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Characterization of a truncated metabotropic glutamate receptor in a primitive metazoan, the parasitic flatworm Schistosoma mansoni.

ABSTRACT: A novel glutamate-binding protein was identified in Schistosoma mansoni. The protein (SmGBP) is related to metabotropic glutamate receptors from other species and has a predicted glutamate binding site located within a Venus Flytrap module but it lacks the heptahelical transmembrane segment that normally characterizes these receptors. The SmGBP cDNA was cloned, verified by 5' and 3' Rapid Amplification of cDNA Ends (RACE) and shown to be polyadenylated at the 3'end, suggesting the transcript is full-length. The cloned cDNA was subsequently expressed in bacteria and shown to encode a functional glutamate-binding protein. Other studies, using a specific peptide antibody, determined that SmGBP exists in two forms, a monomer of the expected size and a stable but non-covalent dimer. The monomer and dimer are both present in the membrane fraction of S. mansoni and are resistant to extraction with high-salt, alkaline pH and urea, suggesting SmGBP is either an integral membrane protein or a peripheral protein that is tightly associated with the membrane. Surface biotinylation experiments combined with western blot analyses and confocal immunolocalization revealed that SmGBP localized to the surface membranes of adult male schistosomes, especially the dorsal tubercles. In contrast, we detected little or no expression of SmGBP either in the females or larval stages. A comparative quantitative PCR analysis confirmed that the level of SmGBP expression is several-fold higher in male worms than cercariae, and it is barely detectable in adult females. Together, the results identify SmGBP as a new type of schistosome glutamate receptor that is both gender- and stage-specific. The high-level expression of this protein in the male tubercles suggests a possible role in host-parasite interaction.

SUBMITTER: Taman A

PROVIDER: S-EPMC3206071 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Characterization of a truncated metabotropic glutamate receptor in a primitive metazoan, the parasitic flatworm Schistosoma mansoni.

Taman Amira A Ribeiro Paula P

PloS one 20111101 11

A novel glutamate-binding protein was identified in Schistosoma mansoni. The protein (SmGBP) is related to metabotropic glutamate receptors from other species and has a predicted glutamate binding site located within a Venus Flytrap module but it lacks the heptahelical transmembrane segment that normally characterizes these receptors. The SmGBP cDNA was cloned, verified by 5' and 3' Rapid Amplification of cDNA Ends (RACE) and shown to be polyadenylated at the 3'end, suggesting the transcript is ...[more]

PMID: 22069494

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Project description:Over 200 million people have, and another 600 million are at risk of contracting, schistosomiasis, one of the major neglected tropical diseases. Transmission of this infection, which is caused by helminth parasites of the genus Schistosoma, depends upon the release of parasite eggs from the human host. However, approximately 50% of eggs produced by schistosomes fail to reach the external environment, but instead become trapped in host tissues where pathological changes caused by the immune responses to secreted egg antigens precipitate disease. Despite the central importance of egg production in transmission and disease, relatively little is understood of the molecular processes underlying the development of this key life stage in schistosomes. Here, we describe a novel parasite-encoded TGF-beta superfamily member, Schistosoma mansoni Inhibin/Activin (SmInAct), which is key to this process. In situ hybridization localizes SmInAct expression to the reproductive tissues of the adult female, and real-time RT-PCR analyses indicate that SmInAct is abundantly expressed in ovipositing females and the eggs they produce. Based on real-time RT-PCR analyses, SmInAct transcription continues, albeit at a reduced level, both in adult worms isolated from single-sex infections, where reproduction is absent, and in parasites from IL-7R(-/-) mice, in which viable egg production is severely compromised. Nevertheless, Western analyses demonstrate that SmInAct protein is undetectable in parasites from single-sex infections and from infections of IL-7R(-/-) mice, suggesting that SmInAct expression is tightly linked to the reproductive potential of the worms. A crucial role for SmInAct in successful embryogenesis is indicated by the finding that RNA interference-mediated knockdown of SmInAct expression in eggs aborts their development. Our results demonstrate that TGF-beta signaling plays a major role in the embryogenesis of a metazoan parasite, and have implications for the development of new strategies for the treatment and prevention of an important and neglected human disease.

