Identification and Characterization of a Smad2 Homologue fromSchistosoma mansoni, a Transforming Growth Factor-β Signal Transducer

Abstract

Smad proteins are essential intracellular signal transducers of the transforming growth factor-␤ (TGF-␤) superfamily. The TGF-␤ superfamily signals through phosphorylation and activation of R-Smad proteins, receptor-regulated Smads, by heteromeric complexes of ligand-specific type I and type II serine/threonine kinase receptors. R-Smads receive a signal from the activated receptor complex and transmit it to the nucleus. A cDNA was isolated that encodes a 649-amino acid protein found to be homologous to members of R-Smad subfamily with highest homology scored to clawed African frog and human Smad2. The Schistosoma mansoni homologue (SmSmad2) was overexpressed in bacteria as a Sj26-GST fusion protein and used to raise specific antibodies. The IgG fraction of the immunized rabbit serum identified 70-and 72-kDa protein bands in Western analysis of schistosome extracts. Treatment with alkaline phosphatase removed the 72-kDa band, which indicates that this band represents the phosphorylated form of schistosome Smad2. SmSmad2 was localized in the subtegument, parenchymal cells, and sex organs in both male and female worm cryosections. Similar results were also obtained from the analysis of the Smad2 mRNA distribution pattern revealed by in situ hybridization of adult worm pair paraffin sections. SmSmad2 mRNA levels were determined by reverse transcriptasepolymerase chain reaction in different mammalian host developmental stages and found to be constitutively expressed. SmSmad2 was also found to interact with a previously identified SmT␤R-I, a serine/threonine type I kinase receptor. Furthermore, SmSmad2 was shown to undergo phosphorylation by constitutively active forms of SmT␤R-I in vitro. In addition, SmSmad2 localized in the nuclei of mink lung epithelial cells upon treatment with TGF-␤ 1 . These data indicate that the SmSmad2 responds to the TGF-␤ signals by interaction with receptor I, which phosphorylates it, whereupon it translocates into the nucleus presumably to regulate target gene transcription and consequently elicit a specific TGF-␤ effect.

Human Transforming Growth Factor-β Activates a Receptor Serine/Threonine Kinase from the Intravascular ParasiteSchistosoma mansoni

“…The completion of the genome sequencing project for S. mansoni will provide valuable information regarding the presence or absence of additional TGF-␤-signaling components in schistosomes. Whereas our studies and those of others (13) suggest that SmRK1 signals through SmSmad2, there is also an interaction between SmRK1 and SmSmad1. When the two proteins were coexpressed in R1B cells, moderate increases in luciferase expression from the GCCG-Lux reporter plasmid were observed relative to SmSmad1 expressed alone.…”

“…The specificity of interaction between R-Smads and type I receptors is facilitated by specific structural motifs in each protein. The predominant domain in the R-Smad is a region of 17 amino acids referred to as the L3 loop (19), and both SmSmads are highly conserved in this region relative to their mammalian counterparts (12,13). A 9-amino acid segment in the kinase domain of type I receptors referred to as the L45 loop has been reported to define the signaling specificity of type I receptors for intracellular responses (16).…”

“…Evaluating the Signaling Specificity of SmRK1-SmRK1 interacts with both SmSmad1 and SmSmad2 in vitro (12,13). The specificity of interaction between R-Smads and type I receptors is facilitated by specific structural motifs in each protein.…”

“…We have recently identified two schistosome R-Smads (SmSmad1 and SmSmad2) and have shown that both can interact with SmRK1 in vitro as well as signal downstream of vertebrate TGF-␤ receptors expressed in mammalian cells (12). SmSmad2 was recently shown to be phosphorylated by a constitutively active SmRK1 in vitro (13), but the physiological significance of the SmRK1-SmSmad1 interaction and, most importantly, the ligand specificity of SmRK1 are unknown.…”

Growth factor receptors in helminth parasites: Signalling and host–parasite relationships

“…Using a heterologous expression system, SmTbR1 was shown to interact with human TbRII and to activate Smad2 signalling molecules in response to human TGF-b [14]. Two R-Smads (SmSmad1 and SmSmad2) as well as a Co-Smad (SmSmad4) have been identified as downstream partners of SmTbR1 [15][16][17]. The existence of a conserved TGF-b signalling pathway in S. mansoni was further confirmed by Knobloch et al [18] who demonstrated an Fig.…”