Molecular characterization of glycogen storage disease type III

Deficiency of the glycogen debranching enzyme (gene, AGL) causes glycogen storage disease type III (GSD-III), an autosomal recessive disease affecting glycogen metabolism. Most GSD-III patients have AGL deficiency in both the liver and muscle (type IIIa), but some have it in the liver but not muscle (type IIIb). Cloning of human AGL cDNAs and determination of the genomic structure and mRNA isoforms of AGL have allowed for the study of GSD-III at the molecular level. In turn, the resulting information has greatly facilitated our understanding of the molecular basis of this storage disease with remarkable clinical and enzymatic variability. In this review, we summarize all 31 GSD-III mutations in the literature and discuss their clinical and laboratory implications. Most of the mutations are nonsense mutations caused by a nucleotide substitution or small insertion or deletion; only one is caused by a missense amino acid change. Some important genotype-phenotype correlation have emerged, in particular, that exon 3 mutations (17delAG and Q6X) are specifically associated with GSD-IIIb. Three other mutations have appeared to have some phenotype correlation. Specifically, the splice mutation IVS32-12A>G was found in GSD-III patients having mild clinical symptoms, while the mutations 3965delT and 4529insA are associated with a severe phenotype and early onset of clinical manifestations. A molecular diagnostic scheme has been proposed to diagnose GSD-III noninvasively. The characterization of AGL mutations in GSD-III patients has also helped the structure-function analysis of this bifunctional enzyme important for glycogen metabolism.     

Definitive prenatal diagnosis for type III glycogen storage disease.

Prenatal diagnosis for type III glycogen storage disease was performed by using (1) immunoblot analysis with a polyclonal antibody prepared against purified porcine-muscle debranching enzyme and (2) a qualitative assay for debranching-enzyme activity. Cultured amniotic fluid cells from three pregnancies (three families in which the proband had absence of debrancher protein) were subjected to immunoblot analysis. Two unaffected and one affected fetus were predicted. In addition, cultured amniotic fluid cells from nine pregnancies (eight families) were screened with a qualitative assay based on the persistence of a polysaccharide that has a structure approaching that of a phosphorylase limit dextrin when the cells were exposed to a glucose-free medium. This qualitative assay predicted six unaffected and three affected fetuses. All predictions by either method were confirmed postnatally except for one spontaneously aborted fetus. Our data indicate that a definitive diagnosis of type III glycogen storage disease can be made prenatally by these methods.     

Case report: Glycogen storage disease type III in Inuit children

GLYCOGEN STORAGE DISEASE TYPE III (GSD III) was diagnosed in 4 Inuit children (3 confirmed, 1 suspected case) at our institution over the last decade. This rare autosomal recessive disease, which results from a deficiency of the debranching enzyme required for complete degradation of the glycogen molecule, has not been previously described in this population. The possible clinical presentations are heterogeneous, as is the spectrum of severity of this disease. The long-term sequelae can be severe, including recurrent hypoglycemia, hepatic cirrhosis and progressive muscle weakness. These 4 cases would suggest an increased prevalence of GSD III in the Inuit population. Therefore, it is important for health care providers caring for this population to consider and recognize this rare but serious disease.Glycogen storage disease type III (GSD III) is part of a rare group of inherited enzyme defects that affect the glycogen synthesis and degradation cycle. It is caused by an autosomal recessive deficiency of the glycogen debranching enzyme, amylo-1,6-glucosidase, which results in incomplete degradation of the glycogen molecule. This enzyme is critical in both liver and muscle tissue. Deficiency of the enzyme in both these tissues produces a variant known as GSD IIIa, which can involve skeletal and cardiac muscle. Another common variant, known as GSD IIIb, is caused by a deficiency of the enzyme in the liver only and is not associated with any muscle involvement.¹ In their severest forms, both variants can present in infancy and early childhood with hepatomegaly, hypoglycemia and growth retardation. Milder cases may present only in adulthood, with asymptomatic hepatomegaly, occult liver disease or myopathy.² Although fasting tolerance for hypoglycemia generally improves with age and hepatomegaly frequently regresses,³ chronic fibrosis leading to overt cirrhosis and end-stage liver disease may occur in a small portion of patients with GSD III.^4,5,6To our knowledge, there are no published cases of GSD III in the Aboriginal population of North America. We describe 3 confirmed cases and 1 suspected case of GSD III in Inuit children from northern Quebec and eastern Nunavut, with different clinical presentations.     

