Identification of latent procathepsins B and L in microsomal lumen: characterization of enzymatic activation and proteolytic processing in vitro

Procathepsins B and L in the hepatic endoplasmic lumen were identified as having a molecular weight of 39,000 by immunoblot analysis. The proenzymes were then purified to remove the mature enzymes by concanavalin A-Sepharose chromatography. The concanavalin A-adsorbed fractions containing the proenzymes showed no appreciable activities of cathepsins B and L. When those fractions were incubated at pH 3.0, the enzymatic activities markedly increased: the activities of cathepsins B and L after 36 h incubation were 60 and 210 times those of the controls, respectively. Immunoblot analysis showed that after 36 h incubation the proenzymes disappeared and the mature enzymes increased. Thus the proenzymes were processed to the mature enzymes under acidic conditions of pH 3.0. The marked increases of enzymatic activities and the conversion of the proenzymes to the mature forms were completely blocked with pepstatin, which is a potent inhibitor of aspartic proteases. The results strongly suggested that a processing protease for procathepsins B and L might be cathepsin D, a major lysosomal aspartic protease. Indeed, lysosomal cathepsin D could convert microsomal procathepsin B to the mature enzyme in vitro. Therefore, procathepsins B and L seem first to be synthesized as enzymatically inactive forms in endoplasmic reticulum and successively may be converted into active forms by cathepsin D in lysosomal compartments.     

Identification of a precursor form of cathepsin D in microsomal lumen: characterization of enzymatic activation and proteolytic

A precursor form of cathepsin D with 45 kDa was demonstrated in the rat liver microsomal lumen by immunoblotting analysis. The microsomal fraction containing procathepsin D which passed through a pepstatin-Sepharose resin showed no appreciable activity of cathepsin D. The in vitro incubation of this fraction at pH 3.0 resulted in a gradual increase of proteolytic activity toward hemoglobin as substrate and also, the proteolytic conversion of procathepsin D to the mature form was concomitantly observed. The proteolytic processing step was sensitive to pepstatin. These results suggest that procathepsin D is inactive in the endoplasmic reticulum and may be converted to the active forms by autoproteolytic processing mechanism at acidic pH during biosynthesis.     

Evidence that aspartic proteinase is involved in the proteolytic processing event of procathepsin L in lysosomes

Our recent studies have shown that cathepsin L is first synthesized as an enzymatically inactive proform in endoplasmic reticulum and is successively converted into an active form during intracellular transport and we postulated that aspartic proteinases would be responsible for the intracellular propeptide-processing step of procathepsin L accompanied by the activation of enzyme (Y. Nishimura, T. Kawabata, and K. Kato (1988) Arch. Biochem. Biophys. 261, 64-71). To better understand this proposed mechanism, we investigated the effect of pepstatin, a potent inhibitor of aspartic proteinases, on the intracellular processing kinetics of cathepsin L analyzed by pulse-chase experiments in vivo with [35S]methionine in the primary cultures of rat hepatocytes. In the pepstatin-treated cells, the proteolytic conversion of cellular procathepsin L of 39 kDa to the mature enzyme was significantly inhibited and considerable amounts of proenzyme were found in the cell after 5-h chase periods. Further, the subcellular fractionation experiments demonstrated that the intracellular processing of procathepsin L in the high density lysosomal fraction was significantly inhibited and that considerable amounts of the procathepsin L form were still observed in the light density microsomal fraction after 2 h of chase. These results suggest that pepstatin treatment caused a significant inhibitory effect on the intracellular processing and also on the intracellular movement of procathepsin L from the endoplasmic reticulum to the lysosomes. These findings provide the first evidence showing that aspartic proteinase may play an important role in the intracellular proteolytic processing and activation of lysosomal cathepsin L in vivo. Therefore, we suggest that cathepsin D, a major lysosomal aspartic proteinase, is more likely to be involved in this proposed model in the lysosomes.     

