Glycogen metabolism in tissues from a mouse model of Lafora disease

Laforin, encoded by the EPM2A gene, by sequence is a member of the dual specificity protein phosphatase family. Mutations in the EPM2A gene account for around half of the cases of Lafora disease, an autosomal recessive neurodegenerative disorder, characterized by progressive myoclonus epilepsy. The hallmark of the disease is the presence of Lafora bodies, which contain polyglucosan, a poorly branched form of glycogen, in neurons, muscle and other tissues. Glycogen metabolizing enzymes were analyzed in a transgenic mouse over-expressing a dominant negative form of laforin that accumulates Lafora bodies in several tissues. Skeletal muscle glycogen was increased 2-fold as was the total glycogen synthase protein. However, the -/+glucose-6-P activity of glycogen synthase was decreased from 0.29 to 0.16. Branching enzyme activity was increased by 30%. Glycogen phosphorylase activity was unchanged. In whole brain, no differences in glycogen synthase or branching enzyme activities were found. Although there were significant differences in enzyme activities in muscle, the results do not support the hypothesis that Lafora body formation is caused by a major change in the balance between glycogen elongation and branching activities.     

Dimerization of Vaccinia virus VH1 is essential for dephosphorylation of STAT1 at tyrosine 701

The gene product of Vaccinia virus gene H1, VH1, is the first identified dual specificity phosphatase (DSP). The human genome encodes 38 different VH1-like DSPs, which include major regulators of signaling pathways, highly dysregulated in disease states. VH1 down-regulates cellular antiviral response by dephosphorylating activated STAT1 in the IFN-γ/STAT1 signaling pathway. In this report, we have investigated the molecular basis for VH1 catalytic activity. Using small-angle x-ray scattering (SAXS), we determined that VH1 exists in solution as a boomerang-shaped dimer. Targeted alanine mutations in the dimerization domain (aa 1-27) decrease phosphatase activity while leaving the dimer intact. Deletion of the N-terminal dimer swapped helix (aa 1-20) completely abolishes dimerization and severely reduces phosphatase activity. An engineered chimera of VH1 that contains only one active site retains wild-type levels of catalytic activity. Thus, a dimeric quaternary structure, as opposed to two cooperative active sites within the same dimer is essential for VH1 catalytic activity. Together with laforin, VH1 is the second DSP reported in literature for which dimerization via an N-terminal dimerization domain is necessary for optimal catalytic activity. We propose that dimerization may represent a common mechanism to regulate the activity and substrate recognition of DSPs, often assumed to function as monomers.     

Protein Degradation and Quality Control in Cells from Laforin and Malin Knockout Mice

Lafora disease is a progressive myoclonus epilepsy caused by mutations in the EPM2A or EPM2B genes that encode a glycogen phosphatase, laforin, and an E3 ubiquitin ligase, malin, respectively. Lafora disease is characterized by accumulation of insoluble, poorly branched, hyperphosphorylated glycogen in brain, muscle, heart, and liver. The laforin-malin complex has been proposed to play a role in the regulation of glycogen metabolism and protein quality control. We evaluated three arms of the protein degradation/quality control process (the autophago-lysosomal pathway, the ubiquitin-proteasomal pathway, and the endoplasmic reticulum (ER) stress response) in mouse embryonic fibroblasts from Epm2a^−/−, Epm2b^−/−, and Epm2a^−/− Epm2b^−/− mice. The levels of LC3-II, a marker of autophagy, were decreased in all knock-out cells as compared with wild type even though they still showed a slight response to starvation and rapamycin. Furthermore, ribosomal protein S6 kinase and S6 phosphorylation were increased. Under basal conditions there was no effect on the levels of ubiquitinated proteins in the knock-out cells, but ubiquitinated protein degradation was decreased during starvation or stress. Lack of malin (Epm2b^−/− and Epm2a^−/− Epm2b^−/− cells) but not laforin (Epm2a^−/− cells) decreased LAMP1, a lysosomal marker. CHOP expression was similar in wild type and knock-out cells under basal conditions or with ER stress-inducing agents. In conclusion, both laforin and malin knock-out cells display mTOR-dependent autophagy defects and reduced proteasomal activity but no defects in the ER stress response. We speculate that these defects may be secondary to glycogen overaccumulation. This study also suggests a malin function independent of laforin, possibly in lysosomal biogenesis and/or lysosomal glycogen disposal.     

