Lysosomal cystine transport. Effect of intralysosomal pH and membrane potential

The regulation of lysosomal cystine transport was studied using cystine dimethyl ester-loaded lysosomes isolated from human diploid fibroblasts. Net efflux from normal fibroblast lysosomes was compared to that from lysosomes of cystinotic fibroblasts, which contain an inherited mutation decreasing lysosomal cystine transport. This exodus of cystine from normal fibroblast lysosomes was greater than from cystinotic fibroblast lysosomes. When lysosomes were incubated with both 5 mM MgCl2 and 2 mM ATP (Mg/ATP), the amount of lysosomal cystine lost from normal lysosomes doubled, but the amount of cystine lost from cystinotic lysosomes remained small. This effect of Mg/ATP on cystine loss from lysosomes isolated from normal fibroblasts was abolished when either carbonyl cyanide m-chlorophenylhydrazone or N-ethylmaleimide was present, suggesting that the effect of Mg/ATP was mediated by the action of a lysosomal proton-translocating ATPase. Addition of KCl, RbCl, or NaCl to normal lysosomes caused smaller increases in cystine exodus. A variety of experimental conditions altered lysosomal pH, membrane potential, and the cystine lost from normal fibroblast lysosomes. These same experimental conditions produced similar alterations in the lysosomal pH and membrane potential of cystinotic fibroblast lysosomes without a comparable alteration in cystine loss. These results have led us to propose a model in which the transport of cystine out of the normal lysosome is regulated by both the lysosomal membrane potential gradient and the transmembrane pH gradient.     

Relations between plasma membrane and lysosomal membrane. 2. Quantitative evaluation of plasma membrane marker enzymes in the lysosomes

We have quantified, in cultured rat fibroblasts, the association to the lysosomal membrane of two classical plasma membrane markers, 5'-nucleotidase and alkaline phosphodiesterase I. To isolate highly purified lysosomal preparations, lysosomes were loaded with horseradish peroxidase (2-h cell uptake, 16-h chase) and isolated by isopycnic centrifugation in linear Percoll gradients, followed by a 3,3'-diaminobenzidine-induced density shift in sucrose gradients. Purified lysosomal preparations contained up to 50% of N-acetyl-beta-glucosaminidase of the homogenate. This lysosomal enzyme was enriched 33-fold in the most purified preparations. In the electron microscope, these preparations appeared to be highly purified and only contained organelles filled with diaminobenzidine reaction products. Analysis of purified preparations indicates that 0.5-0.8% of 5'-nucleotidase, but as much as 10.9-14.3% of alkaline phosphodiesterase I activities of the homogenate, are associated with lysosomes. After freezing-thawing, these activities remained essentially membrane-associated. The larger value obtained for alkaline phosphodiesterase I could not be ascribed to other lysosomal enzymes, as no such activity was detected at acidic pH. These two plasma membrane markers are thus unevenly distributed in the lysosomal compartment.     

Biochemical mechanisms of aminoglycoside cell toxicity. II. Accumulation of phospholipids during myeloid body formation and histological studies on myeloid bodies using twelve aminoglycoside antibiotics

Cultured human skin fibroblasts take up aminoglycoside antibiotics into lysosomes to form myeloid bodies. Gentamicin (GM), one such antibiotic, was taken up until the cellular concentration reached an estimated 64 mM on the 3rd day when cells were incubated with 2 mM gentamicin. The rate of release of intracellular GM was high on the first day of incubation and gradually slowed down over the next 4 d. About 50% of the GM remained in the cells even on longer incubation in GM-free medium, suggesting it may irreversibly bind to cellular components. With myeloid body formation, the cellular phospholipid content increased 1.5 times. Bis(monoacyl-glyceryl)phosphate, which is known as a marker of lysosomal phospholipid, phosphatidylcholine and phosphatidylserine showed 250, 162, and 153% increases, respectively. Sphingomyelin was not accumulated, while lysosomal sphingomyelinase was dramatically inhibited. Of 12 different aminoglycoside antibiotics, paromomycin is the most prominent myeloid body-forming antibiotic. The myeloid body-formation is not directly correlated to human nephrotoxicity. On the other hand, the number of myeloid bodies is well correlated to the affinity to the brush border membrane, suggesting that such aminoglycoside antibiotics are taken up easily through cellular endocytosis. The cytotoxic effects of aminoglycoside antibiotics may be due to by their binding to cellular organelles other than lysosomes.     

