Affinity labelling of enzymes with triazine dyes. Isolation of a peptide in the catalytic domain of horse-liver alcohol dehydrogenase using Procion blue MX-R as a structural probe

The incorporation of [3H]leucine and [32P]phosphate into three lysosomal enzymes, cathepsin D, beta-hexosaminidase and arylsulfatase A by fibroblasts from six patients affected with mucolipidosis III was determined. In the mutant cells the incorporation of 32P in the enzymes was reduced by 70-97% as compared to controls. The residual phosphorylation of lysosomal enzymes is definitely higher than in fibroblasts from patients with mucolipidosis II, where apparently non-phosphorylated enzymes are formed. In mucolipidosis III the major part of the newly formed enzymes accumulated extracellularly and the cellular enzymes were recovered mainly in their processed forms. In mucolipidosis III arylsulfatase A and the processed forms of cathepsin D exhibited a heterogeneity that was not observed in controls. beta-Hexosaminidase and cathepsin D secreted by mucolipidosis III fibroblasts contained only a small amount of phosphorylated oligosaccharides with either one or two phosphate groups per oligosaccharide. As in controls the major fraction of phosphate was present as acid-labile phosphodiester resistant to alkaline phosphatase. The residual phosphorylation of lysosomal enzymes may be related to the partial intracellular retention and processing of these enzymes in fibroblasts from patients with mucolipidosis III.     

Mucolipidoses II and III variants with normal N-acetylglucosamine 1-phosphotransferase activity toward alpha-methylmannoside are due to nonallelic mutations.

Normal N-acetylglucosamine 1-phosphotransferase activity toward mono- and oligosaccharide acceptor substrates was detected in cultured skin fibroblasts from mucolipidoses II and III patients who were designated as variants (one of four mucolipidosis II and three out of six mucolipidosis III patients examined). The activity toward natural lysosomal protein acceptors was absent or deficient in cell preparations from all patients with classical as well as variant forms of mucolipidoses II and III. Complementation analysis, using fused and cocultivated mutant fibroblast combinations, revealed that, while cell lines with variant mucolipidosis III constituted a complementation group distinct from that of classical forms of mucolipidoses II and III, the variant mucolipidosis II cell line belonged to the same complementation group as did the classical forms. In contrast to the mutant enzyme from variant mucolipidosis III patients that failed to recognize lysosomal proteins as the specific acceptor substrates, the activity toward alpha-methylmannoside in the variant mucolipidosis II patient could be inhibited by exogenous lysosomal enzyme preparations (bovine beta-glucuronidase and human hexosaminidase A). These findings suggest that N-acetylglucosamine 1-phosphotransferase is composed of at least two distinct polypeptides: (1) a recognition subunit that is defective in the mucolipidosis III variants and (2) a catalytic subunit that is deficient or altered in the classical forms of mucolipidoses II and III as well as in the mucolipidosis II variant.     

Demonstration of the heterozygous state for I-cell disease and pseudo-Hurler polydystrophy by assay of N-acetylglucosaminylphosphotransferase in white blood cells and fibroblasts

The biochemical abnormalities of I-cell disease (mucolipidosis II) and pseudo-Hurler polydystrophy (mucolipidosis III) can be explained by a deficiency of the enzyme UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase. We demonstrate here that obligate heterozygotes for these autosomal recessive diseases have intermediate levels of this enzymatic activity in homogenates of peripheral blood white cells and in extracts from cultured fibroblasts. This finding provides further evidence that the enzyme deficiency is the primary genetic defect in these diseases. In addition, the previous observation that obligate heterozygotes for mucolipidosis III have elevations of total serum beta-hexosaminidase outside the range of normal was confirmed. In studies of three pedigrees of patients with mucolipidosis III, these techniques were used to score individuals at risk for the carrier state.     

