Biosynthesis of acid alpha-glucosidase in late-onset forms of glycogenosis type II (Pompe's disease)

Cultured human skin fibroblasts from control persons and from patients with the generalized and late-onset forms of Pompe's disease were labelled with radioactive leucine and the incorporation of radioactivity into acid alpha-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 NH4Cl, the labelling of secreted alpha-glucosidase relative to that of secreted cathepsin D in fibroblasts from patients with the late-onset form of Pompe's disease was less than 15% of that in fibroblasts from control persons. However, when the fibroblasts were labelled for less than 1 h, the relative rate of incorporation of radioactivity into acid alpha-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 alpha-glucosidase could be detected.     

Myophosphorylase deficiency (glycogenosis type V; McArdle disease)

McArdle disease, one of the most common metabolic causes of exercise intolerance and recurrent myoglobinuria, is due to biochemical defects of the muscle isoform of glycogen phosphorylase. The gene for myophosphorylase (PGYM) is on chromosome 11, and 33 distinct mutations have been identified in patients from all over the world. In Caucasians, a nonsense mutation in exon 1 (R49X) is common enough to warrant screening of genomic DNA from blood before considering muscle biopsy. Other mutations are prevalent in different ethnic groups or are "private". Mutations are spread throughout the gene and there is no clear genotype:phenotype correlation. High-protein diet and aerobic exercise are beneficial, and gene therapy appears promising.     

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.     

Towards a molecular therapy for glycogen storage disease type II (Pompe disease)

Glycogen storage disease type II (GSD-II), also known as Pompe disease, is a fatal genetic muscle disorder caused by a deficiency of acid alpha-glucosidase, a glycogen-degrading lysosomal enzyme. Currently, there is no treatment for this fatal disorder. However, several lines of research suggest the possibility of future treatment. Enzyme replacement strategies hold the greatest hope for patients currently affected by GSD-II, but future strategies could include in vivo or ex vivo gene therapy approaches and/or mesenchymal stem cell or bone-marrow transplantation approaches. Each of the approaches might eventually be combined to further improve the overall clinical efficacy of any one treatment regimen. The lessons learned from GSD-II research will also benefit a great number of individuals affected by other genetic disorders.     

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.     

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.     

Bovine generalised glycogenosis type II. Uptake of lysosomal alpha-glucosidase by cultured skeletal muscle and reversal of glycogen accumulation

acid alpha-glucosidase (EC 3.2.1.20) was purified from fetal bovine muscle by affinity chromatography on concanavalin A and Sephadex G-100 and added to the culture medium of mature muscle cultures from animals affected by glycogenosis type II. The enzyme activity in homogenates of treated cultures was significantly increased within 4 h of the addition of enzyme, was maximal by 18 h and the internalised activity was stable for at least 48 h after the removal of the enzyme from the culture medium. The acid alpha-glucosidase activity was internalised with an uptake constant of 300 nM and a Vmax of uptake of 133 nmol/h per mg protein. The glycogen concentration in affected cultures treated with exogenous acid alpha-glucosidase for 24 h had decreased by 20% and after a further 24 h of culture was comparable to the concentration observed in cultures from non-affected animals.     

Cell-free translation of human lysosomal alpha-glucosidase: evidence for reduced precursor synthesis in an adult patient with glycogenosis type II

Early events in the biosynthesis of alpha-glucosidase (EC 3.2.1.20) were studied in a wheat-germ cell-free translation system, using control and mutant RNA. In vitro, the primary translation product of the alpha-glucosidase mRNA is a 100 kDa protein. When canine microsomal membranes are added to the translation system, the nascent alpha-glucosidase precursor is cotranslationally transported across the microsomal membranes, yielding a 110 kDa glycosylated form. This protein has the same electrophoretic characteristics as the alpha-glucosidase precursor observed after in vivo labeling of control fibroblasts. Inhibition of glycosylation in vivo by tunicamycin or deglycosylation of the in vivo synthesized alpha-glucosidase precursor by glycopeptidase F reveals a core protein similar in molecular mass to the primary translation product. Total RNA from a patient with the adult form of glycogenosis type II is not able to direct the synthesis of normal amounts of alpha-glucosidase in vitro. Northern blot analysis of the RNA, using cloned alpha-glucosidase cDNA sequences as a probe, demonstrates that in this patient the amount of the 3.4 kb alpha-glucosidase mRNA is highly reduced. The results indicate that the synthesis or stability of the mRNA is affected.     

