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Molecular mechanisms of activity-dependent synapse reduction were studied in an in vitro mammalian neuromuscular preparation. Synapse reduction in this model is activity-dependent and is substantially reduced by the broad-spectrum protease inhibitor, leupeptin, suggesting the role of activity-dependent proteolytic action in the process. Our present experiments show that a potent and specific thrombin inhibitor, hirudin, at nanomolar concentration completely blocked the activity-dependent synapse reduction. Furthermore, a naturally occurring serine protease inhibitor, protease nexin I (PNI), which closely colocalizes with acetylcholine receptors at the neuromuscular junction, inhibited the synapse reduction at the same low concentration. In contrast, neither cystatin, a cysteine protease inhibitor, nor aprotinin, a serine protease inhibitor that does not inhibit thrombin, blocked the synapse reduction. Similarly, neither of the inhibitors of the calcium-activated proteases calpain I and II prevented the reduction of synapses. These results strongly suggest that serine proteolytic action by thrombin or thrombin-like molecules is required for synapse reduction in our in vitro model of the mammalian neuromuscular junction.

Two mutants of the cysteine proteinase inhibitor, stefin B, were prepared by ligating the amino-terminal region from cystatin C and kininogen, members of two other families of cystatin superfamily. The mutant proteins were expressed in Escherichia coli and purified to homogeneity. Inhibition and kinetic constants were determined for authentic and mutated stefins against the four different cysteine proteinases, papain and human cathepsins B, L and H. Inhibition of both amino-terminal elongated stefin B mutants was decreased particularly for cathepsin H. A model of the tertiary structure of cathepsin H and its complex with stefin B was constructed. The framework for the model of cathepsin H consisted of structurally conserved regions from tertiary structures of three cysteine proteinases. Variable regions were selected from fragments of other proteins from the protein data base. We suggest that reduced binding of stefins with elongated amino termini is caused by the mini chain of cathepsin H which is probably in close proximity to the amino termini in the complexes. This mini chain is bridged to Cys214 and has already been proposed to be responsible for the aminopeptidase activity of cathepsin H. We conclude that the amino-terminal region of stefin B plays an important role in determining the strength of inhibition of cathepsin H.

Two deletion variants of chicken cystatin were produced after cassette mutagenesis of the recombinant Arg-Glu-Phe-[Met1, Ile29, Leu89]-chicken egg white cystatin gene in Escherichia coli. The variant des-Ser1-Pro11-[Ala12, Glu13, Phe14, Met15, Ile29, Leu89]-chicken cystatin (N-del 2) and the variant Arg-Glu-Phe-[Met1, Ile29]-des-Cys71-Met89-chicken cystatin (del-helix II) were purified and characterized by inhibition kinetics, far-ultraviolet-CD and fluorescence spectroscopy, and their folding in guanidine hydrochloride (Gdn/HCl) was studied. The del-helix II variant, shortened by 19 amino acids, is a basic, stefin-like mini-cystatin with one disulfide bridge. Its inhibitory properties are identical to chicken cystatin and its stability against Gdn/HCl is similar. The folding of the del-helix II variant corresponds best to a single step process. In contrast to this, the reversible folding of natural and recombinant chicken cystatin is more complex when recorded by either tryptophan fluorescence or far-ultraviolet-CD. With increasing Gdn/HCl concentration, a stabilization of secondary-structural elements is initially observed, followed by unfolding with minor but distinct intermediate states. The N-del 2 variant has a neutral pI and shows folding behaviour very similar to natural and recombinant chicken cystatin. However its inhibition constants with papain, actinidin and cathepsin B and L are 1000-100,000-fold higher than those obtained with natural and recombinant chicken cystatin.

Single-chain urokinase-type plasminogen activator (scu-PA) is inactivated by thrombin, which cleaves the peptide bond between Arg156 and Phe157. In a search for potential activators of thrombin-cleaved two-chain urokinase-type plasminogen activator (tcu-PA/T), we found that the lysosomal aminopeptidase dipeptidyl-peptidase I or cathepsin C efficiently activates tcu-PA/T. Cathepsin C was as active towards tcu-PA/T as the bacterial proteinase thermolysin and about 300-times more active than plasmin. The activation by cathepsin C proceeded in a concentration-dependent and time-dependent manner with a pH optimum between 5 and 7. Furthermore, the effect of cathepsin C was inhibited by cystatin and stimulated by cysteine, typical for the action of a thiol proteinase. As no degradation of the tcu-PA/T molecule by cathepsin C was visible on SDS/PAGE, we suggest that activation of tcu-PA/T occurs by cleavage between Lys158-Ile159 and removal of the two N-terminal amino acid residues (Phe157-Lys158) of the B chain of tcu-PA/T. We conclude that both thrombin and dipeptidyl-peptidases like cathepsin C might play a regulatory role in the plasminogen-plasmin system by inactivating scu-PA and activating tcu-PA/T, respectively.

