Evidence for lysosomal enzyme recognition by human fibroblasts via a phosphorylated carbohydrate moiety

Adsorptive endocytosis of five different lysosomal enzymes from various human and non-human sources was susceptible to inhibition by mannose and l-fucose, methyl α-d-mannoside, α-anomeric p-nitrophenyl glycosides of mannose and l-fucose, mannose 6-phosphate and fructose 1-phosphate. A few exceptions from this general scheme were observed for particular enzymes, particularly for β-glucuronidase from human urine. The inhibition of α-N-acetylglucosaminidase endocytosis by mannose, p-nitrophenyl α-d-mannoside and mannose 6-phosphate was shown to be competitive. The loss of endocytosis after alkaline phosphatase treatment of lysosomal enzymes supports the hypothesis that the phosphorylated sugars compete with a phosphorylated carbohydrate on the enzymes for binding to the cell-surface receptors [Kaplan, Achord & Sly (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 2026–2030]. Endocytosis of `low-uptake' forms of α-N-acetylglucosaminidase and α-mannosidase was likewise susceptible to inhibition by sugar phosphates and by alkaline phosphatase treatment, suggesting that `low-uptake' forms are either contaminated with `high-uptake' forms or are internalized via the same route as `high-uptake' forms. The existence of an alternative route for adsorptive endocytosis of lysosomal enzymes is indicated by the unaffected adsorptive endocytosis of rat liver β-glucuronidase in the presence of phosphorylated sugars and after treatment with alkaline phosphatase.     

Histochemical evidence for lysosomal uptake of lead in tissue cultured fibroblasts

Summary Lysosomes of embryonic rat fibroblasts cultivated in vitro normally contain heavy metals, as shown with a modified sulfide-silver method (SSM). Cultures which received lead added to the cultivation medium showed an enhanced SSM-positivity. However, since the SSM demonstrates several different heavy metals the sulfides of which are weakly soluble in water, it was not possible to distinguish between naturally occurring heavy metals—largely iron—and added lead (supposed to have been taken up by the cells).By treating the cells with a 0.2 M TCA solution after an initial exposure to HS^– it was possible to dissolve FeS without affecting the PbS to any noticeable extent. When subsequently the development process of the SSM was applied to the cells, no metals could be demonstrated in the control cells whereas those exposed to lead showed presence of granules, most of which were identical with lysosomes as visualized with a Gomori type reaction. A lysosomal uptake of lead could thus be demonstrated with the modified SSM when combined with a simple dissolving process.Supported by the Swedish Medical Research Council grants nos. K70-12X-2037-05A and B71-12X-2037-06B.     

Inhibition of receptor-mediated uptake of a lysosomal enzyme into fibroblasts by chloroquine, procaine and ammonia

Cultured human skin fibroblasts take up α- -iduronidase by receptor-mediated pinocytosis. Certain lysosomotropic amines such as chloroquine, ammonia and procaine inhibit this process, without affecting the fluid endocytosis of dextran. In contrast to the competitive inhibition by mannose 6-phosphate, the inhibition by amines is non-competitive and is therefore presumed not to affect binding of the enzyme to receptors. The dose response curves are very steep, and equations that best fit the data use a power of inhibitor concentration (i² for procaine, i⁴ for chloroquine), indicating interaction of several amine molecules at the inhibitory site(s). The inhibition is reversed by removal of the amine from the medium and does not result from accelerated efflux of endocytosed enzyme. We suggest that the amines interfere with delivery of receptor-bound enzyme to lysosomes.     

Concanavalin a promotes the uptake of lysosomal hydrolases by human fibroblasts

Human placental hexosaminidase B and β-galactosidase are taken up very poorly by human fibroblasts in culture. However, if fibroblasts manifesting genetically determined deficiencies of these lysosomal hydrolases are first treated with concanavalin A, then enzyme uptake is markedly increased. Enzyme activity which becomes associated with concanavalin A-treated fibroblasts maintained at 4°C can be greatly removed by treatment with haptene sugar, while enzyme activity which becomes associated with cells maintained at 37°C is refractory to haptene treatment. These results are interpreted as an initial binding of enzyme to concanavalin A molecules located at the cell surface, followed by an active cellular process leading to internalization of the lectin-enzyme complexes.     

