A specific acid alpha-glucosidase in lamellar bodies of the human lung

In the present investigation, we have demonstrated that three lysosomal-type hydrolases, alpha-glucosidase, alpha-mannosidase and a phosphatase, are present in lamellar bodies isolated from adult human lung. The hydrolase activities that were studied, all showed an acidic pH optimum, which is characteristic for lysosomal enzymes. The properties of acid alpha-glucosidase in the lamellar body fraction and that in the lysosome-enriched fraction were compared. Using specific antibodies against lysosomal alpha-glucosidase from human placenta, two alpha-glucosidases could be distinguished in the lamellar body fraction: one with a high affinity to the antibodies as found in the lysosome-enriched fraction and another with a much lower affinity. Both forms showed an acidic pH optimum. The same heterogeneity of alpha-glucosidase in the lamellar body fraction could be observed using immobilized concanavalin A. The lectin was able to precipitate nearly all alpha-glucosidase activity of the lysosome-enriched fraction. In contrast, 30% of the alpha-glucosidase activity in the lamellar body fraction was not precipitable. Furthermore, the lamellar body alpha-glucosidase with the low antibody affinity could not be bound to concanavalin A. The results suggest that lamellar bodies contain at least two acid alpha-glucosidases: one similar to the lung lysosomal alpha-glucosidase, and another lamellar body-specific isoenzyme with a different immunoreactivity and lectin affinity. The lamellar body-specific alpha-glucosidase should prove useful as a lamellar body-specific marker enzyme.     

An improved procedure for the isolation of lamellar bodies from human lung. Lamellar bodies free of lysosomes contain a spectrum of lysosomal-type hydrolases

We have recently shown that lamellar body fractions purified from human lung contain a distinct acid alpha-glucosidase distinguishable from lysosomal acid alpha-glucosidase in that it does not cross-react with antibodies raised against the lysosomal enzyme and does not bind to concanavalin A (De Vries, A.C.J., Schram, A.W., Tager, J.M., Batenburg, J.J. and Van Golde, L.M.G. (1985) Biochim. Biophys. Acta 837, 230-238). In order to study the relationship between the non-concanavalin A-binding alpha-glucosidase and lamellar bodies more closely a method was developed for the further purification of the organelles. A purified lamellar body preparation isolated from human lung homogenate by discontinuous sucrose density centrifugation was subjected to gel filtration with Sepharose 4B followed by Percoll density gradient centrifugation, which yielded a lamellar body preparation with a phospholipid phosphorus/protein ratio of 12.57 +/- 0.38 (mumol/mg) (n = 3) as compared to a ratio of 3.34 +/- 0.16 (mumol/mg) (n = 3) in the sucrose density gradient preparation. Concomitantly there was a 3.3 +/- 0.1 (n = 3)-fold enrichment in the content of total acid alpha-glucosidase and a 3.2 +/- 0.1 (n = 3) -fold enrichment of non-concanavalin A-binding acid alpha-glucosidase. The new purification method removes adhering proteins without changing the phospholipid composition. During the successive purification steps the concanavalin A-sensitive and -insensitive alpha-glucosidases remained fully lamellar body fraction associated. Differences between a lysosome-enriched fraction and a lamellar body preparation at varying stages of purification with respect to the ratio between soluble acid hydrolases and the membrane-associated lysosomal enzyme glucocerebrosidase indicate that the purified lamellar bodies were not contaminated with lysosomes. The absence of lysosomes in the purified lamellar body fraction was confirmed by experiments with the weak base glycyl-L-phenylalanine-beta-naphthylamide, which is an artificial substrate for the lysosomal enzyme cathepsin C and brings about lysis of lysosomes. Morphological examination by electron microscopy endorses the absence of contaminating vesicles and organelles and showed a structural integrity of the lamellar bodies in the final preparation. The improved isolation procedure strongly suggests that the concanavalin A-insensitive acid alpha-glucosidase is endogenous to lamellar bodies and supports our earlier idea that it can be used as a lamellar body-specific marker enzyme. In addition, the experiments show that lamellar bodies free of lysosomes contain a spectrum of lysosomal-type enzymes.     