Effects of flavonoids on enzyme secretion and endocytosis in normal and mucolipidosis II fibroblasts

The effect of flavonoids on beta-hexosaminidase transport and endocytosis has been studied in cultured human skin fibroblasts. In mucolipidosis II fibroblast cultures, characterized by their preferential secretion of most newly synthesized hydrolases, quercetin and phloretin (200 microM) inhibited beta-hexosaminidase synthesis as well as total culture-associated enzyme activity. Taxifolin induced a 2.4-fold increase in the total enzyme activity without altering the intra- and extracellular distribution of the enzyme. Rutin, although less effective, also stimulated an overall increase in total enzyme. The flavonoid effects were all concentration-dependent. Very little effect was observed in either the distribution or the total beta-hexosaminidase activity in normal fibroblast cultures. Taxifolin and hesperitin inhibited receptor-mediated endocytosis of beta-hexosaminidase by fibroblasts up to 50% of control uptake. Naringin, quercetin, and phloretin moderately inhibited uptake by 30% while rutin and fisetin had no effect. The results demonstrate that certain naturally occurring flavonoids affect the secretion of lysosomal enzymes as well as their endocytosis by fibroblasts. Since most individuals ingest up to one gram per day of these substances, flavonoids may prove to have significant effects on normal lysosomal enzyme physiology.     

Neurologic Abnormalities in Mouse Models of the Lysosomal Storage Disorders Mucolipidosis II and Mucolipidosis III γ

UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase is an α₂β₂γ₂ hexameric enzyme that catalyzes the synthesis of the mannose 6-phosphate targeting signal on lysosomal hydrolases. Mutations in the α/β subunit precursor gene cause the severe lysosomal storage disorder mucolipidosis II (ML II) or the more moderate mucolipidosis III alpha/beta (ML III α/β), while mutations in the γ subunit gene cause the mildest disorder, mucolipidosis III gamma (ML III γ). Here we report neurologic consequences of mouse models of ML II and ML III γ. The ML II mice have a total loss of acid hydrolase phosphorylation, which results in depletion of acid hydrolases in mesenchymal-derived cells. The ML III γ mice retain partial phosphorylation. However, in both cases, total brain extracts have normal or near normal activity of many acid hydrolases reflecting mannose 6-phosphate-independent lysosomal targeting pathways. While behavioral deficits occur in both models, the onset of these changes occurs sooner and the severity is greater in the ML II mice. The ML II mice undergo progressive neurodegeneration with neuronal loss, astrocytosis, microgliosis and Purkinje cell depletion which was evident at 4 months whereas ML III γ mice have only mild to moderate astrocytosis and microgliosis at 12 months. Both models accumulate the ganglioside GM2, but only ML II mice accumulate fucosylated glycans. We conclude that in spite of active mannose 6-phosphate-independent targeting pathways in the brain, there are cell types that require at least partial phosphorylation function to avoid lysosomal dysfunction and the associated neurodegeneration and behavioral impairments.     

Deficiency of neuraminidase in the sialidoses and the mucolipidoses

Summary Neuraminidase activity in cultured fibroblasts from patients either with various forms of sialidosis or with I-cell disease (ICD) or mucolipidosis (ML) III has been determined by both a colorimetric and a fluorometric method. The former applied to frozen fibroblast pellets demonstrated a specific deficiency of neuraminidase in patients with the sialidoses. The enzyme was also deficient in I-cells, as were other lysosomal hydrolases. With the fluorogenic substrate these data could be confirmed and extended, and elementary kinetics of neuraminidase studied. In unfrozen freshly harvested fibroblasts, neuraminidase activity was severalfold that in frozen aliquots. A comparative and simultaneous study could not reveal substantial differences between the residual neuraminidase activity found in the various clinical forms of sialidosis. And, in fibroblasts from patients with ICD, also called ML II, the deficiency of this enzyme is quantitatively similar to that in the sialidoses, but the residual activity in ML III is three times higher. In both ML II and ML III the defect is probably secondary to the unknown metabolic error.     

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.     

