Saxiphilin, a saxitoxin-binding protein with two thyroglobulin type 1 domains, is an inhibitor of papain-like cysteine proteinases

The type 1 domain of thyroglobulin is a protein module (Thyr-1) that occurs in a variety of secreted and membrane proteins. Several examples of Thyr-1 modules have been previously identified as inhibitors of the papain family of cysteine proteinases. Saxiphilin is a neurotoxin-binding protein from bullfrog and a homolog of transferrin with a pair of such Thyr-1 modules located in the N-lobe. Saxiphilin is now characterized as a potent inhibitor of three cysteine proteinases as follows: papain, human cathepsin B, and cathepsin L. The stoichiometry of enzyme inhibition reveals that both Thyr-1 domains of saxiphilin inhibit papain (apparent K(i) = 1. 72 nm), but only one of these domains inhibits cathepsin B (K(i) = 1. 67 nm) and cathepsin L (K(i) = 0.02 nm). Physical association of saxiphilin and papain blocked from turnover at the active-site cysteine residue can be detected by cross-linking with glutaraldehyde. The rate of association of saxiphilin and cathepsin B is strongly pH-dependent with an optimum at pH 5.2, reflecting control by at least two H(+)-titratable groups. These results further demonstrate that various Thyr-1 domains are selective inhibitors of cysteine proteinases with utility in the study of protein interactions and degradation.     

3-hydroxy-3-methylglutaryl coenzyme A reductase is sterol-dependently cleaved by cathepsin L-type cysteine protease in the isolated endoplasmic reticulum

We have recently shown that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, an endoplasmic reticulum (ER) membrane protein, is degraded in ER membranes prepared from sterol pretreated cells and that such degradation is catalyzed by a cysteine protease within the reductase membrane domain. The use of various protease inhibitors suggested that degradation of HMG-CoA reductase in vitro is catalyzed by a cathepsin L-type cysteine protease. Purified ER contains E-64-sensitive cathepsin L activity whose inhibitor sensitivity was well matched to that of HMG-CoA reductase degradation in vitro. CLIK-148 (cathepsin L inhibitor) inhibited degradation of HMG-CoA reductase in vitro. Purified cathepsin L also efficiently cleaved HMG-CoA reductase in isolated ER preparations. To determine whether a cathepsin L-type cysteine protease is involved in sterol-regulated degradation of HMG-CoA reductase in vivo, we examined the effect of E-64d, a membrane-permeable cysteine protease inhibitor, in living cells. While lactacystin, a proteasome-specific inhibitor, inhibited sterol-dependent degradation of HMG-CoA reductase, E-64d failed to do so. In contrast, degradation of HMG-CoA reductase in sonicated cells was inhibited by E-64d, CLIK-148, and leupeptin but not by lactacystin. Our results indicate that HMG-CoA reductase is degraded by the proteasome under normal conditions in living cells and that it is cleaved by cathepsin L leaked from lysosomes during preparation of the ER, thus clarifying the apparently paradoxical in vivo and in vitro results. Cathepsin L-dependent proteolysis was observed to occur preferentially in sterol-pretreated cells, suggesting that sterol treatment results in conformational changes in HMG-CoA reductase that make it more susceptible to such cleavage.     

Purification and characterization of a recombinant human testican-2 expressed in baculovirus-infected Sf9 insect cells

Testican-2 is a member of the testican group of brain extracellular proteoglycans where a 45 kDa modular protein core is composed of a follistatin-like domain, a calcium-binding domain, a thyroglobulin type-1 (Tg1) domain and an acid C-terminal region with glycosaminoglycan attachment sites. The modular structure suggests that it could participate in various interactions. The aim of the present study was to express and characterize a recombinant human testican-2 in quantities sufficient for structural and functional studies. Human cDNA coding for a 422 amino acid testican-2 protein was cloned into the pFastBac1 vector and expressed in the Spodoptera frugiperda (Sf9) insect cell expression system. The protein was purified to homogeneity by three chromatographic steps using the His(6) tag in the first two steps and ion exchange chromatography as last one. The final yield of purified recombinant testican-2 was up to 3.5 mg/L culture medium and its molecular mass determined by SDS-PAGE was approximately 55 kDa. Analysis by enzymatic deglycosylation revealed presence of N-linked sugars with a total mass of 4 kDa. In contrast to the Tg1 domain of testican-1, which acts as an inhibitor of the lysosomal cysteine peptidase cathepsin L, the Tg1 domain of testican-2 did not inhibit cathepsins L, B, K and S. We identified the C1q subcomponent of complement component C1 as a potential interacting partner of testican-2. The C1q subcomponent is a recognition molecule which acts in concert with other C1 subcomponents to activate the classical pathway of complement activation. The reported new interaction could be of importance in various complement-mediated inflammatory and other immune processes.     

