Intracellular and extracellular activity of cathepsin D in the liver in cirrhosis and its involution

The distribution of cathepsin D in liver with CCl4 induced cirrhosis and its involution in rats was investigated by ultrastructural cytochemistry. Besides intracellular, it was revealed the extracellular activity of cathepsin D. The reaction product was on collagen fibers near the hepatocytes and connective tissue cells as well as on the hepatocytes microvilli and on the outside part of cellular membrane of connective tissue cells (macrophage, fibroblast, Ito cells). Hence the source of extracellular cathepsin D in liver are the parenchymatous as well as nonparenchymal cell elements. The results testify that under the cirrhosis and its involution, the cathepsin D takes part in intracellular proteolysis and is secreted by hepatocytes and connective tissue cells in the intracellular space; it also takes part in extracellular catabolism of connective tissue.     

Localization of cathepsin D in rat liver

Summary Light and electron microscopic localization of cathepsin D in rat liver was investigated by post-embedding immunoenzyme and protein A-gold techniques. By light microscopy, cytoplasmic granules of parenchymal cells and Kupffer cells were stained for cathepsin D. Weak staining was also noted in sinusoidal endothelial cells. In the parenchymal cells many of positive granules located around bile canaliculi. In the Kupffer cells and the endothelial cells, diffuse staining was noted in the cytoplasm in addition to granular staining. By electron microscopy, gold particles representing the antigenic sites for cathepsin D were seen in typical secondary lysosomes and some multivesicular bodies of the parenchymal cells and Kupffer cells. The lysosomes of the endothelial cells and fat-storing cells were weakly labeled. Quantitative analysis of the labeling density in the lysosomes of these three types of cells demonstrated that the lysosomes of parenchymal cells and Kupffer cells are main containers of cathepsin D in rat liver. The results suggest that cathepsin D functions in the intracellular digestive system of parenchymal cells and Kupffer cells but not so much in that of the endothelial cells.     

Specificity of rat liver cathepsin D

The specificity of highly purified rat liver cathepsin D was investigated by analyzing the digests of denatured proteins. At the P1 site, cathepsin D prefers hydrophobic residues except Ile and Val, that are branched at the beta-carbon. Strong and weak hydrophobicities are required at P1' and P2 sites, respectively. A lower protency for beta-turn formation is essential for the sequence around the P1 site.     

Changes in rat liver immunoreactive cathepsin D after cycloheximide

A rocket immunoelectrophoretic procedure has been developed for the assay of cathepsin D (EC 3.4.23.5) immunoreactive protein, in a 10-100 ng range, directly on crude soluble liver homogenate extracts. By this method, the drop in activity of rat liver cathepsin D effected by repeated doses of cycloheximide, a protein synthesis inhibitor, reflects a parallel change in total enzyme protein content, the specific activity being stable in the course of the treatment. These observations are compatible with the hypothesis that ongoing enzyme degradation, coupled with impaired synthesis, accounts for such a decline of cathepsin D.     

Postnatal changes of cathepsin D activity in rat liver and brain

Total and specific activity of cathepsin D (EC. 3.4.23.5) were measured in rat liver and brain from 1 to 98 days of age. The activity of cathepsin D in the liver of adult and newborn rats was the same while in the rat brain it was higher in adult than in newborn rats. In the liver maximum specific activity of cathepsin D occurred on the 10th postnatal day and minimum on the fourth day of age. In the brain maximum specific activity of the enzyme occurred on the 14th postnatal day. Total activity of cathepsin D increased after birth in rat liver and brain. These results are discussed in relation to the functional role of cathepsin D in the rat liver and the brain.     