Oligosaccharides in lysosomal enzymes. Distribution of high-mannose and complex oligosaccharides in cathepsin D and beta-hexosaminidase

The distribution of the different types of oligosaccharides in cathepsin D and in beta-hexosaminidase synthesized in cultured human fibroblasts was studied by using endo-beta-N-acetylglucosaminidase H as a probe for high-mannose oligosaccharides. The enzymes were specifically labelled in the protein or the carbohydrate moiety. In both enzymes, resistant and cleavable oligosaccharides were found. The resistant oligosaccharides prevailed in the secreted enzymes. Precursor molecules of cathepsin D contained two oligosaccharide side chains. Multiple forms of the precursor are synthesized with both, one or none of two oligosaccharides sensitive to the action of the endo-beta-N-acetylglucosaminidase H. In fibroblasts unable to phosphorylate lysosomal enzymes (mucolipidosis II) the excessively secreted lysosomal enzymes contained predominantly oligosaccharides resistant to endo-beta-N-acetylglucosaminidase H.     

Cystic fibrosis: leakage of lysosomal enzymes and of alkaline phosphatase into the extracellular space

Nine lysosomal enzymes and alkaline phosphatase have been assayed with two different ultramicro techniques in the intra- and extracellular space of fibroblast cultures derived from the skin of cystic fibrosis patients, cystic fibrosis carriers, and normal controls, respectively. Evidence has been obtained for a multiple leakage of lysosomal enzymes and of alkaline phosphatase into the medium of fibroblast cultures from cystic fibrosis patients and carriers. The situation is comparable to a certain extent, to that observed in I-cell-disease (mucolipidosis II). This multiple leakage results in the decrease of intracellular activity of several lysosomal enzymes in cultures from cystic fibrosis patients and carriers and due to the coordinate regulation of the synthesis of the “leaky enzymes” in an overshooting of the intracellular alkaline phosphatase activity in cultures from cystic fibrosis patients. It also explains the retarded catabolism of certain molecules, such as the Tamm-Horsfall glycoprotein, in cystic fibrosis cells. It is speculated that the basic defect in cystic fibrosis leads to abnormal recognition sites on lysosomal enzymes and on alkaline phosphatase, and in consequence to the leakage of these enzymes into the extracellular space. The present findings allow one to develop methods for the pre- and postnatal diagnosis of cystic fibrosis with cell cultures, and for the detection of cystic fibrosis carriers with the peripheral blood.     

Effect of the co-existence of galactosyl and phosphomannosyl residues on β-hexosaminidase on the processing and transport of the enzyme in mucolipidosis I fibroblasts

Cultured fibroblasts from patients with the lysosomal storage disease, mucolipidosis II, produce complex glycosylated lysosomal enzymes which are preferentially excreted presumably due to the absence of specific phosphomannosyl recognition residues needed for intracellular retention. Complex glycosylated hydrolases are also produced by fibroblasts from patients with mucolipidosis I but an abnormal excretion is not apparent in this disorder. Intra- and extracellular distribution, lectin binding, and specific endocytosis were criteria used to compare the properties of intra- and extracellular β-hexosaminidase derived from mucolipidosis I and normal fibroblast cultures. Mucolipidosis I fibroblasts did not hyperexcrete β-hexosaminidase when maintained in serum-free medium. Using the specifity of ricin binding to terminal galactosyl residues, the most galactosylated forms of the enzyme derived from mucolipidosis I cell extracts and culture fluids were found in the mucolipidosis I cell extracts (50% of total enzyme). Mucolipidosis I-excreted β-hexosaminidase which was eluted from ricin-120-Sepharose, was a high-uptake form in endocytosis experiments while unbound enzyme was a low-uptake form. These data suggest that β-hexosaminidase molecules contained phosphomanosyl residues necessary for receptor-mediated endocytosis as well as galactosyl residues on the same molecule. The co-existence of complex chains with high-mannose chains did not interfere with the phosphomannose-mediated endocytosis of β-hexosaminidase nor with the retention of endogenous enzyme. We can speculate that since complex oligosaccharide chains in the mucolipidosis I cellular enzyme persist due to a sialidase deficiency, more extensive sialylation of cellular enzyme in normal fibroblasts probably occurs at some point during post-translational processing. However, the presence of sialidase in normal cells initiates complex chain trimming in the lysosomes resulting in a less glycosylated end product.     