Chymopapain M, the monothiol cysteine proteinase component of the chymopapain band eluted after chymopapains A and B in cation-exchange chromatography, was isolated from the dried latex of Carica papaya and characterized by kinetic and chromatographic analysis. This late-eluted chymopapain is probably a component of the cysteine proteinase fraction of papaya latex discovered by Schack [(1967) Compt. Rend. Trav. Lab. Carlsberg 36, 67-83], named papaya peptidase B by Lynn [(1979) Biochim. Biophys. Acta 569, 193-201] and partially characterized by Polgár [(1981) Biochim. Biophys. Acta 658, 262-269] and is the enzyme with unusual specificity characteristics (papaya proteinase IV) that Buttle, Kembhavi, Sharp, Shute, Rich and Barrett [Biochem. J. (1989) 261, 469-476] claimed to be a previously undetected cysteine proteinase eluted from a cation-exchange column near to the early-eluted chymopapains. A study of the time-dependent chromatographic consequences of thiol-dependent proteolysis of the components of papaya latex is reported. Chymopapain M was isolated by (i) affinity chromatography followed by separation from papain using cation-exchange f.p.l.c. on a Mono S HR5/5 column and (ii) cation-exchange chromatography followed by an unusual variant of covalent chromatography by thiol-disulphide interchange. The existence in chymopapain M of a nucleophilic interactive Cys/His catalytic-site system analogous to those in papain (EC 3.4.22.2) and other cysteine proteinases was deduced from the characteristics shape of the pH-second-order rate constant (k) profiles for its reactions with 2,2'-dipyridyl disulphide and ethyl 2-pyridyl disulphide. Analysis of the pH-k data for the reactions of chymopapain M with the 2-pyridyl disulphides and with 4,4'-dipyridyl disulphide permits the assignment of molecular pKa values of 3.4 and 8.7 to the formation and subsequent dehydronation of the Cys-S-/His-Im+H state of the catalytic site and reveals three other kinetically influential ionizations with pKa values 3.4, 4.3 and 5.6. The pH-dependences of kcat./Km for the hydrolysis of N-acetyl-L-Phe-Gly-4-nitroanilide at 25.0 degrees C and I0.1 M catalysed by chymopapain M and papain were determined. For both enzymes, little catalytic activity (5-7% of the maximal) develops consequent on formation of the catalytic site Cys-S-/His-Im+H ion-pair state (across pKa 3.4 for both enzymes). For papain, full expression of Kcat./Km for the uncharged substrate requires only the additional hydronic dissociation with pKa 4.2. By contrast, full expression of kcat./Km for chymopapain M requires additional hydronic dissociation with pKa values of 4.3 and 5.6.(ABSTRACT TRUNCATED AT 400 WORDS)

The kinetics of the complex formation between bovine cathepsin S and bovine stefin B was studied by conventional and stopped-flow techniques. The inhibition at low inhibitor concentrations was tight and reversible (kass = 5.8 x 10(7) M-1.s-1, kdiss = 4.9 x 10(-4) s-1 at pH 6.0 and 25 degrees C), whereas at higher inhibitor concentrations it was pseudo-irreversible (kass = 6.14 x 10(7) M-1.s-1). The complex was formed directly lacking the fast pre-equilibrium step with the dissociation equilibrium constant of approximately 8 pM. The competitive nature of inhibition was confirmed. The kass was found to be pH-independent between pH 6.0 and 7.5 and decreased at lower or higher pH values in a way that strongly suggests involvement of two ionizable groups in the interaction (pKi = 5.2, pK2 = 8.3). The enzyme-substrate interaction seems to be influenced by different ionizable groups (pKi = 4.4, pK2 = 7.8).

New data on the mechanism of interaction between proteolytic enzymes and protein inhibitors are presented. Most of known serine proteinase inhibitors bind to proteolytic enzymes through a substrate-like mechanism. A different mechanism is observed in the case of the inhibition of thrombin by hirudin and of papain by cystatin (from egg white) and stephin B. Protopeptides released during activation of some proteinase zymogens may act as efficient enzyme inhibitors. Such zymogens can be considered as covalently bound enzyme-inhibitor complexes.

Five monoclonal antibodies (MAbs), 77, 114, 138, 175 and 187, were established for rat cystatin A. MAbs 77, 114, 138 and 175 were shown to belong to the IgG1 subclass, whereas MAb 187 was an IgM. These MAbs partially suppressed inhibitory activity of rat cystatin A to papain. Their epitopes were mapped in detail on the molecule by examining the reactivities of the MAbs with NH2-terminally truncated forms and fragments of rat cystatin A by an enzyme-linked immunosorbent assay (ELISA), and by reactivity with the inhibitor on immunoblotting. In competitive binding assays the MAbs did not compete with each other, indicating that the epitopes recognized by these MAbs were substantially different. The conformational epitope recognized by the three MAbs 114, 138 and 175 belonged to one group that was highly sensitive to denaturation, but those epitopes were unchanged by NH2-terminal truncation. MAb 187 was able to recognize a linear epitope present in amino acid residues 15-50 in the NH2-terminal region. MAbs 77 and 114 reacted weakly with mouse cystatin A but not at all with human cystatin A, whereas MAb 187 reacted similarly with mouse cystatin A but at about half that level with human. The MAbs produced in this study should be useful tools for detecting conformational changes in the rat cystatin A molecule.

The use of oryzacystatins I and II, two cysteine proteinase inhibitors naturally produced in rice grains, represents an attractive way for the control of Coleoptera insect pests. The present study was done to analyze the inhibitory effect of recombinant oryzacystatins produced in Escherichia coli as fusion proteins against digestive proteinases of the major pest Colorado potato beetle (Leptinotarsa decemlineata Say). Both inhibitors had a significant effect on total proteolytic activity, but maximal inhibitions ranged from 20 to 80% for pHs varying from 5.0 to 7.0, respectively. This pH-dependent efficiency of plant cystatins was due to the selective inactivation of potato beetle cathepsin H, as demonstrated by the use of inhibitors with different specificities against cathepsins B and H. These results demonstrate the importance of having an adequate knowledge of insect proteinases specifically recognized by the inhibitors to be used in pest control strategies.

The effect of different protease inhibitors on the proteolytic processing of the plum pox potyvirus (PPV) polyprotein has been analyzed. Human cystatin C, an inhibitor of cysteine proteases, interfered with the autoprocessing of the viral papain-like cysteine protease HCPro. Unexpectedly, it also had an inhibitory effect on the autocatalytic cleavage of the Nla protease which, although it has a Cys residue in its active center, has been described as structurally related to serine proteases. Other protease inhibitors tested had no effect on any of the cleavage events analyzed.

