Mapping the binding site of C1-inhibitor for polyanion cofactors.

The serpin, C1-inhibitor (also known as SERPING1), plays a vital anti-inflammatory role in the body by controlling pro-inflammatory pathways such as complement and coagulation. The inhibitor's action is enhanced in the presence of polyanionic cofactors, such as heparin and polyphosphate, by increasing the rate of association with key enzymes such as C1s of the classical pathway of complement. The cofactor binding site of the serpin has never been mapped. Here we show that residues Lys284, Lys285 and Arg287 of C1-inhibitor play key roles in binding heparin and delivering the rate enhancement seen in the presence of polyanions and thus most likely represent the key cofactor binding residues for the serpin. We also show that simultaneous binding of the anion binding site of C1s by the polyanion is required to deliver the rate enhancement. Finally, we have shown that it is unlikely that the two positively charged zones of C1-inhibitor and C1s interact in the encounter complex between molecules as ablation of the charged zones did not in itself deliver a rate enhancement as might have been expected if the zones interacted. These insights provide crucial information as to the mechanism of action of this key serpin in the presence and absence of cofactor molecules.

Determination of the crystal structure and substrate specificity of ananain.

Ananain (EC 3.4.22.31) accounts for less than 10% of the total enzyme in the crude pineapple stem extract known as bromelain, yet yields the majority of the proteolytic activity of bromelain. Despite a high degree of sequence identity between ananain and stem bromelain, the most abundant bromelain cysteine protease, ananain displays distinct chemical properties, substrate preference and inhibitory profile compared to stem bromelain. A tripeptidyl substrate library (REPLi) was used to further characterize the substrate specificity of ananain and identified an optimal substrate for cleavage by ananain. The optimal tripeptide, PLQ, yielded a high kcat/Km value of 1.7 x 106 M-1s-1, with cleavage confirmed to occur after the Gln residue. Crystal structures of unbound ananain and an inhibitory complex of ananain and E-64, solved at 1.73 and 1.98 Å, respectively, revealed a geometrically flat and open S1 subsite for ananain. This subsite accommodates diverse P1 substrate residues, while a narrow and deep hydrophobic pocket-like S2 subsite would accommodate a non-polar P2 residue, such as the preferred Leu residue observed in the specificity studies. A further illustration of the atomic interactions between E-64 and ananain explains the high inhibitory efficiency of E-64 toward ananain. These data reveal the first in depth structural and functional data for ananain and provide a basis for further study of the natural properties of the enzyme.

Keratinocyte-specific ablation of protease-activated receptor 2 prevents gingival inflammation and bone loss in a mouse model of periodontal disease.

Chronic periodontitis is characterised by gingival inflammation and alveolar bone loss. A major aetiological agent is Porphyromonas gingivalis, which secretes proteases that activate protease-activated receptor 2 (PAR2 ). PAR2 expressed on oral keratinocytes is activated by proteases released by P. gingivalis, inducing secretion of interleukin 6 (IL-6), and global knockout of PAR2 prevents bone loss and inflammation in a periodontal disease model in mice. To test the hypothesis that PAR2 expressed on gingival keratinocytes is required for periodontal disease pathology, keratinocyte-specific PAR2 -null mice were generated using K14-Cre targeted deletion of the PAR2 gene (F2rl1). These mice were subjected to a model of periodontitis involving placement of a ligature around a tooth, combined with P. gingivalis infection ("Lig + Inf"). The intervention caused a significant 44% decrease in alveolar bone volume (assessed by microcomputed tomography) in wildtype (K14-Cre:F2rl1wt/wt ), but not littermate keratinocyte-specific PAR2 -null (K14-Cre:F2rl1fl/fl ) mice. Keratinocyte-specific ablation of PAR2 prevented the significant Lig + Inf-induced increase (2.8-fold) in the number of osteoclasts in alveolar bone and the significant up-regulation (2.4-4-fold) of the inflammatory markers IL-6, IL-1ß, interferon-γ, myeloperoxidase, and CD11b in gingival tissue. These data suggest that PAR2 expressed on oral epithelial cells is a critical regulator of periodontitis-induced bone loss and will help in designing novel therapies with which to treat the disease.

The Structural Basis for Complement Inhibition by Gigastasin, a Protease Inhibitor from the Giant Amazon Leech.