Detection of procathepsin D in rat milk

The presence of procathepsin D, a zymogen of the soluble lysosomal aspartic proteinase cathepsin D, was detected in rat milk using Western blot analysis and assay of proteolytic activity in acidic buffers. No other forms of cathepsin D were found. Two different polyclonal anti-procathepsin D antibodies were used for immunochemical detection of procathepsin D. Both antibodies we found to recognize rat procathepsin D. Proteolytic activity in acidic buffers was detected using a fluorogenic substrate specific for cathepsin D and was abolished by pepstatin A, a specific inhibitor of aspartic proteinases. This study represents third demonstration of presence of procathepsin D in mammal breast milk. Potential sources and physiological functions are discussed.     

Protein inhibitors form complexes with procathepsin L and augment cleavage of the propeptide

The proregion fits tightly into the active site in the tertiary structure of procathepsin L and prevents its activity. We show that complexes between enzyme precursor and its endogenous protein inhibitors-the cystatins-can be formed without prior proteolytic removal of the propeptide. Complexes between cystatins and procathepsin L are formed at acidic pH and their formation is facilitated by acidic oligosaccharides. Binding of the inhibitor to the proenzyme is reversible and the slow dissociation of complex around neutral pH may serve as a pool for the sustained release of the enzyme. Formation of the complex between cystatin and procathepsin L increases the susceptibility of the proregion to proteolytic cleavage. This process may constitute an alternative mechanism of formation of the complex between enzyme and inhibitor without prior activation of the proenzyme.     

Procathepsin L self-association as a mechanism for selective secretion

The lysosomal cysteine pro-protease procathepsin L was enriched in dense vesicles detectable when microsomes prepared from wild-type or transformed mouse fibroblasts were resolved on sucrose gradients. These dense vesicles did not comigrate with proteins characteristic of the endoplasmic reticulum, Golgi, endosomes or lysosomes. When gradient fraction vesicles were lysed at acidic pH in the presence of excess mannose 6-phosphate to prevent binding to mannose phosphate receptors, the majority of the procathepsin L was associated with the membrane, not the soluble, fraction. Immunogold labeling of procathepsin L in thin sections of cells or gradient fractions, using antibodies directed against the propeptide to avoid detection of the mature enzyme in dense lysosomes, revealed that the proenzyme was concentrated in dense cores localized in small vesicles near the plasma membrane and in multivesicular bodies. Consistent with the density of the gradient fraction and the electron density of the cores, yeast two-hybrid assays indicated the proenzyme could bind itself but could not interact with the aspartic proprotease procathepsin D. The data suggest that in mouse fibroblasts procathepsin L may self-associate into aggregates, initiating the formation of dense vesicles that could mediate the selective secretion of procathepsin L independent of mannose phosphate receptors.     

In vitro biosynthesis of the lysosomal cathepsin H

A lysosomal thiol protease cathepsin H has been synthesized in vitro and shown to undergo co-translational segregation into the lumen of microsomal vesicles. Using cell-free synthesis, a 36 K Da cathepsin H was found to be synthesized exclusively on membrane-bound polysomes. When the microsomal membrane were present during translation, a glycosylated 41 K Da proenzyme appeared in the microsomal lumen. This proenzyme was converted to a 34 K Da protein by endoglycosidase H treatment. These results suggest that the nascent chain of cathepsin H has a transient N-terminal prepropeptide.     

An alternate targeting pathway for procathepsin L in mouse fibroblasts

In transformed mouse fibroblasts, a significant proportion of the lysosomal cysteine protease cathepsin L remains in cells as an inactive precursor which associates with membranes by a mannose phosphate-independent interaction. When microsomes prepared from these cells were resolved on sucrose gradients, this procathepsin L was localized in dense vesicles distinct from those enriched for growth hormone, which is secreted constitutively when expressed in fibroblasts. Ultrastructural studies using antibodies directed against the propeptide to avoid detection of the mature enzyme in lysosomes revealed that the proenzyme was concentrated in dense cores within small vesicles and multivesicular endosomes which labeled with antibodies specific for CD63. Consistent with the resemblance of these cores to those of regulated secretory granules, secretion of procathepsin L from fibroblasts was modestly stimulated by phorbol, 12-myristate, 13-acetate. When protein synthesis was blocked with cycloheximide and lysosomal proteolysis inhibited with leupeptin, procathepsin L was found to gradually convert to the active single-chain protease. The data suggest that when synthesis levels are high, a portion of the procathepsin L is packaged in dense cores within multivesicular endosomes localized near the plasma membrane. Gradual activation of this proenzyme achieves targeting of the proenzyme to lysosomes by a mannose phosphate receptor-independent pathway.     