Ubiquitin conjugating enzyme E2-N and sequestosome-1 (p62) are components of the ubiquitination process mediated by the malin–laforin E3-ubiquitin ligase complex

Lafora disease (LD, OMIM254780, ORPHA501) is a rare neurodegenerative form of epilepsy related to mutations in two proteins: laforin, a dual specificity phosphatase, and malin, an E3-ubiquitin ligase. Both proteins form a functional complex, where laforin recruits specific substrates to be ubiquitinated by malin. However, little is known about the mechanism driving malin–laforin mediated ubiquitination of its substrates. In this work we present evidence indicating that the malin–laforin complex interacts physically and functionally with the ubiquitin conjugating enzyme E2-N (UBE2N). This binding determines the topology of the chains that the complex is able to promote in the corresponding substrates (mainly K63-linked polyubiquitin chains). In addition, we demonstrate that the malin–laforin complex interacts with the selective autophagy adaptor sequestosome-1 (p62). Binding of p62 to the malin–laforin complex allows its recognition by LC3, a component of the autophagosomal membrane. In addition, p62 enhances the ubiquitinating activity of the malin–laforin E3-ubiquitin ligase complex. These data enrich our knowledge on the mechanism of action of the malin–laforin complex as an E3-ubiquitin ligase and reinforces the role of this complex in targeting substrates toward the autophagy pathway.     

Relationship between glycogen accumulation and the laforin dual specificity phosphatase

Laforin, encoded by the EPM2A gene, is a dual specificity protein phosphatase that has a functional glycogen-binding domain. Mutations in the EPM2A gene account for around half of the cases of Lafora disease, an autosomal recessive neurodegenerative disorder, characterized by progressive myoclonus epilepsy. The hallmark of the disease is the presence of Lafora bodies, which contain polyglucosan, a poorly branched form of glycogen, in neurons and other tissues. We examined the level of laforin protein in several mouse models in which muscle glycogen accumulation has been altered genetically. Mice with elevated muscle glycogen have increased laforin as judged by Western analysis. Mice completely lacking muscle glycogen or with 10% normal muscle glycogen had reduced laforin. Mice defective in the GAA gene encoding lysosomal alpha-glucosidase (acid maltase) overaccumulate glycogen in the lysosome but did not have elevated laforin. We propose, therefore, that laforin senses cytosolic glycogen accumulation which in turn determines the level of laforin protein.     

Glycogen and related polysaccharides inhibit the laforin dual-specificity protein phosphatase

Lafora disease, a progressive myoclonus epilepsy, is an autosomal recessive disease caused in approximately 80% of cases by mutation of the EPM2A gene, which encodes a dual specificity protein phosphatase called laforin. In addition to its phosphatase domain, laforin contains an N-terminal carbohydrate-binding domain (CBD). Mouse laforin was expressed as an N-terminally polyHis tagged protein in Escherichia coli and purified close to homogeneity. The enzyme was active towards p-nitrophenylphosphate (50-80mmol/min/mg, K(m) 4.5mM) with maximal activity at pH 4.5. Laforin binds to glycogen, as previously shown, and caused potent inhibition, half maximally at approximately 1mug/ml. Less branched glucose polymers, amylopectin and amylose, were even more potent, with half maximal inhibition at 10 and 100ng/ml, respectively. With all polysaccharides, however, inhibition was incomplete and laforin retained 20-30% of its native activity at high polysaccharide concentrations. Glucose and short oligosaccharides did not affect activity. Substitution of Trp32 in the CBD by Gly, a mutation found in a patient, caused only a 30% decrease in laforin activity but abolished binding to and inhibition by glycogen, indicating that impaired glycogen binding is sufficient to cause Lafora disease.     