Concanavalin A promotes the uptake of lysosomal hydrolases by human fibroblasts.

Human placental hexosaminidase B and beta-galactosidase are taken up very poorly by human fibroblasts in culture. However, if fibroblasts manifesting genetically determined deficiencies of these lysosomal hydrolases are first treated with concanavalin A, then enzyme uptake is markedly increased. Enzyme activity which becomes associated with concanavalin A-treated fibroblasts maintained at 4 degrees C can be greatly removed by treatment with haptene sugar, while enzyme activity which becomes associated with cells maintained at 37 degrees C is refractory to haptens treatment. These results are interpreted as an initial binding of enzyme to concanvalin A molecules located at the cell surface, followed by an active cellular process leading to internalization of the lectin-enzyme complexes.     

Presence of a lysosomal enzyme, arylsulfatase-A, in the prelysosome-endosome compartments of human cultured fibroblasts

Although endosomes and lysosomes are associated with different subcellular functions, we present evidence that a lysosomal enzyme, arylsulfatase-A, is present in prelysosomal vesicles which constitute part of the endosomal compartment. When human cultured fibroblasts were subfractionated with Percoll gradients, arylsulfatase-A activity was enriched in three subcellular fractions: dense lysosomes, light lysosomes, and light membranous vesicles. Pulsing the cells for 1 to 10 min with the fluid-phase endocytic marker, horseradish peroxidase, showed that endosomes enriched with the marker were distributed partly in the light lysosome fraction but mainly in the light membranous fraction. By pulsing the fibroblasts for 10 min with horseradish peroxidase conjugated to colloidal gold and then staining the light membranous and light lysosomal fractions for arylsulfatase-A activity with a specific cytochemical technique, the endocytic marker was detected under the electron microscope in the same vesicles as the lysosomal enzyme. The origin of the lysosomal enzyme in this endosomal compartment was shown not to be acquired through mannose 6-phosphate receptor-mediated endocytosis of enzymes previously secreted from the cell. Together with our recent finding that the light membranous fraction contains prelysosomes distinct from bona fide lysosomes and was highly enriched with newly synthesized arylsulfatase-A molecules, these results demonstrate that prelysosomes also constitute part of the endosomal compartment to which intracellular lysosomal enzymes are targeted.     

Biochemical mechanisms of aminoglycoside cell toxicity. I. The uptake of gentamicin by cultured skin fibroblasts and the alteration of lysosomal enzyme activities

In order to study the toxicity of aminoglycoside, human skin fibroblasts were used as a model for basic studies, since they are known to have a specific aminoglycoside-binding site and to translocate the drug into the cells. Following the exposure of fibroblasts to gentamicin for 3 days, the cells formed many osmiophilic lamellar materials (myeloid bodies) in the lysosomes, while the other cellular structures appeared to remain normal. Although gentamicin was intensively accumulated within the lysosomes, intralysosomal pH, determined by the fluorescence intensity ratio method using fluorescein-isothiocyanate-labeled dextran, did not alter. Among the lysosomal enzymes, the activities of six different glycosidases were unchanged. On the other hand, sphingomyelinase and acid lipase activities were greatly decreased, while phospholipase A activity was increased. These results indicate that the lipid metabolism of fibroblasts is altered by gentamicin treatment, and that perturbation of intralysosomal pH can not be the cause of the changes observed in cell lysosomal enzyme activities.     

Age-related changes in hyaluronate uptake by cultured fibroblasts

Skin fibroblasts from chick embryos of 7 and 14 days incubation have been tested for their ability to bind, internalize and degrade [3H]-hyaluronan. Seven-day fibroblasts internalize and degrade a greater amount of labelled hyaluronan than 14-day cells. The implications of these findings in the regulation by the fibroblasts of extracellular matrix composition in glycosaminoglycans are discussed.     

Lectin-mediated uptake of lysosomal hydrolases by genetically deficient human fibroblasts.