Acidic protease from human seminal plasma. Purification and some properties of active enzyme and of proenzyme.

A procedure to purify to homogeneity the active form as well as the proenzyme form of the acidic protease of human seminal plasma is described. This involved precipitation with ammonium sulfate, chromatography on diethylaminoethylcellulose, Sephadex G-200, and Sephadex G-100. The molecular weights of the active form and of the proenzyme were determined by electrophoresis and gel filtration to be 35,000 and 42,000, respectively. The proenzyme was more stable than the active form in alkaline solution and can be converted into the active enzyme under acidic conditions. The active form of the acidic protease can hydrolyze hemoglobin, N,N'-dimethylcasein, N-acetyl-L-phenylalanyl-L-diiodotyrosine, and N-benzyloxycarbonyl-L-glutamyl-L-phenylalanine, but cannot hydrolyze bovine serum albumin, ovalbumin, N-benzyloxycarbonyl-L-glutamyl-L-tyrosine. The active form was also inhibited by p-bromophenacyl bromide and 1,2-epoxy-3-(p-nitrophenoxy)propane.     

棉铃虫组织蛋白酶B酶原在毕赤酵母中的表达

棉铃虫组织蛋白酶B（ Helicoverpa armigera Cathepsin B ,HCB）属于半胱氨酸蛋白酶类,参与胚胎发育中卵黄蛋白水解供给胚胎发育的氨基酸。本研究将HCB基因克隆到pPIC9K载体并转化毕赤酵母KM71菌株,经甲醇诱导,HCB表达并分泌到培养上清中。表达产物经SDS-PAGE测定分子量为38 kD, 与HCB基因编码的蛋白质分子量一致。用HCB的特异性抗体检测表明重组表达产物为棉铃虫组织蛋白酶B,原位水解实验显示重组表达的蛋白酶具有蛋白水解活性,表明在毕赤酵母中表达了有活性的棉铃虫组织蛋白酶B, 可用于组织蛋白酶B酶原活化机理研究及开发新蛋白酶产品。     

Biosynthesis,glycosylation, and enzymatic processing in vivo of human tripeptidyl-peptidase I

Human tripeptidyl-peptidase I (TPP I, CLN2 protein) is a lysosomal serine protease that removes tripeptides from the free N termini of small polypeptides and also shows a minor endoprotease activity. Due to various naturally occurring mutations, an inherited deficiency of TPP I activity causes a fatal lysosomal storage disorder, classic late infantile neuronal ceroid lipofuscinosis (CLN2). In the present study, we analyzed biosynthesis, glycosylation, transport, and proteolytic processing of this enzyme in stably transfected Chinese hamster ovary cells as well as maturation of the endocytosed proenzyme in CLN2 lymphoblasts, fibroblasts, and N2a cells. Human TPP I was initially identified as a single precursor polypeptide of approximately 68 kDa, which, within a few hours, was converted to the mature enzyme of approximately 48 kDa. Compounds affecting the pH of intracellular acidic compartments, those interfering with the intracellular vesicular transport as well as inhibition of the fusion between late endosomes and lysosomes by temperature block or 3-methyladenine, hampered the conversion of TPP I proenzyme into the mature form, suggesting that this process takes place in lysosomal compartments. Digestion of immunoprecipitated TPP I proenzyme with both N-glycosidase F and endoglycosidase H as well as treatment of the cells with tunicamycin reduced the molecular mass of TPP I proenzyme by approximately 10 kDa, which indicates that all five potential N-glycosylation sites in TPP I are utilized. Mature TPP I was found to be partially resistant to endo H treatment; thus, some of its N-linked oligosaccharides are of the complex/hybrid type. Analysis of the effect of various classes of protease inhibitors and mutation of the active site Ser(475) on human TPP I maturation in cultured cells demonstrated that although TPP I zymogen is capable of autoactivation in vitro, a serine protease that is sensitive to AEBSF participates in processing of the proenzyme to the mature, active form in vivo.     

