Abstract: Cathepsin B-like cysteine protease genes (cbls) constitute large multigene families in parasitic and nonparasitic nematodes. Although expressed in the intestine of some nematodes, the biological and biochemical functions of the CBL proteins remain unresolved. Di- and tetra-oligopeptides were used as fluorogenic substrates and irreversible/competitive inhibitors to establish CBL functions in the intestine of the parasitic nematode Haemonchus contortus. Cysteine protease activity was detected against diverse substrates including the cathepsin B/L substrate FR, the caspase 1 substrate YVAD, the cathepsin B substrate RR, but not the CED-3 (caspase 3) substrate DEVD. The pH at which maximum activity was detected varied according to substrate and ranged from pH 5.0 to 7.0. Individual CBLs were affinity isolated using FA and YVAD substrates. pH influenced CBL affinity isolation in a substrate-specific manner that paralleled pH effects on individual substrates. N-terminal sequencing identified two isolated CBLs as H. contortus GCP-7 (33 kDa) and AC-4 (37 kDa). N termini of each began at a position consistent with proregion cleavage and protease activation. Isolation of the GCP-7 band by each peptide was preferentially inhibited when competed with a diazomethane-conjugated inhibitor, Z-FA-CHN2, demonstrating one functional difference among CBLs and among inhibitors. Substrate-based histological analysis placed CBLs on the intestinal microvilli. Data indicate that CBLs are responsible for cysteine protease activity described from H. contortus intestine. Results also support a role of CBLs in nutrient digestion. Cathepsin B-like cysteine protease genes (cbls) constitute large multigene families in parasitic and nonparasitic nematodes. Although expressed in the intestine of some nematodes, the biological and biochemical functions of the CBL proteins remain unresolved. Di- and tetra-oligopeptides were used as fluorogenic substrates and irreversible/competitive inhibitors to establish CBL functions in the intestine of the parasitic nematode Haemonchus contortus. Cysteine protease activity was detected against diverse substrates including the cathepsin B/L substrate FR, the caspase 1 substrate YVAD, the cathepsin B substrate RR, but not the CED-3 (caspase 3) substrate DEVD. The pH at which maximum activity was detected varied according to substrate and ranged from pH 5.0 to 7.0. Individual CBLs were affinity isolated using FA and YVAD substrates. pH influenced CBL affinity isolation in a substrate-specific manner that paralleled pH effects on individual substrates. N-terminal sequencing identified two isolated CBLs as H. contortus GCP-7 (33 kDa) and AC-4 (37 kDa). N termini of each began at a position consistent with proregion cleavage and protease activation. Isolation of the GCP-7 band by each peptide was preferentially inhibited when competed with a diazomethane-conjugated inhibitor, Z-FA-CHN2, demonstrating one functional difference among CBLs and among inhibitors. Substrate-based histological analysis placed CBLs on the intestinal microvilli. Data indicate that CBLs are responsible for cysteine protease activity described from H. contortus intestine. Results also support a role of CBLs in nutrient digestion. cathepsin B-like cysteine protease gene and protein, respectively 7-amino-4-methylcoumarin acetyl-Tyr-Val-Ala-Asp acetyl-Asp-Glu-Val-Asp aldehyde 4-methoxy-2-naphthalamine benzyloxycarbonyl-Phe-Arg benzyloxycarbonyl-Arg-Arg diazomethane biotin fluoromethyl ketone trans-epoxysuccinyl-l-leucylamide-(4-guanidino)-butane excretory and secretory products dithiothreitol phosphate-buffered saline 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid monoclonal antibody Cathepsin B-like cysteine protease (cbl)1 genes occur as large multigene families in a wide range of parasitic and free-living nematodes (1Pratt D. Armes L.G. Hageman R. Reynolds V. Boisvenue R.J. Cox G.N. Mol. Biochem. Parasitol. 