Brain cathepsin B cleaves a caspase substrate

We show that an enzyme exists in rat brain capable of cleaving the caspase-3 specific peptide substrate Ac-DEVD-AMC at low pH. The enzyme shows properties of a cysteine protease and is localized, predominantly, in lysosomes. We have purified this enzyme from rat brain and identified it by MALDI-TOF MS. The enzyme possessing “acidic” DEVDase activity in rat brain appears to be cathepsin B. It remains obscure, whether cathepsin B participates in cleavage of caspase-3 substrates in vivo. We suggest that under certain conditions (e.g. in hypoxia) cathepsin B participates in cleavage of caspase-3 substrates in brain cells. Published in Russian in Biokhimiya, 2008, Vol. 73, No. 3, pp. 408–413.     

Evidence for lysosomal reduction of cystine residues.

Previously reported evidence for the existence of a thiol: protein disulphide oxidoreductase in rat liver lysosomes has been re-examined and ambiguous results obtained. However, incubation of purified rat liver lysosomes with ¹²⁵I-labelled insulin at pH 5.5 shows that cathepsin D and a thiol-dependent enzyme other than cathepsin B or L are important in its digestion. The latter enzyme is most probably a thiol: protein disulphide oxidoreductase.     

Species variations amongst lysosomal cysteine proteinases

Properties of cathepsin L from rat liver lysosomes were compared with those of a similar enzyme, cathepsin S from beef spleen. Major characteristics of cathepsin L are the high activity against Z-Phe-Arg-methylcoumarylamide and sensitivity to the fast reacting irreversible inhibitor Z-Phe-Phe-diazomethane. In contrast, cathepsin S hydrolyzes Z-Phe-Arg-methylcoumarylamide only slowly and Z-Phe-Phe-diazomethane cannot be regarded as a potent inhibitor of this enzyme. The differences in the substrate specificity of cathepsin L from rat liver and cathepsin S from beef spleen are discussed in comparison with the substrate specificity of cathepsin B from rat and human liver and beef spleen.     

Characteristic distribution of cathepsin E which immunologically cross-reacts with the 86-kDa acid proteinase from rat gastric mucosa

The antiserum raised against the high-molecular-weight acid proteinase from rat gastric mucosa, termed 86-kDa acid proteinase, has been shown to recognize rat cathepsin E, but not cathepsin D (Muto, N. et al. (1987) J. Biochem. 101, 1069-1075). Using this specific antiserum, characteristic distribution of cathepsin E in rats was demonstrated. The enzyme was detected in a limited number of tissues, such as stomach, thymus, spleen, bladder, and erythrocyte membranes. Among them, the highest activity was observed in the stomach. In contrast, cathepsin D immunoreactive with the antiserum specific to rat gastric cathepsin D was demonstrated in all the tissues examined. Cathepsin E-type enzymes partially purified from these five tissues were precipitated in the same manner by the specific antiserum, and they had the same molecular weight, electrophoretic mobility, and resistance against denaturation by 4 M urea. These results indicate that they could be exactly classified as cathepsin E. This type of enzyme was also detectable in mice and guinea pigs, but they showed relatively weak immunoreactivities with the antiserum. Thus, it is concluded that the distribution of cathepsin E is intrinsically different from ordinary cathepsin D, suggesting that it has a different physiological role from cathepsin D.     

An immunocytochemical study on co-localization of cathepsin B and atrial natriuretic peptides in secretory granules of atrial myoendocrine cells of rat heart

To examine localization of cathepsin B, a representative lysosomal cysteine protease, in atrial myoendocrine cells of the rat heart, immunohistochemistry at the light and electron microscopic level was applied to the atrial tissue, using a monospecific antibody for rat liver cathepsin B. In serial semi-thin sections, immunoreactivity for cathepsin B and atrial natriuretic peptides (ANP) was detected in the para-nuclear region of atrial myoendocrine cells. Several large granules and many fine granules in the region of the cells were positively stained by the cathepsin B antibody. Gold particles indicating cathepsin B antigenicity labeled secretory granules in the cells, which were also labeled by those indicating ANP, using thin sections of the Lowicryl K4M-embedded material. Moreover, some granules labeled densely by immunogold particles for cathepsin B seemed to be lysosomes. By double immunostaining using thin sections of the Epon-embedded material, gold particles indicating cathepsin B and ANP antigenicities were co-localized in secretory granules of the cells. By enzyme assay, activity of cathepsin B was three times higher in atrial tissue than ventricular tissue. The results suggest that co-localization of cathepsin B and ANP in secretory granules is compatible with the possibility that cathepsin B participates in the maturation process of ANP.     

