Secretion and uptake of beta-N-acetylglucosaminidase by fibroblasts. Effect of chloroquine and mannose 6-phosphate.

The effects of chloroquine and mannose 6-hosphate on the secretion and uptake of the lysosomal enzyme, beta-N-acetylglucosaminidase (EC 3.2.1.30), by human fibroblasts have been compared. There was a reciprocal relationship between intracellular depletion, and extracellular accumulation, of enzyme at chloroquine concentrations ranging from 5 micrometers to 100 micrometers. A loss of enzyme activity from the system (intra- plus extracellular activity) with increasing concentrations of chloroquine was due to inhibition of the beta-N-acetylglucosaminidase. At a concentration of 50 micrometers, chloroquine elicited a three fold increase in the extracellular accumulation of beta-N-acetylglucosaminidase in 24 h whereas the addition of 5 micrometers mannose 6-phosphate (a competitive inhibitor of receptor-mediated uptake) resulted in only a 13% increase. Uptake of beta-N-acetylglucosaminidase by enzyme-deficient fibroblasts was completely inhibited by 5 micrometers mannose 6-phosphate. In the presence of chloroquine there was also no uptake of enzyme, however ther was a marked decrease in the residual activity of the cells. The results suggest that the effect of chloroquine on fibroblasts is to stimulate secretion rather than to inhibit uptake as previously reported. The isoenzyme pattern of the beta-N-acetylglucosaminidase from normal culture medium was compared with that accumulating in the medium following exposure of the cells to 50 micrometers chloroquine. In the presence of chloroquine, there was an increase in the A isoenzyme, however the activity was eluted in a broad peak which probably represents several closely related forms of the enzyme. There was an almost total loss of the A isoenzyme of beta-N-acetylglucosaminidase from fibroblasts cultured in the presence of chloroquine. A small peak of activity eluting at a similar position to the secreted, As, isoenzyme was present in extracts of chloroquine-treated fibroblasts, suggesting that the As isoenzyme is formed and/or stored at a site distinct from the intracellular isoenzyme.     

Purification and characterization of lysosomal β-glucuronidase from human placenta

1. Subcellular fractions of human placenta were prepared by nitrogen-bomb homogenization and differential centrifugation. 2. beta-Glucuronidase from placental lysosomes was purified 2100-fold on a protein basis. 3. The lysosomal enzyme, at different stages of purification, was characterized by using 4-methylumbelliferyl beta-d-glucuronide and phenolphthalein beta-d-glucuronide as substrates. 4. Only one isoenzyme of beta-glucuronidase was found in placenta; the enzyme in the endoplasmic reticulum appeared to be the same as the lysosomal enzyme. 5. The isoenzyme contained in normal plasma was different from that of the placenta. 6. The elevated beta-glucuronidase activity found in plasma obtained during pregnancy was due to increased activity of the normal plasma isoenzyme; no contribution was made by placental isoenzyme. 7. Plasma contained a heat-stable, non-diffusible activator of placental beta-glucuronidase. 8. A heat-stable competitive inhibitor of placental and plasma beta-glucuronidase was also present in plasma.     

Purification and Properties of beta-N-Acetylhexosaminidase from Mucor fragilis Grown in Bovine Blood

Mucor fragilis grown on bovine blood powder as the sole carbon source abundantly produced beta-N-acetylhexosaminidase. The enzyme activity was several times higher than that of a culture obtained with glucose medium. The enzyme had two different molecular weight forms. The high-molecular-weight form had somewhat higher beta-N-acetylgalactosaminidase activity than the lower-molecular-weight enzyme which had beta-N-acetylgalactosaminidase activity equivalent to about 40% of its beta-N-acetylglucosaminidase activity. Bovine blood seemed to induce both enzymes, but N-acetylamino sugars specifically induced the low-molecular-weight form. N-Acetylgalactosamine had an especially marked effect on activity. The low-molecular-weight form of enzyme was purified from the culture filtrate by fractionation with ammonium sulfate and various column chromatographies. The purified enzyme was found to be homogeneous by polyacrylamide gel electrophoresis. The optimum pH was 4.0 to 5.0 for beta-N-acetylglucosaminidase activity and 5.5 to 6.5 for beta-N-acetylgalactosaminidase activity. The enzyme hydrolyzed natural substrates such as di-N-acetylchitobiose, tri-N-acetylchitotriose, and a glycopeptide obtained by modification of fetuin.     