A protein factor named S-II that stimulates RNA polymerase II was previously purified from Ehrlich ascites tumor cells [1]. In this work using an antibody prepared against purified S-II, the localization of S-II in the cell was investigated by an indirect immunofluorescence technique. In 3T3 cells, specific immunofluorescence was detected only in the nucleoplasm where RNA polymerase II is located, and not in the nucleoli where RNA polymerase I is present. In Ehrlich ascites tumor cells fluorescence was detected mainly in the nucleoplasm, although some fluorescence was also detectable in the cytoplasm, possibly due to leak of S-II from the nuclei during preparation of the immunofluorescent samples. In metaphase cells fluorescent was not found on chromosomes but throughout the cytoplasm. These findings suggest that S-II is a nuclear protein and that it spreads into the cytoplasm without being attached to chromosomes in metaphase, but is reassembled into the nucleoplasm in the interphase. Specific immunofluorescence was also detected in the nuclei of HeLa cells and salivary glands cells of flesh-fly larvae, suggesting that the nucleoplasm of these heterologous cells contains proteins immunologically cross-reactive with the antibody against S-II.     

Endocytosis of lysosomal alpha-galactosidase A by cultured fibroblasts from patients with Fabry disease.

The endocytosis of alpha-galactosidase A was studied in cultured fibroblasts from patients with Fabry disease. Alpha-galactosidase A was purified from human placenta by chromatography on concanavalin A-Sepharose, DEAE-cellulose, and N-epsilon-aminocaproyl-alpha-D-galactosylamine-Sepharose. Separation of the high-uptake form of the enzyme from the low-uptake form was accomplished by chromatography on ECTEOLA-cellulose. With the high-uptake form of the enzyme, the uptake was linear at low concentrations of enzyme and had a Kuptake of 0.01 U/ml of medium that corresponds to a Km of 5.0 x 10(-9) M. At high concentrations of enzyme, it became saturated. The high-uptake form could be converted to the low-uptake form by treatment with acid phosphatase. Mannose-6-P strongly inhibited the active uptake of the enzyme. Once taken up into the lysosomes of Fabry disease fibroblasts, alpha-galactosidase A activity was rapidly lost in the first 2 days of incubation at 37 degrees C, but was fairly stable for the next 6 days. The half-life of internalized alpha-galactosidase A activity was calculated to be 4 days. Crosslinking of the enzyme with hexamethylene diisocyanate did not increase the intracellular stability of alpha-galactosidase A activity.     

Energization of amino acid uptake by system A in cultured human fibroblasts

The energization of System A in cultured human fibroblasts has been studied by measuring the energy transfer from the electrochemical gradient of Na+ to the chemical gradient of the site A-specific substrate amino acid 2-methylaminoisobutyric acid. The co-transport Na+/amino acid, studied by kinetic analysis and radiochemical measurements, showed a coupling ratio of 1:1. The assessment of the Na+ electrochemical gradient in cultured adherent cells relied on the development of noninvasive procedures as follows: the membrane electrical potential was estimated from the accumulation of L-arginine at equilibrium (Bussolati, O., Laris, P. C., Nucci, F. A., Dall'Asta, V., Longo, N., Guidotti, G. G., and Gazzola, G. C. (1987) Am. J. Physiol. 253, C391-C397); the chemical gradient of Na+ was determined from spectrometric measurements of Na+. The accumulation of 2-methylaminoisobutyric acid was strongly sensitive to changes of Na+ gradient and of membrane electrical potential, indicating that the electrochemical gradient of Na+ contributed energy for the uphill transport of the amino acid through System A. Changes in the Na+ electrochemical gradient were obtained by: (i) alterations of extracellular concentration of Na+; (ii) changes of membrane electrical potential obtained by variation of extracellular [K+]; and (iii) changes of [Na+]in and membrane electrical potential upon incubation of the cells in serum-free saline solutions (Dall'Asta, V., Gazzola, G. C., Longo, N., Bussolati, O., Franchi-Gazzola, R., and Guidotti, G. G. (1986) Biochim. Biophys. Acta 860, 1-8). The correlation between the chemical gradient of 2-methylaminoisobutyric acid and the Na+ electrochemical potential followed a straight line with a yield close to the thermodynamic equilibrium, thus suggesting that the energy stored in the gradient of Na+ electrochemical potential is fully adequate to energize the intracellular accumulation of site A-reactive amino acids in human fibroblasts.     

