Major Cysteine Proteinase Research Articles

Expression and characterization of a recombinant cysteine proteinase of Leishmania mexicana

A major cysteine proteinase (CPB) of Leishmania mexicana, that is predominantly expressed in the form of the parasite that causes disease in mammals, has been overexpressed in Escherichia coli and purified from inclusion bodies to apparent homogeneity. The CPB enzyme, CPB2.8, was expressed as an inactive pro-form lacking the characteristic C-terminal extension (CPB2.8∆CTE). Pro-region processing was initiated during protein refolding and proceeded through several intermediate stages. Maximum enzyme activity accompanied removal of the entire pro-region. This was facilitated by acidification. Purified mature enzyme gave a single band on SDS/PAGE and gelatin SDS/PAGE gels, co-migrated with native enzyme in L. mexicana lysates, and had the same N-terminal sequence as the native enzyme. The procedure yielded > 3.5 mg of active enzyme per litre of E. coli culture.

Nitric Oxide Inhibits Cruzipain, the Major Papain-like Cysteine Proteinase from Trypanosoma cruzi

Nitric oxide (NO) is a pluripotent regulatory molecule showing, among others, an antiparasitic activity. Moreover, NO inhibits cysteine proteinase action by nitrosylating the Cys catalytic residue. In the present study, the inhibitory effect of the substrate N-α-benzyloxycarbonyl-l-phenylalanyl-l-arginine-(7-amino-4-methylcoumarin) and of NO on the catalytic activity of cruzipain, the major papain-like cysteine proteinase from Trypanosoma cruzi (the hemoflagellate protozoan parasite which causes the American trypanosomiasis), is reported. In particular, NO-donors S-nitroso-glutathione (GSNO), (±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR-3), 3-morpholinosydnonimine (SIN-1), S-nitroso-acetyl-penicillamine (SNAP), and sodium nitroprusside (SNP) dose-dependently inhibited cruzipain, this effect being likely attributable to the S-nitrosylation of the Cys25 catalytic residue. These results were analyzed in parallel with those concerning the inhibitory effect of the substrate and of NO on the catalytic activity of falcipain, the cruzipain-homologous cysteine proteinase from Plasmodium falciparum. The modulation of the cruzipain and falcipain activity by NO may be relevant in developing new strategies against T. cruzi and P. falciparum in human host. As a whole, the NO-mediated S-nitrosylation of pathogenic viral, bacterial, fungal, and parasitic cysteine proteinases may represent a general mechanism of antimicrobial and antiparasitic host defences.

Decreased metastatic spread in mice homozygous for a null allele of the cystatin C protease inhibitor gene.

AIMS: Increased or altered activities of cysteine proteases have been implicated in serious human disorders such as cancer, rheumatoid arthritis, sepsis, and osteoporosis. To improve the current knowledge of the...

Differential in vitro activation and deactivation of cysteine proteinases isolated during spore germination and vegetative growth of Dictyostelium discoideum.

Acid-activatable cysteine proteinases of Dictyostelium discoideum were first identified in spore extracts of strain SG1 using gelatin/SDS/PAGE, followed by acid treatments. Here we utilized the technique of acid activation to identify cryptic cysteine proteinases throughout auto-induced and heat-induced spore germination of D. discoideum strain SG2 and SG1. The major acid-activatable cysteine proteinase identified in SG2 and SG1 spore extracts was ddCP38 (D. discoideum cysteine proteinase with a molecular mass of 38 kDa) and ddCP48, respectively. Further investigation of these enzymes revealed that they were also base deactivatable with a treatment of ammonium chloride directly following acid activation. However, the most intriguing observation was the reversibility of the effects of base deactivation on the enzymes following a second treatment with acetic acid. Thus, we hypothesize that, unlike most mammalian cysteine proteinases which generally require the cleavage of a pro-peptide region for activation, these cysteine proteinases of D. discoideum likely undergo reversible conformational changes between latent and active forms. Moreover, we were able to detect these cryptic cysteine proteinases in the vegetative cells and early aggregates of both strains SG1 and SG2. Studies using 4-[(2S, 3S)-3-carboxyoxiran-2-ylcarbonyl-L-leucylamido]buty lguanidine, a cysteine proteinase inhibitor, revealed that acid activation of a portion of these proteinases was still achievable even after incubation with the inhibitor, further supporting the concept of two stable and reversible conformational arrangements of the enzymes. Thus, we speculate that the pH shuffles that modulate proteinase conformation and activity in vitro may be a reflection of the in vivo regulation of these enzymes via H+-ATPases and ammonia.