Identification of a novel protein interacting with laforin,the EPM2a progressive myoclonus epilepsy gene product

We have identified an interacting partner protein (encoded by the human EPM2AIP1 gene (approved symbol)) for laforin, the product of the EPM2A gene, which is mutated in an autosomal recessive form of adolescent progressive myoclonus epilepsy. The EPM2AIP1 gene was identified in a screen for laforin-interacting proteins with a human brain cDNA library using the yeast two-hybrid system. The specificity of the interaction was confirmed by coimmunoprecipitation of in vivo-transfected protein and by using EPM2A deletion constructs. Subcellular colocalization of laforin and EPM2AIP1 protein was also demonstrated. The human EPM2AIP1 gene, corresponding to the KIAA0766 cDNA clone in the databases, was characterized and shown, like EPM2A, to be ubiquitously expressed. The gene, which comprises one large exon 1824 nucleotides in length and has alternative 3' untranslated regions, maps to human chromosome 3p22.1. The function is currently not known and extensive analyses do not reveal any homology to other proteins or any obvious structural motifs. Because genetic heterogeneity in Lafora disease has been described, mutational analysis of the EPM2AIP1 gene was performed on non-EPM2A patients, but no mutations were found. The identification of this first binding partner for laforin promises to be an important step toward unraveling the underlying pathogenesis of this severest form of teenage-onset epilepsy.     

Dimeric Quaternary Structure of Human Laforin

The phosphatase laforin removes phosphate groups from glycogen during biosynthetic activity. Loss-of-function mutations in the gene encoding laforin is the predominant cause of Lafora disease, a fatal form of progressive myoclonic epilepsy. Here, we used hybrid structural methods to determine the molecular architecture of human laforin. We found that laforin adopts a dimeric quaternary structure, topologically similar to the prototypical dual specificity phosphatase VH1. The interface between the laforin carbohydrate-binding module and the dual specificity phosphatase domain generates an intimate substrate-binding crevice that allows for recognition and dephosphorylation of phosphomonoesters of glucose. We identify novel molecular determinants in the laforin active site that help decipher the mechanism of glucan phosphatase activity.     

Genetic Depletion of the Malin E3 Ubiquitin Ligase in Mice Leads to Lafora Bodies and the Accumulation of Insoluble Laforin

Approximately 90% of cases of Lafora disease, a fatal teenage-onset progressive myoclonus epilepsy, are caused by mutations in either the EPM2A or the EPM2B genes that encode, respectively, a glycogen phosphatase called laforin and an E3 ubiquitin ligase called malin. Lafora disease is characterized by the formation of Lafora bodies, insoluble deposits containing poorly branched glycogen or polyglucosan, in many tissues including skeletal muscle, liver, and brain. Disruption of the Epm2b gene in mice resulted in viable animals that, by 3 months of age, accumulated Lafora bodies in the brain and to a lesser extent in heart and skeletal muscle. Analysis of muscle and brain of the Epm2b^−/− mice by Western blotting indicated no effect on the levels of glycogen synthase, PTG (type 1 phosphatase-targeting subunit), or debranching enzyme, making it unlikely that these proteins are targeted for destruction by malin, as has been proposed. Total laforin protein was increased in the brain of Epm2b^−/− mice and, most notably, was redistributed from the soluble, low speed supernatant to the insoluble low speed pellet, which now contained 90% of the total laforin. This result correlated with elevated insolubility of glycogen and glycogen synthase. Because up-regulation of laforin cannot explain Lafora body formation, we conclude that malin functions to maintain laforin associated with soluble glycogen and that its absence causes sequestration of laforin to an insoluble polysaccharide fraction where it is functionally inert.