Translationally controlled tumor protein is a novel heat shock protein with chaperone-like activity

Translationally controlled tumor protein (TCTP) is often designated as a stress-related protein because of its highly regulated expression in stress conditions. Following a thermal shock, TCTP expression is highly upregulated in a variety of cells. However, at present it is not known whether this upregulation has any cell protective function similar to other heat shock proteins. In this study human TCTP (HuTCTP) and a TCTP homolog (SmTCTP) from Schistosoma mansoni were evaluated for heat shock protein-like function and molecular chaperone activity. Our results show that similar to other molecular chaperones, both human and parasite TCTPs can bind to a variety of denatured proteins and protect them from the harmful effects of thermal shock. An important observation was the ability of both HuTCTP and SmTCTP to bind to native protein and protect them from thermal denaturation. Over expression of TCTP in bacterial cells protected them from heat shock-induced death. These findings suggest that TCTP may belong to a novel small molecular weight heat shock protein.     

日本血吸虫中国大陆株抱雌沟蛋白编码基因的克隆和表达

根据曼氏血吸虫抱雌沟蛋白SmGcp序列和日本血吸虫编码抱雌沟蛋白保守区的基因片段jGcp1分别设计三对引物,以日本血吸虫中国大陆株成虫mRNA为模板 ,用RT－PCR法扩增出大小为1949bp的基因片段。经序列分析推断该基因片段含编码日本血吸虫抱雌沟蛋白基因的阅读框,与SmGcp碱基一致性为85％,其理论推测氨基酸组成与曼氏血吸虫抱雌沟蛋白的一致性为83．7％。将上述扩增的基因片段克隆到表达载体pET28c( )中,在大肠杆菌BL21中获得表达,融合表达产物分子量约为80kD。利用日本血吸虫成虫抗原免疫血清对该表达产物进行Western印迹检测,在预测位置出现了明显的识别条带,说明该编码日本血吸虫中国大陆株抱雌沟蛋白基因的表达产物具有抗原性。     

Paragonimus westermani: biochemical and immunological characterizations of paramyosin

Paramyosin of the helminth parasite is a muscle protein that plays multifunctional roles in host-parasite relationships. In this study, we have cloned a gene encoding Paragonimus westermani paramyosin (PwPmy) and characterized biochemical and immunological properties of the recombinant protein. The recombinant PwPmy (rPwPmy) was shown to bind both human immunoglobulin G (IgG) and collagen. The protein was constitutively expressed in various developmental stages of the parasite and its expression level increased progressively as the parasite matured. Immunohistological analysis revealed that PwPmy was mainly localized in subtegumental muscle, tegument and cells surrounding the oral sucker, intestine, and ovary of the parasite. Sera from patients with paragonimiasis showed antibody reactivity against rPwPmy, and IgG1 and IgG4 were predominant. Immunization of mice with rPwPmy also induced high IgG responses. Biochemical and immunological characterization of PwPmy may provide valuable information for the further study to develop a vaccine or a chemotherapeutic agent for paragonimiasis.     

An immunogenic Mr 23,000 integral membrane protein of Schistosoma mansoni worms that closely resembles a human tumor-associated antigen

A Mr 23,000 Ag of the human trematode parasite, Schistosoma mansoni, has been identified by immunoscreening an adult worm cDNA library with antibody affinity purified on the Mr 23,000 to 25,000 integral membrane protein fraction of the parasite. This Ag is immunogenic in infected humans as well as in rabbits exposed to S. mansoni. The protein sequence of the Ag as deduced from cloned DNA sequences is 218 amino acids long and contains four putative transmembrane regions. Of particular significance, the Ag is strikingly similar, with respect to both amino acid sequence (36% identity) and putative domain structure to ME491, a human stage-specific melanoma-associated Ag.     

Identification of Schistosoma mansoni candidate antigens for diagnosis of schistosomiasis

The development of a more sensitive diagnostic test for schistosomiasis is needed to overcome the limitations of the use of stool examination in low endemic areas. Using parasite antigens in enzyme linked immunosorbent assay is a promising strategy, however a more rational selection of parasite antigens is necessary. In this study we performed in silico analysis of the Schistosoma mansoni genome, using SchistoDB database and bioinformatic tools for screening immunogenic antigens. Based on evidence of expression in all parasite life stage within the definitive host, extracellular or plasmatic membrane localization, low similarity to human and other helminthic proteins and presence of predicted B cell epitopes, six candidates were selected: a glycosylphosphatidylinositol-anchored 200 kDa protein, two putative cytochrome oxidase subunits, two expressed proteins and one hypothetical protein. The recognition in unidimensional and bidimensional Western blot of protein with similar molecular weight and isoelectric point to the selected antigens by sera from S. mansoni infected mice indicate a good correlation between these two approaches in selecting immunogenic proteins.     

