Conversion of proinsulin into insulin by cathepsins B and L from rat liver lysosomes

Conversion of proinsulin and intermediate forms of proinsulin into insulin were studied with rat liver cell fractions and purified lysosomal proteinases by using the technique of polyacrylamide disc-electrophoresis. Both substrates were degraded very rapidly by homogenates and crude lysosomal fractions to split products not detectable on disc-electropherograms. Neither breakdown nor conversion were detected with the cytosol and the microsomal fraction. With partially purified lysosomal fractions (mol. wt. approx. 25 000) or with highly purified cathepsin L or cathepsin B (B1) proinsulin was converted into products migrating like the intermediate forms and insulin, and the intermediates were converted into products migrating like insulin and deoctapeptide-insulin in disc-electropherograms. The mechanism of conversion seems to be different for both enzymes. The results force us to conclude that lysosomal cathepsins, especially cathepsins L and B might be involved in the process of conversion of proinsulin into insulin and perhaps also of other precursors into biologically active proteins in vivo.     

Immunocytochemical localization of cathepsins B, H, and L in the rat gastro-duodenal mucosa

Cathepsins B, H, and L are representative cysteine proteinases in lysosomes of a large variety of cells. Previous immunochemical studies indicated the presence of these enzymes also in the gastrointestinal wall. Using specific antisera, the cellular and subcellular distribution of cathepsins B, H, and L in rat gastric (oxyntic and pyloric part) and duodenal mucosa was investigated by light and electron microscopical immunocytochemistry. The subtypes of cathepsins were distributed differently in the cellular constituents of the epithelia: Cathepsin B was localized to lysosomes of all cells except goblet cells. Cathepsin H was found predominantly in gastric parietal cells (lysosomes) and in secretion granules of pyloric gastrin and duodenal cholecystokinin cells. Cathepsin L immunoreactivities were weak and restricted to a minority of cells (gastric mucous cells, enterocytes). Interstitial cells of the lamina propria immunoreactive for cathepsins H and L were identified as macrophages. The present findings suggest a dual function of cathepsins in the gastro-duodenal mucosa. They (1) cleave enzymatically proteins and peptides ingested in lysosomes, and (2) they may be involved in the processing of biologically active peptides (enteric hormones) from their precursor proteins.     

Immunocytochemical localization of cathepsins B,H, and L in the rat gastro-duodenal mucosa

Summary Cathepsins B, H, and L are representative cysteine proteinases in lysosomes of a large variety of cells. Previous immunochemical studies indicated the presence of these enzymes also in the gastrointestinal wall. Using specific antisera, the cellular and subcellular distribution of cathepsins B, H, and L in rat gastric (oxyntic and pyloric part) and duodenal mucosa was investigated by light and electron microscopical immunocytochemistry. The subtypes of cathepsins were distributed differently in the cellular constituents of the epithelia: Cathepsin B was localized to lysosomes of all cells except goblet cells. Cathepsin H was found predominantly in gastric parietal cells (lysosomes) and in secretion granules of pyloric gastrin and duodenal cholecystokinin cells. Cathepsin L immunoreactivities were weak and restricted to a minority of cells (gastric mucous cells, enterocytes). Interstitial cells of the lamina propria immunoreactive for cathepsins H and L were identified as macrophages. The present findings suggest a dual function of cathepsins in the gastro-duodenal mucosa. They (1) cleave enzymatically proteins and peptides ingested in lysosomes, and (2) they may be involved in the processing of biologically active peptides (enteric hormones) from their precursor proteins.     

