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.

Phylogenetic reconstruction using secondary structures and sequence motifs of ITS2 rDNA of Paragonimus westermani (Kerbert, 1878) Braun, 1899 (Digenea: Paragonimidae) and related species.

BACKGROUND: Most phylogenetic studies using current methods have focused on primary DNA sequence information. However, RNA secondary structures are particularly useful in systematics because they include characteristics that give "morphological" information, not found in the primary sequence. In several mountainous regions of Northeastern India, foci of Paragonimus (lung fluke) infection reportedly involve species that are known to prevail in neighbouring countries. The present study was undertaken to demonstrate the sequence analysis of the ribosomal DNA (ITS2) of the infective (metacercarial) stage of the lung fluke collected from the edible crab hosts that are abundant in a mountain stream of the area (Miao, Changlang District in Arunachal Pradesh) and to construct its phylogeny. Using the approach of molecular morphometrics that is based on ITS2 secondary structure homologies, phylogenetic relationships of the various isolates of Paragonimus species that are prevalent in the neighbouring Near-eastern countries have been discussed. RESULTS: Initially, ten predicted RNA secondary structures were reconstructed and the topology based only on the predicted RNA secondary structure of the ITS2 region resolved most relationships among the species studied. We obtained three similar topologies for seven species of the genus Paragonimus on the basis of traditional primary sequence analysis using MEGA and a Bayesian analysis of the combined data. The latter approach allowed us to include both primary sequence and RNA molecular morphometrics; each data partition was allowed to have a different evolution rate. Paragonimus westermani was found to group with P. siamensis of Thailand; this was best supported by both the molecular morphometrics and combined analyses. P. heterotremus, P. proliferus, P. skrjabini, P. bangkokensis and P. harinasutai formed a separate clade in the molecular phylogenies, and were reciprocally monophyletic with respect to other species. ITS2 sequence motifs allowed an accurate in-silico distinction of lung flukes. CONCLUSION: Data indicate that ITS2 motifs (

Molecular characterization of two myoglobins of Paragonimus westermani.

Myoglobins (Mbs), globin proteins, are present in high concentrations in trematodes. In Paragonimus westermani, 2 cDNAs were found to encode Mbs. The first clone, Pwmyo1, codes a total of 149 amino acids with a calculated mass of 16.6 kDa. The second, Pwmyo2, encodes a 146-amino acid protein with a calculated mass of 16.2 kDa. The predicted secondary structures showed the presence of 8 helices, which is the basic characteristic of Mbs. Sequence alignment revealed a high homology with the other trematode Mbs. The 2 clones contained the characteristic tyrosyl residues at helical positions B10 and distal E7, which are substitutions that have been previously shown to contribute to the high oxygen affinity of Mbs. Polyclonal antibodies against the recombinant Mbs were raised with no cross-reactivity observed. Immunolocalization revealed the proteins to be distributed generally throughout the parenchymal tissues, but absent from the tegument and reproductive organs. The cell mass of the eggs of the worm stained positive to Pwmyo2 but not Pwmyo1, suggesting the stage-specific expression of these Mbs.

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.