Appearance of beta-hexosaminidase and other lysosomal-like enzymes in the uterine lumen of gilts, ewes and mares in response to progesterone and oestrogens

In one experiment, ovariectomized gilts were treated with corn oil (vehicle), progesterone, oestradiol-17 beta or both steroids. While oestradiol treatment did not stimulate enzyme activity in uterine flushings relative to vehicle-treated animals, gilts treated with progesterone had elevated amounts of all enzymes measured. Progesterone was less effective when co-administered with oestradiol-17 beta. Enzymes were not equally stimulated by progesterone. For example, there was a 909-fold increase in acid phosphatase activity in uterine flushings and a 304-fold increase in beta-N-acetylglucosaminidase, but only a 10-fold increase in beta-glucosidase. Endometrial explants from gilts synthesized and secreted radiolabelled beta-N-acetylglucosaminidase, suggesting that at least some lysosomal enzymes enter the uterus through secretory processes. In other experiments, changes in beta-N-acetyglucosaminidase in uterine fluids of mares and ewes treated with hormonal regimens similar to those given to the gilts were evaluated. Treatment with the combination of progesterone and oestrogen stimulated accumulation of the enzyme relative to that in vehicle-treated animals. The biochemical properties of porcine beta-N-acetylglucosaminidase were examined in detail. Properties of the uterine enzyme were similar to reported values for lysosomal hexosaminidase. These included molecular weight (82 000-89 000), pH optimum (pH 4.4), presence of two isomers (isoelectric points of 5.5 and 8.0) and ability to hydrolyse substrates for glucosaminidase and galactosaminidase. We conclude that steroids induce the accumulation of lysosomal enzymes in the uterine lumen. The degree of stimulation differed between enzymes, suggesting that those enzymes stimulated to the greatest extent may play an important role in pregnancy.     

Role of lysosomal and cytosolic pH in the regulation of macrophage lysosomal enzyme secretion.

Rapid and parallel secretion of lysosomal beta-N-acetylglucosaminidase and preloaded fluorescein-labelled dextran was initiated in macrophages by agents affecting intracellular pH (methylamine, chlorpromazine, and the ionophores monensin and nigericin). In order to evaluate the relative role of changes in lysosomal and cytosolic pH, these parameters were monitored by using pH-sensitive fluorescent probes [fluorescein-labelled dextran or 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein]. All agents except chlorpromazine caused large increases in lysosomal pH under conditions where they induced secretion. By varying extracellular pH and ion composition, the changes in lysosomal and cytosolic pH could be dissociated. Secretion was then found to be significantly modulated by changes in cytosolic pH, being enhanced by alkalinization and severely inhibited by cytosolic acidification. However, changes in cytosolic pH in the absence of stimulus were unable to initiate secretion. Dissociation of the effects on lysosomal and cytosolic pH was also achieved by combining stimuli with either nigericin or acetate. Further support for a role of intracellular pH in the control of lysosomal enzyme secretion was provided by experiments where bicarbonate was included in the medium. The present study demonstrates that an increase in lysosomal pH is sufficient to initiate lysosomal enzyme secretion in macrophages and provides evidence for a significant regulatory role of cytosolic pH.     

Lysosomal enzyme secretion by macrophages during intracellular storage of particles.

Cultured mouse peritoneal macrophages containing previously endocytosed zymosan or small-fibre asbestos (but not latex or sucrose) were shown to release selectively into the medium the lysosomal hydrolase beta-N-acetylglucosaminidase. Thus macrophage lysosomal enzyem secretion was experimentally dissociated from endocytosis (as the residual external particles were washed away from the cells). The cells remained viable, and total activities of both N-acetyl-beta-D-glucosaminidase and of lactate dehydrogenase (a cytosol enzyme) rose with time. The relevance of such secretion by macrophages containing stored materials to chronic inflammatory processes is discussed.     