Properties of digestive glycosidases and peptidases and the permeability of the peritrophic membranes of Abracris flavolineata (Orthoptera: Acrididae)

In Abracris flavolineata midguts, cellulose is hydrolyzed by at least three enzymes, whereas the most active hemicellulases are a laminarinase (pH optimum 5.7, M r 146 k) and three lichenases (pH optima in the range 5–7.3; M r values: 22, 71, and 97 k). Digestion of hemicellulose is completed by β-glucosidases described elsewhere and by an α-mannosidase (pH optimum 4.7, K m 1.7 mM). There are a major and two minor amylases (pH optimum 6.5; M r values: 42, 45, and 108 k) activated by chloride. Two of the α-glucosidases ( M r 74 and 94 k) are active on maltose and hence should finish the digestion of starch. It is not clear what is the natural substrate of the remaining α-glucosidase ( M r 93 k). The major α-galactosidase ( M r 112 k) is active on melibiose and raffinose, whereas the minor ( M r 70k) may be active on digalactosyl diglycerides. The effect of pH on azocasein hydrolysis and electrophoresis data suggest that trypsin is a major and chymotrypsin and cysteine proteinase are minor enzymes. The cysteine proteinase may derive from the leaves ingested by the grasshopper, taking into account its activity in leaves. Protein digestion is finished by two soluble aminopeptidases ( M r 92 and 105 k), a major membrane-bound aminopeptidase ( M r 97 k) and two membrane-bound dipeptidases ( M r 87 k). The sizes of the digestive enzymes recovered in A. flavolineata crops suggest that the pores of the semi-fluid caecal peritrophic membrane have diameters larger than 8.6 nm.

Expression and alteration of the S2 subsite of the Leishmania major cathepsin B-like cysteine protease

The mature form of the cathepsin B-like protease of Leishmania major (LmajcatB) is a 243 amino acid protein belonging to the papain family of cysteine proteases and is 54% identical to human-liver cathepsin B. Despite the high identity and structural similarity with cathepsin B, LmajcatB does not readily hydrolyse benzyloxycarbonyl-Arg-Arg-7-amino-4-methyl coumarin (Z-Arg-Arg-AMC), which is cleaved by cathepsin B enzymes. It does, however, hydrolyse Z-Phe-Arg-AMC, a substrate typically cleaved by cathepsin L and B enzymes. Based upon computer generated protein models of LmajcatB and mammalian cathepsin B, it was predicted that this variation in substrate specificity was attributed to Gly234 at the S2 subsite of LmajcatB, which forms a larger, more hydrophobic pocket compared with mammalian cathepsin B. To test this hypothesis, recombinant LmajcatB was expressed in the Pichia pastoris yeast expression system. The quality of the recombinant enzyme was confirmed by kinetic characterization, N-terminal sequencing, and Western blot analysis. Alteration of Gly234 to Glu, which is found at the corresponding site in mammalian cathepsin B, increased recombinant LmajcatB (rLmajcatB) activity toward Z-Arg-Arg-AMC 8-fold over the wild-type recombinant enzyme (kcat/Km = 3740±413 M-1·s-1 versus 472±72.4 M-1·s-1). The results of inhibition assays of rLmajcatB with an inhibitor of cathepsin L enzymes, K11002 (morpholine urea-Phe-homoPhe-vinylsulphonylphenyl, kinact/Ki = 208200±36000 M-1·s-1), and a cathepsin B specific inhibitor, CA074 [N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-ʟ-isoleucyl-L-proline, kinact/Ki = 199200±32900 M-1·s-1], support the findings that this protozoan protease has the P2 specificity of cathepsin L-like enzymes while retaining structural homology to mammalian cathepsin B.