Effect of chloroquine on cultured fibroblasts: release of lysosomal hydrolases and inhibition of their uptake.

Incubation of normal human fibroblasts with 1–5 μM chloroquine at physiological pH for 8 hr produces granular cytoplasmic inclusions, release of lysosomal enzymes into the medium and decrease of intracellular lysosomal enzyme activities. The effects are dose dependent and reversible. The uptake of arylsulfatase A into fibroblasts genetically deficient in arylsulfatase A (grown from skin biopsies of patients with metachromatic leukodystrophy) is completely inhibited by pretreating the cells with 5 μM chloroquine. Arylsulfatase A, which has been taken up as exogenous enzyme from the medium into the cells, is partially released into the culture medium upon incubation with chloroquine. The data suggest that chloroquine competes with the binding of lysosomal enzymes to the cell membrane and to the membranes of pinocytotic vacuoles and causes release of previously internalized exogenous enzyme.     

Mapping and molecular modeling of a recognition domain for lysosomal enzyme targeting.

Lysosomal enzymes contain a common protein determinant that is recognized by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, the initial enzyme in the biosynthesis of mannose-6-P residues. Previously, we generated a lysosomal enzyme recognition domain by substituting two regions (lysine 203 and amino acids 265-292) of the lysosomal hydrolase cathepsin D into a related secretory protein glycopepsinogen. When expressed in Xenopus oocytes, the oligosaccharides of the chimeric protein were efficiently phosphorylated (Baranski, T. J., Faust, P. L., and Kornfeld, S. (1990) Cell 63, 281-291). In the current study, incremental substitutions of cathepsin D residues into glycopepsinogen and alanine-scanning mutagenesis were utilized to define the recognition domain more precisely. A computer-generated model of the cathepsin D/pepsinogen chimeric molecule served as a guide for mutagenesis and for the interpretation of results. These studies indicate that the recognition domain is a surface patch that contains multiple interacting sites. There is a strict positional requirement for the lysine residue at position 203.     

Correction of I-cell defect by hybridization with lysosomal enzyme deficient human fibroblasts

I-cell fibroblasts with a multiple intracellular lysosomal enzyme deficiency were hybridized with cells from patients with different types of single lysosomal enzyme defects. Fusion with G_M2 gangliosidosis, type 2, (Sandhoff disease) fibroblasts resulted in a restoration of the hexosaminidase activity, in a normalization of the electrophoretic mobility of the isoenzymes, and in a decreased activity in the medium. Fusion of I-cells with fibroblasts from G_M1 gangliosidosis, type 1, led to enhancement of β-galactosidase (β-gal) activity. This complementation must be the result of the presence of normal polypeptide chains in I-cells, whereas the other cell types provide a factor that causes the intracellular retention of the enzymes. Restoration of β-gal was also observed in heterokaryons after fusion of I-cells with β-galactosidase/neuraminidase-deficient (β-gal⁻/neur⁻) variants, indicating that the neuraminidase(s) and the posttranslational modification of β-gal are affected in a different way in I-cell disease and in β-gal⁻/neur⁻ variants. Fusion of I-cells with mannosidosis fibroblasts resulted in a restoration of the acidic form of α-mannosidase and in a decrease of the extracellular activity of both this enzyme and the hexosaminidase enzyme, indicating that fusion of I-cells with different types of fibroblasts with a single lysosomal enzyme deficiency not only leads to complementation for one particular enzyme but also to a correction of the basic defect in I-cells.     

Angiotensin I-converting enzyme localization on cultured fibroblasts by immunofluorescence