Compartmental distribution of beta-hexosaminidase isoenzymes in I-cell fibroblasts.

A characteristic of the human lysosomal disorder I-cell disease is an abnormal excretion of most lysosomal hydrolases, including beta-N-acetyl-D-glucosaminidase (EC 3.2.1.30; beta-hexosaminidase) by cultured skin fibroblasts. Treatment of I-cell cultures with cycloheximide or tunicamycin demonstrated that (1) I-cell fibroblasts rapidly excrete all newly synthesized beta-hexosaminidase, (2) two qualitatively distinct pools of beta-hexosaminidase isoenzymes exist inside I-cell fibroblasts, one of which is a rapid-turnover excretory pool, and (3) the induction of an abnormal glycosylation of beta-hexosaminidase by tunicamycin in normal or I-cell fibroblast cultures does not affect subsequent excretion of the enzyme.     

Urinary lysosomal hydrolases in mucolipidosis II and mucolipidosis III.

Investigation of the binding characteristics of acid beta-D-galactosidase, N-acetyl-beta-D-glucosaminidase, alpha-D-galactosidase and alpha-L-fucosidase from patients with mucolipidosis II and mucolipidosis III to concanavalin A--Sepharose 4B revealed a 2--10-fold decrease in the proportion of enzyme activities from patients with mucolipidoses II and III that adsorbed on the lectin. Neuraminidase treatment of the unadsorbed enzyme fraction did not significantly increased the proportion of enzyme activities that bound to the concanavalin A--Sepharose 4B. Characterization of acid beta-D-galactosidase from the adsorbed and unadsorbed enzyme fractions of mucolipidosis II and mucolipidosis III patients demonstrated identical apparent Km values of 0.22 mM with respect to 4-methylumbelliferyl beta-D-galactopyranoside, altered pH--activity profiles and heterogeneous isoelectric-focusing patterns. The results of this study support the suggestion of an alteration of a post-translational modification (possibly glycosylation) occurring in mucolipidosis II and mucolipidosis III common to the lysosomal hydrolases that affects the mannoserelated properties of these enzymes.     

Stimulation of the release of lysosomal and nonlysosomal granular enzymes from macrophages treated with monensin

Mouse peritoneal macrophages that had been treated with a monovalent carboxylic ionophore, monensin, selectively secreted lysosomal and nonlysosomal granular enzymes into the medium. When macrophages were incubated with 1 to 10 microM monensin, the release of beta-glucuronidase, beta-hexosaminidase and beta-galactosidase was stimulated time and does dependently. Neither the beta-glucosidase nor acid phosphatase, enzymes bound to the lysosomal membranes, however, were released by monensin. Neutral alpha-glucosidase, shown recently to be localized in nonlysosomal granules of macrophages (15), was released by monensin at concentrations lower than those required for lysosomal enzyme release. Increased release of lysosomal enzymes also took place in a manner similar to that seen with monensin-treated macrophages after treatment of macrophages with weak bases, chloroquine and ammonium chloride. Neutral alpha-glucosidase, however, was not released when chloroquine was present in concentrations that stimulated the release of lysosomal enzymes. The UDP-galactosyltransferase activity of the Golgi apparatus in the macrophages markedly decreased after treatment with low concentration of monensin.     

Demonstration of altered acidic hydrolases in fibroblasts from patients with mucolipidosis II by lectin titration.

