The Major Secreted Cathepsin L1 Protease of the Liver Fluke, Fasciola hepatica

Stack, Colin M.; Donnelly, Sheila; Lowther, Jonathan; Xu, Weibo; Collins, Peter R.; Brinen, Linda S.; Dalton, John P.

doi:10.1074/jbc.m611501200

Cited by 31 publications

(8 citation statements)

References 56 publications

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“…However, our data indicate that FheCL1 is resistant to this proteolytic degradation, which is consistent with our earlier findings showing that the mature enzyme is highly resistant to proteolytic degradation by endopeptidases (33). We have also shown that FheCL1 can degrade cystatins/serpins such as SCCA1 and SCCA2 (34) and therefore this may protect the enzyme from inhibition by cystatins within the lysosome that are known to regulate the activity of resident endolysosomal cathepsins (35).…”

Section: Discussionsupporting

confidence: 81%

Helminth Cysteine Proteases Inhibit TRIF-dependent Activation of Macrophages via Degradation of TLR3

Donnelly

O’Neill

Stack

et al. 2010

Journal of Biological Chemistry

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121

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Helminth pathogens prepare a Th2 type immunological environment in their hosts to ensure their longevity. They achieve this by secreting molecules that not only actively drive type 2 responses but also suppress type 1 responses. Here, we show that the major cysteine proteases secreted from the helminth pathogens Fasciola hepatica (FheCL1) and Schistosoma mansoni (SmCB1) protect mice from the lethal effects of lipopolysaccharide by preventing the release of inflammatory mediators, nitric oxide, interleukin-6, tumor necrosis factor ␣, and interleukin-12, from macrophages. The proteases specifically block the MyD88-independent TRIF-dependent signaling pathway of Toll-like receptor (TLR)4 and TLR3. Microscopical and flow cytometric studies, however, show that alteration of macrophage function by cysteine protease is not mediated by cleavage of components of the TLR4 complex on the cell surface but occurs by degradation of TLR3 within the endosome. This is the first study to describe a parasite molecule that degrades this receptor and pinpoints a novel mechanism by which helminth parasites modulate the innate immune responses of their hosts to suppress the development of Th1 responses.To establish successful chronic infections, helminth parasites induce potent Th2 immune responses, which can only occur when Th1 cytokines are suppressed (1). To create this anti-inflammatory environment helminths secrete molecules that prevent dendritic cells and macrophages responding to Toll-like receptor (TLR) 2 Th1-stimulating ligands such as LPS and CpG (2-5). It has been proposed that helminth-secreted molecules themselves signal through TLRs, activating a different pattern of kinases compared with LPS, resulting in the extracellular signal-regulated kinase dependent inhibition of interleukin (IL)-12 production (reviewed in Ref. 6). The molecular nature of these Th1-suppressing components, however, remains largely unknown.Enzyme activity ascribable to the papain family of cysteine proteases (clan CA, family C1), such as cathepsin L and B, has been identified as a major component of the secretions of almost all helminth parasites of humans, livestock, and companion animals (7,8). These proteases are pivotal to the parasitic lifestyle by mediating fundamental processes such as host invasion, acquisition of nutrients, and the migration through host tissues (reviewed in Ref. 9). They can also have a systemic effect on the immune responses of their hosts by cleaving immunoglobulins and preventing antibody-mediated immune effector function, which protects the parasites from immune elimination (10, 11). We have shown that the predominant secreted product of the human and animal helminth pathogen Fasciola hepatica, a cathepsin L1 cysteine protease (FheCL1), suppressed the onset of protective Th1 immune responses in mice to infections with the respiratory microbe Bordetella pertussis, making them more susceptible to disease. In addition, injection of the cysteine protease immediately prior to immunization of mice with a B. pertussis whole...

