Autocatalytic processing of recombinant human procathepsin L. Contribution of both intermolecular and unimolecular events in the processing of procathepsin L in vitro

The autocatalytic processing of procathepsin L was investigated in vitro using purified recombinant proenzyme expressed in Pichia pastoris. Pure intermolecular processing was studied by incubating the mutant procathepsin L (C25S), which cannot autoactivate with a small amount of mature active cathepsin L. The results clearly establish that, contrary to recent reports, intermolecular processing of procathepsin L is possible. The main cleavage sites are located at or near the N terminus of the mature enzyme, in an accessible portion of the proregion, which contains sequences corresponding to the known substrate specificity of cathepsin L. Contrary to procathepsins B, K, and S, autocatalytic processing of procathepsin L can generate the natural mature form of the enzyme. A continuous assay using the substrate benzyloxycarbonyl-Phe-Arg 4-methylcoumarinyl-7-amide hydrochloride has also been used to obtain information on the nature of the steps involved in the autocatalytic processing of wild-type procathepsin L. Processing is initiated by decreasing the pH from 8.0 to 5.3. The influence of proenzyme concentration on the rate of processing indicates the existence of both unimolecular and bimolecular steps in the mechanism of processing. The nature of the unimolecular event that triggers processing remains elusive. Circular dichroism and fluorescence measurements indicate the absence of large scale conformational change in the structure of procathepsin L on reduction of pH. However, the bimolecular reaction can be attributed to intermolecular processing of the zymogen.     

Self-activation of recombinant human lysosomal procathepsin D at a newly engineered cleavage junction, "short" pseudocathepsin D

To obtain a recombinant model of human cathepsin D with kinetic properties that are identical with native human liver enzyme, we have addressed the significant differences in structure and catalytic function between naturally occurring enzyme and bacterially derived pseudocathepsin D. Human procathepsin D was expressed in a baculovirus system to obtain correctly folded, glycosylated enzyme that upon acidification completely converts to the active intermediate, pseudocathepsin D. The oligosaccharide moieties of this recombinant enzyme contributed to about 5% of the apparent molecular mass of the enzyme, and the carbohydrate composition was quite similar to the native material. However, specificity constants (kcat/Km) of this glycosylated pseudoform for several synthetic chromogenic substrates were considerably less (33%-50%) than those for the native enzyme and were virtually identical with those observed with nonglycosylated pseudocathepsin D. A cleavable junction suitable for self-processing at the normal maturation point of human cathepsin D was engineered into procathepsin D according to known specificity requirements of this enzyme, and the construct was expressed using baculovirus. Following experiments that demonstrated that the new proenzyme failed to process to the expected point, the new cleavage junction was moved 6 residues toward the amino terminus of procathepsin D and expressed in Escherichia coli. After refolding, the protein containing the newly engineered junction self-processed, generating a shortened mutant form of pseudocathepsin D that is 6 residues longer at the amino terminus than the native material. The kinetic properties of this newly engineered pseudoform proved to be identical with those of the native enzyme, thus establishing an improved recombinant model for this important aspartic proteinase.     

The crystal structure of human procathepsin K

Cathepsin K is a cysteine protease present in human osteoclasts that plays an important role in bone resorption. Cathepsin K is synthesized as an inactive proenzyme and activated under conditions of low pH. Autoproteolytic processing of the N-terminal 99 amino acid propeptide produces the active, mature form of cathepsin K. It is presumed that the activation of procathepsin K in vivo occurs in the bone resorption pit, which has a low-pH environment. We have determined the structure of human procathepsin K at 2.8 A resolution. The structure of the mature enzyme domain within procathepsin K is virtually identical to that of mature cathepsin K. The fold of the propeptide of procathepsin K is similar to that observed in procathepsins B and L despite differences in length and sequence. A portion of the propeptide occupies the active site cleft of cathepsin K. Hydrophobic interactions, salt bridges, and hydrogen-bonding interactions are observed in the structure of the propeptide and between the propeptide and the mature enzyme of procathepsin K. These interactions suggest an explanation for the stability of the proenzyme. The structure of procathepsin K contributes to an understanding of the molecular basis of inhibition by the propeptide portion of the molecule and activation of this important member of the cysteine protease family.     

