Study Of C1q-Binding 12-mer Peptides With C1q-Inhibiting Activities

Complement system plays an important role in the first line of defense against infections, but the disorder of its activation can cause diseases, and hence, the anti-complement therapy has been a major field in the complement research. Through different regulation ways have been developed to inhibit activation of complement system, there still are no clinically available drugs that inhibit complement activation. We advanced a novel strategy to inhibit the activities of Clq, the recognition molecule in the classical activation pathway of complement system, which might effectively block the activation of the classical pathway. A panel of Clq-binding phage clones had been selected by screening Ph.D.-12?Phage Display Peptide Library with Clq. In this research, the peptides displayed on these phage clones were studied.Part I Identification of the Clq-binding peptidesThe Clq-binding phage clones screened from Ph.D.-12 library were identified by U937 cell-ligand-binding inhibitive assay and aggregated IgG (AIgG) competition inhibitive assay. It was found that fourteen phage clones are positive in the two inhibitive assays. The genes of peptides displayed on the phage clones were sequenced and the deduced amino acid sequences of 9 peptides (2 clones have the same sequence, the sequencing of 4 clones was not successful) were obtained, which are HWDPFSLSAYFP, WTPVRTNPF-LLH, NGHLFSLSAYFP, RTQRNSPFFLCP, SPAFHPEHMGRG, SRAFHPF-YRGRA, WYEGPFTLQTWP, LTQHNSPFFLLP, and TSNPFFLWYPQP.Part II Study on activities of Clq-binding 12-mer peptidesFive 12-mer peptides whose inhibitory activities are relatively stronger were synthesized and part of synthetic peptides were conjugated to the BSA, and the synthetic peptides and the conjugates were tested for the biologicalactivities to inhibit the binding of C1q with C1q receptors (C1qR) on U937 cells or AIgG and the C1q-mediated nemo lysis. It was found that the free synthetic peptides do not have effects on the activities of C1q but the synthetic peptide-BSA conjugates inhibit strongly the binding of C1q to C1qR or AIgG and the C1q-mediated hemolysis in vitro. These results indicated that the 5 mimic peptides of C1qR inhibit the binding between C1q and immune complex and thus block the activation of the complement classical pathway.Part III Reform and initial test of the a synthetic peptideAccroding to the results of the experiments above, peptide a, whose sequence is HWDPFSLSAYFP, was chosen for further study. To obtain some peptides with stable structure and C1q-inhibiting activities, the peptide was designed to be reformed: al, GGGHWDPFSLSAYFPSHS and a2,. After synthesized, peptide al was tested forthe biological activities. It was found that this peptide inhibits the binding of C1q to C1qR on U937 cells or AIgG and the C1q-mediated hemolysis in vitro. The data showed that the structure and bioactivities of the reformed peptide can be restored to those of the peptide displayed on the phage particles.In summary, we have identified some C1qR-mimic peptides that may inhibit the activation of the complement classical pathway. They have potential therapeutic use in diseases involving complement-mediated tissue damage, but great research efforts have to be made to develop an effective small-molecule inhibitor of complement.