Effects of dimethylsulfoxide on sphingomyelinase activities in normal and Niemann-Pick type A, B and C fibroblasts

The effects of dimethylsulfoxide (DMSO) on sphingomyelinase activity measured at pH range 3.5-8.0 were examined in normal and Niemann-Pick disease type A, B and C fibroblasts culture. In normal cells, a minor activity was observed at pH 7.5, which was 3- to 4-fold lower than a major one at pH 5.0. Both activities at pH 5.0 and 7.5 were Mg2+-independent and localized to lysosomes. Niemann-Pick type C cells had 30-50% residual sphingomyelinase activity at both pH 5.0 and 7.5, as compared to normal control cells, whereas type A and B cells exhibited virtually no activity over the entire pH range examined. Treatment with 2% DMSO caused a marked increase in sphingomyelinase activities at pH 5.0 and 7.5 in normal and Niemann-Pick disease type C cells, while in type A and B cells, both activities remained virtually unchanged after DMSO treatment. The increase in sphingomyelinase activity at pH 5.0 induced in normal cells by DMSO resulted in an increase in the Vmax without a substantial change in the Km and was inhibited by the simultaneous addition of 10 micrograms/ml of cycloheximide. By comparison, a less than 2-fold increase in other lysosomal hydrolase activities was observed after DMSO treatment in all cell lines examined.     

Synthesis and hydrolysis of cholesteryl esters by isolated rat-liver lysosomes and cell-free extracts of human lung fibroblasts

The objective of this study was to examine and characterize the cholesteryl ester synthesizing [S] and hydrolyzing [H] properties of the acid cholesteryl ester hydrolase (acid cholesteryl ester hydrolase), both in isolated rat liver lysosomes and in cell-free extracts from cultured fibroblasts. For both liver lysosomes and fibroblasts extracts, the major synthesizing activity was found around pH 4 and did not require exogenous ATP. The rate of hydrolysis was measured at pH 4.5. Several different inhibitors were used in order to characterize the reactions. Ammonium chloride did not markedly affect the activity of acid cholesteryl ester hydrolase at pH 4 [S] or 4.5 [H], whereas chloroquine was a potent inhibitor of acid CEase in both liver lysosomes and fibroblast extracts. The [S] activity of the acid cholesteryl ester hydrolase in either material was not affected by the acylCoA:cholesterol acyltransferase inhibitor Compound 58-035 from Sandoz. Progesterone, on the other hand, which is an often used acylCoA:cholesterol acyltransferase inhibitor, markedly blocked both activities of the acid CEase. Our results indicate that the lysosomal compartment of both studied tissues, in addition to hydrolysis activity, also have a significant esterification activity. It appears that both activities are carried out by the same enzyme.     

Processing of human cathepsin D in lysosomes in vitro

The proteolytic maturation of cathepsin D polypeptides was studied in lysosomes isolated from metabolically labeled fibroblasts. In lysosomes isolated from fibroblasts labeled with [35S]methionine, 70-95% of labeled cathepsin D polypeptides were represented by a Mr = 47,000 polypeptide after a 20-min pulse and 75-min chase. When these lysosomes were incubated in vitro, up to 70% of the Mr = 47,000 polypeptide was processed to mature cathepsin D polypeptides. The processing was dependent on the integrity of the lysosomes, had an optimum between pH 6 and 7, and could be stimulated by dithiothreitol and ATP. The noncleavable ATP analogue, adenosine 5'-(beta, gamma-imido)triphosphate, and GTP, CTP, and UTP could not substitute for ATP. The ATP-dependent stimulation was associated with an acidification of lysosomes. It was inhibited by agents that dissipate the lysosomal pH gradient (carbonyl cyanide p-trifluoromethoxyphenylhydrazone, N,N'-dicyclohexylcarbodiimide, nigericin, NH4Cl). A stimulatory effect of ATP was observed also at pH 5.5. The stimulation at pH 5.5 was not associated with acidification of lysosomes and was resistant to protonophores. Inhibitors of lysosomal cysteine proteinases and N-ethylmaleimide inhibited the processing. In the presence of ATP the processing activity was partially protected from inhibition by N-ethylmaleimide. In conclusion, the maturation of cathepsin D in lysosomes depends on cysteine proteinases and is stimulated by the ATP-driven acidification of lysosomes. In addition, ATP stimulates maturation at pH 5.5 by a mechanism not involving the proton pump.     