Binding of glycogen, oligosaccharides, and glucose to glycogen debranching enzyme

The binding of glucose and a series of oligosaccharides to glycogen debranching enzyme was determined by the ability of the saccharides to decrease the rate of reaction of sulfhydryl groups with 5,5'-dithiobis(2-nitrobenzoate) (DTNB). At pH 7.2, the strength of binding increases with chain length from glucose to maltotriose to maltopentaose but not to maltohexaose, and the free energies for binding of the oligosaccharides suggest subsites of equivalent affinities for the four glucose units following the initial reducing moiety. The rate of reaction of DTNB with enzyme saturated with saccharide is the same for all compounds, suggesting that all the saccharides, including glucose, induce the same conformational state. The site of binding may be that which binds the alpha-1,6-linked side chain of the natural limit dextrin substrate. At pH 8.0, this site exhibits similar characteristics, but an additional site, which may bind the four terminal glucose units of the main chain of the natural substrate, is manifested and exhibits different characteristics, including a very low affinity for glucose itself. The binding of glycogen to the debranching enzyme was monitored by centrifugal separation from the protein and exhibits a much lower dissociation constant than that for the oligomers, suggesting that branched polymers have more than one set of subsites.     

Glycogen storage disease type III: A novel Agl knockout mouse model

Glycogen storage disease type III is an autosomal recessive disease characterized by a deficiency in the glycogen debranching enzyme, encoded by AGL. Essential features of this disease are hepatomegaly, hypoglycemia, hyperlipidemia, and growth retardation. Progressive skeletal myopathy, neuropathy, and/or cardiomyopathy become prominent in adults. Currently, there is no available cure. We generated an Agl knockout mouse model by deletion of the carboxy terminus of the protein, including the carboxy end of the glucosidase domain and the glycogen-binding domain. Agl knockout mice presented serious hepatomegaly, but we did not observe signs of cirrhosis or adenomas. In affected tissues, glycogen storage was higher than in wild-type mice, even in the central nervous system which has never been tested in GSDIII patients. The biochemical findings were in accordance with histological data, which clearly documented tissue impairment due to glycogen accumulation. Indeed, electron microscopy revealed the disruption of contractile units due to glycogen infiltrations. Furthermore, adult Agl knockout animals appeared less prompt to move, and they exhibited kyphosis. Three-mo-old Agl knockout mice could not run, and adult mice showed exercise intolerance. In addition, older affected animals exhibited an accelerated respiratory rate even at basal conditions. This observation was correlated with severe glycogen accumulation in the diaphragm. Diffuse glycogen deposition was observed in the tongues of affected mice. Our results demonstrate that this Agl knockout mouse is a reliable model for human glycogenosis type III, as it recapitulates the essential phenotypic features of the disease.     

Escherichia coli DNA topoisomerase III: purification and characterization of a new type I enzyme

A new topoisomerase capable of relaxing negatively supercoiled DNA in Escherichia coli has been identified during chromatography on novobiocin-Sepharose. A simple and reproducible purification procedure is described to obtain this enzyme, called topoisomerase III (topo III), in a homogeneous form. The protein is a single polypeptide with a molecular weight of 74 000 +/- 2000 and is a type I topoisomerase, changing the linking number of DNA circles in steps of one. It is present in deletion strains lacking the topA gene and further differs from the well-studied topoisomerase I (omega protein; Eco topo I) in (1) its requirement for K+ in addition to Mg2+ to exhibit optimal activity and (2) its affinity to novobiocin-Sepharose. Positively supercoiled DNA is not relaxed during exposure to the enzyme. Topo III has no ATPase activity, and ATP does not show any discernible effect on the reduction of superhelical turns. The purified topoisomerase has no supercoiling activity and is unaffected by high concentrations of oxolinic acid and novobiocin in the relaxing reaction. Single-stranded DNA and spermidine strongly inhibit the topoisomerase activity.     

