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.     

Effects of non-contractile inclusions on mechanical performance of skeletal muscle

Glycogen storage disease II is an inherited progressive muscular disease in which the lack of functional acid 1-4 alpha-glucosidase results in the accumulation of lysosomal glycogen. In the present study, we examine the effect of these non-contractile inclusions on the mechanical performance of skeletal muscle. To this end, force developed in an isometrically contracting slice of a muscle was calculated with a finite element model. Force was calculated at several inclusion densities and distributions and compared to muscle lacking inclusions. Furthermore, ankle dorsal flexor torque was measured in situ of alpha-glucosidase null mice of 6 months of age and unaffected litter mates as was inclusion density in the dorsal flexor muscles. The calculated force loss was shown to be almost exclusively dependent on the inclusion density and less on the type of inclusion distribution. The force loss predicted by the model (6%) on the basis of measured inclusion density (3.3%) corresponded to the loss in mass-normalized strength in these mice measured in situ (7%). Therefore, we conclude that the mechanical interaction between the non-contractile inclusions and the nearby myofibrils is a key factor in the loss of force per unit muscle mass during early stages of GSD II in mice. As glycogen accumulation reaches higher levels in humans, it is highly probable that the impact of this mechanical interaction is even more severe in human skeletal muscle.     

Deconstructing Pompe Disease by Analyzing Single Muscle Fibers: “To See a World in a Grain of Sand…”

Autophagy is a major pathway for delivery of proteins and organelles to lysosomes where they are degraded and recycled. We have previously shown excessive autophagy in a mouse model of Pompe disease (glycogen storage disease type II), a devastating myopathy caused by a deficiency of the glycogen-degrading lysosomal enzyme, acid alpha-glucosidase. The autophagic buildup constituted a major pathological component in skeletal muscle and interfered with delivery of the therapeutic enzyme. To assess the role of autophagy in the pathogenesis of the human disease, we have analyzed vesicles of the lysosomal-degradative pathway in isolated single muscle fibers from Pompe patients. Human myofibers showed abundant autophagosome formation and areas of autophagic buildup of a wide range of sizes. In patients, as in the mouse model, the enormous autophagic buildup causes greater skeletal muscle damage than the enlarged, glycogen-filled lysosomes outside the autophagic regions. Clearing or preventing autophagic buildup seems, therefore, a necessary target of Pompe disease therapy.     

Deconstructing Pompe disease by analyzing single muscle fibers: to see a world in a grain of sand..

Autophagy is a major pathway for delivery of proteins and organelles to lysosomes where they are degraded and recycled. We have previously shown excessive autophagy in a mouse model of Pompe disease (glycogen storage disease type II), a devastating myopathy caused by a deficiency of the glycogen-degrading lysosomal enzyme acid alpha-glucosidase. The autophagic buildup constituted a major pathological component in skeletal muscle and interfered with delivery of the therapeutic enzyme. To assess the role of autophagy in the pathogenesis of the human disease, we have analyzed vesicles of the lysosomal-degradative pathway in isolated single muscle fibers from Pompe patients. Human myofibers showed abundant autophagosome formation and areas of autophagic buildup of a wide range of sizes. In patients, as in the mouse model, the enormous autophagic buildup causes greater skeletal muscle damage than the enlarged, glycogenfilled lysosomes outside the autophagic regions. Clearing or preventing autophagic buildup seems, therefore, a necessary target of Pompe disease therapy.     

Autophagy and lysosomes in Pompe disease

In Pompe disease, a deficiency of lysosomal acid alpha-glucosidase, intralysosomal glycogen accumulates in multiple tissues, with skeletal and cardiac muscle most severely affected.(1) Complete enzyme deficiency results in rapidly progressive infantile cardiomyopathy and skeletal muscle myopathy that is fatal within the first two years of life. Patients with partial enzyme deficiency suffer from skeletal muscle myopathy and experience shortened lifespan due to respiratory failure. The major advance has been the development of enzyme replacement therapy, which recently became available for Pompe patients. However, the effective clearance of skeletal muscle glycogen, as shown by both clinical and preclinical studies, has proven more difficult than anticipated.(2-4) Our recent work published in Annals of Neurology(5) was designed to cast light on the problem, and was an attempt to look beyond the lysosomes by analyzing the downstream events affected by the accumulation of undigested substrate in lysosomes. We have found that the cellular pathology in Pompe disease spreads to affect both endocytic (the route of the therapeutic enzyme) and autophagic (the route of glycogen) pathways, leading to excessive autophagic buildup in therapy-resistant skeletal muscle fibers of the knockout mice.     

