Cloning, Expressing And Preliminary Identification Of Gender-associated Gene Tsunagi In Schistosoma Japonicum

Schistosomiasis is known as a debilitating disease in human. The life cycle of schistosomes is complex and involves both a snail intermediate host and a mammalian definitive host in which they become adult worms that are reproductively active for years. The eggs produced by female worms are the agent of schistosomiasis transmission. Meanwhile, the pathogenesis of this disease is the granulomatous reaction evoked by the soluble egg antigens. Controlling sexual maturation, sexual dimorphism and labour division may be effective in prevention of schistosomiasis.Mago nashi was a strict maternal-effect gene important for germ plasm assembly during the physiological development of the Drosophila embryo and also plays important roles in cellular differentiation. The protein encoded by Mago nashi gene interacts avidly and specifically with the Tsunagi protein (also known as Y14) among divergent organisms. In Drosophila oogenesis, both Tsunagi and Mago nashi are required for interpreting the posterior follicle cell-to-oocyte signal to define the major body axes and to localize components necessary for determination of the primordial germ cells. Moreover, the Caenorhabditis elegans homologue Ce-Y14 is essential for embryonic morphogenesis and regulation of germline sex switching. Previous studies in other laboratories have also shown the significance of Tsunagi in sexual development and its conservation among species. We have previously isolated the Mago nashi gene (GenBank accession number BM735619) from a Schistosomulum japonicum cDNA library and investigated its function in schistosomes by RNAi method. However, no information is available regarding the characterization of Tsunagi in Schistosoma japonicum. We hypothesized that because schistosomes undergo sexual differentiation during development, it is probable that the parasites have similar pathways to achieve this. Moreover, because Tsunagi proteins are ubiquitous and highly conserved, we reasoned that a Tsunagi homologue is also present in S. japonicum. This paper describes the identification and preliminary characterization of the Tsunagi protein from S. japonicum.For this purpose, we have isolated the partial gene of Tsunagi protein from adult cDNA of S. japonicum using a degenerate PCR strategy. This strategy employed the CODEHOP primer design algorithm. The sequence amplified from degenerate PCR was about 180bp, displaying strong identity to other Tsunagi proteins. The remaining cDNA sequence, including the 5'and 3'regions, was recovered by anchored PCR from a cDNA library of Schistosomulum japonicum. The whole sequence of the SjTsunagi cDNA containing ORF was amplified from the schistosomulum cDNA library and the adult cDNA, respectively. It encoded a protein of 177 amino acids with an estimated molecular weight of 20.078 KD and isoelectric point of pH6.195. The gene has 55.9% identity and 67.2% similarity at the predicted amino acid level to Drosophila melanogaster and has 40.6% identity and 51.4% similarity to C. elegans. Amino acid sequence analysis and alignment showed significant similarity to other Tsunagi proteins, including the conservation of an RNA recognition motif. The encoding ORF was amplified and inserted into pET-28a expression vector and then pET-28a-SjTsunagi recombinant protein was expressed as a 6xHis-tag fusion protein. Soluble recombinant protein used for in vitro experiments was purified by affinity column chromatography. Specific antiserum was obtained from the mice immunized with the protein. Mouse antiserum (1: 51200) was used to detect the recombinant protein by indirect ELISA. To investigate the distribution of SjTsunagi in tissue, immunofluorescent staining was performed on cryosections prepared from adult worms. SjTsunagi was most abundant in the ovary of female worms. Immunoblot analyses was used to determine the proteins extracted from eggs, adult females, adult males and mixed sex parasites. Proteins extracted from adult females and mixed sex parasites probed with anti-SjTsunagi polyclonal antiserum identified a single protein with an apparent molecular weight of 20 KD and revealed that SjTsunagi protein is detected in adult females only. Besides, by pull-down and co-immunoprecipitation, we therefore concluded that the interaction of Tsunagi with the mago nashi gene product is also conserved in S. japonicum. Taken together, these results suggest that SjTsunagi might be a gender-associated protein in schistosomes.