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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

Multiple sequence alignment of deduced amino acids of the cathepsin F from Opisthorchis viverrini Ov-CF-1 (AAV69023).The alignment includes other members of the Clan CA, family C1 peptidase family, including CsCF-6 from Clonorchis sinensis (ABK918111) and orthologues from Paragonimus westermani (AAY81944), Teladorsagia circumcincta (ABA01328) and Homo sapiens (NP_003784). (The T. circumcincta orthologue was included as a representative of a nematode helminth sequence, while human cathepsin F sequence was included to highlight the large prosegment with a cystatin domain, underlined, that exists in some cathepsins F.) Identical residues in more than 50% of sequences are presented in black boxes. Conserved substitutions are identified by grey boxes. The conserved active site cysteine, asparagine and histidine residues are highlighted (stars). The protease-susceptible region at the junction of the prosegment and mature domain is indicated by the thick black line. This region contains the cleavage sites for release of the prosegment from the mature domain and includes the FhCL1 cleavage site for Ov-CF-1, VT↓MDNSNFD, and the cleavage site in human cathepsin F, DL↓APPEWD, determined by X-ray crystallography [50]. The thin (green) arrow indicates the position of signal peptide cleavage site. The arrow heads indicate six cysteine residues likely involved in disulfide bridges. The box indicates a conserved, putative N-linked glycosylation site, NGS in the mature enzyme domain. The position of the ERFNAQ propeptide motif, conserved in cathepsin F, is indicated.
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pntd-0000398-g001: Multiple sequence alignment of deduced amino acids of the cathepsin F from Opisthorchis viverrini Ov-CF-1 (AAV69023).The alignment includes other members of the Clan CA, family C1 peptidase family, including CsCF-6 from Clonorchis sinensis (ABK918111) and orthologues from Paragonimus westermani (AAY81944), Teladorsagia circumcincta (ABA01328) and Homo sapiens (NP_003784). (The T. circumcincta orthologue was included as a representative of a nematode helminth sequence, while human cathepsin F sequence was included to highlight the large prosegment with a cystatin domain, underlined, that exists in some cathepsins F.) Identical residues in more than 50% of sequences are presented in black boxes. Conserved substitutions are identified by grey boxes. The conserved active site cysteine, asparagine and histidine residues are highlighted (stars). The protease-susceptible region at the junction of the prosegment and mature domain is indicated by the thick black line. This region contains the cleavage sites for release of the prosegment from the mature domain and includes the FhCL1 cleavage site for Ov-CF-1, VT↓MDNSNFD, and the cleavage site in human cathepsin F, DL↓APPEWD, determined by X-ray crystallography [50]. The thin (green) arrow indicates the position of signal peptide cleavage site. The arrow heads indicate six cysteine residues likely involved in disulfide bridges. The box indicates a conserved, putative N-linked glycosylation site, NGS in the mature enzyme domain. The position of the ERFNAQ propeptide motif, conserved in cathepsin F, is indicated.

Mentions: To search for transcripts encoding Clan CA, Family C1 cysteine proteases of O. viverrini, degenerate primers were designed to target conserved domains of papain-like cysteine proteases. The degenerate forward primer, 5′-TGYGGNTCNTGYTGGGCDTTYTCN targeted the conserved CGSCWAFV residues and the reverse primer, 5′-CCARCTRTTYTTBACDATCCARTA targeted the conserved YWIVKNSW residues (Figure 1), where B = C/G/T; D = A/G/T; N = A/C/G/T; R = A/G; Y = C/T. These primers were employed to amplify target sequences from cDNA of adult O. viverrini. In addition, specific primers Ov-cf-spf1 (5′-TGTGGTTCGTGTTGGGCATTTTCT) and Ov-cf-spr1 (5′-CCAACTGTTTTTGACCGTCCAGTA) targeting the same regions were designed based on the nucleotide sequence of Clonorchis sinensis cathepsin F (DQ909018). PCR was performed as follows; 95°C for 5 min followed by 35 cycles of 94°C for 30 sec, 55°C for 30 sec, 72°C for 30 sec and finally, 72°C for 7 min. Fragments of cDNA sequence encoding an O. viverrini cysteine protease were obtained by using rapid PCR amplification of cDNA ends (RACE); 5′- and 3′-RACE were performed using specific primers Ov-cf-spf1 and Ov-cf-spr1 (above) paired with linker specific primers (Invitrogen). Subsequently, a full length transcript termed Ov-cf-1 was obtained by PCR using primers designed from nucleotide sequences obtained from 5′- and 3′-RACE products.


