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Normal human spleen contains two forms of membrane-bound beta-glucosidase, distinguishable by their thermostability and kinetic properties. The spleen from a patient with adult Gaucher's disease was deficient in the major, thermolabile, form of the enzyme.
The bglu1 cDNA for a beta-glucosidase cloned from rice (Oryza sativa L.) seedlings was expressed as a soluble and active protein in Escherichia coli and designated BGlu1. This enzyme hydrolysed beta-1,4-linked oligosaccharides with increasing catalytic efficiency (kcat/Km) values as the DP (degree of polymerization) increased from 2 to 6. In contrast, hydrolysis of beta-1,3-linked oligosaccharides decreased from DP 2 to 3, and polymers with a DP greater than 3 were not hydrolysed. The enzyme also hydrolysed p -nitrophenyl beta-D-glycosides and some natural glucosides but with lower catalytic efficiency than beta-linked oligosaccharides. Pyridoxine 5'-O-beta-D-glucoside was the most efficiently hydrolysed natural glycoside tested. BGlu1 also had high transglucosylation activity towards pyridoxine, producing pyridoxine 5'-O-beta-D-glucopyranoside in the presence of the glucose donor p-nitrophenyl beta-D-glucoside.
To enhance the delivery of rhGAA (recombinant GAA, where GAA stands for acid alpha-glucosidase) to the affected muscles in Pompe disease, the carbohydrate moieties on the enzyme were remodelled to exhibit a high affinity ligand for the CI-MPR (cation-independent M6P receptor, where M6P stands for mannose 6-phosphate). This was achieved by chemically conjugating on to rhGAA, a synthetic oligosaccharide ligand bearing M6P residues in the optimal configuration for binding the receptor. The carbonyl chemistry used resulted in the conjugation of approx. six synthetic ligands on to each enzyme. The resulting modified enzyme [neo-rhGAA (modified recombinant human GAA harbouring synthetic oligosaccharide ligands)] displayed near-normal specific activity and significantly increased affinity for the CI-MPR. However, binding to the mannose receptor was unaffected despite the introduction of additional mannose residues in neo-rhGAA. Uptake studies using L6 myoblasts showed neo-rhGAA was internalized approx. 20-fold more efficiently than the unmodified enzyme. Administration of neo-rhGAA into Pompe mice also resulted in greater clearance of glycogen from all the affected muscles when compared with the unmodified rhGAA. Comparable reductions in tissue glycogen levels in the Pompe mice were realized using an approx. 8-fold lower dose of neo-rhGAA in the heart and diaphragm and an approx. 4-fold lower dose in the skeletal muscles. Treatment of older Pompe mice, which are more refractory to enzyme therapy, with 40 mg/kg neo-rhGAA resulted in near-complete clearance of glycogen from all the affected muscles as opposed to only partial correction with the unmodified rhGAA. These results demonstrate that remodelling the carbohydrate of rhGAA to improve its affinity for the CI-MPR represents a feasible approach to enhance the efficacy of enzyme replacement therapy for Pompe disease.
We studied the effect of the plant alkaloid castanospermine on the biosynthesis and secretion of human hepatoma glycoproteins. The HepG-2 cells, grown in the presence or absence of the alkaloid, were labelled with [2-3H]mannose and then the labelled glycopeptides were prepared by Pronase digestion. This material was analysed by gel filtration on Bio-Gel P-4 before and after treatment with endo-beta-N-acetylglucosaminidase H. Castanospermine caused an accumulation of high-mannose oligosaccharides, by 70-75% over control. The major accumulated product, which could also be labelled with [3H]galactose and was only partially susceptible to alpha-mannosidase digestion, was identified by h.p.l.c. as a Glc3Man9GlcNAc. Thus the alkaloid inhibits glucosidase I in the human hepatoma cells. Analysis of total glycoproteins secreted by the cells into the medium revealed the presence of only complex oligosaccharides in both control and treated cultures, and the amount of the oligosaccharides labelled with radioactive mannose, galactose or N-acetylmannosamine, secreted by treated cells, was decreased by about 60%. The rate of secretion of total protein labelled with [35S]methionine and precipitated from the medium with trichloroacetic acid was inhibited by up to 40% in the presence of castanospermine. Pulse-chase studies utilizing [35S]methionine labelling were performed to study the effect of the alkaloid on secretion of individual plasma proteins. Immunoprecipitation at different chase times with monospecific antisera showed that castanospermine markedly decreased the secretion rates of alpha 1-antitrypsin, caeruloplasmin and, to a lesser extent, that of antithrombin-III. Secretions of apolipoprotein E, a glycoprotein containing only O-linked oligosaccharide(s), and albumin, a non-glycosylated protein, were not affected by the drug. It is suggested that castanospermine inhibits secretion of at least some glycoproteins containing N-linked oligosaccharides, owing to the inhibition of oligosaccharide processing.