Binding of cathepsin D to the mannose receptor on rat peritoneal macrophages

Adherent cultures of rat peritoneal macrophages secrete lysozyme and the lysosomal marker enzymes beta-glucuronidase, beta-N-acetylglucosaminidase and acid phosphatase; the levels of secreted lysosomal cathepsin D, however, were found to be insignificant. Incubation of the cells at 4 degrees C for 15 min with yeast mannan or with 50 mM mannose, methyl alpha-glucopyranoside, or N-acetylglucosamine caused the concentration of cathepsin D in the culture medium to increase 30-40-fold; mannose-6-phosphate had no effect. 125I-labeled cathepsin D was prepared and the binding constant to the macrophage cell surface was determined to be KD = 27 nM. The data suggest that cathepsin D binds to the mannose receptor of macrophages and that binding to this receptor is not in equilibrium with the bulk medium.     

Hyaluronic acid-degrading enzymes in rat alveolar macrophages and in alveolar fluid: stimulation of enzyme activity after oral treatment with the immunomodulator RU 41740

RU 41740 (Biostim) is an immunomodulator clinically used for the treatment of chronic bronchitis and recurrent pulmonary infections. In these diseases large amounts of mucus are produced which congest the bronchi. A major glycosaminoglycan constituent of this mucus is hyaluronic acid, one of the largest molecules in nature; its metabolic degradation is carried out by 3 acid hydrolases: hyaluronidase, beta-N-acetylglucosaminidase, and beta-glucuronidase. In the lung these enzymes are especially synthesized and active in alveolar macrophages. It was thus interesting to study the effect of RU 41740 administration on the hyaluronic acid-degrading activity of these cells. This compound was given by gastric gavage to rats and the activities of lung alveolar macrophage and alveolar fluid hyaluronidase, beta-N-acetylglucosaminidase, beta-glucuronidase, and acid phosphatase as a lysosomal marker were determined. The effect on macrophage proliferation was also examined. The results obtained showed that: (1) unstimulated alveolar macrophages display the remarkable property, compared with other cell types, that hyaluronidase activity is about equally distributed between the inside and the outside of the cell; (2) RU 41740 administration increases the total activity of the 4 enzymes studied in the alveolar macrophages without inducing any increase in the number of macrophages; (3) the intracellular activities of beta-N-acetylglucosaminidase and beta-glucuronidase are markedly increased, whereas intracellular hyaluronidase activity is not changed. However, in the extracellular fluid only hyaluronidase activity is highly increased; (4) even the lysosomal marker enzyme acid phosphatase has only its intracellular activity increased. This would suggest the possibility that other lysosomal enzymes may also be increased by this immunomodulator.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycosidases in normal and regenerating chicken liver, hepatoma Mc-29, Rous sarcoma, in turkey poult liver and hemocytoblastomes, provoked by the leukosis virus strain Mc-31

Four glycosidases (beta-galactosidase, alpha-mannosidase, alpha-fucosidase and beta-N-acetylglucosaminidase) were studied in chicken normal and regenerating liver, in turkey poult liver and in virus induced avian tumors--chicken hepatoma (strain Mc-29), Rous sarcoma (strain Schmidt-Ruppin) and turkey poult hemocytoblastoma nodules (strain Mc-31). The multiple forms of beta-N-acetylglucosaminidase were assayed as well. A particular enzyme pattern was found in the tumor lines under investigation. A characteristic property of hepatoma cells was the elevation of beta-galactosidase activity and of the former enzyme and that of beta-N-acetylglucosaminidase for the hemocytoblastoma. In Rous sarcoma the glycosidase activities (except that of alpha-fucosidase) were much lower, compared to the other two solid tumors. All enzyme activities were compared with those in the normal liver of the corresponding avian species, and with the liver of tumor bearing fowls and with regenerating chicken liver. Unlike the rat liver in the avian normal and tumor tissues the percentual ratio between the multiple forms A and B of beta-N-acetylglucosaminidase was found to be 30:70%.     

