The Investigation Of Functional Domains And Key Amino Acids Of BAFF Recognized By TACI

B cell activating factor (BAFF), also known as B lymphocyte stimulator (BLYS), identified in1999, is an important member of the TNF family of cytokines and it can specifically stimulate B cell survival, proliferation and differentiation and antibody secretion. It is significance for promoting and regulating the survival and development of B lymphocytes. The biological role of BAFF is mediated by three receptors:B-cell maturation antigen (BCMA), transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and BAFF receptor (BAFF-R, also known as BR3). It is through these receptors play immune regulation role in the various stages of B cell differentiation and development.Abnormal levels of BAFF in vivo will result in many immune-related diseases. Numerous studies demonstrate that low expression of BAFF triggers the deficiency of peripheral B cell maturation and the reduction of immunoglobulin, while overproduction of BAFF would thus break the immune tolerance and is associated with autoimmune diseases such as systemic lupus erythematous (SLE), sjogren’s syndrome, rheumatoid arthritis (RA). Thus, BAFF and its receptors receive widespread attention as a new target for the treatment of autoimmune diseases.BAFF-specific antagonists (soluble receptor TACI-Fc or anti-BAFF antibody, etc.) can inhibit the biological activity of BAFF. Atacicept, a fully human recombinant receptor-Ig fusion protein (TACI-Fc) used as a novel BAFF antagonist in the treatment of a variety of autoimmune disease, has entered clinical trials. A phase I trial already completed achieved good results in SLE and RA patients. At the same time, new antagonist developments become recent research focus. However, it is still not clear how BAFF interact with TACI. The binding mode between BAFF and its receptor TACI is becoming the research hot spot, which is very important for the development of new BAFF antagonists.In this study, with the help of computer-aided molecular docking techniques, dynamics simulation technology to build the ligand-receptor interaction of the complex model, the binding mode and the key domains of BAFF interacting with TACI were analyzed by the computer-guided molecular modeling method based on the crystal structure and the theoretical simulation of three-dimensional conformation of ligand (BAFF) and receptor (TACI). The predicted antigen epitope was tested by molecular biology mutant experiment and functional evaluation method.Some details of this research were shown as follows:1. Complete the expression purification and activity identification of human BAFF protein extracellular domain through molecular biology and immunology methods.(1) Carry out Prokaryotic expression for the pET32a(+)/BAFF, pET32a(+)/TACI plasmid. Complete Nickel ion affinity purification through the His label carried by fusion protein. Carry out specificity evaluation for the obtained protein (rhBAFF and rhTACI) through SDS-PAGE and Western Blot.(2) Analyze the modality of rhBAFF in PBS buffer through non-reducing electrophoresis, Western Blot, HPLC.(3) Determine the binding activity of rhBAFF and rhTACI through ELISA and compare the result with commercialized protein.(4)Demonstrate the functional activity through mice B-cell proliferation experiment in vitro.2. Demonstrate the key domains for the interaction between rhBAFF and TACI through biological experiment with computer simulation(1) Using the crystal structures of BAFF and its receptor TACI, selecting the appropriate force field parameters and considering the solvent effect, the space structure of BAFF interaction with receptor were constructed based on the computer-aided molecular docking techniques and dynamics simulations.(2) The rhBAFF and rhBAFF mutants were designed and constructed in prokaryotic expression vector (pET-32a). Then BAFF and its domain mutants are expressed, purified and identified in vitro. And then evaluate the tripolymer structure through non-reducing electrophoresis and HPLC.(3) The binding capacity difference between rhBAFF、rhBAFF and TACI mutants were evaluated by ELISA and Octet protein interaction dynamics analysis.(4) Determine the functional activity difference between rhBAFF and mutants through mice B-cell proliferation experiment and NF-κB activation experiment in vitro. Verify the functional domains of BAFF recognized by TACI. 3. Further theoretical simulations on each of the relevant regional sites were completed after the key domains of rhBAFF interaction with TACI were determined. The theory predictions were proved reasonable by a series of binding activity and functional activity experiments through site-directed mutagenesis techniques.