Recombinant production, crystallization and preliminary structural characterization of Schistosoma japonicum profilin.
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ABSTRACT:
SUBMITTER: Vervaet N
PROVIDER: S-EPMC3818049 | biostudies-literature | 2013 Nov
REPOSITORIES: biostudies-literature
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Project description:Thioredoxin glutathione reductase from Schistosoma japonicum (SjTGR), a multifunctional enzyme, plays a vital role in antioxidant pathways and is considered to be a potential drug target for the development of antischistosomal chemotherapy. In this study, two constructs of a truncated form of SjTGR without the last two residues (Sec597-Gly598) were cloned, overexpressed and purified using wild-type and codon-optimized genes. Only SjTGR from the wild-type gene was found to form a complex with flavin adenine dinucleotide (FAD), which could be crystallized in the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 84.185, b = 86.47, c = 183.164 Å, at 295 K using the hanging-drop vapour-diffusion method. One dimer was present in the crystallographic asymmetric unit and the calculated Matthews coefficient (VM) and solvent content were 2.6 Å(3) Da(-1) and 52.8%, respectively. Structural determination of SjTGR is in progress using the molecular-replacement method.
| S-EPMC3943093 | biostudies-literature
Project description:BackgroundN-acetyltransferase 13 (NAT13) is a probable catalytic component of the ARD1A-NARG1 complex possessing alpha (N-terminal) acetyltransferase activity.ResultsIn this study, a full-length complementary DNA (cDNA) encoding Schistosoma japonicum NAT13 (SjNAT13) was isolated from schistosome cDNAs. The 621 bp open reading frame of SjNAT13 encodes a polypeptide of 206 amino acids. Real-time PCR analysis revealed SjNAT13 expression in all tested developmental stages. Transcript levels were highest in cercariae and 21-day-old worms, and higher in male adult worms than female adult worms. The rSjNAT13 protein induced high levels of anti-rSjNAT13 IgG antibodies. In two independent immunoprotection trials, rSjNAT13 induced 24.23% and 24.47% reductions in the numbers of eggs in liver. RNA interference (RNAi) results showed that small interfering RNA (siRNA) Sj-514 significantly reduced SjNAT13 transcript levels in worms and decreased egg production in vitro.ConclusionsThus, rSjNAT13 might play an important role in the development and reproduction of schistosomes.
| S-EPMC8540080 | biostudies-literature
Project description:BackgroundThe outer-tegument membrane covering the schistosome is believed to maintain via the fusion of membranous vesicles. Fusion of biological membranes is a fundamental process in all eukaryotic cells driven by formation of trans-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes through pairing of vesicle associated v-SNAREs (VAMP) with complementary t-SNAREs on target membranes. The purpose of this study was to characterize Schistosoma japonicum vesicle-associated membrane protein 2 (SjVAMP2) and to investigate its potential as a candidate vaccine against schistosomiasis.Methodology/principal findingsThe sequence of SjVAMP2 was analyzed, cloned, expressed and characterized. SjVAMP2 is a member of the synaptobrevin superfamily harboring the v-SNARE coiled-coil homology domain. RT-PCR analysis revealed that significantly higher SjVAMP2 levels were observed in 14-, 28- and 42-day-old worms, and SjVAMP2 expression was much higher in 42-day-old female worms than in those male worms. Additionally, the expression of SjVAMP2 was associated with membrane recovery in PZQ-treated worms. Immunostaining assay showed that SjVAMP2 was mainly distributed in the sub-tegument of the worms. Western blotting revealed that rSjVAMP2 showed strong immunogenicity. Purified rSjVAMP2 emulsified with ISA206 adjuvant induced 41.5% and 27.3% reductions in worm burden, and 36.8% and 23.3% reductions in hepatic eggs in two independent trials. Besides, significantly higher rSjVAMP2-specific IgG, IgG1, IgG2a levels were detected in rSjVAMP2-vaccinated mice.ConclusionOur study indicated that SjVAMP2 is a potential vaccine candidate against S. japonicum and provided the basis for further investigations into the biological function of SjVAMP2.