Role of alpha-glucosidase in fetal lung maturation

The role of lysosomal enzyme acid alpha-glucosidase in fetal lung development was investigated with the aid of a specific inhibitor, the pseudosaccharide acarbose. The drug was added to a Waymouth culture medium of fetal rat lung explants cultivated for 48 h from gestational stage 19.5 days, an in vitro system previously shown to allow morphological and biochemical maturation of alveolar epithelium. Glycogenolysis was reduced by 40% as compared with tissue cultivated on control medium, which means that alpha-glucosidase could account for as much as 40% of fetal lung glycogenolysis, the remaining 60% being presumably achieved by cytosolic phosphorylase and by a microsomal neutral alpha-glucosidase. By the same time, the increase of phospholipids of surfactant fraction extracted from cultivated explants was partially inhibited: total and saturated phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylinositol were about 30-40% lower than in lungs cultivated on control medium. It should be emphasized that DNA concentration and increases in non-surfactant phospholipids were unchanged by the drug. alpha-Glucosidase activity was evidenced in the lysosomal fraction, in the microsomal fraction and, although in lower amounts, in the surfactant fraction extracted from term fetal lung. The results suggest that lysosomal alpha-glucosidase plays a major role in lung maturation and could facilitate glycogenolysis for the specific use of glycogen stores in providing substrates for surfactant phospholipid biosynthesis.     

Use of immobilized antibodies in investigating acid alpha-glucosidase in urine in relation to Pompe's disease.

(1) A simple method is described for the isolation of the lysosomal enzyme, acid alpha-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) from normal human liver. Antibodies raised against the purified enzyme were immobilized by covalent coupling to Sepharose 4B. (2) Acid alpha-glucosidase can be quantitatively removed from normal urine by incubating with an excess of immobilized antibody. With p-nitrophenyl-alpha-glucoside as substrate, acid alpha-glucosidase accounts for 91 +/- 3% of the total alpha-glucosidase activity at pH 4.0 IN Normal urine. (3) In urine from a patient with the infantile form of Pompe's disease ('acid maltase deficiency'), no alpha-glucosidase activity could be removed by the immobilized antibody, in agreement with the fact that acid alpha-glucosidase is absent in these patients. (4) In urine from patients with the late-onset form of Pompe's disease, 46 +/- 11% of the alpha-glucosidase activity at pH 4.0 can be removed by incubation with immobilized antibodies, indicating that residual acid alpha-glucosidase activity is present in urine of these patients. The residual acid alpha-glucosidase activity amounts to about 5% of that in the urine of control persons. (5) If acid alpha-glucosidase is adsorbed to immobilized antibodies, the activity can still be measured with p-nitrophenyl-alpha-glucoside as substrate. The Km for p-nitrophenyl-alpha-glucoside is not significantly changed by adsorbing purified acid alpha-glucosidase to immobilized antibodies. (6) The properties of acid alpha-glucosidase from urine of patients with late-onset Pompe's disease were compared with those of acid alpha-glucosidase from normal urine, both adsorbed to immobilized antiserum. The pH-activity profile of the enzyme from urine of patients with late-onset Pompe's disease can not be distinguished from that of the normal urinary enzyme. The Km for p-nitro-phenyl-alpha-glucoside of the two enzymes is identical, both at pH 4 and 3. The titration curves of the two enzymes with immobilized antibodies are identical.     

Secretion of a precursor form of lysosomal alpha-glucosidase from the brush border of human kidney proximal tubule cells

We have shown previously (R.P.J. Oude Elferink, E.M. Brouwer-Kelder, I. Surya, A. Strijland, M. Kroos, A.J.J. Reuser, J.M. Tager, Eur. J. Biochem. 139, 489-495 (1984)) that human urine contains considerable amounts of a precursor form of lysosomal alpha-glucosidase (about 50% of the total alpha-glucosidase activity present). We have now purified alpha-glucosidase from human kidney. Only about 5 to 10% of the total lysosomal alpha-glucosidase present in kidney comprises the precursor form of the enzyme. By means of immunocytochemistry using monoclonal antibodies, the precursor of alpha-glucosidase was detected in the brush border of the proximal tubule cells. Taking into account the amount of precursor alpha-glucosidase excreted daily into the urine and the amount present in the kidneys, we conclude that extensive secretion of precursor alpha-glucosidase occurs from the brush border of the proximal tubules.     

