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.     

Glycosaminoglycans facilitate procathepsin B activation through disruption of propeptide-mature enzyme interactions

Lysosomal cysteine cathepsin B participates in numerous diverse cellular processes. In acquiring its activity, the proregion, which blocks the substrate-binding site in the proenzyme, needs to be cleaved off. Here we demonstrate that polyanionic polysaccharides, glycosaminoglycans (GAGs), can accelerate the autocatalytic removal of the propeptide and subsequent activation of cathepsin B. We show that naturally occurring GAGs such as chondroitin sulfates and heparin, as well as the synthetic analog dextran sulfate, accelerate the processing in a concentration-dependent manner. Heparin oligosaccharides down to the size of tetrasaccharides were efficient in accelerating the procathepsin B processing, whereas disaccharides were without effect. Further, the ability of the GAGs to accelerate procathepsin B processing was sensitive to increasing NaCl concentrations, indicating that electrostatic interaction between the GAGs and procathepsin B are operative in the accelerating effect. Also the processing of the catalytic procathepsin B mutant by wild type cathepsin B was enhanced in the presence of GAGs, suggesting that GAGs induce a conformational change in procathepsin B, converting it into a better substrate. Site-directed mutagenesis showed that His(28), Lys(39), and Arg(40), located within the procathepsin B propeptide, have significant roles in the acceleration of procathepsin B activation induced by short GAGs. Because procathepsin B and GAGs often co-localize in vivo, we propose that GAGs may play a physiological role in the activation of procathepsin B.     

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.     

Apoptosis of human endothelial cells is accompanied by proteolytic processing of latent TGF-beta binding proteins and activation of TGF-beta

Transforming growth factors beta (TGF-betas) are multifunctional cytokines that modulate cell growth, differentiation and apoptosis. Numerous effects initiated by TGF-betas in vitro have been described, but the role of TGF-beta targeting and activation under physiological conditions has gained very little attention and understanding. We report here that apoptosis of human umbilical vein endothelial cells (HUVECs) is accompanied by release of truncated large latent TGF-beta complexes from the pericellular matrix followed by activation of TGF-beta. The activation of TGF-beta during apoptosis was accompanied by enhanced secretion of beta1-LAP protein, and apoptotic HUVECs acquired the capacity to induce the release of latent TGF-beta-binding proteins (LTBPs) from extracellular matrices. Activated TGF-beta, in turn, attenuated apoptotic death of HUVECs. Current results indicate that the activation of TGF-beta accompanies the apoptosis of HUVECs, and may play a protective feedback role against apoptotic cell death. The results suggest a role for TGF-beta as a putative extracellular modulator of apoptosis.     

Furin processing and proteolytic activation of Semliki Forest virus

The alphavirus Semliki Forest virus (SFV) infects cells via a low-pH-dependent membrane fusion reaction mediated by the E1 envelope protein. Fusion is regulated by the interaction of E1 with the receptor-binding protein E2. E2 is synthesized as a precursor termed "p62," which forms a stable heterodimer with E1 and is processed late in the secretory pathway by a cellular furin-like protease. Once processing to E2 occurs, the E1/E2 heterodimer is destabilized so that it is more readily dissociated by exposure to low pH, allowing fusion and infection. We have used FD11 cells, a furin-deficient CHO cell line, to characterize the processing of p62 and its role in the control of virus fusion and infection. p62 was not cleaved in FD11 cells and cleavage was restored in FD11 cell transfectants expressing human furin. Studies of unprocessed virus produced in FD11 cells (wt/p62) demonstrated that the p62 protein was efficiently cleaved by purified furin in vitro, without requiring prior exposure to low pH. wt/p62 virus particles were also processed during their endocytic uptake in furin-containing cells, resulting in more efficient virus infection. wt/p62 virus was compared with mutant L, in which p62 cleavage was blocked by mutation of the furin-recognition motif. wt/p62 and mutant L had similar fusion properties, requiring a much lower pH than control virus to trigger fusion and fusogenic E1 conformational changes. However, the in vivo infectivity of mutant L was more strongly inhibited than that of wt/p62, due to additional effects of the mutation on virus-cell binding.     

Mechanisms and kinetics of procathepsin D activation.

In vitro, procathepsin D is activated to pseudocathepsin D by incubation at low pH. To investigate the mechanism of this activation, recombinant human procathepsin D and two mutants were generated in a baculovirus expression system. One mutant carried a point mutation within the catalytic domain, which resulted in a catalytically inactive enzyme form (D77A). The other carried a point mutation within the propeptide, which prevented activation by processing at the 'autoproteolysis-site' (L26P). Neither mutant is capable of processing itself to form pseudocathepsin D, and L26P is not able to process D77A. Despite the inability of L26P to cleave either its own or a wild-type prosequence, it did exhibit activity against a synthetic peptide substrate. The ability of intact precursor (zymogen) to cleave a peptide, but not a protein substrate, offers new insights into the mechanism of inhibition by the propeptide. Mature cathepsin D can process the inactive D77A mutant to the pseudoform, demonstrating that processed species are capable of cleaving zymogen molecules in an intermolecular interaction. In addition, kinetic studies provide evidence for a two-phase mechanism for the conversion of procathepsin D to pseudocathepsin D, one phase where the first molecules of pseudocathepsin D are formed at a low rate and a second phase where the process is autocatalytically accelerated by newly formed pseudocathepsin D molecules. Finally, with the help of the mutants L26P and D77A it was observed that at least two additional proteinase activities, found in conditioned media from insect cell culture, are capable of activating procathepsin D by cleaving it within the proregion. This observation suggests that there are likely to be multiple proteinases in the extracellular matrix that are capable of activating procathepsin D, thereby triggering the second autocatalytic phase. This may also be important for solid tumors, where the presence of cathepsin D has been correlated with tumor growth and invasion.     