1992; 51: 209-218Crossref PubMed Scopus (65) Google Scholar, 2Harrop S.A. Prociv P. Brindley P.J. Trop. Med. Parasitol. 1995; 46: 119-122PubMed Google Scholar, 3Waterston R. Martin C. Craxton M. Huynh C. Coulson A. Hillier L. Durbin R. Green P. Shownkeen R. Halloran N. Metzstein M. Hawkins T. Wilson R. Berks M. Du Z. Thomas K. Thierrymieg J. Sulston J. Nat. Genet. 1992; 1: 114-123Crossref PubMed Scopus (316) Google Scholar). The functions of CBL proteins remain unresolved at both a biochemical and biological level. Severalcbl genes were shown to be expressed in the intestine of the parasitic worm Haemonchus contortus or in the free-living nematode Caenorhabditis elegans (1Pratt D. Armes L.G. Hageman R. Reynolds V. Boisvenue R.J. Cox G.N. Mol. Biochem. Parasitol. 1992; 51: 209-218Crossref PubMed Scopus (65) Google Scholar, 4Larminie C.G. Johnstone I.L. DNA Cell Biol. 1996; 15: 75-82Crossref PubMed Scopus (56) Google Scholar). Accordingly, one function of nematode CBLs might be nutrient digestion. CBLs may have application to control of parasitic nematodes in multiple ways. For instance, CBLs that function in host blood digestion are considered potential anthelmintic targets in schistosome flatworms (5Brindley P.J. Kalinna B.H. Dalton J.P. Day S.R. Wong J.Y. Smythe M.L. McManus D.P. Mol. Biochem. Parasitol. 1997; 89: 1-9Crossref PubMed Scopus (139) Google Scholar,6Wasilewski M.M. Lim K.C. Phillips J. McKerrow J.H. Mol. Biochem. Parasitol. 1996; 81: 179-189Crossref PubMed Scopus (127) Google Scholar). CBLs are also currently considered as vaccine candidates for immune control of parasitic nematodes, including H. contortus(6Wasilewski M.M. Lim K.C. Phillips J. McKerrow J.H. Mol. Biochem. Parasitol. 1996; 81: 179-189Crossref PubMed Scopus (127) Google Scholar, 7Skuce P.J. Redmond D.L. Liddell S. Stewart E.M. Newlands G.F. Smith W.D. Knox D.P. Parasitology. 1999; 119: 405-412Crossref PubMed Scopus (90) Google Scholar, 8Knox D.P. Smith S.K Smith W.D. Parasite Immunol. 1999; 21: 201-210Crossref PubMed Scopus (70) Google Scholar, 9Smith S.K. Pettit D. Newlands G.F.J. Redmond D.L. Skuce P.J. Knox D.P. Smith W.D. Parasite Immunol. 1999; 21: 187-199Crossref PubMed Scopus (86) Google Scholar). Alternatively, digestive proteases may indirectly mediate anthelmintic efficacy involving benzimidazole anthelmintics (10Jasmer D.P. Yao C. Rehman A. Johnson S. Mol. Biochem. Parasitol. 2000; 105: 81-90Crossref PubMed Scopus (52) Google Scholar). Following fenbendazole treatment of lambs, anterior intestinal cells ofH. contortus undergo fragmentation of nuclear DNA and tissue disintegration (10Jasmer D.P. Yao C. Rehman A. Johnson S. Mol. Biochem. Parasitol. 2000; 105: 81-90Crossref PubMed Scopus (52) Google Scholar). The pattern of DNA fragmentation resembled that associated with apoptosis, which can be initiated by caspase and noncaspase cysteine proteases (11Alnemri E.S. J. Cell. Biochem. 1997; 64: 33-42Crossref PubMed Scopus (292) Google Scholar, 12Vaux D.L. Wilhelm S. Hacker G. Mol. Cell. Biol. 1997; 17: 6502-6507Crossref PubMed Scopus (22) Google Scholar), while CED-3 is a prominent nematode caspase (13Xue D. Shaham S. Horvitz H.R. Genes Dev. 1996; 10: 1073-1083Crossref PubMed Scopus (282) Google Scholar). Both tissue disintegration and DNA fragmentation was associated with inhibited transport of secretory vesicles and subsequent cytoplasmic dispersal of vesicle contents in anterior intestinal cells (10Jasmer D.P. Yao C. Rehman A. Johnson S. Mol. Biochem. Parasitol. 