Immunocytochemical localization of lysosomal acid phosphatase in normal and "I-cell" fibroblasts

This study represents the first example of immunological localization of lysosomal acid phosphatase. The intracellular localization of lysosomal acid phosphatase was investigated with immunocytochemical methods at the light and electron microscopical level in cultured fibroblasts obtained from normal subjects and from a patient with I-cell disease. Double-labeling studies using fluorescence microscopy showed that acid phosphatase is present in the same organelles as other hydrolases. At the electron microscopic level in control fibroblasts acid phosphatase was found in the rough endoplasmic reticulum, lysosomes, at the plasma membrane, in vesicles just below the plasma membrane and in multivesicular bodies. This localization was comparable with that of other lysosomal enzymes tested (acid alpha-glucosidase, N-acetyl-beta-hexosaminidase, beta-galactosidase). Acid phosphatase labeling was mainly found in association with the lysosomal membrane and with membranous material present within the lysosome. In I-cell fibroblasts the label was present in the same subcellular organelles but always associated with membranous structures. We suggest that the association of acid phosphatase with membranes might explain the normal enzyme activity found in I-cell fibroblasts.     

The cation-independent mannose 6-phosphate receptor is not involved in the polarized secretion of lysosomal alpha-glucosidase from Caco-2 cells.

In the human adenocarcinoma cell line Caco-2 a substantial amount of a precursor form of the lysosomal enzyme alpha-glucosidase is not segregated into lysosomes, but instead secreted from the apical membrane. In this study we addressed the question whether this process is mediated by mannose 6-phosphate receptors. The subcellular distribution of the cation-independent mannose 6-phosphate receptor was studied by means of electron microscopic immunocytochemistry. The bulk of label was found in the perinuclear region in electron-lucent and dense vesicles, some of the latter bearing a coat. Receptor-containing dense vesicles were also found throughout the cytoplasm. In the apical part of the cells, label for the receptor was present over the surrounding membrane and the interior vesicles of multivesicular bodies, but not over lysosomes. Label on the plasma membrane was mainly restricted to the apical domain. In contrast to alpha-glucosidase, the secreted forms of the lysosomal enzymes cathepsin D, beta-hexosaminidase and beta-glucuronidase are mainly found in the basolateral medium. Enzyme activity measurements and immunoprecipitation of metabolically labeled cells showed that incubation with NH4Cl leads to an enhanced secretion of these enzymes into the basolateral medium, but has no effect on the basolateral secretion of alpha-glucosidase. In addition, NH4Cl caused a minor decrease in the secretion of these enzymes from the apical side and had little or no effect on the secretion of alpha-glucosidase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Primary structure and processing of lysosomal alpha-glucosidase; homology with the intestinal sucrase-isomaltase complex.

Lysosomal alpha-glucosidase (acid maltase) is essential for degradation of glycogen in lysosomes. Enzyme deficiency results in glycogenosis type II. The amino acid sequence of the entire enzyme was derived from the nucleotide sequence of cloned cDNA. The cDNA comprises 3636 nt, and hybridizes with a messenger RNA of approximately 3.6 kb, which is absent in fibroblasts of two patients with glycogenosis type II. The encoded protein has a molecular mass of 104.645 kd and starts with a signal peptide. Sites of proteolytic processing are established by identification of N-terminal amino acid sequences of the 110-kd precursor, and the 76-kd and 70-kd mature forms of the enzyme encoded by the cDNA. Interestingly, both amino-terminal and carboxy-terminal processing occurs. Sites of sugar-chain attachment are proposed. A remarkable homology is observed between this soluble lysosomal alpha-glucosidase and the membrane-bound intestinal brush border sucrase-isomaltase enzyme complex. It is proposed that these enzymes are derived from the same ancestral gene. Around the putative active site of sucrase and isomaltase, 10 out of 13 amino acids are identical to the corresponding amino acids of lysosomal alpha-glucosidase. This strongly suggests that the aspartic acid residue at this position is essential for catalytic function of lysosomal alpha-glucosidase.     