Immunocytochemical localization of angiotensinogen and cathepsins B, H, and L in rat hepatocytes, with special reference to degradation of angiotensinogen in lysosomes after colchicine

We examined the effects of bilateral nephrectomy and colchicine treatment on localization and content of angiotensinogen and cathepsins B, H, and L in rat liver using immunohistochemistry, radioimmunoassay, and enzyme assay. Angiotensinogen content increased in the liver of colchicine-treated rats, whereas a clear-cut increase was not detected in the liver of nephrectomized rats. This tendency was consistent with the immunocytochemical results; only perivenous hepatocytes in control and nephrectomized rats were diffusely immunostained by anti-angiotensinogen, whereas perivenous and periportal hepatocytes of colchicine-treated rats were strongly immunostained. Enzyme assay revealed no significant change in activities of cathepsins B, H, and L in liver extracts under these experimental conditions. Immunocytochemical localization of these cysteine proteinases in hepatocytes after colchicine treatment was more widespread in the cytoplasm than that in the control hepatocytes. By electron microscopy, angiotensinogen was localized in smaller vesicles and some larger vesicles (lysosomes) of hepatocytes after colchicine treatment. Double immunostaining demonstrated co-localization of cathepsins B, H, and L with angiotensinogen in lysosomes. These results suggest that cathepsins B, H, and L play a role in the degradation of excess angiotensinogen in hepatocytes of rats after colchicine treatment.     

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.     

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.     

Different immunolocalizations of cathepsins B, H, and L in the liver

Different localizations of cathepsin B, H, and L in normal rat liver were revealed immunohistochemically with anticathepsin Fab'-horseradish peroxidase conjugates. Staining of cathepsin B was strong in the periportal sinusoids, possibly in Kupffer cells; and weaker in panlobular hepatocytes. Staining of cathepsin H was strong in panlobular hepatocytes, especially in the periphery of the cytoplasm, possibly representing the peribiliary dense bodies; and weaker in periportal sinusoidal cells, possibly Kupffer cells. Staining of cathepsin L was strongest in centrilobular hepatocytes and weaker in periportal sinusoidal cells, possibly Kupffer cells. These findings, revealed for the first time in the present study, show that the histologic and intracellular localizations of the three cathepsins are different, suggesting that they have different roles in degradation of exogenous and endogenous proteins.     

Effect of proteinase inhibitors on intracellular processing of cathepsin B, H and L in rat macrophages

The effects of various proteinase inhibitors on the processing of lysosomal cathepsins B, H and L were investigated in cultured rat peritoneal macrophages. The processing of newly synthesized pro-cathepsins B, H and L to the mature single-chain enzymes was sensitive to a metal chelator,1,10-phenanthroline, and a synthetic metalloendopeptidase substrate, Z-Gly-Leu-NH2, and insensitive to inhibitors of serine proteinases, aspartic proteinases and cysteine proteinases. Inhibitors of cysteine proteinases, E-64-d and leupeptin, inhibited the processing of the single-chain forms of cathepsins B, H and L to the two-chain forms. These results suggest that (a) metal endopeptidase(s) is (are) involved in the propeptide processing of cathepsin B, H and L, and that proteolytic cleavages of the mature single-chain cathepsins are accomplished by cysteine proteinases in lysosomes.     

Effects of cathepsins B and L inhibition on postischemic protein alterations in the brain

The effects of selective inhibition of cathepsins B and L on postischemic protein alterations in the brain were investigated in a rat model of middle cerebral artery occlusion (MCAO). Cathepsin B activity increased predominantly in the subcortical region of the ischemic hemisphere where the levels of collapsing mediator response protein 2, heat shock cognate 70 kDa protein, 60 kDa heat shock protein, protein disulfide isomerase A3 and albumin, were found to be significantly elevated. Postischemic treatment with Cbz-Phe-Ser(OBzl)-CHN₂, cysteine protease inhibitor 1 (CP-1), reduced infarct volume, neurological deficits and cathepsin B activity as well as the amount of heat shock proteins and albumin found in the brain. Our data strongly suggests that the decrease in heat shock protein levels and the significant reduction of serum albumin leakage into the brain following acute treatment with CP-1 is indicative of less secondary ischemic damage, which ultimately, is related to less cerebral tissue loss and improved neurological recovery of the animals.     