Two endopeptidases are present in the seeds of Vigna aconitifolia (moth bean), and their activities increase during germination. One enzyme, which we term "vignain," can be assayed with benzyloxycarbonyl-phenylalanyl-arginyl-7-(4-methyl)coumarylamide as substrate. The second is legumain (EC 3.4.22.34), which can be assayed with benzyloxycarbonyl-alanyl-alanyl-asparaginyl-7-(4-methyl)-coumarylamide. The enzymes were purified, and their specificities for substrates and inhibitors were examined. Vignain has properties expected of a cysteine endopeptidase of the papain family, with the exception of a remarkably low reactivity with iodoacetate. Legumain is a very atypical cysteine endopeptidase, being insensitive to inhibition by chicken cystatin and E-64 (L-3-carboxy-2,3-trans-epoxypropionyl-leucyl-amido(4-guanidino )butane), and reacting more rapidly with iodoacetamide than with iodoacetate. We discuss our findings in relation to the literature on the proteolytic enzymes of legume seeds.

Human cystatin C variants in which the evolutionarily conserved Gly-11 residue has been replaced by residues with positively charged (Arg), negatively charged (Glu), bulky hydrophobic (Trp), or small (Ser or Ala) side-chains have been produced by site-directed mutagenesis and expression in Escherichia coli. The five variants were isolated and structurally verified. Their inhibitory properties were compared with those of wild-type recombinant cystatin C by determination of the equilibrium constants for dissociation (Ki) of their complexes with the cysteine endopeptidases papain and human cathepsin B and with the cysteine exopeptidase dipeptidyl peptidase I. The Ser-11 and Ala-11 cystatin C variants displayed Ki values for the two endopeptidases that were approx. 20-fold higher than those of wild-type cystatin C, while the corresponding values for the Trp-11. Arg-11 and Glu-11 variants were increased by a factor of about 2000. In contrast, the Ki values for the interactions of all five variants with the exopeptidase differed from that of wild-type cystatin C by a factor of less than 10. Wild-type cystatin C and the Ser-11, Ala-11 and Glu-11 variants were incubated with neutrophil elastase, which in all cases resulted in the rapid hydrolysis of a single peptide bond, between amino acid residues 10 and 11. The Ki values for the interactions with papain of these three N-terminal-decapeptide-lacking cystatin C variants were 20-50 nM, just one order of magnitude higher than the value for N-terminally truncated wild-type cystatin C, which in turn was similar to the corresponding values for the full-length Glu-11, Arg-11 and Trp-11 variants. These data indicate that the crucial feature of the conserved Gly residue in position 11 of wild-type cystatin C is that this residue, devoid of a side-chain, will allow the N-terminal segment of cystatin C to adopt a conformation suitable for interaction with the substrate-binding pockets of cysteine endopeptidases, resulting in high-affinity binding and efficient inhibition. The functional properties of the remaining part of the proteinase contact area, which is built from more C-terminal inhibitor segments, are not significantly affected even when amino acids with bulky or charged side-chains replace the Gly-11 residue of the N-terminal segment.

Cathepsin L is known as the most unstable lysosomal cysteine proteinase at neutral or alkaline pH. The kinetics of inactivation of human cathepsin L was studied by mixing the enzyme with a substrate and recording the release of product. The inactivation was found to be a first-order process, and the rate of the process decreased with the substrate concentration. The substrate-independent inactivation rate constant kinact was found to be 0.15 s-1 at pH 7.4 and 37 degrees C and increased 85-fold between pH 7.0 and 8.0. At pH 7.4, kinact increased 3200-fold between 5 and 37 degrees C with an energy of activation 174.7 kJ/mol. Inactive cathepsin L did not reactivate at pH 5.5. The rate of inhibition of cathepsin L by stefin B or chicken cystatin at pH 7.4 was much faster than the rate of spontaneous inactivation of the enzyme. The stefin B-cathepsin L complex incubated at pH 7.4 released active enzyme at pH 5.5, suggesting that the cysteine proteinase inhibitors might act as extracellular carriers of the cysteine proteinases.

Synthetic peptides which tentatively mimic the cystatin inhibitory surface were used to study the mechanism of inhibition of cysteine proteinases by their natural inhibitors. The inhibitory properties of these peptides depend mainly on the presence of the QxVxG consensus sequence. N and C-terminal peptide derivatives bearing large hydrophobic groups showed dramatically improved inhibition. Molecular dynamic studies after energy minimization showed that the non covalent interaction between these hydrophobic groups induced the formation of a loop structure which probably favours inhibition. Antibodies were raised against one of these peptides, which recognized kininogens in the serum of all mammal species tested, but not cystatins from family two.

Antibodies were raised against a synthetic dodecameric peptide KGAGQVVAGPWK (K12K), encompassing sequences thought to be important for the function of the cysteine proteinase inhibitors of the cystatin superfamily. These antibodies specifically recognized molecules of family 3, i.e., kininogens, in the serum of seven mammalian species tested in this study. The only notable exception was that of rat thiostatin (T kininogen) which is structurally related to the kininogen family. The antibodies also discriminated between family 2 (cystatins) and family 3 (kininogens) of the cystatin superfamily, since neither chicken cystatin nor human and rat cystatins C and S, which all belong to family 2 were recognized. The cystatin-like inhibitory domains resulting from fragmentation of human low molecular weight kininogen by bovine trypsin, were still recognized by antibodies, indicating that discrimination does not require two neighbouring inhibitory sites on the kininogen heavy chain. The antibodies blocked the capacity of kininogens to inhibit papain, suggesting that they recognize a conformational epitope at or near the kininogen inhibitory sites. The inhibitory properties of family 2 cystatins remained unchanged, confirming that members of this family do not interact with anti K12K antibodies. These antibodies are thus a new tool able to discriminate functionally and structurally between the members of the cystatin superfamily.