Regulated secretion of mature cathepsin B from rat exocrine pancreatic cells.

By indirect immunofluorescence and immunogold electron microscopy with an antibody that recognizes specifically the two forms of native mature rat cathepsin B (31 kDa and 5:25 kDa) but not the proenzyme, we detected cathepsin B not only in lysosomes of adult rat exocrine pancreatic cells but also in the trans Golgi condensing vacuoles, the zymogen granules and the pancreatic juice in the intralobular ducts. In contrast, immunocytochemistry with an antibody specific for rat cathepsin D showed the latter to be present in the same cells only in lysosomal compartments as expected. The same pattern of labeling with these two antibodies was found in the first zymogen granules to form in 17-day-old fetal rat pancreas. Counts of the extent of immunogold labeling of cathepsin B in the adult exocrine cells showed that the concentration of the enzyme was only two-fold higher in the lysosomal compartments than in the zymogen granules. To confirm these observations, rat pancreatic postnuclear supernatant (PNS), a fraction enriched in zymogen granules and rat pancreatic juice obtained by catheterization of the pancreatic duct, were subjected to 2D gel electrophoresis followed by immunoblotting with the cathepsin B antibody. All three samples contained a 31 kDa protein recognized by the antibody with a pI of about 4.5, the single chain mature form of cathepsin B. We then radiolabeled pancreatic PNS and zymogen granule fractions with benzyloxycarbonyl-Tyr[125I]-Ala-CHN2, an affinity label that covalently binds to the active sites of mature forms of both cathepsin B and cathepsin L. In both PNS and zymogen granule fractions this reagent labeled cathepsin B. Immunoprecipitation experiments showed that the antibody to cathepsin B recognized specifically both the single chain and the double chain mature forms of cathepsin B in the native state. Finally, Northern blots with a cDNA of rat cathepsin B showed that the concentration of cathepsin B mRNA in total pancreatic RNA increased following in vivo stimulation of the exocrine pancreatic cells with optimal doses of cerulein, a cholecystokinin analogue. We conclude that significant amounts of mature cathepsin B are secreted from exocrine pancreatic cells via the apical regulated exocytotic pathway, and we discuss this in terms of models for sorting of proteins to the cores of dense cored secretory granules.     

Properties of a fructose 1,6-bisphosphatase converting enzyme in rat liver lysosomes.

An enzyme present in rat liver lysosomes catalyzes the conversion of neutral rabbit liver fructose 1,6-bisphosphatase (Fru-P₂ase, EC 3.1.3.11) to a form having maximum activity at pH 9.2. The converting enzyme is partly released when lysosomes are subjected to a single freeze-thaw cycle, but a significant fraction tends to remain with the lysosomal membrane fraction even after repeated freezing and thawing. After repeated freezing and thawing hexosaminidase and cathepsin D are also partly membrane-bound, but cathepsins A, B, and C are completely solubilized. The membrane-bound enzymes, unlike those in intact lysosomes, are not cryptic. The converting enzyme activity is inactivated by phenylmethanesulfonyl fluoride, and is almost completely inactive after exposure to iodoacetic acid or tosylamido-2-phenylethyl and N-α-tosyl lysyl chloromethyl ketones. Unlike cathepsin B, it is not inhibited by leupeptin. Converting enzyme is unstable above pH 6.5, and this property also serves to distinguish it from cathepsins B and D. The results suggest that the converting enzyme is not identical to any of the well-characterized cathepsins.     

An improved procedure for the histochemical demonstration of cathepsin D by the mercury-labeled pepstatin method

The desirable fixation conditions for the histochemical demonstration of cathepsin D using mercury-labeled pepstatin as an enzyme inhibitor were examined biochemically and histochemically. Four well known fixatives, namely, glutaraldehyde (GA), paraformaldehyde (PFA), glutaraldehyde with paraformaldehyde (GA-PFA) and periodate-lysine-paraformaldehyde (PLP), were applied to the prefixation of tissues prior to the reaction of the labeled inhibitor to the enzyme-active site. The effects of fixatives on cathepsin D were biochemically examined using subcellular fractionated lysosomes. Cathepsin D from rat liver lysosomes was rapidly inactivated by the fixatives containing glutaraldehyde, i.e., GA and GA-PFA, whereas the activity of cathepsin D was sufficiently maintained after fixing the enzyme in the PFA or PLP preparations. Effects of the PLP fixative on lysosomal cathepsin D in liver tissues using the mercury-labeled pepstatin method were also studied histochemically. The best result for the visualization of lysosomal cathepsin D in liver tissues was obtained using the PLP fixative with the prefixation time of three hours or more.     