Complement is crucial to the immune response, but dysregulation of the system causes inflammatory disease. Complement is activated by three pathways: classical, lectin, and alternative. The classical and lectin pathways are initiated by the C1r/C1s (classical) and MASP-1/MASP-2 (lectin) proteases. Given the role of complement in disease, there is a requirement for inhibitors to control the initiating proteases. In this article, we show that a novel inhibitor, gigastasin, from the giant Amazon leech, potently inhibits C1s and MASP-2, whereas it is also a good inhibitor of MASP-1. Gigastasin is a poor inhibitor of C1r. The inhibitor blocks the active sites of C1s and MASP-2, as well as the anion-binding exosites of the enzymes via sulfotyrosine residues. Complement deposition assays revealed that gigastasin is an effective inhibitor of complement activation in vivo, especially for activation via the lectin pathway. These data suggest that the cumulative effects of inhibiting both MASP-2 and MASP-1 have a greater effect on the lectin pathway than the more potent inhibition of only C1s of the classical pathway.

Polyphosphate is a novel cofactor for regulation of complement by a serpin, C1 inhibitor.

The complement system plays a key role in innate immunity, inflammation, and coagulation. The system is delicately balanced by negative regulatory mechanisms that modulate the host response to pathogen invasion and injury. The serpin, C1-esterase inhibitor (C1-INH), is the only known plasma inhibitor of C1s, the initiating serine protease of the classical pathway of complement. Like other serpin-protease partners, C1-INH interaction with C1s is accelerated by polyanions such as heparin. Polyphosphate (polyP) is a naturally occurring polyanion with effects on coagulation and complement. We recently found that polyP binds to C1-INH, prompting us to consider whether polyP acts as a cofactor for C1-INH interactions with its target proteases. We show that polyP dampens C1s-mediated activation of the classical pathway in a polymer length- and concentration-dependent manner by accelerating C1-INH neutralization of C1s cleavage of C4 and C2. PolyP significantly increases the rate of interaction between C1s and C1-INH, to an extent comparable to heparin, with an exosite on the serine protease domain of the enzyme playing a major role in this interaction. In a serum-based cell culture system, polyP significantly suppressed C4d deposition on endothelial cells, generated via the classical and lectin pathways. Moreover, polyP and C1-INH colocalize in activated platelets, suggesting that their interactions are physiologically relevant. In summary, like heparin, polyP is a naturally occurring cofactor for the C1s:C1-INH interaction and thus an important regulator of complement activation. The findings may provide novel insights into mechanisms underlying inflammatory diseases and the development of new therapies.

Structural basis for substrate specificity of Helicobacter pylori M17 aminopeptidase.

The M17 aminopeptidase from the carcinogenic gastric bacterium Helicobacter pylori (HpM17AP) is an important housekeeping enzyme involved in catabolism of endogenous and exogenous peptides. It is implicated in H. pylori defence against the human innate immune response and in the mechanism of metronidazole resistance. Bestatin inhibits HpM17AP and suppresses H. pylori growth. To address the structural basis of catalysis and inhibition of this enzyme, we have established its specificity towards the N-terminal amino acid of peptide substrates and determined the crystal structures of HpM17AP and its complex with bestatin. The position of the D-phenylalanine moiety of the inhibitor with respect to the active-site metal ions, bicarbonate ion and with respect to other M17 aminopeptidases suggested that this residue binds to the S1 subsite of HpM17AP. In contrast to most characterized M17 aminopeptidases, HpM17AP displays preference for L-Arg over L-Leu residues in peptide substrates. Compared to very similar homologues from other bacteria, a distinguishing feature of HpM17AP is a hydrophilic pocket at the end of the S1 subsite that is likely to accommodate the charged head group of the L-Arg residue of the substrate. The pocket is flanked by a sodium ion (not present in M17 aminopeptidases that show preference for L-Leu) and its coordinating water molecules. In addition, the structure suggests that variable loops at the entrance to, and in the middle of, the substrate-binding channel are important determinants of substrate specificity of M17 aminopeptidases.

The protease cathepsin L regulates Th17 cell differentiation.

