Native and Acid-denatured L-Lactate Dehydrogenase Are Different in the Lysosomal Proteinases Involving in Their Overall Degradation

When native and acid-denatured lactate dehydrogenase (LDH) were incubated with total lysosomal enzymes in vitro, amino acids from their degradation were produced at various acidic pH. The pH profile in the overall degradation of native LDH was markedly different from that of acid-denatured LDH. Disappearance of the 35-kDa subunit of native LDH was markedly suppressed by a low level of cystatin α as well as by a general cysteine proteinase inhibitor, N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucine-3-methylbutylamide (E-64-c). On the other hand, the degradation of acid-denatured LDH was only slightly suppressed by these inhibitors. It was concluded that at least a part of the proteinases involved in the overall degradation of native LDH is different from the proteinases involved in the degradation of acid-denatured form and a role of a cystatin α-sensitive cysteine proteinase is critical in the lysosomal degradation of native LDH, but not in that of acid-denatured form.     

Cathepsin L plays an important role in the lysosomal degradation of L-lactate dehydrogenase

A cystatin alpha-sensitive cysteine proteinase that plays an important role in the lysosomal inactivation and degradation of L-lactate dehydrogenase (LDH) was purified by column chromatography from an ammonium sulfate precipitate of lysosome extract prepared from rat livers. It was eluted with marked delay from cathepsins B and H in a Sephacryl S-200 column by its specific interaction with the gel, and then effectively separated from cathepsins B and H and other proteins. It was eluted with 0.5 M NaCl after washing with 0.2 M NaCl in a CM-Sephadex column, indicating that it showed the same elution behavior as cathepsin L from the CM-Sephadex column. It had activity to hydrolyze z-Phe-Arg-NH-Mec, a synthetic substrate for cysteine proteinases, including cathepsins B and L. The N-terminal sequences of the final preparation of LDH-inactivating enzyme were identical with those of rat cathepsin L. Inactivation and degradation of LDH by the final preparation were observed and effectively inhibited by a low level of cystatin alpha as well as a general cysteine proteinase inhibitor, leupeptin or (L-3-trans-carboxyoxirane-2-carbonyl)-L-leucine (3-methylbutyl)amide (E-64-c). From these results, it is concluded that cathepsin L plays a critical role in the lysosomal degradation of native LDH.     

Cathepsin L Plays an Important Role in the Lysosomal Degradation of L-Lactate Dehydrogenase

A cystatin α-sensitive cysteine proteinase that plays an important role in the lysosomal inactivation and degradation of L-lactate dehydrogenase (LDH) was purified by column chromatography from an ammonium sulfate precipitate of lysosome extract prepared from rat livers. It was eluted with marked delay from cathepsins B and H in a Sephacryl S-200 column by its specific interaction with the gel, and then effectively separated from cathepsins B and H and other proteins. It was eluted with 0.5 M NaCl after washing with 0.2 M NaCl in a CM-Sephadex column, indicating that it showed the same elution behavior as cathepsin L from the CM-Sephadex column. It had activity to hydrolyze z-Phe-Arg-NH-Mec, a synthetic substrate for cysteine proteinases, including cathepsins B and L. The N-terminal sequences of the final preparation of LDH-inactivating enzyme were identical with those of rat cathepsin L. Inactivation and degradation of LDH by the final preparation were observed and effectively inhibited by a low level of cystatin α as well as a general cysteine proteinase inhibitor, leupeptin or (L-3-trans-carboxyoxirane-2-carbonyl)-L-leucine (3-methylbutyl)amide (E-64-c). From these results, it is concluded that cathepsin L plays a critical role in the lysosomal degradation of native LDH.     

Autodegradation of lysosomal cysteine proteinases

Repeated injections of Ep-475, a potent cysteine proteinase inhibitor, into rats caused several-fold increase in the hepatic contents of the lysosomal cysteine proteinases cathepsin B, H and L and in the activities of other lysosomal hydrolases. The rates of degradation of these lysosomal enzymes, estimated by repeated injections of cycloheximide, were found to be retarded in Ep475-treated rats, indicating that lysosomal cysteine proteinases are involved in degradation of lysosomal enzymes including proteinases.     

