Crystal structure of glycogen debranching enzyme and insights into its catalysis and disease-causing mutations.
ABSTRACT: Glycogen is a branched glucose polymer and serves as an important energy store. Its debranching is a critical step in its mobilization. In animals and fungi, the 170?kDa glycogen debranching enzyme (GDE) catalyses this reaction. GDE deficiencies in humans are associated with severe diseases collectively termed glycogen storage disease type III (GSDIII). We report crystal structures of GDE and its complex with oligosaccharides, and structure-guided mutagenesis and biochemical studies to assess the structural observations. These studies reveal that distinct domains in GDE catalyse sequential reactions in glycogen debranching, the mechanism of their catalysis and highly specific substrate recognition. The unique tertiary structure of GDE provides additional contacts to glycogen besides its active sites, and our biochemical experiments indicate that they mediate its recruitment to glycogen and regulate its activity. Combining the understanding of the GDE catalysis and functional characterizations of its disease-causing mutations provides molecular insights into GSDIII.
Project description:Glycogen storage disorder type III (GSDIII), or debranching enzyme (GDE) deficiency, is a rare metabolic disorder characterized by variable liver, cardiac, and skeletal muscle involvement. GSDIII manifests with liver symptoms in infancy and muscle involvement during early adulthood. Muscle biopsy is mainly performed in patients diagnosed in adulthood, as routine diagnosis relies on blood or liver GDE analysis, followed by AGL gene sequencing. The GSDIII mouse model recapitulate the clinical phenotype in humans, and a nearly full rescue of muscle function was observed in mice treated with the dual AAV vector expressing the GDE transgene.In order to characterize GSDIII muscle morphological spectrum and identify novel disease markers and pathways, we performed a large international multicentric morphological study on 30 muscle biopsies from GSDIII patients. Autophagy flux studies were performed in human muscle biopsies and muscles from GSDIII mice. The human muscle biopsies revealed a typical and constant vacuolar myopathy, characterized by multiple and variably sized vacuoles filled with PAS-positive material. Using electron microscopy, we confirmed the presence of large non-membrane bound sarcoplasmic deposits of normally structured glycogen as well as smaller rounded sac structures lined by a continuous double membrane containing only glycogen, corresponding to autophagosomes. A consistent SQSTM1/p62 decrease and beclin-1 increase in human muscle biopsies suggested an enhanced autophagy. Consistent with this, an increase in the lipidated form of LC3, LC3II was found in patients compared to controls. A decrease in SQSTM1/p62 was also found in the GSDIII mouse model.In conclusion, we characterized the morphological phenotype in GSDIII muscle and demonstrated dysfunctional autophagy in GSDIII human samples.These findings suggest that autophagic modulation combined with gene therapy might be considered as a novel treatment for GSDIII.
Project description:Glycogen storage disease type III (GSDIII) is an autosomal recessive disorder caused by a deficiency of glycogen-debranching enzyme (GDE), which results in profound liver metabolism impairment and muscle weakness. To date, no cure is available for GSDIII and current treatments are mostly based on diet. Here we describe the development of a mouse model of GSDIII, which faithfully recapitulates the main features of the human condition. We used this model to develop and test novel therapies based on adeno-associated virus (AAV) vector-mediated gene transfer. First, we showed that overexpression of the lysosomal enzyme alpha-acid glucosidase (GAA) with an AAV vector led to a decrease in liver glycogen content but failed to reverse the disease phenotype. Using dual overlapping AAV vectors expressing the GDE transgene in muscle, we showed functional rescue with no impact on glucose metabolism. Liver expression of GDE, conversely, had a direct impact on blood glucose levels. These results provide proof of concept of correction of GSDIII with AAV vectors, and they indicate that restoration of the enzyme deficiency in muscle and liver is necessary to address both the metabolic and neuromuscular manifestations of the disease.