Expression and alteration of the S2 subsite of the Leishmania major cathepsin B-like cysteine protease

The mature form of the cathepsin B-like protease of Leishmania major (LmajcatB) is a 243 amino acid protein belonging to the papain family of cysteine proteases and is 54% identical to human-liver cathepsin B. Despite the high identity and structural similarity with cathepsin B, LmajcatB does not readily hydrolyse benzyloxycarbonyl-Arg-Arg-7-amino-4-methyl coumarin (Z-Arg-Arg-AMC), which is cleaved by cathepsin B enzymes. It does, however, hydrolyse Z-Phe-Arg-AMC, a substrate typically cleaved by cathepsin L and B enzymes. Based upon computer generated protein models of LmajcatB and mammalian cathepsin B, it was predicted that this variation in substrate specificity was attributed to Gly234 at the S2 subsite of LmajcatB, which forms a larger, more hydrophobic pocket compared with mammalian cathepsin B. To test this hypothesis, recombinant LmajcatB was expressed in the Pichia pastoris yeast expression system. The quality of the recombinant enzyme was confirmed by kinetic characterization, N-terminal sequencing, and Western blot analysis. Alteration of Gly234 to Glu, which is found at the corresponding site in mammalian cathepsin B, increased recombinant LmajcatB (rLmajcatB) activity toward Z-Arg-Arg-AMC 8-fold over the wild-type recombinant enzyme (kcat/Km=3740+/-413 M-1.s-1 versus 472+/-72.4 M-1.s-1). The results of inhibition assays of rLmajcatB with an inhibitor of cathepsin L enzymes, K11002 (morpholine urea-Phe-homoPhe-vinylsulphonylphenyl, kinact/Ki=208200+/-36000 M-1. s-1), and a cathepsin B specific inhibitor, CA074 [N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-l-isoleucyl-l- prolin e, kinact/Ki=199200+/-32900 M-1.s-1], support the findings that this protozoan protease has the P2 specificity of cathepsin L-like enzymes while retaining structural homology to mammalian cathepsin B.

Cysteine Proteinase Inhibitors Kill Cultured Bloodstream Forms ofTrypanosoma brucei brucei

Troeberg, L., Morty, R. E., Pike, R. N., Lonsdale-Eccles, J. D., Palmer, J. T., McKerrow, J. H., and Coetzer, T. H. T. 1999. Cysteine proteinase inhibitors kill cultured bloodstream forms ofTrypanosoma brucei brucei. Experimental Parasitology91, 349–355.Trypanosoma brucei bruceiis a causative agent of bovine trypanosomiasis (nagana), a disease of considerable economic significance in much of Africa. Here we report investigations on the effects of various irreversible cysteine proteinase inhibitors, including vinyl sulfones (VS), peptidyl chloromethylketones (CMK), diazomethylketones, and fluoromethyl ketones, on the major lysosomal cysteine proteinase (trypanopain-Tb) ofT. b. bruceiand onin vitro-cultured bloodstream forms of the parasite. Many of the tested inhibitors were trypanocidal at low micromolar concentrations. Methylpiperazine urea-Phe-homoPhe-VS was the most effective trypanocidal agent, killing 50% of test populations at a work ing concentration of 0.11 μM, while carbobenzoxy-Phe-Phe-CMK was the most trypanocidal of the methylketones with an IC50of 3.6 μM. Labelling of live and lysedT. b. bruceiwith biotinylated inhibitor derivatives suggests that trypanopain-Tb is the likely intracellular target for these inhibitors. Kinetic analysis of the inhibition of purified trypanopain-Tb by the inhibitors showed that most hadkassvalues in the 106M−1s−1range. We conclude that cysteine proteinase inhibitors have potential as trypanocidal agents and that a major target of these compounds is the lysosomal enzyme trypanopain-Tb.

