A conserved retinoid X receptor (RXR) from the mollusk Biomphalaria glabrata transactivates transcription in the presence of retinoids.

Retinoid X receptors (RXR) are members of the nuclear receptor superfamily of ligand-activated transcription factors that have been characterized in a wide variety of metazoan phyla. They act as heterodimer partners of other nuclear receptors, and in vertebrates also activate transcription as homodimers in the presence of a ligand, 9-cis retinoic acid. In order to test the hypothesis that retinoic acid signaling pathways involving RXRs are present in the Lophotrochozoa, we have sought to isolate conserved members of this family from the platyhelminth parasite Schistosoma mansoni and its intermediate host, the mollusk Biomphalaria glabrata. Here we report that an RXR ortholog from B. glabrata (BgRXR) is better conserved, compared with mouse RXRalpha, both in the DNA-binding domain (89% identity) and in the ligand-binding domain (LBD) (81% identity), than are arthropod homologs. In EMSA, BgRXR binds to the direct repeat response element DR1 as a homodimer or as a heterodimer with mammalian RARalpha, LXR, FXR or PPARalpha. When transfected alone into mammalian cell lines, BgRXR transactivated transcription of a reporter gene from the Apo-A1 promoter in the presence of 9-cis retinoic acid or DHA. Constructs with the Gal4 DNA binding domain fused to the hinge and LBDs of BgRXR were used to show that ligand-dependent activation of transcription by BgRXR required its intact AF-2 activation domain, and that the LBD can form homodimers. Finally, the binding of 9-cis retinoic acid preferentially protected the LBD of BgRXR from degradation by trypsin in a proteolysis protection assay. Our results show that BgRXR binds and is activated by retinoids and suggest that retinoid signaling pathways are conserved in the Lophotrochozoa. The nucleotide sequence reported in this paper has been submitted to the GenBank/EBI Data Bank with accession no. AY048663.

Hormones and nuclear receptors in schistosome development.

A substantial but disparate body of evidence suggests that hormones affect the development of schistosomes within their definitive hosts. Here, Raymond Pierce and colleagues review such evidence for host steroid and thyroid hormones, and for ecdysteroids, and link this to the expanding knowledge of the nuclear receptors for these hormones. Phylogenetic analysis of the nuclear receptor superfamily and the characterization of the first schistosome nuclear receptors suggest that steroids and thyroid hormone probably act indirectly, or by pathways not involving the control of gene transcription. However, the probability that schistosome nuclear receptors exist for a variety of unique ligands opens up exciting possibilities for targeted drug development.

Structural and functional divergence of a nuclear receptor of the RXR family from the trematode parasite Schistosoma mansoni.

We describe the cloning and functional characterization of Schistosoma mansoni retinoid-X-receptor (SmRXR; NR2B4-B), a novel member of the nuclear receptor superfamily from S. mansoni, a homologue of vertebrate retinoid-X-receptor. The DNA-binding C domain of SmRXR shows 80% sequence identity to both human RXRalpha and Drosophila ultraspiracle (USP), but a much lower level of conservation of the ligand-binding E domain (22-25% identity). Phylogenetic analysis places SmRXR within the RXR group as an early offshoot of this clade. SmRXR mRNA is expressed at all life-cycle stages but at higher levels in the free-living larval stages. However, the SmRXR protein is expressed at markedly different levels, being almost absent from eggs while present at the highest concentration in schistosomula. Recombinant SmRXR fails to bind to the consensus direct repeat response elements, either alone, or as a heterodimer with mouse retinoic acid receptor alpha or the Drosophila ecdysone receptor. However, the use of chimaeric constructions shows that the C domain of SmRXR will bind to conventional response elements as a heterodimer, and that its specificity is modified by the presence of the D and E domains. In accordance with these results, native SmRXR failed to transactivate the transcription of a reporter gene after cotransfection of mammalian cell lines.

Schistosoma mansoni Ca2+-ATPase SMA2 restores viability to yeast Ca2+-ATPase-deficient strains and functions in calcineurin-mediated Ca2+ tolerance.

The sarco(endo)plasmic reticulum of animal cells contains an ATP-powered Ca2+ pump that belongs to the P-type family of membrane-bound cation-translocating enzymes. In Schistosoma mansoni, the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is encoded by the SMA1 and SMA2 genes. A full-length SMA2 cDNA clone was isolated, sequenced, and expressed into a yeast Ca2+-ATPase-deficient strain requiring plasmid-borne rabbit SERCA1a for viability. The S. mansoni Ca2+-ATPase supports growth of mutant cells lacking SERCA1a, indicating functional expression in yeast and a role in calcium sequestration. Subcellular fractionation showed that the SMA2 ATPase is localized in yeast internal membranes. SMA2 expression was found to be associated with thapsigargin-sensitive, Ca2+-dependent ATPase activity. The activity increased 2-fold upon calcineurin inactivation, which correlates with in vivo stimulated contribution of SMA2 in calcium tolerance. These results suggest that calcineurin controls calcium homeostasis by inhibiting Ca2+-ATPase activity in an internal compartment.

Cloning and characterization of a putative calcium-transporting ATPase gene from Schistosoma mansoni.

Complementary DNA was isolated, encoding a putative Ca(2+)-transport ATPase (SMA1) of the human parasitic trematode Schistosoma mansoni. The cDNA was isolated by a nested polymerase chain reaction based strategy. The oligonucleotides used were designed on the basis of conserved amino-acid regions found in P-type ATPases. The complete nucleotide sequence was determined. The primary structure and topology of the enzyme were deduced. SMA1 has 1022 amino acids and a predicted molecular mass of 113 kDa. This protein is 67% identical and phylogenetically related to several sarco/endoplasmic reticulum Ca(2+)-ATPases but lacks the phospholamban-binding domain that exists in the SERCA isoforms 1 and 2. The membrane topology predicted for SMA1 is characteristic of the P-type ATPases, showing two major cytoplasmic loops and ten conserved hydrophobic segments. Sequences and residues that are important for the function of the SER Ca(2+)-ATPase, such as the high-affinity Ca(2+)-binding sites, the putative fluorescein isothiocyanate binding site, the 5'-(p-fluorosulfonyl)benzoyladenosine binding site and the aspartyl phosphorylation site, are conserved in SMA1, suggesting that the cloned gene is a Ca(2+)-transport ATPase of the SERCA family. In addition, three PCR products were cloned which share homology with another SER Ca(2+)-ATPase, with the yeast secretory pathway Ca(2+)-ATPase PMR1 and its mammalian homologue, and with the alpha subunit of a Na+,K(+)-ATPase.