A transient-kinetic study of beta-glucosidase from soyabean cell walls was performed with the use of a stopped-flow apparatus. The progress curve of the reaction exhibits a 'slow' burst of about 1 s before the steady state is reached. In the time scale investigated this burst may be accounted for by only one exponential, whose time constant varies with the substrate concentration. As this concentration is increased the value of the time constant increases at first, then decreases. Premixing the enzyme with glucose, the last product of the reaction sequence, reverses the 'slow' burst into a 'slow' lag. Taken together, these results are only compatible with a model that involves the existence of a 'slow' conformational transition of the enzyme.
A 59-year-old Japanese farmer with asymptomatic fasting hypoglycemia and with exaggerated hypoglycemic episodes induced by insulin and oral hypoglycemic agent administered for his postprandial hyperglycemia was diagnosed as glycogen storage disease type I. This diagnosis was suggested by unresponsiveness of blood glucose level to glucagon and confirmed by 13% normal level of glucose 6-phosphatase activity in liver biopsy specimen and by the presence of PAS positive amylase digestable glycogen in liver specimen. This case was associated with an incomplete type of Fanconi syndrome characterized by hyperphosphaturic hypophosphatemia, partial aminoaciduria, mild proteinuria and hyperuricosuric normouricemia in spite of the lactic acidemia due to glycogen storage disease type I. The etiology for the absence of hypoglycemia and other typical manifestations of glycogen storage disease type I was studied. The glucose production from glycogen by lysosomal alpha 1,4-glucosidase especially at prolonged fasting and the presence of postprandial hyperglycemia by insulin deficiency are regarded as responsible for keeping this patient free from typical manifestations of glycogen storage disease type I.
Pompe's disease is caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). GAA is synthesized as a 110-kDa precursor containing N-linked carbohydrates modified with mannose 6-phosphate groups. Following trafficking to the lysosome, presumably via the mannose 6-phosphate receptor, the 110-kDa precursor undergoes a series of complex proteolytic and N-glycan processing events, yielding major species of 76 and 70 kDa. During a detailed characterization of human placental and recombinant human GAA, we found that the peptides released during proteolytic processing remained tightly associated with the major species. The 76-kDa form (amino acids (aa) 122-782) of GAA is associated with peptides of 3.9 kDa (aa 78-113) and 19.4 kDa (aa 792-952). The 70-kDa form (aa 204-782) contains the 3.9- and 19.4-kDa peptide species as well as a 10.3-kDa species (aa 122-199). A similar set of proteolytic fragments has been identified in hamster GAA, suggesting that the multicomponent character is a general phenomenon. Rabbit anti-peptide antibodies have been generated against sequences in the proteolytic fragments and used to demonstrate the time course of uptake and processing of the recombinant GAA precursor in Pompe's disease fibroblasts. The results indicate that the observed fragments are produced intracellularly in the lysosome and not as a result of nonspecific proteolysis during purification. These data demonstrate that the mature forms of GAA characterized by polypeptides of 76 or 70 kDa are in fact larger molecular mass multicomponent enzyme complexes.