Evidence for a founder effect in Sicilian patients with glycogen storage disease type II

Glycogen storage disease type II (GSD II) is an autosomal recessive inherited disorder due to the deficiency of the enzyme acid alpha-glucosidase, which causes an accumulation of glycogen in lysosomes. The deletion of exon 18 (delta 18) is a frequent mutation associated with a severe phenotype. We analyzed 25 Italian patients, 5 of whom were found to be delta 18 carriers. All these 5 patients came from Catania, a town in Sicily. We report on the analysis of 5 intragenic single-point polymorphic markers in the delta 18 patients and on the subsequent characterization of a delta 18-associated haplotype. The frequency of this haplotype in GSD II patients and normal individuals was 1 and 0.196, respectively (chi(2) = 20.9; p < 0.001). The high frequency of the delta 18 allele in this Italian subpopulation is likely to be due to a founder effect.     

Infantile acid maltase deficiency

The loss of normal ultrastructure of skeletal muscle during the relentless course of infantile acid maltase deficiency (AMD) is re-examined in the light of the lysosomal rupture hypothesis. This hypothesis suggests that movement and increased myofibril rigidity during contraction cause lysosomes in muscle to rupture and release glycogen and other lysosomal contents to a much greater extent than do lysosomes in other cell types in cases of infantile AMD. Muscle fibers are destroyed, while macrophages and other cells mostly accumulate glycogen in storage lysosomes without being destroyed. When morphological stages of fiber destruction are placed in a sequential series, from fibers most like normal infant muscle to those with only remnants of muscle ultrastructure, the earliest stages seen contain intact storage lysosomes. Intermediate stages exhibit ruptured lysosomal membranes and free glycogen as well as glycogen in lysosomes. Loss of myofibrillar material and loss of glycogen occur in later stages of fiber destruction. Membrane-enclosed glycogen and mitochondria are relatively protected from the process of destruction. The electron-microscopic observations support the lysosomal rupture hypothesis and are compatible with the original proposal of Hers, that the disease results from a deficiency of a single lysosomal enzyme. Secondary changes other than muscle fiber destruction probably relate to disrupted control mechanisms and the nature of muscle as a specialized cell. At least two different mechanisms could contribute to the loss of contractile activity and myofibrillar structure.     

Expression and routeing of human lysosomal alpha-glucosidase in transiently transfected mammalian cells.

Previously isolated lysosomal alpha-glucosidase cDNA clones were ligated to full-length constructs for expression in vitro and in mammalian cells. One of these constructs (pSHAG1) did not code for functional enzyme, due to an arginine residue instead of a tryptophan residue at amino acid position 402. The mutation does not affect the rate of enzyme synthesis, but interferes with post-translational modification and intracellular transport of the acid alpha-glucosidase precursor. Using immunocytochemistry it is demonstrated that the mutant precursor traverses the endoplasmic reticulum and the Golgi complex, but does not reach the lysosomes. Pulse-chase experiments suggest premature degradation. The Trp-402-containing enzyme (encoded by construct pSHAG2) is processed properly, and has catalytic activity. A fraction of the enzyme is localized at the plasma membrane. It is hypothesized that membrane association of the acid alpha-glucosidase precursor, as demonstrated by Triton X-114 phase separation, is responsible for transport to this location. Transiently expressed acid alpha-glucosidase also enters the secretory pathway, since a catalytically active precursor is found in the culture medium. This precursor has the appropriate characteristics for use in enzyme replacement therapy. Efficient uptake via the mannose 6-phosphate receptor results in degradation of lysosomal glycogen in cultured fibroblasts and muscle cells from patients with glycogenosis type II.     

High-resolution light microscopy (HRLM) and digital analysis of Pompe disease pathology.

Pompe disease is an autosomal recessive lysosomal storage disorder caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase, responsible for the degradation of lysosomal glycogen. Absent or low levels of the enzyme leads to lysosomal glycogen accumulation in cardiac and skeletal muscle cells, resulting in progressive muscle weakness and death from cardiac or respiratory failure. Recombinant enzyme replacement and gene therapy are now being investigated as treatment modalities for this disease. A knockout mouse model for Pompe disease, induced by the disruption of exon 6 within the acid alpha-glucosidase gene, mimics the human disease and has been used to evaluate the efficacy of treatment modalities for clearing glycogen. However, for accurate histopathological assessment of glycogen clearance, maximal preservation of in situ lysosomal glycogen is essential. To improve retention of glycogen in Pompe tissues, several fixation and embedding regimens were evaluated. The best glycogen preservation was obtained when tissues fixed with 3% glutaraldehyde and postfixed with 1% osmium tetroxide were processed into epon-araldite. Preservation was confirmed by staining with the Periodic acid-Schiff's reaction and by electron microscopy. This methodology resulted in high-resolution light microscopy (HRLM) sections suitable for digital quantification of glycogen content in heart and skeletal muscle. Combining this method of tissue fixation with computer-assisted histomorphometry has provided us with what we believe is the most objective and reproducible means of evaluating histological glycogen load in Pompe disease.     