Human proteoglycan testican-1 inhibits the lysosomal cysteine protease cathepsin L.

Testican-1, a secreted proteoglycan enriched in brain, has a single thyropin domain that is highly homologous to domains previously shown to inhibit cysteine proteases. We demonstrate that purified recombinant human testican-1 is a strong competitive inhibitor of the lysosomal cysteine protease, cathepsin L, with a Ki of 0.7 nM, but it does not inhibit the structurally related lysosomal cysteine protease cathepsin B. Testican-1 inhibition of cathepsin L is independent of its chondroitin sulfate chains and is effective at both pH 5.5 and 7.2. At neutral pH, testican-1 also stabilizes cathepsin L, slowing pH-induced denaturation and allowing the protease to remain active longer, although the rate of proteolysis is reduced. These data indicate that testican-1 is capable of modulating cathepsin L activity both in intracellular vesicles and in the extracellular milieu.     

Localization of rat cathepsin K in osteoclasts and resorption pits: inhibition of bone resorption and cathepsin K-activity by peptidyl vinyl sulfones.

We have localized cathepsin K in rat osteoclasts and within exposed resorption pits by immuno-fluorescence microscopy. Intracellular staining using an antibody raised against recombinant mouse cathepsin K was vesicular and uniformly distributed throughout the cell. Confocal microscopy analysis did not reveal an accumulation of cathepsin K containing vesicles opposing the ruffled border and the resorption lacuna. Exposed resorption pits exhibited a uniform distribution of cathepsin K, and no differences were observed between the edges and the centers of the pits. The immunostaining of resorption pits with anti-cathepsin K antibodies demonstrates that the protease is secreted into the sub-osteoclastic compartment. Cathepsin K-specific inhibition using peptidyl vinyl sulfones as selective cysteine protease inactivators reduced bone resorption by 80% in a dose-dependent manner at sub-micromolar concentrations. No reduction of bone resorption was observed at those low concentrations using a potent cathepsin L, S, B-specific inhibitor. That the inhibition of bone resorption can be attributed to cathepsin K-like protease inhibition was corroborated by the selective inhibition of the osteoclastic Z-Gly-Pro-Arg-MbetaNA hydrolyzing activity by the cathepsin K, L, S, B-inhibitor, but not by the cathepsin L, B, and S inhibitor. Z-Gly-Pro-Arg-MbetaNA is efficiently hydrolyzed by cathepsin K but only poorly by cathepsins L, S, and B. On the contrary, the intracellular hydrolysis of the cathepsin B-specific substrate, Z-Arg-Arg-MbetaNA, was prevented by both types of inhibitors. The identification of cathepsin K in resorption pits and the inhibition of bone resorption and intracellular cathepsin K activity by selective vinyl sulfone inhibitors indicate the critical role of the protease in osteoclastic bone resorption.     

Bombyx cysteine proteinase inhibitor (BCPI) homologous to propeptide regions of cysteine proteinases is a strong, selective inhibitor of cathepsin L-like cysteine proteinases.

Bombyx cysteine proteinase inhibitor (BCPI) is a novel cysteine proteinase inhibitor. The protein sequence is homologous to the proregions of certain cysteine proteinases. Here we report the mechanism of its inhibition of several cysteine proteinases. BCPI strongly inhibited Bombyx cysteine proteinase (BCP) activity with a K(i) = 5.9 pM, and human cathepsin L with a K(i) = 36 pM. The inhibition obeyed slow-binding kinetics. The inhibition of cathepsin H was much weaker (K(i) = 82 nM), while inhibition of papain (K(i) > 1 microM) and cathepsin B (K(i) > 4 microM) was negligible. Following incubation with BCP, BCPI was first truncated at the C-terminal end, and then gradually degraded over time. The truncation mainly involved two C-terminal amino acid residues. Recombinant BCPI lacking the two C-terminal amino acid residues still retained substantial inhibitory activity. Our results indicate that BCPI is a stable and highly selective inhibitor of cathepsin L-like cysteine proteinases.     

