Cloning, Expression And Biological Function Analysis Of Schistosoma Japonicum Frizzled Family Receptors

Many biological processes including growth, development, sexual maturation and egg laying are controlled by diverse signalling network in cellular communication. In our previous work, we deduced the existence of Wnt signaling pathway in Schistosome japonicum through diverse bioinformatic analysis. Furthermore, we first cloned two potential signal proteins SjWnt4 and SjWnt10a, two potential receptors SjFz5 and SjFz7, and some other elements of S. japonicum Wnt signalling. However, the role and molecular mechanism of Wnt signalling in developmental processes of schistosomes remains poorly understood. In view of the fact that binding of wnt ligands to Frizzled (Fz) receptors is the beginning of Wnt signaling, it is crucial to elucidate the binding mode of Wnt-Fz pairs from S. japonicum. Since one Wnt protein can bind to multiple Frizzled receptors and vice verse, it is necessary to isolate S. japonicum Fz family members as far as possible and investigate their combinations and biological functions in such issues as expression pattern and tissue distribution. In order to discover Fz family members of S. japonicum and identify which Wnt signaling they may be implicated in during the development of S. japonicum, in this study, we carried out a series of the experimental investigations including isolation of two novel Fz gene ESTs from S. japonicum, analysis of stage-specific transcripts pattern of the three cloned S. japonicum Fzs (SjFzs), additionally the tissue localization of SjFz9 and expression of functional domain of SjFz5 in 293T cells.We isolated two novel S. japonicum Frizzled homologue Frizzled 1 and Frizzled 9 by degenerate PCR. With RACE procedure and electronic assembling methods, we successfully cloned the full sequence of S. japonicum Frizzled 9 (SjFz9), which contained a complete ORF with 3670 bp nucleotide, encoding 923 amino acid with relative molecular weight (Mr ) of 103.97 kD. The SjFz9 protein shared general feature of Frizzled family proteins: N-terminal cysteine-rich domain (CRD) and seven transmembrane spanning segments. Sequences alignment showed that SjFz9 was highly similar to Schistosoma mansoni Frizzled-like receptor with identity of 79%, next to Xenopus Frizzled 9 with identity of 33%. We deduced that Frizzled family genes in schistosome were composed of four members, including Fz5, Fz7, Fz9 homologues and the partial Fz1 to be identified.We investigated mRNA expression patterns of the 3 SjFzs (SjFz5, SjFz7 and SjFz9) during different life stages in definitive hosts. Quantitative real-time PCR analysis revealed that SjFz5 mRNA was highly expressed in schistosomulum stages and highest in 13 day, while weakly expressed in adult stages; SjFz7 gene was also highly transcript in schistosomulum stages and degenerate gradually from 7 day to 18 day, while moderately or weakly expressed in adult stages except rising in female of 23 day, 29 day and 42 day; SjFz9 mRNA were highly expressed in the schistosomulum and exhibited high level at day 23 and day 42 in both male and female, compared to other adult stages. The expression profiles of these SjFzs suggest that they may participate in developmental processes of early stage schistosomulum and reproductive organ of schistosoma adult worms.We recombined CRD domain of SjFzs with pET28a and successfully expressed the His-tagged recombinants in prokaryotic system which was identified by Western-blot assay. We prepared polyclonal anti-serum against SjFz5, SjFz7 and SjFz9 respectively by immunizing mice with the expressed proteins. Western-blot detection indicated that the anti-serum could recognize natural SjFz5, SjFz7 and SjFz9 proteins from 18 day schistosomula. Furthermore, we studied the tissue distribution of SjFz9 protein by means of immunohistochemical staining. The result showed that SjFz9 protein was conspicuously localized in the subtegumental musculature and acetabulum musculature of schistosomulum and adult worms. Intriguingly, SjFz9 was also found prominently expressed in the testes of the male and the ovary as well as the vitellarium of the female. The tissue distribution suggested that SjFz9 was likely related to the motility of schistosome and may participate in the development of reproductive organs.Besides, we constructed two recombination plasmids pRK5-SjFz5CRD and pMK33-SjWnt4 and transfected them into 293T cells. The expression of the genes of interest was detected by indirect immunofluorescence (IIF) method. The result showed that pRK5-SjFz5CRD was successfully expressed in 293T cells and pMK33-SjWnt4 was not expressed. That benefits for our further studying of the interplay of schistosoma Wnt-Frizzled pairs.In conclusion, we successfully obtained a new member of S.japonicum Frizzled receptor family , SjFz9, with the Genbank accession number GQ500895. Our primary results suggest that SjFzs likely participate in developmental processes of early stage schistosomulum and reproductive organ of schistosoma adult worms. All the data could provide basis for further studying on the detailed function of SjFz genes and identifying the ligand receptor interaction mode of S.japonicum wnt signalling.