Involvement of carboxy-terminal amino acids in secretion of human lysosomal protease cathepsin L

Cathepsin L, a lysosomal cysteine protease, is overexpressed and secreted by malignantly transformed cells. However, the reason for secretion of this man 6-phosphate-containing lysosomal protease into the extracellular medium is not clear. We wished to determine whether there is a region within the primary sequence of the proenzyme form of cathepsin L which affects its subcellular and extracellular localization. High-level transient expression of human procathepsin L in mouse NIH 3T3 cells results in the secretion of most of this protein into the extracellular medium. At the same time, the endogenous mouse procathepsin L in these nontransformed cells is found in its usual location in lysosomes. Mutants of human procathepsin L with carboxy-terminus deletions involving the last 11 amino acids are not secreted into the medium. Deletion of as little as two amino acids, Thr and Val, from the carboxy terminus, blocked the secretion of the protein but did not affect its enzyme activity, posttranslational processing, or subcellular distribution. Replacement of Thr-Val by two bulky amino acids Tyr-Asn allowed secretion of the procathepsin L, but the replacement of these two amino acids by nonbulky alanines prevented its secretion. Single alanine substitutions of the last six amino acids (ASYPTV) indicated that substitution by alanine of Y or T does not affect the secretion of hproCAT L, but alanine substitutions of S, P, or V completely blocked its secretion into the culture medium. We therefore conclude that the carboxy terminus of procathepsin L contains a sequence essential for its secretion.     

Radiofrequency capacitive hyperthermia for unresectable hepatic cancers

To determine the involvement of proteinases with hydrolytic activity towards extracellular matrix and basement membrane, in invasion and metastasis of tumour cells, the expression of cathepsin D, an aspartic proteinase, and cathepsin B, a cysteine proteinase, was studied. Formalin-fixed paraffin-embedded specimens from 13 patients who had squamous cell carcinomas (SCC) with local recurrence, skin and/or lymph node metastasis were examined. Cathepsin D stained intensely as a granular pattern (mature enzyme) in tumour cells of 69% of primary lesions and all the secondary lesions of the patients with SCC. Cathepsin B stained more intensely in SCC cells of all of the primary and secondary lesions than in normal epidermis; staining patterns were almost diffuse (procathepsin B). Granular and diffuse patterns (mature enzyme of cathepsin D and procathepsin B, respectively) appeared in the outer and inner parts of tumour islands, respectively. The presence of the active mature form of cathepsin D and procathepsin B in metastatic skin lesions of SCC was confirmed by Western blotting analysis. The presence and localization of the active mature form of cathepsin D suggests that activated cathepsin D may be involved in the invasion and metastasis of SCC.     

Recombinant expression and localization of Schistosoma mansoni cathepsin L1 support its role in the degradation of host hemoglobin

Cysteine proteinases expressed by schistosomes appear to play key roles in the digestion of host hemoglobin, the principal source of amino acid nutrients utilized by these parasites. We have shown previously that the predominant cysteine proteinase activity in soluble extracts and excretory/secretory (ES) products of adults of Schistosoma mansoni and S. japonicum is cathepsin L-like in its substrate specificity. However, biochemical analysis of the cathepsin L activity in extracts and ES products of schistosomes has been complicated by the presence of at least two distinct forms of schistosome cathepsin L, termed SmCL1 and SmCL2. We now report the purification and enzyme characteristics of active, recombinant SmCL1 which was obtained by transforming Saccharomyces cerevisiae with an expression plasmid encoding the preproenzyme of SmCL1. Recombinant SmCL1 was secreted by the transformed yeast into the culture media from which it was purified by gel filtration and ion-exchange chromatography. The purified enzyme exhibited substrate specificity against synthetic peptidyl substrates (e.g., Boc-Val-Leu-Lys-NHMec and Z-Phe-Arg-NHMec; kcat/Km = 17.25 and 6.24 mM-1 s-1, respectively) and against gelatin and hemoglobin, characteristic of cathepsin L. Immunoblot analysis using antiserum raised against recombinant SmCL1 demonstrated that native SmCL1 of 33 kDa was present in ES products and soluble extracts of S. mansoni. Using this antiserum and thin tissue sections, we localized the native SmCL1 to the gastrodermis and to the tegument of adult schistosomes. Recombinant SmCL1 was capable of degrading human hemoglobin at pH 4.0 to 4.5 but not higher, suggesting that denaturation of hemoglobin by low pH, as found in the cecum of the adult schistosome, may be necessary for its catalysis by cathepsin L and other gut-associated proteinases. Together, these results support a role for SmCL1 in the degradation of host hemoglobin within the gut of the schistosome.     

