Protein phosphorylation networks in spargana of Spirometra erinaceieuropaei revealed by phosphoproteomic analysis.

BACKGROUND: Sparganosis caused by Spirometra erinaceieuropaei spargana is a zoonotic parasitic infection that has been reported in many countries, including China, Japan, Thailand and Korea, as well as European countries and the USA. The biological and clinical significance of the parasite have previously been reported. Although the genomic and transcriptomic analysis of S. erinaceieuropaei provided insightful views about the development and pathogenesis of this species, little knowledge has been acquired in terms of post-translational regulation that is essential for parasite growth, development and reproduction. Here, we performed site-specific phosphoproteomic profiling, with an aim to obtain primary information about the global phosphorylation status of spargana. RESULTS: A total of 3228 phosphopeptides and 3461 phosphorylation sites were identified in 1758 spargana proteins. The annotated phosphoproteins were involved in a variety of biological pathways, including cellular (28%), metabolic (20%) and single-organism (17%) processes. The functional enrichment of phosphopeptides by Gene Ontology analysis indicated that most spargana phosphoproteins were related to the cytoskeleton cellular compartment, signaling molecular function, and a variety of biological processes, including a molecular function regulator, guanyl-nucleotide exchange factor activity, protein kinase activities, and calcium ion binding. The highly enriched pathways of phosphorylation proteins include the phosphatidylinositol signaling system, phagosome, endocytosis, inositol phosphate metabolism, terpenoid backbone biosynthesis, and peroxisome. Domain analysis identified an EF-hand domain and pleckstrin homology domain among the key domains. CONCLUSIONS: To our knowledge, this study performed the first global phosphoproteomic analysis of S. erinaceieuropaei. The dataset reported herein provides a valuable resource for future studies on the signaling pathways of this important zoonotic parasite.

Molecular cloning and characterization of a Spirometra erinacei casein kinase I.

The Spirometra erinacei casein kinase I (SeCKI) gene was cloned and expressed in Escherichia coli, and its characteristics were investigated in this study. The recombinant SeCP protein (rSeCKI) was purified. The vaccination of mice with rSeCKI induced the Th1/Th2-mixed type of immune response with Th2 predominant (high levels of IgG1). Western blotting analysis showed that rSeCP was recognized by the sera of plerocercoid-infected mice, and anti-rSeCP serum recognized the native SeCP protein of plerocercoid crude antigens. Transcription and expression of SeCP was observed at the plerocercoid and adult stages of S. erinacei. Immunolocalization identified SeCKI in the tegument and parenchymal tissues of plerocercoids and in the teguments of adults. SeCKI appeared to be essential indispensable for the S. erinacei development and survival in host, but its biological functions need to be further investigated.

Mitochondrial Genome Sequences of Spirometra erinaceieuropaei and S. decipiens (Cestoidea: Diphyllobothriidae).

The present study was performed to compare the mitochondrial genomes between 2 Spirometra tapeworms, Spirometra erinaceieuropaei and Spirometra decipiens (Cestoidea: Diphyllobothriidae), which larval stages are important etiological agents of sparganosis in humans. For each species, the full mitochondrial genome was amplified in 8 overlapping fragments using total genomic DNA purified from a single worm as the template. The mitochondrial genomes were 13,643 bp (S. erinaceieuropaei) and 13,641 bp (S. decipiens) in length and contained 36 genes; 12 protein-coding genes, 2 ribosomal RNA (rRNA, small and large subunits), and 22 transfer RNAs (tRNAs). The 12 protein-coding genes constituted 10,083 bp (S. erinaceieuropaei) and 10,086 bp (S. decipiens) of their respective mitochondrial genomes. The tRNA genes, ranging in length from 56 to 70 bp, were identified based on putative secondary structures such as the typical cloverleaf shape. A total of 23 intergenic sequences, varying from 1 to 204 bp in size, were interspersed in S. erinaceieuropaei (total, 504 bp) and S. decipiens (total, 496 bp) mtDNA. The 12 protein-coding genes of S. erinaceieuropaei and S. decipiens differed by 12.4%, whereas the overall difference in mtDNA sequence between S. erinaceieuropaei and S. decipiens was 12.9%. Thus, from the standpoint of the mitochondrial genome, S. decipiens represents a valid species that can be distinguished from S. erinaceieuropaei.