Release of beta-hexosaminidase after administration of different agents affecting the reticuloendothelial system. An experimental study in rats

Plasma levels of a lysosomal enzyme, beta-hexosaminidase (beta-N-acetylglucosaminidase, EC 3.2.1.30) were studied in Wistar rats after administration of 99mTc -sulfur colloid, 198Au colloid, gelatine (Haemaccel), alcohol, methylpalmitate and zymosan. The activity of beta-hexosaminidase was increased 10, 30 and 60 min after the zymosan injection. After 24 and 48 h, enzyme levels had returned to those at outset. The transient release of beta-hexosaminidase probably occurred only during the phagocytosis of zymosan which was evaluated by histological examination of lung, liver and spleen. After the injection of all other agents tested, no significant aberration of beta-hexosaminidase levels was seen. Activity distribution of the radio-labeled colloids revealed differences in organ uptake which were attributed to a difference in colloid particle size. Although the colloids tested have been used extensively for determination of reticuloendothelial function and histological studies suggest phagocytosis of the particles, their administration did not affect plasma beta-hexosaminidase levels. Since lysosomal enzymes are cleared from the blood predominantly by liver macrophages, the primary location of particle phagocytosis may explain the present findings.     

Effect of lectins on endocytosis and secretion of lysosomal enzymes by cultured fibroblasts.

1. Pretreatment of cultured human skin fibroblasts with convanavalin A and wheat germ agglutinin inhibited endocytosis of alpha-N-acetylglucosaminidase and increased extracellular accumulation of beta-N-acetylglucosaminidase. 2. These effects were dose-dependent, reversible and could be prevented by haptenic carbohydrates, such as methyl alpha-D-mannoside or N-acetylglucosamine. 3. Pretreatment of fibroblasts with di- and monovalent succinylated concanavalin A inhibited alpha-N-acetylglucosaminidase endocytosis, but had no effect on extracellular beta-N-acetylglucosaminidase accumulation. 4. Concanavalin A-alpha-N-acetylglucosaminidase complexes become internalized via the recognition of the lectin. Complex formation prevents recognition of the phosphorylated carbohydrate on lysosomal enzymes that interacts with cell surface receptors specific for lysosomal enzymes. The inhibitory effect of all lectins tested on lysosomal enzyme endocytosis suggests that the cell surface receptors for lysosomal enzymes interact either directly with lectins or are closely linked to lectin receptors. The effect of polyvalent lectins on extracellular lysosomal enzyme accumulation is ascribed to their alteration of membrane fluidity.     

Effects of insulin on cardiac lysosomes and protein degradation

Hearts perfused in the absence of added insulin had 1) accelerated rates of protein degradation, as assessed by release of phenylalanine and tyrosine; 2) increased rates of release of seven other amino acids; 3) decreased lysosomal latency and sedimentable lysosomal enzyme activity; 4) increased numbers of autophagic vacuoles in cardiac muscle cells; and 5) decreased activity of beta-N-acetylglucosaminidase in dense lysosomes (1.06-1.09 g/ml), as compared to hearts perfused in the presence of the hormone. After 3 h of perfusion in the absence of insulin, the changes that developed in protein degradation, lysosomal latency, and sedimentability, and in enzyme activity in dense lysosomes, were reversed by insulin addition during 90 min of subsequent perfusion. These studies suggest a role for insulin in controlling the activity of the lysosomal system and the involvement of this system in protein degradation, particularly in insulin-deprived tissue.     

Glycoprotein catabolism in rat liver. Lysosomal digestion of iodinated asialo-fetuin

