Mass spectrometric quantification of glycogen to assess primary substrate accumulation in the Pompe mouse (original) (raw)
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International Journal of Translational Medicine, 2021
Glycogen is present in all tissues, but it is primarily stored in the liver and in muscle. As a branched chain carbohydrate, it is broken down by phosphorylase and debrancher enzymes, which are cytoplasmic. It is also degraded by a lysosomal α-glucosidase (GAA) also known as acid α-glucosidase and lysosomal acid α-glucosidase. The deficiency of GAA in patients is known as Pompe disease, and the phenotypes as infantile, juvenile and later onset forms. Pompe disease is treated by enzyme replacement therapy (ERT) with a recombinant form of rhGAA. Following ERT in Pompe mice and human patients there is residual carbohydrate material present in the cytoplasm of cells. The goal of this work is to improve ERT and attempt to identify and treat the residual cytoplasmic carbohydrate. Initial experiments were to determine if rhGAA can completely degrade glycogen. The enzyme cannot completely degrade glycogen. There is a residual glycosylated protein as well as a soluble glycosylated protein, w...
Journal of Biological Chemistry, 1998
We have used gene targeting to create a mouse model of glycogen storage disease type II, a disease in which distinct clinical phenotypes present at different ages. As in the severe human infantile disease (Pompe Syndrome), mice homozygous for disruption of the acid ␣-glucosidase gene (6 neo /6 neo) lack enzyme activity and begin to accumulate glycogen in cardiac and skeletal muscle lysosomes by 3 weeks of age, with a progressive increase thereafter. By 3.5 weeks of age, these mice have markedly reduced mobility and strength. They grow normally, however, reach adulthood, remain fertile, and, as in the human adult disease, older mice accumulate glycogen in the diaphragm. By 8-9 months of age animals develop obvious muscle wasting and a weak, waddling gait. This model, therefore, recapitulates critical features of both the infantile and the adult forms of the disease at a pace suitable for the evaluation of enzyme or gene replacement. In contrast, in a second model, mutant mice with deletion of exon 6 (⌬6/⌬6), like the recently published acid ␣-glucosidase knockout with disruption of exon 13 (Bijvoet, A. G., van de Kamp, E. H.
Human Molecular Genetics, 1999
Pompe's disease or glycogen storage disease type II (GSDII) belongs to the family of inherited lysosomal storage diseases. The underlying deficiency of acid α α α α-glucosidase leads in different degrees of severity to glycogen storage in heart, skeletal and smooth muscle. There is currently no treatment for this fatal disease, but the applicability of enzyme replacement therapy is under investigation. For this purpose, recombinant human acid α α α α-glucosidase has been produced on an industrial scale in the milk of transgenic rabbits. In this paper we demonstrate the therapeutic effect of this enzyme in our knockout mouse model of GSDII. Full correction of acid α α α α-glucosidase deficiency was obtained in all tissues except brain after a single dose of i.v. enzyme administration. Weekly enzyme infusions over a period of 6 months resulted in degradation of lysosomal glycogen in heart, skeletal and smooth muscle. The tissue morphology improved substantially despite the advanced state of disease at the start of treatment. The results have led to the start of a Phase II clinical trial of enzyme replacement therapy in patients.
Generalized glycogen storage and cardiomegaly in a knockout mouse model of Pompe disease
Human Molecular Genetics, 1998
Glycogen storage disease type II (GSDII; Pompe disease), caused by inherited deficiency of acid α-glucosidase, is a lysosomal disorder affecting heart and skeletal muscles. A mouse model of this disease was obtained by targeted disruption of the murine acid α-glucosidase gene (Gaa) in embryonic stem cells. Homozygous knockout mice (Gaa-/-) lack Gaa mRNA and have a virtually complete acid α-glucosidase deficiency. Glycogen-containing lysosomes are detected soon after birth in liver, heart and skeletal muscle cells. By 13 weeks of age, large focal deposits of glycogen have formed. Vacuolar spaces stain positive for acid phosphatase as a sign of lysosomal pathology. Both male and female knockout mice are fertile and can be intercrossed to produce progeny. The first born knockout mice are at present 9 months old. Overt clinical symptoms are still absent, but the heart is typically enlarged and the electrocardiogram is abnormal. The mouse model will help greatly to understand the pathogenic mechanism of GSDII and is a valuable instrument to explore the efficacy of different therapeutic interventions.
Biosynthesis of acid α-glucosidase in late-onset forms of glycogenosis type II (Pompe's disease)
FEBS Letters, 1982
Cultured human skin fibroblasts from control persons and from patients with the generalized and lateonset forms of Pompe's disease were labelled with radioactive leucine and the incorporation of radioactivity into acid cY-glucosidase and cathepsin D was analysed by immunoprecipitation, gel electrophoresis and fluorography. When the labelling was carried out for 6-12 h in the presence of NH&l, the labelling of secreted cu-glucosidase relative to that of secreted cathepsin D in fibroblasts from patients with the lateonset form of Pompe's disease was < 15% of that in fibroblasts from control persons. However, when the fibroblasts were labelled for < 1 h, the relative rate of incorporation of radioactivity into acid a-glucosidase was rather similar in the two types of fibroblasts. In fibroblasts from patients with the generalized form of Pompe's disease no incorporation of radioactivity into acid cy-glucosidase could be detected.