Cloning and characterization of the glycogen branching enzyme gene existing in tandem with the glycogen debranching enzyme from Pectobacterium chrysanthemi PY35

The glycogen branching enzyme gene (glgB) from Pectobacterium chrysanthemi PY35 was cloned, sequenced, and expressed in Escherichia coli. The glgB gene consisted of an open reading frame of 2196bp encoding a protein of 731 amino acids (calculated molecular weight of 83,859Da). The glgB gene is upstream of glgX and the ORF starts the ATG initiation codon and ends with the TGA stop codon at 2bp upstream of glgX. The enzyme was 43-69% sequence identical with other glycogen branching enzymes. The enzyme is the most similar to GlgB of E. coli and contained the four regions conserved among the alpha-amylase family. The glycogen branching enzyme (GlgB) was purified and the molecular weight of the enzyme was estimated to be 84kDa by SDS-PAGE. The glycogen branching enzyme was optimally active at pH 7 and 30 degrees C.     

Branching enzyme activity of cultured amniocytes and chorionic villi: prenatal testing for type IV glycogen storage disease.

Although type IV glycogen storage disease (Andersen disease; McKusick 23250) is considered to be a rare, autosomally recessive disorder, of the more than 600 patients with glycogenosis identified in our laboratory by enzymatic assays, 6% have been shown to be deficient in the glycogen branching enzyme. Most of the 38 patients with type IV glycogen storage disease who are known to us have succumbed at a very early age, with the exception of one male teenager, an apparently healthy 7-year-old male, and several 5-year-old patients. Fourteen pregnancies at risk for branching enzyme deficiency have been monitored using cultured amniotic fluid cells, and four additional pregnancies have been screened using cultured chorionic villi. Essentially no branching enzyme activity was detectable in eight samples (amniocytes); activities within the control range were found in five samples (three amniocyte and two chorionic villi samples); and five samples appeared to have been derived from carriers. In two of the cases lacking branching enzyme activity, in which the pregnancies were terminated and fibroblasts were successfully cultured from the aborted fetuses, no branching enzyme activity was found. Another fetus, which was predicted by antenatal assay to be affected, was carried to term. Skin fibroblasts from this baby were deficient in branching enzyme. Pregnancies at risk for glycogen storage disease due to the deficiency of branching enzyme can be successfully monitored using either cultured chorionic villi or amniocytes.     

Donor substrate specificity of 4-alpha-glucanotransferase of porcine liver glycogen debranching enzyme and complementary action to glycogen phosphorylase on debranching

Glycogen debranching enzyme (GDE) has both 4-alpha-glucanotransferase and amylo-alpha-1,6-glucosidase activities. Here, we examined 4-alpha-glucanotransferase action of porcine liver GDE on four 6(4)-O-alpha-maltooligosyl-pyridylamino(PA)-maltooctaoses, in the presence or absence of an acceptor, maltohexaose. HPLC analysis of digested fluorogenic branched dextrins revealed that in the presence or absence of acceptor, 6(4)-O-alpha-glucosyl-PA-maltooctaose (B4/81) was liberated from 6(4)-O-alpha-maltopentaosyl-PA-maltooctaose (B4/85), 6(4)-O-alpha-maltotetraosyl-PA-maltooctaose (B4/84) and 6(4)-O-alpha-maltotriosyl-PA-maltooctaose (B4/83), whereas 6(4)-O-alpha-maltosyl-PA-maltooctaose (B4/82) was resistant to the enzyme. The fluorogenic product was further hydrolyzed by amylo-alpha-1,6-glucosidase to PA-maltooctaose (G8PA) and glucose. The ratio of the rates of 4-alpha-glucanotransferase actions on B4/85, B4/84 and B4/83 in the absence of the acceptor was 0.15, 0.42 and 1.00, respectively. The rates increased with increasing amounts of acceptor, changing the ratio of the rates to 0.09, 1.00 and 0.60 (with 0.5 mM maltohexaose) and 0.10, 1.00 and 0.58 (with 1.0 mM maltohexaose), respectively. Donor substrate specificity of GDE 4-alpha-glucanotransferase suggests complementary action of GDE and glycogen phosphorylase on glycogen degradation in the porcine liver. Glycogen phosphorylase degrades the maltooligosaccharide branches of glycogen by phosphorolysis to form maltotetraosyl branches, and phosphorolysis does not proceed further. GDE 4-alpha-glucanotransferase removes a maltotriosyl residue from the maltotetraosyl branch such that the alpha-1,6-linked glucosyl residue is retained.     