Mitochondrial Genome Sequences of Spirometra erinaceieuropaei and S. decipiens (Cestoidea: Diphyllobothriidae)

The present study was performed to compare the mitochondrial genomes between 2 Spirometra tapeworms, Spirometra erinaceieuropaei and Spirometra decipiens (Cestoidea: Diphyllobothriidae), which larval stages are important etiological agents of sparganosis in humans. For each species, the full mitochondrial genome was amplified in 8 overlapping fragments using total genomic DNA purified from a single worm as the template. The mitochondrial genomes were 13,643 bp (S. erinaceieuropaei) and 13,641 bp (S. decipiens) in length and contained 36 genes; 12 protein-coding genes, 2 ribosomal RNA (rRNA, small and large subunits), and 22 transfer RNAs (tRNAs). The 12 protein-coding genes constituted 10,083 bp (S. erinaceieuropaei) and 10,086 bp (S. decipiens) of their respective mitochondrial genomes. The tRNA genes, ranging in length from 56 to 70 bp, were identified based on putative secondary structures such as the typical cloverleaf shape. A total of 23 intergenic sequences, varying from 1 to 204 bp in size, were interspersed in S. erinaceieuropaei (total, 504 bp) and S. decipiens (total, 496 bp) mtDNA. The 12 protein-coding genes of S. erinaceieuropaei and S. decipiens differed by 12.4%, whereas the overall difference in mtDNA sequence between S. erinaceieuropaei and S. decipiens was 12.9%. Thus, from the standpoint of the mitochondrial genome, S. decipiens represents a valid species that can be distinguished from S. erinaceieuropaei.

Bioinformatic analysis for structure and function of TCTP from Spirometra mansoni.

OBJECTIVE: To predict structure and function of translationally controlled tumor protein (TCTP) from Spirometra mansoni by bioinformatics technology, and to provide a theoretical basis for further study. METHODS: Open reading frame (ORF) of EST sequence from Spirometra mansoni was obtained by ORF finder and was translated into amino acid residue by DNAclub. The structure domain was analyzed by Blast. By the method of online analysis tools: Protparam, InterProScan, protscale, SignalP-3.0, PSORT II, BepiPred, TMHMM, VectorNTI Suite 9 packages and Phyre2, the structure and function of the protein were predicted and analyzed. RESULTS: The results showed that the EST sequence was Sm TCTP with 173 amino acid residues, theoretical molecular weight was 19 872.0 Da. The protein has the closest evolutionary status with Clonorchis sinensis, Schistosoma mansoni, and Schistosoma japonicum. Then it had no signal peptide site and transmembrane domain. Secondary structure of TCTP contained two α -helices and eight ß -strands. CONCLUSIONS: Sm TCTP was a variety of biological functions of protein that may be used as a vaccine candidate molecule and drug target.

Identification, structure and function of a novel tetraspaninhomologue from Spirometra erinaceieuropaei.

OBJECTIVE: To identify a full length c DNA sequence of a novel tetraspanin (TSP) homologue from Spirometra erinaceieuropaei and to predict the structure and function of its encoding protein using bioinformatics methods. METHODS: Using the NCBI, EMBI, Expasy and other online sites, the open reading frame (ORF), conserved domain, physical and chemical parameters, signal peptide, transmembrane domain, epitope, topological structures of the protein sequences were predicted. And Vector NTI software was used for multiple sequence alignment and phylogenetic tree construction. RESULTS: The target sequence was 1132 bp length with a 681 bpbiggest ORF encoding 226 amino acids protein with typical TSP conserved domain. It was confirmed as full length c DNA of TSP16 from Spirometra erinaceieuropaei and named as SeTSP16 (GenBank accession number: JF728872). The predicted molecular weight and isoelectric point of the deduced protein were 24 750.5 Da and 7.88 Da, respectively. Compared with TSP16s from Schistosoma japonicum and Schistosoma mansoni, it showed similarity of 59% and 59%, respectively. SeTSP16 contained four transmembrane domains (TM1-4), intracellular N and C-termini, one short small extracellular loop and one large extracellular loop. Four major epitopes that were significant different from the corresponding epitope regions of TSP16 from Schistosoma mansoni and Schistosoma japonicum were predicted. CONCLUSIONS: The full length c DNA sequences of SeTSP16 are identified. It encodes a transmembrane protein which might be an ideal diagnosis antigen and target molecule for antiparasitic drugs.

[Soluble proteins of the plerocercoid of Spirometra mansoni].

