Molecular Characterization Of An Inhibitor Of Apoptosis Protein(SjBIRP) In Schistosoma Japonicum

Dioecious flatworms of the genus schistosoma cause schistosomiasis, which is a major public health problem in developing countries and this disease is one of the most important public health concerns in China. However, no vaccine is available for preventing schistosomiasis. Treatment of schistosomiasis and controlling morbidity relies mainly on a single drug, praziquantel, which is ineffective against the young developing stages of schistosomes and reinfection. Therefore, it is urgent for developing novel strategies for controlling this disease.We previously found that the apoptosis inhibitors may play important roles in schistosoma parasitism and development. Among them, inhibitor of apoptosis proteins(IAP) may play important roles in the regulation of schistosome ce ll survival via baculovirus IAP repeat domains(BIR) thus may be potential drug targets for schistosomiasis control. In this study, we found another schistosoma apoptosis inhibitor Sj BIRP via bioinformatics analysis. We further cloned and analyzed the function of this apoptosis inhibitor in following three sections:1) Cloning, expression and bioinformatics analysis of S. japonicum of apoptosis inhibitor Sj BIRPBy analyzing protein database of S. japonicum, a murine ortholog of schistosome BIRP(Gen Bank ID: AAW27178.1) was identif ied. The corresponding c DNA fragment of Sj BIRP was then cloned. Sequence analyses indicated that the amplif ied c DNA fragment contained an open reading frame of 765 bp, encoding a BIRP protein with a predicted molecular weight of 20. 14 k Da. Multiple alignments revealed that the BIRP sequence of S. japonicum shared 50 % identity with that of Homo sapiens, 44.0 % with Rattus norvegicus, and 46 % with Mus musculus. Phylogenetic tree of analysis indicated that the Sj BIRP sequence was localized in the invertebrate group and was distal to the vertebrate group. Subsequently, the recombinant Sj BIRP was successfully expressed as a His fusion protein with an expected molecular weight of 25 k Da and further confirmed by ESI-Q-TOF mass spectrometry.2) Sj BIRP expression profiles at different stages, sex, and hosts of S. japonicumThe transcript levels of Sj BIRP at different stages(7, 14, 21, 35, and 42 days), different sex(adult male and female), and different hosts(Rats, mice, and New Zealand rabbits) were determined using q RT-PCR. Results indicated that Sj BIRP m RNA was readily detected at each of the several stages investigated, and exhibited a relatively higher level in 7-day-old schistosomula. Additionally, the level of Sj BIRP m RNA in adult females was significantly higher than in adult males. Furthermore, the level of Sj BIRP m RNA in rats was relatively higher than that in the rabbits and BALB/c mice. All these indicated that schistosoma Sj BIRP inhibitor of apoptosis may play important roles in the development and schistosome parasitism.3) Functional studies of S. japonicum apoptosis factor Sj BIRPThe resulting Sj BIRP PCR products were purified, ligated into digested prokaryotic expression vector p ET28a(+), and the recombinant Sj BIRP(r Sj BIRP) plasmids were then transformed into E. coli BL21(DE3) cells for protein expression. After induction, the soluble r Sj BIRP was purified and the fusionprotein was injected to BALB/c mice by subcutaneous to prepare polyclonal antibody, the serum titer was then measure by using ELIS A method, and an appropriate proportion 1:6400 was found. Western blot analysis showed that the polyclonal antibody of r Sj BIRP have better specificity. The recombinant plasmids pc DNA3.1(+)-Sj BIRP and control plasmids were subsequently transfected into the He La cells using Lipofectamine 2000. We found that 1.0 um Act D can induce significant apoptosis in Hela cells. Overexpressing of Sj BIRP attenuated Act D-induced caspase activity. In addition, purified r Sj BIRP was also added to schistosome lysates to explore the effect of r Sj BIRP on caspase activity. A remarkable reduction in caspase activity was observed in schistosome lysates upon r Sj BIRP addition, implying that the Sj BIRP may functionally suppress caspase activity in vivo in sc histosomes.Taken together, our studies preliminary suggest that Sj BIRP may play a functional role in the regulation of schistosoma development and parasitism that targeting Sj BIRP might be a potential for schistosomiasis control.