Rabbit microsomal epoxide hydrolase: isolation and characterization of the xenobiotic metabolizing enzyme cDNA

Many endogenous and xenobiotic chemicals are metabolized to epoxides which may be enzymatically hydrated, via microsomal epoxide hydrolase (mEH), to less reactive dihydrodiol derivatives. On the basis of the reported rabbit mEH amino acid sequence [F. S. Heinemann and J. Ozols (1984) J. Biol. Chem. 259, 797-804], we constructed a 35 base oligonucleotide which was used to screen rabbit liver cDNA libraries. Overlapping rabbit mEH clones were isolated and the full-length cDNA sequence of 1653 bp was determined. The rabbit nucleotide sequence has a high degree of similarity (greater than 75%) with cDNA sequences reported for rat and human mEH. Northern blot analyses with fragments of the rabbit cDNA demonstrate that mEH messenger RNA (mRNA) is expressed constitutively in the liver and induced following exposure to phenobarbital or polychlorinated biphenyls. Constitutive expression of mEH mRNA is also observed in rabbit kidney, testes, and lung. Using benzo[alpha]pyrene-4,5-oxide as substrate, mEH enzymatic activity is shown to correlate closely with tissue levels of mEH mRNA. Southern blot analyses of rabbit DNA suggest that the mEH gene exists as a single copy per haploid genome. The mEH amino acid sequences of the human and rat were compared to that of the deduced rabbit protein in order to analyze the degree of conservation and hydropathy profiles in these species. This comparison permitted the formulation of a computer-assisted model of mammalian mEH as it may relate to the microsomal membrane.     

Identification of the human hepatic microsomal glucose-6-phosphatase enzyme

The glucose-6-phosphatase enzyme protein of the human hepatic microsomal glucose-6-phosphatase system was identified as a 36.5 kDa polypeptide. The 36.5 kDa glucose-6-phosphatase enzyme protein was shown to be absent in the microsomes isolated from a patient previously diagnosed as having a type 1a glycogen storage disease.     

Purification and characterization of a new proteolytic enzyme produced by Aeromonas salmonicida

A proteolytic enzyme produced by the fish pathogen Aeromonas salmonicida was isolated and purified. It showed the following characteristics: temperature optimum at 48°C, pH optimum at pH 9 and a molecular weight of 87 500. The enzyme was inhibited by phenylmethanesulphonylfluoride (PMSF) indicating it to be a serine protease.     

Evidence for the proteolytic processing of dentin matrix protein 1. Identification and characterization of processed fragments and cleavage sites

Full-length cDNA coding for dentin matrix protein 1 (DMP1) has been cloned and sequenced, but the corresponding complete protein has not been isolated. In searching for naturally occurring DMP1, we recently discovered that the extracellular matrix of bone contains fragments originating from DMP1. Shortened forms of DMP1, termed 37K and 57K fragments, were treated with alkaline phosphatase and then digested with trypsin. The resultant peptides were purified by a two-dimensional method: size exclusion followed by reversed-phase high performance liquid chromatography. Purified peptides were sequenced by Edman degradation and mass spectrometry, and the sequences compared with the DMP1 sequence predicted from cDNA. Extensive sequencing of tryptic peptides revealed that the 37K fragments originated from the NH2-terminal region, and the 57K fragments were from the COOH-terminal part of DMP1. Phosphate analysis indicated that the 37K fragments contained 12 phosphates, and the 57K fragments had 41. From 37K fragments, two peptides lacked a COOH-terminal lysine or arginine; instead they ended at Phe173 and Ser180 and were thus COOH termini of 37K fragments. Two peptides were from the NH2 termini of 57K fragments, starting at Asp218 and Asp222. These findings indicated that DMP1 is proteolytically cleaved at four bonds, Phe173-Asp174, Ser180-Asp181, Ser217-Asp218, and Gln221-Asp222, forming eight fragments. The uniformity of cleavages at the NH2-terminal peptide bonds of aspartyl residues suggests that a single proteinase is involved. Based on its reported specificity, we hypothesize that these scissions are catalyzed by PHEX protein. We envision that the proteolytic processing of DMP1 plays a crucial role during osteogenesis and dentinogenesis.     

Purification and characterization of a new proteolytic enzyme produced by Aeromonas salmonicida

A proteolytic enzyme produced by the fish pathogen Aeromonas salmonicida was isolated and purified. It showed the following characteristics: temperature optimum at 48 degrees C, pH optimum at pH 9 and a molecular weight of 87500. The enzyme was inhibited by phenylmethanesulphonylfluoride (PMSF) indicating it to be a serine protease.     