Molecular and biochemical characterization of a novel intronic single point mutation in a Tunisian family with glycogen storage disease type III

Genetic deficiency of the glycogen debranching enzyme causes glycogen storage disease type III, an autosomal recessive inherited disorder. The gene encoding this enzyme is designated as AGL gene. The disease is characterized by fasting hypoglycemia, hepatomegaly, growth retardation, progressive myopathy and cardiomyopathy. In the present study, we present clinical features and molecular characterization of two consanguineous Tunisian siblings suffering from Glycogen storage disease type III. The full coding exons of the AGL gene and their corresponding exon–intron boundaries were amplified for the patients and their parents. Gene sequencing identified a novel single point mutation at the conserved polypyrimidine tract of intron 21 in a homozygous state (IVS21-8A>G). This variant cosegregated with the disease and was absent in 102 control chromosomes. In silico analysis using online resources showed a decreased score of the acceptor splice site of intron 21. RT-PCR analysis of the AGL splicing pattern revealed a 7 bp sequence insertion between exon 21 and exon 22 due to the creation of a new 3′ splice site. The predicted mutant enzyme was truncated by the loss of 637 carboxyl-terminal amino acids as a result of premature termination. This novel mutation is the first mutation identified in the region of Bizerte and the tenth AGL mutation identified in Tunisia. Screening for this mutation can improve the genetic counseling and prenatal diagnosis of GSD III.     

Purification of glycogen debranching enzyme from porcine brain: evidence for glycogen catabolism in the brain

Amylo-1,6-glucosidase from porcine brain was purified to homogeneity by ammonium sulfate fractionation, followed by sequential steps of liquid chromatography on DEAE-Sephacel, Sephacryl S-300, and Super Q. The purified enzyme had both maltooligosaccharide transferase and amylo-1,6-glucosidase activities within a single polypeptide chain, and the combination of these two activities removed the branches of phosphorylase limit dextrin. Based on these results, the purified enzyme was identified as a glycogen debranching enzyme (GDE). The molecular weight of the brain GDE was 170,000 by gel-filtration and 165,000 by reducing SDS-PAGE. The pH profile of maltooligosaccharide transferase activity coincided with that of the amylo-1,6-glucosidase activity (pH optimum at 6.0). The existence of GDE as well as glycogen phosphorylase in the brain explains brain glycogenolysis fully and supports the hypothesis that glycogen is a significant source of energy in this organ.     

Molecular cloning and nucleotide sequence of cDNA encoding human muscle glycogen debranching enzyme.

cDNA comprising the entire length of the human muscle glycogen debranching enzyme was cloned and its nucleotide sequence determined. The debrancher mRNA includes a 4545-base pair coding region and a 2371-base pair 3'-nontranslated region. The calculated molecular mass of the debrancher protein derived from cDNA sequence is 172,614 daltons, consistent with the estimated size of purified protein (Mr 165,000 +/- 500). A partial amino acid sequence (13 internal tryptic peptides with a total of 213 residues) determined on peptides derived from purified porcine muscle debrancher protein confirmed the identity of the cDNA clone. Comparison of the amino acid sequence predicted from the human glycogen debrancher cDNA with the partial protein sequence of the porcine debrancher revealed a high degree (88%) of interspecies sequence identity. RNA blot analysis showed that debrancher mRNA in human muscle, lymphoblastoid cells, and in porcine muscle are all similar in size (approximately 7 kilobases). Two patients with inherited debrancher deficiency had a reduced level of debrancher mRNA, whereas two other patients had no detectable abnormality in RNA blots. The isolation of the debrancher cDNA and determination of its primary structure is an important step toward defining the structure-function relationship of this multifunctional enzyme and in understanding the molecular basis of the type III glycogen storage disease.     

Isolation and characterization of the gene encoding yeast debranching enzyme.

Using a genetic screen aimed at identifying cellular factors involved in Ty1 transposition, we have identified a mutation in a host gene that reduces Ty1 transposition frequency. The mutant, dbr1, is also defective in the process of intron turnover. In dbr1 cells, excised introns derived from a variety of pre-mRNAs are remarkably stable and accumulate to levels exceeding that of the corresponding mRNA. The stable excised introns accumulate in the form of a lariat that is missing the linear sequences 3' of the branchpoint. The DBR1 gene has been isolated by complementation of the transposition phenotype. DBR1 is shown to encode debranching enzyme, an RNA processing activity that hydrolyzes the 2'-5' phosphodiester linkage at the branchpoint of excised intron lariats. In Saccharomyces cerevisiae, debranching enzyme plays a requisite role in the rapid turnover of excised introns, yet its function is not essential for viability.     