The heterogeneity of rat kidney lysosomes revealed by immunoelectron microscopic staining for cathepsins B and H

The heterogeneity of lysosomes was studied by analyzing the immunostaining behavior of cathepsins B and H in rat kidney proximal tubule cells. Rat kidneys were fixed by perfusion and embedded in Lowicryl K4M. A protein A-gold technique was applied to serial sections and a double labeling technique to conventional sections. By analyzing the immunostaining behavior of cathepsins B and H in the same lysosomes which were cut into separate sections, four types of lysosomes were found: Type 1 positive for both proteinases; type 2 strongly positive for cathepsin B, but weakly or negative for cathepsin H; type 3 strongly positive for cathepsin H, but weakly or negative for cathepsin B; and type 4 negative for both proteinases. The double labeling by two different sizes of the protein A-gold probes showed these four types of lysosomes. The results indicate that there exists the lysosomal heterogeneity of the proteinase content in the kidney proximal tubule cells.     

An immunocytochemical study on distinct intracellular localization of cathepsin E and cathepsin D in human gastric cells and various rat cells.

Immunocytochemical localization of two distinct intracellular aspartic proteinases, cathepsins E and D, in human gastric mucosal cells and various rat cells was investigated by immunogold technique using discriminative antibodies specific for each enzyme. Cathepsin D was exclusively confined to primary or secondary lysosomes in almost all the cell types tested, whereas cathepsin E was not detected in the lysosomal system. The localization of cathepsin E varied with different cell types. Microvillous localization of cathepsin E was found in the intracellular canaliculi of human and rat gastric parietal cells, rat renal proximal tubule cells, and the bile canaliculi of rat hepatic cells. The immunolocalization of each enzyme in gastric cells were essentially the same in humans and rats. In the gastric feveolar epithelial cells and parietal cells, definite immunolabeling for cathepsin E was observed in the cytoplasmic matrix, the cisternae of the rough endoplasmic reticulum, and the dilated perinuclear envelope. In rat kidney, cathepsin E was detected only in the proximal tubule cells, while cathepsin D was found mainly in the lysosomes of the distal tubule cells but not in those of the proximal tubule cells. These results clearly indicate the distinct intracytoplasmic localization of cathepsins E and D and suggest the possible involvement of cathepsin E in extralysosomal proteolysis that is related to specialized functions of each cell type.     

An Improved Procedure for the Histochemical Demonstration of Cathepsin D by the Mercury-Labeled Pepstatin Method

The desirable fixation conditions for the histochemical demonstration of cathepsin D using mercury-labeled pepstatin as an enzyme inhibitor were examined biochemically and histochemically. Four well known fixatives, namely, glutaraldehyde (GA), paraformaldehyde (PFA), glutaraldehyde with paraformaldehyde (GA-PFA) and periodate-lysine-paraformaldehyde (PLP), were applied to the prefixation of tissues prior to the reaction of the labeled inhibitor to the enzyme-active site. The effects of the fixatives on cathepsin D were biochemically examined using subcellular fractionated lysosomes. Cathepsin D from rat liver lysosomes was rapidly inactivated by the fixatives containing glutaraldehyde, i.e., GA and GA-PFA, whereas the activity of cathepsin D was sufficiently maintained after fixing the enzyme in the PFA or PLP preparations. Effects of the PLP fixative on lysosomal cathepsin D in liver tissues using the mercury-labeled pepstatin method were also studied histochemically. The best result for the visualization of lysosomal cathepsin D in liver tissues was obtained using the PLP fixative with the prefixation time of three hours or more.     