Physiological adaptation explains the insensitivity of Baris coerulescens to transgenic oilseed rape expressing oryzacystatin I

Larvae of Baris coerulescens Scop. (Coleoptera: Curculionidæ) exhibit a complex array of gut proteinase activities comprising cysteine and serine proteinases. The major cysteine proteinase activity, showing an optimum at pH 6.0, corresponds to at least 4 different proteinases. On the contrary, the minor serine proteinase activity, with an optimum at pH 9.0, seems to be due essentially to a single proteinase. The cysteine proteinase inhibitor oryzacystatin I (OC-I) inhibits completely the cysteine proteinase activity in vitro. However, larval growth and survival were not significantly different on control and transgenic oilseed rape plants expressing high levels of active OC-I. In larvae grown on transgenic plants, cysteine proteinase activity was dramatically decreased, whereas serine proteinase activity was increased by more than 2-fold, when compared to larvae raised on control plants. For both activities, no new proteinase was detected in insects fed plants expressing OC-I. These results suggest that partial compensation of the inhibition of cysteine proteinase activity by the increase in serine proteinase activity allowed the larvae to overcome the effects of OC-I consumption. This case illustrates problems that could arise when trying to achieve high levels of protection for plants against Coleopteran pests possessing a complex digestive proteinase pool.

Determinants of electron transfer in the nitric oxide synthases: [formula omitted], R. Timothy Miller, and Bettie Sue MastersU. Texas Health Science Center at San Antonio

Nitric oxide (NO) is a pluripotent regulatory molecule showing, among others, an antiparasitic activity. Moreover, NO inhibits cysteine proteinase action by nitrosylating the Cys catalytic residue. In the present study, the inhibitory effect of the substrate N-α-benzyloxycarbonyl-l-phenylalanyl-l-arginine-(7-amino-4-methylcoumarin) and of NO on the catalytic activity of cruzipain, the major papain-like cysteine proteinase from Trypanosoma cruzi (the hemoflagellate protozoan parasite which causes the American trypanosomiasis), is reported. In particular, NO-donors S-nitroso-glutathione (GSNO), (±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR-3), 3-morpholinosydnonimine (SIN-1), S-nitroso-acetyl-penicillamine (SNAP), and sodium nitroprusside (SNP) dose-dependently inhibited cruzipain, this effect being likely attributable to the S-nitrosylation of the Cys25 catalytic residue. These results were analyzed in parallel with those concerning the inhibitory effect of the substrate and of NO on the catalytic activity of falcipain, the cruzipain-homologous cysteine proteinase from Plasmodium falciparum. The modulation of the cruzipain and falcipain activity by NO may be relevant in developing new strategies against T. cruzi and P. falciparum in human host. As a whole, the NO-mediated S-nitrosylation of pathogenic viral, bacterial, fungal, and parasitic cysteine proteinases may represent a general mechanism of antimicrobial and antiparasitic host defences.

Activities of the Porphyromonas gingivalis PrtP proteinase determined by construction of prtP-deficient mutants and expression of the gene in Bacteroides species.

PrtP is a major cysteine proteinase of Porphyromonas gingivalis. The gene encoding this proteinase, prtP, was cloned into the Escherichia coli-Bacteroides shuttle vectors pFD288 and pFD340 and was expressed in Bacteroides cells, apparently under the control of its own promoter, when in pFD288, or a Bacteroides promoter present on pFD340. Proteolytically active PrtP was detected by fibrinogen zymography in cells or spent growth medium of several Bacteroides species harboring the recombinant plasmids. The proteinase was recovered from Bacteroides fragilis ATCC 25285(pFD340-prtP) cells by 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS) extraction and characterized with regard to exopeptidase specificity and sensitivity to proteinase inhibitors. Lys-amidolytic activity, but not Arg-amidolytic activity, was detected. PrtP was activated by cysteine and, to a lesser extent, dithiothreitol, and it was stimulated by glycine-containing compounds. It also was inhibited by Nalpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) and, to a lesser extent, H-D-Tyr-L-Pro-L-arginyl chloromethyl ketone (YPRCK) and was relatively insensitive to EDTA and leupeptin. Neither B. fragilis ATCC 25285(pFD340-prtP) cells nor the CHAPS extract effected hemagglutination of sheep red blood cells or collagen cleavage, but the cells did cleave gelatin. Furthermore, P. gingivalis W12, ATCC 33277, KDP110, and HG66 with knockout mutations in prtP were constructed by allelic replacement. Unlike the parent strains, the mutant strains produced beige colonies on plates containing sheep blood. These strains also were affected in their ability to effect hemagglutination, cleave collagen, and cleave a Lys-specific peptide substrate. This report presents the results of the first characterization of the PrtP proteinase clearly in the absence of any influence by other P. gingivalis proteins and describes the properties of P. gingivalis cells defective in the production of PrtP.

