Cathepsin F cysteine protease of the human liver fluke, Opisthorchis viverrini

The liver fluke Opisthorchis viverrini is classified as a class I carcinogen due to the association between cholangiocarcinoma and chronic O. viverrini infection. During its feeding activity within the bile duct, the parasite secretes several cathepsin F cysteine proteases that may induce or contrib...

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Published in:PLoS neglected tropical diseases 2009, Vol.3 (3), p.e398
Main Authors: Pinlaor, Porntip, Kaewpitoon, Natthawut, Laha, Thewarach, Sripa, Banchob, Kaewkes, Sasithorn, Morales, Maria E, Mann, Victoria H, Parriott, Sandi K, Suttiprapa, Sutas, Robinson, Mark W, To, Joyce, Dalton, John P, Loukas, Alex, Brindley, Paul J
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Biochemistry/Bioinformatics
Cancer
Carcinogens
Cathepsin F - genetics
Cathepsin F - metabolism
Cricetinae
Helminth Proteins - genetics
Helminth Proteins - metabolism
Humans
Immunization
Immunohistochemistry
Infections
Liver
Liver - parasitology
Molecular Sequence Data
Opisthorchiasis - parasitology
Opisthorchis - enzymology
Opisthorchis - genetics
Parasites
Parasitic diseases
Phylogenetics
Phylogeny
Scholarships & fellowships
Sequence Alignment
Tropical diseases
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Summary:The liver fluke Opisthorchis viverrini is classified as a class I carcinogen due to the association between cholangiocarcinoma and chronic O. viverrini infection. During its feeding activity within the bile duct, the parasite secretes several cathepsin F cysteine proteases that may induce or contribute to the pathologies associated with hepatobiliary abnormalities. Here, we describe the cDNA, gene organization, phylogenetic relationships, immunolocalization, and functional characterization of the cathepsin F cysteine protease gene, here termed Ov-cf-1, from O. viverrini. The full length mRNA of 1020 nucleotides (nt) encoded a 326 amino acid zymogen consisting of a predicted signal peptide (18 amino acids, aa), prosegment (95 aa), and mature protease (213 aa). BLAST analysis using the Ov-CF-1 protein as the query revealed that the protease shared identity with cathepsin F-like cysteine proteases of other trematodes, including Clonorchis sinensis (81%), Paragonimus westermani (58%), Schistosoma mansoni and S. japonicum (52%), and with vertebrate cathepsin F (51%). Transcripts encoding the protease were detected in all developmental stages that parasitize the mammalian host. The Ov-cf-1 gene, of approximately 3 kb in length, included seven exons interrupted by six introns; the exons ranged from 69 to 267 bp in length, the introns from 43 to 1,060 bp. The six intron/exon boundaries of Ov-cf-1 were conserved with intron/exon boundaries in the human cathepsin F gene, although the gene structure of human cathepsin F is more complex. Unlike Ov-CF-1, human cathepsin F zymogen includes a cystatin domain in the prosegment region. Phylogenetic analysis revealed that the fluke, human, and other cathepsin Fs branched together in a clade discrete from the cathepsin L cysteine proteases. A recombinant Ov-CF-1 zymogen that displayed low-level activity was expressed in the yeast Pichia pastoris. Although the recombinant protease did not autocatalytically process and activate to a mature enzyme, trans-processing by Fasciola hepatica cathepsin L cleaved the prosegment of Ov-CF-1, releasing a mature cathepsin F with activity against the peptide Z-Phe-Arg-NHMec >50 times that of the zymogen. Immunocytochemistry using antibodies raised against the recombinant enzyme showed that Ov-CF-1 is expressed in the gut of the mature hermaphroditic fluke and also in the reproductive structures, including vitelline glands, egg, and testis. Ov-CF-1 was detected in bile duct epithelial cells
ISSN:1935-2735
1935-2727
1935-2735
DOI:10.1371/journal.pntd.0000398