(125)I-labelled asialo-fetuin, administered intravenously, rapidly accumulates in rat liver and the radioactivity is subsequently cleared from the liver within 60min. Plasma radioactivity reaches a minimum between 10 and 15 min after injection and rises slightly during the period of liver clearance. Free iodide is the only radioactive compound found in plasma during this latter period. Fractionation of rat liver at 5 and 13min after injection of (125)I-labelled asialo-fetuin supports the hypothesis that asialo-glycoprotein is taken into liver by pinocytosis after binding to the plasma membrane and is then hydrolysed by lysosomal enzymes. At 5min, radioactivity was concentrated 23-fold in a membrane fraction similarly enriched in phosphodiesterase I, a plasma-membrane marker enzyme, whereas at 13min the radioactivity appeared to be localized within lysosomes. Separation of three liver fractions (heavy mitochondrial, light mitochondrial and microsomal) on sucrose gradients revealed the presence of two populations of radioactive particles. One population banded in a region coincident with a lysosomal marker enzyme. The other, more abundant, population of radioactive particles had a density of 1.13 and contained some phosphodiesterase, but very little lysosomal enzyme. These latter particles appear to be pinocytotic vesicles produced after uptake of the asialo-fetuin bound by the plasma membrane. Lysosomal extracts extensively hydrolyse asialo-fetuin during incubation in vitro at pH4.7 and iodotyrosine is completely released from the iodinated glycoprotein. Protein digestion within lysosomes was demonstrated by incubating intact lysosomes containing (125)I-labelled asialo-fetuin in iso-osmotic sucrose, pH7.2. The radioactive hydrolysis product, iodotyrosine, readily passed through the lysosomal membrane and was found in the external medium. These results are not sufficient to account for the presence of free iodide in plasma, but this was explained by the observation that iodotyrosines are deiodinated by microsomal enzymes in the presence of NADPH.     

Cell-specific beta-N-acetylglucosaminidase activity in cultures and field populations of eukaryotic marine phytoplankton.

It is widely appreciated that eukaryotic marine phytoplankton can hydrolyze a variety of compounds within the dissolved organic matter (DOM) pool in marine environments. Herein, cultures and field populations of marine phytoplankton were assayed for beta-N-acetylglucosaminidase activity, a terminal enzyme of chitin degradation. A traditional bulk assay, which can assess hydrolytic rate, but is not cell-specific, was complemented with a cell-specific assay that images the activity associated with single cells using an enzyme labeled fluorescence (ELF) substrate. beta-N-acetylglucosaminidase activity was widespread across various taxa of marine phytoplankton, and activity was observed both under controlled culture conditions and in field populations. The number of cells with enzyme activity varied with the nutritional physiology of the test species in three of the 17 cultures tested. In these three cases the number of cells with activity in the low nutrient medium was higher than in nutrient replete medium. Taken together, these data suggest that a broad group of marine phytoplankton may be a relevant part of chitin-like DOM degradation and should be incorporated into conceptual models of chitin cycling in marine systems.     

Purification and characterization of an extracellular beta-n-acetylhexosaminidase from Paecilomyces persicinus.

Both beta-N-acetylglucosaminidase nad beta-N-acetylgalactosaminidase activities were detected in the culture fluids of Paecilomyces persicinus P-10 after growth in a soybean meal-corn meal medium. The active material was purified by means of protamine sulfate fractionation and ultrafiltration, followed by ion exchange and gel chromatography. The ratio of the two activities remained constant throughout the purification, and the final product was shown to migrate as a single band by using gel isoelectric focusing, disc electrophoresis, and detergent gel electrophoresis. Temperature, pH, inhibition, and kinetic studies were performed to characterize both activities. The molecular weight of the enzyme was estimated to be about 100,000 by high-resolution gel chromatography. Based on the data obtained, it is suggested that both beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities reside in the same protein.     

Two mutations produce intron insertion in mRNA and elongated beta-subunit of human beta-hexosaminidase

An elongated beta-subunit of the lysosomal enzyme beta-hexosaminidase was found in fibroblast strains derived from two patients with juvenile Sandhoff disease and two asymptomatic individuals sharing an unusual isoenzyme pattern: a low level of residual A (alpha beta) isoenzyme activity (3-6% of normal for the juvenile Sandhoff and 9-10% for the asymptomatic strains) without B (beta beta) isoenzyme activity. The elongated beta-subunit was abnormal in other ways: It reacted with antiserum against the unfolded polypeptide, it was not phosphorylated on mannose residues, it was not processed to the mature form, and it was degraded rapidly. The increased length of the beta-subunit was caused by two different mutations. Cells from two juvenile Sandhoff and one asymptomatic individuals had the previously described G----A transition in intron 12 that creates a splice site, causing an in-frame insertion of 24 intronic nucleotides into mRNA (Nakano, T., and Suzuki, K. (1989) J. Biol. Chem. 264, 5155-5158). The second mutation was found in cells from the asymptomatic girl whose A+B- isoenzyme pattern had been designated "Hexosaminidase Paris" (Dreyfus, J. C., Poenaru, L., Vibert, M., Ravise, N., and Boue, J. (1977) Am. J. Hum. Genet. 29, 287-293); duplication of a region straddling the junction of intron 13 and exon 14 generates an alternate splice site, causing an in-frame insertion of 18 nucleotides into mRNA. Although the two new splice sites are used preferentially, the normal sites may be used to some extent, accounting for the residual A isoenzyme activity.     