The effect of human recombinant cystatin C, a cysteine proteinase inhibitor, on bone resorption in vitro was evaluated. Bone resorption was assessed by analyzing the release of 45Ca and 3H from mouse calvarial bones prelabeled in vivo by injections with 45Ca or [3H]proline, respectively. In 24 h cultures, cystatin C (50 micrograms/ml) significantly inhibited the release of 45Ca and 3H stimulated by parathyroid hormone (PTH, 15 nmol/liter) or parathyroid hormone-related peptide of malignancy (PTHrP, 15 nmol/liter). The degree of inhibition caused by cystatin C in these 24 h cultures was similar to that caused by calcitonin (30 ng/ml). The inhibitory effect of cystatin C on 45Ca release induced by PTH was sustained in 96 h cultures, whereas the initial inhibition caused by calcitonin was transient. Cystatin C, 10-100 micrograms/ml, caused a dose-dependent inhibition of PTH (15 nmol/liter), and PTHrP (15 nmol/liter) stimulated 45Ca release. Addition of 50 micrograms/ml of cystatin C to mouse bone cultures inhibited the release of 45Ca induced by PTH and PTHrP at a wide range of submaximal and maximal concentrations of hormones (0.01-10 nmol/liter). No effect of cystatin C on 45Ca release in dead bones could be observed, nor did the inhibitor decrease the release of calcium in control bones. The inhibition by cystatin C on PTH-induced mineral mobilization was reversible. Cystatin C (1-100 micrograms/ml) did not affect protein synthesis or mitotic activities in mouse calvarial bones as assessed by the incorporation of [3H]proline and [3H]thymidine, respectively. These data show that cystatin C is a potent inhibitor of mineral mobilization and matrix degradation in cultured bones stimulated to resorb by PTH and PTHrP and that this effect is not due to general cytotoxicity.

Poliovirus, a picornavirus family member, requires the processing of its poly-protein by its own cysteine proteinase for replication. Oryzacystatin-I and oryzacystatin-II, proteinaceous cysteine proteinase inhibitors (cystatins) of rice seed origin, were found to inhibit the replication of poliovirus effectively in infected Vero cells in vitro. Truncated oryzacystatin-I, which lacks the NH2-terminal 25 amino acid residues of the intact protein, is an even more effective inhibitor, eliciting its effect at concentrations of less than 0.25 nmol/ml. The low molecular weight cysteine proteinase inhibitors, E-64, E-64C and loxistatin, showed no anti-viral effect at any concentration investigated.

In the near future salivary research will most likely play a rather important role in daily dental practice. The dentist will be expected to gather data concerning the patient's nutrition, medical history, oral complaints and xerostomia as perceived by the patient. In addition he may perform the following plain measurements: secretion rate of whole saliva in resting condition and after stimulation, and some parameters of importance for caries. The biochemical analysis of whole saliva and glandular salivas can be performed only in specialized laboratory. New developments are to be expected on the field of biotechnical synthesis of salivary protective factors, that is to say, histatins and cystatins in the nearby future and mucins on the longer term. New saliva substitutes and mouth rinses will be made on the basis of these artificially made proteins, not only to relieve xerostomia, but also to prevent oral infection and inflammation.

Previously, we suggested the participation of a hemocyte proteinase in the dissociation of fat body of Sarcophaga peregrina (flesh fly) at metamorphosis. We have now purified this proteinase to near homogeneity from pupal hemocytes. It is a cysteine proteinase with a molecular mass of 29 kDa and has a unique substrate specificity hydrolyzing both Suc-Leu-Leu-Val-Tyr-MCA and Z-Phe-Arg-MCA (Suc, succinyl; MCA, methylcoumaryl-7-amide; Z, carbobenzoxy), which are substrates for chymotrypsin and cathepsin B, respectively. Partial similarity was found between the amino-terminal sequence of this proteinase and that of cathepsin B, including Pro, Glu and Arg residues conserved in the papain superfamily of enzymes.

Cystatin C, the major inhibitor of the cysteine proteinases found in human and rat body fluids, is particularly abundant in seminal plasma and cerebrospinal fluid. In a precedent report, we have evidenced noteworthy levels of cystatin C in rat kidney cortex. In the present study, we show that rat mesangial glomerular cells produce cystatin C. Immunoprecipitation of extracts of metabolically labeled cells and culture media showed that the synthesized cystatin C is a 15.5 +/- 0.5 kDa protein. The protein was released into the culture supernatant (1.6 +/- 0.26 micrograms/10(6) cells/24 h). Urinary rat cystatin C and PPPR synthetic peptide (5-8 N-terminal sequence of rat cystatin C) increased mesangial cell proliferation. Affinity chromatography on Ultrogel-avidin-biotin-PPPR of extracts of metabolically labeled cells indicate the existence of a PPPR binding protein of 46 kDa. The results described in this work suggest, for glomerular rat mesangial cells in vitro, an autocrine regulation of proliferation by cystatin C.

A cDNA clone that encodes oryzacystatin, a cysteine protease inhibitor from rice, was isolated and expressed in Escherichia coli BL-21 (DE3) using an expression plasmid under the control of a T7 RNA polymerase promoter. The construct pT7OC 9b encoded a fusion protein containing 11 amino acid residues of the NH2 terminus of the bacterial protein phi 10 and 79 residues of oryzacystatin lacking 23 NH2-terminal residues of the wild-type protein. Recombinant oryzacystatin (ROC) constituted approximately 10% of the total bacterial protein mass and was purified in a single step by anion-exchange chromatography. The inhibitory activity of ROC toward papain (Ki = 3 x 10(-8) M) was comparable with that of the naturally occurring protein isolated from rice. Caseinolytic activity in midgut homogenates from seven species of stored product insects was inhibited from 18 to 85% by ROC, whereas the same activity was inhibited from 14 to 69% by the serine proteinase inhibitor phenylmethylsulfonyl fluoride. Midguts of stored product insects apparently contain both cysteine proteinases and serine proteinases, but the relative amounts vary with the species. When fed to the red flour beetle, Tribolium castaneum, 10 wt% ROC in the diet suppressed growth approximately 35% relative to that of the control group of insects.