Project description:Schistosomiasis is endemic in over 70 countries, in which more than 200 million people are infected with the various schistosome species. Understanding the physiological processes underlying key developmental events could be useful in developing novel chemotherapeutic reagents or infection intervention strategies. Calmodulin is a small, calcium-sensing protein found in all eukaryotes and, although the protein has been previously identified in various Schistosoma mansoni stages and implicated in egg hatching and miracidia transformation, few molecular and functional data are available for this essential protein. Herein, we report the molecular cloning, expression, and functional characterization of calmodulin in the miracidia and primary sporocyst stages of S. mansoni. Two transcripts, SmCaM1 and SmCaM2, were cloned and sequenced, and a recombinant SmCaM1 protein was expressed in Escherichia coli and used to generate anti-CaM antibodies. The 2 protein sequences were highly conserved when compared to other model organisms. The alignment of the predicted proteins of both SmCaM1 and SmCaM2 exhibited 99% identity to each other and 97-98% identity with mammalian calmodulins. Analysis of steady-state transcript abundance indicate that the 2 calmodulin transcripts differ in their stage-associated expression patterns, although the CaM protein isotype appears to be constitutively expressed during early larval development. Application of RNAi to larval parasites results in a "stunted growth" phenotype in sporocysts with 30 and 35% reduction in transcript abundance for SmCaM1 and SmCaM2, respectively, and a corresponding 35% reduction in protein level after incubation in double-stranded RNA. Differential expression of CaM transcripts during early larval development and a growth defect-inducing effect associated with partial transcript and protein inhibition as a result of RNAi suggest a potentially important role of calmodulin during early larval development.

Project description:Cys-loop ligand-gated ion channels (LGICs) mediate fast ionotropic neurotransmission. They are proven drug targets in nematodes and arthropods, but are poorly characterized in flatworms. In this study, we characterized the anion-selective, non-acetylcholine-gated Cys-loop LGICs from Schistosoma mansoni. Full-length cDNAs were obtained for SmGluCl-1 (Smp_096480), SmGluCl-2 (Smp_015630) and SmGluCl-3 (Smp_104890). A partial cDNA was retrieved for SmGluCl-4 (Smp_099500/Smp_176730). Phylogenetic analyses suggest that SmGluCl-1, SmGluCl-2, SmGluCl-3 and SmGluCl-4 belong to a novel clade of flatworm glutamate-gated chloride channels (GluCl) that includes putative genes from trematodes and cestodes. The flatworm GluCl clade was distinct from the nematode-arthropod and mollusc GluCl clades, and from all GABA receptors. We found no evidence of GABA receptors in S. mansoni. SmGluCl-1, SmGluCl-2 and SmGluCl-3 subunits were characterized by two-electrode voltage clamp (TEVC) in Xenopus oocytes, and shown to encode Cl⁻-permeable channels gated by glutamate. SmGluCl-2 and SmGluCl-3 produced functional homomers, while SmGluCl-1 formed heteromers with SmGluCl-2. Concentration-response relationships revealed that the sensitivity of SmGluCl receptors to L-glutamate is among the highest reported for GluCl receptors, with EC₅₀ values of 7-26 µM. Chloride selectivity was confirmed by current-voltage (I/V) relationships. SmGluCl receptors are insensitive to 1 µM ivermectin (IVM), indicating that they do not belong to the highly IVM-sensitive GluClα subtype group. SmGluCl receptors are also insensitive to 10 µM meclonazepam, a schistosomicidal benzodiazepine. These results provide the first molecular evidence showing the contribution of GluCl receptors to L-glutamate signaling in S. mansoni, an unprecedented finding in parasitic flatworms. Further work is needed to elucidate the roles of GluCl receptors in schistosomes and to explore their potential as drug targets.

Dataset Information

Characterization of a truncated metabotropic glutamate receptor in a primitive metazoan, the parasitic flatworm Schistosoma mansoni.

Publications

Characterization of a truncated metabotropic glutamate receptor in a primitive metazoan, the parasitic flatworm Schistosoma mansoni.

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