2000; 105: 81-90Crossref PubMed Scopus (52) Google Scholar). The monoclonal antibody (mAb 42/10.6.1) used to monitor secretory vesicle contents binds to a periodate-sensitive determinant found on numerous intestinal membrane and secretory proteins (14Jasmer J.P. Perryman L.E. Conder G.A. Crow S. McGuire T.C. J. Immunol. 1993; 151: 5450-5460PubMed Google Scholar). Proteins that were immunoaffinity isolated by this mAb included an intestinal CBL, among other proteases (15Rehman A. Jasmer D.P. Mol. Biochem. Parasitol. 1998; 97: 55-68Crossref PubMed Scopus (54) Google Scholar). Therefore, CBLs might mediate some of the intestinal pathology induced by fenbendazole treatment. Despite these connections with basic aspects of parasite biology, CBLs remain enigmatic. The complexity of the family (16Rehman A. Jasmer D.P. Mol. Biochem. Parasitol. 1999; 102: 297-310Crossref PubMed Scopus (25) Google Scholar) has hindered biochemical and functional characterization of CBLs. Intestinal CBLs have predicted signal peptides and are expected to be secreted. CBLs also have predicted proregions, and N termini of some isolated CBLs indicated propeptide cleavage (4Larminie C.G. Johnstone I.L. DNA Cell Biol. 1996; 15: 75-82Crossref PubMed Scopus (56) Google Scholar, 16Rehman A. Jasmer D.P. Mol. Biochem. Parasitol. 1999; 102: 297-310Crossref PubMed Scopus (25) Google Scholar, 17Cox G.N. Pratt D. Hageman R. Boisvenue R.J. Mol. Biochem. Parasitol. 1990; 41: 25-34Crossref PubMed Scopus (94) Google Scholar). Cysteine protease activity was detected in H. contortus intestine and excretory-secretory products (ESP). This activity was characterized as cathepsin l-like, based on hydrolysis of Phe-Arg, but not Arg-Arg, dipeptide substrates (18Rhoads M.L. Fetterer R.H. J. Parasitol. 1995; 81: 505-512Crossref PubMed Scopus (79) Google Scholar). Glu245 was implicated in determining the ability of cathepsin B to degrade substrates with an Arg in the P2 position, a characteristic that is distinct from cathepsins L and S (16Rehman A. Jasmer D.P. Mol. Biochem. Parasitol. 1999; 102: 297-310Crossref PubMed Scopus (25) Google Scholar, 19Musil D. Zucic D. Turk D. et al.EMBO J. 1991; 10: 2321-2330Crossref PubMed Scopus (551) Google Scholar). Most predicted CBL sequences from H. contortus lack a Glu corresponding to position 245 in cathepsin B (16Rehman A. Jasmer D.P. Mol. Biochem. Parasitol. 1999; 102: 297-310Crossref PubMed Scopus (25) Google Scholar), which theoretically could compromise the ability of H. contortus CBLs to hydrolyze Arg-Arg substrates. This simple picture is complicated by several observations. In H. contortus ESP, distinct zymogram bands of cysteine protease activity were restricted to acidic pH, while others were active from acidic to neutral conditions (20Karanu F.N. Rurangirwa F.R. McGuire T.C. Jasmer D.P. Exp. Parasitol. 1993; 77: 362-371Crossref PubMed Scopus (74) Google Scholar). Phe-Arg substrate-based affinity probes identified four protease bands from ESP. This number was more restricted than expected according to the size of the CBL gene family reported. Substantial amino acid diversity was observed among CBL sequences at multiple positions within predicted S2 and S2′ subsite residues, and then, between CBLs and cathepsin B (16Rehman A. Jasmer D.P. Mol. Biochem. Parasitol. 