Hox genes from the parasitic flatworm Schistosoma japonicum

Hox genes are characterized by a highly conserved peptide domain and contribute to antero-posterior axis patterning during embryogenesis. These genes have been widely studied in a variety of animal species due to their central role in evolutionary developmental biology. Based on the published genome assembly and unpublished re-sequencing project data, we present the first genome-wide characterization and comparative genomic analysis of the Hox gene family within Schistosoma japonicum. Eight Hox genes were identified and validated in our investigation. Phylogenetic analysis revealed that these genes are distributed among seven orthology groups of the Hox gene family. Our study further suggested that differences in the Lox5 gene copy number existed between the two closely related species, S. japonicum and Schistosoma mansoni. Semi-quantitative real-time polymerase chain reaction experiments revealed that Lox5 and Hox4 gene expression was high in the schistosomulum stage, and all four genes investigated showed highest expression within the eggs.     

Stromal cell-derived receptor 2 and cytochrome b561 are functional ferric reductases

Iron has a variety of functions in cellular organisms ranging from electron transport and DNA synthesis to adenosine triphosphate (ATP) and neurotransmitter synthesis. Failure to regulate the homeostasis of iron can lead to cognition and demyelination disorders when iron levels are deficient, and to neurodegenerative disorders when iron is in excess. In this study we show that three members of the b561 family of predicted ferric reductases, namely mouse cytochrome b561 and mouse and fly stromal cell-derived receptor 2 (SDR2), have ferric reductase activity. Given that a fourth member, duodenal cytochrome b (Dcytb), has previously been shown to be a ferric reductase, it is likely that all remaining members of this family also exhibit this activity. Furthermore, we show that the rat sdr2 message is predominantly expressed in the liver and kidney, with low expression in the duodenum. In hypotransferrinaemic (hpx) mice, sdr2 expression in the liver and kidney is reduced, suggesting that it may be regulated by iron. Moreover, we demonstrate the presence of mouse sdr2 in the choroid plexus and in the ependymal cells lining the four ventricles, through in situ hybridization analysis.     

Involvement of 101F6, a homologue of cytochrome b561, in the reduction of ferric ions

We have isolated and characterized a small transmembrane protein, called 101F6, showing high sequence homology to cytochrome b(561), a protein containing two binding sites for haem. The newly identified 101F6 contains six membrane spanning domains in which conserved histidine residues are located, and has a molecular mass of 25 kDa. When the haem-binding with bacterial expressed 101F6 was examined, the protein bound haem and the deletion of one histidine residue at 149 caused a loss of the binding. 101F6 mRNA was expressed widely in various tissues, and especially abundant in liver, kidney and lung. It was also expressed in several cultured cell lines. The protein expressed from the 101F6 cDNA in Balb/3T3 cells was about 25 kDa in size and was localized in small vesicles, including endosomes and endoplasmic reticulum of the perinuclear region. Comparison of the location of 101F6 with that of transferrin receptor-1 revealed that the localization of 101F6 in small vesicles was not always the same as the localization of transferrin receptor-1, but was similar to that of haem oxygenase-1. The other homologue to cytochrome b(561), SDR-2 was also expressed in the small vesicles similar to the location of 101F6. Finally, reduction of ferric ions as well as of azo-dye increased with 101F6- or SDR-2-expressing cells. Thus, both 101F6 and SDR-2 were localized in small vesicles of cells and played roles in the reduction of ferric ions.     

The cellular distribution and stage-specific expression of two dynein light chains from the human blood fluke Schistosoma japonicum

Schistosomes are pathogenic helminth parasites of human portal veins. Their body wall is a highly active syncytial tegument involved in an array of host interactions. The cytoskeletal organization and dynamics of this syncytium are poorly understood, but predominant motor components are the LC8 class of cytoplasmic dynein light chains (DLC). Four LC8 members occur in schistosomes, two of which are expressed in the tegument. Here, we describe the cytoplasmic distribution, stage-specific expression and cellular location of two diverse LC8 molecules of Schistosoma japonicum. SjDLC1 was detected in surface-membrane specific extracts of adult worms and was shown by quantitative immuno-electron microscopy to predominate along heptalaminate membranes of the worm surface. SjDLC3 also occurs in the tegument, but was shown to be present in basal layers of the tegument and did not preferentially co-localize with particular membrane components. SjDLC3 was also detected in the gastrodermis. SjDLC1 is expressed only in mammalian-parasitic stages, whereas SjDLC3 is expressed throughout the life-cycle. The data suggest that SjDLC1 is preferentially located to the host-interactive distal parasite membrane, and plays a role in surface membrane dynamics, while SjDLC3 is a ubiquitous motor component of schistosome epithelia of all stages.     

