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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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Related in: MedlinePlus

Trans-processing of Ov-CF-1 by Fasciola hepatica cathepsin L1 (FhCL1).(A) (Top) Purified recombinant Ov-CF-1 was incubated at pH 4.5 for 180 min. Aliquots of the reaction mixtures were removed at time 0, 2, 5, 10, 20, 30, 60 and 180 min and analyzed on 4–12% Bis-Tris NuPage gels (Invitrogen). Recombinant Ov-CF-1 is not capable of auto-activation at pH 4.5. The two bands shown are likely due to differential glycosylation as the recombinant was produced in yeast; (bottom) Purified recombinant Ov-CF-1 (50 µg) was incubated with fully activated mature FhCL1 (5 µg) for up to 180 min in 0.1 M sodium acetate, pH 4.5 (reaction volume 150 µl). Aliquots (15 µl) were removed from the mixtures at time 0, 2, 5, 10, 20, 30, 60 and 180 min and analyzed by 4–12% Bis-Tris NuPage gels. By 180 min marked trans-processing of Ov-CF-1 by FhCL1 had occurred. (B) Profiles of the Ov-CF-1 (time 0 min) and at 180 min of trans-processing with FhCL1 time showing the peptide bands (1–4) that were analyzed by N-terminal sequencing. The position of exogenously added FhCL1 and the released Ov-CF-1 prosegment are also shown. (C) N-terminal sequences obtained for each of the Ov-CF-1 peptides before and after trans-processing by FhCL1. (D) The cleavage sites identified by N-terminal sequences were also mapped onto the primary amino acid sequence of the Ov-CF-1 prosegment. The EF found at the N-terminal was introduced by the EcoR I cloning site used in the pPicZα expression vector.
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pntd-0000398-g007: Trans-processing of Ov-CF-1 by Fasciola hepatica cathepsin L1 (FhCL1).(A) (Top) Purified recombinant Ov-CF-1 was incubated at pH 4.5 for 180 min. Aliquots of the reaction mixtures were removed at time 0, 2, 5, 10, 20, 30, 60 and 180 min and analyzed on 4–12% Bis-Tris NuPage gels (Invitrogen). Recombinant Ov-CF-1 is not capable of auto-activation at pH 4.5. The two bands shown are likely due to differential glycosylation as the recombinant was produced in yeast; (bottom) Purified recombinant Ov-CF-1 (50 µg) was incubated with fully activated mature FhCL1 (5 µg) for up to 180 min in 0.1 M sodium acetate, pH 4.5 (reaction volume 150 µl). Aliquots (15 µl) were removed from the mixtures at time 0, 2, 5, 10, 20, 30, 60 and 180 min and analyzed by 4–12% Bis-Tris NuPage gels. By 180 min marked trans-processing of Ov-CF-1 by FhCL1 had occurred. (B) Profiles of the Ov-CF-1 (time 0 min) and at 180 min of trans-processing with FhCL1 time showing the peptide bands (1–4) that were analyzed by N-terminal sequencing. The position of exogenously added FhCL1 and the released Ov-CF-1 prosegment are also shown. (C) N-terminal sequences obtained for each of the Ov-CF-1 peptides before and after trans-processing by FhCL1. (D) The cleavage sites identified by N-terminal sequences were also mapped onto the primary amino acid sequence of the Ov-CF-1 prosegment. The EF found at the N-terminal was introduced by the EcoR I cloning site used in the pPicZα expression vector.

Mentions: The recombinant Ov-CF-1 purified from yeast culture medium resolved as two major protein bands migrating at 41 kDa and 47 kDa on 4–12% Bis-Tris NuPage gels (Figure 7A). Given the theoretical molecular mass of the recombinant Ov-CF-1 zymogen (including the c-myc and His6 tags) of ∼37.5 kDa, and given that both the 41 kDa and 47 kDa peptides share identical N-termini (EFRTT; Figure 7C), it is likely that the yeast-expressed enzyme exhibits differential addition of N-linked glycans. This is consistent with the smearing observed around both protein bands (Ov-CF-1 contains a single predicted N-linked glycosylation site, NGS, at residue 109; Figure 1). The yeast-expressed Ov-CF-1 zymogen displayed modest activity against the protein substrates hemoglobin and gelatin at acidic pH (4.5–6.0) (not shown). Assays undertaken with the fluorogenic substrate Z-Phe-Arg-NHMec revealed that the enzyme was active over a broad pH range, 4.5–8.0, with optimal activity at pH 5.5 (not shown). However, the apparent activity of the zymogen was very low, which is not surprising since the gel analysis did not reveal evidence of a fully processed and activated enzyme (Figure 7A).