1999; 102: 297-310Crossref PubMed Scopus (25) Google Scholar, 19Musil D. Zucic D. Turk D. et al.EMBO J. 1991; 10: 2321-2330Crossref PubMed Scopus (551) Google Scholar). Such diversity might translate into CBL properties that are distinct from related mammalian enzymes. Specific properties have not been attributed to any individual H. contortus CBL, which has hindered more directed research on recombinant forms of these enzymes. In this study, H. contortus intestinal CBLs were directly linked to known cysteine protease activity from this worm. Substrates for known cathepsin L, cathepsin B, caspase 1, and CED-3 (caspase 3) enzymes proved effective for dissecting distinct CBL activities. The results provide guidance to investigate these CBLs in relation to normal biological functions, functional diversity among individual CBLs and CBL contributions to anthelmintic-induced intestinal pathology. Fluorogenic peptide substrates included Ac-YVAD.AMC, Ac-DEVD.AMC, Z-FR.MNA, and Z-RR.MNA. Competitive peptide inhibitors included Ac-YVAD.CHO and Ac-DEVD.CHO. Irreversible peptide inhibitors included Z-FA.CHN2, Bt-YVAD.FMK, and Bt-FA.FMK. All of these substrates were obtained from Enzyme Systems Products (Dublin, CA). Approximately 10,000 viable L3 larvae of a Beltsville isolate of H. contortus (21Jasmer D.P. McGuire T.C. Infect. Immun. 1991; 59: 4412Crossref PubMed Google Scholar) were used to orally infect 4–8-month-old parasite-free lambs, which were killed 25–27 days after infection. Adult worms were harvested for dissection of intestines and for culture to obtain ESP (20Karanu F.N. Rurangirwa F.R. McGuire T.C. Jasmer D.P. Exp. Parasitol. 1993; 77: 362-371Crossref PubMed Scopus (74) Google Scholar). Intestines thawed from −80 °C were homogenized in PBS containing 1% Triton X-100. Protein concentration of lysates was determined by using the bicinchoninic acid protein assay reagent (Pierce). The extracts were stored in 200-μl aliquots at −80 °C. Protease activity was initially determined using the fluorogenic di- and tetrapeptide substrates, Z-FR.MNA, Z-RR.MNA, Ac-YVAD.AMC, and Ac-DEVD.AMC at 50 μm concentrations. Reactions were performed in 1.5-ml volumes with 10 μg of intestinal extracts or ESP and incubated at 37 °C for 2 h. Samples were evaluated at 30-min intervals for 2 h and activity reported for the 2-h incubations. Worm samples without substrates served as negative control. Inhibition studies incorporated protease-specific class inhibitors including 1 mm phenylmethylsulfonyl fluoride, 10 μm E-64, 100 μm leupeptin, 1 μm pepstatin, 100 μm iodoacetic acid, 10 mm 1,10-phenanthroline, or 5 mm EDTA. In addition, substrate-based dipeptide (Z-FA.CHN2) and tetrapeptide (Ac-YVAD.CHO, Ac-YVAD.FMK) protease inhibitors were used. The pH activity profiles were established using the following buffers: 100 mm citrate phosphate (pH 3.0–7.0) or 100 mm phosphate (pH 8.0) and 50 mm glycine-NaOH (pH 9.0) containing 10 mm DTT. Classic caspase activity was evaluated under conditions of 10 mm DTT, 312.5 mm HEPES, 31.25% sucrose, 0.3125% CHAPS (pH 7.5). Liberation of the leaving fluorescent groups, AMC and MNA, was monitored with a fluorescence spectrophotometer (MPF 42A, Perkin Elmer Life Sciences) using excitation and emission wavelengths of 360 and 460 nm (AMC), respectively, or 340 and 425 nm (MNA), respectively. Specific activity was determined in picomoles of AMC or MNA liberated per minute per microgram of protein at 37 °C. A standard curve produced with free AMC or MNA was used for these measurements. Enzyme reactions were done by incubating 64 μg of intestinal extract with biotinylated irreversible substrate inhibitors Bt-YVAD.FMK or Bt-FA.FMK (5 μm) in 0.1 m citrate-phosphate buffer, pH 5.0, containing 10 mm DTT. The reaction mixtures were incubated for 15 min at 37 °C. Following this incubation, a mixture of protease inhibitors (100 μm leupeptin, 1 mm pepstatin, 10 mm 1,10-phenanthroline, and 10 μm E-64) was added to the solution. The mixture was adjusted to 0.2% SDS and incubated for an additional 15 min