Purification and characterization of a cysteine proteinase from silkworm eggs

Eggs of the silkworm, Bombyx mori, contain a high level of a proteinase which is most active in acidic pH region. The proteinase was purified from an extract of eggs by a six-step procedure which included conventional chromatographic fractionations. The molecular mass of the proteinase was estimated to be 350 kDa by gel filtration and 47 kDa by electrophoresis on sodium dodecyl sulfate/polyacrylamide gels, suggesting an octameric structure. The amino acid composition was found to resemble that of mammalian lysosomal cysteine proteinases, in particular cathepsin L. The NH2-terminal 10-residue sequence is Val-Gln-Phe-Phe-Asp-Leu-Val-Lys-Glu-Glu-. The enzyme appears to be a member of the class of cysteine proteinases since it was strongly inhibited by sulfhydryl-reactive compounds and N-[N-(1,3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine (E-64). The enzyme hydrolyzed various protein substrates, such as hemoglobin, vitellogenin, vitellin, and lipophorin, with maximal activity around pH 3-3.5. The specificity of the cleavage sites in the oxidized B chain of insulin was rather well defined and there was high affinity for hydrophobic residues at the P2 and P3 positions. The cysteine proteinase is thought to be involved in protein degradation during embryonic development of silkworm eggs.     

Analysis of where and which types of proteinases participate in lysosomal proteinase processing using bafilomycin A1 and Helicobacter pylori Vac A toxin

Lysosomal proteinases are translated as preproforms, transported through the Golgi apparatus as proforms, and localized in lysosomes as mature forms. In this study, we analyzed which subclass of proteinases participates in the processing of lysosomal proteinases using Bafilomycin A1, a vacuolar ATPase inhibitor. Bafilomycin A1 raises lysosomal pH resulting in the degradation of lysosomal proteinases such as cathepsins B, D, and L. Twenty-four hours after the withdrawal of Bafilomycin A1, NIH3T3 cells possess these proteinases in amounts and activities similar to those in cells cultured in DMEM and 5% BCS. In the presence of various proteinase inhibitors, procathepsin processing is disturbed by E-64-d, resulting in abnormal processing of cathepsins D and L, but not by APMSF, Pepstatin A, or CA-074. In the presence of Helicobacter pylori Vac A toxin, which prevents vesicular transport from late endosomes to lysosomes, the processing of procathepsins B and D occurs, while that of procathepsin L does not. Thus, procathepsins B and D are converted to their mature forms in late endosomes, while procathepsin L is processed to the mature form after its arrival in lysosomes by some cysteine proteinase other than cathepsin B.     

中华鲟半胱氨酸蛋白酶抑制剂在毕赤酵母中的表达和活性分析

为研究鱼类半胱氨酸蛋白酶抑制剂（Cystatin）的功能并探索其在水产加工和病害防治中的应用潜力,将PCR改造后的编码成熟肽中华鲟（Acipenser sinensis）cystatin 基因亚克隆到毕赤酵母整合型表达载体pPICZαA,氯化锂法转化毕赤酵母菌株GS115,构建表达cystatin的酵母基因工程菌。经甲醇诱导、SDSPAGE检测培养基上清液,表明中华鲟cystatin在毕赤酵母中实现了高效表达,重组cystatin表达量约为215mg·L^-1。纯化后重组蛋白纯度达94.2%。生物活性检测结果表明,1μg重组中华鲟cystatin约能抑制15μg木瓜蛋白酶的水解活性。     

Identification of the probable inhibitory reactive sites of the cysteine proteinase inhibitors human cystatin C and chicken cystatin

When an excess of human cystatin C or chicken cystatin was mixed with papain, an enzyme-inhibitor complex was formed immediately. The residual free cystatin was then progressively converted to a form with different electrophoretic mobility and chromatographic properties. The modified cystatins were isolated and sequenced, showing that there had been cleavage of a single peptide bond in each molecule: Gly11-Gly12 in cystatin C, and Gly9-Ala10 in chicken cystatin. The residues Gly11 (cystatin C) and Gly9 (chicken cystatin) are among only three residues conserved in all known sequences of inhibitory cystatins. The modified cystatins were at least 1000-fold weaker inhibitors of papain than the native cystatins. An 18-residue synthetic peptide corresponding to residues 4-21 of cystatin C did not inhibit papain but was cleaved at the same Gly-Gly bond as cystatin C. When iodoacetate or L-3-carboxy-trans-2,3-epoxypropionyl-leucylamido-(4-guanidin o)butane was added to the mixtures of either cystatin with papain, modification of the excess cystatin was blocked. Papain-cystatin complexes were stable to prolonged incubation, even in the presence of excess papain. We conclude that the peptidyl bond of the conserved glycine residue in human cystatin C and chicken cystatin probably is part of a substrate-like inhibitory reactive site of these cysteine proteinase inhibitors of the cystatin superfamily and that this may be true also for other inhibitors of this superfamily. We also propose that human cystatin C and chicken cystatin, and probably other cystatins as well, inhibit cysteine proteinases by the simultaneous interactions with such proteinases of the inhibitory reactive sites and other, so far not identified, areas of the cystatins. The cleavage of the inhibitory reactive site glycyl bond in mixtures of papain with excess quantities of cystatins is apparently due to the activity of a small percentage of atypical cysteine proteinase molecules in the papain preparation that form only very loose complexes with cystatins under the conditions employed and degrade the free cystatin molecules.     

