The Function Of MASP3 In The Activation Of Complement Alternative Pathway

Objectives:The abnormal activation or the dysregulation of complement alternative pathway（AP）is involved in the pathogenesis of many diseases.The aim of this study is to investigate whether mannose-binding lectin associated serine protease-3（MASP3）is a key molecule in the activation of the complement alternative pathway,and to further verify whether MASP3 can be a therapeutic target in the AP-mediated diseases.Methods:1.The generation of MASP3 knock out mice(MASP3^-/-)and the preliminary analysis of the phenotypic characteristics:MASP3^-/-mice were generated using CRISPR/Cas9.We designed the binding sequence of crRNA that targeted at the 12^thh exon of the MASP1/3 gene.After delivering the mixture of crRNA、tracrRNA and Cas9 nuclease into mouse’s embryos,the embryos were transferred into the ampulla of the oviduct of pseudopregnant C57BL/6 females.After getting the pups,we did PCR and DNA sequence to screen for the MASP3^-/-mice.Then we checked the expression level of MASP3 protein by western blot and further tested the activity of alternative pathway of all the pups.To study erythrophagocytosis,Crry/C3^-/-erythrocytes were labeled ex vivo with carboxyfluorescein diacetate succinimidyl ester（CFSE,Molecular Probes）and were transfused into wild-type（WT）or MASP3^-/-recipient mice.The percentage of labeled erythrocytes present was calculated after FACS.We also tried to reconstitute the AP activity of MASP3^-/-mice using recombined mouse MASP3 protein.2.The production of anti-mouse MASP3 monoclonl antibody（mAb）:Recombined mouse MASP3 were expressed in ExpiCHO cells and purified by affinity chromatography in vitro.To generate anti-mouse MASP3 mAb,a 7-week old MASP3^-/-mouse was immunized with recombined mouse MASP3 protein.When the serum antibody titer was high enough,we euthanized the mouse to collect the spleen cells and fused the spleen cells with the myeloma cells to produce hybridoma cells.To get the cells that can produce antibodies,we did the screening by ELISA and enlarged the culture scale of 12 positive clones.After purifying the anibodies from the culture supernatant,we tested the binding affinity of the 12 antibodies to the recombinant mouse MASP3 protein and we also established the functional aasay through which we screened out the antibody that can block the enzyme activity of mouse MASP3 in vitro.In addition,we also checked the cross-reactivity of the anti-mouse MASP3 mAb with the recombined human MASP3 protein.Results:1.The activity of alternative pathway in the serum of MASP3^-/-mice was lower than that of wild type mice to varying degrees but not absent.After being transfused into WT mice,the Crry/C3^-/-erythrocytes were eliminated quickly by the activation of complement.However,the MASP3^-/-mice can protect the Crry/C3^-/-erythrocytes from spontaneous elimination.Besides that,recombinant mouse MASP3 can obviously reconstitute the activity of alternative pathway in the serum of MASP3^-/-mice.2.The monoclonal antibodies generated in this study have high binding affinity to recombinant mouse MASP3 and most of them can cross react with human MASP3.By the assay conducted in vitro,we found that the mAb 7C8-26 can block the function of both mouse and human MASP3.Conclusions:MASP3 plays an important role in the activation of complement alternative pathway by cleavage of pro-factor D to mature factor D.However,under the deficient condition of MASP3,the alternative pathway can still be activated to a certain extent by other enzymes.In vitro,the activation of alternative pathway can be suppressed efficiently by blocking the function of MASP3.