Previously we reported that IL-17(+) T cells, primarily IL-17(+) γδ cells, are increased in mice lacking the protease inhibitor serpinB1 (serpinb1(-/-) mice). Here we show that serpinB1-deficient CD4 cells exhibit a cell-autonomous and selective deficiency in suppressing T helper 17 (Th17) cell differentiation. This suggested an opposing role for one or more protease in promoting Th17 differentiation. We found that several SerpinB1-inhibitable cysteine cathepsins are induced in Th17 cells, most prominently cathepsin L (catL); this was verified by peptidase assays, active site labeling and Western blots. Moreover, Th17 differentiation was suppressed by both broad cathepsin inhibitors and catL selective inhibitors. CatL is present in Th17 cells as single chain (SC)- and two-chain (TC)-forms. Inhibiting asparagine endopeptidase (AEP) blocked conversion of SC-catL to TC-catL and increased generation of serpinb1(-/-) Th17 cells, but not wild-type Th17 cells. These findings suggest that SC-catL is biologically active in promoting Th17 generation and is counter-regulated by serpinB1 and secondarily by AEP. Thus, in addition to regulation by cytokines and transcription factors, differentiation of CD4 cells to Th17 cells is actively regulated by a catL-serpinB1-AEP module. Targeting this protease regulatory module could be an approach to treating Th17 cell-driven autoimmune disorders.

The x-ray crystal structure of mannose-binding lectin-associated serine proteinase-3 reveals the structural basis for enzyme inactivity associated with the Carnevale, Mingarelli, Malpuech, and Michels (3MC) syndrome.

The mannose-binding lectin associated-protease-3 (MASP-3) is a member of the lectin pathway of the complement system, a key component of human innate and active immunity. Mutations in MASP-3 have recently been found to be associated with Carnevale, Mingarelli, Malpuech, and Michels (3MC) syndrome, a severe developmental disorder manifested by cleft palate, intellectual disability, and skeletal abnormalities. However, the molecular basis for MASP-3 function remains to be understood. Here we characterize the substrate specificity of MASP-3 by screening against a combinatorial peptide substrate library. Through this approach, we successfully identified a peptide substrate that was 20-fold more efficiently cleaved than any other identified to date. Furthermore, we demonstrated that mutant forms of the enzyme associated with 3MC syndrome were completely inactive against this substrate. To address the structural basis for this defect, we determined the 2.6-Å structure of the zymogen form of the G666E mutant of MASP-3. These data reveal that the mutation disrupts the active site and perturbs the position of the catalytic serine residue. Together, these insights into the function of MASP-3 reveal how a mutation in this enzyme causes it to be inactive and thus contribute to the 3MC syndrome.

PROSPER: an integrated feature-based tool for predicting protease substrate cleavage sites.

The ability to catalytically cleave protein substrates after synthesis is fundamental for all forms of life. Accordingly, site-specific proteolysis is one of the most important post-translational modifications. The key to understanding the physiological role of a protease is to identify its natural substrate(s). Knowledge of the substrate specificity of a protease can dramatically improve our ability to predict its target protein substrates, but this information must be utilized in an effective manner in order to efficiently identify protein substrates by in silico approaches. To address this problem, we present PROSPER, an integrated feature-based server for in silico identification of protease substrates and their cleavage sites for twenty-four different proteases. PROSPER utilizes established specificity information for these proteases (derived from the MEROPS database) with a machine learning approach to predict protease cleavage sites by using different, but complementary sequence and structure characteristics. Features used by PROSPER include local amino acid sequence profile, predicted secondary structure, solvent accessibility and predicted native disorder. Thus, for proteases with known amino acid specificity, PROSPER provides a convenient, pre-prepared tool for use in identifying protein substrates for the enzymes. Systematic prediction analysis for the twenty-four proteases thus far included in the database revealed that the features we have included in the tool strongly improve performance in terms of cleavage site prediction, as evidenced by their contribution to performance improvement in terms of identifying known cleavage sites in substrates for these enzymes. In comparison with two state-of-the-art prediction tools, PoPS and SitePrediction, PROSPER achieves greater accuracy and coverage. To our knowledge, PROSPER is the first comprehensive server capable of predicting cleavage sites of multiple proteases within a single substrate sequence using machine learning techniques. It is freely available at http://lightning.med.monash.edu.au/PROSPER/.

Novel scabies mite serpins inhibit the three pathways of the human complement system.