Modulation of interleukin-8 activity by gingipains from Porphyromonas gingivalis: implications for pathogenicity of periodontal disease

Gingipains are the major cysteine proteinases synthesized by Porphyromonas gingivalis which, in soluble form, are able to initially convert IL-8 (77 amino acid residues) to a more potent species truncated at the amino terminus, followed by slow degradation and destruction of chemokine biological activity. In contrast, the same enzymes when associated with bacterial outer-membrane blebs (vesicles), instantly degrade this chemokine. This division of enhancing and inactivating activity between soluble and membrane-bound gingipains can cause the compartmentalization of pro- and anti-inflammatory reactions to distal and proximal positions from bacterial plaque, respectively, which may explain why, despite the massive neutrophil accumulation at periodontitis sites, there is no elimination of infection.

Expression of a Recombinant Fab Antibody Fragment against Cruzipain, the Major Cysteine Proteinase ofTrypanosoma cruzi

Cruzipain, the major proteinase ofTrypanosoma cruzi,plays an important role in the biology of this parasite. This study reports the development of a recombinant Fab antibody, using RNA isolated from the anti-Ag163B6 hybridoma against cruzipain. This procedure involves the use of cDNAs obtained with the aid of a specific set of primers complementary to the complete light kappa chain (Lκ) and the first two domains of the IgG1 heavy chain (VH/CH1). These products were subsequently cloned in the pComb3 system, from which the gIII gene had been removed, and expressed inEscherichia colicells. The recombinant Fab molecule recognized cruzipain by ELISA, in a fashion similar to the original mAb anti-Ag163B6. Nucleotide sequence analysis of the recombinant molecule, together with its immunological recognition by specific anti-mouse IgG (Fab)2, indicated the immunoglobulin nature of the recombinant product. Moreover, both the mAb anti-Ag163B6 and the soluble Fab fragment described here react similarly with the intact parasite surface, as observed in an indirect immunofluorescence assay. In conclusion, our recombinant Fab anti-Ag 163B6 allows the possible use of this molecule for diagnosis, antigen purification, and eventually treatment of Chagas-afflicted individuals.

Structure-based design, synthesis and evaluation of conformationally constrained cysteine protease inhibitors

The inhibition of cysteine proteases is being studied as a strategy to combat parasitic diseases such as Chagas' disease, leishmaniasis, and malaria. Cruzain is the major cysteine protease of Trypanosoma cruzi, the etiologic agent of Chagas' disease. A crystal structure of cruzain, covalently inactivated by fluoromethyl ketone inhibitor 1 (Cbz-Phe-Ala-FMK), was used as a template to design potential inhibitors. Conformationally constrained γ-lactams containing electrophilic aldehyde ( 12, 17, 18, 25, 26, and 29) or vinyl sulfone ( 43, 44, and 46) units were synthesized. Constrained lactam 26 had IC 50 values of ca. 20 nM against the Leishmania major protease and ca. 50 nM versus falcipain, an important cysteine protease isolated from Plasmodium falciparum. However, all of the conformationally constrained inhibitors were weak inhibitors of cruzain, compared to unconstrained peptide aldehyde (e.g. 5) and vinyl sulfone inhibitors (e.g. 48, which proved to be an excellent inhibitor of cruzain with an apparent second order inhibition rate constant ( k inact/ K i) of 634,000 s −1M −1). A significant reduction in activity was also observed with acyclic inhibitors 30 and 51 containing α-methyl phenylalanine residues at the P 2 position. These data indicate that the pyrrolidinone ring, especially the quarternary center at P 2, interferes with the normal substrate binding mode with cruzain, but not with falcipain or the leishmania protease.

Two stable unfolding intermediates of the disease-causing L68Q variant of human cystatin C.