Effect of helicobacter pylori vacuolating toxin on maturation and extracellular release of procathepsin D and on epidermal growth factor degradation

The effect of vacuolating toxin (VacA) from Helicobacter pylori on endosomal and lysosomal functions was studied by following procathepsin D maturation and epidermal growth factor (EGF) degradation in HeLa cells exposed to the toxin. VacA inhibited the conversion of procathepsin D (53 kDa) into both the intermediate (47 kDa) and the mature (31 kDa) form. Nonprocessed cathepsin D was partly retained inside cells and partly secreted in the extracellular medium via the constitutive secretion pathway. Intracellular degradation of EGF was also inhibited by VacA with a similar dose-response curve. VacA did not alter endocytosis, cell surface recycling, and retrograde transport from plasma membrane to trans-Golgi network and endoplasmic reticulum, as estimated by using transferrin, diphtheria toxin, and ricin as tracers. Subcellular fractionation of intoxicated cells showed that procathepsin D and nondegraded EGF accumulate in lysosomes. Measurements of intracellular acidification with fluorescein isothiocyanate-dextran revealed a partial neutralization of the lumen of endosomes and lysosomes, sufficient to account for both mistargeting of procathepsin D outside the cell and the decreased activity of lysosomal proteases.     

Importance of the Arg-Gly-Asp triplet in human thrombin for maintenance of structure and function

Site-directed mutagenesis was employed to assess the importance of the Arg-Gly-Asp triplet that comprises residues 197 to 199 in the B-chain of thrombin. Properties of the R197E and the D199E variants were compared with those of zeta-thrombin and the inactive S205A variant wherein the active site Ser is replaced by Ala. Relative to zeta-thrombin, the R197E thrombin variant under the assay conditions used exhibits 26% activity toward a small chromogenic substrate, 13% activity in the activation of protein C in the presence of thrombomodulin, < 3% activity in processing fibrinogen, and 1% activity in inducing platelet activation. Thus, the substrate specificity of thrombin was altered by the R197-->E replacement. The D199E variant was essentially inactive. It exhibited only 0.02% of the activity of thrombin toward the chromogenic substrate and its reactivity toward the active site-directed alkylating agent D-Phe-Pro-Arg-CH2Cl was 10,000-fold lower than that of thrombin. Like the inactive S205A thrombin variant, the D199E variant antagonized the interactions of thrombin with hirudin and thrombomodulin, but was a less effective antagonist. The dependence of the antagonism of the thrombin-thrombomodulin interaction on the concentration of D199E thrombin variant provided evidence suggesting the presence of two or more domains in thrombin that independently interact with their counterparts in thrombomodulin. Although the S205A thrombin variant antagonized the action of thrombin on platelets no such activity could be demonstrated for the D199E variant in the concentration range studied (< 800 nm). Comparison of the circular dichroism spectra of zeta-thrombin, the D199E, R197E, and S205A variants indicated that subtle differences in conformation exist between the D199E variant and the other thrombins. These differences in conformation might well account for the altered behavior of the D199E variant with respect to its interactions toward thrombomodulin, hirudin, and platelets.     