Glycogen storage diseases in animals and their potential value as models of human disease
Journal of Inherited Metabolic Disease, 1983
Glycogen storage diseases (GSD) are inborn errors of glycogen metabolism. Of the eight human GSD types in which the enzymatic deficiency has been identified, spontaneous animal counterparts have been reported for GSD I (glucose-6-phosphatase deficiency) in the mouse, for GSD II (acid ~-glucosidase deficiency) in the dog, in cattle and in the quail, for GSD III (debrancher enzyme deficiency) in the dog and for GSD VIII (phosphorylase kinase deficiency) in the rat and the mouse. Experimentally induced GSD-like conditions have been described in the rat (Acarbose-induced GSD II-like conditions, iodoacetate-induced symptoms of myophosphorylase (GSD V) and myophosphofructokinase (GSD VII) deficiency) and the chicken (ochratoxin A-induced symptoms of cyclic AMP-dependent protein kinase deficiency). Enzymatic defects that are typical of the human GSD types have not been clearly identified in the induced animal conditions. The homology of animal and human GSD types is discussed. It is concluded that clinical, pathogenic and therapeutic studies of GSD may benefit from the use of animal models. For genetic studies of human GSD these models may prove to be of limited value, as the picture of several human GSD types is already obscured by genetic heterogeneity.
Journal of Clinical Investigation, 1987
The molecular basis of clinical diversity in glycogenosis type II (Pompe's disease) was investigated by comparing the nature of acid a-glucosidase deficiency in cultured fibroblasts from 30 patients. Biosynthetic forms of acid a-glucosidase with different molecular mass were separated electrophoretically and identified by immunoblotting. Immuno-electron microscopy was employed to determine the intracellular localization of mutant enzyme. Our studies illustrate that maturation of acid a-glucosidase is associated with transport to the lysosomes. Deficiency of catalytically active mature enzyme in lysosomes is common to all clinical phenotypes but, in the majority of cases, is more profound in early onset than in late onset forms of the disease. Thus, the results suggest that the clinical course of glycogenosis type II is primarily determined by the amount of functional acid a-glucosidase. The role of secondary factors can, however, not be excluded because three adult patients were identified with very low activity and little enzyme in the lysosomes.
Folia neuropathologica / Association of Polish Neuropathologists and Medical Research Centre, Polish Academy of Sciences, 2007
Glycogen storage disease type II (GSD II) is an autosomal recessive deficiency of acidalpha-1,4-glucosidase(GAA) caused by mutations in the GAA gene located on human chromosome 17 (17q 25.2-q 25.3). Although its pathophysiology is partially understood, it has not yet been elucidated whether the level of GAA deficiency is directly proportional to the level of glycogen storage, vacuolar degeneration and/or GSD II severity. Muscle and skin biopsies were taken from three female patients with symptoms of progressive muscle weakness and respiratory failure: patient 1 aged 19, as well as patients 2 and 3 (two sisters) aged 31 and 29, respectively. Initial clinical manifestations, respiratory failure and muscle weakness, were similar in all the examined patients, while their character and intensity differed. For each examined patient, the activity of lysosomal GAA (at pH 3.8) was measured fluorometrically in isolated blood leukocytes (L) and dried blood spots (DBS). Biopsy samples were stud...
Molecular Therapy, 2007
We investigated the use of pharmacological chaperones for the therapy of Pompe disease, a metabolic myopathy due to mutations of the gene encoding the lysosomal hydrolase a-glucosidase (GAA) and characterized by generalized glycogen storage in cardiac and skeletal muscle. We studied the effects of two imino sugars, deoxynojirimycin (DNJ) and N-butyldeoxynojirimycin (NB-DNJ), on residual GAA activity in fibroblasts from eight patients with different forms of Pompe disease (two classic infantile, two non-classic infantile onset, four late-onset forms), and with different mutations of the GAA gene. We demonstrated a significant increase of GAA activity (1.3-7.5-fold) after imino sugar treatment in fibroblasts from patients carrying the mutations L552P (three patients) and G549R (one patient). GAA enhancement was confirmed in HEK293T cells where the same mutations were overexpressed. No increase of GAA activity was observed for the other mutations. Western blot analysis showed that imino sugars increase the amount of mature GAA molecular forms. Immunofluorescence studies in HEK293T cells overexpressing the L552P mutation showed an improved trafficking of the mutant enzyme to lysosomes after imino sugar treatment. These results provide a rationale for an alternative treatment, other than enzyme replacement, to Pompe disease.