sequence corresponding to 680-689 residues and adopts a stableβ-sheet structure.Conformation change did not prevent the binding of1D10 with COMP molecular,suggesting that the reaction are independent with tertiary structure of epitope.Competitive ELISA experiments showed that synthetic peptide derived from aa 680-689 of COMP can be specifically prevented from binding to mAb 1D10 by selected phage and by COMP molecule.Conclusion:This study identifies the epitope recognized by 1D10 has,heretofore,directed against residue 680-689determinant rather than native conformation,this region of COMP is embedded into the molecule,this anti-COMP autoantibody is more likely a secondary response to the exposure of auo-antigens during cartilage destruction by other antibodies directed to surface antigens.The findings give insight into the mechanisms controlling the formation of cartilaginous protein autoantibodies in RA.更多还原

【Abstract】 Introduction:Cartilage oligomeric matrix protein(COMP) is a joint-specific arthritogenic antigen associated with rheumatoid arthritis and experimental arthritis animal model.We recently developed a mAb 1D10 recognizes the C-terminal globular domain of COMP and proved that mAb 1D10 is pathogenic.The present study was aimed to identify the antigenic epitope of monoclonal anti-cartilage oligomeric matrix protein antibody 1D10.Methods:A filamentous phage library that displays random linear dodecapeptides was employed to identify epitope of mAb 1D10,and the selected phages binding specificity were confirmed by ELISA.Confirmation relevantion of interaction between mAb 1D10 and COMP molecule was tested by calcium chelation analysis and by western blot.Synthetic peptides derived from COMP sequence were designed and analyzed for inhibitory activity of selected phages and COMP molecule with mAb 1D10.Results:We selected two similar peptide-presenting phages(HSFKWLDSPRLR-phage and HSFQWLDSPRLR-phage)which interact specifically with mAb 1D10.Homology search and structure analysis identified the peptide sequence corresponding to 680-689 residues and adopts a stable p-sheet structure.Conformation change did not prevent the binding of1D10 with COMP molecular,suggesting that the reaction are independent with tertiary structure of epitope.Competitive ELISA experiments showed that synthetic peptide derived from aa 680-689 of COMP can be specifically prevented from binding to mAb 1D10 by selected phage and by COMP molecule.Conclusion:This study identifies the epitope recognized by 1D10 has,heretofore,directed against residue 680-689 determinant rather than native conformation,this region of COMP is embedded into the molecule,this anti-COMP autoantibody is more likely a secondary response to the exposure of auo-antigens during cartilage destruction by other antibodies directed to surface antigens.The findings give insight into the mechanisms controlling the formation of cartilaginous protein autoantibodies in RA.更多还原