Abnormal Metabolism of Glycogen Phosphate as a Cause for Lafora Disease

Abstract

Lafora disease is a progressive myoclonus epilepsy with onset in the teenage years followed by neurodegeneration and death within 10 years. A characteristic is the widespread formation of poorly branched, insoluble glycogen-like polymers (polyglucosan) known as Lafora bodies, which accumulate in neurons, muscle, liver, and other tissues. Approximately half of the cases of Lafora disease result from mutations in the EPM2A gene, which encodes laforin, a member of the dual specificity protein phosphatase family that is able to release the small amount of covalent phosphate normally present in glycogen. In studies of Epm2a ؊/؊ mice that lack laforin, we observed a progressive change in the properties and structure of glycogen that paralleled the formation of Lafora bodies. At three months, glycogen metabolism remained essentially normal, even though the phosphorylation of glycogen has increased 4-fold and causes altered physical properties of the polysaccharide. By 9 months, the glycogen has overaccumulated by 3-fold, has become somewhat more phosphorylated, but, more notably, is now poorly branched, is insoluble in water, and has acquired an abnormal morphology visible by electron microscopy. These glycogen molecules have a tendency to aggregate and can be recovered in the pellet after low speed centrifugation of tissue extracts. The aggregation requires the phosphorylation of glycogen. The aggregrated glycogen sequesters glycogen synthase but not other glycogen metabolizing enzymes. We propose that laforin functions to suppress excessive glycogen phosphorylation and is an essential component of the metabolism of normally structured glycogen.Glycogen is a branched polymer of glucose that acts as a repository of energy used in times of need (1). Liver and skeletal muscle contain the two largest deposits in mammals, but heart, brain, adipose, as well as many other tissues synthesize the polymer. Polymerization occurs via ␣-1,4-glycosidic linkages between glucose residues, with branch points introduced by ␣-1,6-glycosidic linkages. The frequency of branching determines the topology of glycogen and distinguishes it from the carbohydrate moiety of starch (2). A unique three-dimensional structure of glycogen cannot be determined experimentally because of the polydispersity of the molecule, but a widely accepted model of its structure has been proposed (3). Glycogen is composed of successive layers, or tiers, of glucose residues; a full size molecule consists of 12 tiers, with M r ϭ ϳ10 7 and a diameter of ϳ40 nm (4). Glycogen has been reported to contain small amounts of covalently linked phosphate, on the order of 0.064% by weight or 0.121% mol/mol (5-7). The mechanism by which the phosphate is introduced remains unclear. One suggestion (5) has been the existence of a glucose-1-phosphate transferase that would form C1-C6 bridging phosphodiesters, but this enzyme has not been further defined at the molecular level. In plant amylopectin, C1 and C3 phosphomonoesters have been shown to be formed by dikinase enzymes (8), but...

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

“…Defects in either of these pathways have been proposed to play a role in the pathogenesis of Lafora disease. Previous work has shown that laforin dephosphorylates glycogen (14) and that the absence of laforin in Epm2a Ϫ/Ϫ mice causes hyperphosphorylated glycogen, which correlates with abnormalities in glycogen structure, decreased branching, and solubility in water (15), consistent with the formation of Lafora bodies. However the role of malin to reduce glycogen phosphorylation is not fully understood.…”

“…Extensive studies have been conducted using cell culture and animal models in attempts to understand the etiopathogenesis of Lafora disease. We have previously shown that laforin acts as a glycogen phosphatase in vitro and in vivo, and as a result of increased phosphorylation of glycogen there are disturbances in glycogen structure and defects in branching and water solubility, consistent with Lafora body formation (14,15). Malin contains a RING finger domain characteristic of E3 ubiquitin ligases, and several proteins involved in glycogen metabolism, laforin (16), glycogen synthase (17), protein targeted to glycogen (PTG) (18), and AGL (19) have been proposed to be malin substrates.…”

Structural basis for the glucan phosphatase activity of Starch Excess4

“…Laforin contains a CBM and DSP, like SEX4, but in the opposite orientation. Similar to SEX4, laforin dephosphorylates phospho-glucans in vitro (28), and LBs from LD patients and a LD mouse model have increased phosphate compared to glycogen (29)(30)(31)(32). Strikingly, mutations in the genes encoding both laforin and SEX4 result in the accumulation of insoluble glucans (19)(20)(21).…”

Starch Binding Domain-containing Protein 1/Genethonin 1 Is a Novel Participant in Glycogen Metabolism

“…PP1G enzymes exist as a PP1 catalytic subunit associated with a glycogen-targeting regulatory subunit (20), of which R GL /G M (PPP1R3A), G L (PPP1R3B), and PTG (PPP1R3C) are the best studied. A more recently identified glycogen-associated protein is laforin, by sequence similarity a member of the atypical dual specificity protein phosphatase subfamily, but which is in fact a glycogen phosphatase involved in maintaining glycogen structural integrity (21,22). Laforin binds to glycogen via a CBM20 carbohydrate-binding module (23).…”