Study Of C1q-binding Peptides With C1q-inhibiting Activity

Complement system shows an important role in the first line of defense against infections,but the disorder of its activation can cause diseases. Anti-complement therapy is a very key field in the complement research. Through different regulation pathway have been developed to inhibit the activation of complement,there is still not a satisfactory way which can make reasonable regulation to the activation of complement. The development of genetic engineering complement regulation proteins (CRPs) is a better strategy appeared recently,but for its trivial procedures,it is very hard to get its action. In this study,a novel strategy to inhibit the activation of the complement system has been developed.To get the purified Clq as the target molecule for biopanning,the first step of our purification was to isolate the Clq from Clr2Cls2 by affinity chromatography on IgG-Sepharose 4B column. In the second step,we separated Clr from Cls by DEAE-Sephacel by ion exchange chromatography. The final step of purification was to improve further the purity of Clq by affinity chromatography on Clq McAb-Sepharose 4B column. In all steps of purification,protease inhibitor PMSF and NPGB were added to protect Clq from lysis,the temperature was keep in 4C and the pH was adjusted to 6.1,no catholyte exist in the buffer solutions. After three steps of purification,purified Clq,and Clr and Cls in zymogen form were obtained. Screening Ph.D.-C7C?Phage Display Peptide Library and Ph.D.-12?Phage Display Peptide Library,a panel of C1 q-targeting peptides was recognized. By ELISA analysis,inhibition of binding of Clq with the C!q receptors on U937 cells and competitive inhibition of binding of Clq with aggregated immunoglobulin G by selected phage clones,and DNA sequencing,a number of similar,but not identical,sets of sequences of Clq-binding clones were identified. The deduced amino acid sequences of selected 9 peptides are WYEGPFTLYTWP,HWDPFSLSAYFP,LTQHNSPFFLLP,TSNPFFLWYPQP,QTPFQLW,NPFNWTS,SPFXLTS,FLTWLDP and FSTFLYP. They show significant efficiency to inhibit the binding of Clq with the Clq receptors on U937 cells and/or aggregated immunoglobulin G,which suggest that the selected peptides contain the modeling epitopes of Clq receptor to bind the collage-like region or IgG to bind the head domain of Clq.