at room temperature with gentle agitation. This solution was used for affinity isolation procedures described below. Inhibition reactions were run by incubating the intestinal extracts with nonbiotinylated irreversible inhibitors, Ac-YVAD.FMK (5 μm), Z-FA.CHN2 (5 μm), or E-64 (10 μm) for 5 min (37 °C) prior to incubation with biotinylated substrate inhibitors. Proteases conjugated to inhibitors were isolated by incubation with 100 μl of streptavidin-agarose beads (Sigma, St Louis, MO.) for 2 h at room temperature with gentle rotation. Bound beads were washed 5 times with 0.1 m glycine and 1% SDS (pH 8.0). The streptavidin-agarose beads were boiled in 3X-sample buffer for 15 min. Eluted proteins were fractionated on 7.5–17.5% gradient SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes as described previously (22Towbin H. Gordon H. J. Immunol. Methods. 1984; 72: 313-340Crossref PubMed Scopus (802) Google Scholar). The membranes were blocked overnight at 4 °C in PBS, Tween 20 (0.1%), and nonfat dry milk (5%). Membranes were incubated with streptavidin-horseradish peroxidase conjugate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) diluted 1:1000 in blocking buffer. Bound streptavidin was visualized using an enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia Biotech, Amersham Place, United Kingdom). TheM r of stained proteins was estimated using prestained Rainbow molecular mass standards ranging from 14.3 to 200 kDa (Amersham Pharmacia Biotech). This mAb (IgG2a) was used on immunoblots of affinity isolated proteases to evaluate glycosylation of CBLs and purity of the preparations. This mAb also provided enhanced sensitivity for evaluating affinity isolated proteins from ESP. Immunoblots were done with mAb 42/10.6.1 (2 μg/ml) as described (14Jasmer J.P. Perryman L.E. Conder G.A. Crow S. McGuire T.C. J. Immunol. 1993; 151: 5450-5460PubMed Google Scholar). An isotype control mAb (Babb) against an irrelevant determinant was used as a control on replicate blots. Bound antibodies were detected with horseradish peroxidase-conjugated goat anti-mouse IgG diluted 1:3000 (Kirkegaard & Perry Laboratories). The blot was developed using ECL. Proteases were isolated using Bt-YVAD.FMK (pH 5.0) or Bt-FA.FMK (pH 5.0 and 7.0). Affinity isolation procedures were optimized in standard assays by titrating the following components: peptide inhibitors, DTT, streptavidin-agarose beads, and the incubation time. Affinity isolation assays were then scaled up 50-fold to produce an estimated 450 pmol of proteins. Isolated proteases were fractionated on 7.5–17.5% gradient SDS-polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene difluoride Immobilon-P membranes (Millipore, Bedford, MA). Transblotted proteins were visualized by staining with 0.1% Coomassie Blue for 10 min, followed by rapid destaining. Destained bands were excised and their N-terminal amino acid sequence determined using an Applied Biosystems 494/HT Procise sequencing system (University of Texas Medical Branch, Protein Chemistry Laboratory, Galveston, TX). Location of H. contortus intestinal proteases was determined using the biotinylated peptide inhibitors Bt-YVAD.FMK and Bt-FA.FMK. Whole worms were placed in OCT compound (Tissue-TekR, Miles Inc., Elkhart, IN) and rapidly frozen in liquid nitrogen. Five micrometer tissue sections were cut using a cryotome, attached to ProbeOn™ Plus microscope slides (Fisher Scientific, Pittsburgh, PA), and air-dried for 30 min. Tissue sections were fixed in 30% methanol, 60% acetone, and 10% distilled H2O for 30 s at room temperature. Subsequently, the tissues were rinsed in PBS and incubated at 37 °C for 15 min in 0.1m citrate-phosphate pH 5.0 buffer, 10 