A beta-glucosidase was isolated from Candida guilliermondii, a yeast capable of growth on cellobiose. The enzyme was partially purified by treatment with polyethyleneimine and ammonium sulfate precipitation. Further purification was achieved by affinity chromatography using a Sepharose 4B matrix to which oxidized salicin was coupled through adipic dihydrazide. The final product was a 12.5-fold purification of the crude extract with a recovery of 27% of the initial enzyme activity. Polyacrylamide disc electrophoresis of the purified enzyme gave a single band. A km of 1.25 x 10(-4)M was obtained using p-nitrophenyl beta-D-glucopyranoside as the substrate. The optimum pH for enzyme activity was 6.8. Maximum activity was observed at temperature of 37 degrees C. Enzyme activity was completely inhibited by Hg++, Pb++, and Zn++ ions. The molecular weight of the enzyme is 48,000 as estimated by sucrose density gradient centrifugation.
Glycogen storage disease type II (GSD-II) is a lethal, autosomal recessive metabolic myopathy caused by a lack of acid-alpha-glucosidase (GAA) activity in the cardiac and skeletal muscles. Absence of adequate intralysosomal GAA activity results in massive amounts of glycogen accumulation in multiple muscle groups, resulting in morbidity and mortality secondary to respiratory embarrassment and/or cardiomyopathy. In a mouse model of GSD-II, we demonstrate that infection of the murine liver with a modified adenovirus (Ad) vector encoding human GAA (hGAA) resulted in long-term persistence of the vector in liver tissues for at least 6 months. Despite both a rapid shutdown of hGAA mRNA expression from the vector, as well as the elicitation of anti-hGAA antibody responses (hGAA is a foreign antigen in this model), the hGAA secreted by the liver was taken up by all muscle groups analyzed and, remarkably, persisted in them for at least 6 months. The persistence of the protein also correlated with long-term correction of pathologic intramuscular glycogen accumulations in all muscle groups tested, but most notably the cardiac tissues, which demonstrated a significantly decreased glycogen content for at least 190 days after a single vector injection. The results suggest that gene therapy strategies may have the potential to significantly improve the clinical course for GSD-II patients.
Genetic deficiency of acid alpha glucosidase (GAA) results in glycogen storage disease type II. To study the disease at the molecular level, we have previously isolated and sequenced the cDNA (3.6 kb) for human GAA. We have now isolated the structural gene, mapped and determined the position and size of the exons containing the entire cDNA, and determined the sequence of the intron-exon junctions. The structural gene is approximately 28 kb and contains 20 exons. The first exon has only 5' untranslated sequence and is separated by an approximately 2.7-kb intron from the second exon that contains the initiation ATG. The second as well as the last exon are quite large (578 and 607 bp) with the remainder of the exons ranging from 85-187 bp. Additionally, two new restriction fragment length (RFLPs) for Xba I and Stu I are described at the GAA locus, one of which is most 5' of the eight RFLPs we have previously described.
Myoblasts have properties that make them suitable vehicles for gene replacement therapy, and lysosomal storage diseases are attractive targets for such therapy. Type II Glycogen Storage Disease, a deficiency of acid alpha-glucosidase (GAA), results in the abnormal accumulation of glycogen in skeletal and cardiac muscle lysosomes. The varied manifestations of the enzyme deficiency in affected patient are ultimately lethal. We used a retroviral vector carrying the cDNA encoding for GAA to replace the enzyme in deficient myoblasts and fibroblasts and analyzed the properties of the transduced cells. The transferred gene was efficiently expressed, and the de novo-synthesized enzyme reached lysosomes where it digested glycogen. In enzyme-deficient myoblasts after transduction, enzyme activity rose to more than 30-fold higher than in normal myoblasts and increased about five-fold more when the cells were allowed to differentiate into myotubes. The transduced cells secreted GAA that was endocytosed via the mannose-6-phosphate receptor into lysosomes of deficient cells and digested glycogen. Moreover, the transduced myoblasts were able to fuse with and provide enzyme for GAA-deficient fusion partners. Thus, the gene-corrected cells, which appear otherwise normal, may ultimately provide phenotypic correction to neighboring GAA-deficient cells by fusion and to distant cells by secretion and uptake mechanisms.