A blood fluke serine protease inhibitor regulates an endogenous larval elastase

The larvae of Schistosoma mansoni invade their mammalian host by utilizing a serine protease, cercarial elastase (SmCE), to degrade macromolecular proteins in host skin. The catalytic activity of serine and cysteine proteases can be regulated after activation by serpins. SmSrpQ, one of two S. mansoni serpins found in larval secretions, is only expressed during larval development and in the early stages of mammalian infection. In vitro, (35)S-SmSrpQ was able to form an SDS-stable complex with a component of the larval lysate, but no complex was detected when (35)S-SmSrpQ was incubated with several mammalian host proteases. Formation of a complex was sensitive to the protease active site inhibitors PMSF, Z-AAPF-CMK, and Z-AAPL-CMK. Western blot analysis of parasite lysates from different life stages detected a complex of comparable size to SmCE bound to SmSrpQ using anti-SmSrpQ or anti-SmCE antibodies. SmSrpQ and SmCE are located in adjacent but discrete compartments in the secretion glands of the parasite. Fluorescence immunohistochemical analysis of simulated infection showed co-localization of SmCE and SmSrpQ in host tissue suggesting a post release regulation of parasite protease activity during skin transversal. The results of this study suggest that cercarial elastase degradation of skin tissue is carefully regulated by SmSrpQ.     

Biochemical characterization of 2-Cys peroxiredoxins from Schistosoma mansoni

Peroxiredoxins are a large family of peroxidases that have important antioxidant and cell signaling functions. Genes encoding two novel 2-cysteine peroxiredoxin proteins were identified in the expressed sequence tag data base of the helminth parasite Schistosoma mansoni, a causative agent of schistosomiasis. The recombinant proteins showed peroxidase activity in vitro with a variety of hydroperoxides and used both the thioredoxin and the glutathione systems as electron donors. Steady-state kinetic analysis indicated that the new peroxiredoxins had saturable kinetics, whereas a previously identified schistosome peroxiredoxin was found to function with more typical unsaturable (ping-pong) kinetics. The catalytic efficiencies S. mansoni peroxiredoxins were similar to those for other peroxiredoxins studied (10(4)-10(5) m(-1) s(-1)). Mutagenesis of S. mansoni peroxiredoxins indicated that glutathione dependence and kinetic differences were conferred by the C-terminal alpha-helix forming 22 amino acids. This is the first report of 2-cysteine peroxiredoxins efficiently utilizing reducing equivalents from both the thioredoxin and glutathione systems. Studies to determine the resistance to oxidative inactivation, important in regulating cell signaling pathways, showed that S. mansoni possess both bacterial-like resistant and mammalian-like sensitive peroxiredoxins. The susceptibility to oxidative inactivation was conferred by the C-terminal tail containing a tyrosine-phenylalanine motif. S. mansoni is the first organism shown to possess both robust and sensitive peroxiredoxins. The ability of schistosome peroxiredoxins to use alternative electron donors, and their variable resistance to overoxidation may reflect their presence in different cellular sites and emphasizes the significant differences in overall redox balance mechanisms between the parasite and its mammalian host.     

Clustering of Schistosoma mansoni mRNA sequences and analysis of the most transcribed genes: implications in metabolism and biology of different developmental stages

The study of the Schistosoma mansoni genome, one of the etiologic agents of human schistosomiasis, is essential for a better understanding of the biology and development of this parasite. In order to get an overview of all S. mansoni catalogued gene sequences, we performed a clustering analysis of the parasite mRNA sequences available in public databases. This was made using softwares PHRAP and CAP3. The consensus sequences, generated after the alignment of cluster constituent sequences, allowed the identification by database homology searches of the most expressed genes in the worm. We analyzed these genes and looked for a correlation between their high expression and parasite metabolism and biology. We observed that the majority of these genes is related to the maintenance of basic cell functions, encoding genes whose products are related to the cytoskeleton, intracellular transport and energy metabolism. Evidences are presented here that genes for aerobic energy metabolism are expressed in all the developmental stages analyzed. Some of the most expressed genes could not be identified by homology searches and may have some specific functions in the parasite.     