mm DTT, containing 5 μm Bt-YVAD.FMK or 5 μmBt-FA.FMK. Inhibitor control tissue sections were pre-incubated with nonbiotinylated protease inhibitors or 5 μm E-64 for 15 min at 37 °C, followed by addition of biotinylated inhibitors. Negative control tissues were incubated without the peptides. The slides were then washed twice, for 5 min each, in wash buffer and blocked for 30 min at room temperature using PBS, Tween 20 (0.1%), and 10% normal sheep serum. Endogenous peroxidase inhibitor was included in reactions (Peroxidase Suppressor, ImmunoPureR; Pierce). Bound peptides were detected using streptavidin-horseradish peroxidase conjugate diluted 1:500 in blocking buffer. Streptavidin and biotin complexes were visualized using the ImmunoPure Metal Enhanced DAB (3,3′-diaminobenzidine tetrahydrochloride) substrate kit (Pierce). Protease activity was evaluated in intestinal extracts and ESP using four peptide substrates with the following sequences (designations): FR (cathepsins B/L), RR (cathepsin B), YVAD (caspase 1), and DEVD (caspase 3/CED-3). Initial interest in evaluating caspase substrates arose from the DNA fragmentation that can be induced by fenbendazole in intestinal cell nuclei of this parasite (10Jasmer D.P. Yao C. Rehman A. Johnson S. Mol. Biochem. Parasitol. 2000; 105: 81-90Crossref PubMed Scopus (52) Google Scholar). Preliminary experiments at pH 5.0 demonstrated activity against each substrate, with the exception of DEVD (Fig.1). When further evaluated, optimal pH for activity varied according to the substrate used. For example, activity against the YVAD substrate was restricted predominately to acidic pH, while activity against the RR substrate was highest around pH 6.0, and relatively high activity against the FR substrate occurred from pH 4.5 to 7.5 (Fig. 2,A–C). No activity against the DEVD substrate was detected under any of the described pH conditions, including preferred caspase conditions (data not shown). Significant activity against the RR substrate was not expected in H. contortus intestine based on a previous report (18Rhoads M.L. Fetterer R.H. J. Parasitol. 1995; 81: 505-512Crossref PubMed Scopus (79) Google Scholar). In addition, the preferential activity against YVAD under acidic conditions is inconsistent for caspases, which have neutral pH requirements (11Alnemri E.S. J. Cell. Biochem. 1997; 64: 33-42Crossref PubMed Scopus (292) Google Scholar).Figure 2pH effects on intestinal and ESP protease activity. Protease activities in intestinal extracts (int) and ESP of H. contortus were assayed at pH 3.0–9.0 with Z-FR.MNA (A), Z-RR.MNA (B), and Ac-YVAD.AMC (C). Means of triplicate assays are shown.Bars represent standard deviations.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Inhibitory effects of leupeptin and E-64 indicated that the majority of the activities against FR, RR, and YVAD substrates from intestine and ESP are due to cysteine proteases (TableI). Some variation on inhibition was observed among substrates with iodoacetic acid. Similar inhibitory results were obtained for FR and RR substrates at pH 5.0–6.0 (data not shown). Although 1,10-phenanthroline had moderate inhibitory effect on activities (57–87%), cysteine protease inhibition by 1,10-phenanthroline may be ascribed to a possible metal ion chelate. Although the relatively low inhibitory effect of EDTA supports this possibility, activity contributed by noncysteine proteases cannot be completely ruled out by these experiments. In contrast to the relative insensitivity of caspases to E-64 and leupeptin, intestinal activity against the YVAD substrate was ablated by both inhibitors. This result and observations on pH requirements suggested that activity against the YVAD substrate is conferred by noncaspase cysteine protease activity.Table IMean percentage of inhibition of protease activity from extracts of adult H. contortus intestineClass InhibitorSubstrates 1-aFluorogenic peptide substrates Z-FR.MNA, Z-RR.MNA, and Ac-YVAD.AMC.YVADFRRRCysteine Leupeptin98.0 1-bPercentage of inhibition (S.D.) (n = 3), compared to non-inhibited controls. (2.1)100 (0.0)100 (0.0) E-6499.0 (1.2)100 (0.0)100 (0.0) IAA78.0 (4.0)82.0 (2.1)100 (0.0)Metallo 1-cMetalloprotease. 