The heterogeneity of rat kidney lysosomes revealed by immunoelectron microscopic staining for cathepsins B and H

Summary The heterogeneity of lysosomes was studied by analyzing the immunostaining behavior of cathepsins B and H in rat kidney proximal tubule cells. Rat kidneys were fixed by perfusion and embedded in Lowicryl K4M. A protein A-gold technique was applied to serial sections and a double labeling technique to conventional sections. By analyzing the immunostaining behavior of cathepsins B and H in the same lysosomes which were cut into separate sections, four types of lysosomes were found: Type 1 positive for both proteinases; type 2 strongly positive for cathepsin B, but weakly or negative for cathepsin H; type 3 strongly positive for cathepsin H, but weakly or negative for cathepsin B; and type 4 negative for both proteinases. The double labeling by two different sizes of the protein A-gold probes showed these four types of lysosomes. The results indicate that there exists the lysosomal heterogeneity of the proteinase content in the kidney proximal tubule cells.     

Cathepsin L. A new proteinase from rat-liver lysosomes.

1. Cathepsin L was purified from rat liver lysosomes by cell fractionation, osmotic disruption of the lysosomes in the lysosomal mitochondrial pellet, gel filtration of the lysosomal extract and chromatography on CM-Sephadex. 2. Cathepsin L is a thiol proteinase and exists in several multiple forms visible on the disc electropherogram. By polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate its molecular weight was found to be 23000-24000. The isoelectric points of the multiple forms of cathepsin L extended from pH 5.8-6.1 ascertained by analytical isoelectric focusing. 3. Using various protein substrates, cathepsin L was found to be the most active endopeptidase from rat liver lysosomes acting at pH 6-7. In contrast to cathepsin B1, its capability of hydrolyzing N-substituted derivatives of arginine is low and it does not split esters. 4. Greatest activity is obtained close to pH 5.0 with 70-90% of maximal activity at pH 4.0 and pH 6.0 and 30-40% at pH 7.0. 5. The enzyme is strongly inhibited by leupeptin and the chloromethyl ketone of tosyl-lysine. Leupeptin acts as a pseudo-irreversible inhibitor. 6. The enzyme is stable for several months at slightly acid pH values in the presence of thiol compounds in a deep-frozen state.     

Intracellular transport and processing of lysosomal cathepsin B

Intracellular transport and processing of lysosomal cathepsin B was investigated in the subcellular fractions of rat liver by pulse-labeling experiments with [35S]methionine in vivo. A newly synthesized procathepsin B with a molecular weight of 39 kDa firstly appeared in the rough microsomal fraction at 10 min postinjection of label. This procathepsin B moved from the microsomal fractions to the Golgi subfractions at 30 min postinjection, and then a processed mature enzyme appeared in the lysosomal fraction at 60 min. These results suggest that the propeptide-processing of procathepsin B takes place in lysosomes in the course of intracellular transport from endoplasmic reticulum through Golgi complex to lysosomes.     

Immunocytochemical localization of cathepsin B in rat kidney. II. Electron microscopic study using the protein A-gold technique

Thin sections of Lowicryl K4M-embedded materials were labeled with protein A-gold complex. Gold particles representing the antigen sites for cathepsin B were exclusively confined to lysosomes of each segment of the nephron. The heaviest labeling was noted in the lysosomes of the S1 segment of the proximal tubules. Labeling intensity varied considerably with the individual lysosomes. Lysosomes of the other tubular segments, such as the S2 and S3 segments of the proximal tubules, distal convoluted tubules, and collecting tubules were weakly labeled by gold particles. Quantitative analysis of labeling density also confirmed that lysosomes in the S1 segment have the highest labeling density and that approximately 65% of labeling in the whole renal segments, except for the glomerulus, was found in the S1 segment. These results indicate that in rat kidney the lysosomes of the S1 segment are a main location of cathepsin B. Further precise observations on lysosomes of the S1 segment revealed that apical vesicles, tubules, and vacuoles were devoid of gold particles, but when the vacuoles contained fine fibrillar materials, gold labeling was detectable in such vacuoles. As the lysosomal matrix becomes denser, the labeling density is increased. Some small vesicles around the Golgi complex were also labeled. These results indicate that the endocytotic apparatus including the apical vesicles, tubules, and vacuoles contains no cathepsin B. When the vacuoles develop into phagosomes, they acquire this enzyme to digest the absorbed proteins.     

A cysteine proteinase secreted from human breast tumours is immunologically related to cathepsin B.

The stable cathepsin B-like cysteine (thiol) proteinase secreted from human breast tumours in culture was shown to be destabilized by mercurial compounds. After such treatment the enzyme cross-reacts in a radioimmunoassay with a monospecific antiserum to human liver cathepsin B. It is suggested that the secreted enzyme may be a precursor form of lysosomal cathepsin B.     