Expression and activation of the vacuolar processing enzyme in Saccharomyces cerevisiae

Vacuolar processing enzymes (VPEs) are cysteine proteinases responsible for maturation of various vacuolar proteins in plants. A larger precursor to VPE synthesized on rough endoplasmic reticulum is converted to an active enzyme in the vacuoles. In this study, a precursor to castor bean VPE was expressed in a pep4 strain of the yeast Saccharomyces cerevisiae to examine the mechanism of activation of VPE. Two VPE proteins of 59 and 46 kDa were detected in the vacuoles of the transformant. They were glycosylated in the yeast cells, although VPE is not glycosylated in plant cells in spite of the presence of two N-linked glycosylation sites. During the growth of the transformant, the level of the 59 kDa VPE increased slightly until a rapid decrease occurred after 9 h. By contrast, the 46 kDa VPE appeared simultaneously with the disappearance of the 59 kDa VPE. Vacuolar processing activity increased with the accumulation of the 46 kDa VPE, but not of the 59 kDa VPE. The specific activity of the 46 kDa VPE was at a similar level to that of VPE in plant cells. The 46 kDa VPE instead of proteinase A mediated the conversion of procarboxypeptidase Y to the mature form. This indicates that proteinase A responsible for maturation of yeast vacuolar proteins can be replaced functionally by plant VPE. These findings suggest that an inactive VPE precursor synthesized on the endoplasmic reticulum is transported to the vacuoles in the yeast cells and then processed to make an active VPE by self-catalytic proteolysis within the vacuoles.     

Identification of dermcidin in human gestational tissue and characterization of its proteolytic activity

Dermcidin (DCD) is a gene for an antimicrobial peptide DCD-1 in human sweat glands. It has become evident that the gene products of DCD exhibit a wide range of biological functions. In addition to its antimicrobial function, it is reported to be a neuronal survival factor, a putative oncogene in breast cancer and a proteolysis-inducing factor (PIF) that induces skeletal muscle proteolysis to cause cancer cachexia. Here we identified DCD in human placental tissue and determined its previously uncharacterized proteolytic activity. We also show that recombinant DCD induced an invasive phenotype in a human choriocarcinoma cell line JAR in vitro. This work suggests that DCD might participate in the regulation of placental function by means of modulating the proteolytic cascades on the trophoblastic cell surface, and might be involved in the pathophysiology of pregnancy-related disorders, as well as cancer and neuronal diseases.     

Translation of encephalomyocarditis virus RNA in vitro yields an active proteolytic processing enzyme.

In contrast to other cell-free translation systems, the mRNA-dependent reticulocyte lysate can translate encephalomyocarditis virus RNA efficiently and completely when supplemented with heterologous tRNA. Cleavage of the nascent polypeptide chain occurs, and one of the translation products appears to be a specific proteolytic enzyme which correctly processes the primary products. The identity of the proteins made in vitro was verified by comparison with infected cell proteins on dodecylsulphate/polyacrylamide gels, and by mapping their coding sequences on the viral genome.     

Identification of cleavage sites involved in proteolytic processing of Pseudomonas aeruginosa preproelastase.

The extracellular elastase (33 kDa) of Pseudomonas aeruginosa is synthesized as a 53.6 kDa preproenzyme containing a long, N-terminal propeptide. The free propeptide and the elastase precursor generated upon propeptide removal were isolated from P. aeruginosa cells and subjected to N-terminal amino acid sequence analysis. The results identified Ala-174 and Ala+1 as the amino terminal residues of the propeptide and the elastase precursor, respectively, indicating that: (1) the signal peptide consists of 23 amino acid residues and its molecular weight is 2.4 kDa, (2) the propeptide contains 174 amino acid residues and is of 18.1 kDa molecular weight, and (3) no additional N-terminal proteolytic cleavage is required for elastase maturation.     

Isolation and characterization of a proteolytic enzyme from the adult hookworm Ancylostoma caninum

The adult hookworm Ancylostoma caninum releases a proteolytic enzyme which is thought to be essential for its adaption to parasitism. The protease was purified from parasite extracts by ion-exchange chromatography followed by gel filtration and hydrophobic interaction chromatography. The purified enzyme exhibited a molecular weight of 37,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had an NH2-terminal sequence of Arg-His-His-Gln-Pro-Lys-Val-Ala-Leu-Leu-Gly-Ala-His-Gly-Gly-Ile. Using 125I-fibrin as substrate, the enzyme displayed optimal activity at pH 9-11 and was inactivated by dialysis against EDTA. The enzyme degraded [3H]elastin and both elastin and trypsin-labile glycoproteins in a rat vascular smooth muscle extracellular matrix. Antiserum raised to the protease in rabbits cross-reacted with extracts from the infective larval stage of A. caninum, suggesting that the production of the enzyme begins in an earlier developmental stage of the parasite life cycle. The role of the protease in the histolytic and anticlotting processes of the hookworm and its importance in immunity to ancylostomiasis is discussed.