Cathepsin L1, the Major Protease Involved in Liver Fluke (Fasciola hepatica) Virulence

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205

Proteases frequently function not only as individual enzymes but also in cascades or networks. A notable evolutionary switch occurred in one such protease network that is involved in protein digestion in the intestine. In vertebrates, this is largely the work of trypsin family serine proteases, whereas in invertebrates, cysteine proteases of the papain family and aspartic proteases assume the role. Utilizing a combination of protease class-specific inhibitors and RNA interference, we deconvoluted such a network of major endopeptidases functioning in invertebrate intestinal protein digestion, using the parasitic helminth, Schistosoma mansoni as an experimental model. We show that initial degradation of host blood proteins is ordered, occasionally redundant, and substrate-specific. Although inhibition of parasite cathepsin D had a greater effect on primary cleavage of hemoglobin, inhibition of cathepsin B predominated in albumin degradation. Nevertheless, in both cases, inhibitor combinations were synergistic. An asparaginyl endopeptidase (legumain) also synergized with cathepsin B and L in protein digestion, either by zymogen activation or facilitating substrate cleavage. This protease network operates optimally in acidic pH compartments either in the gut lumen or in vacuoles of the intestinal lining cells. Defining the role of each of these major enzymes now provides a clearer understanding of the function of a complex protease network that is conserved throughout invertebrate evolution. It also provides insights into which of these proteases are logical targets for development of chemotherapy for schistosomiasis, a major global health problem.

Section: Discussionmentioning

confidence: 99%

A Multienzyme Network Functions in Intestinal Protein Digestion by a Platyhelminth Parasite

Delcroix

Sajid

Caffrey

et al. 2006

214

205

“…Enzyme Preparation and Animals-Functionally active recombinant F. hepatica cathepsin L1 (FheCL1) and S. mansoni cathepsin B1 (SmB1) were expressed in Pichia pastoris and purified by affinity chromatography on nickel-nitrilotriacetic acid-agarose as described (15,16). A conformationally intact but proteolytically inactive variant of FheCL1, termed FheCL1Gly 26 , was prepared by substituting the active site cysteine with a glycine as described (17).…”

Section: Methodsmentioning

confidence: 99%

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

Donnelly

O’Neill

Stack

et al. 2010

Self Cite

125

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...

“…Virulence is associated with the secretion of a variety of proteins, the most predominant of which are cathepsin L proteases. The cathepsin L proteases are synthesized by epithelial cells that line the parasite gut and are stored as inactive zymogens in specialized secretory vesicles prior to extrusion into the parasite gut lumen (2,3). Here the proteases mix with blood derived from the host and catabolize proteins to peptides and amino acids that are absorbed and used as nutrient by the parasite (3).…”

Section: (Cysmentioning

confidence: 99%

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

Stack

Donnelly

Lowther

et al. 2007

Self Cite

A protease secreted by the parasitic helminth Fasciola hepatica, a 37-kDa procathepsin L1 (FheproCL1), autocatalytically processes and activates to its mature enzyme (FheCL1) over a wide pH range of 7.3 to 4.0, although activation is more rapid at low pH. Maturation initiates with cleavages of a small proportion of molecules within the central region of the prosegment, possibly by intramolecular events. However, activation to fully mature enzymes is achieved by a precise intermolecular cleavage at a Leu ؊12 -Ser ؊11 2His ؊10 sequence within the nonconserved C-terminal region of the prosegment. ؊12 , was very slow but was increased 40-fold in the presence of FheCL2. These studies provide a molecular insight into the regulation of FheproCL1 autocatalysis.