Immunohistochemical localization of cathepsins B,D and L in the rat osteoclast

Immunohistochemical localization of cathepsins B, D and L in the osteoclasts of rat alveolar and femoral bones was investigated by using the avidin-biotin-peroxidase complex method for semithin, 1-m-thick cryosections. Extracellular immunoreactivity for cathepsins B and L was clearly demonstrated along the bone resorption lacunae; the intensity of the extracellular immunoreactivity of cathepsin L was stronger than that of cathepsin B. However, the intracellular immunoreactivity of both cathepsins was weak compared with that of cathepsin D. The intracellular immunoreactivity of cathespin D in the osteoclasts was clearly observed in the granules and/or vacuoles, but extracellular cathepsin D immunoreactivity was either negligible or not detected along the resorption lacunae. In the adjacent sections stained with anti-cathepsin L or D, extensive extracellular deposition of cathepsin L was found along the bone resorption lacunae, with or without osteoclasts, although the intracellular reactivity of cathepsin L was weak. This is the first morphological study in which cathepsins B and L have been demonstrated to be produced in the osteoclasts and extensively secreted into resorption lacunae, and in which cathepsin D was found to be present in the cells but scantily secreted into the lacunae. These findings suggest that cathepsins B and L directly and effectively participate in the degradation of the bone matrix.     

Immunocytochemical localization of cathepsins B and H in rat liver

Summary Light and electron microscopic localization of cathepsins B and H in rat liver was investigated by immunoenzyme and protein A-gold techniques. For light microscopy (LM), semi-thin sections of the Epon-embedded material were stained by the immunoenzyme technique after removal of epoxy resin. For electron microscopy (EM), ultrathin sections of the Lowicryl K4M-embedded material were stained by the protein A-gold technique. By LM, reaction deposits for cathepsins B and H were present in the cytoplasmic granules of parenchymal cells and endothelial cells, and Kupffer cells. The sinus-lining cells and the parenchymal cells showed the similar staining intensity. By EM, gold particles were present exclusively in lysosomes of all the cell types cited above. The same results were obtained from quantitative analysis. In addition, Golgi complexes themselves were mostly negative but some small vesicles on the trans side of them were labeled for these proteinases. The results indicate that cathepsins B and H are present in the lysosomes of rat liver and that these enzymes seem to be transported by small vesicles from endoplasmic reticulum to lysosomes via tubuloreticular network of the trans Golgi region.     

Immunocytochemical localization of cathepsins B and H in rat liver

Light and electron microscopic localization of cathepsins B and H in rat liver was investigated by immunoenzyme and protein A-gold techniques. For light microscopy (LM), semi-thin sections of the Epon-embedded material were stained by the immunoenzyme technique after removal of epoxy resin. For electron microscopy (EM), ultra-thin sections of the Lowicryl K4M-embedded material were stained by the protein A-gold technique. By LM, reaction deposits for cathepsins B and H were present in the cytoplasmic granules of parenchymal cells and endothelial cells, and Kupffer cells. The sinus-lining cells and the parenchymal cells showed the similar staining intensity. By EM, gold particles were present exclusively in lysosomes of all the cell types cited above. The same results were obtained from quantitative analysis. In addition, Golgi complexes themselves were mostly negative but some small vesicles on the trans side of them were labeled for these proteinases. The results indicate that cathepsins B and H are present in the lysosomes of rat liver and that these enzymes seem to be transported by small vesicles from endoplasmic reticulum to lysosomes via tubuloreticular network of the trans Golgi region.     

Immunocytochemical localization of cathepsins B, H, L, and T4 in follicular cells of rat thyroid gland

To localize cathepsins B, H, and L in follicular cells of rat thyroid gland, we applied immunocytochemistry to the thyroid tissue using their respective monospecific antibodies. On serial semi-thin sections, cathepsins B, H, and L were localized in granules of various sizes located throughout the cytoplasm, whereas T4 was detected in larger granules located in the apical and supranuclear regions. By electron microscopy, cathepsins B, H, and L were localized in large less-dense granules (so-called colloid droplets) and in dense bodies of various sizes, whereas T4 was localized more intensely in large less-dense granules than in smaller dense bodies. By double immunostaining using an immunogold method, cathepsins H and B or L were co-localized in the same cytoplasmic granules. Moreover, immunoblotting demonstrated that proteins similar to cathepsins B, H, and L in the liver are present in the thyroid gland. These results suggest that cathepsins B, H, and L participate not only in degradation of thyroglobulin but in maturation of thyroid hormones, although it remains unknown whether all of them participate in the maturation process.     