Alzheimer's disease amyloid beta-clipping enzyme (APP secretase): identification, purification, and characterization of the enzyme.

Alzheimer's disease (AD) is the most frequent cause of dementia, although no genetic abnormality has been identified. Recent studies have elucidated the molecular defect in AD, including the abnormal deposition of amyloid beta peptide (beta/A4) in senile plaques of affected individuals. Normal brain contains the enzyme, APP secretase, which cleaves inside the beta/A4 portion of the precursor protein (APP); abnormal processing of APP occurs in AD brain. Until now, no evidence has been provided that APP secretase is an intracellular proteinase. We have now prepared two synthetic substrates of APP secretase, both of which contain the cleavage point and are much more sensitive than substrates previously available to identify APP secretase. Using these substrates, we found an intracellular proteinase that has APP secretase activity. This proteinase has been identified as cathepsin B.     

Glucose-free/high-protein diet improves hepatomegaly and exercise intolerance in glycogen storage disease type III mice

Glycogen disease type III (GSDIII), a rare incurable autosomal recessive disorder due to glycogen debranching enzyme deficiency, presents with liver, heart and skeletal muscle impairment, hepatomegaly and ketotic hypoglycemia. Muscle weakness usually worsens to fixed myopathy and cardiac involvement may present in about half of the patients during disease. Management relies on careful follow-up of symptoms and diet. No common agreement was reached on sugar restriction and treatment in adulthood.We administered two dietary regimens differing in their protein and carbohydrate content, high-protein (HPD) and high-protein/glucose-free (GFD), to our mouse model of GSDIII, starting at one month of age. Mice were monitored, either by histological, biochemical and molecular analysis and motor functional tests, until 10?months of age.GFD ameliorated muscle performance up to 10?months of age, while HPD showed little improvement only in young mice. In GFD mice, a decreased muscle glycogen content and fiber vacuolization was observed, even in aged animals indicating a protective role of proteins against skeletal muscle degeneration, at least in some districts. Hepatomegaly was reduced by about 20%. Moreover, the long-term administration of GFD did not worsen serum parameters even after eight months of high-protein diet. A decreased phosphofructokinase and pyruvate kinase activities and an increased expression of Krebs cycle and gluconeogenesis genes were seen in the liver of GFD fed mice.Our data show that the concurrent use of proteins and a strictly controlled glucose supply could reduce muscle wasting, and indicate a better metabolic control in mice with a glucose-free/high-protein diet.     

Molecular diagnosis of type 1c glycogen storage disease

Glycogen storage disease type 1 (GSD 1) results from deficiency of the microsomal multicomponent glucose-6-phosphatase system. Malfunction of the catalytic subunit characterises GSD 1a. GSD 1b and GSD 1c are characterised by defective microsomal glucose-6-phosphate or pyrophosphate/phosphate transport, respectively. Recently, a gene encoding a microsomal transporter protein has been found to be mutated in GSD 1b and 1c patients. Here, we report the genomic sequence of the transporter gene and the detection of a homozygous 2-bp deletion (1211delCT) and a homozygous donor splice site mutation (317+1G→T) in two GSD 1c patients, confirming that GSD 1c is allelic to GSD 1b. Received: 16 October 1998 / Accepted: 11 January 1998     

Cell death and stress signaling in glycogen storage disease type I

Cell death has been traditionally classified in apoptosis and necrosis. Apoptosis, known as programmed cell death, is an active form of cell death mechanism that is tightly regulated by multiple cellular signaling pathways and requires ATP for its appropriate process. Apoptotic death plays essential roles for successful development and maintenance of normal cellular homeostasis in mammalian. In contrast to apoptosis, necrosis is classically considered as a passive cell death process that occurs rather by accident in disastrous conditions, is not required for energy and eventually induces inflammation. Regardless of different characteristics between apoptosis and necrosis, it has been well defined that both are responsible for a wide range of human diseases. Glycogen storage disease type I (GSD-I) is a kind of human genetic disorders and is caused by the deficiency of a microsomal protein, glucose-6-phosphatase-α (G6Pase-α) or glucose-6-phosphate transporter (G6PT) responsible for glucose homeostasis, leading to GSD-Ia or GSD-Ib, respectively. This review summarizes cell deaths in GSD-I and mostly focuses on current knowledge of the neutrophil apoptosis in GSD-Ib based upon ER stress and redox signaling.