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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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Immunolocalization of cathepsin F cysteine protease in adult Opisthorchis viverrini using thin sections of paraffin embedded worms probed with rabbit antiserum.(A) Representative section spanning the gut, vitellaria, parenchyma and tegument, probed with pre-immunization serum (negative control). Sections of adults, probed with the rabbit anti-cathepsin F serum, the vicinity of the tegument and vitellaria (B), tegument and gut (C), and testis, seminal receptacle, parenchyma and eggs (D). Gut (g), vitelline glands (v), egg (e) and testis (T) all showed strong positive reactions whereas the sperm seminal receptacle (s) was negative. The tegument (t) was faintly positive but the tegumental cells were negative for the cathepsin F cysteine protease. Immunoperoxidase staining, original magnification, ×100.
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pntd-0000398-g005: Immunolocalization of cathepsin F cysteine protease in adult Opisthorchis viverrini using thin sections of paraffin embedded worms probed with rabbit antiserum.(A) Representative section spanning the gut, vitellaria, parenchyma and tegument, probed with pre-immunization serum (negative control). Sections of adults, probed with the rabbit anti-cathepsin F serum, the vicinity of the tegument and vitellaria (B), tegument and gut (C), and testis, seminal receptacle, parenchyma and eggs (D). Gut (g), vitelline glands (v), egg (e) and testis (T) all showed strong positive reactions whereas the sperm seminal receptacle (s) was negative. The tegument (t) was faintly positive but the tegumental cells were negative for the cathepsin F cysteine protease. Immunoperoxidase staining, original magnification, ×100.

Mentions: Immunohistochemical localization of Ov-CF-1 revealed strong expression in the gut, vitellaria, egg and testis (Figure 5B–5D). By contrast, control sections of flukes probed with pre-immunization serum showed no or little reactivity (Figure 5A). The intense immunolocalization to the luminal margin of the gut (Figure 5C) indicated strongly that the cathepsin F participates in proteolysis of ingested host tissues. In addition, sections through hamster bile ducts containing adult O. viverrini flukes confirmed the localization of Ov-CF-1 in organs and tissues of the fluke, including the gut and, notably, revealed the presence of Ov-CF-1 in epithelial cells lining the infected bile duct (Figure 6D). Ov-CF-1 also had accumulated in secondary bile ducts too small in internal diameter to include an adult fluke (Figure 6B) and in Kupffer cells and mononuclear cells lining sinuses of the liver (Figure 6C). Control sections from the same infected hamster probed with pre-immunization serum showed no or little reactivity (Figure 6A).


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)

Immunolocalization of cathepsin F cysteine protease in adult Opisthorchis viverrini using thin sections of paraffin embedded worms probed with rabbit antiserum.(A) Representative section spanning the gut, vitellaria, parenchyma and tegument, probed with pre-immunization serum (negative control). Sections of adults, probed with the rabbit anti-cathepsin F serum, the vicinity of the tegument and vitellaria (B), tegument and gut (C), and testis, seminal receptacle, parenchyma and eggs (D). Gut (g), vitelline glands (v), egg (e) and testis (T) all showed strong positive reactions whereas the sperm seminal receptacle (s) was negative. The tegument (t) was faintly positive but the tegumental cells were negative for the cathepsin F cysteine protease. Immunoperoxidase staining, original magnification, ×100.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0000398-g005: Immunolocalization of cathepsin F cysteine protease in adult Opisthorchis viverrini using thin sections of paraffin embedded worms probed with rabbit antiserum.(A) Representative section spanning the gut, vitellaria, parenchyma and tegument, probed with pre-immunization serum (negative control). Sections of adults, probed with the rabbit anti-cathepsin F serum, the vicinity of the tegument and vitellaria (B), tegument and gut (C), and testis, seminal receptacle, parenchyma and eggs (D). Gut (g), vitelline glands (v), egg (e) and testis (T) all showed strong positive reactions whereas the sperm seminal receptacle (s) was negative. The tegument (t) was faintly positive but the tegumental cells were negative for the cathepsin F cysteine protease. Immunoperoxidase staining, original magnification, ×100.
Mentions: Immunohistochemical localization of Ov-CF-1 revealed strong expression in the gut, vitellaria, egg and testis (Figure 5B–5D). By contrast, control sections of flukes probed with pre-immunization serum showed no or little reactivity (Figure 5A). The intense immunolocalization to the luminal margin of the gut (Figure 5C) indicated strongly that the cathepsin F participates in proteolysis of ingested host tissues. In addition, sections through hamster bile ducts containing adult O. viverrini flukes confirmed the localization of Ov-CF-1 in organs and tissues of the fluke, including the gut and, notably, revealed the presence of Ov-CF-1 in epithelial cells lining the infected bile duct (Figure 6D). Ov-CF-1 also had accumulated in secondary bile ducts too small in internal diameter to include an adult fluke (Figure 6B) and in Kupffer cells and mononuclear cells lining sinuses of the liver (Figure 6C). Control sections from the same infected hamster probed with pre-immunization serum showed no or little reactivity (Figure 6A).

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