OBJECTIVE: To analyze soluble proteins of the plerocercoid of Spirometra mansoni. METHODS: The total protein of the plerocercoid of S. mansoni was separated by SDS-PAGE and two-dimensional electrophoresis (2-DE). Western blotting was performed to find out distinct antigens by sera of plerocercoid-infected mice. RESULTS: A total of 33 protein bands were separated by SDS-PAGE (Mr 13,800-145,400). Four of them were high-abundance proteins with Mr 26,500, Mr 37,600, Mr 88,200, and Mr 130,200, respectively. At least two protein bands of Mr 31,600 and 37,600 reacted with the infected mice sera. 367 protein spots were detected on 2-DE gel, among which. about 67% proteins were found within Mr 18,840-46,800 and isoelectric point (pI) 4-7. Western blotting showed 30 antigen spots specifically reacting with sera from mice infected by S. mansoni. CONCLUSION: Two protein bands and thirty protein spots are specific acidic proteins of S. mansoni plerocercoid.

Production of polyclonal antibodies against the tegument of sparganum (plerocercoid of Spirometra mansoni) and its immunolocalization.

In a previous study, the author developed a method for separation of the tegument of spargana (plerocercoids of Spirometra mansoni) from the parenchyme using urea. The present study, as a next step, was performed to evaluate which molecules are present in the outer tegument. Two major proteins, 180 and 200 kDa, are present in the tegument and we could make polyclonal antibodies against these molecules. Their immunolocalization was processed and the outermost layer of the spargana showed strong positive staining. Conclusively, we could confirm that the 180 and 200 kDa molecules might be tightly bound membrane proteins in the tegument of spargana.

Differential protein expression in Spirometra erinacei according to its development in its final host.

This study was undertaken to identify genes involved in the growth and development of Spirometra erinacei larvae, an intestinal tapeworm of cats and dogs, within the final host. The differential protein expression at three different stages of S. erinacei, the plerocercoid larvae, 8-day-old juveniles, and adults, was compared using two-dimensional electrophoresis. Specifically or highly expressed proteins in juvenile worms were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MS)/MS. The proteome map of larvae showed fewer protein spots than juveniles or adults, whereas juveniles or adults revealed a similar protein expression profile. Eight juvenile-specific and five upregulated proteins of juveniles were identified and matched to proteins of known biological functions. These were grouped into several categories of functionally related proteins: DNA/RNA metabolism, cell trafficking, cytoskeleton, protein processing and degradation, energy metabolism, and oxidative stress. Our results give an overview of the growth and development mechanisms of cestodes within the final host and extend our understanding of parasite biology in the host-parasite relationship.

Separation of the syncytial layer of spargana using urea.

The tegument of tapeworms is known to be composed of an outer syncytial cytoplasm layer which includes microtriches and cytoplasmic organelles (= syncytial layer), and a parenchymatous cytoplasm layer that contains subtegumental cell nuclei (= subtegumental layer) and organelles. In the present study, separation of the syncytial layer of the sparganum, the plerocercoid stage of Spirometra mansoni, was tried using urea as the chemical reagent. Histological sections were prepared to visualize the status of separation after staining with hematoxylin and eosin. The results showed that the syncytial layer of the sparganum tegument which includes microtriches and cytoplasmic organelles were successfully separated from the parenchyma using 3 M urea.

Separation of the Syncytial Layer of Spargana using Urea

The tegument of tapeworms is known to be composed of an outer syncytial cytoplasm layer which includes microtriches and cytoplasmic organelles (= syncytial layer), and a parenchymatous cytoplasm layer that contains subtegumental cell nuclei (= subtegumental layer) and organelles. In the present study, separation of the syncytial layer of the sparganum, the plerocercoid stage of Spirometra mansoni, was tried using urea as the chemical reagent. Histological sections were prepared to visualize the status of separation after staining with hematoxylin and eosin. The results showed that the syncytial layer of the sparganum tegument which includes microtriches and cytoplasmic organelles were successfully separated from the parenchyma using 3 M urea.

A glycoprotein from Spirometra erinaceieuropaei plerocercoids suppresses osteoclastogenesis and proinflammatory cytokine gene expression.

Various parasites modify the immune-reactions of the host. We have previously shown that crude excretory/secretory (ES) products from plerocercoids of Spirometra erinaceieuropaei, the plerocercoids of which cause sparganosis in humans, suppress the expression of tumor necrosis factor (TNF)-alpha and IL-1beta in lipopolysaccharide (LPS)-stimulated macrophages. As osteoclasts are cells of the monocyte/macrophage lineage, we hypothesised that ES products might suppress receptor activator of nuclear factor kappaB ligand-induced osteoclastogenesis. Crude ES products from plerocercoids suppressed osteoclastogenesis, judged by tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cell counting, and the mature osteoclast-specific gene expression (calcitonin receptor and TRAP). Second, we purified the inhibitory factor for osteoclastogenesis from the crude ES products. The factor was a trypsin-sensitive glycoprotein and had a relative molecular mass of 90 kDa. The glycoprotein, plerocercoid-immunosuppressive factor, from crude ES products could suppress the gene expression of TNF-alpha, IL-1beta and NO synthesis in LPS-stimulated RAW264.7 macrophages.