Regulation of collagenase activities of human cathepsins by glycosaminoglycans

Cathepsin K, a lysosomal papain-like cysteine protease, forms collagenolytically highly active complexes with chondroitin sulfate and represents the most potent mammalian collagenase. Here we demonstrate that complex formation with glycosaminoglycans (GAGs) is unique for cathepsin K among human papain-like cysteine proteases and that different GAGs compete for the binding to cathepsin K. GAGs predominantly expressed in bone and cartilage, such as chondroitin and keratan sulfates, enhance the collagenolytic activity of cathepsin K, whereas dermatan, heparan sulfate, and heparin selectively inhibit this activity. Moreover, GAGs potently inhibit the collagenase activity of other cysteine proteases such as cathepsins L and S at 37 degrees C. Along this line MMP1-generated collagen fragments in the presence of GAGs are stable against further degradation at 28 degrees C by all cathepsins but cathepsin K, whereas thermal destabilization at 37 degrees C renders the fragments accessible to all cathepsins. These results suggest a novel mechanism for the regulation of matrix protein degradation by GAGs. It further implies that cathepsin K represents the only lysosomal collagenolytic activity under physiologically relevant conditions.     

Identification and characterization of the interactive proteins with cytotoxic T-lymphocyte antigen-2α

Cytotoxic T-lymphocyte antigen-2α (CTLA-2α) is a potent inhibitor of cathepsin L-like cysteine proteases. Recombinant CTLA-2α is known to be a potent, competitive inhibitor of cathepsin L-like cysteine proteases. In this study, cathepsin L, cathepsin C, and tubulointerstitial nephritis antigen-related protein 1 (TINAGL1) were identified as novel interactive proteins of CTLA-2α by the yeast two-hybrid screening system. The direct interactions and co-localization of these proteins with CTLA-2α were confirmed using co-immunoprecipitation and immunofluorescence staining, respectively. The disulfide-bonded CTLA-2α/cathepsin L complex was isolated from mouse tissue. CTLA-2α was found to be specific and consistently expressed on the maternal side of the mouse placenta. Double immunofluorescence analysis showed that CTLA-2α was co-localized with cathepsin L, cathepsin C, and TINAGL1 in placenta. A simple cell-based fluorescence assay revealed that CTLA-2α exhibited inhibitory activity toward cathepsin C in live cells, which indicated that CTLA-2α is a novel endogenous inhibitor of cathepsin C.     

Inhibitory selectivity of canecystatin: a recombinant cysteine peptidase inhibitor from sugarcane

The cDNA of a cystein peptidase inhibitor was isolated from sugarcane and expressed in Escherichia coli. The protein, named canecystatin, has previously been shown to exert antifungal activity on the filamentous fungus Trichoderma reesei. Herein, the inhibitory specificity of canecystatin was further characterized. It inhibits the cysteine peptidases from plant source papain (Ki =3.3nM) and baupain (Ki=2.1x10(-8)M), but no inhibitory effect was observed on ficin or bromelain. Canecystatin also inhibits lysosomal cysteine peptidases such as human cathepsin B (Ki=125nM), cathepsin K (Ki=0.76nM), cathepsin L (Ki=0.6nM), and cathepsin V (Ki=1.0nM), but not the aspartyl peptidase cathepsin D. The activity of serine peptidases such as trypsin, chymotrypsin, pancreatic, and neutrophil elastases, and human plasma kallikrein is not affected by the inhibitor, nor is the activity of the metallopeptidases angiotensin converting enzyme and neutral endopeptidase. This is the first report of inhibitory activity of a sugarcane cystatin on cysteine peptidases.     

The role of aspartic and cysteine proteinases in albumin degradation by rat kidney cortical lysosomes

