Structure And Function Study Of Annexin B2

Annexins were a large family of calcium-dependent phospholipid-binding proteins widely distributed in eukaryotes. They could reversibly bind to acidic phospholipid membranes in the presence of Ca2+. Annexin B2 (AnxB2) was a novel member of the annexin family of Ca2+- and phospholipid-binding proteins from Cysticercus cellulosae. It contained four typical annexin repeats, each repeat was composed of 70-80 amino acids and contained a canonical motif'G-X-G-T (38 residues)-D/E'defined as type II Ca2+ binding site.Bioinformatics analysis revealed that AnxB2 contained 15 antigenic determinants and AnxB2 also contained two unique"Insert"fragments, a Lys-Gly-Asp (KGD) motif and a unique glycosaminoglycan attachment site (G-AS). According to the structure analyses of AnxB2, we studied the function of AnxB2, which set an important base for further characterization of the protein.Our research contained three aspects:1. AnxB2 was cloned from oncosphere, which indicated that AnxB2 could be expressed in oncosphere. Subsequently, we constructed four domain-deleted mutants of AnxB2, M234, M134, M124 and M123 by homology modeling. The DNA vaccine plasmids of AnxB2 and its mutants were constructed by DNA recombination technology. And, the plasmids were transfected into HEK293 cells in vitro by lipofectamine 2000 (lip2000). The expression of correlated antigens was detected by Western blot. Additionally, we also constructed the prokaryotic expression vector of AnxB2 mutants and purified the target protein. Plasmid pJLA503-AnxB2 was constructed,the protein was expressed as soluble form in E. coli. A modified purification method for AnxB2 had been developed and established. The method made good use of the calcium-dependent phospholipids binding activity which was one of the typical features of annexin superfamily. The Ca2+-triggered precipitation method was fast, easy to manipulate with only a few centrifugation steps followed by resolubilization of purified protein. We produced sufficient AnxB2 with high purity and bioactivity.2. We used indirect enzyme linked immunosorbent assay (ELISA) to assess the specific antibody IgG induced by six DNA vaccine plasmids, pcDNA3.1-AnxB2; pcDNA3.1-M234; pcDNA3.1-M134; pcDNA3.1-M124; pcDNA3.1-M123 and"DNA prime-protein boost"and used the commercial ELISA kits to assayed IFN-γsecreted by the separated splenocytes from plasmids immunized mice. The results indicated that six DNA vaccine plasmids could elicit the prominent Th1 response but the levels of immune response induced were different: the lowest levels of sera antibody titers were detected in immunized mice by using pcDNA3.1-M234 and the lowest levels of IFN-γwere detected in immunized mice by using pcDNA3.1-M123;"DNA prime-protein boost"group induced the highest levels of sera antibody titers and IFN-γ. Therefore, we could infer that the epitodes deleted by M234 were B cell epitodes and the epitodes deleted by M123 were T cell epitodes;"DNA prime-protein boost"group could enhance the specific immune response.3. To AnxB2, we processed the research of anticoagulant activity. The results indicated that AnxB2 retained extrinsic anticoagulant activity; the affinity of AnxB2 for activated platelets was more potent (versus AnxB1). Therefore, the purification procedure we reported is a rapid and effective method to obtain AnxB2 with high purity and bioactivity. Subsequently, we studied the anticoagulant activity and immunogenicity of M123 and M234. The results showed that M234 not only retained the anticoagulant activity but also had the lower immunogenicity, which set an important stage for producing highly effective thrombus targeting agent.