Glycogen storage disease type II (GSDII) is a lysosomal storage disease caused by a deficiency in acid alpha-glucosidase (GAA), and leads to cardiorespiratory failure by the age of 2 years. In this study, we investigate the impact of anti-GAA antibody formation on cross-correction of the heart, diaphragm, and hind-limb muscles from liver-directed delivery of recombinant adeno-associated virus (rAAV)5- and rAAV8-GAA vectors. GAA(-/-) mice receiving 1 x 10(12) vector genomes of rAAV5- or rAAV8-DHBV-hGAA were analyzed for anti-GAA antibody response, GAA levels, glycogen reduction, and contractile function. We demonstrate that restoration of GAA to the affected muscles is dependent on the presence or absence of the antibody response. Immune-tolerant mice had significantly increased enzyme levels in the heart and skeletal muscles, whereas immune-responsive mice had background levels of GAA in all tissues except the diaphragm. The increased levels of activity in immune-tolerant mice correlated with reduced glycogen in the heart and diaphragm and, overall, contractile function of the soleus muscle was significantly improved. These findings highlight the importance of the immune response to rAAV-encoded GAA in correcting GSDII and provide additional understanding of the approach to treatment of GSDII.
In rats, the period of refeeding after a fast is associated with accelerated weight gain without a concomitant increase in food intake. In this study the alpha-glucosidase inhibitor, acarbose, was used to delay carbohydrate absorption in normal adult rats, and the effects on body weight, food intake, and intestinal enzyme activities were determined. Refeeding with acarbose in the food (500 mg/kg) reduced the rate of weight gain compared to refeeding without acarbose but did not change food intake. Acarbose also lowered midjejunal mass and blunted the refeeding-induced rise in certain brush border disaccharidase and intracellular glycolytic enzymes. However, acarbose refed rats still had accelerated weight gain compared to nonfasted rats, implying that the refeeding response was not totally abolished. These studies suggest that inhibition of carbohydrate absorption during refeeding might have a role in the maintenance of diet-induced weight loss.
Glycogenosis type II (GSD II) is a lysosomal disorder affecting skeletal and cardiac muscle. In the infantile form of the disease, patients display cardiac impairment, which is fatal before 2 years of life. Patients with juvenile or adult forms can present diaphragm involvement leading to respiratory failure. The enzymatic defect in GSD II results from mutations in the acid alpha-glucosidase (GAA) gene, which encodes a 76 kDa protein involved in intralysosomal glycogen hydrolysis. We previously reported the use of an adenovirus vector expressing GAA (AdGAA) for the transduction of myoblasts and myotubes cultures from GSD II patients. Transduced cells secreted GAA in the medium, and GAA was internalized by receptor-mediated capture, allowing glycogen hydrolysis in untransduced cells. In this study, using a GSD II mouse model, we evaluated the feasibility of GSD II gene therapy using muscle as a secretary organ. Adenovirus vector encoding AdGAA was injected in the gastrocnemius of neonates. We detected a strong expression of GAA in the injected muscle, secretion into plasma, and uptake by peripheral skeletal muscle and the heart. Moreover, glycogen content was decreased in these tissues. Electron microscopy demonstrated the disappearance of destruction foci, normally present in untreated mice. We thus demonstrate for the first time that muscle can be considered as a safe and easily accessible organ for GSD II gene therapy.
Gaucher disease (GD), which results from mutations in the human acid beta-glucosidase (beta-Glc) gene, was used as a model system to compare the utility of three methods capable of detecting single base substitutions. PCR-amplified beta-Glc exon 9 sequences of GD patients were screened for single base mutations by GC-clamped denaturing gradient gel electrophoresis (DGGE) and RNase A cleavage of RNA-DNA heteroduplexes, and by chemical (hydroxylamine/osmium tetroxide) cleavage of dsDNA heteroduplexes. PCR products showing abnormal behaviour were cloned and sequenced. Three new point mutations were detected by this strategy. A G to C (Asp409 to His409) substitution was present in two Type 1 and one Type 3 GD patients; an A to T transversion (Asp409 to Val409) was detected in only a single Type 3 individual, and a G to T mutation (Val394 to Leu394) was present in one Type 1 and one Type 3 patient. GD thus exhibits extensive molecular heterogeneity, with at least five single base mutations in beta-Glc exon 9. In every case verified by ASO hybridization, DGGE had correctly identified the presence of the three new mutations, as well as the two previously described exon 9 mutations. In comparison, although RNase A and the chemical method were both able to detect some of these mutations, neither method reproducibly detected all of them. Additionally, DGGE was the only method that was able to reliably determine whether a given mutation was present homozygously or heterozygously. These results suggest that GC-clamped DGGE may be a more reliable and informative screening method for point mutation detection.