Vitellogenesis-related ovary cathepsin D from Xenopus laevis: Purification and properties in comparison with liver cathepsin D

Cathepsin D was purified from ovaries of Xenopus laevis by both QAE-cellulose and pepstatin-Sepharose chromatography and then characterized and compared with Xenopus liver cathepsin D. Ovary cathepsin D appeared predominantly as a 43-kilodalton (kDa) molecular mass, as revealed by SDS-polyacrylamide gel electrophoresis, whereas the liver enzyme was obtained exclusively as a 36-kDa protein. The purified 43-kDa ovary enzyme cleaved vitellogenin limitedly to produce yolk proteins at pH 5.6. The specific activity of ovary cathepsin D was five to six times lower than that of the liver enzyme, as measured by hemoglobin-hydrolysis at pH 3, but the ovary enzyme was shown to be superior to the liver enzyme in terms of vitellogenin-cleaving activity, as examined at pH 5.6. Ovarian enzyme preparations contained variable amounts of 36-kDa species; this form was considered to be an autolytic product of the 43-kDa form arising during purification, because it was not detected in oocyte extracts but was generated by incubation of the purified 43-kDa enzyme alone in an acid solution. The conversion of the 43-kDa form by hepatic factors was accompanied by a marked increase in hemoglobin-hydrolytic activity.     

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.     

Biosynthesis and intracellular transport of alpha-glucosidase and cathepsin D in normal and mutant human fibroblasts

In order to study the intracellular localization of the proteolytic processing steps in the maturation of alpha-glucosidase and cathepsin D in cultured human skin fibroblasts we have used incubation with glycyl-L-phenylalanine-beta-naphthylamide (Gly-Phe-NH-Nap) as described by Jadot et al. [Jadot, M., Colmant, C., Wattiaux-de Coninck, S. & Wattiaux, R. (1984) Biochem. J. 219,965-970] for the specific lysis of lysosomes. When a homogenate of fibroblasts was incubated for 20 min with 0.5 mM Gly-Phe-NH-Nap, a substrate for the lysosomal enzyme cathepsin C, the latency of the lysosomal enzymes alpha-glucosidase and beta-hexosaminidase decreased from 75% to 10% and their sedimentability from 75% to 20-30%. In contrast, treatment with Gly-Phe-NH-Nap had no significant effect on the latency of galactosyltransferase, a marker for the Golgi apparatus, and on the sedimentability of glutamate dehydrogenase and catalase, markers for mitochondria and peroxisomes, respectively. The maturation of alpha-glucosidase and cathepsin D in fibroblasts was studied by pulse-labelling with [35S]methionine, immunoprecipitation, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and fluorography. When homogenates of labelled fibroblasts were incubated with Gly-Phe-NH-Nap prior to immunoprecipitation, 70-80% of all proteolytically processed forms of metabolically labelled alpha-glucosidase and cathepsin D was recovered in the supernatant. The earliest proteolytic processing steps in the maturation of alpha-glucosidase and cathepsin D appeared to be coupled to their transport to the lysosomes. Although both enzymes are transported via the mannose-6-phosphate-specific transport system, the velocity with which they arrived in the lysosomes was consistently different. Whereas newly synthesized cathepsin D was found in the lysosomes 1 h after synthesis, alpha-glucosidase was detected only after 2-4 h. When a pulse-chase experiment was carried out in the presence of 10 mM NH4Cl there was a complete inhibition of the transport of cathepsin D and a partial inhibition of that of alpha-glucosidase to the lysosomes. Leupeptin, an inhibitor of lysosomal thiol proteinases, had no effect on the transport of labelled alpha-glucosidase to the lysosomes. However, the early processing steps in which the 110-kDa precursor is converted to the 95-kDa intermediate form of the enzyme were delayed, a transient 105-kDa form was observed and the conversion of the 95-kDa intermediate form to the 76-kDa mature form of the enzyme was completely inhibited.(ABSTRACT TRUNCATED AT 400 WORDS)     

On inactive precursor of cathepsin D from chicken liver

Two enzyme preparations (components 1 and 11) were isolated from chicken liver by means of DEAE-cellulose chromatography. The highest proteolytic activity was seen in the zone of comparatively low protein concentration. Storage of the isolated first component in acid medium at pH 3 resulted in considerable increase of its activity, as well as in increase of its ability to bind to diazocarboxyl inhibitor. In the course of this reaction an additional, highly active component and an inactive, low-molecular-weight fraction were formed.     

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.     

