Construction Of A Recombinant Complement Receptor Type 1 Composed Of Two Functional Domains And Analysis Of Its Bioactivity

The complement system is a major component of the innate immune system, on the front line of the fight against infection, but itcan cause substantial cell and tissue damage when activated inappropriately. For this reason, activation of the complement system is under delicate regulation that involves a set of membrane and plasma inhibitors. A reduced level of component inhibition can result in a variety of diseases such as hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria, glomerulonephritis,and hereditary angioedema, while overactivation of the complement cascade may be a contributing factor to ischemia reperfusion injury. Therefore, rational strategies are needed to design therapeutic agents that modulate complement activity, and are based on the mechanisms of the complement system's natural inhibitors.The complement system can be activated by three pathways, known as the classical, alternative, and mannose-binding lectin pathways. All three converge at the point of cleavage of C3 into C3a and C3b. C3b then participates in the formation of C3 convertase in the alternative pathway, and C5 convertase. Thus, C3b is central to complement regulation. Complement receptor type 1 (CR1) is a complement activation family regulator, and is also known as the immune adherence receptor because it binds to C3b and C4b . CR1 can regulate the activation of the complement cascades by serving as a co-factor for factor I-mediated cleavage of C3b and C4b, and by accelerating the decay of both the classical and alternative pathway C3 and C5 convertases . For this reason, CR1 is the most potent and versatile complement inhibitor of the complement activation family . CR1 is composed of multiple of short consensus repeat (SCR) domains, a transmembrane domain and a small cytoplasmic domain, with each SCR containing about 60–70 amino acids. Based on degree of internal homology, all except the two carboxyl terminal SCRs can be classified into larger units, called long homologous repeats (LHR) A, B, C, and D, each of which is composed of seven SCRs. In CR1, three domains interact with C3b/C4b and with the convertases. One domain is located in LHR A, B, and C, more precisely in the first three SCRs of each LHR. The active domain in SCRs 1–3, called domain 1, is unique. The active domain in SCRs 8–10 is nearly identical to the domain in SCRs 15–17, with the exception of three amino acids. These are both called domain 2 . Structure and function relationship studies have demonstrated that domain 1 binds C4b and, very weakly, C3b. It has high decay-accelerating activity (DAA) for the C3 convertases, but low co-factor activity (CA) for factor I-dependent cleavage of C3b and C4b. Domain 2 binds C3b and C4b efficiently, with an affinity for C4b that is lower than for C3b, but comparable to the affinity for C4b of domain 1. It has high co-factor activity for both C3b and C4b, but low DAA for the C3 convertases . Efficient DAA for the C5 convertases by CR1 is achieved only if both domains 1 and 2 are present . Thus, combining the active sites within the N-terminal three SCRs of the LHR A and those of either LHR B or LHR C, could result in a smaller and more potent CR1-based inhibitor .In the present study, based on the potential complement inhibitory activities in the LHRs described above, we combined domain 1 of LHR A with domain 2 from LHR C using an overlap extension PCR method. We expressed the constructed CR1-based protein fused to thioredoxin in Escherichia coli, where it was mainly in inclusion bodies. After release and subsequent refolding, enterokinase cleavage, and purification, we verified its protective effects in an in vitro model of blood group alloimmune incompatibility, and in a mouse model of transfusion incompatibility. We demonstrated the CR1-based molecule could prolong red blood cells (RBC) survival in vivo, demonstrating its potential to be used as a potent and cost-effective complement inhibitor.The research mainly achieved the following results:1. RNA was isolated from fresh human peripheral blood mononuclear cells (PBMC) and the functional fragment1 was successfully amplified using the reverse transcription PCR method.The fragment was coloned into the pMD18-T simple plasmid.The sequencing result show the fragment sequence was completely agree with the corresponded sequence in the GeneBank.2.the blunt end fragment 1 was successfully amplifed using the above construct plasmid as template. The blunt end fragment 2 was succesfully amplfied using the plasmid PET32a-CR1-SCR15-18 that was constructed before. The fusion fragment was amplifed using the splicing overlap extention PCR(SOE-PCR) method,the fused frament was ligated into the PET32a plasmid. The constructed plasmid was verified by enzyme digestion and sequencing, The verified plasmid was named as PET32a-CR1-2D.3.the tansformed PET32a-CR1-2D/Rosetta(DE3) was induced by IPTG and analyzed by SDS-PAGE. Result show the fusion protein was highly expressed in E.coli in inclusion bodies form and the molecule mass of the expressed protein was 63 kD. Further research show the protein has the optimal expression level when induced for 4 hours at the concentration of 1mM IPTG under 37℃.western blot against 6xHis antibody further confirmed the expressed protein.4.The protein can be isolated by on-step affinity purification. The purity of the recombinat protein was up to 92% after Ni-NTA column affinity chromatography. We screened the dialysis refolding procedure and found the CR1-2D ptotein formed the least aggregates and had best bioactivity when refolded under the GSH and GSSG concentrations of 3.5 and1.5 mM, respectively. The gradual removal of the denaturing agent over time is essential in the refolding process. After clevege by enterokinase to remove the Trx Tag and subsequent Ni-NTA column affinity chromatography, we obtained the purified CR1-2D protein without extra amino acid.5.In vitro and in vivo functional experiments analysised the recombinant protein bioactivity. In the in vitro transfusion imcompatible model, Spectrophotometer measured the optical density of hemoglobin, result show the recombinant protein can inhibit hemolysis; flow cytometry method found the recombinant CR1-2D protein can reduce the deposition of C3b on the red blood cell membrane. Elisa method measured the C3a in the supernatant and found the recombinant protein can reduce C3a release. These results indicated the protein could inhibit hemolysis intravascularly and extravascularly to a certain extent. In mouse transfusion imcomptible model, flow cytometry counted the red blood cell marked by fluorescence dye, PKH67, at different time point, result show: for the initial 2 h post-transfusion, CR1-2D protein prolonged transfused RBC survival in the mouse circulation, compared to buffer-treated mice .In conclusion,we combined functional domain 1, located in the long homologous repeat (LHR) A, with functional domain 2,located in LHR C. We expressed the two-domain, two-function protein with an enterokinase site at the Nterminus and a termination codon at the C-terminus in Escherichia coli.We verified the bioactivity of the molecule in vitro and in vivo experiment model. Our results indicate that the CR1-based protein may be a model for developing smaller and more potent complement inhibitors for future therapeutics.