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Section: Discussionsupporting

confidence: 81%

Helminth Cysteine Proteases Inhibit TRIF-dependent Activation of Macrophages via Degradation of TLR3

Donnelly

O’Neill

Stack

et al. 2010

Journal of Biological Chemistry

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121

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“…1 B ). To determine whether the native zymogen packaged within EVs could undergo pH-dependent autocatalytic activation similar to our purified recombinant cathepsin Ls ( 35 – 37 ), soluble extracts of the 15 K vesicle pellet were incubated at low pH (pH 4.5) for up to 3 h at 37 °C. Western blot analysis of the in vitro auto-activation process shows that the 37 kDa zymogen diminishes over time, concomitant with the appearance of a band corresponding to the processed 24 kDa mature enzyme ( Fig.…”

Section: Resultsmentioning

confidence: 99%

The Extracellular Vesicles of the Helminth Pathogen, Fasciola hepatica: Biogenesis Pathways and Cargo Molecules Involved in Parasite Pathogenesis*

Cwiklinski

Torre-Escudero

Trelis

et al. 2015

Molecular & Cellular Proteomics

195

227

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Extracellular vesicles (EVs) released by parasites have important roles in establishing and maintaining infection. Analysis of the soluble and vesicular secretions of adult Fasciola hepatica has established a definitive characterization of the total secretome of this zoonotic parasite. Fasciola secretes at least two subpopulations of EVs that differ according to size, cargo molecules and site of release from the parasite. The larger EVs are released from the specialized cells that line the parasite gastrodermus and contain the zymogen of the 37 kDa cathepsin L peptidase that performs a digestive function. The smaller exosome-like vesicle population originate from multivesicular bodies within the tegumental syncytium and carry many previously described immunomodulatory molecules that could be delivered into host cells. By integrating our proteomics data with recently available transcriptomic data sets we have detailed the pathways involved with EV biogenesis in F. hepatica and propose that the small exosome biogenesis occurs via ESCRT-dependent MVB formation in the tegumental syncytium before being shed from the apical plasma membrane. Furthermore, we found that the molecular “machinery” required for EV biogenesis is constitutively expressed across the intramammalian development stages of the parasite. By contrast, the cargo molecules packaged within the EVs are developmentally regulated, most likely to facilitate the parasites migration through host tissue and to counteract host immune attack.

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“…The initial event might be performed by the same protease or a different protease, such as asparaginyl endopeptidase or cathepsin B or L. Our data showing that recombinant Ov -CF-1 did not undergo autocatalytic activation, despite displaying low-level activity, suggested that native Ov -CF-1 requires trans -processing. Stack and co-workers described how the junction between the prosegment and mature enzyme of human and helminth cysteine proteases consists of a non-conserved, randomly-structured motif that is susceptible to cleavage at several sites, depending on the processing enzyme [24]. Based on the three-dimensional structure of human cathepsin F [50] and multiple sequence alignments with helminth orthologues (Figure 1), we predict that the protease-susceptible region of Ov -CF-1 prosegment is the peptide PTPQEDVTMD.…”

Section: Discussionmentioning

confidence: 99%

“…The reaction products were analyzed by 4–12% Bis-Tris NuPage gel (Invitrogen) electrophoresis. Trans -processing of recombinant Ov -CF-1 was undertaken by mixing 50 µg purified recombinant enzyme with 5.0 µg of recombinant O. viverrini asparaginyl endopeptidase [17] or an activated recombinant Fasciola hepatica cathepsin L (FhCL1) [24] in 0.1 M sodium acetate, pH 4.5, 1 mM DTT , 1.25 mM EDTA in a total reaction volume of 150 µl. The mixture was incubated for 180 min at 37°C, and samples (10 µl) were removed at intervals for analysis (as above).…”

Section: Methodsmentioning

confidence: 99%

Cathepsin F Cysteine Protease of the Human Liver Fluke, Opisthorchis viverrini

et al. 2009

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BackgroundThe 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 FindingsHere, 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 ∼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/SignificanceA 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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The Major Secreted Cathepsin L1 Protease of the Liver Fluke, Fasciola hepatica

Cited by 31 publications

References 56 publications

Helminth Cysteine Proteases Inhibit TRIF-dependent Activation of Macrophages via Degradation of TLR3

Helminth Cysteine Proteases Inhibit TRIF-dependent Activation of Macrophages via Degradation of TLR3

The Extracellular Vesicles of the Helminth Pathogen, Fasciola hepatica: Biogenesis Pathways and Cargo Molecules Involved in Parasite Pathogenesis*

Cathepsin F Cysteine Protease of the Human Liver Fluke, Opisthorchis viverrini

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