Scabies is a parasitic infestation of the skin by the mite Sarcoptes scabiei that causes significant morbidity worldwide, in particular within socially disadvantaged populations. In order to identify mechanisms that enable the scabies mite to evade human immune defenses, we have studied molecules associated with proteolytic systems in the mite, including two novel scabies mite serine protease inhibitors (SMSs) of the serpin superfamily. Immunohistochemical studies revealed that within mite-infected human skin SMSB4 (54 kDa) and SMSB3 (47 kDa) were both localized in the mite gut and feces. Recombinant purified SMSB3 and SMSB4 did not inhibit mite serine and cysteine proteases, but did inhibit mammalian serine proteases, such as chymotrypsin, albeit inefficiently. Detailed functional analysis revealed that both serpins interfered with all three pathways of the human complement system at different stages of their activation. SMSB4 inhibited mostly the initial and progressing steps of the cascades, while SMSB3 showed the strongest effects at the C9 level in the terminal pathway. Additive effects of both serpins were shown at the C9 level in the lectin pathway. Both SMSs were able to interfere with complement factors without protease function. A range of binding assays showed direct binding between SMSB4 and seven complement proteins (C1, properdin, MBL, C4, C3, C6 and C8), while significant binding of SMSB3 occurred exclusively to complement factors without protease function (C4, C3, C8). Direct binding was observed between SMSB4 and the complement proteases C1s and C1r. However no complex formation was observed between either mite serpin and the complement serine proteases C1r, C1s, MASP-1, MASP-2 and MASP-3. No catalytic inhibition by either serpin was observed for any of these enzymes. In summary, the SMSs were acting at several levels mediating overall inhibition of the complement system and thus we propose that they may protect scabies mites from complement-mediated gut damage.

Analysis of Fasciola cathepsin L5 by S2 subsite substitutions and determination of the P1-P4 specificity reveals an unusual preference.

Fasciola parasites (liver flukes) express numerous cathepsin L proteases that are believed to be involved in important functions related to host invasion and parasite survival. These proteases are evolutionarily divided into clades that are proposed to reflect their substrate specificity, most noticeably through the S(2) subsite. Single amino acid substitutions to residues lining this site, including amino acid residue 69 (aa69; mature cathepsin L5 numbering) can have profound influences on subsite architecture and influence enzyme specificity. Variations at aa69 among known Fasciola cathepsin L proteases include leucine, tyrosine, tryptophan, phenylalanine and glycine. Other amino acids (cysteine, serine) might have been expected at this site due to codon usage as cathepsin L isoenzymes evolved, but C69 and S69 have not been observed. The introduction of L69C and L69S substitutions into FhCatL5 resulted in low overall activity indicating their expression provides no functional advantage, thus explaining the absence of such variants in Fasciola. An FhCatL5 L69F variant showed an increase in the ability to cleave substrates with P(2) proline, indicating F69 variants expressed by the fluke would likely have this ability. An FhCatL2 Y69L variant showed a decreased acceptance of P(2) proline, further highlighting the importance of Y69 for FhCatL2 P(2) proline acceptance. Finally, the P(1)-P(4) specificity of Fasciola cathepsin L5 was determined and, unexpectedly, aspartic acid was shown to be well accepted at P(2,) which is unique amongst Fasciola cathepsins examined to date.

Effect of O-glycosylation and tyrosine sulfation of leech-derived peptides on binding and inhibitory activity against thrombin.

Synthesis of sulfated and unsulfated (glyco)peptide fragments of Hirudin P6 (a potent anticoagulant from the leech Hirudinaria manillensis) is described. The effect of O-glycosylation and tyrosine sulfation on thrombin binding and peptidolytic activity was investigated, together with the inhibition of fibrinogen cleavage.

The lysine-specific gingipain of Porphyromonas gingivalis : importance to pathogenicity and potential strategies for inhibition.

Periodontitis is a disease affecting the supporting structures of the teeth. The most severe forms of the disease result in tooth loss and have recently been strongly associated with systemic diseases, including cardiovascular and lung diseases and cancer. The disease is caused by biofilms of predominantly anaerobic bacteria. A major pathogen associated with severe, adult forms of the disease is Porphyromonas gingivalis. This organism produces potent cysteine proteases known as gingipains, which have specificity for cleavage after arginine or lysine residues. The lysine-specific gingipain, Kgp, appears to be the major virulence factor of this organism and here we describe its structure and function. We also discuss the inhibitors of the enzyme produced to date and the potential pathways to newer versions of such molecules that will be required to combat periodontitis.

Cathepsin B proteases of flukes: the key to facilitating parasite control?

Cysteine proteases are important virulence factors for parasites. This review will focus on the cathepsin B proteases of trematodes (also known as flukes) which are abundant in juvenile and immature flukes. Recent research, primarily in Fasciola, using inhibitors, RNA interference (RNAi) and vaccination studies indicates that cathepsin Bs play a key role in the biology of trematodes. As these proteases are largely expressed by infective parasite stages, their inactivation by chemotherapy or vaccination will greatly reduce the damage wrought by flukes as they invade host tissues. This validates cathepsin Bs as key strategic targets for fluke control.

