Cloning and characterization of a bone morphogenetic protein homologue of Schistosoma japonicum

Abstract

Bone morphogenetic proteins (BMPs) are known to play an important role in the regulation of cell proliferation, survival, differentiation and apoptosis in many vertebrates and invertebrates through the TGF-β signaling pathway. Although the TGF-β signaling pathway exists in schistosomes, BMP homologue, a ligand of TGF-β in Schistosoma japonicum, has not yet been identified. In this study, a BMP homologue of S. japonicum was cloned and characterized. The full length SjBMP cDNA is 3,020 bp and encodes 928 amino acids, which include a TGF-β superfamily conserved domain at the C-terminus. BLAST analysis showed that, SjBMP has 68%, 51% and 43% homology with BMP from Schistosoma mansoni, Schmidtea mediterranea and Dugesia japonica at the amino acid level, respectively. According to data from real-time PCR, SjBMP was expressed in lung-stage schistosomula, 21-day liver-stage schistosomula, 50-day adult worms (the male and female), and eggs. The PCR data also indicated that, there was a ≈ 27- and ≈ 37-fold increase of SjBMP transcripts in the lung-stage schistosomula and eggs, respectively, and that there was relatively more SjBMP transcript in the adult male worm than in the adult female, in which the hepatic schistosomula was set as the calibrator for calculation. In situ hybridization based on FITC-labeled specific antisense oligonucleotide probes showed that SjBMP mRNA localized to the ovary of female worms and the integument and epithelium of female and male worms. After treatment with double-stranded RNA (dsRNA) at a concentration of 8 × 10(-2) μg/ml, which was added to the culture medium every other day for a week, the level of SjBMP mRNA in the cultured adult mixed-sex S. japonicum decreased at a range of ≈ 25-98% within 7 days compared with the level of SjBMP mRNA in the blank control group. On the 2nd day, the number of eggs produced per pair of worms decreased 28.7%, and the percent of normal eggs also decreased (12.7% vs. 4.3%) in the SjBMP dsRNA-treated group when compared with the eggs laid by the blank control group. No difference was detected between the two groups on the 7th day of treatment, because the eggs of the untreated worms were also mostly abnormal, similar to the eggs laid by the treated group. In addition, no significant difference in the morphological structure of the adult worms was observed. Thus, the preliminary in vitro experiment indicated that SjBMP may be involved in the oviposition behavior of S. japonicum, and further studies based on the recombinant virus vector-induced steady knockdown of SjBMP or in vivo experiments are required for more in-depth investigation.

Keywords: Bone morphogenetic protein; Oviposition; RNA interference; Schistosoma japonicum.