Adult forms of glycogenosis type II. A defect in an early stage of acid alpha-glucosidase realization

The activity of acid alpha-glucosidase in cultured fibroblasts from adult patients with the lysosomal storage disease glycogenosis type II is only 10% of normal. A normal activity per molecule is found for the mature as well as for the precursor form of acid alpha-glucosidase in adult mutant fibroblasts. Excessive lysosomal breakdown of mature enzyme purified from mutant fibroblasts and taken up by acceptor cells does not occur. However, the NH4Cl-stimulated secretion of a precursor form of acid alpha-glucosidase by adult mutant fibroblasts is markedly reduced. The results are indicative of a defect during the production of acid alpha-glucosidase.     

Change in the alpha-glucosidase activity of cattle muscle tissue during slow autolysis]

Changes in the alpha-glucosidase activity of the cattle muscular tissue (oxen eye muscle of loin) were evaluated during storage at 2 degrees. Under these conditions both lysosomal and extralysosomal alpha-glucosidase activities underwent no significant changes during a long period of time (12 days). Activity of lysosome-bound alpha-glucosidase was about 10% of total enzyme activity in the homogenate; the remaining part of alpha-glucosidase was contained outside lysosomes.     

Purification and characterization of lysosomal alpha-glucosidase secreted by eukaryote Tetrahymena

A large amount of lysosomal acid hydrolases was released into the medium by Tetrahymena pyriformis strain W during growth. An extracellular lysosomal acid alpha-glucosidase has been purified 500-fold with a 41% yield to homogeneity, as judged by polyacrylamide gel electrophoresis. It was found to be a glycoprotein and to consist of a single 110,000-dalton polypeptide chain. The carbohydrate content of the alpha-glucosidase was equivalent to 2.8% of the total protein content, and the oligosaccharide moiety was composed of mannose and N-acetylglucosamine in a molar ratio of 6.7:2. The optimal pHs for hydrolysis of maltose and p-nitrophenyl-alpha-glucopyranoside, maltose, isomaltose, and glycogen were 1.1 mM, 2.5 mM, 33.0 mM, and 18.5 mg/ml, respectively. This purified enzyme appears to have alpha-1,6-glucosidase as well as alpha-1,4-glucosidase activity. Turanose has a noncompetitive inhibitory effect on the hydrolysis of maltose. The antibody raised against Tetrahymena acid alpha-glucosidase inhibited the hydrolysis of all substrates tested. These properties of Tetrahymena acid alpha-glucosidase were found to be similar to those of the human liver lysosomal alpha-glucosidase.     

The subcellular localization of soluble and membrane-bound lysosomal enzymes in I-cell fibroblasts: a comparative immunocytochemical study

Using electron microscopic immunocytochemistry with gold probes, we have studied the localization of acid alpha-glucosidase, N-acetyl-beta-hexosaminidase and beta-glucocerebrosidase in cultured skin fibroblasts from control subjects and patients with mucolipidosis II (I-cell disease). In control fibroblasts, a random distribution of acid alpha-glucosidase and N-acetyl-beta-hexosaminidase within the lysosomes was observed, whereas beta-glucocerebrosidase was found to be localized on or near the lysosomal membrane. The observations confirm the soluble character of acid alpha-glucosidase and N-acetyl-beta-hexosaminidase and the membrane-bound character of beta-glucocerebrosidase. In I-cell fibroblasts an abnormal localization of the two soluble enzymes was found. Labeling in lysosomes was very weak, but instead, small 'presumptive' vesicles containing both enzymes were detected throughout the cytoplasm and close to the plasma membrane. These vesicles could be involved in the secretion of the two enzymes. In contrast, a normal membrane-bound lysosomal localization was observed for beta-glucocerebrosidase. It is concluded that the intracellular transport of beta-glucocerebrosidase to the lysosomes can occur even when the mannose-6-phosphate recognition system is defective. This explains the normal activity of beta-glucocerebrosidase in I-cells in contrast to the deficiency of most other lysosomal enzymes.     