Vector-based RNA interference of cathepsin B1 in Schistosoma mansoni.

In helminth parasites, proteolytic enzymes have been implicated in facilitating host invasion, moulting, feeding, and evasion of the host immune response. These key functions render them potential targets for anti-parasite chemotherapy and immunotherapy. Schistosomes feed on host blood and the digested haemoglobin is their major source of amino acids. Haemoglobin digestion is essential for parasite development, growth, and reproduction. We recently reported the use of pseudotyped Moloney murine leukaemia virus to accomplish transformation of Schistosoma mansoni. Here, we report the design of a viral vector expressing a dsRNA hairpin to silence expression of the schistosome cathepsin B1 (SmCB1) gene. We observed 80% reduction in transcript level 72 h after virus exposure and complete silencing of enzyme activity in transduced worms. This is the first report using this technology in any helminth parasite. It will facilitate the evaluation of potential drug targets and biochemical pathways for novel interventions in schistosomes.

Protease-activated receptor 2 has pivotal roles in cellular mechanisms involved in experimental periodontitis.

The tissue destruction seen in chronic periodontitis is commonly accepted to involve extensive upregulation of the host inflammatory response. Protease-activated receptor 2 (PAR-2)-null mice infected with Porphyromonas gingivalis did not display periodontal bone resorption in contrast to wild-type-infected and PAR-1-null-infected mice. Histological examination of tissues confirmed the lowered bone resorption in PAR-2-null mice and identified a substantial decrease in mast cells infiltrating the periodontal tissues of these mice. T cells from P. gingivalis-infected or immunized PAR-2-null mice proliferated less in response to antigen than those from wild-type animals. CD90 (Thy1.2) expression on CD4(+) and CD8(+) T-cell-receptor beta (TCRbeta) T cells was significantly (P < 0.001) decreased in antigen-immunized PAR-2-null mice compared to sham-immunized PAR-2-null mice; this was not observed in wild-type controls. T cells from infected or antigen-immunized PAR-2-null mice had a significantly different Th1/inflammatory cytokine profile from wild-type cells: in particular, gamma interferon, interleukins (interleukin-2, -3, and -17), granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor alpha demonstrated lower expression than wild-type controls. The absence of PAR-2 therefore appears to substantially decrease T-cell activation and the Th1/inflammatory response. Regulation of such proinflammatory mechanisms in T cells and mast cells by PAR-2 suggests a pivotal role in the pathogenesis of the disease.

Proteinase-activated receptor-2 (PAR2) and mouse osteoblasts: regulation of cell function and lack of specificity of PAR2-activating peptides.

1. Using synthetic proteinase-activated receptor-2 (PAR(2))-activating peptides (PAR(2)APs) corresponding to the tethered ligand domain of the extracellular N-terminus of PAR(2) to mimic the actions of activating proteinases and using primary cultures of calvarial osteoblasts derived from both wild-type (WT) and PAR(2)-null (KO) mice, we investigated the potential role of PAR(2) in regulating osteoblast function. 2. Primary calvarial osteoblasts from WT and KO mice were evaluated for their growth kinetics and mineralization in the absence of PAR(2) agonists and for their responses in a variety of functional assays to the PAR(2)APs Ser-Leu-Ile-Gly-Arg-Leu-amide (SLIGRL-NH(2)) and 2-furoyl-Leu-Ile-Gly-Arg-Leu-Orn-amide (2-fLIGRLO-NH(2)), as well as to trypsin. 3. In contrast with WT cells, PAR(2)-KO osteoblasts did not exhibit increased collagen Type I mRNA expression in response to SLIGRL-NH(2). When grown in serum-containing medium, KO cells increased in number more rapidly than WT cells, an effect that could be attributed to decreased apoptosis rather than increased proliferation. Surprisingly, in both WT and KO osteoblasts, the two PAR(2)APs induced mobilization of intracellular calcium stores. Similarly, the PAR(2)APs inhibited serum deprivation-induced apoptosis and parathyroid hormone-, 1,25-dihydroxyvitamin D(3)- or interleukin-11-induced mineralization in WT and KO cells. 4. We conclude that PAR(2) plays a role in osteoblast survival and collagen Type I mRNA induction and that osteoblasts can respond to the PAR(2)APs via both PAR(2)-dependent and -independent mechanisms.

The gingipains: scissors and glue of the periodontal pathogen, Porphyromonas gingivalis.