Purification and localization of a 10 kDa calcareous corpuscle binding protein of Spirometra mansoni plerocercoid.

In cestode parasites, calcareous corpuscles are thought to be associated with a number of intracellular physiologies by regulating the trafficking of mineral components. We previously separated these particular components by Ficoll, and their binding proteins of 10 kDa and 35 kDa in the Spirometra mansoni plerocercoid (sparganum). In the present study, we purified a 10 kDa protein employing octyl-Sepharose CL-4B affinity chromatography. Anti-serum raised against the purified protein showed specific reactions to the calcareous corpuscles of the worm section by immunohistochemistry, and recognized the 10 kDa protein by immunoblotting.

Amino acid sequence of a trypsin inhibitor from a Spirometra (Spirometra erinaceieuropaei).

A trypsin inhibitor that is highly homologous with bovine pancreatic trypsin inhibitor (BPTI) was co-purified along with RNase from Spirometra (Spirometra erinaceieuropaei). The amino acid sequence of this inhibitor (SETI) and the nucleotide sequence of the cDNA encoding this protein were determined by protein chemistry and gene technology. SETI contains 68 amino acid residues and has a molecular mass of 7,798 Da. SETI has 31 amino acid residues that are identical with BPTI's sequence, including 6 half-cystine and 5 aromatic amino acid residues. The active site Lys residue in BPTI is replaced by an Arg residue in SETI. SETI is an effective inhibitor of trypsin and moderately inhibits a-chymotrypsin, but less inhibits elastase or subtilisin. SETI was expressed by E. coli containing a PelB vector carrying the SETI encoding cDNA; an expression yield of 0.68 mg/l was obtained. The phylogenetic relationship of SETI and the other BPTI-like trypsin inhibitors was analyzed using most likelihood inference methods.

Synthetic studies on glycosphingolipids from protostomia phyla: synthesis of a glycosphingolipid analogue from the parasite Spirometra erinacei.

Novel neutral glycosphingolipids isolated from the plerocercoids of a tapeworm, Spirometra erinacei, may be expected to be involved in host-parasite interactions. We have synthesized this glycosphingolipid analogue containing 2-branched fatty alkyl residue in place of ceramide. Glycosylation of nonreducing-end trisaccharide derivative 15 with the reducing-end disaccharide derivative 17 in the presence of trimethylsilyl triflate (TMSOTf) gave the desired oligosaccharide derivative in good yield. The fully per-O-acylated 2-(trimethylsilyl)ethyl glycoside 19 was converted to glycosylimidate 20, which was condensed with 2-(tetradecyl)hexadecanol and subsequently deacylated to give the target glycosphingolipid analogue 22.

Effect of temperature on fatty acid composition in each lipid fraction of Spirometra erinaceieuropaei plerocercoids.

The effects of temperature and host fatty acids on the fatty acid contents of Spirometra erinaceieuropaei plerocercoids were investigated to clarify their role in sparganosis. After 24 hr incubation at 18 C in host snake serum, omega6 series fatty acids, especially arachidonic acid in the phospholipid fraction of the plerocercoids, increased compared with those of plerocercoids incubated at 37 C. The changes in the ratio of polyunsaturated to saturated fatty acids in the phospholipid fraction of plerocercoids incubated in physiological saline for 6 hr at 10 C were almost the same as the changes at 37 C. The ratio of polyunsaturated to saturated fatty acids of the triglyceride fraction showed almost opposite change versus the phospholipid fraction. The percentage of arachidonic acid in the phospholipid fraction of plerocercoids increased during the first 3 hr of incubation and then decreased, regardless of temperature. At 37 C, the percentage of arachidonic acid in the free fatty acid fraction fell for the first 3 hr of incubation and was significantly elevated at the end of the 6-hr incubation. At 10 C, however, arachidonic acid in the free fatty acid fraction decreased for the first hour of incubation, increased at 3 hr of incubation, then decreased again. These results suggest that fatty acids of the plerocercoids are frequently exchanged between fractions. Plerocercoids can mobilize arachidonic acid to the free fatty acid fraction more quickly at lower temperature than at higher temperature. They may utilize mobilized arachidonic acid early in the infection stage to produce prostaglandins. Alternatively, they can incorporate arachidonic acid into the phospholipid fraction again when arachidonic acid is readily available in the environment.

A monoclonal antibody against a glycolipid SEGLx from Spirometra erinaceieuropaei plerocercoid.

