Identification and characterization of a Smad8 homologue from Schistosoma mansoni
Carlo, Joelle M
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Schistosoma mansoni is a parasitic blood fluke with a complex life cycle. Of the growth-related pathways in parasitic heminths, the schistosome TGFβ (Transforming Growth Factor β) pathway has been the most extensively studied. An EST demonstrating homology to Smad8, a TGFβ superfamily member, was identified and characterized in this research. SmSmad8 is the third Smad protein to be identified in S. mansoni . The SmSmad8 gene is encoded by 5 exons. The SmSmad8 protein is composed of a 380 amino acids and contains a highly conserved MH1 and MH2 domain separated by a short linker region. Based on sequence and phylogenetic analysis, SmSmad8 demonstrates homology to Smads belonging to the BMP/GDF pathway, a TGFβ sub-family. SmSmad8 transcript is expressed throughout all the stages of schistosome development as determined by RT-PCR analysis and exhibits the highest expression level in cercariae, the infective stage of S. mansoni . By immunolocalization experiments, the SmSmad8 protein was detected in the sub-tegument of adult schistosomes as well as in the female reproductive tissues, including the vitellaria and reproductive ducts. The SmSmad8 protein was not detected in the testes of the male worms. Through in vivo yeast two-hybrid experiments, an interaction was detected between SmSmad8 and the Co-Smad SmSmad4 and also with the wild type and mutant Type I receptor SmTBRI. As determined by yeast three-hybrid assays, the presence of the wild type or mutated SmTBRI receptor resulted in a decreased interaction between SmSmad8 and SmSmad4. Protein interaction results were confirmed in vitro by MBP pull-down assays. SmSmad8, as well as the schistosome BMP/GDF-related Smad SmSmad1 also interacted with the schistosome coactivator protein GCN5 in GST pull-down assays. Lastly, a pilot RNAi experiment has suggested that the gene silencing of SmSmad8 may impair schistosome infection/host-penetration or development within the mammalian host. In all, these data suggest the involvement of SmSmad8 in critical biological processes such as schistosome reproductive development.