Purification, cloning, and sequence analysis of beta-N-acetylglucosaminidase from the chitinolytic bacterium Aeromonas hydrophila strain SUWA-9

A chitinolytic bacterium was isolated from Lake Suwa and identified as Aeromonas hydrophila strain SUWA-9. The strain grew well on a synthetic medium containing colloidal chitin as sole carbon source. Chitin-degrading activity was induced by colloidal chitin or N-acetylglucosamine (GlcNAc). Most of the activity, however, was not detected in culture fluid but was associated with cells. A beta-N-acetylglucosaminidase was purified after it was solubilized from cells by sonication. The purified enzyme hydrolyzed N-acetylchitooligomers from dimer to pentamer and produced GlcNAc as a final product. The enzyme also hydrolyzed synthetic substrates such as p-nitrophenyl (pNP)-N-acetyl-beta-D-glucosaminide and pNP-N-acetyl-beta-D-galactosaminide. A gene coding for the purified beta-N-acetylglucosaminidase was isolated. The ORF identified is 2661 nucleotides long and encodes a precursor protein of 887 amino acids including a signal peptide of 22 amino acid residues. The amino acid sequence deduced showed a high similarity to those of bacterial beta-N-acetylhexosaminidases classified in family 20 of glycosyl hydrolases.     

Cytoplasmic enzyme patterns in isolated hamster pulmonary alveolar type II cells

Three cytoplasmic enzyme patterns were studied in pulmonary alveolar type II cells isolated from normal adult hamster lung: lactate dehydrogenase (total and isoenzymes), peroxidase, and beta-N-acetylglucosaminidase. Enzyme patterns of freshly-isolated type II cells were found to be different from those of freshly-isolated pulmonary hamster fibroblasts. After both types of cells had been cultured for seven days, no difference in cytoplasmic enzyme patterns remained. Lactate dehydrogenase isoenzyme patterns for type II cells were different from those obtained from polymorphonuclear leukocytes and alveolar macrophages. These data may be useful in detecting sources of lung injury by assessment of enzyme patterns in bronchoalveolar lavage fluid.     

Toxic effects of different concentrations of dimethoate on lysosomal enzymes of female albino rats.

The effect of three different concentrations of dimethoate on the activity of certain lysosomal enzymes, viz. beta-glucuronidase, beta-N-acetylglucosaminidase, cathepsin B and cathepsin D in serum, skin, liver, kidney and spleen and the stability of liver and kidney lysosomes was studied in female albino rats. The activity of beta-glucuronidase, beta-N-acetylglucosaminidase, cathepsin D was found to increase in serum and tissues in higher concentration (2.25 mg/100 g body weight) of dimethoate treated rats. A significant increase in the rate of release of beta-glucuronidase was found in the liver and kidney of higher concentration of dimethoate treated rats compared to controls. The results demonstrate that the activity of lysosomal enzymes increased in higher concentration of dimethoate treated rats than the lower concentration (0.56 mg/100 g body weight) of dimethoate treated rats.     

Plasma alpha-galactosidase A:properties and comparisons with tissue alpha-galactosidases.

The human plasma form of alpha-galactosidase A (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) was highly purified and exhibited apparent Km values of 1.9 mM with 4-methylumbelliferyl-alpha-D-galactopyranoside and 0.23 mM with globotriglycosylceramide. Its inhibition with myo-inositol (Ki = 0.29 M) was similar to that observed with alpha-galactosidase A from various tissues. The plasma form of this lysosomal enzyme has a lower molecular weight of 96 600, a lower pI of 3.7 and faster electrophoretic mobility in polyacrylamide gels than the enzyme obtained from human liver. These data and the increased pI obtained after neuraminidase treatment suggest that the plasma form is an isoenzyme with a more highly sialylated carbohydrate moiety than the tissue isoenzymes.