Treatment of subdural effusion with hydrocephalus after ruptured intracranial aneurysm clipping

This study examined the role of cysteine proteinases and their inhibitor in the development of emphysema in comparison with neutrophil elastase (NE) complexed with alpha1-protease inhibitor (NE-alpha1-PI), which was previously demonstrated to be increased in bronchoalveolar lavage (BAL) fluid from subjects with subclinical emphysema. Eight nonsmokers and 31 current smokers with (n=17) and without (n=14) emphysema, as evidenced by lung computed tomographic scans, were studied. The concentrations of immunologically detected cathepsin L and cystatin C, but not cathepsin B, were significantly increased in BAL fluid from the smokers with emphysema compared with those without emphysema, although the activity of cathepsin L, measured using a synthetic substrate and cathepsin L, released from cultured alveolar macrophages at 24 h, did not show any significant difference between the two groups. When comparison was made only for the subjects aged <60 yrs, the difference between the two groups disappeared for cathepsin L, but remained for NE-alpha1-PI. There was no significant correlation between the level of cathepsin L and that of NE-alpha1-PI in BAL fluid from the subjects with emphysema. In conclusion, increased levels of cathepsin L and cystatin C were demonstrated in bronchoalveolar lavage fluid from subjects with subclinical emphysema. However, the roles of cathepsin L and neutrophil elastase in the development of emphysema may vary between subjects and between the young and the old.     

Cathepsin D associates with lysosomal membranous protein

The membrane-association of early biosynthetic form of cathepsin D has been demonstrated in hepatoma cells, and this membrane-association is not mediated by mannose 6-phosphate residues, implying that a mannose 6-phosphate receptor-independent mechanism operates in the sorting of cathepsin D. In this paper, to demonstrate whether cathepsin D is associated with the lysosomal membranes, an in vitro binding experiment was carried out employing lysosomal cathepsin D or microsomal procathepsin D isolated from rat liver. Immunoblotting analysis revealed that an intermediate form of cathepsin D was associated with the lysosomal membranes; this lysosomal membrane-associated cathepsin D was released from the membranes by washing with Na2CO3 (pH 10.6) but not with solutions containing mannose 6-phosphate. This suggested that cathepsin D associates with the membranes by ionic-interaction, and that the membrane-associated cathepsin D resides as a peripheral membrane protein in the lysosomal membrane fraction. To confirm that the intermediate form of cathepsin D specifically interacts with the lysosomal integral membrane proteins, the lysosomal membrane fraction was treated with trypsin and the binding experiment was conducted. The result showed that the binding capacity of cathepsin D to the lysosomal membranes was apparently abolished and cathepsin D did not rebind to the membranes. These data suggest that the intermediate form of cathepsin D is preferentially recognized by the lysosomal membranous protein which complements the mannose 6-phosphate receptor-dependent intracellular sorting mechanism.     

Modification of ribonuclease T1 specificity by random mutagenesis of the substrate binding segment

Attempts to modify the guanine specificity of ribonuclease T1 (RNase T1) by rationally designed amino acid substitutions failed so far. Therefore, we applied a semirational approach by randomizing the guanine binding site. A combinatorial library of approximately 1.6 million RNase T1 variants containing permutations of 6 amino acid positions within the recognition loop was screened on RNase indicator plates. The specificity profiles of 180 individual clones showing RNase activity revealed that variant K41S/N43W/N44H/Y45A/E46D (RNaseT1-8/3) exhibits an altered preference toward purine nucleotides. The ApC/GpC preference in the cleavage reaction of this variant was increased 4000-fold compared to wild-type. Synthesis experiments of dinucleoside monophosphates from cytidine and the corresponding 2'3'-cyclic diesters using the reverse reaction of the transesterification step showed a 7-fold higher ApC synthesis rate of RNase 8/3 than wild-type, whereas the GpC synthesis rates for both enzymes were comparable. This study shows that site-directed random mutagenesis is a powerful additional tool in protein design in order to achieve new enzymatic specificities.     

Mechanism-based inhibitors of serine proteinases based on the Gabriel-Colman rearrangement

Neutrophil-derived mediators such as, for example, the serine proteinase elastase, cathepsin G and proteinase 3, play a critical role in inflammatory lung disease. This report describes the design, synthesis and in vitro inhibitory activity of some novel mechanism-based inhibitors of human leukocyte elastase and cathepsin G. The design of the inhibitors is based on the Gabriel-Colman rearrangement. The behavior of the synthesized compounds toward elastase and cathepsin G with respect to inhibitory prowess, mode of interaction, specificity, etc., has been found to be dependent on the recognition and reactivity elements present in each inhibitor.     