Immunocytochemical localization of prorenin, renin, and cathepsins B, H, and L in juxtaglomerular cells of rat kidney

To examine the correlation of localization of prorenin, renin, and cathepsins B, H, and L, immunocytochemistry was applied to rat renal tissue, using a sequence-specific anti-body (anti-prorenin) that recognizes the COOH terminus of the rat renin prosegment. In serial semi-thin sections, immunodeposits for prorenin, renin, and cathepsins B, H, and L were localized in the same juxtaglomerular (JG) cells. Immunodeposits for renin were detected throughout the cytoplasm of the cells, whereas those for prorenin were detected in the perinuclear region. Immunoreactivity for cathepsin B was stronger than that for cathepsins H and L. By electron microscopy, prorenin was localized in small (immature) granules but not in large mature granules, whereas renin was localized mainly in mature granules. In serial thin sections, prorenin, renin, and cathepsin B were colocalized in the same immature granules containing heterogeneously dense material (intermediate granules). By double immunostaining, co-localization of renin with cathepsins B, H, or L was demonstrated in mature granules. The results suggest the possibility that processing of prorenin to renin occurs in immature granules of rat JG cells, and cathepsin B detected in JG cells may be a major candidate for the maturation of renin.     

Breast cancer cells express cathepsins B and L but not cathepsins K or H

Lysosomal cysteine proteinases (cathepsins) are considered to play a role in bone degradation mediated by metastatic breast cancers. To evaluate which cathepsin contributes to the osteolysis, we quantitatively determined the expression levels of four cathepsins in two breast cancer cell lines, MCF-7 and MDA-MB-231, by competitive RT-PCR. Cathepsin K, which is the most abundant cathepsin in osteoclasts, was not detected in either cell lines. We also failed to detect cathepsin H mRNA. By contrast, we found significant expression of cathepsins B and L in both cell lines. By Northern blot analysis cathepsin B mRNA was detected in a single form in these cells, whereas osteoclasts contained multiple forms of the mRNA. Cathepsin B protein was also detected by Western blotting as a single immunoreactive band corresponding to its mature enzyme. These findings suggest that osteolysis associated with metastatic breast cancers takes place in a different way from osteoclast-mediated bone resorption.     

Structural studies on the carbohydrate moieties of rat liver cathepsins B and H

Cathepsins B and H from rat liver contain one asparagine-linked sugar chain in each molecule. The sugar chains were liberated from the polypeptide portions by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. Paper electrophoresis of the radioactive oligosaccharide fractions revealed that they were mixtures of neutral oligosaccharides only. After fractionation by gel filtration the structure of each oligosaccharide was studied by sequential exoglycosidase digestion in combination with methylation analysis. The sugar chain of cathepsin H was a high mannose type oligosaccharide which varied in size from 5 to 9 mannose residues; on the other hand the major oligosaccharide of cathepsin B was a tetrasaccharide whose structure was Manalpha 1----6Manbeta 1----4GlcNAcbeta 1----4GlcNAc.     