Project description:Glycogen storage disease type III (GSDIII) is a metabolic disorder characterized by a deficiency in the glycogen debranching enzyme, amylo-1,6-glucosidase,4-alpha-glucanotransferase (AGL). Patients with GSDIII commonly exhibit hypoglycemia, along with variable organ dysfunction of the liver, muscle or heart tissues. The AGL protein binds to glycogen through its C-terminal region, and possesses two separate domains for the transferase and glucosidase activities. Most causative mutations are nonsense, and how they affect the enzyme is not well understood. Here we investigated four rare missense mutations to determine the molecular basis of how they affect AGL function leading to GSDIII. The L620P mutant primarily abolishes transferase activity while the R1147G variant impairs glucosidase function. Interestingly, mutations in the carbohydrate-binding domain (CBD; G1448R and Y1445ins) are more severe in nature, leading to significant loss of all enzymatic activities and carbohydrate binding ability, as well as enhancing targeting for proteasomal degradation. This region (Y1445-G1448R) displays virtual identity across human and bacterial species, suggesting an important role that has been conserved throughout evolution. Our results clearly indicate that inactivation of either enzymatic activity is sufficient to cause GSDIII disease and suggest that the CBD of AGL plays a major role to coordinate its functions and regulation by the ubiquitin-proteasome system.
Project description:Deficiency of debrancher enzyme causes Glycogen Storage Disease (GSD) type III, an autosomal recessive disorder, characterized by tissue accumulation of abnormally structured glycogen. This report reviews current clinical and molecular knowledge about this disorder and describes the variability at phenotype and genotype levels of a large group of Italian GSDIII patients.
Project description:Amylo-alpha-1-6-glucosidase-4-alpha-glucanotransferase (AGL) is an enzyme primarily responsible for glycogen debranching. Inactivating germline mutations lead to glycogen storage disease III (GSDIII). AGL was found to be a tumor suppressor in xenograft models of bladder cancer (BC) and low levels of expression in BC were associated with poor patient prognosis. However, the impact of AGL levels in normal tissues on susceptibility to carcinogenesis is unknown. We address this gap by developing a full body/germline AGL knock-out (AGL-FBKO) that recapitulates biochemical and histological features of GSDIII. AGL-FBKO mice exposed to N-Butyl-N-(4-hydroxybutyl) nitrosamine (BBN) had higher BC incidence at several time points following exposure, compared to wild type littermates (AGL-FBWT). To determine if this increased BC prevalence was due to decreased expression of AGL in the urothelium, we derived a urothelium specific conditional AGL knock-out (AGL-cKO) mouse driven by a Uroplakin II-Cre/loxP system. BBN experiments with these mice revealed AGL-cKO had higher BC incidence than mice without AGL knock-out (AGL-cWT). RNA-sequencing revealed that tumors from AGL-FBKO had 19 differentially expressed genes compared to AGL-FBWT mice. An “AGL Loss” gene signature was developed and found to successfully stratify normal and tumor samples in two BC patient datasets. These results support the role of AGL as a tumor suppressor. Our work suggests that evaluating AGL expression levels, or AGL KO gene signature scores, in normal urothelium may serve as a predictor for increased risk of BC development in individuals with carcinogenic exposures, such as smoking. Overall design: RNA-seq of BBN induced tumors in AGL-FBWT and AGL-FBKO mice
Project description:BACKGROUND:Branched polymers of glucose are universally used for energy storage in cells, taking the form of glycogen in animals, fungi, Bacteria, and Archaea, and of amylopectin in plants. Some enzymes involved in glycogen and amylopectin metabolism are similarly conserved in all forms of life, but some, interestingly, are not. In this paper we focus on the phylogeny of glycogen branching and debranching enzymes, respectively involved in introducing and removing of the ?(1-6) bonds in glucose polymers, bonds that provide the unique branching structure to glucose polymers. RESULTS:We performed a large-scale phylogenomic analysis of branching and debranching enzymes in over 400 completely sequenced genomes, including more than 200 from eukaryotes. We show that branching and debranching enzymes can be found in all kingdoms of life, including all major groups of eukaryotes, and thus were likely to have been present in the last universal common ancestor (LUCA) but have been lost in seemingly random fashion in numerous single-celled eukaryotes. We also show how animal branching and debranching enzymes evolved from their LUCA ancestors by acquiring additional domains. Furthermore, we show that enzymes commonly perceived as orthologous, such as human branching enzyme GBE1 and E. coli branching enzyme GlgB, are in fact related by a gene duplication and consequently paralogous. CONCLUSIONS:Despite being usually associated with animal liver glycogen and plant starch, energy storage in the form of branched glucose polymers is clearly an ancient process and has probably been present in the last universal common ancestor of all present life. The evolution of the enzymes enabling this form of energy storage is more complex than previously thought and illustrates the need for explicit phylogenomic analysis in the study of even seemingly "simple" metabolic enzymes. Patterns of conservation in the evolution of the glycogen/starch branching and debranching enzymes hint at some as yet unknown mechanisms, as mutations disrupting these patterns lead to a variety of genetic diseases in humans and other mammals.