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)

Multiple sequence alignment of deduced amino acids of the cathepsin F from Opisthorchis viverrini Ov-CF-1 (AAV69023).The alignment includes other members of the Clan CA, family C1 peptidase family, including CsCF-6 from Clonorchis sinensis (ABK918111) and orthologues from Paragonimus westermani (AAY81944), Teladorsagia circumcincta (ABA01328) and Homo sapiens (NP_003784). (The T. circumcincta orthologue was included as a representative of a nematode helminth sequence, while human cathepsin F sequence was included to highlight the large prosegment with a cystatin domain, underlined, that exists in some cathepsins F.) Identical residues in more than 50% of sequences are presented in black boxes. Conserved substitutions are identified by grey boxes. The conserved active site cysteine, asparagine and histidine residues are highlighted (stars). The protease-susceptible region at the junction of the prosegment and mature domain is indicated by the thick black line. This region contains the cleavage sites for release of the prosegment from the mature domain and includes the FhCL1 cleavage site for Ov-CF-1, VT↓MDNSNFD, and the cleavage site in human cathepsin F, DL↓APPEWD, determined by X-ray crystallography [50]. The thin (green) arrow indicates the position of signal peptide cleavage site. The arrow heads indicate six cysteine residues likely involved in disulfide bridges. The box indicates a conserved, putative N-linked glycosylation site, NGS in the mature enzyme domain. The position of the ERFNAQ propeptide motif, conserved in cathepsin F, is indicated.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0000398-g001: Multiple sequence alignment of deduced amino acids of the cathepsin F from Opisthorchis viverrini Ov-CF-1 (AAV69023).The alignment includes other members of the Clan CA, family C1 peptidase family, including CsCF-6 from Clonorchis sinensis (ABK918111) and orthologues from Paragonimus westermani (AAY81944), Teladorsagia circumcincta (ABA01328) and Homo sapiens (NP_003784). (The T. circumcincta orthologue was included as a representative of a nematode helminth sequence, while human cathepsin F sequence was included to highlight the large prosegment with a cystatin domain, underlined, that exists in some cathepsins F.) Identical residues in more than 50% of sequences are presented in black boxes. Conserved substitutions are identified by grey boxes. The conserved active site cysteine, asparagine and histidine residues are highlighted (stars). The protease-susceptible region at the junction of the prosegment and mature domain is indicated by the thick black line. This region contains the cleavage sites for release of the prosegment from the mature domain and includes the FhCL1 cleavage site for Ov-CF-1, VT↓MDNSNFD, and the cleavage site in human cathepsin F, DL↓APPEWD, determined by X-ray crystallography [50]. The thin (green) arrow indicates the position of signal peptide cleavage site. The arrow heads indicate six cysteine residues likely involved in disulfide bridges. The box indicates a conserved, putative N-linked glycosylation site, NGS in the mature enzyme domain. The position of the ERFNAQ propeptide motif, conserved in cathepsin F, is indicated.
Mentions: To search for transcripts encoding Clan CA, Family C1 cysteine proteases of O. viverrini, degenerate primers were designed to target conserved domains of papain-like cysteine proteases. The degenerate forward primer, 5′-TGYGGNTCNTGYTGGGCDTTYTCN targeted the conserved CGSCWAFV residues and the reverse primer, 5′-CCARCTRTTYTTBACDATCCARTA targeted the conserved YWIVKNSW residues (Figure 1), where B = C/G/T; D = A/G/T; N = A/C/G/T; R = A/G; Y = C/T. These primers were employed to amplify target sequences from cDNA of adult O. viverrini. In addition, specific primers Ov-cf-spf1 (5′-TGTGGTTCGTGTTGGGCATTTTCT) and Ov-cf-spr1 (5′-CCAACTGTTTTTGACCGTCCAGTA) targeting the same regions were designed based on the nucleotide sequence of Clonorchis sinensis cathepsin F (DQ909018). PCR was performed as follows; 95°C for 5 min followed by 35 cycles of 94°C for 30 sec, 55°C for 30 sec, 72°C for 30 sec and finally, 72°C for 7 min. Fragments of cDNA sequence encoding an O. viverrini cysteine protease were obtained by using rapid PCR amplification of cDNA ends (RACE); 5′- and 3′-RACE were performed using specific primers Ov-cf-spf1 and Ov-cf-spr1 (above) paired with linker specific primers (Invitrogen). Subsequently, a full length transcript termed Ov-cf-1 was obtained by PCR using primers designed from nucleotide sequences obtained from 5′- and 3′-RACE products.

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