Delineating distinct heme-scavenging and -binding functions of domains in MF6p/helminth defense molecule (HDM) proteins from parasitic flatworms.

MF6p/FhHDM-1 is a small protein secreted by the parasitic flatworm (trematode) Fasciola hepatica that belongs to a broad family of heme-binding proteins (MF6p/helminth defense molecules (HDMs)). MF6p/HDMs are of interest for understanding heme homeostasis in trematodes and as potential targets for the development of new flukicides. Moreover, interest in these molecules has also increased because of their immunomodulatory properties. Here we have extended our previous findings on the mechanism of MF6p/HDM-heme interactions and mapped the protein regions required for heme binding and for other biological functions. Our data revealed that MF6p/FhHDM-1 forms high-molecular-weight complexes when associated with heme and that these complexes are reorganized by a stacking procedure to form fibril-like and granular nanostructures. Furthermore, we showed that MF6p/FhHDM-1 is a transitory heme-binding protein as protein·heme complexes can be disrupted by contact with an apoprotein (e.g. apomyoglobin) with higher affinity for heme. We also demonstrated that (i) the heme-binding region is located in the MF6p/FhHDM-1 C-terminal moiety, which also inhibits the peroxidase-like activity of heme, and (ii) MF6p/HDMs from other trematodes, such as Opisthorchis viverrini and Paragonimus westermani, also bind heme. Finally, we observed that the N-terminal, but not the C-terminal, moiety of MF6p/HDMs has a predicted structural analogy with cell-penetrating peptides and that both the entire protein and the peptide corresponding to the N-terminal moiety of MF6p/FhHDM-1 interact in vitro with cell membranes in hemin-preconditioned erythrocytes. Our findings suggest that MF6p/HDMs can transport heme in trematodes and thereby shield the parasite from the harmful effects of heme.

Paragonimus westermani: identification and characterization of the fasciclin I domain-containing protein.

Paragonimus westermani is a trematode parasite that causes inflammatory lung disease as well as systemic infections in carnivorous mammals. The interaction of the parasite with host cells and paired worms is initiated by adhesion and plays an important role in parasite proliferation and differentiation. In this study, we isolated a cDNA encoding a P. westermani fasciclin I domain-containing protein (Pwfas-I). The fasiclin-I domain is suggested to be involved in cell adhesion, migration, and differentiation. Immunohistochemical analysis of P. westermani adult worms with polyclonal anti-Pwfas-I serum revealed immunoreactivity in the egg shells and the cells lining the sub-tegumental layer of adult worm throughout the contact regions of the cyst wall and paired worms. Using cell adhesion and spreading assays, we showed that Pwfas-I supports cell adhesion and spreading. Furthermore, we determined that the alphanubeta5 integrin was a functional receptor for the Pwfas-I. Taken together, these results suggest that Pwfas-I may be functional for the modulation of cell adhesion via binding with alphanubeta5 integrin in the extracellular matrix of Paragonimus.

Molecular characterization and kinetic properties of a novel two-domain taurocyamine kinase from the lung fluke Paragonimus westermani.

Taurocyamine kinase (TK) was previously reported to be restricted to certain marine annelids; however, the present study has proven otherwise. The lung fluke Paragonimus westermani has a contiguous two-domain TK with a mass of 80216 Da consisting of 713 amino acid residues sharing higher sequence identity with molluscan arginine kinase (AK). Both domains of P. westermani TK have significant activity for the substrate taurocyamine and exhibited synergism during substrate binding. Since TK plays a key role in energy metabolism and is not present in mammals, inhibitors against P. westermani TK could be effective novel chemotherapeutic agents and could be utilized for the development of specific diagnostic tools for the detection of paragonimiasis.

Excretory-secretory products from Paragonimus westermani increase nitric oxide production in microglia in PKC-dependent and -independent manners.

Excretory-secretory products (ESP) from helminthic parasites may play pivotal roles in the immune regulation in hosts. Previously, we reported that ESP produced from Paragonimus westermani induced morphological activation of microglial cells and markedly stimulated nitric oxide (NO) production via activation of mitogen-activated protein kinases (MAPKs). In the present study, we investigated the role of protein kinase C and protein kinase A in MAPKs-dependent NO production by ESP. We found that treatment with protein kinase C inhibitor Go6976 strongly inhibited the phosphorylation of p38 and JNK, but not ERK, of MAPKs and decreased the production of NO in ESP-stimulated microglial cells. Inhibition of ERK, p38 or PKC decreased the ESP-induced activation of NF-kappaB, an important transcription factor for iNOS expression. Furthermore, ESP increased the level of p-CREB in microglial cells. However, adenylyl cyclase activator (forskolin), adenylyl cyclase inhibitor (SQ22536), cAMP analogue (db-cAMP) or protein kinase A inhibitor (H89) was not able to change iNOS expression and NO production in ESP-treated microglial cells. It implies that the cAMP-PKA-CREB pathway is not implicated in the ESP-evoked NO production in microglial cells. Thus, our results indicate that ESP stimulates microglial expression of iNOS via both PKC-dependent and -independent MAPKs phosphorylation and NF-kappaB activation.