The anaerobic bacterium, Porphyromonas gingivalis, is associated with chronic periodontal disease (periodontitis or gum disease). The disease is not only the leading cause of tooth loss in the developed world, but is associated with a number of systemic diseases, such as cardiovascular disease and diabetes. The most potent virulence factors of this bacterium are the gingipains, three cysteine proteases that bind and cleave a wide range of host proteins. This article summarizes current knowledge of the structure and function of the enzymes, with a particular focus on what remains to be elucidated regarding the structure and function of the nonenzymatic adhesin domains of the high-molecular-weight forms of the proteases.

Thrombin-stimulated growth factor and cytokine expression in osteoblasts is mediated by protease-activated receptor-1 and prostanoids.

Thrombin exerts multiple effects upon osteoblasts including stimulating proliferation, and inhibiting osteoblast differentiation and apoptosis. Some of these effects are believed to be mediated by the synthesis and secretion of autocrine factors such as growth factors and cytokines. Many but not all cellular responses to thrombin are mediated by members of the protease-activated receptor (PAR) family of G protein-coupled receptors. The current study was undertaken to investigate the nature of thrombin's induction of autocrine factors by analysing the expression of twelve candidate genes in thrombin-stimulated primary mouse osteoblasts. Analysis by quantitative reverse transcription polymerase chain reaction (qRT-PCR) demonstrated that thrombin induced transforming growth factor beta, cyclooxygenase-2, tenascin C, fibroblast growth factor-1 and -2, connective tissue growth factor and interleukin-6 expression in wild type osteoblasts, but not PAR-1 null mouse osteoblasts. Induction of all the thrombin-responsive genes was blocked by the presence of the non-selective cyclooxygenase inhibitor indomethacin. Further studies were conducted on interleukin-6, which was the gene that showed the greatest increase in expression following stimulation of osteoblast-like cells with thrombin. A PAR-1-specific activating peptide, but neither a PAR-4-activating peptide nor catalytically inactive thrombin induced release of interleukin-6 by osteoblasts. Furthermore, in the presence of the selective cyclooxygenase-1 and -2 inhibitors SC-560 and NS-398 thrombin-induced interleukin-6 release was prevented. Levels of both prostaglandin E(2) and interleukin-6 in medium conditioned by thrombin-stimulated osteoblast-like cells were found to be significantly increased compared to medium conditioned by non-stimulated cells, however release of prostaglandin E(2) was found to precede release of interleukin-6. Treatment of isolated osteoblast-like cells with a number of synthetic prostanoids stimulated secretion of interleukin-6 with differing potencies. These studies suggest that activation of PAR-1 on osteoblasts by thrombin induces cyclooxygenase activity, which in turn results in the increased expression of multiple secreted factors. The induction of these secreted factors may act in an autocrine fashion to alter osteoblast function, allowing these cells to participate in the earliest stages of bone healing by both autocrine and paracrine mechanisms.

A major cathepsin B protease from the liver fluke Fasciola hepatica has atypical active site features and a potential role in the digestive tract of newly excysted juvenile parasites.

The newly excysted juvenile (NEJ) stage of the Fasciola hepatica lifecycle occurs just prior to invasion into the wall of the gut of the host, rendering it an important target for drug development. The cathepsin B enzymes from NEJ flukes have recently been demonstrated to be crucial to invasion and migration by the parasite. Here we characterize one of the cathepsin B enzymes (recombinant FhcatB1) from NEJ flukes. FhcatB1 has biochemical properties distinct from mammalian cathepsin B enzymes, with an atypical preference for Ile over Leu or Arg residues at the P(2) substrate position and an inability to act as an exopeptidase. FhcatB1 was active across a broad pH range (optimal activity at pH 5.5-7.0) and resistant to inhibition by cystatin family inhibitors from sheep and humans, suggesting that this enzyme would be able to function in extracellular environments in its mammalian hosts. It appears, however, that the FhcatB1 protease functions largely as a digestive enzyme in the gut of the parasite, due to the localization of a specific, fluorescently labeled inhibitor with an Ile at the P(2) position. Molecular modelling and dynamics were used to predict the basis for the unusual substrate specificity: a P(2) Ile residue positions the substrate optimally for interaction with catalytic residues of the enzyme, and the enzyme lacks an occluding loop His residue crucial for exopeptidase activity. The unique features of the enzyme, particularly with regard to its specificity and likely importance to a vital stage of the parasite's life cycle, make it an excellent target for therapeutic inhibitors or vaccination.