Reconstruction and in silico analysis of the MAPK signaling pathways in the human blood fluke, Schistosoma japonicum

At present, little is known about signal transduction mechanisms in schistosomes, which cause the disease of schistosomiasis. The mitogen-activated protein kinase (MAPK) signaling pathways, which are evolutionarily conserved from yeast to Homo sapiens, play key roles in multiple cellular processes. Here, we reconstructed the hypothetical MAPK signaling pathways in Schistosoma japonicum and compared the schistosome pathways with those of model eukaryote species. We identified 60 homologous components in the S. japonciumMAPK signaling pathways. Among these, 27 were predicted to be full-length sequences. Phylogenetic analysis of these proteins confirmed the evolutionary conservation of the MAPK signaling pathways. Remarkably, we identified S. japonicum homologues of GTP-binding protein beta and alpha-I subunits in the yeast mating pathway, which might be involved in the regulation of different life stages and female sexual maturation processes as well in schistosomes. In addition, several pathway member genes, including ERK, JNK, Sja-DSP, MRAS and RAS, were determined through quantitative PCR analysis to be expressed in a stage-specific manner, with ERK, JNK and their inhibitor Sja-DSP markedly upregulated in adult female schistosomes.     

Sjalpha elements, short interspersed element-like retroposons bearing a hammerhead ribozyme motif from the genome of the oriental blood fluke Schistosoma japonicum

Smalpha is a short interspersed element (SINE)-like retroposon that occurs in high copy number of the genome of the human blood fluke Schistosoma mansoni. The sequence of the consensus Smalpha element includes the hallmark features of SINE-like elements including a promoter region for RNA polymerase III, an AT-rich stretch at its 3'-terminus, a short length of 500 bp or less, and short direct repeat sequences flanking the insertion site. Interestingly, the sequence of Smalpha also encodes an active ribozyme bearing a hammerhead domain. Contrary to the recent findings of Ferbeyre et al. (Mol. Cell. Biol. 18 (1998) 3880-8) that indicated that Smalpha-like elements were absent from the genome of the Oriental blood fluke Schistosoma japonicum, we report here that the genome of S. japonicum does contain a family of Smalpha-like retroposons, elements that we have named the Sjalpha family. Like Smalpha, Sjalpha elements are SINE-like in structure and sequence, are present at high copy number interspersed throughout the S. japonicum genome, and contain an ostensibly functional, hammerhead ribozyme motif. The presence of these elements in all species of Schistosoma so far examined suggests that the hammerhead domain was acquired by vertical transmission from a common schistosome ancestor.     

An Arabidopsis cytochrome b561 with trans-membrane ferrireductase capability

Ascorbate-reducible cytochromes b561 (Cyts-b561) are a class of intrinsic trans-membrane proteins. Tonoplast Cyt-b561 (TCytb), one of the four Cyt-b561 isoforms in Arabidopsis was localized to the tonoplast. We demonstrate here that the optical spectra, EPR spectra and redox potentials of recombinant TCytb are similar to those of the well characterized bovine chromaffin granule Cyt-b561. We provide evidence for the reduction of ferric-chelates by the reduced TCytb. It is also shown that TCytb is capable of trans-membrane electron transport from intracellular ascorbate to extracellular ferric-chelates in yeast cells.     

Patterns of relatedness and genetic diversity inferred from whole genome sequencing of archival blood fluke miracidia (Schistosoma japonicum)

Genomic approaches hold great promise for resolving unanswered questions about transmission patterns and responses to control efforts for schistosomiasis and other neglected tropical diseases. However, the cost of generating genomic data and the challenges associated with obtaining sufficient DNA from individual schistosome larvae (miracidia) from mammalian hosts have limited the application of genomic data for studying schistosomes and other complex macroparasites. Here, we demonstrate the feasibility of utilizing whole genome amplification and sequencing (WGS) to analyze individual archival miracidia. As an example, we sequenced whole genomes of 22 miracidia from 11 human hosts representing two villages in rural Sichuan, China, and used these data to evaluate patterns of relatedness and genetic diversity. We also down-sampled our dataset to test how lower coverage sequencing could increase the cost effectiveness of WGS while maintaining power to accurately infer relatedness. Collectively, our results illustrate that population-level WGS datasets are attainable for individual miracidia and represent a powerful tool for ultimately providing insight into overall genetic diversity, parasite relatedness, and transmission patterns for better design and evaluation of disease control efforts.     