Differential expression of Paragonimus westermani eggshell proteins during the developmental stages.

Eggs of trematode parasites are comprised of numerous vitelline cells and one fertilized ovum, and are encapsulated within a protein shell provided by the vitellocytes. In this study, we isolated two full-length cDNA clones that showed substantial levels of sequence identity with trematode-specific eggshell precursor proteins from the human lung fluke, Paragonimus westermani. These cDNAs, designated Pw-Vit20 (868-bp-long) and Pw-Vit36 (883-bp-long), shared a 76% identity with one another at the nucleotide level, and each encoded a 261-amino acid (aa) polypeptide. The deduced aa sequences contained a N-terminal hydrophobic segment, as well as a sequence motif of Gly-Gly-Gly-Tyr-Asp-Asn/Thr-Tyr-Gly-Lys/Gln, which is highly homologous with the eggshell proteins of Fasciola hepatica. With the high frequencies of tyrosine, glycine and lysine, the positions occupied by tyrosine, which has been proved to be converted into dihydroxyphenylalanine, were well preserved. Pw-Vit20 and Pw-Vit36 were found to be monoexonic genes with variably diverged variants scattered into multiple genomic loci. Their protein products were localized in the vitelline follicles and eggshells. Expression of Pw-Vit20 was restricted to the egg and adult stages, thus suggesting a critical involvement of Pw-Vit20 in the parasite's fecundity activity. Conversely, Pw-Vit36 was constitutively expressed in the metacercariae and juvenile stages in the vitelline follicles and ducts, which suggested that the prepositioning of stem or primordial vitelline cells within the juveniles prior to sexual maturation. Pw-Vit36 might acquire a unique or additional function relevant to the maturation and/or development of the vitelline cells/follicles during the evolutionary period of P. westermani. Differential biological implications of multiple eggshell precursor proteins may provide insight into the molecular mechanism of eggshell formation and the developmental process of the vitelline follicles in the parasitic trematode.

Comparison of gene representation between diploid and triploid Paragonimus westermani by expressed sequence tag analyses.

Expressed sequence tag (EST) analysis of the diploid and triploid Paragonimus westermani genes was done to have a rapid and informative outlook of the gene-expression profiles of the parasites. Totals of 506 and 505 ESTs were generated from the diploid and triploid P. westermani cDNA libraries. Based on the BLASTx search results of the diploid P. westermani ESTs, 308 (60.9%) matched significantly with formerly identified genes and 198 (39.1%) showed no significant homology in the GenBank database. A similar homology pattern was shown from the triploid EST BLASTx search results with 346 (68.5%) sharing homology with previously identified genes and 159 (31.5%) showing no significant homology. The EST data from both libraries were analyzed and grouped into 9 categories. Comparison of the 2 EST pools revealed high similarities among the categories of the significantly matched genes. Single genes matched repeatedly were also observed in the 2 EST data. Some genes were found that are not yet characterized in P. westermani; these genes were matched by both the diploid and triploid ESTs. Further study of these genes may provide us with more understanding on the parasite's biology and their specific functions in the 2 strains.

Excretory-secretory products produced by paragonimus westermani differentially regulate the nitric oxide production and viability of microglial cells.

BACKGROUND: Tissue-invading helminth parasites secrete a large amount of cysteine proteases that may play critical roles in tissue invasion and immune modulation. However, roles of excretory-secretory products (ESP) secreted by Paragonimus westermani in the activation and death of microglial cells in brain are poorly understood. OBJECTIVES: In the present study, we investigated whether ESP could regulate microglial nitric oxide (NO) production and viability. METHODS: The NO production and cell viability were assessed by respectively measuring the formation of nitrite and the release of lactate dehyrogenase. RESULTS: At a low (0.2 microg/ml) concentration, ESP significantly stimulated NO production with no apparent cell injury or death in cultured microglial cells. However, at high (> or =2 microg/ml) concentrations, ESP induced severe cell death. Inhibition of inducible NO synthase significantly reduced the NO productivity, but not cytotoxicity, of ESP. Similarly, inhibitors of the extracellular signal-regulated kinase, p38 and nuclear factor kappa B also blocked only the NO productivity of ESP. Interestingly, heat inactivation did not hamper the ability of ESP to stimulate microglial NO production. Similarly, pretreatment with thiol-crosslinking reagents dramatically reduced both proteolytic activity and cytotoxicity of ESP, but did not alter NO production in microglial cells. Interestingly, although cysteine protease competitive inhibitors and thiol-alkylating reagents markedly reduced the proteolytic activity of ESP, they did not influence the NO productivity and cytotoxicity of ESP. CONCLUSION: The present results indicate that the NO production and cytotoxicity by ESP may be differentially regulated via unknown mechanisms, not related with cysteine protease activity.