Decreased binding by the lectins concanavalin A and wheat-germ agglutinin was found for a number of acidic hydrolases from skin fibroblasts of three unrelated patients with mucolipidosis II. This decreased binding as compared with normal controls was demonstrated by titration of hydrolase activities with increasing amounts of immobilized lectins. Neuraminidase treatment slightly improved the binding of enzymes from mucolipidosis-II patients, in contrast with the diminished binding found or hydrolases from control cell lines. The abnormality in binding by lectins of hydrolases of mucolipidosis-II patients was observed for enzymes with various degrees of intracellular deficiency as well as for enzymes with normal intracellular activities. These findings suggest a generalized alteration of fibroblast acidic hydrolase molecules in mucolipidosis II.     

Deficient phosphorylation of mannose residues of mannan in fibroblasts of patients with mucolipidoses II and III.

Incorporation of 32P from [gamma 32P]ATP into mannan could not be detected in homogenates of cultivated skin fibroblasts from patients with mucolipidosis II, and accounted for only up to 10% of normal control activity in cell lysates from patients with mucolipidosis III. Parents of patients with mucolipidosis II demonstrated 60-70% of normal control activity. On high-voltage electrophoresis, the hydrolysed mannan from reactions performed with normal cells, over the pH range 5.5-7.5, yielded a radioactive band migrating with the same mobility as mannose 6-phosphate, whereas no such product could be demonstrated in fibroblasts of patients with mucolipidosis II.     

Multiple molecular forms of lysosomal enzymes in mucolipidosis II

Summary The multiple molecular forms of selected lysosomal enzymes, as determined by analytical isoelectric focusing electrophoresis, from mucolipidosis II fibroblasts have a highly simplified pattern demonstrating a failure to undergo normal oligosaccharide processing. On the other hand, the multiple molecular forms of these same enzymes in mucolipidosis II sera and culture media are indistinguishable from controls.     

Study of lysosomal enzymes in chick embryo fibroblasts infected with microorganisms of family Halprowiaceae (Chlamydiaceae)

It is shown that infection of chick embryo fibroblasts with agents of paratrachoma and meningopneumonia Halprowiaceae (Chlamydiaceae) causes a sharp decrease of the activities of lysosomal enzymes, e.g. acidic alpha-glucosidase, beta-glucuronidase, beta-galactosidase, alpha-mannosidase, acid phosphatase, etc. The activity of cytosol enzymes (neutral alpha-glucosidase, amylo-1,6-glucosidase) does not change, however. A decrease in the activities of lysosomal enzymes in infected fibroblasts occurs some time later after inoculation and is due to a release of lysosomal enzymes from the fibroblasts into the culture medium, without loss of cell integrity. No changes in the activity of lysosomal enzymes in fibroblasts and culture medium is observed in the case of inoculation of cells with a killed agents, as well as after contact of cells with a suspension of normal chick embryo yolk sacs. The release of lysosomal enzymes from halprowiae-infected chick embryo fibroblasts probably occurs by the exocytosis.     

Cell type dependent inhibition of transport of cathepsin D in HepG2 cells and fibroblasts exposed to deoxy-manno-nojirimycin and deoxynojirimycin

The synthesis, transport and processing of lysosomal enzymes was examined in human hepatoma HepG2 cells and in human fibroblasts exposed to the Golgi alpha-mannosidase I inhibitor 1-deoxy-manno-nojirimycin. In HepG2 cells cathepsin D, beta-hexosaminidase and arylsulfatase B synthesized in the presence of 5 mM 1-deoxy-manno-nojirimycin contained exclusively endo-beta-N-acetylglucosaminidase H-cleavable oligosaccharides, indicating that alpha-mannosidase I had been inhibited efficiently. The proteolytic processing of intracellularly retained cathepsin D was retarded and the fraction of secreted cathepsin D was increased two-fold. In fibroblasts neither segregation nor maturation of cathepsin D were affected by 1-deoxy-manno-nojirimycin in spite of the inhibition of oligosaccharide processing. In the presence of the glucosidase I inhibitor 1-deoxynojirimycin, the precursor of cathepsin D (larger by about 1 kDa than the secreted form) accumulated transiently in light membranes in HepG2 cells. Release from the site of accumulation was accompanied by a decrease in size by about 1 kDa. This change was attributed to the removal of glucose residues. In fibroblasts the transient accumulation of larger precursors in the presence of 1-deoxynojirimycin was more pronounced than in HepG2 cells. The differential effects of alpha-mannosidase I and glucosidase I inhibitors on the transport of cathepsin D in HepG2 cells and fibroblasts may indicate that different intermediates in the biosynthetic pathway of asparagine-linked oligosaccharides participate in the transport of lysosomal enzymes in the two cell types.     