A mouse monoclonal antibody AK97 (IgM) was established against a new type of glycosphingolipid, SEGLx, isolated from plerocercoids of tapeworm, Spirometra erinaceieuropaei. The chemical structure of SEGLx (Gal beta1-4(Fuc alpha1-3)(Glc beta1-3Gal beta1-ceramide) had been previously characterized. The specificity of AK97 was determined by thin-layer chromatography-immunostaining and enzyme-linked immunosorbent assay. AK97 was found to be directed to SEGLx and GalSEGLx (Gal beta1-4(Fuc alpha1-3)Glc beta1-3(Gal beta1-6)Gal beta1-ceramide) and also showed cross-reactivity with the stage specific embryonic antigen-1 (SSEA-1), the epitope being defined to be the non-reducing terminal trisaccharide sequence. On immunohistochemical examination, AK97 predominantly stained the tegument, the external surfaces of worms which have a brush border-like organization. Based on the immunohistochemical findings for the staining liability as to organic solvents and the results of Western blot analysis of the plerocercoid glycoproteins, it was proved that the antigens in the tapeworm were glycolipids. Considering that the tapeworm is in direct contact with its host's tissue through the tegument, the membrane surface of which is exposed to the external environment, it is suspected that SEGLx and GalSEGLx on the tegument play functionally important roles in the host parasite interaction.

A novel fucosyltetrahexosylceramide in plerocercoids of the parasite Spirometra erinacei.

This glycosphingolipid was tentatively designated as GalSEGLx, in which the carbohydrate structure is characterized by an additional galactose molecule attached to the reducing-end galactose of SEGLx [Gal beta-4 (Fuc alpha-3) Glc beta-3 Gal beta Cer], which was previously determined by us [Kawakami, Y., Nakamura, K., Kojima, H., Suzuki, M., Inagaki, F., Suzuki, A., Sonoki, S., Uchida, A., Murata, Y. & Tamai, Y. (1993) J. Biochem. 114, 677-683], through a beta 1-6 linkage. The ceramide contained sphinganine and 4D-hydroxysphinganine in an about equimolar ratio, and a non-hydroxy fatty acid with carbon atoms ranging from 16 to 28, 26:0, 28:0 and 28:1 being major components. Based on the finding that a novel carbohydrate structure. Gal beta-4 Glc beta-3 Gal, was commonly found in glycosphingolipids from the parasite, S. erinacei, we here propose the terms, spirometo series, for this core structure series, and spirometosides, for glycosphingolipids having this carbohydrate structure.

The growth factor from plerocercoids of Spirometra mansonoides is both a growth hormone agonist and a cysteine proteinase.

Plerocercoids of the tapeworm Spirometra mansonoides produce a substance that stimulates growth of experimental hosts. We report purification of plerocercoid growth factor (PGF) to homogeneity by a process involving isolation and solubilization of plerocercoid membranes, isoelectric point selection by chromatofocusing chromatography or preparative isoelectric focusing, and anion-exchange chromatography. A radioreceptor assay (RRA) for human growth hormone (hGH) was used to detect PGF and purity of the 27.5-kDa protein was judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteolytic activity was detected in the 27.5-kDa protein by gelatin substrate PAGE. Characterization of PGF as a neutral cysteine proteinase was based on substrate and inhibitor specificities and dependence on pH and thiol-containing reagents. The association of hGH agonist and proteinase activities was shown by comparing RRA and hydrolytic activities in the presence and absence of the cysteine proteinase inhibitor E-64.

Molecular cloning and expression of the gene encoding a cysteine proteinase of Spirometra erinacei.

A cDNA library constructed from plerocercoid of Spirometra erinacei (SEP) was immunoscreened using rabbit anti-plerocercoid proteinase polyclonal antibody. A 1.0-kb cDNA clone encoding a cysteine proteinase composed of 336 amino acids was isolated. The amino acid sequence predicted from the cDNA showed significant homology with human and mouse cathepsin L. N-terminal amino acid sequence of the native cysteine proteinase extracted from SEP was the same as that of mature proteinase predicted from the cloned gene. The gene encoding the proteinase was characterized by Southern and Northern blot analysis using the cDNA as a probe. The proteinase with a molecular mass of 34 kDa was demonstrated in in vitro translation products using anti-proteinase polyclonal antibody. A fusion protein derived from the cDNA synthesized by Escherichia coli (TB1) using the expression vector, pMAL-c2 was identified as an immunodominant antigen by epitope-selection method and had no cross-reactivity with other parasite-infected sera. A genomic DNA library derived from SEP was screened by the colony hybridization technique using the cDNA probe. A gene with 4.5 kb encoding the proteinase was obtained, which comprised three exons and two introns.