Identification and characterization of an R-Smad ortholog (SmSmad 1B) from Schistosoma mansoni

Smad proteins are the cellular mediators of the transforming growth factor-beta superfamily signals. Herein, we describe the isolation of a fourth Smad gene from the helminth Schistosoma mansoni, a receptor-regulated Smad (R-Smad) gene termed SmSmad1B. The SmSmad1B protein is composed of 380 amino acids, and contains conserved MH1 and MH2 domains separated by a short 42 amino acid linker region. The SmSmad1B gene (> 10.7 kb) is composed of five exons separated by four introns. On the basis of phylogenetic analysis, SmSmad1B demonstrates homology to Smad proteins involved in the bone morphogenetic protein pathway. SmSmad1B transcript is expressed in all stages of schistosome development, and exhibits the highest expression level in the cercariae stage. By immunolocalization experiments, the SmSmad1B protein was detected in the cells of the parenchyma of adult schistosomes as well as in female reproductive tissues. Yeast two-hybrid experiments revealed an interaction between SmSmad1B and the common Smad, SmSmad4. As determined by yeast three-hybrid assays and pull-down assays, the presence of the wild-type or mutated SmT beta RI receptor resulted in a decreased interaction between SmSmad1B and SmSmad4. These results suggest the presence of a nonfunctional interaction between SmSmad1B and SmT beta RI that does not give rise to the phosphorylation and the release of SmSmad1B to form a heterodimer with SmSmad4. SmSmad1B, as well as the schistosome bone morphogenetic protein-related Smad SmSmad1B and the transforming growth factor-beta-related SmSmad2, interacted with the schistosome coactivator proteins SmGCN5 and SmCBP1 in pull-down assays. In all, these data suggest the involvement of SmSmad1B in critical biological processes such as schistosome reproductive development.