Opisthorchis viverrini: identification of a glycine-tyrosine rich eggshell protein and its potential as a diagnostic tool for human opisthorchiasis

A cDNA encoding a novel eggshell protein (OvESP) with high-glycine (49.2%) and -tyrosine (27.8%) content was cloned from the human liver fluke Opisthorchis viverrini. In the adult parasite, the RNA products of the OvESP gene are limited to the vitelline follicles. They have a size of 800 nucleotides and are already present in 2-week-old juveniles. Immune sera of hamsters, experimentally infected, and humans, naturally infected with O. viverrini, detect bacterially expressed recombinant OvESP (rOvESP). A rabbit anti-rOvESP antiserum only reacts with the shells of intrauterine eggs in tissue sections of the parasite. Comparison of rOvESP with the parasite's excretion/secretion products as diagnostic tools for human opisthorchiasis shows a higher sensitivity (0.82-0.48) and specificity (0.97-0.91) of the recombinant protein in the ELISA technique. But the observed weak cross-reactivity of immune sera from mice infected with Schistosoma mansoni, Schistosoma mekongi, and Fasciola gigantica in Western blots of rOvESP indicates that the diagnostic quality of this protein might be compromised if infections by other trematodes are present.     

Characterization of an omega-class glutathione S-transferase from Schistosoma mansoni with glutaredoxin-like dehydroascorbate reductase and thiol transferase activities.

Glutathione S-transferases (EC 2.5.1.18) (GSTs), are a family of multifunctional enzymes present in all living organisms whose main function is the detoxification of electrophilic compounds. GSTs are considered the most prominent detoxifying class II enzymes in helminths. We describe here the characterization of novel dehydroascorbate reductase and thiol transferase activities that reside in the human parasite Schistosoma mansoni GSTx. Protein sequence analysis of this parasite product showed lower identity to known GSTs. However, phylogenic analysis placed SmGSTx among the recently described omega class GSTs (GSTO1-1). We report here that SmGSTO protein is a 28-kDa polypeptide, detected in all life stages of the parasite, being highly expressed in adult worms. Like other omega class GSTs, SmGSTO showed very low activity toward classical GSTs substrates as 1-chloro-2,4-dinitrobenzene, and no binding affinity to glutathione-agarose matrix but showed some biochemical characteristics related with thioredoxins/glutaredoxins. Interestingly, SmGSTO was able to bind S-hexyl glutathione matrix and displayed significant glutathione-dependent dehydroascorbate reductase and thiol transferase enzymatic activities.     

日本血吸虫中国株次黄嘌呤鸟嘌呤磷酸核糖转移酶的克隆、表达及其免疫保护性研究

根据基因库中日本血吸虫次黄嘌呤鸟嘌呤磷酸核糖转移酶 (HGPRT) EST (BU803192) 以及日本血吸虫成虫cDNA文库载体λgt11多克隆位点邻近核苷酸序列设计引物,以日本血吸虫成虫cDNA文库为模板,采用锚式PCR对SjHGPRT基因不完整的3′端和5′端进行扩增、测序,用电子软件拼接,获得SjHGPRT全长cDNA (1 270 bp),经序列分析,推断该片段含有编码SjHGPRT基因的完整阅读框,其编码基因与曼氏血吸虫次黄嘌呤鸟嘌呤磷酸核糖转移酶 (SmHGPRT) 全长编码基因碱基一致性为82%,其理论推导的氨基酸组成与曼氏血吸虫次黄嘌呤鸟嘌呤磷酸核糖转移酶的一致性约为83%. 将其编码基因克隆到表达载体pQE30上,在大肠杆菌M15中获得准确、高效表达,表达产物分子质量约为28 ku. 用日本血吸虫成虫抗原免疫血清对表达产物进行蛋白质印迹检测,在预测位置上出现明显的识别条带. 重组蛋白动物免疫保护性结果显示：在虫荷、每克肝卵、每克粪卵和雌子宫内卵数方面,疫苗组与对照组比较差异均具有显著性 (P < 0.05,P < 0.01). 结果表明,日本血吸虫次黄嘌呤鸟嘌呤磷酸核糖转移酶 (SjHGPRT) 全长cDNA成功克隆并在大肠菌中得到表达,表达产物具有良好的抗原性和动物免疫保护效果,是一种潜在的具有部分免疫保护性的抗血吸虫病疫苗候选分子.     