In hereditary cystatin C amyloid angiopathy (HCCAA), presence of the Leu68 --> Gln substitution in cystatin C is coupled to a decreased concentration of this major cysteine proteinase inhibitor in cerebrospinal fluid and leads to its amyloid deposition in the brain. We established a high-yield expression system for L68Q cystatin C in Escherichia coli resulting in inclusion body accumulation at a level of 40% of the total cellular protein. Refolding of protein from purified inclusion bodies yielded a pure, almost completely monomeric and active inhibitor. CD and NMR spectroscopy demonstrated that so produced L68Q cystatin C is folded, conformationally homogeneous, and structurally very similar to wild-type cystatin C. Incubation at pH 7.0-5.5 caused the cystatin C variant to dimerize rapidly. The molecular form present at pH 6.0 displayed a slightly increased amount of hydrophobic parts on the surface as measured by 1-anilinonaphthalene-8-sulfonic acid (ANS) binding. NMR results showed that the dimer has a structure similar to that of the wild-type cystatin C dimer formed as a result of slight denaturation. Under more acidic conditions, at pH 4.5, another stable unfolding intermediate of L68Q cystatin C was identified. This molecular form exists in a monomeric state, is characterized by changes in secondary structure according to far UV CD spectroscopy, and shows an altered ANS binding resembling that of a molten globule state. The acidic pH also caused an almost complete monomerization of preformed dimers. The state of denaturation of L68Q cystatin C in vivo is thus a critical factor for the concentration of active cysteine proteinase inhibitor in cerebrospinal fluid and likely also for the development of amyloidosis, in HCCAA patients.

Inhibition of Trypanosomal Cysteine Proteinases by Their Propeptides

The ability of the prodomains of trypanosomal cysteine proteinases to inhibit their active form was studied using a set of 23 overlapping 15-mer peptides covering the whole prosequence of congopain, the major cysteine proteinase of Trypanosoma congolense. Three consecutive peptides with a common 5-mer sequence YHNGA were competitive inhibitors of congopain. A shorter synthetic peptide consisting of this 5-mer sequence flanked by two Ala residues (AYHNGAA) also inhibited purified congopain. No residue critical for inhibition was identified in this sequence, but a significant improvement in Ki value was obtained upon N-terminal elongation. Procongopain-derived peptides did not inhibit lysosomal cathepsins B and L but did inhibit native cruzipain (from Dm28c clone epimastigotes), the major cysteine proteinase of Trypanosoma cruzi, the proregion of which also contains the sequence YHNGA. The positioning of the YHNGA inhibitory sequence within the prosegment of trypanosomal proteinases is similar to that covering the active site in the prosegment of cysteine proteinases, the three-dimensional structure of which has been resolved. This strongly suggests that trypanosomal proteinases, despite their long C-terminal extension, have a prosegment that folds similarly to that in related mammal and plant cysteine proteinases, resulting in reverse binding within the active site. Such reverse binding could also occur for short procongopain-derived inhibitory peptides, based on their resistance to proteolysis and their ability to retain inhibitory activity after prolonged incubation. In contrast, homologous peptides in related cysteine proteinases did not inhibit trypanosomal proteinases and were rapidly cleaved by these enzymes.

Substrate inhibition of cruzipain is not affected by the C-terminal domain

Endogenous and recombinant cruzipain, the major cysteine proteinase from the protozoan parasite Trypanosoma cruzi, exhibit differences in the protein and circular dichroism spectra probably attributed to the absence of the C-terminal domain in the recombinant enzyme. Substrate hydrolysis of both molecules at 25°C and neutral pH obeyed Michaelis-Menten kinetics whereas significant substrate inhibition was observed above neutral pH. The results suggest that substrate inhibition of cruzipain is pH-dependent, and that the C-terminal domain does not play an essential role in this process.

The group A streptococcal dipeptide permease (Dpp) is involved in the uptake of essential amino acids and affects the expression of cysteine protease.

