Echinococcus multilocularis: molecular characterization of EmSmadE, a novel BR-Smad involved in TGF-β and BMP signaling.

Smad transcription factors are central components of transforming growth factor-β (TGF-β)/bone morphogenetic protein (BMP) signaling pathways in metazoans, and regulate key developmental processes such as body axis formation or regeneration. In the present study, we have identified and characterized a novel member of this protein family, EmSmadE, in the human parasitic cestode Echinococcus multilocularis, the causative agent of alveolar echinococcosis. The cDNA of the corresponding gene, emsmadE, was fully sequenced and shown to encode a protein with considerable homologies to known members of the receptor regulated Smad (R-Smad) family of a wide variety of organisms. EmSmadE contains highly conserved MH1- and MH2-domains and, on the basis of sequence features around the L3 loop region, could be assigned to the BR-Smad subfamily that typically transmits BMP signals. RT-PCR analyses indicated expression of emsmadE in all larval stages that are involved in the infection of the intermediate host. Yeast two-hybrid interaction studies demonstrated that EmSmadE can form homodimers, and is capable of heterodimer formation with the previously identified common Smad (Co-Smad) EmSmadD and the R-Smads, EmSmadA, and EmSmadB. In a heterologous expression system, EmSmadE was specifically phosphorylated at a conserved C-terminal SSVS motif by the human BMP type I receptor and, despite being structurally a BR-Smad, also by the human TGF-β type I receptor. Taken together, these data indicate that EmSmadE is a functionally active R-Smad that is involved in larval Echinococcus development. The data presented herein will be important for further analyses on the role of TGF-β/BMP signaling pathways in Echinococcus pattern formation and differentiation.