Expression and refolding of recombinant human fibroblast procathepsin D

Procathepsin D is a precursor of the human lysosomal protease cathepsin D. Due to its short half-life, procathepsin D is difficult to obtain in quantities sufficient to allow structural and enzymatic studies. To obtain large quantities of this precursor, procathepsin D was expressed using the T7 promoter vector pET3a in bacteria that carry a chromosomal copy of the T7 RNA polymerase gene under the control of the lac promoter. At high cell density in rich medium, basal levels of T7 RNA polymerase were sufficient to express recombinant procathepsin D without addition of an exogenous inducer of the lac promoter. The recombinant protein, constituting almost half of the total cell protein, accumulated in intracytoplasmic inclusion bodies and was isolated from the insoluble fraction of lysed cells. Antibodies prepared against the purified recombinant protein were shown to crossreact with native human placental and porcine spleen cathepsin D. Recombinant procathepsin D was solubilized in denaturants and was refolded. After extended preincubation of the denatured protein at acid pH to allow folding and activation of the zymogen, pepstatin inhibitable catalytic proteolysis was detected. These data demonstrated that the glycosylated aspartic protease, procathepsin D can be refolded and activated in an unglycosylated form and thus provides a system for the study of procathepsin D structure and function.     

Activation of proenzyme of acidic protease from human seminal plasma.

The kinetics and the extent of the conversion of the proenzyme into the active acidic protease (EC 3.4.23.--) of human seminal plasma were dependent on acidic pH. Between pH 2 and 4, the initial rate of the activation was first-order with respect to the proenzyme. Between pH 4.5 and 5, the rate deviated from the first-order with an initial lag period which can be abolished by adding an excess amount of the acidic protease or pepsin. The extent of the activation was complete between pH 2 and 3 and became incomplete between pH 4 and 5. Addition of the acidic protease or pepsin did not alter the extent of the activation at the high pH values. According to the chromatographic profile on a Sephadex G-75 column, the activation products (namely active acidic protease and an activation peptide) obtained at pH 3 and those obtained at pH 4.5 were identical. The molecular weight of the activation peptide obtained at pH 3 was 6900; its amino acid composition was analyzed and compared with those of the proenzyme and the acidic protease. Remarkable similarity between the amino acid composition of the acidic protease and that of human pepsin was observed. In the presence of an excess amount of hemoglobin, the conversion of the proenzyme was self-activated and showed an initial lag period. Addition of acidic protease did not change the rate of self activation or the lag period.     

Mannose 6-phosphate-independent targeting of cathepsin D to lysosomes in HepG2 cells.

We have studied the role of N-linked oligosaccharides and proteolytic processing on the targeting of cathepsin D to the lysosomes in the human hepatoma cell line HepG2. In the presence of tunicamycin cathepsin D was synthesized as an unglycosylated 43-kDa proenzyme which was proteolytically processed via a 39-kDa intermediate to a 28-kDa mature form. Only a small portion was secreted into the culture medium. During intracellular transport the 43-kDa procathepsin D transiently became membrane-associated independently of binding to the mannose 6-phosphate receptor. Subcellular fractionation showed that unglycosylated cathepsin D was efficiently targeted to the lysosomes via intermediate compartments similar to the enzyme in control cells. The results show that in HepG2 cells processing and transport of cathepsin D to the lysosomes is independent of mannose 6-phosphate residues. Inhibition of the proteolytic processing of 53-kDa procathepsin D by protease inhibitors caused this form to accumulate intracellularly. Subcellular fractionation revealed that the procathepsin D was transported to lysosomes, thereby losing its membrane association. Procathepsin D taken up by the mannose 6-phosphate receptor also transiently became membrane-associated, probably in the same compartment. We conclude that the mannose 6-phosphate-independent membrane-association is a transient and compartment-specific event in the transport of procathepsin D.     

Structure of human procathepsin L reveals the molecular basis of inhibition by the prosegment.