Purification and properties of rat lung soluble glutathione peroxidase

Gluthathione peroxidase (gluthatione:hydrogen-peroxide oxidoreductase, EC 1.11.1.9) has been purified approximately 2700-fold from rat lung soluble fraction. The purified enzyme was shown to be homogeneous by sodium dodecyl sulfate/urea polyacrylamide gel electrphoresis. Selenium-75 tracer cochromatographed with the enzyme activity, indicating that rat lung soluble gluthathione peroxidase is a selenium enzyme. The enzyme had an approximate molecular weight of 80000 and contained four identical subunits. The optimal activity of the enzyme was at between pH 8.8 and 9.1. The enzyme had general specificity toward hydroperoxides, and high specificity for reduced glutathione. The kinetic behavior or the purified lung soluble glutathione peroxidase followed a ping-pong-like mechanism; the enzyme first reduced the lipid hydroperoxide substrate to the corresponding hydroxy fatty acid, then was regenerated to the native form by reduced glutathione.     

Major increase in endopeptidase activity of human cathepsin B upon removal of occluding loop contacts

The main feature distinguishing cathepsin B from other cysteine proteases of the papain family is the presence of a large insertion loop, termed the occluding loop, which occupies the S' subsites of the enzyme. The loop is held in place mainly by two contacts with the rest of the enzyme, involving residues His110 and Arg116 on the loop that form salt bridges with Asp22 and Asp224, respectively. The influence of this loop on the endopeptidase activity of cathepsin B has been investigated using site-directed mutagenesis and internally quenched fluorogenic (IQF) substrates. Wild-type cathepsin B displays poor activity against the substrates Abz-AFRSAAQ-EDDnp and Abz-QVVAGA-EDDnp as compared to cathepsin L and papain. Appreciable increases in kcat/KM were observed for cathepsin B containing the single mutations D22A, H110A, R116A, and D224A. The highest activity however is observed for mutants where both loop to enzyme contacts are disrupted. For the triple-mutant D22A/H110A/R116A, an optimum kcat/KM value of 12 x 10(5) M-1 s-1 was obtained for hydrolysis of Abz-AFRSAAQ-EDDnp, which corresponds to a 600-fold increase relative to wild-type cathepsin B and approaches the level of activity observed with cathepsin L or papain. By comparison, the mutations have little effect on the hydrolysis of Cbz-FR-MCA. The influence of the mutations on the pH dependency of activity also indicates that the complexity of pH activity profiles normally observed for cathepsin B is related to the presence of the occluding loop. The major increase in endopeptidase activity is attributed to an increase in loop "flexibility" and suggests that the occluding loop might move when an endopeptidase substrate binds to the enzyme. The possible contribution of these interactions in regulating endopeptidase activity and the implications for cathepsin B activity in physiological or pathological conditions are discussed.     

Processing of procathepsin from Musca domestica eggs

The major source of amino acids for insect embryos are yolk proteins which accumulate in developing oocytes and are hydrolyzed during embryogenesis. Studies on Musca domestica embryogenesis indicated that a cathepsin B-like proteinase is responsible for yolk protein degradation (Ribolla et al., 1993). In this study, we report the purification of mature cathepsin and show that it is made up of a single 41 kDa polypeptide chain. The Musca domestica cathepsin NH2-terminal 11-residue sequence was determined (Ala-Pro-Lys-Tyr-Val-Asp-Tyr-Gly-Glu-Asn-Glu) and reveals homology with other cathepsins of the papain family. Experiments using serum anti-cathepsin show that the enzyme is stored in oocytes as a 55 kDa zymogen. The activation of the zymogen occurs in vitro only at low pH. In vitro activation in the presence of cysteine protease inhibitors is blocked at an intermediary polypeptide of 48 kDa. Kinetic studies of this activation process at pH 3.5 and 4.6 show that the zymogen is processed in a manner similar to that of pepsin (Foltmann, 1986) and papain (Vernet et al., 1991). We propose that Musca domestica cathepsin zymogen activation occurs in two steps. First, an intramolecular cleavage of the procathepsin polypeptide chain (55,000), induced by low pH gives rise to an intermediary polypeptide (48,000) which then undergoes autolysis to produce the mature enzyme (41,000).     