| S-EPMC4671580 | biostudies-literature
Project description:BackgroundSchistosomiasis japonica remains a major public health problem in China. Its pathogen, Schistosoma japonicum has a complex life cycle and a unique repertoire of genes expressed at different life cycle stages. Exploring schistosome gene regulation will yield the best prospects for new drug targets and vaccine candidates. MicroRNAs (miRNAs) are a highly conserved class of noncoding RNA that control many biological processes by sequence-specific inhibition of gene expression. Although a large number of miRNAs have been identified from plants to mammals, it remains no experimental proof whether schistosome exist miRNAs.Methodology and resultsWe have identified novel miRNAs from Schistosoma japonicum by cloning and sequencing a small (18-26 nt) RNA cDNA library from the adult worms. Five novel miRNAs were identified from 227 cloned RNA sequences and verified by Northern blot. Alignments of the miRNAs with corresponding family members indicated that four of them belong to a metazoan miRNA family: let-7, miR-71, bantam and miR-125. The fifth potentially new (non conserved) miRNA appears to belong to a previously undescribed family in the genus Schistosome. The novel miRNAs were designated as sja-let-7, sja-miR-71, sja-bantam, sja-miR-125 and sja-miR-new1, respectively. Expression of sja-let-7, sja-miR-71 and sja-bantam were analyzed in six stages of the life cycle, i.e. egg, miracidium, sporocyst, cercaria, schistosomulum, and adult worm, by a modified stem-loop reverse transcribed polymerase chain reaction (RT-PCR) method developed in our laboratory. The expression patterns of these miRNAs were highly stage-specific. In particular, sja-miR-71 and sja-bantam expression reach their peaks in the cercaria stage and then drop quickly to the nadirs in the schistosomulum stage, following penetration of cercaria into a mammalian host.ConclusionsAuthentic miRNAs were identified for the first time in S. japonicum, including a new schistosome family member. The different expression patterns of the novel miRNAs over the life stages of S. japonicum suggest that they may mediate important roles in Schistosome growth and development.
| S-EPMC2603315 | biostudies-literature
Project description:BACKGROUND: Tetraspanins (TSPs), also known as members of the trans-membrane 4 super-family (TM4SF), comprise an assemblage of surface antigens reported in eukaryotic organisms. In the work presented here, six novel TSP proteins from the human blood fluke Schistosoma japonicum (S. japonicum) were produced and analyzed through a combination of bioinformatics and experimental approaches. RESULTS: Six novel TSP proteins of Schistosoma japonicum (designated as Sj-TSP-#1~6) contained four trans-membrane regions and one large extracellular loop (LEL) with a conserved CCG motif. Size of the proteins varied from 227 to 291 amino acid residues. All the six proteins were produced in E.coli and immune sera to each protein were prepared. Analysis of transcription profiles of the proteins by RT-PCR showed that Sj-TSP-#4 was transcribed only in the egg stage while transcription of the Sj-TSP-#2 was detected in female worms but not in males. The similar results were obtained by Western blot. Immunolocalization of the TSP proteins by immunofluorescence assay showed that the Sj-TSP-#2, Sj-TSP-#5 and Sj-TSP-#6 were located in the tegument of worms. CONCLUSIONS: This study provided six novel TSP members of S. japonicum including their sequences and recombinant proteins. Availability of the novel proteins and information on their expression profile and location provided a basis for further investigation of the TSP proteins for their biological functions and as vaccine candidates.
| S-EPMC3203850 | biostudies-literature
Project description:BackgroundSmall endogenous non-coding RNAs (sncRNAs) such as small interfering RNA (siRNA), microRNA and other small RNA transcripts are derived from distinct loci in the genome and play critical roles in RNA-mediated gene silencing mechanisms in plants and metazoa. They are approximately 22 nucleotides long; regulate mRNA stability through perfect or imperfect match to the targets. The biological activities of sncRNAs have been related to many biological events, from resistance to microbe infections to cellular differentiation. The development of the zoonotic parasite Schistosoma japonicum parasite includes multiple steps of morphological alterations and biological differentiations, which provide a unique model for studies on the functions of small RNAs. Characterization of the genome-wide transcription of the sncRNAs will be a major step in understanding of the parasite biology. The objective of this study is to investigate the transcriptional profile and potential function of the small non-coding RNAs in the development of S. japanicum.ResultsThe endogenous siRNAs were found mainly derived from transposable elements (TE) or transposons and the natural antisense transcripts (NAT). In contrast to other organisms, the TE-derived siRNAs in S. japonicum were more predominant than other sncRNAs including microRNAs (miRNAs). Further, there were distinct length and 3'end variations in the sncRNAs, which were associated with the developmental differentiation of the parasite. Among the identified miRNA transcripts, there were 38 unique to S. japonicum and 16 that belonged to 13 miRNA families are common to other metazoan lineages. These miRNAs were either ubiquitously expressed, or they exhibited specific expression patterns related to the developmental stages or sex. Genes that encoded miRNAs are mainly located in clusters within the genome of S. japonicum. However, genes within one cluster could be differentially transcribed, which suggested that individual genes might be regulated by distinct mechanisms during parasite development.ConclusionsMany miRNA and endogenous siRNA transcripts were identified in S. japonicum and the amount of siRNA was at least 4.4 and 1.6 times more than that of miRNA in both schistosomulum and adult worm stages respectively. SiRNAs are mainly derived from transposable elements (or transposons); while natural antisense transcripts (NAT)-derived siRNAs were much less. A majority of miRNA transcripts identified in the parasite were species-specific and the expression of certain miRNAs was found developmentally regulated. Both miRNA and siRNAs are potentially important regulators in the development of schistosomal parasites.