We have investigated the degradation of 125I-labeled bovine serum albumin by lysates of rat kidney cortical lysosomes. Maximal degradation of albumin occurred at pH 3.5-4.2, with approximately 70% of the maximal rate occurring at pH 5.0. Degradation was proportional to lysosomal protein concentration (range 100-600 micrograms) and time of incubation (1-5 h). Dithioerythritol (2 mM) stimulated albumin degradation 5- to 10-fold. Albumin degradation was not inhibited by phenylmethanesulfonyl fluoride (1 mM) or EDTA (5 mM), indicating that neither serine nor metalloproteinases are involved to a significant extent. Pepstatin (5 micrograms/ml), an inhibitor of aspartic proteinases, inhibited albumin degradation by approximately 50%. Leupeptin (10 microM) and N-ethylmaleimide (10 mM), inhibitors of cysteine proteinases, decreased albumin degradation by 34 and 65%, respectively. Combinations of aspartic and cysteine proteinase inhibitors produced nearly complete inhibition of albumin degradation. Taken together, these data indicate that aspartic and cysteine proteinases are primarily responsible for albumin degradation by renal cortical lysosomes under these conditions. In keeping with the above data, we have measured high activities of the cysteine proteinases, cathepsins B, H, and L, in cortical tubules, the major site of renal protein degradation. Using the peptidyl 7-amino-4-methylcoumarin (NHMec) substrates (Z-Arg-Arg-NHMec, for cathepsin B; Arg-NHMec for cathepsin H; and Z-Phe-Phe-CHN2-inhibitable hydrolysis of Z-Phe-Arg-NHMec corrected for inhibition of cathepsin B activity for cathepsin L) values obtained were (means +/- SE, mU/mg protein, 1 mU = production of 1 nM product/min, n = 6): cathepsin B, 2.1 +/- 0.34; cathepsin H, 1.35 +/- 0.19; cathepsin L, 14.49 +/- 1.26. In comparison, the activities of cathepsins B, H, and L in liver were: 0.56 +/- 0.03, 0.28 +/- 0.04, and 1.27 +/- 0.16, respectively.     

Matrigel® invasion by the prostate cancer cell lines, PC3 and DU145, and cathepsin L+B activity

Cathepsins L and B are lysosomal cysteine proteinases whose activities and cellular location are altered in many types of cancers and cancer cell lines. Cathepsins L and B play an unspecified role in cancer invasion and metastasis. The purpose of our study was to determine whether cathepsins L and B are important for the ability of two prostate cancer cell lines, PC3 and DU 145, to invade the basement membrane-like preparation, Matrigel®. Exposure of PC3 and DU145 to the irreversible cysteine proteinase inhibitor, E64, decreases the invasive ability of DU145, but not PC3. PC3 and DU145 were treated with the phorbol ester analogue, phorbol 12-myristate 13-acetate (PMA), a known tumor promoter that activates protein kinase C and contributes to the metastatic phenotype. PMA increased secreted cathepsin L+B activity and the invasive ability of PC3 and DU145; co-exposure to E64 and PMA decreased both cathepsin L+B activity and invasion. We conclude that DU145 requires cathepsin L+B activity more than PC3 for the invasion of the Matrigel®. When the amount of secreted cathepsin L+B activity is increased by PMA treatment, however, PC3 becomes dependent on cathepsin L+B for invasion. Our study demonstrates that modulation of the amount of secreted cathepsin L+B activity influences the invasive phenotype of PC3 and DU145.     

Matrigel® invasion by the prostate cancer cell lines,PC3 and DU145, and cathepsin L+B activity

Cathepsins L and B are lysosomal cysteine proteinases whose activities and cellular location are altered in many types of cancers and cancer cell lines. Cathepsins L and B play an unspecified role in cancer invasion and metastasis. The purpose of our study was to determine whether cathepsins L and B are important for the ability of two prostate cancer cell lines, PC3 and DU 145, to invade the basement membrane-like preparation, Matrigel®. Exposure of PC3 and DU145 to the irreversible cysteine proteinase inhibitor, E64, decreases the invasive ability of DU145, but not PC3. PC3 and DU145 were treated with the phorbol ester analogue, phorbol 12-myristate 13-acetate (PMA), a known tumor promoter that activates protein kinase C and contributes to the metastatic phenotype. PMA increased secreted cathepsin L+B activity and the invasive ability of PC3 and DU145; co-exposure to E64 and PMA decreased both cathepsin L+B activity and invasion. We conclude that DU145 requires cathepsin L+B activity more than PC3 for the invasion of the Matrigel®. When the amount of secreted cathepsin L+B activity is increased by PMA treatment, however, PC3 becomes dependent on cathepsin L+B for invasion. Our study demonstrates that modulation of the amount of secreted cathepsin L+B activity influences the invasive phenotype of PC3 and DU145.     