Schistosoma mansoni: the dicer gene and its expression

RNA interference (RNAi) is a gene silencing mechanism that plays an important role in regulating gene expression in many eukaryotes and has become a valuable molecular tool for analyzing gene function. Multi-domain nucleases called Dicer proteins play pivotal roles in RNAi. In this paper, we characterize the structure and expression of the Dicer gene from the platyhelminth parasite Schistosoma mansoni. The gene (SmDicer) is over 54kb long and comprises 30 exons that potentially encode a 2641 amino acid protein. This is the largest Dicer protein yet described. SmDicer contains all domains that are characteristic of metazoan dicers including an amino terminal helicase domain, DUF283, a PAZ domain, two RNAse III domains and an RNA binding domain. An examination of the available S. mansoni genome sequence suggests that the Dicer gene described here is the only Dicer gene in the parasite genome. SmDicer is expressed throughout schistosome development suggesting that RNAi technologies might be employed in deciphering gene function in all life stages of this parasite.     

Schistosoma japonicum: analysis of eggshell protein genes, their expression, and comparison with similar genes from other schistosomes.

As the egg of Schistosoma japonicum plays a central role in transmission and in pathogenesis, we sought to understand the molecular biology of egg formation. In this study we characterized an eggshell protein gene of S. japonicum and compared it with similar genes from S. mansoni and S. haematobium. To initiate studies on the eggshell protein genes of S. japonicum, a cloned genomic fragment containing an entire copy of a S. haematobium eggshell protein gene was used to identify three EcoRI hybridizing fragments of 2.6, 2.0, and 1.3 kbp in S. japonicum genomic DNA and to isolate three independent genomic clones from a S. japonicum genomic library. Two genomic clones, SJ 4-1 and SJ 3-1, contain at least two copies of the gene. The DNA sequence of a 2.0-kbp EcoRI fragment of clone SJ 3-1 showed two open reading frames (ORF), one of which showed a strong homology to the chorion proteins of insects. This ORF had 207 amino acids with a calculated molecular size of 18.5 kDa. The predicted peptide was glycine (50%) and tyrosine (10%) rich like other described schistosome eggshell proteins. Primer extension and the dideoxynucleotide sequence of the mRNA defined the cap site of the RNA and positioned the putative TATA and CAAAT elements and other cis-acting elements. Northern analysis demonstrated that eggshell protein mRNA was only detected in mature female parasites. The appearance of the female-specific mRNA was dependent on pairing with the male parasite and increased with egg production (as determined by hybridization intensity). A comparison of the DNA and deduced protein sequences of eggshell protein genes from S. japonicum with those of similar genes from S. mansoni and S. haematobium indicated that the genes are highly conserved, with S. mansoni and S. haematobium genes being more similar to each other than either is to S. japonicum.     

Novel translationally controlled tumor protein homologue in the buccal gland secretion of Lampetra japonica

We have cloned a homologue of the translationally controlled tumor protein (TCTP) from the buccal gland of Lampetra japonica according to information from a cDNA library and primary analysis of expressed sequence tags. Sequence analysis of L. japonica TCTP showed that it had two signature regions of high sequence homology termed TCTP-1 and TCTP-2, respectively. TCTP is highly conserved in evolution. It showed more than 40% identification similarities with parasite TCTPs that had effect on immune responses of host. Phylogeny of 31 TCTP sequences showed that lamprey was closer to jawed vertebrates than to Amphioxus and was a sister group of gnathostomes. TCTP gene from L. japonica was expressed in a pET23b vector and purified by using His Bind affinity chromatography. Polyclonal antibody to recombinant protein was generated in New Zealand Rabbit. Immunoblot analysis to localize the recombinant protein in buccal gland secretion proves that recombinant TCTP is a secretion protein, which may be secreted through a non-classical secretion pathway. A characterization study shows that recombinant TCTP has histamine-releasing function in vitro. It mediated histamine release from rat basophilic leukemia (RBL-2H3) cells. TCTP links both the innate and the adaptive immune responses by modulating the secretion of cytokines from mast cells, basophils, eosinophils, and T and B lymphocytes. These may indicate a potential role of TCTP in the inflammatory process and immune regulation between L. japonica and host.