The majority of characterized bacterial dipeptide permeases (Dpp) are membrane-associated complexes of five proteins belonging to the ABC-transporter family. They have been found to be involved in the uptake of essential amino acids, haem production, chemotaxis and sporulation. A 5.8 kb genomic DNA fragment of the serotype M49 group A streptococcal (GAS) strain CS101 was sequenced and found to contain five putative GAS Dpp genes (dppA to dppE). Deduced amino acid sequences exhibited 17-54% similarity to corresponding ABC-transporter sequences. The operon organization of the five genes was confirmed by transcriptional analysis, and a shorter, more abundant, dppA-only transcript was detected similar to that found in the GAS oligopeptide permease (Opp) system. Insertional inactivation was used to create serotype M2 and M49 strains that did not express the dppD and dppEATPase genes or nearly the entire operon. In feeding experiments with di- to hexapeptides, the wild-type strain grew with each peptide tested. The dpp mutants were unable to grow on dipeptides, whereas hexapeptides did not sustain the growth of opp mutants. Expression of the dpp operon was induced approximately fourfold in late exponential growth phase. In addition, a striking increase in the dppA to dppA-E ratio from 5:1 to more than 20:1 occurred during late exponential growth phase in complex medium. Growth in chemically defined medium (CDM) supplemented with various dipeptides specifically induced the expression of dpp and reduced both the dppA to dppA-E and oppA to oppA-F mRNA ratios. Expression of the virulence factor SpeB (major cysteine protease) was reduced eightfold in dpp mutants, whereas dpp expression was decreased about fourfold in a Mga virulence regulator mutant. Taken together, these data indicate a correlation between levels of intracellular essential amino acids and the regulation of virulence factor expression.

Cathepsins B and D are dispensable for major histocompatibility complex class II-mediated antigen presentation.

Antigen presentation by major histocompatibility complex (MHC) class II molecules requires the participation of different proteases in the endocytic route to degrade endocytosed antigens as well as the MHC class II-associated invariant chain (Ii). Thus far, only the cysteine protease cathepsin (Cat) S appears essential for complete destruction of Ii. The enzymes involved in degradation of the antigens themselves remain to be identified. Degradation of antigens in vitro and experiments using protease inhibitors have suggested that Cat B and Cat D, two major aspartyl and cysteine proteases, respectively, are involved in antigen degradation. We have analyzed the antigen-presenting properties of cells derived from mice deficient in either Cat B or Cat D. Although the absence of these proteases provoked a modest shift in the efficiency of presentation of some antigenic determinants, the overall capacity of Cat B-/- or Cat D-/- antigen-presenting cells was unaffected. Degradation of Ii proceeded normally in Cat B-/- splenocytes, as it did in Cat D-/- cells. We conclude that neither Cat B nor Cat D are essential for MHC class II-mediated antigen presentation.

Fluorescence microscopic demonstration of cathepsin K activity as the major lysosomal cysteine proteinase in osteoclasts.

The enzyme activity of lysosomal cysteine proteinases in vital rabbit osteoclasts and mouse osteoclast-like cells was visualized with Z-Leu-Arg-4-methoxy-beta-naphthylamide (Z-LR-MNA) as the enzyme substrate. The MNA liberated by proteolysis forms a fluorescent insoluble Schiff-base product in the presence of 5-nitrosalicylaldehyde. Many small fluorescent particles, endproducts of the Z-LR-MNA hydrolysis, were observed in proximity to the bone surface underneath the actively resorbing osteoclasts, as well as in the cytoplasm. The Z-LR-MNA hydrolase activity was markedly diminished by bafilomycin A1 and chloroquine treatment. Moreover, the activity was completely inhibited by cysteine proteinase inhibitors such as leupeptin and E-64d, but not by other classes of proteinase inhibitors. About 60% of the hydrolase activity in mouse osteoclast-like cells was immunoabsorbed by anti-cathepsin K antibody-coupled Sepharose CL-4B beads, and about 10% of the activity was absorbed with the anti-cathepsin L antibody-coupled beads. Thus, the majority of the Z-LR-MNA hydrolase activity in osteoclasts was derived from cathepsin K. In contrast, using the same substrate in the assay, no detectable cathepsin K activity was observed in mouse peritoneal macrophages. The abundant cathepsin K activity in osteoclasts would therefore indicate a significant role of this enzyme in bone matrix degradation.