Evidence that extracellular cathepsin D is not responsible for the resorption of cartilage matrix in culture

Cathepsin D, the major lysosomal aspartic proteinase, is responsible for the autolysis of cartilage at slightly acidic pH, and it has been suspected of making a significant contribution to the breakdown of the living tissue, such as is stimulated by retinol. Our finding, however, has been that neither inhibitory antibodies against cathepsin D, nor chemical inhibition with pepstatin, significantly decreases the rate of degradation of proteoglycan in the organ culture system. Most of the other proteinase inhibitors tested were similarly ineffective, although EDTA and 1,10-phenanthroline inhibited the resorption by a cytotoxic effect. We conclude that although cartilage matrix degradation has clear characteristics of a proteolytic process, the identity of the enzyme(s) responsible remains obscure.     

Defective acidification of intracellular organelles results in aberrant secretion of cathepsin D in cancer cells

Aberrant secretion of lysosomal hydrolases such as (pro)cathepsin D (proCD) is a common phenotypic change in many human cancers. Here we explore the underlying molecular defect(s) and find that MCF-7 breast and CaCo-2 colorectal cancer cells that are unable to acidify their endosomal compartments secreted higher amounts of proCD than did acidification-competent cancer cell types. The latter secreted equivalent amounts of proCD only after dissipation of their organellar pH gradients with NH(4)Cl. Assessing the critical steps that resulted in proCD secretion revealed that the Golgi-associated sorting receptor for CD, i.e. the cation-independent mannose-6-phosphate receptor (MPR300), was aberrantly distributed in acidification-defective MCF-7 cells. It accumulated mainly in late endosomes and/or lysosomes as a complex with its ligand (proCD or intermediate CD), as evidenced by its co-localization with both CD and LAMP-2, a late endosome/lysosome marker. Our immunoprecipitation analyses also showed that MCF-7 cells possessed 7-fold higher levels of receptor-enzyme complexes than did acidification-competent cells. NH(4)Cl induced similar receptor redistribution into LAMP-2-positive structures in acidification-competent cells but not in MCF-7 cells. The receptor also recovered its normal Golgi localization upon drug removal. Based on these observations, we conclude that defective acidification results in the aberrant secretion of proCD in certain cancer cells and interferes mainly with the normal disassembly of the receptor-enzyme complexes and efficient receptor reutilization in the Golgi.     

Self-quenched fluorogenic protein substrates for the detection of cathepsin D and other protease activities

Self-quenched fluorogenic substrates for proteolytic enzymes have been prepared by alkylation of thiol groups in reduced bovine serum albumin with iodoacetamidofluorescein or iodoacetamidoeosin. Substrates immobilized by adsorption onto nitrocellulose membranes or by incorporation into agarose gel slabs are suitable for fluorescence zymography after electrophoretic separation of catalytically active proteases, including cathepsin D.     

Fluorescent histochemical demonstration of cathepsin B in the rat yolk sac

Fluorescence demonstration of cathepsin B (E.C. 3.4.22.1) activity was performed in the yolk sac of rats near term (18th day of gestation). The enzyme demonstration was performed on freeze-dried and celloidin mounted yolk-sac sections using different substituted beta-naphthylamide derivatives as substrates and nitrosalicylaldehyde as coupling agent. The discrete reaction products are localized preferentially in the apical part of the visceral yolk-sac epithelium. There is little doubt that cathepsin B is contained here in the well developed lysosomal apparatus of the yolk-sac epithelium.     

Fluorescent histochemical demonstration of cathepsin B in the rat yolk sac

Summary Fluorescence demonstration of cathepsin B (E.C. 3.4.22.1) activity was performed in the yolk sac of rats near term (18th day of gestation). The enzyme demonstration was performed on freeze-dried and celloidin mounted yolk-sac sections using different substituted -naphthylamide derivatives as substrates and nitrosalicylaldehyde as coupling agent. The discrete reaction products are localized preferentially in the apical part of the visceral yolk-sac epithelium. There is little doubt that cathepsin B is contained here in the well developed lysosomal apparatus of the yolk-sac epithelium.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)