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

Pinlaor P, Kaewpitoon N, Laha T, Sripa B, Kaewkes S, Morales ME, Mann VH, Parriott SK, Suttiprapa S, Robinson MW, To J, Dalton JP, Loukas A, Brindley PJ - PLoS Negl Trop Dis (2009)

Bottom Line: 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.A cathepsin F cysteine protease of the human liver fluke O. viverrini has been characterized at the gene and protein level.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Khon Kaen University, Khon Kaen, Thailand.

ABSTRACT

Background: 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.

Methodology/principal findings: 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 surrounding the flukes several weeks after infection of hamsters with O. viverrini and, in addition, had accumulated in the secondary (small) bile ducts where flukes cannot reach due to their large size.

Conclusions/significance: A cathepsin F cysteine protease of the human liver fluke O. viverrini has been characterized at the gene and protein level. Secretion of this protease may contribute to the hepatobiliary abnormalities, including cholangiocarcinogenesis, observed in individuals infected with this parasite.

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Neighbor joining tree.The tree revealed the phylogenetic relationship between the cathepsin F cysteine protease of Opisthorchis viverrini and homologous enzymes from ∼75 other informative species. Species names and GenBank accessions are provided on the branches. Bootstrap values of 1,000 replicates are provided at the nodes of the branches (bootstrap values less than 500 were omitted).
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pntd-0000398-g003: Neighbor joining tree.The tree revealed the phylogenetic relationship between the cathepsin F cysteine protease of Opisthorchis viverrini and homologous enzymes from ∼75 other informative species. Species names and GenBank accessions are provided on the branches. Bootstrap values of 1,000 replicates are provided at the nodes of the branches (bootstrap values less than 500 were omitted).

Mentions: Phylogenetic analysis using >75 related sequences (primarily cathepsin F and cathepsin L like enzymes) confirmed the relationship of Ov-CF-1 with other cathepsin F proteases (Figure 3). Ov-CF-1 clustered with more than ten C. sinensis cysteine protease mRNA sequences. It also clustered with five or more cathepsin F-like sequences from the lung fluke, P. westermani, although the Paragonimus orthologues branched separately from the Opisthorchis and Clonorchis cathepsins F. Further, it clustered with the cathepsins F of the schistosomes S. japonicum and S. mansoni, although the schistosome orthologues branched separately from both the Opisthorchis/Clonorchis and the Paragonimus cathepsins F. Together the branches that included these fluke cathepsins F branched separately from mammalian cathepsins F, including human cathepsin F. The branch that included the mammalian cathepsins F clustered with another branch of cathepsin F-like enzymes from a diverse group of eukaryotes including potato, mosquito, C. elegans and trypanosomes. An unusual cathepsin F-like enzyme from the nematode Brugia malayi (AAT07059) [28] formed a basal branch of the cathepsin F-like sequences. Cathepsin L like sequences from Fasciola species, schistosomes, tapeworms, Haemonchus contortus, Aedes aegypti, human, and other species formed a completely distinct clade to the cathepsins F. The entire tree was rooted with the cathepsin B of S. japonicum.


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

Pinlaor P, Kaewpitoon N, Laha T, Sripa B, Kaewkes S, Morales ME, Mann VH, Parriott SK, Suttiprapa S, Robinson MW, To J, Dalton JP, Loukas A, Brindley PJ - PLoS Negl Trop Dis (2009)

Neighbor joining tree.The tree revealed the phylogenetic relationship between the cathepsin F cysteine protease of Opisthorchis viverrini and homologous enzymes from ∼75 other informative species. Species names and GenBank accessions are provided on the branches. Bootstrap values of 1,000 replicates are provided at the nodes of the branches (bootstrap values less than 500 were omitted).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654340&req=5

pntd-0000398-g003: Neighbor joining tree.The tree revealed the phylogenetic relationship between the cathepsin F cysteine protease of Opisthorchis viverrini and homologous enzymes from ∼75 other informative species. Species names and GenBank accessions are provided on the branches. Bootstrap values of 1,000 replicates are provided at the nodes of the branches (bootstrap values less than 500 were omitted).
Mentions: Phylogenetic analysis using >75 related sequences (primarily cathepsin F and cathepsin L like enzymes) confirmed the relationship of Ov-CF-1 with other cathepsin F proteases (Figure 3). Ov-CF-1 clustered with more than ten C. sinensis cysteine protease mRNA sequences. It also clustered with five or more cathepsin F-like sequences from the lung fluke, P. westermani, although the Paragonimus orthologues branched separately from the Opisthorchis and Clonorchis cathepsins F. Further, it clustered with the cathepsins F of the schistosomes S. japonicum and S. mansoni, although the schistosome orthologues branched separately from both the Opisthorchis/Clonorchis and the Paragonimus cathepsins F. Together the branches that included these fluke cathepsins F branched separately from mammalian cathepsins F, including human cathepsin F. The branch that included the mammalian cathepsins F clustered with another branch of cathepsin F-like enzymes from a diverse group of eukaryotes including potato, mosquito, C. elegans and trypanosomes. An unusual cathepsin F-like enzyme from the nematode Brugia malayi (AAT07059) [28] formed a basal branch of the cathepsin F-like sequences. Cathepsin L like sequences from Fasciola species, schistosomes, tapeworms, Haemonchus contortus, Aedes aegypti, human, and other species formed a completely distinct clade to the cathepsins F. The entire tree was rooted with the cathepsin B of S. japonicum.

Bottom Line: 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.A cathepsin F cysteine protease of the human liver fluke O. viverrini has been characterized at the gene and protein level.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Khon Kaen University, Khon Kaen, Thailand.

ABSTRACT

Background: 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.

Methodology/principal findings: 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 surrounding the flukes several weeks after infection of hamsters with O. viverrini and, in addition, had accumulated in the secondary (small) bile ducts where flukes cannot reach due to their large size.

Conclusions/significance: A cathepsin F cysteine protease of the human liver fluke O. viverrini has been characterized at the gene and protein level. Secretion of this protease may contribute to the hepatobiliary abnormalities, including cholangiocarcinogenesis, observed in individuals infected with this parasite.

Show MeSH
Related in: MedlinePlus