Conversion of tyrosine phenol-lyase to dicarboxylic amino acid beta-lyase, an enzyme not found in nature

Tyrosine phenol-lyase (TPL), which catalyzes the beta-elimination reaction of L-tyrosine, and aspartate aminotransferase (AspAT), which catalyzes the reversible transfer of an amino group from dicarboxylic amino acids to oxo acids, both belong to the alpha-family of vitamin B6-dependent enzymes. To switch the substrate specificity of TPL from L-tyrosine to dicarboxylic amino acids, two amino acid residues of AspAT, thought to be important for the recognition of dicarboxylic substrates, were grafted into the active site of TPL. Homology modeling and molecular dynamics identified Val-283 in TPL to match Arg-292 in AspAT, which binds the distal carboxylate group of substrates and is conserved among all known AspATs. Arg-100 in TPL was found to correspond to Thr-109 in AspAT, which interacts with the phosphate group of the coenzyme. The double mutation R100T/V283R of TPL increased the beta-elimination activity toward dicarboxylic amino acids at least 10(4)-fold. Dicarboxylic amino acids (L-aspartate, L-glutamate, and L-2-aminoadipate) were degraded to pyruvate, ammonia, and the respective monocarboxylic acids, e.g. formate in the case of L-aspartate. The activity toward L-aspartate (kcat = 0.21 s-1) was two times higher than that toward L-tyrosine. beta-Elimination and transamination as a minor side reaction (kcat = 0.001 s-1) were the only reactions observed. Thus, TPL R100T/V283R accepts dicarboxylic amino acids as substrates without significant change in its reaction specificity. Dicarboxylic amino acid beta-lyase is an enzyme not found in nature.     

Brain aging in normotensive and hypertensive strains of rats. III. A quantitative study of cerebrovasculature

The mechanism of degradation of fructose-1,6-bisphosphate aldolase from rabbit muscle by the lysosomal proteinase cathepsin B was determined. Treatment of aldolase with cathepsin B destroys up to 90% of activity with fructose 1,6-bisphosphate as substrate, but activity with fructose 1-phosphate is slightly increased. Cathepsin L, another lysosomal thiol proteinase, and papain are also potent inactivators of aldolase, whereas inactivation is not caused by cathepsins D or H even at high concentrations, or by cathepsin B inhibited by leupeptin or iodoacetate. The cathepsin-B-treated aldolase shows no detectable change in subunit molecular weight, oligomer molecular weight or subunit interactions. Cathepsin B cleaves dipeptides from the C-terminus of th aldolase subunits. Four dipeptides are released sequentially: Ala-Tyr, Asn-His, Ile-Ser and Leu-Phe, and a maximum of five additional dipeptides may be released. There are indications that this peptidyldipeptidase activity of cathepsin B may be an important aspect of its action on protein substrates generally.     

Influence of cholesterol and fat feeding on hepatic lysosomal enzymes in guinea pigs

The activity of four lysosomal enzymes have been examined in liver cytoplasmic extract from guinea pigs fed three different diets: a) an ordinary diet, low in fat, high in carbohydrates; b) a semisynthetic diet containing 10% cottonseed oil (by weight) without and c) with 1% cholesterol. The cholesterol content in the liver was similar in control-fed and fat-fed animals, while there was a 10-fold increase in cholesterol + fat-fed animals, and most of this cholesterol was present as ester. We observed increased activity of beta-glucuronidase, beta-acetyl-glucosaminidase and cathepsin D during fat cholesterol feeding (diet c) while the activity of acid phosphatase decreased compared to control-fed animals. These findings probably mirror the increased hepatic accumulation of lipids and lipoproteins observed in cholesterol + fat-fed guinea pigs.