Uptake and stability of human and bovine acid alpha-glucosidase in cultured fibroblasts and skeletal muscle cells from glycogenosis type II patients

Acid alpha-glucosidase (EC 3.2.1.20) was purified from human placenta and bovine testis by affinity chromatography using concanavalin A (conA) and Sephadex G 200. When added to the culture medium of human fibroblasts, the enzyme purified from bovine testis is taken up with a 200-fold higher efficiency than the enzyme from human placenta. Uptake of acid alpha-glucosidase from bovine testis is mediated by the mannose-6-phosphate receptor, whereas only a minor fraction of placental enzyme appears to be equipped with the mannose-6-phosphate recognition marker. Once internalized, both human and bovine acid alpha-glucosidase demonstrate a half-life of about 10 days in fibroblasts from control individuals and patients with different clinical forms of glycogenosis type II (Pompe's disease, acid alpha-glucosidase deficiency). Evidence is presented that the mannose-6-phosphate receptor is also present on the plasma membrane of the clonal myogenic skeletal muscle cell lines G8-1 and L6J1 (respectively from mouse and rat origin) and on cultured human skeletal muscle cells derived from a muscle biopsy. Addition of bovine testis acid alpha-glucosidase to skeletal muscle cell cultures from an adult patient with glycogenosis type II leads to complete correction of the enzyme deficiency.     

Biochemical, immunological, and cell genetic studies in glycogenosis type II.

Fibroblasts from patients with the adult, juvenile, and infantile form of glycogenosis type II (Pompe disease) were cultured under standardized conditions, and the activity of acid alpha-glucosidase (E.C.3.2.1.20) towards glycogen, maltose, and 4-methylumbelliferyl-alpha-D-glucopyranoside was measured. Glycogen levels in muscle biopsies and in cultured fibroblasts from patients were determined. Residual enzyme activities varying from 7%-22% were detected in fibroblasts from patients with the adult form but not from patients with the infantile form of glycogenosis II. An inverse correlation was found between the severity of the clinical manifestation and the degree of residual enzyme activity in the fibroblasts. The kinetic and electrophoretic properties of acid alpha-glucosidase in fibroblasts from the adult patients and from control individuals were similar. Immunological studies suggested that the decrease of acid alpha-glucosidase activity is caused by a mutation that affects the production or degradation of the enzyme rather than its catalytic activity. Complementation studies were carried out by fusing fibroblasts from patients with the adult, juvenile, and infantile form of glycogenosis II, but neither conventional assays on multikaryons nor enzyme assays on single binuclear heterokaryons gave any evidence for genetic heterogeneity among these forms.     

Acid alpha-glucosidase deficiency: Identification and expression of a missense mutation (S529V) in a Japanese adult phenotype

We report a missense mutation in an adult Japanese patient with acid alpha-glucosidase (GAA) deficiency. A TC to GT transition at nucleotides 1585–1586, was identified. This transition resulted in an amino acid substitution of Ser-529 to Val (S529V) in exon 11. We also have demonstrated that the S529V mutation abolishes the catalytic activity of the enzyme. Our data suggest that this mutation is the cause of the clinical manifestation known as adult-onset GAA deficiency. The missense mutation described here is a new mutation, and the first identified in Japanese patients with GAA deficiency. Received: 23 May 1995     

A newly identified c.1824_1828dupATACG mutation in exon 13 of the GAA gene in infantile-onset glycogen storage disease type II (Pompe disease)

Pompe disease or glycogen storage disease type II is a glycogen storage disorder associated with malfunction of the acid α-glucosidase enzyme (GAA; EC.3.2.1.3) leading to intracellular aggregations of glycogenin muscles. The infantile-onset type is the most life-threatening form of this disease, in which most of patients suffer from cardiomyopathy and hypotonia in early infancy. In this study, a typical case of Pompe disease was reported in an Iranian patient using molecular analysis of the GAA gene. Our results revealed a new c.1824_1828dupATACG mutation in exon 13 of the GAA gene. In conclusion, with the finding of this novel mutation, the genotypic spectrum of Iranian patients with Pompe disease has been extended, facilitating the definition of disease-related mutations.     

Leukocyte adhesion deficiency type II

Leukocyte adhesion deficiency type II (LAD II) is a rare disorder characterized by recurrent infections, persistent leukocytosis, and severe mental and growth retardation. LAD II neutrophils are deficient in expression of selectin ligand activity, and exhibit a correspondingly diminished ability to roll on endothelium and to traffic to inflammatory sites in vivo. LAD II patients exhibit a deficiency in the expression of cell surface fucosylated glycan structures that include the H and Lewis blood group determinants and the sialyl Lewis x epitope, yet the corresponding fucosyltransferase activities responsible for synthesis of these structures are expressed at normal levels. The molecular defect in LAD II has been localized to the pathway that synthesizes GDP-fucose from GDP-mannose. However, the two known component enzymes in this GDP-fucose biosynthetic pathway are normal in sequence and in expression levels in LAD II cells. The genetic lesion in LAD II that accounts for the generalized fucosylation defect in LAD II patients remains to be determined.     

alpha-Glucosidase deficiency (Pompe's disease)

alpha-Glucosidase is deficient (less than 30% of control) in Pompe's disease, but the extent of the deficiency does not always correlate with the severity of the clinical symptoms. The defects that lead to a deficiency of alpha-glucosidase include synthesis of catalytically inactive protein, absence of mRNA for the enzyme, decreased synthesis of the precursor, lack of phosphorylation of the precursor, impaired conversion of the precursor to the mature enzyme and synthesis of unstable precursor. A single type of defect can lead to different clinical phenotypes. The precursor of alpha-glucosidase is present in the brush border of the polarized epithelial cells of small intestine and kidney and is secreted into urine.