Project description:Glycogen storage disease type III (GSDIII) is a rare disorder of glycogenolysis due to AGL gene mutations, causing glycogen debranching enzyme deficiency and storage of limited dextrin. Patients with GSDIIIa show involvement of liver and cardiac/skeletal muscle, whereas GSDIIIb patients display only liver symptoms and signs. The International Study on Glycogen Storage Disease (ISGSDIII) is a descriptive retrospective, international, multi-centre cohort study of diagnosis, genotype, management, clinical course and outcome of 175 patients from 147 families (86 % GSDIIIa; 14 % GSDIIIb), with follow-up into adulthood in 91 patients. In total 58 AGL mutations (non-missense mutations were overrepresented and 21 novel mutations were observed) were identified in 76 families. GSDIII patients first presented before the age of 1.5 years, hepatomegaly was the most common presenting clinical sign. Dietary management was very diverse and included frequent meals, uncooked cornstarch and continuous gastric drip feeding. Chronic complications involved the liver (hepatic cirrhosis, adenoma(s), and/or hepatocellular carcinoma in 11 %), heart (cardiac involvement and cardiomyopathy, in 58 % and 15 %, respectively, generally presenting in early childhood), and muscle (pain in 34 %). Type 2 diabetes mellitus was diagnosed in eight out of 91 adult patients (9 %). In adult patients no significant correlation was detected between (non-) missense AGL genotypes and hepatic, cardiac or muscular complications. This study demonstrates heterogeneity in a large cohort of ageing GSDIII patients. An international GSD patient registry is warranted to prospectively define the clinical course, heterogeneity and the effect of different dietary interventions in patients with GSDIII.
Project description: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.
Project description:BACKGROUND:Glycogen storage disease type III (GSDIII) is caused by mutations of AGL gene with debranching enzyme deficiency. Patients with GSDIII manifest fasting hypoglycemia, hepatomegaly, hepatopathy, myopathy, and cardiomyopathy. We report on an 18-year-old boy with a profound growth retardation (<3 SD) besides typical clinical features of GSDIII, whereby endocrinological studies were negative. METHODS AND RESULTS:Molecular analysis of AGL gene revealed the homozygous reported variant c.3903_3904insA. Since discordant results from segregation studies showed the carrier status in one parent only, SNP array and short tandem repeats analyses were performed, revealing a paternal disomy of chromosome 1 (UPD1). CONCLUSION:This study describes the first case of GSDIII resulting from UPD1. UPD can play an important role even in case of imprinted genes. DIRAS3 is a maternally imprinted tumor suppressor gene, located on chromosome 1p31, and implicated in growth and oncogenesis. It can be speculated that DIRAS3 overexpression might have a role in the severe short stature of our patient. The study emphasizes the importance of parental segregation analysis especially in patients with recessive conditions to look for specific genetic causes of disease and to estimate properly the risk of family recurrence.
Project description:BACKGROUND:Bladder cancer is the most common malignancy of the urinary system, yet our molecular understanding of this disease is incomplete, hampering therapeutic advances. METHODS:Here we used a genome-wide functional short-hairpin RNA (shRNA) screen to identify suppressors of in vivo bladder tumor xenograft growth (n = 50) using bladder cancer UMUC3 cells. Next-generation sequencing was used to identify the most frequently occurring shRNAs in tumors. Genes so identified were studied in 561 patients with bladder cancer for their association with stratification of clinical outcome by Kaplan-Meier analysis. The best prognostic marker was studied to determine its mechanism in tumor suppression using anchorage-dependent and -independent growth, xenograft (n = 20), and metabolomic assays. Statistical significance was determined using two-sided Student t test and repeated-measures statistical analysis. RESULTS:We identified the glycogen debranching enzyme AGL as a prognostic indicator of patient survival (P = .04) and as a novel regulator of bladder cancer anchorage-dependent (P < .001), anchorage-independent (mean ± standard deviation, 180 ± 23.1 colonies vs 20±9.5 in control, P < .001), and xenograft growth (P < .001). Rescue experiments using catalytically dead AGL variants revealed that this effect is independent of AGL enzymatic functions. We demonstrated that reduced AGL enhances tumor growth by increasing glycine synthesis through increased expression of serine hydroxymethyltransferase 2. CONCLUSIONS:Using an in vivo RNA interference screen, we discovered that AGL, a glycogen debranching enzyme, has a biologically and statistically significant role in suppressing human cancer growth.