Leucine aminopeptidase of the human blood flukes, Schistosoma mansoni and Schistosoma japonicum

An array of schistosome endoproteases involved in the digestion of host hemoglobin to absorbable peptides has been described, but the exoprotease responsible for catabolising these peptides to amino acids has yet to be identified. By searching the public databases we found that Schistosoma mansoni and Schistosoma japonicum express a gene encoding a member of the M17 family of leucine aminopeptidases (LAPs). A functional recombinant S. mansoni LAP produced in insect cells shared biochemical properties, including pH optimum for activity, substrate specificity and reliance on metal cations for activity, with the major aminopeptidase activity in soluble extracts of adult worms. The pH range in which the enzyme functions and the lack of a signal peptide indicate that the enzyme functions intracellularly. Immunolocalisation studies showed that the S. mansoni LAP is synthesised in the gastrodermal cells surrounding the gut lumen. Accordingly, we propose that peptides generated in the lumen of the schistosome gut are absorbed into the gastrodermal cells and are cleaved by LAP to free amino acids before being distributed to the internal tissues of the parasite. Since LAP was also localised to the surface tegument it may play an additional role in surface membrane re-modelling.     

Characterization of a novel NADH-specific, FAD-containing, soluble reductase with ferric citrate reductase activity from maize seedlings

A novel NADH-dependent, soluble flavoreductase of 60 kDa, active toward ferric chelates and quinones, has been purified from maize seedlings. Two closely related isoforms were separated. The two isoforms are similar in several biochemical features, with the exception of the apparent molecular mass of their subunits (29 and 31 kDa, respectively). They are homodimers in the native state, they bind FAD as the prosthetic group and show strong preference for NADH over NADPH as the electron donor. Ferric chelates (chiefly ferric citrate, Km 3-5 x 10(-5) M; kcat/Km 3.4-3.7 x 10(5) M-1 s-1), and some quinones (benzoquinone, coenzyme Q-0, and juglone) are used as electron acceptors. Enzymatic reduction of benzoquinone occurs with formation of radical semiquinones. Both soluble ferric chelate reductase isoforms are strongly inhibited by p-hydroxymercuribenzoic acid (I50 5 nM) and by cibachron blue, the latter giving nonlinear inhibition. It is suggested that soluble ferric chelate reductase might be involved in the symplastic reduction of iron chelates which is required for the assembly of iron-containing macromolecules such as cytochromes and ferritin.     

Characterization of a cytochrome b(558) ferric/cupric reductase from rabbit duodenal brush border membranes

Iron and probably also copper are absorbed by the intestine in their reduced form. A b-type cytochrome, Dcytb, has recently been cloned from mouse and has been proposed to be the corresponding reductase. However, the nature of the cytochrome and the reduction reaction remain unknown. Here we describe the isolation and functional characterization of a novel b-type cytochrome from rabbit enterocytes. The 33 kDa heme protein was solubilized from brush border membranes with Triton X-100 and purified by successive ion exchange chromatography and hydrophobic interaction chromatography. Spectroscopic analysis of the heme revealed a b(558) cytochrome. The purified hemoprotein exhibited ascorbate-stimulated reduction of iron(III) and copper(II). The rate constants, k(1), for these reactions were 1.38 +/- 0.12 and 0.64 +/- 0.16 min(-1), respectively. Cytochrome b(558) may be the rabbit Dcytb homologue. A novel mechanism of how cytochrome b(558) could shuttle electrons from cytoplasmic ascorbate to luminal dehydroascorbate is proposed.     

Structure prediction for the di-heme cytochrome b561 protein family

Summary.  Atomic models possessing the common structural features identified for the cytochrome b ₅₆₁ (cyt b ₅₆₁) protein family are presented. A detailed and extensive sequence analysis was performed in order to identify and characterize protein sequences in this family of transmembrane electron transport proteins. According to transmembrane helix predictions, all sequences contain 6 transmembrane helices of which 2–6 are located closely in the same regions of the 26 sequences in the alignment. A mammalian (Homo sapiens) and a plant (Arabidopsis thaliana) sequence were selected to build 3-dimensional structures at atomic detail using molecular modeling tools. The main structural constraints included the 2 pairs of heme-ligating His residues that are fully conserved in the family and the lipid-facing sides of the helices, which were also very well conserved. The current paper proposes 3-dimensional structures which to our knowledge are the first ones for any protein in the cyt b ₅₆₁ family. The highly conserved His residues anchoring the two hemes on the cytoplasmic side and noncytoplasmic side of the membrane are in all proteins located in the transmembrane helices 2, 4 and 3, 5, respectively. Several highly conserved amino acids with aromatic side chain are identified between the two heme ligation sites. These residues may constitute a putative transmembrane electron transport pathway. The present study demonstrates that the structural features in the cyt b ₅₆₁ family are well conserved at both the sequence and the protein level. The central 4-helix core represents a transmembrane electron transfer architecture that is highly conserved in eukaryotic species. Received May 12, 2002; accepted September 20, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Institute of Biophysics, Biological Research Centre, P.O. Box 521, 6701 Szeged, Hungary. E-mail: tpali@nucleus.szbk.u-szeged.hu