Heterogeneity of N-acetylglucosamine 1-phosphotransferase within mucolipidosis III

The primary defect responsible for mucolipidosis III is a deficiency of UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine 1-phosphotransferase activity (GlcNAc phosphotransferase). Genetic complementation analysis of cultured fibroblasts derived from 12 patients with mucolipidosis III identified complementation groups A, B, and C (Honey, N. K., Mueller, O. T., Little, L. E., Miller, A. L., and Shows, T. B. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7420-7424). The GlcNAc phosphotransferase activity present in the cell lines comprising the complementation groups was characterized with respect to endogenous substrates and two exogenous acceptors, alpha-methyl-D-mannoside and high mannose glycopeptides. All group C cell lines and one group A cell line were found to have normal GlcNAc phosphotransferase activity levels at 37 degrees C when screened with these exogenous acceptors. The enzyme activity in group A cell lines was within normal range when assayed at 23 degrees C. Inhibition of the phosphorylation of alpha-methyl-D-mannoside in the presence of increasing amounts of endogenous substrate N-acetyl-beta-D-hexosaminidase B was demonstrated in normal cell lines at 23 and 37 degrees C and in group A cells at 23 degrees C. However, group C cell lines did not show any inhibition at either temperature. This suggests that the alteration of the GlcNAc phosphotransferase from individuals in group C affects the recognition site for the protein portion of lysosomal enzymes, whereas group A individuals have mutations which result in a temperature-sensitive enzyme.     

Mucolipidosis III beta-N-acetyl-D-hexosaminidase A. Purification and properties

Mucolipidosis III acid hydrolases possess an altered carbohydrate recognition marker needed for their lysosomal localization. As a result of this alteration, a portion of these enzymes is secreted from the cell to the extracellular spaces. The structural changes that may have occurred to one of these secreted enzymes, beta-N-acetyl-d-hexosaminidase A (EC 3.2.1.52) were investigated. Normal and mucolipidosis III urinary beta-N-acetyl-d-hexosaminidase A were purified to apparent homogeneity by using affinity [Sepharose-2-acetamido-N-(epsilon-aminocaproyl)-2-deoxy-beta- d-glucopyranosylamine] and ion-exchange (DEAE- and CM-cellulose) chromatography. Sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis showed that both enzymes had similar subunit patterns consisting of apparent mol.wts. of 68000, 60000-58000, 55000 and 29000. Differences, however, were noted in the relative proportions of the protein bands where the normal urinary beta-N-acetyl-d-hexosaminidase A contained predominantly the smaller subunits, whereas the mucolipidosis III enzyme had a predominance of the larger subunits. The binding of mucolipidosis III beta-N-acetyl-d-hexosaminidase A to Ricinus communis lectin and concanavalin A with and without endo-beta-N-acetyl-d-glucosaminidase H treatment indicated that the mutation leads to a modification of a portion of the normally occurring high-mannose-type oligosaccharide units to the complex-type. This was further supported by carbohydrate compositional analysis, which revealed a mannose/galactose ratio of 2.1 for the mucolipidosis III beta-N-acetyl-d-hexosaminidase A compared with a ratio of 3.5 for the normal enzyme. Our results indicate that as a result of their inability to be properly localized to the lysosome the majority of the mucolipidosis III lysosomal hydrolase high-mannose oligosaccharide units are further processed to the complex-type before secretion of predominantly higher-molecular-weight subunits from the cell.