Cathepsin L is a member of the papain superfamily of cysteine proteases and, like many other proteases, it is synthesized as an inactive proenzyme. Its prosegment shows little homology to that of procathepsin B, whose structure, the first for a cysteine protease proenzyme, has been determined recently. We report here the 3-D structure of a mutant of human procathepsin L determined at 2.2 A resolution, describe the mode of binding employed by the prosegment and discuss the molecular basis for other possible roles of the prosegment. The N-terminal part of the prosegment is globular and contains three alpha-helices with a small hydrophobic core built around aromatic side chains. This domain packs against a loop on the enzyme's surface, with the aromatic side chain from the prosegment being located in the center of this loop and providing a large contact area. The C-terminal portion of the prosegment assumes an extended conformation and follows along the substrate binding cleft toward the N-terminus of the mature enzyme. The direction of the prosegment in the substrate binding cleft is opposite to that of substrates. The previously described role of the prosegment in the interactions with membranes is supported by the structure of its N-terminal domain. The fold of the prosegment and the mechanism by which it inhibits the enzymatic activity of procathepsin L is similar to that observed in procathepsin B despite differences in length and sequence, suggesting that this mode of inhibition is common to all enzymes from the papain superfamily.     

Gastric procathepsin E and progastricsin from guinea pig. Purification, molecular cloning of cDNAs, and characterization of enzymatic properties, with special reference to procathepsin E.

Procathepsin E and progastricsin were purified from the gastric mucosa of the guinea pig. They were converted to the active form autocatalytically under acidic conditions. Each active form hydrolyzed protein substrates maximally at around pH 2.5. Pepstatin inhibited cathepsin E very strongly at an equimolar concentration, whereas the inhibition was much weaker for gastricsin. Molecular cloning of the respective cDNAs permitted us to deduce the complete amino acid sequences of their pre-proforms; preprocathepsin E and preprogastricsin consisted of 391 and 394 residues, respectively. Procathepsin E has unique structural and enzymatic features among the aspartic proteinases. Lys at position 37, which is common to various aspartic proteinases and is thought to be important for stabilizing the activation segment, was absent at the corresponding position, as in human procathepsin E. The rate of activation of procathepsin E to cathepsin E is maximal at around pH 4.0. It is very different from the pepsinogens and may be correlated with the absence of Lys37. Native procathepsin E is a dimer, consisting of two monomers covalently bound by a disulfide bridge between 2 Cys37. Interconversion between the dimer and the monomer was reversible and regulated by low concentrations of a reducing reagent. Although the properties of the dimeric and monomeric cathepsins E are quite similar, a marked difference was found between them in terms of their stability in weakly alkaline solution: monomeric cathepsin E was unstable at weakly alkaline pH whereas the dimeric form was stable. The generation of the monomer was thought to be the process leading to inactivation, hence degradation of cathepsin E in vivo.     

Properties of soluble fusions between mammalian aspartic proteinases and bacterial maltose-binding protein

The mammalian aspartic proteinases procathepsin D and pepsinogen form insoluble inclusion bodies when expressed in bacteria. They become soluble but nonnative when synthesized as fusions to the carboxy terminus of E. coli maltose-binding protein (MBP). Since these nonnative states of the two aspartic proteinases showed no tendency to form insoluble aggregates, their biophysical properties were analyzed. The MBP portions were properly folded as shown by binding to amylose, but the aspartic proteinase moieties failed to bind pepstatin and lacked enzymatic activity, indicating that they were not correctly folded. When treated with proteinase K, only the MBP portion of the fusions was resistant to proteolysis. The fusion between MBP and cathepsin D had increased hydrophobic surface exposure compared to the two unfused partners, as determined by bis-ANS binding. Ultracentrifugal sedimentation analysis of MBP–procathepsin D and MBP–pepsinogen revealed species with very large and heterogeneous sedimentation values. Refolding of the fusions from 8 M urea generated proteins no larger than dimers. Refolded MBP–pepsinogen was proteolytically active, while only a few percent of renatured MBP–procathepsin D was obtained. The results suggest that MBP–aspartic proteinase fusions can provide a source of soluble but nonnative folding states of the mammalian polypeptides in the absence of aggregation.     