Cleavage site specificity of cathepsin K toward cartilage proteoglycans and protease complex formation

Cathepsin K is a potent extracellular matrix-degrading protease that requires interactions with soluble glycosaminolycans for its collagenolytic activity in bone and cartilage. The major sources of glycosaminoglycans in cartilage are aggrecan aggregates. Therefore, we investigated whether cathepsin K activity is capable to hydrolyze aggrecan into fragments allowing the formation of glycosaminoglycan-cathepsin K complexes and determined the cleavage site specificity of cathepsin K toward the cartilage-resident link protein and aggrecan. The cleavage site specificity was compared with those of cathepsins S and L. All three cathepsins released glycosaminoglycans from native bovine cartilage at lysosomal pH and to a lesser degree at neutral extracellular pH. Cathepsin-predigested aggrecan complexes and cartilage provided suitable glycosaminoglycan fragments that allowed the formation of collagenolytically active cathepsin K complexes. A detailed analysis of the degradation of aggrecan aggregates revealed two cathepsin K cleavage sites in the link protein and several sites in aggrecan, including one site within the interglobular domain E1. In summary, these results demonstrate that cathepsin K is capable to degrade aggrecan complexes at specific cleavage sites and that cathepsin K activity alone is sufficient to self-provide the glycosaminoglycan fragments required for the formation of its collagenolytically active complex.     

Influx of bivalent cations can be independent of receptor stimulation in human endothelial cells.

The major active forms of cathepsins B and L were identified in Kirsten-virus-transformed mouse fibroblasts by the use of a specific radiolabelled inhibitor, benzyloxycarbonyl-Tyr(-125I)-Ala-CHN2. No other proteins were labelled, demonstrating the specificity of this inhibitor for cysteine proteinases. Cathepsins B and L were distinguished by the use of specific antibodies. One active form of cathepsin B, Mr 33,000-35,000, and two active forms of cathepsin L, Mr 30,000 and 23,000, were identified. The intracellular precursors of these proteins had higher Mr values of 39,000 and 36,000 for cathepsins B and L respectively, as shown by pulse-chase experiments with [35S]methionine-labelled proteins. These did not react with the inhibitor under our culture conditions. The precursor of cathepsin L was secreted whereas the precursor of cathepsin B was not, demonstrating that secretions of the two enzymes are regulated differently. In contrast with results found previously for the purified protein [Mason, Gal & Gottesman (1987) Biochem. J. 248, 449-454], the secreted precursor form of cathepsin L did not react with the inhibitor either, indicating that it is not active and therefore, as such, cannot be directly involved in tumour invasion. The secreted protein did react with the inhibitor when incubated at pH 3.0, showing that the protein can be activated, although this did not occur under our culture conditions.     

A noninvasive cell-based assay for monitoring proteolytic activity within a specific subcellular compartment

A noninvasive cell-based assay has been developed to monitor the proteolytic activity of cathepsin L within a specific subcellular compartment, the lysosome. The green fluorescent protein (GFP) of Aequorea victoria was selected as a substrate. Targeting to lysosomes was achieved by fusing GFP to preprocathepsin L, which also ensures colocalization of the enzyme and the substrate. Stably transfected HeLa-rtTA (reverse tetracycline-controlled transactivator) cells were induced with doxycycline and cultured in the presence of various concentrations of cysteine protease inhibitors for 48 h. In the absence of inhibitor, proteolytic degradation of GFP leads to loss of fluorescence, which is due almost exclusively to the action of recombinant cathepsin L. However, a dose-dependent increase of GFP fluorescence is observed for cells treated with the potent cathepsin L inhibitor benzyloxycarbonyl-LeuLeuTyr-CHN(2). Fluorescence is also observed when GFP is fused to an inactive preprocathepsin L (C25A mutant). Targeting of GFP to an acidic cellular compartment can destabilize the protein and render it susceptible to proteolytic degradation. The approach should be generally applicable for proteases localized in acidic environments. Such an assay can be of great value in validating the participation of a specific enzyme in a given process or in testing the ability of putative inhibitors to reach their intracellular target.     