Common antigenicity for two glycosidases

Enzyme replacement therapy (ERT) has proven to be an effective therapy for some lysosomal storage disorder (LSD) patients. A potential complication during ERT is the generation of an immune response against the replacement protein. We have investigated the antigenicity of two distantly related glycosidases, alpha-glucosidase (Pompe disease or glycogen storage disease type II, GSD II), and alpha-L-iduronidase (Hurler syndrome, mucopolysaccharidosis type I, MPS I). The linear sequence epitope reactivity of affinity purified polyclonal antibodies to recombinant human alpha-glucosidase and alpha-L-iduronidase was defined, to both glycosidases. The polyclonal antibodies exhibited some cross-reactive epitopes on the two proteins. Moreover, a monoclonal antibody to the active site of alpha-glucosidase showed cross-reactivity with a catalytic structural element of alpha-L-iduronidase. In a previous study, in MPS I patients who developed an immune response to ERT, this same site on alpha-L-iduronidase was highly antigenic and the last to tolerise following repeated enzyme infusions. We conclude that glycosidases can exhibit cross-reactive epitopes, and infer that this may relate to common structural elements associated with their active sites.     

Lysosomal glycogen accumulation in rat liver and its in vivo kinetics after a single intraperitoneal injection of acarbose, an alpha-glucosidase inhibitor

A single intraperitoneal injection of acarbose (400 mg/kg) into rats caused lysosomal accumulation of glycogen in the liver, mimicking the cytological characteristics of human glycogen storage disease type II (Pompe's disease). The animal model is therefore useful for studying the pathogenesis of the disease. In the present study, we applied this model to examine the lysosomal hydrolytic pathway of glycogen in vivo. To quantify the lysosomal glycogen, the lysosome-rich fraction was rapidly prepared from liver homogenate by agglutination in the presence of Ca2+. Then the fraction was treated with alpha-amylase in isotonic medium to remove cytosolic glycogen, followed by transfer to hypotonic conditions in the presence of Triton X-100 to destroy total glycogen. The amount of lysosomal glycogen was calculated from the difference between the glycogen levels measured before and after the treatment under hypotonic conditions, and then it was corrected based on measurements of the intactness (%) of lysosomes and the recovery (%) of the lysosomal marker enzyme (beta NAGase). We observed no measurable lysosomal glycogen in normal liver by this method, and this was confirmed by electron microscopy. After administration of acarbose, the lysosomal glycogen level increased to 2.5 mg/g liver within 2 days, and then decreased gradually at a rate of 0.4 mg/day/g. The accumulation of glycogen in the lysosomes at an initial velocity of 1.5 mg/day/g liver may be considered as the amount of glycogen that would normally be degraded by acid alpha-glucosidase. Therefore, assuming that the liver breaks down about 40 mg glycogen/day/g, we estimated that about 3% of the glycogen would be hydrolyzed by the lysosomal pathway.     

Autophagy and mitochondria in Pompe disease: nothing is so new as what has long been forgotten

Macroautophagy (often referred to as autophagy) is an evolutionarily conserved intracellular system by which macromolecules and organelles are delivered to lysosomes for degradation and recycling. Autophagy is robustly induced in response to starvation in order to generate nutrients and energy through the lysosomal degradation of cytoplasmic components. Constitutive, basal autophagy serves as a quality control mechanism for the elimination of aggregated proteins and worn-out or damaged organelles, such as mitochondria. Research during the last decade has made it clear that malfunctioning or failure of this system is associated with a wide range of human pathologies and age-related diseases. Our recent data provide strong evidence for the role of autophagy in the pathogenesis of Pompe disease, a lysosomal glycogen storage disease caused by deficiency of acid alpha-glucosidase (GAA). Large pools of autophagic debris in skeletal muscle cells can be seen in both our GAA knockout model and patients with Pompe disease. In this review, we will focus on these recent data, and comment on the not so recent observations pointing to the involvement of autophagy in skeletal muscle damage in Pompe disease.     