The binding of glucono-1,5-lactone (gluconolactone) with almond beta-glucosidase was studied at pH 5.0 and 25 degrees C, in the absence and presence of glucose, by monitoring the enzyme fluorescence as a probe. From the results of fluorometric titration, the dissociation constant Kd and the maximum fluorescence intensity increase (percent) of the enzyme-gluconolactone complex relative to the enzyme alone, delta Fmax, were determined to be 12.7 microM and 14.7%, respectively. From the study of the temperature dependence of Kd, delta G degrees, delta H degrees and delta S degrees for the binding were evaluated to be -6.7 kcal mol-1, -3.5 kcal mol-1, and 10.8 e.u. (cal mol-1 deg-1), respectively, at 25 degrees C. The analysis of the fluorometric titration data in the presence of glucose revealed that these ligands bind competitively to the enzyme, probably at the same site. The results of a stopped-flow kinetic study are consistent with the following two-step mechanism: (formula; see text) which indicates that gluconolactone (L) and the enzyme (E) transiently form a loosely bound complex, ELtr (k-1/k+1 = 4.5 mM), in the first rapid bimolecular association step, and ELtr is converted into a more tightly bound complex EL (k+2 = 94 s-1, k-2 = 0.36 s-1) in the subsequent slow unimolecular process. The fluorescence intensity increase occurs solely in the latter step.
A p-nitrophenyl-alpha-D-glucopyranoside-hydrolyzing alpha-glucosidase from an obligate thermophile, Bacillus thermoglucosidius KP 1006, gave a triphasic relationship at pH 6.8 in the van't Hoff plot of Km, in the Arrhenius plot of the first order rate constant of inactivation with 0.1% sodium dodecyl sulfate, and in the logarithmic plot of the maximal fluorescence intensity at 346 nm versus reciprocal of temperature. The respective plots exhibited two breaks at 40 and 61 degrees C, 43 and 62 degrees C, and 40 and 61 degrees C. However, the Arrhenius plot of the molecular activity at pH 6.8 had a single discontinuity at 64 degrees C. These findings, together with thermodynamic quantities for the enzyme, suggest that the thermal conformation changes in the enzyme protein occur around 40--43 degrees C and 61--64 degrees C. The Arrhenius plot of the rate constant of heat inactivation at pH 6.8 was bent at 73 degrees C. Thermodynamic data indicate that the enzyme is transformed from a heat stable form into a heat unstable form at 73 degrees C with temperature elevation. The critical points localized near the minimum, optimum, and maximum temperatures (40, 60, and 72 degrees C) of the cell growth, respectively.
Acid alpha-glucosidase [EC 3.2.1.3] was purified from pig liver by a procedure including Sephadex G-100 affinity chromatography. Electrophoresis on SDS-polyacrylamide gel of the purified enzyme indicated the presence of two components with molecular weights of 73K and 64K. The two components of the enzyme were completely separated, in reasonable yield, by chromatography on a DEAE-5PW column. Both components catalyzed the hydrolysis of the alpha-1,4 and alpha-1,6 linkages of glycogen, maltose, isomaltose, dextrin, and a synthetic glucoside at acid pH. The pH optima of both components were 4.3 for maltase and glucoamylase, and 4.8 for isomaltase and dextrinase. But as to the activity on 4MU-alpha-Glc, the pH optimum of the larger component was 4.8 and that of the smaller component 5.3. The Km values of both components for 4MU-alpha-Glc, maltose, glycogen, isomaltose, and dextrin were 1.0 X 10(-4) M, 9.1 X 10(-3) M, 16.7 mg/ml, 6.7 X 10(-2) M, and 12.5 mg/ml, respectively. Erythritol, Tris, and turanose inhibited the two components competitively. The Ki values of the larger component were 5.0 X 10(-2) M, 13.3 X 10(-3) M, and 3.2 X 10(-3) M, and those of the smaller component were 2.5 X 10(-2) M, 6.1 X 10(-3) M, and 4.7 X 10(-3) M, for erythritol, Tris, and turanose, respectively.