The Functional Study Of The N-terminal Mutants Of The Novel Human C5a Receptor, C5L2

The anaphylatoxin C5a is an important mediator and chemotactic factor in inflammatory responses.It plays an important role in acute lung inflammation,sepsis,meningitis, rheumatic arthritis and glomerular nephritis.The C-terminal Arg of C5a is rapidly cleaved in vivo to form C5a des Arg⁷⁴.The complement C5a exerts biologic activities through its receptors-CD88 and C5L2.The CD88 interacts with the C5a following a two-site model of receptor binding,which causes a series of inflammatory responses,e.g.leukocyte chemoattraction,degranulation,increases vascular permeability and stimulates cytokine secretion.C5L2 is a novel receptor found to interact with the human anaphylatoxins complement factor C5a.C5L2 has a high affinity for C5a and C5a-des Arg⁷⁴,but is not coupled to G proteins.It is a nonsignaling decoy receptor for C5a and plays an important anti-inflammatory role by binding to anaphylatoxin C3a/C4a/C5a.Currently,the precise binding domains and mode of interaction of C5a with C5L2 have not been determined yet,researchers presume it is probably to be a two-site binding model and a N-terminal domain of C5L2 is essential to bind C5a/C5a des Arg⁷⁴.Therefore,it is helpful to understand this issue thoroughly through researching the essential amino acid residues for binding and it is possible to block or inhibit the interaction of C5a with CD88 and then alleviate excessive inflammatory responses induced by C5a through activating or promoting the binding of C5a to C5L2.Our studies analyzed the potential binding sites by the technology of bioinformatics, and the three-dimensional structure of C5L2 protein was determined by homologous modeling.We select the N-terminus of C5L2 as target of investigation,wild-type and mutant N-terminus of C5L2 protein fragments were expressed in prokaryotic cells,the affinity of wild-type and mutant N-terminal of C5L2 proteins with rhC5a was determined by using biosensors,which preliminarily determined the key amino acid sequence to bind to C5a.At last,synthesizing the polypeptide containing binding-site,the bioactivities of the polypeptide was verified through cell cultures and animal experiments.Our studies provide foundation for further study of the physiological functions of C5L2 and treatment for diseases caused by C5a.Methods1.Based on the sequence of amino acid sequence of full-length C5L2,the parameters of secondary structure,hydrophilicity,accessibility and flexibility were assessed by bioinformatics technology.The three-dimensional structure of C5L2 protein was predicted by homologous modeling.2.Prokaryotic expression vector for wild-type and mutant-type of N-terminus of C5L2 were constructed by gene recombination.Target protein fragments were obtained following induced expression by IPTG,purified by affinity chromatography,and digested by thrombin.3.Affinity of wild-type and mutants N-terminal of C5L2 proteins with rhC5a were determined by using rhC5a-coated biosensors.4.The polypeptide containing the key binding-site domain was synthesized,and named as D3.The D3 was incubated together with rhC5a and peripheral blood PMN for 4 hours.It was assayed that the contents of cytokines(IL-6) receased from PMN stimulated by rhC5a with ELISA.The endotoxin shock model of mouse was established,and observed the survival rate of mouse in 48 hours.Results1.Domains between 3～22,179～183,190～195,265～275 are most likely to be the binding domains of C5L2.The N-terminal domains of C5L2 show better hydrophilicity, accessibility,and flexibility.These domains are regarded as the target of study.Surface potential analysis reveals that amino acid residues of C5a protein fragment are rich in positive charges,while that of the C5L2 are rich in negative charges;therefore,they attracts to each other and can be bound together.According to the above results,5 mutants, i.e.point mutants(S-1,S-2,S-3) and deletion mutants(D-1,D-2),are determined,which provide foundation for further study of the molecular biology.2.The constructed expression vector carrying wild-type gene is preliminarily identified by digestion and PCR.Sequencing results also indicate the correctness of the insertion of target fragments.Sequencing of the mutant expression vectors shows that the sites are mutated as designed.Following successful transformation of wild-type and mutant expression vectors,33kDa wild-type and mutant fusion proteins are obtained.Fusion proteins are purified by affinity chromatography,with a purity of approximately 80%. Following thrombin digestion,4kDa target proteins are obtained.3.The results of affinity show that R4,W,S-1 and S-2 can be bound to rhC5a,binding rate are 88%,86%,85%and 81%,respectively(P＞0.05);the binding rate of S-3 decreases obviously,is 21%(P＜0.01);and the deletion mutants(D-1,D-2) can not be bound to rhC5a. Therefore,we search key domains related with binding tightly.4.The synthesized polypeptide-D3 shows significantly decrease of the cytokines IL-6 released from PMN stimulated by rhC5a(P＜0.01).When mice are treated with the D3,the survival rate of them are significant improved(p＜0.01).It suggests that the D3 has antagonistic effects against C5a in vivo.Conclusions1.ASP^22,25,32 are not essential for N-terminus of C5L2 to bind to rhC5a.2.Amino acid residues 10 to 22 of N-terminus of C5L2:NH2-YGDYSDDRPVD-COOH may be the essential domain for the N-terminus of C5L2 to bind to rhC5a,and ASP^12,15,18 play an important role in binding of C5L2 and rhC5a.3.Obtained a polypeptide with anti-inflammation activities.