Molecular characterisation of a second structurally unusual AR-Smad without an MH1 domain and a Smad4 orthologue from...

Members of the transforming growth factor-beta/bone morphogenetic protein (TGF-beta/BMP) family of cytokines play crucial roles in animal development and are candidate molecules for host-parasite cross-communication in helminth diseases. TGF-beta/BMP-signalling involves binding of the cytokines to receptor kinases which subsequently activate intracellular transcription factors of the Smad family. We have previously characterized two members of the receptor-regulated Smad (R-Smad) family, EmSmadA and EmSmadB, from the human parasitic cestode Echinococcus multilocularis and now present evidence for two additional Smads that are expressed by the larval stages of the parasite. The full-length cDNAs coding for a third R-Smad, EmSmadC, and a common mediator Smad (Co-Smad), EmSmadD, were characterized. While EmSmadD displayed a typical Co-Smad structure, EmSmadC lacked the N-terminal MH1 domain which is typically found in Smads. In yeast two-hybrid analyses, EmSmadC and EmSmadD were capable of homo- and heterodimer formation with other Echinococcus Smads. Furthermore, EmSmadC displayed autonomous transcription activation activity and interacted with EmSkip, a member of the SNW/SKIP family of transcriptional regulators. In a heterologous expression system, EmSmadC was specifically phosphorylated by mammalian TGF-beta receptors, indicating that it is a member of the AR-Smad sub-family. Finally, in activity assays, the parasite's Erk-like kinase EmMPK1 phosphorylated EmSmadD, indicating cross-regulation between mitogen-activated protein kinase cascade- and TGF-beta/BMP-signalling in Echinococcus. The data presented herein significantly broaden our knowledge of Smad-signalling factors in E. multilocularis and will facilitate studies on TGF-beta/BMP-regulated genes in the parasite as well as TGF-beta/BMP mediated host-parasite cross-interaction during alveolar echinococcosis.