The Physical Association Of Human C5a Receptor And Chemotaxis Inhibitory Protein Secreted By Staphyloccous Aureu And The Effect Of Tyrosine Sulfation Of C5aR On The Binding Of C5aR To CHIPs

The physical association of human C5a receptor and chemotaxis inhibitory protein secreted by Staphyloccous Aureu and the effect of tyrosine sulfation of C5aR on the binding of C5aR to CHIPsObjective:To investigate the physical association between CHIPs secretd by Staphylococcus aureu and human C5aR, and the orphan receptor C5L2. To study on the key role of sulfrotyrosines at N-terminal of C5aR in associating with CHIPs.Methods:1. CHIPs, chp was amplified templated by Genomic DNA of methicillin resistant Staphylococcus aureus, ATCC29213, and subclone into pET32a(+) expression vector. CHIPs protein was expression in E. coli, purified and tested the concentration.2. Expression plasmids encoding the human C3aR, C5aR, C5L2, CXCR3 and PTAFR with the addition of a ten amino-acid tag at its amino terminus (myc tag) was generated by PCR amplification of human genomic DNA, and subcloned into the pcDNA 3.1(+) expression vector (Invitrogen). HEK 293T cells were transfected with plasmids encoding the abovementioned genes using calcium phosphate. Western blot was used to test the expression of these genes to ensure them at the comparable level.3. Purified CHIPs was incubated with C3aR, C5aR, C5L2, CXCR3 or PTAFR respectively. Binding signal was detected by western blot.4. HEK 293T cells overexpressing C5aR was treated with non-specific sulfation inhibitor-NaClO3, the effect of the variuos concentrations of NaC103 on binding ability of C5aR and CHIPs was detected. Difference in the binding affinity between CHIPs and C5aR cotransfected with or without TPST or shRNA was compared. All the C5aR variants in which one or more tyrosines were mutated to phenylalanine were made by the PCR-based QuickChange method and confirmed by sequencing the entire reading frame. Tyrosine at positions 11 or (and) 14 was mutated into phenylalanine to test the role of sulfated tyrosines on the binding affinity of C5aR and CHIPs. Results:In this study, we constructed expression vectors for CHIPs and chemokine receptors, purified CHIPs protein at a concentration of 96μg/ml. C3aR, C5aR, C5L2, CXCR3, and PTAFR were expressed respectively on 293T cells. Western blot showed that CHIPs bound to C5aR solely, but not C5L2, C3aR, CXCR3 or PTAFR. The inhibition of NaClO3 on binding of C5aR and CHIPs was in a concentration-dependent manner. The concentration of NaClO3 was 50mmol/L, the binding capacity of C5aR and CHIPs was obviously decreased by 76.47% compared with other experimental concentrations. The binding affinity of cells cotransfected with C5aR and plasmid encoding TPSTs was increased, while, the binding affinity of cells cotransfected with shRNAs to CHIPs was not decreased markedly. When tyrosine at position 14 of C5aR was mutated into phenylalanine, the binding capacity of cells expressing C5aR to CHIPs was decreased by 76.32% compared with cells expressing wild type C5aR, furthermore, as both of tyrosines at positions 11 and 14 of C5aR were mutated into phenylalanine, the binding signal was not visible.Conclusion:C5aR is the natural receptor of CHIPs. Furthermore, our data demonstrated that sulfated tyrosine at position 14 of N-terminal C5aR formed the binding site for CHIPs. This study elucidates the fine structral characteristics of human C5aR to associate with CHIPs, implying a promising pharmacological target to block the association between human C5aR and CHIPs.