The metastasis of malignant tumour cells depends on their rapid replication, and their ability to adhere to the matrix of a biological barrier such as basement membrane, to degrade the matrix, and to migrate through this more permeable barrier. Secreted enzymes, including the cysteine proteinases cathepsins B and L, are known to degrade basement membrane components. Using a barrier-free substratum we studied the possible role of cysteine proteinases in influencing the motility per se of metastatic cells. We found that stefins, the natural inhibitors of cysteine proteinases, markedly decreased the stimulated motility of both human melanoma cells and W256 carcinosarcoma cells at low concentrations (0.5 microM). A stefin also inhibited melanoma cell adherence, but to a lesser extent than motility. Additionally, synthetic inhibitors (E-64, diazomethyl ketones) of cysteine proteinases were found to depress stimulated motility of W256 cells. These results suggest that cysteine proteinases and their inhibitors may have a direct role in the development of a migratory response per se in tumour cells.

Cystatin C, a potent inhibitor of cysteine proteases such as papain and cathepsin B, was examined for its effect on human coronaviruses OC43 and 229e. Both viruses were greater than 99% inhibited by 0.1 mM inhibitor. Endpoint titrations showed that inhibiting activity paralleled that of leupeptin, a serine and cysteine protease inhibitor, and indicated that 1 to 2 microM inhibitor, slightly above physiologic levels, was effective.

Reversible GuHCl denaturation of human stefin A (25 degrees C, pH 8) was monitored by the tyrosine fluorescence, by circular dichroism in the near UV and by circular dichroism in the far UV. In each case a midpoint of 2.8 +/- 0.1 M GuHCl was obtained, demonstrating the cooperativity of the denaturation. Kinetics of the slow folding on diluting the protein from the GuHCl denatured state, was also measured by the three spectroscopic probes (10 degrees C, pH 8). Results conform to a sequential mechanism. Denaturant concentration and temperature dependence of the slow folding were measured by fluorescence. From a linear Arrhenius plot the Ea of 100 +/- 5 kJ/mol was read. 'Double mixing' experiments revealed a slow reaction going on in the unfolded state which influenced the amplitude of the fluorescence changes. 'Double mixing' experiments performed by FPLC have shown that the folding itself, i.e., the formation of a compact state, was not dependent on the time spent under unfolding conditions.

The high-Mr alkali-stable form of cathepsin B was purified from purulent human sputum. It was shown to solubilize proteoglycan monomer entrapped in polyacrylamide at a rate comparable with that of human lysosomal cathepsin B. Like the enzyme from lysosomes, sputum cathepsin B was bound by human alpha 2-macroglobulin, which inhibited its action on proteoglycan. Cystatin C in purulent sputum was shown to be the N-terminally truncated form generated by neutrophil elastase cleavage, and sputum cathepsin B was only weakly inhibited by recombinant cystatin C that had been cleaved by neutrophil elastase in vitro. Addition of neutrophil elastase to mucoid sputum led to a 5-fold increase in cathepsin B activity concomitant with a lowering in Mr of the cysteine proteinase from 40,000 to 37,000, i.e. the size of the active enzyme purified from purulent sputum. It is concluded that the high-Mr form of cathepsin B present in purulent sputum is a functional proteinase, unlike similar forms of the enzyme secreted by mammary gland in organ culture. The activity of cathepsin B in sputum is modulated by neutrophil elastase, by a combination of inhibitor inactivation and zymogen activation.

Three rat salivary cystatins (designated as RSC-1, RSC-2 and RSC-3) induced by chronic isoproterenol (IPR) treatment, but not detected in normal rats, were purified from submandibular saliva of chronically IPR-treated rats by Mono-Q, hydroxyapatite and TSKgel Phenyl-5PW chromatographies. Their molecular weights (Mr) and isoelectric points (pI) differed from each other as follows: RSC-1 (Mr 16,500, pI 4.4), RSC-2 (Mr 15,500, pI 4.4) and RSC-3 (Mr 14,500, pI 4.5). The amino acid compositions of these inhibitors were very similar and the three forms showed complete immunological identity in a double immunodiffusion system. The partial amino acid sequence results showed that these inhibitors belonged to family 2 of the cystatin superfamily. These three forms strongly inhibited the enzyme activities of ficin and papain, but not of cathepsin B and trypsin. The inhibition constants (Ki) of RSC-1, RSC-2 and RSC-3 for ficin were 0.19, 0.50 and 0.012 nM, and for papain were 1.5, 0.93 and 0.03 nM, respectively. RSC-3 inhibited ficin and papain more strongly than did RSC-1 and RSC-2.

Two cyclic peptides, Ac-CTKSQPNLDTC-NH2 (SA-LOOP1) and Ac-CSFQIYEVPWE DRMSLVNSRC-NH2 (SA-LOOP2) were prepared. These sequences are respectively found in the second and third exons of cystatin SA and are well conserved among the cystatins of family II. In addition, these sequences are extremely homologous to the inhibitory regions of several serine-proteinase inhibitors. The peptides were assayed for their inhibiting properties towards serine- and cysteine-proteinases. SA-LOOP1 inhibited porcine pancreatic trypsin (Ki = 370 microM), but did not inhibit cysteine-proteinases. SA-LOOP2 inhibited not only porcine pancreatic alpha-chymotrypsin (Ki = 23 microM) but also papain (Ki = 24 microM) and ficin (Ki = 52 microM). These data indicate that the exon-intron organization of the cystatin genes coinside with the structural and/or functional domains of the protein, and may have significant implications for understanding the active sites of cystatins.