Polyamines stimulate lysosomal cystine transport

Lysosomal cystine transport is a carrier-dependent process that, in isolated lysosomes, is stimulated by proton gradients, membrane potential, and millimolar concentrations of divalent cations. The importance of these regulatory factors in vivo is not well established. Polyamines were found to stimulate cystine transport in Percoll gradient purified rat liver lysosomes with spermidine greater than putrescine = cadaverine greater than spermine in order of effectiveness. Maximal stimulation was achieved with 500 microM spermidine. The effects of optimal concentrations of polyamines and divalent cations on cystine transport were not additive. Spermidine stimulated cystine efflux from lysosomes of cultured human diploid fibroblasts, but had no effect on lysosomes of cystinotic fibroblasts which have defective cystine transport. Spermidine did not accumulate within lysosomes in exchange for cystine, had no effect on lysosomal pH, had only slight effects on the lysosomal membrane potential, and had little effect on either methionine or tyrosine efflux. Polyamines are cellular cytoplasmic components that, in physiologic concentrations, stimulate lysosomal cystine transport.     

Cystic fibrosis transmembrane conductance regulator contributes to reacidification of alkalinized lysosomes in RPE cells

The role of the cystic fibrosis transmembrane conductance regulator (CFTR) in lysosomal acidification has been difficult to determine. We demonstrate here that CFTR contributes more to the reacidification of lysosomes from an elevated pH than to baseline pH maintenance. Lysosomal alkalinization is increasingly recognized as a factor in diseases of accumulation, and we previously showed that cAMP reacidified alkalinized lysosomes in retinal pigmented epithelial (RPE) cells. As the influx of anions to electrically balance proton accumulation may enhance lysosomal acidification, the contribution of the cAMP-activated anion channel CFTR to lysosomal reacidification was probed. The antagonist CFTR(inh)-172 had little effect on baseline levels of lysosomal pH in cultured human RPE cells but substantially reduced the reacidification of compromised lysosomes by cAMP. Likewise, CFTR activators had a bigger impact on cells whose lysosomes had been alkalinized. Knockdown of CFTR with small interfering RNA had a larger effect on alkalinized lysosomes than on baseline levels. Inhibition of CFTR in isolated lysosomes altered pH. While CFTR and Lamp1 were colocalized, treatment with cAMP did not increase targeting of CFTR to the lysosome. The inhibition of CFTR slowed lysosomal degradation of photoreceptor outer segments while activation of CFTR enhanced their clearance from compromised lysosomes. Activation of CFTR acidified RPE lysosomes from the ABCA4(-/-) mouse model of recessive Stargardt's disease, whose lysosomes are considerably alkalinized. In summary, CFTR contributes more to reducing lysosomal pH from alkalinized levels than to maintaining baseline pH. Treatment to activate CFTR may thus be of benefit in disorders of accumulation associated with lysosomal alkalinization.     

Antibody to mannose 6-phosphate specific receptor induces receptor deficiency in human fibroblasts.

Polyclonal antibodies to the mannose 6-phosphate specific receptor from human liver inhibited the endocytosis of lysosomal enzymes in fibroblasts by greater than 95% and enhanced 3-20-fold the secretion of precursors of lysosomal enzymes in these cells. Exposing fibroblasts for 4 h to antibody resulted in loss of greater than 90% of the membrane-bound receptors. If fibroblasts were treated with the antibody in the presence of CBZ-Phe-Ala-CHN2, an inhibitor of lysosomal cysteine proteinases, the receptor and smaller degradation products are recovered in dense lysosomes. In treated cells 18-58% of total receptor-related polypeptides were recovered in dense lysosomes. In control cells less than 4% of the receptor was found in the lysosomal fraction. We conclude from these results that normally the receptor is spared from lysosomal degradation. When tagged with antibody, however, the receptor is transported into lysosomes and degraded. The loss of intracellular receptors involved in segregation of newly synthesized lysosomal enzymes indicates an exchange between the former and the plasma membrane-bound receptors.     

Inhibition of proteolytic degradation of low density lipoprotein in human fibroblasts by chloroquine, concanavalin A, and Triton WR 1339.