Correction of glycogen storage disease type II by enzyme replacement with a recombinant human acid maltase produced by over-expression in a CHO-DHFR(neg) cell line

Inherited genetic deficiency of lysosomal acid alpha glucosidase or acid maltase (GAA) results in the autosomal recessive glycogen storage disease type II (GSD II). To investigate whether we could generate a functional recombinant human GAA (rhGAA) for enzyme replacement therapy, we subcloned the cDNAs for human GAA and mouse dihydrofolate reductase (DHFR) into DHFR(neg) Chinese hamster ovary cells and established a stable cotransformant that expressed rhGAA. We cultured the recombinant cells in media with progressively increasing concentrations of methotrexate and found that human GAA enzyme activity increased to over 2,000 IU per gram protein. Importantly, the human GAA enzyme activity correlated to equivalent amounts of human GAA protein by rocketimmunoelectrophoresis. We confirmed that the human GAA enzyme activity corresponded to an amplification in human GAA mRNA by Northern analysis and human GAA cDNA copy number by Southern analysis. Exposing the rhGAA to human GSDII fibroblast cells or patient's lymphocytes or monocytes resulted in uptake of the rhGAA and reversal of the enzymatic defect. Mannose-6-phosphate in the media blocked uptake. GAA -/- mice were treated with the rhGAA at 1 mg/kg, which resulted in heterozygous levels of GAA in tissues, most notably skeletal muscle, heart and diaphragm after two infusions. More importantly, after multiple infusions, hind, and fore-limb muscle weakness was reversed. This rhGAA would be ideal for enzyme replacement therapy in GSD II.     

Autophagy and Lysosomes in Pompe Disease

In Pompe disease, a deficiency of lysosomal acid alpha-glucosidase, intralysosomal glycogen accumulates in multiple tissues, with skeletal and cardiac muscle most severely affected.¹ Complete enzyme deficiency results in rapidly progressive infantile cardiomyopathy and skeletal muscle myopathy that is fatal within the first two years of life. Patients with partial enzyme deficiency suffer from skeletal muscle myopathy and experience shortened lifespan due to respiratory failure. The major advance has been the development of enzyme replacement therapy, which recently became available for Pompe patients. However, the effective clearance of skeletal muscle glycogen, as shown by both clinical and pre-clinical studies, has proven more difficult than anticipated.^2-4 The work published in Annals of Neurology⁵ was designed to cast light on the problem, and was an attempt to look beyond the lysosomes by analyzing the downstream events affected by the accumulation of undigested substrate in lysosomes. We have found thatthe cellular pathology in Pompe disease spreads to affect both endocytic (the route of the therapeutic enzyme) and autophagic (the route of glycogen) pathways, leading to excessive autophagic buildup in therapy-resistant skeletal muscle fibers of the knockout mice.

Addendum to:

Dysfunction of Endocytic and Autophagic Pathways in a Lysosomal Storage Disease

Tokiko Fukuda, Lindsay Ewan, Martina Bauer, Robert J. Mattaliano, Kristien Zaal,Evelyn Ralston, Paul H. Plotz and Nina Raben

Ann Neurol 2006; 59:700-8     

Post mortem glycogenolysis is a combination of phosphorolysis and hydrolysis

1. Glycogen, glucose, lactate and glycogen phosphorylase concentrations and the activities of glycogen phosphorylase a and acid 1,4-alpha-glucosidase were measured at various times up to 120 min after death in the liver and skeletal muscle of Wistar and gsd/gsd (phosphorylase b kinase deficient) rats and Wistar rats treated with the acid alpha-glucosidase inhibitor acarbose. 2. In all tissues glycogen was degraded rapidly and was accompanied by an increase in tissue glucose and lactate concentrations and a lowering of tissue pH. In the liver of Wistar and acarbose-treated Wistar rats and in the skeletal muscle of all rats glycogen loss proceeded initially very rapidly before slowing. In the gsd/gsd rat liver glycogenolysis proceeded at a linear rate throughout the incubation period. Over 120 min 60, 20 and 50% of the hepatic glycogen store was degraded in the livers of Wistar, gsd/gsd and acarbose-treated Wistar rats, respectively. All 3 types of rat degraded skeletal muscle glycogen at the same rate and to the same extent (82% degraded over 2 hr). 3. In Wistar rat liver and skeletal muscle glycogen phosphorylase was activated soon after death and the activity of phosphorylase a remained well above the zero-time level at all later time points, even when the rate of glycogenolysis had slowed significantly. Liver and skeletal muscle acid alpha-glucosidase activities were unchanged after death. 4. The decreased rate and extent of hepatic glycogenolysis in both the gsd/gsd and acarbose-treated rats suggests that this process is a combination of phosphorolysis and hydrolysis. 5. Glycogen was purified from Wistar liver and skeletal muscle at various times post mortem and its structure investigated. Fine structural analysis revealed progressive shortening of the outer chains of the glycogen from both tissues, indicative of random, lysosomal hydrolysis. Analysis of molecular weight distributions showed inhomogeneity in the glycogen loss; in both tissues high molecular weight glycogen was preferentially degraded. This material is concentrated in lysosomes of both skeletal muscle and liver. These results are consistent with a role for lysosomal hydrolysis in glycogen degradation.     