Electrophoresis of midgut extracts from the rice weevil, Sitophilus oryzae, and the red flour beetle, Tribolium castaneum, in polyacrylamide gels containing sodium dodecyl sulfate and gelatin revealed there was one major proteinase (apparent molecular mass = 40,000) in the rice weevil and two major proteinases (apparent molecular masses = 20,000 and 17,000) in the red flour beetle. The pH optima using [3H]casein as substrate were about pH 6.8 for the rice weevil and pH 5.2 for the red flour beetle. Use of specific inhibitors, including L-trans-epoxysuccinyl-leucylamino-(4- guanidino)-butane (E-64), p-chloromercuriphenylsulfonic acid (PCMS), and oryzacystatin, indicated that nearly all of the proteinase activity against casein was contributed by cysteine proteinases. The estimated IC50 values for oryzacystatin were 2 x 10(-6) M and 4 x 10(-7) M when tested against midgut extracts from T. castaneum and S. oryzae, respectively.

The adsorption, at hydroxyapatite surfaces of neutral cystatin SN, acidic cystatin S and the phosphoserine-containing acidic cystatin S1 was compared to that of statherin. The effects of these adsorbed proteins on the constant-composition growth kinetics of hydroxyapatite were also studied. The neutral cystatin SN had a higher adsorption maximum than the acidic cystatins S and S1. Although the affinity of cystatin for hydroxyapatite surfaces was lower than that of statherin, their influence on the growth kinetics of hydroxyapatite was considerably greater, with the acidic cystatin S1 being the most active. At a surface concentration of 7.0 x 10(-8) mol m-2 hydroxyapatite, the cystatins decreased the rate of crystal growth by 80-95% as compared to that in the absence of protein. At this concentration, statherin showed a growth inhibition of 40%.

A cloned synthetic gene coding for (AEF S1M M29I M89L) chicken egg white cystatin was modified site-specifically at position Q53 by cassette mutagenesis. Two recombinant variants were isolated from a pIN-III-ompA E. coli expression system and purified by Cm-papain affinity chromatography. The mutations at the position 53 were confirmed by amino acid composition and amino acid sequence analysis of the appropriate tryptic peptides. The complexes of both cystatin variants, the Q53E- and Q53N-variant with papain, display Ki values similar to those determined with native chicken cystatin. However, the Ki values of the complexes with actinidin are hundredfold and with cathepsin B three hundredfold higher than with the native chicken cystatin. The different inhibition kinetics of these variants compared to wild type chicken cystatin emphasizes the specificity of single amino acid substitutions for optimal contacts between the binding segments of enzyme and inhibitor.

Human cystatins A, B and C were purified, and their inhibition efficiency was tested with the cysteine proteinase cathepsin S. Cathepsin S was strongly inhibited by cystatins A and B in the subnanomolar range and by cystatin C in the picomolar range. Two steps of inhibition of cathepsin S by the cystatins which involve slow binding are discussed.

Natural and recombinant variants of the cysteine proteinase inhibitors chicken cystatin and human stefin B were characterized by determination of their inhibition constants for papain, actinidin and human cathepsins B and H. The individual contributions of the three contact regions to the binding energy of the chicken cystatin-papain complex were calculated as 36% for the N-terminal segment, 51% for the first and 13% for the second hairpin loop. Removal of the N-terminal contact region of chicken cystatin resulted in a 10000-fold lower affinity for papain. In contrast, stefin B remained a tight-binding inhibitor of papain and actinidin without its N-terminal segment. Affinity of stefin B for papain was only slightly affected by exchange of the residue predicted to bind in the S2 subsite of papain. The essential contribution of the first hairpin loop to inhibitor binding was confirmed by the 240-fold lower affinity for papain of a Val48----Asp mutant of stefin B. Inhibition of cathepsin B by stefins A and B is slow-binding. Binding of stefin B, not of stefin A, follows a two-step mechanism involving a slow isomerisation of the enzyme-inhibitor complex.

Seeds of Wisteria floribunda contain several kinds of cysteine proteinase inhibitor (cystatin). We purified and characterized one of these inhibitors, named WCPI-3. The molecular weight of WCPI-3 was estimated to be 17,500 and 15,700 by gel filtration and SDS-PAGE, respectively. The isoelectric point was 5.7. WCPI-3 formed an equimolar complex with native papain and the dissociation constant was estimated to be 6.1 nM. Complex formation between WCPI-3 and Cys25-modified papain, such as S-carboxy-methylated or S-carbamoylmethylated papain, could not be observed by gel filtration or native PAGE analysis. A peptide fragment derived from WCPI-3 digested by Achromobacter proteinase (lysyl endopeptidase) had the amino acid sequence of VVAGVNYRFVLK. The VVAG sequence in this fragment corresponds to the conserved sequence QVVAG which is considered to be one of binding regions to cysteine proteinases. The amino acid sequence of the amino-terminal portion (34 residues) of WCPI-3 was highly homologous to that of oryzacystatin from rice seeds.

Oryzacystatin (oryzacystatin-I) is a proteinaceous cysteine proteinase inhibitor (cystatin) in rice seeds and is the first well defined cystatin of plant origin. In this study we isolated cDNA clones for a new type of cystatin (oryzacystatin-II) in rice seeds by screening with the oryzacystatin-I cDNA probe. The newly isolated cDNA clone encodes 107 amino acid residues whose sequence is similar to that of oryzacystatin-I (approximately 55% of identity). These oryzacystatins have no disulfide bonds, and so could be classified as family-I cystatins; however, the amino acid sequences resemble those of family-II members more than family-I members. Oryzacystatin-I and -II are remarkably distinct in two respects: 1) their specificities against cysteine proteinases; and 2) the expression patterns of their mRNAs in the ripening stage of rice seeds. Oryzacystatin-I inhibits papain more effectively (Ki 3.0 x 10(-8) M) than cathepsin H (Ki 0.79 x 10(-6) M), while oryzacystatin-II inhibits cathepsin H (Ki 1.0 x 10(-8) M) better than papain (Ki 0.83 x 10(-6) M). The mRNA for oryzacystatin-I is expressed maximally at 2 weeks after flowering and is not detected in mature seeds, whereas the mRNA for oryzacystatin-II is constantly expressed throughout the maturation stages and is clearly detected in mature seeds. Western blotting analysis using antibody to oryzacystatin-II showed that, as is the case with oryzacystatin-I, oryzacystatin-II occurs in mature rice seeds. Thus, these two oryzacystatin species are believed to be involved in the regulation of proteolysis caused by different proteinases.