The proteolytic degradation of 125I-labeled low density lipoprotein by monolayers of cultured human fibroblasts was prevented by exposure of the cells to chloroquine, an agent that has been reported previously to inhibit lysosomal degradative processes. Chloroquine did not inhibit the binding of low density lipoprotein to its cell surface receptor. However, the two regulatory actions that normally follow low density lipoprotein binding to its receptor, namely suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and stimulation of cholesteryl ester formation, were both prevented when degradation of the lipoprotein was inhibited by chloroquine. Two other agents affecting lysosomal function, Triton WR 1339 and concanavalin A, also inhibited the proteolytic degradation of low density lipoprotein in intact fibroblasts and simultaneously prevented low density lipoprotein-mediated suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and stimulation of cholesteryl ester formation. Unlike chloroquine, however, these two agents also affect the binding of low density lipoprotein to the cells. The inhibitory action of chloropuine, concanavalin A, and Triton WR 1339 could each be reversed by removal of the agent from the culture medium. These in vivo culture data, together with the observation that cell-free extracts of fibroblasts maximally degrade 125I-labeled low density lipoprotein at pH 4 and do not form acid-soluble material above pH 6, are consistent with the hypothesis that the proteolytic degradation of low density lipoprotein by monolayers of fibroblasts occurs within lysosomes. The data also suggest that normal lysosomal function is required in order for low density lipoprotein to regulate cholesterol synthesis and cholesteryl ester formation in the fibroblast system.     

Effect of acidotropic amines on the accumulation of newly synthesized membrane and luminal proteins in Chinese-hamster ovary (CHO) cell lysosomes.

Lysosomes were isolated by sequential gradient centrifugation [Madden, Wirt & Storrie (1987) Arch. Biochem. Biophys. 257, 27-38] from control or acidotropic-amine-treated Chinese-hamster ovary (CHO) cells. By marker-enzyme analysis, the preparation from chloroquine or NH4Cl-treated cells was about 25-fold enriched for lysosomes compared with the postnuclear supernatant and contained little or no marker activities for the plasma membrane, rough endoplasmic reticulum, Golgi apparatus, mitochondria, cytosol and peroxisomes. The yield of amine-treated lysosomes was about 60% relative to the postnuclear supernatant. Electron microscopy and cytochemistry demonstrated that the amine-treated preparation was highly purified. Cytochemical analyses after a short-term pulse of horseradish peroxidase revealed that endosomal contamination of the lysosomal preparation was less than 1%. Lysosomal polypeptides were biosynthetically labelled with [35S]methionine and identified by SDS/polyacrylamide-gel electrophoresis. As expected, the bulk accumulation of luminal proteins into lysosomes was decreased. The bulk accumulation of membrane proteins was increased by acidotropic amine treatment. There were also several qualitative differences in each lysosomal compartment, with new species observed and other species absent. These data suggest that a low pH is not necessary for the normal accumulation of the bulk of membrane proteins in lysosomes and that membrane trafficking from Golgi apparatus to lysosomes occurs at a high rate in acidotropic-amine-treated CHO cells.     

Role of oxidative stress in lysosomal membrane permeabilization and apoptosis induced by gentamicin, an aminoglycoside antibiotic

Gentamicin, an aminoglycoside antibiotic used to treat severe bacterial infections, may cause acute renal failure. At therapeutic concentrations, gentamicin accumulates in lysosomes and induces apoptosis in kidney proximal tubular cells. In gentamicin-treated renal LLC-PK1 cells, acridine orange release from lysosomes, previously interpreted as lysosomal membrane permeabilization, precedes the apoptotic cascade that develops during incubation with gentamicin. However, the link between gentamicin lysosomal accumulation and apoptosis remains unclear. We here examined if reactive oxygen species (ROS) production could account for gentamicin-induced acridine orange release and apoptosis, and the implication of iron in these events. We found that gentamicin induced ROS production prior to, and at lower drug concentrations than required for, acridine orange release and apoptosis. ROS antioxidant or scavenger, catalase, and N-acetylcysteine largely prevented these events. Vital confocal imaging revealed that gentamicin-induced ROS production occurs in lysosomes. Deferoxamine, an iron chelator, which is endocytosed and accumulates in lysosomes, largely prevented gentamicin-induced ROS production as well as apoptosis. Direct evidence for gentamicin-induced permeabilization of lysosomal membrane was provided by showing the release into the cytosol of Lucifer yellow, a membrane-impermeant endocytic tracer with a comparable molecular weight as gentamicin. Altogether, our data demonstrate a key role of lysosomal iron and early ROS production in gentamicin-induced lysosomal membrane permeabilization and apoptosis.     