Inhibition of cysteine proteinases by a protein inhibitor from potato

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.     

Decreased activity of cathepsins L+B and decreased invasive ability of PC3 prostate cancer cells

Cancer metastasis involves multiple factors, one of which is the production and secretion of matrix degrading proteases by the cancer cells. Many metastasizing cancer cells secrete the lysosomal proteases, cathepsins L and B, which implicates them in the metastatic process. Cathepsins L and B are regulated by endogenous cysteine proteinase inhibitors (CPI) known as cystatins. An imbalance between cathepsin L and/or B and cystatin expression/activity may be a characteristic of the metastatic phenotype. To determine whether cystatins can attenuate the invasive ability of PC3 prostate cancer cells, cells were transfected with a cDNA coding for chicken cystatin. Expression of chicken cystatin mRNA was determined by PCR analysis. Total cysteine proteinase inhibitory activity, cathepsins L+B activity, and invasion through a Matrigel® matrix were assessed. Stably transfected cells expressed the chicken cystatin mRNA and exhibited a significant decrease in secreted cathepsin L+B activity and a small increase in secreted cysteine proteinase inhibitor activity. The ability of cystatin transfected cells to invade the reconstituted basement membrane, Matrigel®, was attenuated compared to nontransfected cells or cells transfected with vector alone. We have demonstrated that the cysteine proteinases cathepsins L and B participate in the invasive ability of the PC3 prostate cancer cell line, and we discuss here the potential of using cysteine proteinase inhibitors such as the cystatins as anti-metastatic agents.     

Decreased activity of cathepsins L+B and decreased invasive ability of PC3 prostate cancer cells

Cancer metastasis involves multiple factors, one of which is the production and secretion of matrix degrading proteases by the cancer cells. Many metastasizing cancer cells secrete the lysosomal proteases, cathepsins L and B, which implicates them in the metastatic process. Cathepsins L and B are regulated by endogenous cysteine proteinase inhibitors (CPI) known as cystatins. An imbalance between cathepsin L and/or B and cystatin expression/activity may be a characteristic of the metastatic phenotype. To determine whether cystatins can attenuate the invasive ability of PC3 prostate cancer cells, cells were transfected with a cDNA coding for chicken cystatin. Expression of chicken cystatin mRNA was determined by PCR analysis. Total cysteine proteinase inhibitory activity, cathepsins L+B activity, and invasion through a Matrigel® matrix were assessed. Stably transfected cells expressed the chicken cystatin mRNA and exhibited a significant decrease in secreted cathepsin L+B activity and a small increase in secreted cysteine proteinase inhibitor activity. The ability of cystatin transfected cells to invade the reconstituted basement membrane, Matrigel®, was attenuated compared to nontransfected cells or cells transfected with vector alone. We have demonstrated that the cysteine proteinases cathepsins L and B participate in the invasive ability of the PC3 prostate cancer cell line, and we discuss here the potential of using cysteine proteinase inhibitors such as the cystatins as anti-metastatic agents.     

Proteolysis of isolated mitochondria by myocardial lysosomal enzymes.

1. Solubilized mitochondria and lysosomal fractions were obtained from guinea-pig heart by differential centrifugation and selective membrane disruption. 2. Mitochondria incubated at 37 degrees C in the presence of lysosomal enzymes underwent proteolysis. The rate of protein degradation was inversely dependent on pH. 3. The use of proteinase inhibitors showed that at low pH the major enzyme involved in mitochondrial digestion was cathepsin D. 4. At neutral pH carboxyl proteinases were still active, but thiol proteinases accounted for most of the protein breakdown. 5. The role of lysosomal enzymes as mediators of mitochondrial damage in ischaemic myocardium is discussed.     

Effects of selective inhibition of cathepsin B and general inhibition of cysteine proteinases on lysosomal proteolysis in rat liver in vivo and in vitro.