Summary Angiotensin I-converting enzyme is responsible for the activation of angiotensin I and the inactivation of bradykinin. It has been localized by immunofluorescence on the endothelium of a variety of tissues and has been considered to be a specific marker for endothelial cells in culture. The present paper demonstrates, by immunofluorescence, the presence of angiotensin I-converting enzyme in monolayer cultures of fibroblasts derived from adult rat lung, bovine calf pulmonary artery, and human foreskin (CF-3 cells). Fluorescent localization of angiotensin I-converting enzyme was observed over the cytoplasm of adult rat lung and bovine calf pulmonary artery fibroblasts and as distinct areas overlying the nuclei of human foreskin fibroblasts. Determination of angiotensin I-converting enzyme activity by fluorimetric assay in parallel studies confirmed the presence of angiotensin I-converting enzyme activity in cultured fibroblasts. Immunofluorescent studies with antibody to Factor VIII demonstrated the presence of Factor VIII on cultured endothelial cells but not on fibroblasts. These results indicate that angiotensin I-converting enzyme is not confined to endothelial cells, and thus may not serve as a specific marker for endothelial cells in culture. Factor VIII may be a more specific marker for these cells. Presented in part at the 31st Annual Meeting of the Histochemical Society, April 11–15, 1980, New Orleans, Louisiana. Wendy Baur and Ms. Jane Aghajanian for expert assistance in the preparation of the cell cultures. This work was supported by Research Grant HL 14456 and Training Grant HL 07053 from the National Institutes of Health, Bethesda, MD.     

Angiotensin I-converting enzyme localization on cultured fibroblasts by immunofluorescence

Angiotensin I-converting enzyme is responsible for the activation of angiotensin I and the inactivation of bradykinin. It has been localized by immunofluorescence on the endothelium of a variety of tissues and has been considered to be a specific marker for endothelial cells in culture. The present paper demonstrates, by immunofluorescence, the presence of angiotensin I-converting enzyme in monolayer cultures of fibroblasts derived from adult rat lung, bovine calf pulmonary artery, and human foreskin (CF-3 cells). Fluorescent localization of angiotensin I-converting enzyme was observed over the cytoplasm of adult rat lung and bovine calf pulmonary artery fibroblasts and as distinct areas overlying the nuclei of human foreskin fibroblasts. Determination of angiotensin I-converting enzyme activity by fluorimetric assay in parallel studies confirmed the presence of angiotensin I-converting enzyme activity in cultured fibroblasts. Immunofluorescent studies with antibody to Factor VIII demonstrated the presence of Factor VIII on cultured endothelial cells but not on fibroblasts. These results indicate that angiotensin I-converting enzyme is not confined to endothelial cells, and thus may not serve as a specific marker for endothelial cells in culture. Factor VIII may be a more specific marker for these cells.     

Receptor-dependent uptake of human chylomicron remnants by cultured skin fibroblasts

Human chylomicrons were isolated from plasma from a subject with familial hypertriglyceridemia and converted to chylomicron remnants by incubation with postheparin plasma. The interaction of these apolipoprotein E-containing, cholesterol-rich human chylomicron remnants with cultured skin fibroblasts was studied. Chylomicron remnants were internalized by skin fibroblasts as a unit, mainly via the low density lipoprotein (LDL)-receptor pathway, resulting in increased cell cholesterol content. After entering the fibroblast, chylomicron remnants stimulated cholesterol esterification, suppressed 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, and down-regulated LDL receptor activity similar to the action of LDL. As a function of increasing lipolysis, remnant particles were progressively more effectively taken up by skin fibroblasts, despite a decrease in the apolipoprotein E content per lipoprotein particle. Remnant particles produced after hydrolysis of 70 to 80% of chylomicron triglyceride increased cell cholesterol content to an amount nearly identical to that observed with LDL when the two lipoproteins were incubated at an equal cholesterol concentration. However, when incubated on the basis of equal particle number, chylomicron remnants were 2 to 3 times more effective than LDL in delivering cholesterol to the cells. These results suggest that chylomicron remnants play a role in the regulation of postabsorptive cholesterol homeostasis in nonhepatic cells, and possibly in the pathogenesis of atherosclerosis.     