Stabilising normal and mis-sense variant alpha-glucosidase

alpha-Glucosidase (EC 3.2.1.3) is a lysosomal enzyme that hydrolyses alpha-1,4- and alpha-1,6-linkages of glycogen to produce free glucose. A deficiency in alpha-glucosidase activity results in glycogen storage disorder type II (GSD II), also called Pompe disease. Here, d-glucose was shown to be a competitive inhibitor of alpha-glucosidase and when added to culture medium at 6.0 g/L increased the production of this protein by CHO-K1 expression cells and stabilised the enzyme activity. D-Glucose also prevented alpha-glucosidase aggregation/precipitation and increased protein yield in a modified purification scheme. In fibroblast cells, from adult-onset GSD II patients, D-glucose increased the residual level of alpha-glucosidase activity, suggesting that a structural analogue of d-glucose may be used for enzyme enhancement therapy.     

Intracellular transport of acid alpha-glucosidase in human fibroblasts: evidence for involvement of phosphomannosyl receptor-independent system

Intracellular transport of two lysosomal enzymes, acid alpha-glucosidase and beta-hexosaminidase, was analyzed in human fibroblasts. The precursors of beta-hexosaminidase in normal fibroblasts were released from the membrane fraction by treatment with mannose 6-phosphate, but the precursor of alpha-glucosidase was not. Percoll density gradient centrifugation revealed a normal amount of acid alpha-glucosidase activity in heavy lysosomes in I-cell disease fibroblasts despite impaired maturation and defective phosphorylation, and beta-hexosaminidase activity was markedly reduced in lysosomes. It was concluded that the membrane-bound precursor of acid alpha-glucosidase is transported to lysosomes by a phosphomannosyl receptor-independent system although the enzyme lacks the recognition marker for the phosphomannosyl receptor and processing of an intermediate form to mature forms does not occur in this disease.     

Defects in synthesis, phosphorylation, and maturation of acid alpha-glucosidase in glycogenosis type II

Glycogenosis type II is an inherited lysosomal storage disease with acid alpha-glucosidase deficiency as the primary defect. Using cultured skin fibroblasts, we have studied the biosynthesis of acid alpha-glucosidase in clinically different forms of this disease. Three unrelated patients were identified (one with an infantile, one with a juvenile, and one with an adult form of the disease) producing normal quantities of the 110-kDa precursor form of acid alpha-glucosidase. However, post-translational modification to mature 76-kDa enzyme protein was either completely deficient or extremely inefficient. No abnormalities were observed in glycosylation of the mutant precursors, as measured by the incorporation of [3H]mannose, but phosphorylation was only detectable for the precursor synthesized by fibroblasts from the juvenile patient. In three other patients (one with a juvenile and two with adult forms of glycogenosis type II) apparently reduced synthesis of precursor protein was observed, but the processing to mature enzyme seemed to be undisturbed. Finally, neither precursor nor mature forms of acid alpha-glucosidase were detectable in one particular case of infantile glycogenosis type II. The studies reveal an unexpected degree of genetic heterogeneity in this disease and identify various mutants which could be of importance to further elucidate the biosynthetic events during lysosomal enzyme formation.     

Human lysosomal alpha-glucosidase. Characterization of the catalytic site.