Intraperitoneal administration of N-(L-trans-propylcarbamoyloxirane-2-carbonyl)-L-isoleucyl-L-prolin e (CA-074) to rats at a dose of 4 mg/100 g greatly inhibited cathepsin-B activity in both liver and kidney for at least 4 h. Its inhibitory effect was selective for cathepsin-B activity in the liver but not in the kidney. The effects of selective inhibition of cathepsin-B activity by CA-074 treatment, and general inhibition of cysteine proteinases by N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl-3-methylbutylamid e (E-64-c) on the degradation of fluorescein isothiocyanate (FITC)-labeled asialofetuin in liver lysosomes, were examined in vivo. Undegraded or partially degraded FITC-labeled asialofetuin and its FITC-labeled degradation products were both found in the lysosomes and were easily separated by Sephadex G-25' column chromatography. The FITC-labeled degradation products were mainly lysine with an FITC-labeled epsilon-amino group. Accumulation of undegraded or partially degraded FITC-labeled asialofetuin in the lysosomes was marked after E-64-c treatment, but slight after CA-074 treatment. Under the marked inhibition of general lysosomal cysteine-proteinase activity by E-64-c or marked selective inhibition of cathepsin-B activity by CA-074 in vitro, degradation of FITC-labeled asialofetuin by disrupted lysosomes was analyzed on the basis of measurement of FITC-labeled degradation products by Sephadex G-25 column chromatography. It was suppressed markedly but incompletely by E-64-c as well as by CA-074, but more weakly than by E-64-c. These results shows that E-64-sensitive cysteine proteinases are important in lysosomal protein degradation, but cathepsin B has only a role in part and that an E-64-resistant proteinase(s) may also be important.     

Increase of cyclin B by overexpression of cystatin α

Degradation of cyclin B was effectively suppressed when cells were treated with ALLN (N-acetylleucylleucylnorleucinal) which inhibits proteasome, calpain and cysteine proteinase cathepsins. In order to examine which protease degrades cyclin B, the effect of a cathepsin inhibitor, cystatin α, was investigated. The cystatin α gene was inserted into an inducible expression vector, pMSG, and transfected into NIH3T3 mouse fibroblasts. The expression of cystatin α was induced effectively in the transfected cells after treatment with dexamethasone. Overexpression of cystatin α resulted in an increase of the amount of cyclin B, suggesting that cysteine proteinase cathepsins might be involved in the degradation of cyclin B.     

Production of the cysteine proteinase inhibitor cystatin C by rat Sertoli cells.

The Sertoli cells of the rat testis produce cystatin C, a cysteine proteinase inhibitor. Primary culture of Sertoli cells secreted both unglycosylated and glycosylated forms of rat cystatin C. Despite the low concentration of cystatin C in rete testis fluid, equilibrium dissociation constants (Ki) for the interaction between cystatin C and lysosomal cathepsins indicate that this molecule could be involved in the local regulation of testicular cysteine proteinase activity which may be necessary for spermatogenesis and spermiogenesis.     

A group-specific inhibitor of lysosomal cysteine proteinases selectively inhibits both proteolytic degradation and presentation of the antigen dinitrophenyl-poly-L-lysine by guinea pig accessory cells to T cells

A limited intralysosomal proteolytic degradation is probably a key event in the accessory cell processing of large protein antigens before their presentation to T cells. With the aid of highly specific inhibitors of proteinases, we have examined the role of proteolysis in the presentation of antigens by guinea pig accessory cells. The proteinase inhibitor benzyloxycarbonyl-phenylalanylalanine-diazomethyl-ketone, which selectively inhibits cysteine proteinases, was used to block this set of enzymes in cultured cells. We demonstrate that the selective inhibition of the cysteine proteinases of antigen-presenting cells causes a profound inhibition of both the proteolytic degradation and the presentation of the synthetic antigen dinitrophenyl-poly-L-lysine. In contrast, the presentation of another synthetic antigen, the copolymer of L-glutamic acid and L-alanine, was enhanced by the same inhibitor. Another inhibitor, pepstatin A, which selectively blocks aspartic proteinases, did not block the presentation of dinitrophenyl-poly-L-lysine. The results identify cysteine proteinases, probably lysosomal, as one of the groups of enzymes involved in antigen processing.     