Phosphorylation, glycosylation, and proteolytic activity of the 52-kD estrogen-induced protein secreted by MCF7 cells

We have studied the posttranslational modifications of the 52-kD protein, an estrogen-regulated autocrine mitogen secreted by several human breast cancer cells in culture (Westley, B., and H. Rochefort, 1980, Cell, 20:353-362). The secreted 52-kD protein was found to be phosphorylated mostly (94%) on high-mannose N-linked oligosaccharide chains, and mannose-6-phosphate signals were identified. The phosphate signal was totally removed by alkaline phosphatase hydrolysis. The secreted 52-kD protein was partly taken up by MCF7 cells via mannose-6-phosphate receptors and processed into 48- and 34-kD protein moieties as with lysosomal hydrolases. By electron microscopy, immunoperoxidase staining revealed most of the reactive proteins in lysosomes. After complete purification by immunoaffinity chromatography, we identified both the secreted 52-kD protein and its processed cellular forms as aspartic and acidic proteinases specifically inhibited by pepstatin. The 52-kD protease is secreted in breast cancer cells under its inactive proenzyme form, which can be autoactivated at acidic pH with a slight decrease of molecular mass. The enzyme of breast cancer cells, when compared with cathepsin D(s) of normal tissue, was found to be similar in molecular weight, enzymatic activities (inhibitors, substrates, specific activities), and immunoreactivity. However, the 52-kD protein and its cellular processed forms of breast cancer cells were totally sensitive to endo-beta-N-acetylglucosaminidase H (Endo H), whereas several cellular cathepsin D(s) of normal tissue were partially Endo H-resistant. This difference, in addition to others concerning tissue distribution, mitogenic activity and hormonal regulation, strongly suggests that the 52-kD cathepsin D-like enzyme of breast cancer cells is different from previously described cathepsin D(s). The 52-kD estrogen-induced lysosomal proteinase may have important functions in facilitating the mammary cancer cells to proliferate, migrate, and metastasize.     

RGD-dependent binding of procathepsin X to integrin alphavbeta3 mediates cell-adhesive properties

Secreted lysosomal cysteine proteases (cathepsins) are involved in degradation and remodeling of the extracellular matrix, thus contributing to cell adhesion and migration. Among the eleven human lysosomal cysteine proteases, only procathepsin X contains an RGD motif located in a highly exposed region of the propeptide, which may allow binding of the proenzyme to RGD-recognizing integrins. Here, we have tested procathepsin X for cell-adhesive properties and found that it supports integrin alpha(v)beta(3)-dependent attachment and spreading of human umbilical vein endothelial cells. Using site-directed mutants of procathepsin X, we proved that this effect is mediated by the RGD sequence within the proregion of the protease. Endogenous procathepsin X is transported to the plasma membrane, accumulates in vesicles at lamellipodia of the human umbilical vein endothelial cell, and is partly associated with the cell surface, as shown by immunofluorescence. In addition, procathepsin X is partly co-localized with integrin beta(3), as detected by immunogold electron microscopy. A direct interaction between endogenous procathepsin X and alpha(v)beta(3) was demonstrated by co-immunoprecipitation. Moreover, surface plasmon resonance analysis revealed significant and RGD-dependent binding of procathepsin X to integrin alpha(v)beta(3). Our results provide for the first time evidence that the extracellular function of cathepsin X may include binding to integrins thereby modulating the attachment of migrating cells to ECM components.     

A simple and efficient protocol for the production of recombinant cathepsin V and other cysteine cathepsins in soluble form in Escherichia coli

Cysteine cathepsins are major players in numerous physiologic and pathologic processes and important drug targets. Several different expression systems have been developed for the production of these enzymes. Here we describe a novel, simple and efficient protocol for the production of recombinant cathepsin V and other cysteine cathepsins. Recombinant procathepsin V was expressed in soluble form in the cytoplasm of Escherichia coli and purified in one step by immobilized nickel ion-affinity chromatography, yielding approximately 0.7 mg procathepsin V per liter bacterial culture. The recombinant proenzyme was then autocatalytically activated in vitro by incubation at pH 4.0 and 30 °C. The yield of proenzyme conversion was over 95% and the mature enzyme exhibited potent activity towards several commonly used synthetic substrates. The same protocol also proved successful in the production of several other cysteine procathepsins, such as cathepsin B, demonstrating that this procedure is widely applicable for the production of recombinant papain-like cysteine peptidases.