Inhibition of cathepsin D prevents free-radical-induced apoptosis in rat cardiomyocytes

Apoptosis was inhibited in rat cardiomyocytes pretreated with the aspartic protease inhibitor pepstatin A and subsequently exposed to naphthazarin (5,8-dihydroxy-1,4-naphthoquinone). Cathepsin D was released from lysosomes to the cytosol upon exposure to naphthazarin, and the enzyme activity decreased simultaneously. Later, cathepsin D reappeared in granules of increased size, and enzyme activity was restored. Activation of caspase-3-like proteases was detected, and the number of cells showing apoptotic morphology increased with time. Pepstatin A pretreatment did not prevent release of cathepsin D from lysosomes but did significantly inhibit subsequent naphthazarin-induced caspase activation and apoptotic morphology. This suggests that cathepsin D exerts its apoptosis-stimulating effect upstream of caspase-3-like activation.     

Immunocytochemical localization of cathepsin D in lysosomes of cortical collecting tubule cells of the rat kidney

Immunocytochemical localization of cathepsin D in rat renal tubules was investigated by means of indirect immunoenzyme and protein A--gold techniques. By light microscopy, fine granular staining was seen in the mesangial cells of glomeruli. Heavy reaction deposits were present in the cortical tubular segments and some of the medullary collecting tubules. The proximal tubules contained a few positive granules. Other segments were negative for cathepsin D. By electron microscopy, gold particles representing the antigenic sites for cathepsin D were present in cytoplasmic granules and multivesicular bodies of the segment of the cortical collecting tubule. These cytoplasmic granules were presumed to be digestive vacuoles (secondary lysosomes) from their morphological profile. The proximal tubule cells contained the very weakly labeled secondary lysosomes. No specific labeling was noted in other segments of the nephron. Control experiments confirmed the specificity of the immunostaining. Quantitative analysis of the labeling density in each subcellular compartment also confirmed that the main subcellular sites for cathepsin D are the secondary lysosomes and multivesicular bodies. The labeling density in these granules of the lysosomal system varied widely with the individual granules, suggesting that there is a considerable heterogeneity of enzyme content among the granules of the lysosomal system. The prominent presence of cathepsin D in the cortical collecting tubule suggests a certain segment-specific function of this proteinase.     

Unique cathepsin D-type proteases in rat thoracic duct lymphocytes and in rat lymphoid tissues.

Two unique cathepsin D-type proteases apparently present only in rat thoracic duct lymphocytes and in rat lymphoid tissues are described. One, termed H enzyme, has an apparent molecular weight of similar to95,000; the other, termed L enzyme, has an apparent molecular weight of similar to45,000, in common with that of most cathepsins D from other tissues and species. Both enzymes differ from cathepsin D, however, by a considerably greater sensitivity to inhibition by pepstatin and by a smaller degree of inhibition by an antiserum which inhibits rat liver cathepsin D. H enzyme is converted to L enzyme by treatment with beta-mercaptoethanol; the relationship between the two enzymes remains unknown. H and L enzyme have been detected in rat lymphoid tissues and in mouse spleen, but they are not present in other rat tissues (liver, kidney, adrenals), rabbit tissues, calf thymus, bovine spleen, or human tonsils. As measured on acid-denatured bovine hemoglobin as substrate, both enzymes have pH activity curves identical with that of rat liver cathepsin D, with optimal activity at pH 3.6. Activity on human serum albumin is much less and also shows an optimum at pH 3.6; hence, neither enzyme has the properties of cathepsin E. Thiol-reactive inhibitiors have no effect on the activity of H and L enzyme; thus they do not belong to the B group of cathepsins. Additional information, discussed in this paper, leads us to conclude that partially purified H and L enzymes are cathepsin D-type proteases.     

Species variations amongst proteinases in liver lysosomes

Cathepsin B, H, L and D activities in liver lysosomes were compared between species. Although cathepsin B and D were detected in bovine, pig, chicken and rat liver, striking species differences were evident for cathepsin H and L. Cathepsin L activity was particularly high in chicken lysosomal extracts, but could not be detected in bovine and pig extracts. Whereas there was no significant cathepsin H activity in bovine extracts, rat liver lysosomal extracts contained large amounts of cathepsin H activity.