The inhibitory specificity of a protein from potato tubers that inhibits cysteine proteinases (potato cysteine proteinase inhibitor, PCPI) has been compared with that of chicken egg-white cystatin. Most proteinases that are inhibited by cystatin were also inhibited by PCPI, but the potato inhibitor inhibited stem bromelain and fruit bromelain, which are not inhibited by cystatin, and for which no protein inhibitor of comparable potency has previously been described. In contrast, papaya proteinase IV was unaffected by PCPI as it is by the cystatins, and the exopeptidase, dipeptidyl peptidase I, is inhibited by cystatins, but was unaffected by PCPI. The differences in inhibitory specificity between these proteins may well reflect differences between superfamilies of cysteine proteinase inhibitors.

Recent work has demonstrated that a tripeptide derivative mimicking the active proteinase-binding site of cystatin C, a human cysteine proteinase inhibitor, can block growth of group A streptococci and replication of herpes simplex virus (HSV). In the case of HSV, intact cystatin C was also found to inhibit replication of the virus. Many streptococcal strains and HSV-infected cells produce immunoglobulin (Ig)-binding proteins, and a possible connection between such proteins and proteolytic activity was indicated by the finding that bacterial Ig-binding proteins also show affinity for proteinase inhibitors. The significance of these various observations is not clear, but available data suggest that proteinases play a role in vital microbial functions (e.g. viral replication) and may be utilized as targets for antimicrobial agents. The results discussed here also indicate that peptide derivatives based on the structure of proteinase inhibitors occurring in nature could be used as such agents.

Two isoelectric forms of human cystatin C with pI 9.2 and 7.8 have been isolated from urine of patients with different nephrological disorders. Treatment of both forms with alkaline phosphatase revealed that the difference between them is not due to the phosphorylation of some amino-acid residue. Further purification of cystatin C with pI 9.2 by hydrophobic chromatography and N-terminal sequencing showed that it consists predominantly of the full-length form of cystatin C with the N-terminal sequence SSPG-. Cystatin C with pI 7.8 was separated into two peaks. The first represented a pure form truncated by an octapeptide and beginning with the N-terminal sequence LVGG-. The second was a mixture containing 33% of the first peak and 66% of a truncated form with the N-terminal sequence VGGP-. Inhibitory activity of the full-length cystatin C and the pure truncated form has been measured against cathepsins B, H and L and show no significant differences in Ki values. These results further support the proposed mechanism of interaction of cysteine proteinases with their inhibitors cystatins (Bode, W., Engh, R., Musil, D., Thiele, U., Huber, R., Karshikow, A., Brzin, J., Kos, J. & Turk, V. (1988) EMBO J. 7, 2593-2599).

Cystatin domains or homologous sequences were synthesized and tested as inhibitors of papain, and rat brain cathepsins B and L. These domains included: I, an enzyme substrate binding site containing a -GG- cleavage site (YGGFL); II, known cystatin consensus sequences (-QVVAG- or -QLVSG-); and III, the proposed ancillary site for binding of chicken cystatin to papain (-IPWLN-). A Domain II analog QVVAG(K-NH2) inhibited cathepsin L and papain with Ki 1-4 X 10(-4) M but was inactive towards cathepsin B. A peptide containing Domains I and II, YGGFL-QVVAG(K-NH2), inhibited papain and cathepsin B with Ki 10(-4)-10(-5) M, and cathepsin L with Ki 10(-6) M. The presence of Domain III in the analog YGGFL-QVVAG-IPWLN(K-NH2) resulted in a 10-fold increase in potency towards papain. These data demonstrated that putative cystatin domains are: 1) probably proximal in the intact cystatins; 2) can be linked directly to each other to yield smaller peptides active as inhibitors; 3) showed some specificity towards the three cysteine proteinases.

Cystatin C, a cysteine proteinase inhibitor has recently been suggested to be a potent regulator of inflammatory processes and may act in defense against viral and bacterial infections. Two common forms of the protein were purified from the urine of a patient having received a renal transplant. The slow form of cystatin C possessed the N-terminal tetrapeptide Lys Pro Pro Arg, which was cleaved in the fast form. This peptide sequence, called postin, was synthesized. The three molecules, slow and fast forms of cystatin and the synthetic peptide, were tested for their effects on the migration activity of human polymorphonuclear neutrophils (PMNs). The slow form was found to display both chemotactic and chemokinetic activities, while the fast form and postin were only chemokinetic. Nevertheless, all the substances could induce a "motile" morphology. In addition, the two forms of cystatin C were powerful inhibitors of PMN chemotaxis induced by complement-derived chemotactic factors. This suggests that cystatin C in its two different cleaved forms and the N-terminal tetrapeptide can modulate PMN locomotion. Cysteine proteases may therefore play a role in neutrophil migration activity.