Studies of lysosomal sialic acid metabolism: retention of sialic acid by Salla disease lysosomes

Purified rat liver lysosomes were incubated in 0.2 M sialic acid resulting in an increase in lysosomal free sialic acid of 3.8 +/- 1.5 nmol/unit beta hexosaminidase. Sialic acid loss by these lysosomes was stimulated 2-3 fold by 25 mM sodium phosphate. Loss of sialic acid by lysosomes from cultured human diploid fibroblasts was similar to that observed in rat liver lysosomes while loss of sialic acid by lysosomes from cultured fibroblasts from a patient with infantile Salla disease occurred much more slowly. Salla disease appears to be the consequence of defective lysosomal transport of sialic acid and is analogous to cystinosis, a disorder of lysosomal amino acid transport.     

Physicochemical characteristics of the glycosaminoglycan-lysosomal enzyme interaction in vitro. A model of control of leucocytic lysosomal activity.

1. The activities of 30 different lysosomal enzymes were determined in vitro in the presence of the sulphated glycosaminoglycans, heparin and chondroitin sulphate, all the enzymes being measured on a density-gradient-purified lysosomal fraction. 2. Each enzyme was studied as a function of the pH of the incubation medium. In general the presence of sulphated glycosaminoglycans induced a strong pH-dependent inhibition of lysosomal enzymes at pH values lower than 5.0, with full activity at higher pH values. However, in the particular case of lysozyme and phospholipase A2 the heparin-induced inhibition was maintained in the pH range 4.0-7.0. 3. For certain enzymes, such as acid beta-glycerophosphatase, alpha-galactosidase, acid lipase, lysozyme and phospholipase A2, the pH-dependent behaviour obtained in the presence of heparin was quite different to that obtained with chondroitin sulphate, suggesting the existence of physicochemical characteristic factors playing a role in the intermolecular interaction for each of the sulphated glycosaminoglycans studied. 4. Except in the particular case of peroxidase activity, in all other lysosomal enzymes measured the glycosaminoglycan-enzyme complex formation was a temperature-and time-independent phenomenon. 5. The effects of the ionic strength and pH on this intermolecular interaction reinforce the concept of an electrostatic reversible interaction between anionic groups of the glycosaminoglycans and cationic groups on the enzyme molecule. 6. As leucocytic primary lysosomes have a very acid intragranular pH and large amounts of chondroitin sulphate, we propose that this glycosaminoglycan might act as molecular regulator of leucocytic activity, by inhibiting lysosomal enzymes when the intragranular pH is below the pI of lysosomal enzymes. This fact, plus the intravacuolar pH changes described during the phagocytic process, might explain the unresponsiveness of lysosomal enzymes against each other existing in primary lysosomes as well as its full activation at pH values occurring in secondary lysosomes during the phagocytic process.     

Defective oligomerization of arylsulfatase a as a cause of its instability in lysosomes and metachromatic leukodystrophy.

In one of the most common mutations causing metachromatic leukodystrophy, the P426L-allele of arylsulfatase A (ASA), the deficiency of ASA results from its instability in lysosomes. Inhibition of lysosomal cysteine proteinases protects the P426L-ASA and restores the sulfatide catabolism in fibroblasts of the patients. P426L-ASA, but not wild type ASA, was cleaved by purified cathepsin L at threonine 421 yielding 54- and 9-kDa fragments. X-ray crystallography at 2.5-A resolution showed that cleavage is not due to a difference in the protein fold that would expose the peptide bond following threonine 421 to proteases. Octamerization, which depends on protonation of Glu-424, was impaired for P426L-ASA. The mutation lowers the pH for the octamer/dimer equilibrium by 0.6 pH units from pH 5.8 to 5.2. A second oligomerization mutant (ASA-A464R) was generated that failed to octamerize even at pH 4.8. A464R-ASA was degraded in lysosomes to catalytically active 54-kDa intermediate. In cathepsin L-deficient fibroblasts, degradation of P426L-ASA and A464R-ASA to the 54-kDa fragment was reduced, while further degradation was blocked. This indicates that defective oligomerization of ASA allows degradation of ASA to a catalytically active 54-kDa intermediate by lysosomal cysteine proteinases, including cathepsin L. Further degradation of the 54-kDa intermediate critically depends on cathepsin L and is modified by the structure of the 9-kDa cleavage product.     

Accumulation of sialic acid in endocytic compartments interferes with the formation of mature lysosomes. Impaired proteolytic processing of cathepsin B in fibroblasts of patients with lysosomal sialic acid storage disease.