Cystatin B as an intracellular modulator of bone resorption

Degradation of organic bone matrix requires proteinase activity. Cathepsin K is a major osteoclast proteinase needed for bone resorption, although osteoclasts also express a variety of other cysteine- and matrix metalloproteinases that are involved in bone remodellation. Cystatin B, an intracellular cysteine proteinase inhibitor, exhibits a lysosomal distribution preferentially in osteoclasts but it's role in osteoclast physiology has remained unknown. The current paper describes a novel regulatory function for cystatin B in bone-resorbing osteoclasts in vitro. Rat osteoclasts were cultured on bovine bone and spleen-derived cystatin B was added to the cultures. Nuclear morphology was evaluated and the number of actively resorbing osteoclasts and resorption pits was counted. Intracellular cathepsin K and tartrate-resistant acid phosphatase (TRACP) activities were monitored using fluorescent enzyme substrates and immunohistology was used to evaluate distribution of cystatin B in rat metaphyseal bone. Microscopical evaluation showed that cystatin B inactivated osteoclasts, thus resulting in impaired bone resorption. Cathepsin K and TRACP positive vesicles disappeared dose-dependently from the cystatin B-treated osteoclasts, indicating a decreased intracellular trafficking of bone degradation products. At the same time, cystatin B protected osteoclasts from experimentally induced apoptosis. These data show for the first time that, in addition to regulating cysteine proteinase activity and promoting cell survival in the nervous system, cystatin B inhibits bone resorption by down-regulating intracellular cathepsin K activity despite increased osteoclast survival.     

Autolysis in the tail muscles of metamorphosing tadpoles

Degradation of muscle homogenate from the metamorphosing tadpole tail of bullfrog, Rana catesbeiana, was examined at acid and neutral pHs. More rapid and complete degradation was observed at acid pH. Proteinases working at acid pH were not inhibited by pepstatin but were inhibited by leupeptin. However, the inhibition by leupeptin was enhanced by pepstatin. These results show that lysosomal proteinases, a thiol proteinase(s) rather than cathepsin D, are involved in the degradation of tail muscle proteins.     

Protein inhibitors of cysteine proteinases. III. Amino-acid sequence of cystatin from chicken egg white

Cystatin, the protein inhibitor of cysteine proteinases from chicken egg white was purified by a new method. The two major forms with pI 6.5 (Peak I) and 5.6 (Peak II) were separated. Molecular masses of both forms are approx. 12700 Da as determined by gel chromatography; Form A from Peak I has a molecular mass of 12191 Da as calculated from its amino-acid sequence. The complete amino-acid sequence of Form A was determined by automated solid-phase Edman degradation of the whole inhibitor and its cyanogen bromide fragments. It contains 108 amino-acid residues. Form B from Peak II represents an elongation of Form A by 8 amino-acid residues at the N-terminus. Cystatin contains four cysteine residues, presumably forming two disulphide bridges. Comparison of the amino-acid sequences and near ultraviolet circular dichroism spectra of stefin, the cysteine proteinase inhibitor from human granulocytes, and cystatin shows that the two proteins are entirely different. According to the primary structures, probably neither proteinase inhibitor is involved in a thiol-disulphide exchange mechanism in the interaction with its target enzyme.     

Invasion of ras-transformed breast epithelial cells depends on the proteolytic activity of cysteine and aspartic proteinases

It has been suggested that the lysosomal proteinases cathepsin B, L and D participate in tumour invasion and metastasis. Whereas for cathepsins B and L the role of active enzyme in invasion processes has been confirmed, cathepsin D was suggested to support tumour progression via its pro-peptide, rather than by its proteolytic activity. In this study we have compared the presence of active cathepsins B, L and D in ras-transformed human breast epithelial cells (MCF-10A neoT) with their ability to invade matrigel. In this cell line high expression of all three cathepsins was detected by immunofluorescence microscopy. The effect of proteolytic activity on cell invasion was studied by adding various natural and synthetic cysteine and aspartic proteinase inhibitors. The most effective compound was chicken cystatin, a general natural inhibitor of cysteine proteinases, (82.8+/-1.6% inhibition of cell invasion), followed by the synthetic inhibitor trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64). CLIK-148, a specific inhibitor of cathepsin L, showed a lower effect than chicken cystatin and E-64. Pepstatin A weakly inhibited invasion, whereas the same molar concentrations of squash aspartic proteinase (SQAPI)-like inhibitor, isolated from squash Cucurbita pepo, showed significant inhibition (65.7+/-1.8%). We conclude that both cysteine and aspartic proteinase activities are needed for invasion by MCF-10A neoT cells in vitro.