(1) Using the structure of compound from the interaction of TACI and BAFF, carry out point wise virtual mutant scanning in the203-211,233-238domain with the computer-assisted virtual mutant technique. Predict the key site of recognition of BAFF by TACI according to the change of interaction energy.(2) Construct the prokaryotic expression vector of rhBAFF single point mutants by molecular biological method. Complete the expression, purification and evaluation of the single point mutants.(3) The binding capacity difference between rhBAFF and rhBAFF mutants recognized by TACI were evaluated by ELISA and Octet protein interaction dynamics analysis.(4) Determine the functional activity difference between rhBAFF and mutants through mice B-cell proliferation experiment and NF-κB activation experiment in vitro. Verify the functional domain of BAFF identified by TACI.The results are as follows:1. The exacellular recombination fusion proteins of BAFF and its receptor TACI were got with high-purity and activity(1) After prokaryotic expression, affinity purification, the rhBAFF and rhTACI soluble expression protein were got. SDS-PAGE and Western Blot showed the fusion proteins were got with high purity and specificity(2) After non-reducing electrophoresis, Western Blot, HPLC analysis, we found that rhBAFF exist in tripolymer structure with a purity of more than90%in PBS buffer(3) Double sandwich of ELISA method showed that rhBAFF can be specifically integrated with rhTACI, which is not significantly different from commercialized protein.(4) The activity of rhBAFF for promotion of proliferation was demonstrated by mice B-cell proliferation experiment.2. Confirmed the key functional domain of the interaction between BAFF and TACI (1) Using the3-D stable complex structures of BAFF and TACI obtained, the theories of the formation of intermolecular hydrogen bonds, distance geometry and computer graphics technology, the function sites of BAFF interaction with TACI are discriminated reasonably; Considering the secondary structure and the intermolecular hydrogen bond of BAFF, five series of domain mutants of BAFF were designed based on computer-aided molecular modeling.(2) The prokaryotic expression vector of rhBAFF (mutants)/pET32a(+) was constructed. rhBAFF fragment mutants were expressed succefully,such as including M1(from Ile158to Phe165), M2(from Asp203to Leu211), M3(from Ser225to Arg231) and M4(from Ile233to Glu238), M5(264-269). After expression and purification by Ni-NTA sepharose, we got the target protein with high-purity. rhBAFF and mutants were identified by SDS-PAGE,Western blot and HPLC were used to confirm the tripolymer forms of our proteins with molecular weight of110bp under non-reducing condition.(3) It was shown that the mutants M2, M4and M5lost the binding capacity with TACI through ELISA and Octet protein interaction dynamics analysis. Mice B-cell proliferation experiment and NF-κB activation experiment showed that M2,M4lost biologic activity significantly compared with rhBAFF, so we initially confirm that the mutant field203-211,233-238of rhBAFF mutants M2,M4is the functional domain of BAFF.3. Confirmed the key sites in the functional domain of the interaction between rhBAFF and TACI(1) The key domains (M2, M4) of BAFF interaction with TACI were predicted by virtual single point mutation. The influence of the single point mutation on the3-D structure of BAFF and interaction of BAFF with TACI were analyzed through dynamic changes of the interaction energy, which showed that the key sites for the recognition of BAFF by TACI are M208, G209, H210, Q234, M236and P237.(2) Construct the prokaryotic expression vector with virtual key sites through molecular biological Site-Directed Mutagenesis; We got target protein with high-purity through expression and purification in vitro.The same method demonstrate that single point mutant still exsist in tripolymer in buffer.(3) ELISA and protein interaction dynamics analysis showed that after mutation of M208, G209, H210, M236and P237, the interaction between rhBAFF and TACI is losing significantly. Meanwhile, activity analysis experiment result is in accord with combination experiment, which showed that M208, G209, M236and P237played a clear role in the biological function of BAFF and TACI-Mediated combination. And we confirmed that P237is the key functional site of BAFF.Conclusion:Based on computer-aided molecular modeling and biological experiments, a series of mutants of BAFF were designed by the theoretical predictions of the dynamic identification and3-D structural characteristics of BAFF interaction with TACI and evaluated with biological experiments. The binding mode and domains between BAFF and its receptor TACI were completely understood by our research, which will highlight the clues for further developments of novel BAFF inhibitors.