Clitocypin, a new type of cysteine proteinase inhibitor from fruit bodies of mushroom clitocybe nebularis

A novel inhibitor of cysteine proteinases has been isolated from fruit bodies of a mushroom Clitocybe nebularis. The inhibitor was purified to homogeneity by affinity chromatography and gel filtration, followed by reverse-phase high pressure liquid chromatography. The active inhibitor has an apparent molecular mass of about 34 kDa by gel filtration and by SDS-polyacrylamide gel electrophoresis without prior boiling of the sample. Boiling in 2.5% SDS or incubation in 6 m guanidine hydrochloride resulted in a single band of 17 kDa, indicating homodimer composition with no intersubunit disulfide bonds. The inhibitor in nondenaturing buffer is resistant to boiling in water, retaining its activity and dimer composition. The mushroom protein is a tight binding inhibitor of papain (K(i) = 0.59 nm), cathepsin L (K(i) = 0.41 nm), cathepsin B (K(i) = 0.48 micrometer), and bromelain (K(i) = 0.16 micrometer) but is inactive toward cathepsin H, trypsin, and pepsin. Its isoelectric point is 4.4, and sugar analysis indicates the absence of carbohydrate. A single protein sequence of 150 amino acids, containing no cysteine or methionine residues, was obtained by amino acid sequencing. The calculated molecular mass of 16854 Da corresponds well with the value obtained by mass spectrometry. A major part of this sequence was verified by molecular cloning. The monomer sequence is clearly devoid of typical cystatin structure elements and has no similarity to any other known cysteine proteinase inhibitors but bears some similarity to a lectin-like family of proteins from mushrooms. The inhibitor, which is present in at least two other members of the Clitocybe genus, has been named clitocypin (Clitocybe cysteine proteinase inhibitor).     

Regulation of collagenolytic cysteine protease synthesis by estrogen in osteoclasts

In ovariectomized (Ovx) mice, collagenolytic cysteine protease (CCP) activity in calvaria significantly increased 7 days after ovariectomy and was about 50% of that observed in sham-operated (Sham) mice 3 weeks later. In Ovx mice, subcutaneously (s.c.) administered estradiol-17beta (E2) (10 microg/kg) for 2 weeks led to a decrease in CCP activity in calvaria to the level observed in Sham mice. In Ovx mice, though the amount of cathepsin L increased more than that of cathepsin K, cathepsin K and cathepsin L content increased by 200-400% compared with the Sham mice; cathepsin K was detected in larger amounts than cathepsin L in calvaria from both Sham and Ovx mice. The amounts of cathepsin K and cathepsin L in Ovx mice were reduced to the values seen with Sham mice after administration (s.c.) of E2 (10 microg/kg) for 2 weeks. In mouse calvarial organ culture, the increase of CCP activity and release of hydroxyproline, an indicator of degradation of type-I collagen, in the presence of 1alpha,25-(OH)(2)D(3), parathyroid hormone, interleukin (IL)-1alpha, IL-6, or tumor necrosis factor-alpha was suppressed by E2 (10(-9)-10(-7) M). In all cases, secretion of both cathepsin K and cathepsin L were suppressed by E2. In osteoclasts, expression of cathepsin K and cathepsin L was suppressed by E2 at the mRNA level. Cathepsin B was detected faintly or not at all. These results suggest that synthesis of cathepsin K and cathepsin L was negatively regulated by E2 at the mRNA level. In Ovx mice, deficiency of E2 resulted in an augmentation of cathepsin K and cathepsin L synthesis, and the cathepsins might share roles in bone resorption in vivo.     

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.     

Role of cathepsin B and L in anti-glomerular basement membrane nephritis in rats

We have examined the potential role of the cysteine proteinases, cathepsin B and L, in renal tubular protein degradation and increased permeability of the glomerular basement membrane (GBM) which occurs in a neutrophil- and complement-independent model of anti-GBM antibody disease. The specific activity of cathepsin L, but not cathepsin B, was significantly increased (157%, p greater than 0.01) in cortical homogenates (85-90% tubules) prepared from anti-GBM-treated rats compared to saline-treated controls. Using highly purified cathepsin B and L, we documented the ability of these proteinases to degrade albumin in vitro (Km 5.92 and 0.22 microM for B and L, respectively). In two separate studies, treatment of rats with trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, (E-64), a specific and irreversible inhibitor of cysteine proteinases, significantly reduced proteinuria (-45 and -41%, p less than 0.01) in the 24-hour period following injection of the anti-GBM IgG. Taken together, these data suggest an important role for cysteine proteinases in the increased tubular protein degradation which occurs in response to increased filtered protein loads and in the increased GBM permeability (proteinuria) characteristic of glomerular disease.