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)

Trans-processing of Ov-CF-1 by Fasciola hepatica cathepsin L1 (FhCL1).(A) (Top) Purified recombinant Ov-CF-1 was incubated at pH 4.5 for 180 min. Aliquots of the reaction mixtures were removed at time 0, 2, 5, 10, 20, 30, 60 and 180 min and analyzed on 4–12% Bis-Tris NuPage gels (Invitrogen). Recombinant Ov-CF-1 is not capable of auto-activation at pH 4.5. The two bands shown are likely due to differential glycosylation as the recombinant was produced in yeast; (bottom) Purified recombinant Ov-CF-1 (50 µg) was incubated with fully activated mature FhCL1 (5 µg) for up to 180 min in 0.1 M sodium acetate, pH 4.5 (reaction volume 150 µl). Aliquots (15 µl) were removed from the mixtures at time 0, 2, 5, 10, 20, 30, 60 and 180 min and analyzed by 4–12% Bis-Tris NuPage gels. By 180 min marked trans-processing of Ov-CF-1 by FhCL1 had occurred. (B) Profiles of the Ov-CF-1 (time 0 min) and at 180 min of trans-processing with FhCL1 time showing the peptide bands (1–4) that were analyzed by N-terminal sequencing. The position of exogenously added FhCL1 and the released Ov-CF-1 prosegment are also shown. (C) N-terminal sequences obtained for each of the Ov-CF-1 peptides before and after trans-processing by FhCL1. (D) The cleavage sites identified by N-terminal sequences were also mapped onto the primary amino acid sequence of the Ov-CF-1 prosegment. The EF found at the N-terminal was introduced by the EcoR I cloning site used in the pPicZα expression vector.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0000398-g007: Trans-processing of Ov-CF-1 by Fasciola hepatica cathepsin L1 (FhCL1).(A) (Top) Purified recombinant Ov-CF-1 was incubated at pH 4.5 for 180 min. Aliquots of the reaction mixtures were removed at time 0, 2, 5, 10, 20, 30, 60 and 180 min and analyzed on 4–12% Bis-Tris NuPage gels (Invitrogen). Recombinant Ov-CF-1 is not capable of auto-activation at pH 4.5. The two bands shown are likely due to differential glycosylation as the recombinant was produced in yeast; (bottom) Purified recombinant Ov-CF-1 (50 µg) was incubated with fully activated mature FhCL1 (5 µg) for up to 180 min in 0.1 M sodium acetate, pH 4.5 (reaction volume 150 µl). Aliquots (15 µl) were removed from the mixtures at time 0, 2, 5, 10, 20, 30, 60 and 180 min and analyzed by 4–12% Bis-Tris NuPage gels. By 180 min marked trans-processing of Ov-CF-1 by FhCL1 had occurred. (B) Profiles of the Ov-CF-1 (time 0 min) and at 180 min of trans-processing with FhCL1 time showing the peptide bands (1–4) that were analyzed by N-terminal sequencing. The position of exogenously added FhCL1 and the released Ov-CF-1 prosegment are also shown. (C) N-terminal sequences obtained for each of the Ov-CF-1 peptides before and after trans-processing by FhCL1. (D) The cleavage sites identified by N-terminal sequences were also mapped onto the primary amino acid sequence of the Ov-CF-1 prosegment. The EF found at the N-terminal was introduced by the EcoR I cloning site used in the pPicZα expression vector.
Mentions: The recombinant Ov-CF-1 purified from yeast culture medium resolved as two major protein bands migrating at 41 kDa and 47 kDa on 4–12% Bis-Tris NuPage gels (Figure 7A). Given the theoretical molecular mass of the recombinant Ov-CF-1 zymogen (including the c-myc and His6 tags) of ∼37.5 kDa, and given that both the 41 kDa and 47 kDa peptides share identical N-termini (EFRTT; Figure 7C), it is likely that the yeast-expressed enzyme exhibits differential addition of N-linked glycans. This is consistent with the smearing observed around both protein bands (Ov-CF-1 contains a single predicted N-linked glycosylation site, NGS, at residue 109; Figure 1). The yeast-expressed Ov-CF-1 zymogen displayed modest activity against the protein substrates hemoglobin and gelatin at acidic pH (4.5–6.0) (not shown). Assays undertaken with the fluorogenic substrate Z-Phe-Arg-NHMec revealed that the enzyme was active over a broad pH range, 4.5–8.0, with optimal activity at pH 5.5 (not shown). However, the apparent activity of the zymogen was very low, which is not surprising since the gel analysis did not reveal evidence of a fully processed and activated enzyme (Figure 7A).

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