Effect Of 187 Q Mutation On The Function Of Human STALL-2

TALL-2 (TNF and apoptosis ligand-related leukocyte-expressed ligand 2), also known as APRIL (a proliferation inducing ligand), is a new member of the tumor necrosis factor (TNF) family. TALL-2 shows a unique maturation pathway among the TNF ligand family member as it is processed in Goili apparatus prior to its secretion(soluble TALL-2, sTALL-2. It is virtually undetectable in normal tissues but is strongly expressed in adenocarcinomas and can accelerate the growth of malignant cells in vitro and in vivo. According to the close relationship between TALL-2 and malignant tumors, and the current status of the TALL-2-inhibition molecules, we may develope a new strategy targeting against TALL-2 for tumor therapy based on TALL-2 mutants.Liu et al developed a mutant of sTALL-1(also called BAFF, BLyS) which still bound its receptor but could not active NF-kB and did not stimulate B lymphocyte proliferation. TALL-2 is a close homolog to TALL-1, so we want to use the same method to generate mutants of sTALL-2 which should has the same effect as the mutant of sTALL-1. According to structure-based sequence alignments of TALL-1 and TALL-2, we selected 187 residue as the mutational site. Three mutants of sTALL-2 were successfully constructed by one-step opposite direction PCR. The expressed proteins were purified, then, the biological activities of the prepared proteins were analyzed. The results and conclusions are summarized as follows:1. Three sTALL-2 mutant cDNAs were amplified by one-step opposite direction PCR from the plasmid pUC18/sTALL-2 (previously constructed by our laboratory). The 187th Gln-encoding sequences of sTALL-2 were replaced by Ser-encoding sequences, Asp-encoding sequence and Arg-encoding sequence in sTALL-2 mutants (named as pUC18/msTALL-2-S, pUC18/ms TALL-2-D and pUC18/ms TALL-2-R).2. After sequenced, the three sTALL-2 mutant cDNAs were transformed on pQE-80L. The recombinant vectors pQE-80L/msTALL-2-S, pQE-80L/msTALL-2-D and pQE-80L/ms TALL-2-R were successfully constructed.3. The recombinant vectors pQE-80L/msTALL-2-S, pQE-80L/msTALL-2-D and pQE-80L/msTALL-2-R were transformed into E.coli DH5αrespectively and expressed in the present of IPTG, and the three corresponding proteins were about 19 kDa identified by SDS-PAGE.4. The expressed proteins were purified by Ni2+-NTA chromatography and refolded by dialysis against urea-buffer. The proteins were further purified by Sephadex G-75 chromatography to get homotrimers.5. The biological activities of the prepared proteins were analyzed by immunohisto- chemistry technique, MTT method and BudU incorporation. The datas showed that sTALL-2 mutants could bind to the receptors on A549 cells and HepG2 cells but had lost proliferation-inducing acticity and even could inhibit the proliferation–inducing acticity of sTALL-2. In summary, the functional recombinant human sTALL-2 mutans were successfully produced, which may pave a way for further study on the mechanism of TALL-2 and novel developing malignant tumor therapeutic agents based on TALL-2 mutants .