| S-EPMC2820009 | biostudies-literature
Project description:Myoferlin is a member of the ferlin family of proteins, which are involved in plasma membrane repair, and has been identified as one of the tegument proteins of Schistosoma japonicum. The tegument proteins are potential candidates for vaccines and new drug targets. In this study, myoferlin of S. japonicum (SjMF) was cloned, expressed and characterized, the potential of SjMF recombinant protein (rSjMF) as a vaccine candidate was evaluated, and the effect of praziquantel on SjMF was detected by Real-time PCR. Immunofluorescence showed that this protein was mainly distributed on the surface of worms at different stages. Sequence analysis revealed that the SjMF open reading frame was conserved at all stages of the S. japonicum life cycle. And SjMF transcription was upregulated in 42-day-old worms, and was significantly higher in female worms. Western blotting revealed that rSjMF showed strong immunogenicity. The cytokine profile and IgG isotype analysis demonstrated that rSjMF plus ISA206 immunization induced a mixed T helper (Th)1/Th2 response. Purified rSjMF emulsified with ISA206 adjuvant significantly reduced worm burden from 21.8% to 23.21% and liver egg number from42.58% to 28.35%. Besides, SjMF transcription was downregulated when worms were exposed to low-dose praziquantel (PZQ) and upregulated when PZQ was degraded, accompanied by recovery of damaged tegument. When worms were exposed to high-dose PZQ, SjMF transcription was downregulated all the time and the damaged tegument did not recover. These findings indicated that SjMF is a potential vaccine against S. japonicum and provides the basis for further investigations into the biological function of SjMF.
| S-EPMC3688920 | biostudies-literature
Project description:The stromal-cell-derived factor 2-like protein of Arabidopsis thaliana (AtSDL) has been shown to be highly up-regulated in response to unfolded protein response (UPR) inducing reagents, suggesting that it plays a crucial role in the plant UPR pathway. AtSDL has been cloned, overexpressed, purified and crystallized using the vapour-diffusion method. Two crystal forms have been obtained under very similar conditions. The needle-shaped crystals did not diffract X-rays, while the other form diffracted to 1.95 A resolution using a synchrotron-radiation source and belonged to the hexagonal space group P6(1), with unit-cell parameters a = b = 96.1, c = 69.3 A.
| S-EPMC2805525 | biostudies-literature
Project description:BACKGROUND:Schistosomiasis is a neglected tropical disease with high morbidity and mortality in the world. Currently, the treatment of this disease depends almost exclusively on praziquantel (PZQ); however, the emergence of drug resistance to PZQ in schistosomes makes the development of novel drugs an urgent task. Aldose reductase (AR), an important component that may be involved in the schistosome antioxidant defense system, is predicted as a potential drug target. METHODS:The tertiary structure of Schistosoma japonicum AR (SjAR) was obtained through X-ray diffraction method and then its potential inhibitors were identified from the Maybridge HitFinder library by virtual screening based on this structural model. The effects of these identified compounds on cultured adult worms were evaluated by observing mobility, morphological changes and mortality. To verify that SjAR was indeed the target of these identified compounds, their effects on recombinant SjAR (rSjAR) enzymatic activity were assessed. The cytotoxicity analysis was performed with three types of human cell lines using a Cell Counting Kit-8. RESULTS:We firstly resolved the SjAR structure and identified 10 potential inhibitors based on this structural model. Further in vitro experiments showed that one of the compounds, renamed as AR9, exhibited significant inhibition in the activity of cultured worms as well as inhibition of enzymatic activity of rSjAR protein. Cytotoxicity analysis revealed that AR9 had relatively low toxicity towards host cells. CONCLUSIONS:The work presented here bridges the gap between virtual screening and experimental validation, providing an effective and economical strategy for the development of new anti-parasitic drugs. Additionally, this study also found that AR9 may become a new potential lead compound for developing novel antischistosomal drugs against parasite AR.
| S-EPMC3691639 | biostudies-literature
Project description:Malaria is a devastating disease caused by apicomplexan parasites of the genus Plasmodium that use a divergent actin-powered molecular motor for motility and invasion. Plasmodium actin differs from canonical actins in sequence, structure and function. Here, the purification, crystallization and secondary-structure analysis of the two Plasmodium actin isoforms are presented. The recombinant parasite actins were folded and could be purified to homogeneity. Plasmodium actins I and II were crystallized in complex with the gelsolin G1 domain; the crystals diffracted to resolutions of 1.19 and 2.2?Å and belonged to space groups P2?2?2? and P2?, respectively, each with one complex in the asymmetric unit.
| S-EPMC3792683 | biostudies-literature