The impact of an altered endocytic environment on the biogenesis of lysosomes was studied in fibroblasts of patients suffering from sialic acid storage disease (SASD). This inherited disorder is characterized by the accumulation of acidic monosaccharides in lysosomal compartments and a concomitant decrease of their buoyant density. We demonstrate that C-terminal trimming of the lysosomal cysteine proteinase cathepsin B is inhibited in SASD fibroblasts. This late event in the biosynthesis of cathepsin B normally takes place in mature lysosomes, suggesting an impaired biogenesis of these organelles in SASD cells. When normal fibroblasts are loaded with sucrose, which inhibits transport from late endosomes to lysosomes, C-terminal cathepsin B processing is prevented to the same extent. Further characterization of the terminal endocytic compartments of SASD cells revealed properties usually associated with late endosomes/prelysosomes. In addition to a decreased buoyant density, SASD "lysosomes" show a reduced acidification capacity and appear smaller than their normal counterparts. We conclude that the accumulation of small non-diffusible compounds within endocytic compartments interferes with the formation of mature lysosomes and that the acidic environment of the latter organelles is a prerequisite for C-terminal processing of lysosomal hydrolases.     

Proton-translocating ATPase and lysosomal cystine transport

A proton-translocating ATPase was identified in highly purified lysosomes from Epstein-Barr virus-transformed human lymphoblasts. Activity of this ATPase caused acidification of highly purified, fluorescein isothiocyanate dextran-loaded lysosomes and correlated with the ATP-dependent efflux of lysosomal cystine. The lysosomal ATPase was distinct from mitochondrial F1-ATPase in its responses to a variety of inhibitors. Although ATP-dependent lysosomal cystine efflux is not demonstrable in cultured lymphoblasts from individuals with nephropathic cystinosis, ATPase activity and acidification in lysosomes from these cells is comparable to that in noncystinotic lysosomes. ATPase activity in lymphoblasts from normal individuals was 543 +/- 79 nmol/mg/min while in lymphoblasts from cystinotic individuals this activity was 541 +/- 25 nmol/mg/min. ATP-dependent acidification of lysosomes from normals was -0.5 +/- 0.1 pH units compared to -0.5 +/- 0.1 pH units in cystinotic lysosomes. Activity of the lysosomal proton-translocating ATPase is a necessary, but not sufficient, condition for lysosomal cystine efflux.     

Intravital determination of pH gradient between the cytoplasm and lysosomes in human fibroblasts in the normal state and in hereditary storage diseases

Presented are the results of measurements of pH in cytoplasm and lysosomes of skin fibroblasts of healthy donors and patients with lysosomal storage diseases, mannosidosis, Fabry, Krabbe disease. The pH value was estimated in the stationary phase of growth using neutral red (lysosomes) and fluorescein diacetate (cytoplasm). It was shown that the cytoplasmic pH value in pathological cells didn't virtually differ from the control values. The intralysosomal pH value in fibroblasts of patients with mannosidosis and Fabry disease was essentially increased, which correlated with the size increase of these organelles upon the accumulation of unsplit compounds. This led to the decrease in pH gradient between the cytoplasm and lysosomes in the pathological cells, an increase in intralysosomal pH along with hereditary deficiency of enzymes could bring about the retardation of catabolic processes in lysosomes.     

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

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

Cell growth and substrate effects on characteristics of a lysosomal enzyme (cathepsin C) in Duchenne muscular dystrophy fibroblasts

Skin fibroblasts from normal males and males suffering from Duchenne muscular dystrophy were studied in culture over a 10-week period. The lysosomal enzyme cathepsin C (dipeptidyl aminopeptidase I; EC 3.4.14.1), defined by the chloride-dependent hydrolysis of dipeptide-beta-naphthylamide (dipeptide-beta-NA) substrates at pH 5.1, was significantly lower in Duchenne cell sonicates and cell lysosomal preparations. The apparent difference in activity tended to increase with in vitro cell culture age, with the Duchenne cells being found also to grow faster and yield a greater number of cells at confluence. An analysis of all 10 cell lines as a group indicated that cathepsin C activity was related to growth rate. In addition, while analyses of cell homogenization and fractionation showed that the yield of cathepsin C was not different in Duchenne lysosomal preparations, the enzyme showed significantly lower latent activity in the Duchenne lysosomes with Gly-Phe-NA used as substrate. However, despite significant differences in specific activity compared with normal lysosomal preparations, no latency difference was observed if three other substrates were used (Gly-Arg-, Pro-Arg-, and Pro-Phe-NAs). The expression of this enzyme can thus be differentially influenced by cell growth and its latency characteristics can be influenced by the substrate used in assays.