The substrate analogue conduritol B epoxide (CBE) is demonstrated to be an active site-directed inhibitor of human lysosomal alpha-glucosidase. A competitive mode of inhibition is obtained with glycogen as natural and 4-methylumbelliferyl-alpha-D-glucopyranoside as artificial substrate. The inactivation of the enzyme is time and concentration dependent and results in the covalent binding of CBE. Catalytic activity is required for binding to occur. CBE-labeled peptides containing the catalytic residue of lysosomal alpha-glucosidase were isolated and identified by microsequencing and amino acid analysis. The peptides appeared to originate from a protein domain which is highly conserved among alpha-amylases, maltase, glucoamylases, and transglucanosylases. Based on the sequence similarity and the mechanism of CBE binding, Asp-518 is predicted to be the essential carboxylate in the active site of lysosomal alpha-glucosidase. The functional importance of Asp-518 and other residues around the catalytic site was studied by expression of in vitro mutagenized alpha-glucosidase cDNA in transiently transfected COS cells. Substitution of Asp-513 by Glu-513 is shown to interfere with the posttranslational modification and the intracellular transport of the alpha-glucosidase precursor. The residues Trp-516 and Asp-518 are demonstrated to be critical for catalytic function.     

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.     

Identification of a point mutation in the human lysosomal alpha-glucosidase gene causing infantile glycogenosis type II

Two patients in a consanguineous Indian family with infantile glycogenosis type II were found to have a G to A transition in exon 11 of the human lysosomal alpha-glucosidase gene. Both patients were homozygous and both parents were heterozygous for the mutant allele. The mutation causes a Glu to Lys substitution at amino acid position 521, just three amino acids downstream from the catalytic site at Asp-518. The mutation was introduced in wild type lysosomal alpha-glucosidase cDNA and the mutant construct was expressed in vitro and in vivo. The Glu to Lys substitution is proven to account for the abnormal physical properties of the patients lysosomal alpha-glucosidase precursor and to prevent the formation of catalytically active enzyme. In homozygous form it leads to the severe infantile phenotype of glycogenosis type II.     

Biochemical microanalysis of alpha-glucosidase activity in preneoplastic and neoplastic hepatic lesions induced in rats by N-nitrosomorpholine

Preneoplastic and neoplastic hepatic lesions were induced in male Sprague-Dawley rats by oral administration of N-nitrosomorpholine (NNM) for 7 weeks at a concentration of 200 mg/l of drinking-water (stop model). Using a laser dissection technique and biochemical microanalysis, the activity of the lysosomal enzyme alpha-glucosidase was measured in glycogen storage foci emerging early, and in mixed or basophilic cell populations (foci and carcinomas) appearing later during hepatocarcinogenesis. In the liver tissue of normal appearance in both untreated controls and NNM-treated animals a slight gradient of alpha-glucosidase activity was observed leading from relatively high activities in zone 1 to lower activities in zone 3 of the liver lobule. In preneoplastic glycogen storage foci a considerable relative reduction in alpha-glucosidase activity was detected, suggesting that a decrease in the hydrolytic glycogen degradation contributes to the disturbance in phosphorylytic glycogen breakdown observed earlier in the majority of the glycogenotic foci. In contrast with glycogen storage foci, mixed and basophilic cell foci and particularly hepatocellular carcinomas showed a marked increase in alpha-glucosidase activity compared with that of normal liver tissue. The gradual enhancement in enzyme activity appeared to be closely related to the reduction in glycogen initially stored in excess during the later stages of hepatocarcinogenesis. The results support the concept that a fundamental shift in carbohydrate metabolism is characteristic of neoplastic transformation of hepatocytes.     

Synthesis and intracellular localization of chick acid alpha-glucosidase in chick erythrocyte-human fibroblast heterokaryons. A model system for the study of lysosomal enzyme synthesis

The synthesis and localization of chick acid alpha-glucosidase has been studied in chick erythrocyte-human fibroblast heterokaryons. Monospecific antibodies raised against purified chick liver acid alpha-glucosidase were used. It was found that the acid alpha-glucosidase in the heterokaryons is of chick origin, and is localized in the same lysosomes as the human lysosomal enzymes. It is concluded that chick erythrocyte-human fibroblast heterokaryons provide a useful model system for the study of lysosomal enzyme synthesis and routing.