Cell surface-associated lysosomal enzymes in cultured human skin fibroblasts

Trypsin released from the surface of intact human skin fibroblasts β-N-acetylglucosaminidase. The amount of trypsin removable β-N-acetylglucosaminidase in 4 control and 14 mucopolysaccharidosis cell lines was equivalent to 1.5% (range 0.5–4.3%) of the intracellular activity. Cell surface-associated β-N-acetylglucosaminidase was absent in mucolipidosis II and III fibroblasts that form lysosomal enzymes defective in binding to the cell surface receptors of fibroblasts and in β-N-acetylglucosaminidase deficient fibroblasts (Sandhoff's disease). Indirect immunofiuorescence with monospecific antisera allowed the demonstration of β-N-acetylglucosaminidase, α-N-acetylglucosaminidase, α-mannosidase and β-glucuronidase on the cell surface of fibroblasts, whereas these enzymes were absent on the cell surface of mucolipidosis II and III fibroblasts. Simultaneous staining for β-glucuronidase and β-N-acetylglucosaminidase showed presence of both enzymes in almost identical areas of the same cell. Cross-reacting material was present on the cell surface of fibroblasts with a deficiency of β-N-acetylglycosaminidase, α-N-acetylglucosaminidase (mucopolysaccharidosis III B), α-mannosidase (mannosidosis) and β-glucuronidase (mucopolysaccharidosis VII). The demonstration of lysosomal enzymes on the cell surface is in agreement with the hypothesis that in fibroblasts transport of lysosomal enzymes to the lysosomal apparatus involves cycling of lysosomal enzymes via the cell surface.     

A photoreactive competitive inhibitor of the human lysosomal neuraminidase in cultured skin fibroblasts

A photoreactive, potent, competitive inhibitor of the human lysosomal neuraminidase in cultured skin fibroblasts has been prepared. The starting material, 2,3 dehydro-N-acetyl neuraminic acid methyl ester, was selectively tosylated at the C-9 position with tosyl chloride and subsequently peracetylated with acetic anhydride. The tosyl group was displaced with potassium thio acetate in dimethylformamide at 60 degrees C for 80 min. 4-fluoro-3-nitrophenylazide was incorporated by reaction with the thio acetate product and equimolar sodium methoxide in methanol followed by reacetylation. Base hydrolysis gave the final product, 9-S-(4-azido-2-nitrophenyl)-5-acetamido-2,6 anhydro-2,3,5,9-tetradeoxy-9-thio-D-glycero-D-galacto-non-2-enonic acid (W5). The yields at each step were 50-70%. Competitive inhibition kinetics were observed when W5 was tested with the fibroblast neuraminidase using 4-methylumbelliferyl-N-acetyl-neuraminic acid as substrate giving an apparent Ki of about 10 microM. These results suggest that the terminal hydroxyl group at C-9 may not be important in the recognition and binding of the substrate by the enzyme. Also, the compounds prepared here may be useful as photoaffinity probes or ligands for affinity chromatography for purification.     

Two species of lysosomal organelles in cultured human fibroblasts.

Cultured diploid human skin fibroblasts were fractionated by a procedure that maximizes recovery of particles containing acid hydrolases. The cells were detached by controlled trypsinization, disrupted by N2 cavitation at low pressure and fractionated at 18,000 x g on a self-generating gradient of colloidal silica. This procedure separated two species of particles that could be consisered lysosomal. The denser one (peak density 1.11) was apparently free of other contaminants, but the more buoyant one (peak density 1.085) sedimented with or close to the peaks of other organelles, including mitochondria, Golgi, endoplasmic reticulum and plasma membranes. The two populations of particles contained acid hydrolases (phosphatase, six glycosidases and four cathepsins) in roughly equal proportions, displayed latency, had similar turnover of 35S-mucopolysaccharide in normal as well as in iduronidase-deficient cells, and were recipients of alpha-L-iduronidase, previously shown to be acquired by receptor-mediated endocytosis. Acid phosphatase staining of the intact fibroblasts showed residual bodies scattered throughout the cytoplasm and, near the nucleus, a prominent network of tubules and associated dilatations and knob-like enlargements. In both thin and thick sections, these appeared continuous, as if forming a three-dimensional network similar to the network described by Novikoff (1976) as GERL. Ultrastructural studies of the isolated fractions showed the denser lysosomal peak to be composed of small round or oblong acid phosphatase-positive bodies. The more buoyant peak contained the nonlysosomal organelles predicted from the biochemical markers, small acid phosphatase-positive bodies and large multivesiculated structures in which acid phosphatase was localized in a matrix surrounding apparently empty vesicles. These large structures may represent fragments of GERL. We suggest that the dense and buoyant lysosomal organelles originate primarily from residual bodies and the GERL network, respectively.