Cystatin C, a cysteine protease inhibitor, has recently been suggested to be a potent regulator in inflammatory processes. Human cystatin C was isolated from the urine of one patient suffering from tubular disorders and was tested for its effects on two functions of human polymorphonuclear neutrophils (PMN): O2- release and phagocytosis. Slow-form or (des 1-4) cystatin C and fast-form or (des 1-8) cystatin C differed by the presence in (des 1-4) cystatin C only of the N-terminal tetrapeptide Lys-Pro-Pro-Arg. Whereas (des 1-8) cystatin C did not seem to interfere with PMN functions at physiological concentrations, (des 1-4) cystatin C induced an inhibition of PMN phagocytosis-associated respiratory burst in response to opsonized zymosan particles. The inhibition may be attributed to the tetrapeptide Lys-Pro-Pro-Arg which has been synthesized and shown to have the same inhibitor effects, at concentrations similar to those required for (des 1-4) cystatin C. These results support a potential role for cystatin C as a modulator during inflammation.

The pineapple plant (Ananas comosus) was shown to contain at least four distinct cysteine proteinases, which were purified by a procedure involving active-site-directed affinity chromatography. The major proteinase present in extracts of plant stem was stem bromelain, whilst fruit bromelain was the major proteinase in the fruit. Two additional cysteine proteinases were detected only in the stem: these were ananain and a previously undescribed enzyme that we have called comosain. Stem bromelain, fruit bromelain and ananain were shown to be immunologically distinct. Enzymic characterization revealed differences in both substrate-specificities and inhibition profiles. A study of the cysteine proteinase derived from the related bromeliad Bromelia pinguin (pinguinain) indicated that in many respects it was similar to fruit bromelain, although it was found to be immunologically distinct.

Papaya proteinase IV (PPIV) is not inhibited by chicken cystatin, or human cystatins A or C, unlike most other proteinases of the papain superfamily. The enzyme inactivates chicken cystatin and human cystatin C by limited proteolysis of the glycyl bond previously shown to be involved in the inhibitory inactivity of the cystatins, but has no action on cystatin A. Contamination of commercial crystalline papain with PPIV accounts for the limited proteolysis of cystatins by 'papain' reported previously. PPIV is slowly bound by human alpha 2-macroglobulin. The enzyme is irreversibly inactivated by E-64, and by peptidyl diazomethanes containing glycine in P1 and a hydrophobic side-chain in P2. The reaction of PPIV with iodoacetate is extremely slow. PPIV is inhibited by peptide aldehydes despite the presence of bulky sidechains in P1, suggesting that these reversible inhibitors do not bind as substrate analogues.

Cystatin C is a human cysteine proteinase inhibitor present in extracellular fluids. Cystatin C and a tripeptide derivative (Z-LVG-CHN2) that mimics its proteinase-binding center, were tested for possible antiviral activity against herpes simplex virus type 1 (HSV) and poliovirus type 1. Both recombinant cystatin C and Z-LVG-CHN2 displayed strong inhibitory effects on HSV replication, whereas no significant effect on poliovirus replication was seen. The molar concentration of cystatin C that gave total inhibition of HSV replication was lower than that of either Z-LVG-CHN2 or of acyclovir, the drug currently most used against HSV infections. These results suggest that cysteine proteinase inhibitors might play a physiological role as inhibitors of viral replication and that such proteinase inhibitors, or peptide derivatives that mimic their proteinase-binding centers, might be used as antiviral agents.

Elevated activities of cysteine proteinases such as cathepsins B and L and cancer procoagulant have been linked to tumor malignancy. In the present study we examined the hypothesis that these elevated activities could be due to impaired regulation by the endogenous low molecular mass cysteine proteinase inhibitors (cystatins). Inhibitors from human sarcoma were compared to those from human liver, a normal tissue in which the inhibitors had been characterized previously. An extract of cystatins from sarcoma was less effective against papain and cathepsin B (liver or tumor) than was an extract from liver. This reduced inhibitory capacity in sarcoma was not due to a reduction in either the concentrations or specific activities of the cystatins or an absence of any family or isoform of cystatins. We purified two members of the cystatin superfamily (stefin A and stefin B) to homogeneity and determined their individual inhibitory properties. Stefins B from liver and sarcoma exhibited comparable inhibition of papain and cathepsin B. In contrast, stefin A from sarcoma exhibited a reduced ability to inhibit papain, human liver cathepsins B, H and L and human and murine tumor cathepsin B. The Ki for inhibition of liver cathepsin B by sarcoma stefin A was 10-fold higher than that for inhibition of liver cathepsin B by liver stefin A, reflecting a reduction in the rate constant for association and an increase in the rate constant for dissociation. Cancer is now the third pathologic condition reported to be associated with alterations in cystatins, the other two being amyloidosis and muscular dystrophy.

Rat cystatin C was purified to apparent homogeneity from rat urine after induction of a tubular dysfunction with sodium chromate. Twentyfold concentrated urine was chromatographed by a rapid purification procedure. A two-step purification including affinity chromatography on carboxymethyl papain- Sepharose and high-resolution anion exchange chromatography was developed. The purified protein has an apparent molecular mass of 15 kDa and pI of 10.2; its aminoacid composition was similar to human cystatin C. As opposed to previous data, purified urinary rat cystatin C did not contain significant amounts of carbohydrate. Antisera against rat cystatin C, raised in rabbits, partially cross-reacted with human and mouse cystatin C, indicating their antigenic similarities. Like human cystatin C, native rat cystatin C, named slow form, is degraded into a more acidic form, called fast form, by a loss of N-terminal amino acids; fast form displayed a pI of 9.4.

Stefin A, a cysteine proteinase inhibitor of the cystatin superfamily, has been found to be most abundant in epidermal cells. In order to determine its cellular function, we have expressed human stefin A in Escherichia coli using plasmid expression vectors under the control of bacteriophage T7 RNA polymerase. The heat-stable, antibody-positive bacterial product was isolated using a papain-Sepharose affinity column and was shown to inhibit two cysteine proteinases, papain and human cathepsin B. Recombinant stefin A may have commercial and therapeutic potential in situations requiring inhibition of cysteine proteinase activities, and in cosmetics, as an ingredient in skin creams.