1,10-Phen 1-d1,10-Phenanthroline.57.0 (4.6)87.0 (2.3)80.0 (1.8) EDTA51.2 (4.9)7.2 (2.4)38.1 (5.5)Aspartic Pepstatin1.7 (2.9)0.0 (0.0)10.7 (3.6)Serine PMSF 1-ePhenylmethylsulfonyl fluoride.0.0 (0.0)3.9 (3.7)9.5 (2.1)1-a Fluorogenic peptide substrates Z-FR.MNA, Z-RR.MNA, and Ac-YVAD.AMC.1-b Percentage of inhibition (S.D.) (n = 3), compared to non-inhibited controls.1-c Metalloprotease.1-d 1,10-Phenanthroline.1-e Phenylmethylsulfonyl fluoride. Open table in a new tab The possibility that related cysteine proteases confer activity against each of the substrates was evaluated by cross-inhibition experiments. Fluorogenic peptide substrates and peptide-based irreversible inhibitors were used in these experiments. Since no inhibitor construct was available for the RR substrate, experiments focused on the YVAD- and FR-based inhibitors. Additionally, a FA substrate modified by diazomethane (CHN2) was previously shown to inhibit H. contortus cysteine protease activity (20Karanu F.N. Rurangirwa F.R. McGuire T.C. Jasmer D.P. Exp. Parasitol. 1993; 77: 362-371Crossref PubMed Scopus (74) Google Scholar) and was used here. Activity against each substrate was inhibited at pH 5.0 by the irreversible inhibitors Ac-YVAD.FMK and Z-FA.CHN2 (Fig. 3). The inhibitor Ac-YVAD.CHO had similar effects on both YVAD and FR hydrolysis (data not shown). The high level of cross-inhibition suggested that a major portion of the activity against all substrates resided in the same enzymes at pH 5.0. In contrast to YVAD, hydrolysis of the FR substrate was relatively high at pH 7.0. However, this activity was inhibited by Z-FA.CHN2 (92%), Ac-YVAD.FMK (94%), and Ac-YVAD.CHO (78%). This result suggests that pH 7.0 inhibited protease binding by YVAD to a much lesser extent than protease hydrolysis of this substrate, which will be discussed below. Nevertheless, the higher level of inhibition by the irreversible inhibitor Ac-YVAD.FMK compared with Ac-YVAD.CHO may indicate low level hydrolysis of this substrate at pH 7.0. Biotinylated peptide substrates modified by a fluoromethylketone reactive group were used to isolate, characterize, and identify proteases that hydrolyze the peptide substrates described. Use of Bt-YVAD.FMK led to isolation of multiple prominent bands ranging from 29 to 37 kDa (Fig.4 A), with a 42-kDa band that was weakly visible (lane 2). A 33-kDa band was most prominent in multiple experiments. A similar profile of proteins was isolated by Bt-FA.FMK (lane 6), although the signal was always reduced compared with Bt-YVAD.FMK. A 33-kDa band was also most prominent in this lane, followed by a 37-kDa band. E-64 eliminated bands isolated by each of the biotinylated substrates, indicating they are cysteine proteases. Use of the nonbiotinylated inhibitor Ac-YVAD.FMK produced similar results. A Bt-DEVD.FMK probe failed to isolate any detectable bands from intestinal lysates (data not shown). The diazomethane inhibitor Z-FA.CHN2 was more discriminating, causing obvious reduction of only the 33-kDa band in preparations isolated by both the Bt-YVAD.FMK and Bt-FA.FMK. The two discernible weaker bands remaining at this position may reflect incomplete inhibition. Since Z-FA.CHN2 reduced activity against the FR and YVAD fluorogenic substrates by 100% and 94%, respectively (Fig. 3), proteases from the 33-kDa band may be largely responsible for hydrolyzing these substrates. Alternatively, Z-FA.CHN2 could function in fluorogenic assays as a competitive inhibitor against proteases that are otherwise resistant to irreversible binding by this inhibitor. Previous results suggested that CBLs are modified by the periodate-sensitive determinant recognized by mAb 42/10.6.1 (16Rehman A. Jasmer D.P. Mol. Biochem. Parasitol. 