Biosynthesis of lysosomal cathepsins B and H in cultured rat hepatocytes

The biosynthesis of lysosomal cysteine proteases, cathepsins B and H, was investigated by using pulse-chase experiments in vivo in primary cultures of rat hepatocytes. Cathepsins B and H were isolated from either total cell extracts or culture medium labeled with [35S]methionine by immunoprecipitation and analyzed for their molecular forms. Within 60 min of chase, cellular proforms of cathepsins B of 39 kDa and H of 41 kDa were converted to single-chain form cathepsins B of 29 kDa and H of 28 kDa, respectively, and persisted as these forms even after 12-h chase periods. The proforms of cathepsins B and H derived from pulse-labeling experiments showed complete susceptibility to endoglycosidase H treatment, indicating that these proenzymes bear high-mannose-type oligosaccharides at the stage of initial events of biosynthesis. In the presence of tunicamycin, unglycosylated proenzymes of cathepsins B of 35 kDa and H of 34 kDa were found to be secreted into the extracellular medium without undergoing proteolytic processing. Furthermore, in the presence of swainsonine, a potent inhibitor of Golgi mannosidase II, considerable amounts of the proenzymes were secreted and accumulated in the medium during chasing periods. These results suggest that the oligosaccharide moiety of these enzymes would be necessary for the intracellular sorting mechanism. In monensin-treated cells, the conversion of intracellular proenzymes to mature enzymes was significantly inhibited and the proenzymes were secreted into the medium. In the presence of chloroquine or ammonium chloride, proteolytic processing of the proenzymes was completely prevented and the enhanced secretion of proenzymes was observed. These results suggest that in the presence of lysosomotropic amines the intracellular sorting of proenzymes might not occur properly during biosynthesis.     

Modulation of the endosomal and lysosomal distribution of cathepsins B,L and S in human monocytes/macrophages

Endosomal and lysosomal fractions of human monocytes/macrophages prepared from buffy coats were analyzed for activities of cathepsins B, L and S, and expression of cathepsin proteins along with major histocompatibility complex class I and class II molecules under control and immunomodulatory conditions. While the total activity of cathepsins B, L, and S together remained unchanged in lysates of control cells during culture for 72 h, the subcellular distribution of cathepsin activities underwent a shift from a predominantly endosomal localization in freshly isolated cells to a lysosomal pattern after 72 h of culture. Interferon-gamma treatment for 72 h resulted in an upregulation of both major histocompatibility complex proteins and cathepsins with differential changes in cathepsin B, L and S activities in endosomes versus lysosomes. These changes suggest a remodeling of the endocytic machinery and imply different functions of cathepsins B, L and S during monocyte differentiation.     

Localization of cathepsins B,D, and L in the rat osteoclast by immuno-light and -electron microscopy

The localization of cathepsins B, D, and L was studied in rat osteoclasts by immuno-light and-electron microscopy using the avidin-biotin-peroxidase complex (ABC) method. In cryosections prepared for light microscopy, immunoreactivity for cathepsin D was found in numerous vesicles and vacuoles but was not detected along the resorption lacunae of osteoclasts. However, immunoreactivity for cathepsins B and L occurred strongly along the lacunae, and only weak intracellular immunoreactivity was observed in the vesicles and peripheral part of the vacuoles near the ruffled border. In control sections that were not incubated with the antibody, no cathepsins were found in the osteoclasts or along the resorption lacunae of osteoclasts. At the electron microscopic level, strong intracellular reactivity of cathepsin D was found in numerous vacuoles and vesicles, while extracellular cathepsin D was only slightly detected at the base of the ruffled border but was not found in the eroded bone matrix. Most osteoclasts showed strong extracellular deposition of cathepsins B and L on the collagen fibrils and bone matrix under the ruffled border. The extracellular deposition was stronger for cathepsin L than for cathepsin B. Furthermore cathepsins B and L immunolabled some pits and part of the ampullar extracellular spaces, appearing as vacuoles in the sections. Conversely, the intracellular reactivity for cathepsins B and L was weak: cathepsin-containing vesicles and vacuoles as primary and secondary lysosomes occurred only sparsely. These findings suggest that cathepsins B and L, unlike cathepsin D, are rapidly released into the extracellular matrix and participate in the degradation of organic bone matrix containing collagen fibrils near the tip of the ruffled border. Cathepsin L may be more effective in the degradation of bone matrix than cathepsin B.