Suppression of autophagy permits successful enzyme replacement therapy in a lysosomal storage disorder—murine Pompe disease

Autophagy, an intracellular system for delivering portions of cytoplasm and damaged organelles to lysosomes for degradation/recycling, plays a role in many physiological processes and is disturbed in many diseases. We recently provided evidence for the role of autophagy in Pompe disease, a lysosomal storage disorder in which acid alpha-glucosidase, the enzyme involved in the breakdown of glycogen, is deficient or absent. Clinically the disease manifests as a cardiac and skeletal muscle myopathy. The current enzyme replacement therapy (ERT) clears lysosomal glycogen effectively from the heart but less so from skeletal muscle. In our Pompe model, the poor muscle response to therapy is associated with the presence of pools of autophagic debris. To clear the fibers of the autophagic debris, we have generated a Pompe model in which an autophagy gene, Atg7, is inactivated in muscle. Suppression of autophagy alone reduced the glycogen level by 50–60%. Following ERT, muscle glycogen was reduced to normal levels, an outcome not observed in Pompe mice with genetically intact autophagy. The suppression of autophagy, which has proven successful in the Pompe model, is a novel therapeutic approach that may be useful in other diseases with disturbed autophagy.Key words: Pompe disease, lysosomal glycogen storage, myopathy, Atg7, enzyme replacement therapy     

Latency of some glycosidases of rat liver lysosomes.

The latency of the alpha-glucosidase activity of intact rat liver lysosomes was studied by using four substrates (glycogen, maltose, p-nitrophenyl, alpha-glucoside, alpha-fluoroglucoside) at a range of substrate concentrations. The results indicate that the entire lysosome population is impermeable to glycogen and maltose, but a proportion of lysosomes are permeable to alpha-fluoroglucoside and a still higher proportion permeable to p-nitrophenyl alpha-glucoside. Incubation at 37 degrees C in an osmotically protected buffer of of pH 5.0 caused lysosomes to become permeable to previously impermeant substrates and ultimately to release their alpha-glucosidase into the medium. The latencies of lysosomal beta-glucosidase and beta-galactosidase were examined by using p-nitrophenyl beta-glucoside and beta-galactoside as substrates. The results indicate permeability properties to these substrates similar to that to p-nitrophenyl alpha-glucoside. On incubation in an osmotically protected buffer of pH 5, lysosomes progressively released their beta-galactosidase in soluble form, but beta-glucosidase remained attached to sedimentable material. Lysosomal beta-glucosidase was inhibited by 0.1% Triton X-100; alpha-glucosidase and beta-galactosidase were not inhibited.     

Inhibitory effect of pseudo-aminosugars on oligosaccharide glucosidases I and II and on lysosomal alpha-glucosidase from rat liver

We examined the inhibitory effect of three pseudo-aminosugars (validamine, valienamine, and valiolamine), which were isolated from the broth of Streptomyces hygroscopicus, on the oligosaccharide-processing glucosidases I and II involved in glycoprotein biosynthesis in rat liver. Both glucosidases I and II were inhibited to the same extent by the pseudoaminosugars, and valiolamine had a more potent inhibitory activity than validamine or valienamine. A 50% inhibition of valiolamine was observed at 12 microM for glucosidase I and glucosidase II activities acting respectively on the substrates Glc3Man9GlcNAc2 and p-nitrophenyl alpha-D-glucopyranoside. Further, in order to investigate further the ability of valiolamine to inhibit glucosidase I, reaction products were analyzed by gel filtration on a Bio-Gel P-4 column. We also compared the inhibitory action of these pseudo-aminosugars on the acid alpha-glucosidase of rat liver lysosomes. They competitively inhibited the hydrolysis of both substrates, maltose and glycogen. Valiolamine again had a more potent lysosomal alpha-glucosidase inhibitory activity than the other two. The Ki values of valiolamine for the hydrolysis of maltose and glycogen were 8.1 and 11 microM, respectively. Valiolamine is a particularly effective inhibitor of oligosaccharide glucosidases I and II and of lysosomal alpha-glucosidase. Hence valiolamine might be useful as a research tool in investigations of carbohydrate metabolism.