1999; 102: 297-310Crossref PubMed Scopus (25) Google Scholar). This determinant occurs on a multitude of membrane/secreted/excreted proteins from H. contortus (14Jasmer J.P. Perryman L.E. Conder G.A. Crow S. McGuire T.C. J. Immunol. 1993; 151: 5450-5460PubMed Google Scholar). Immunoblot analysis showed that most, if not all, proteins affinity-purified by Bt-YVAD.FMK and Bt-FA.FMK are modified by the mAb 42/10.6.1 determinant. In addition, the restricted size range of isolated proteins, compared with whole intestinal lysates, demonstrated a high level of purity in these preparations (Fig. 4 B). pH effects were evaluated on affinity isolation at pH 5.0 and 7.0. The bands isolated by Bt-YVAD.FMK at pH 5.0 were eliminated at pH 7.0 (Fig. 5). In contrast, a similar profile of bands was isolated by Bt-FA.FMK regardless of the pH conditions used. These results are consistent with the pH effects on hydrolysis of fluorogenic substrates. Characteristics established for presumptive intestinal proteases were next applied to evaluate proteins in ESP. Streptavidin-horseradish peroxidase lacked necessary sensitivity for this purpose. Alternatively, mAb 42/10.6.1 provided for a sensitive detection method. A size range of proteins was isolated from ESP by each substrate that paralleled those found in intestine (Fig. 5). The proteins isolated behaved in a substrate- and pH-dependent manner that paralleled intestinal proteins. The results also establish that the isolated proteins are modified by the mAb determinant. Collectively, results agree with a previous report (18Rhoads M.L. Fetterer R.H. J. Parasitol. 1995; 81: 505-512Crossref PubMed Scopus (79) Google Scholar) that the ESP proteins most likely represent intestinal cysteine proteases excreted from the parasite. N-terminal sequencing was done on two isolated intestinal protein bands. The 33-kDa band was chosen based on evidence of activity against both YVAD and FR substrates. The 37-kDa band was chosen due to the relative resolution of this band and the resistance of the band to inhibition by Z-FA.CHN2. N-terminal sequences were obtained for bands isolated at pH 5.0 (FA, YVAD) and 7.0 (FA). Current data bases contain an estimated 11 published H. contortus CBL sequences (25Schotte P. Declercq W. Huffel S. Vandenabeele P. Beyaert R. FEBS Lett. 1999; 442: 117-121Crossref PubMed Scopus (285) Google Scholar) and expressed sequence tags for an additional 11 unique CBLs. 2S. Shompole and D. P. Jasmer, unpublished data.The N-terminal sequences from each band had greatest similarity withH. contortus CBLs (Fig. 6). The sequences began at a uniform site consistent for pro-region cleavage, which is expected for the active proteases. Each sequence (20–24 residues) of the 37-kDa protein band isolated by each inhibitor, regardless of pH, exactly matched the H. contortus CBL AC-4 (Fig. 6). The residue corresponding to a single predicted cysteine residue was ambiguous in the protein sequences. The AC-4 sequence has multiple amino acids that are distinct from other CBLs, especially from consensus residues 11–17, providing a high degree of confidence in this identification. Of the sequences from the 33-kDa band