SUMMARY Glycogen is the major mammalian glucose storage cache and is critical for energy homeostasis. Glycogen synthesis in neurons must be tightly controlled, due to neuronal sensitivity to perturbations in glycogen metabolism. Lafora disease (LD) is a fatal, congenital, neurodegenerative epilepsy. Mutations in the gene encoding the glycogen phosphatase laforin result in hyperphosphorylated glycogen that forms water-insoluble inclusions called Lafora bodies (LBs). LBs induce neuronal apoptosis and are the causative agent of LD. The mechanism of glycogen dephosphorylation by laforin and dysfunction in LD is unknown. We report the crystal structure of laforin bound to phosphoglucan product, revealing its unique integrated tertiary and quaternary structure. Structure-guided mutagenesis combined with biophysical and biochemical analyses reveal the basis for normal function of laforin in glycogen metabolism. Analyses of LD patient mutations define the mechanism by which subsets of mutations disrupt laforin function. These data provide fundamental insights connecting glycogen metabolism to neurodegenerative disease.

Living organisms utilize carbohydrates as essential energy storage molecules. Starch is the predominant carbohydrate storage molecule in plants while glycogen is utilized in animals. Starch is a water-insoluble polymer that requires the concerted activity of kinases and phosphatases to solubilize the outer surface of the glucan and mediate starch catabolism. All known plant genomes encode the glucan phosphatase Starch Excess4 (SEX4). SEX4 can dephosphorylate both the starch granule surface and soluble phosphoglucans and is necessary for processive starch metabolism. The physical basis for the function of SEX4 as a glucan phosphatase is currently unclear. Herein, we report the crystal structure of SEX4, containing phosphatase, carbohydrate-binding, and C-terminal domains. The three domains of SEX4 fold into a compact structure with extensive interdomain interactions. The C-terminal domain of SEX4 integrally folds into the core of the phosphatase domain and is essential for its stability. The phosphatase and carbohydratebinding domains directly interact and position the phosphatase active site toward the carbohydrate-binding site in a single continuous pocket. Mutagenesis of the phosphatase domain residue F167, which forms the base of this pocket and bridges the two domains, selectively affects the ability of SEX4 to function as a glucan phosphatase. Together, these results reveal the unique tertiary architecture of SEX4 that provides the physical basis for its function as a glucan phosphatase.carbohydrate | Lafora disease | laforin | phosphorylation P lants and animals store carbohydrates as starch and glycogen, respectively. Starch is produced in diurnal cycles and is composed of <10% w∕w amylose and >80% w∕w amylopectin in Arabidopsis thaliana leaves (1). Amylose is a linear molecule composed of glucose moieties linked by α-1,4-glycosidic linkages with very few branches. Amylopectin, which is similar to glycogen, is composed of α-1,4-glycosidic linkages with α-1,6-glycosidic branches, but amylopectin branches are arranged in clusters at regular intervals and the branches form double helices that pack together to form crystalline lamellae (2, 3). The decreased branching and crystalline lamellae of amylopectin are key contributors to the insolubility of starch, while glycogen has more branches and is water-soluble.Starch is a water-insoluble polymer whose surface is inaccessible to most enzymes. Recent work convincingly demonstrates that reversible starch phosphorylation and dephosphorylation is essential for processive starch metabolism (reviewed in refs. 4-7). An essential signal triggering starch catabolism is phosphorylation on the C6 position of glucose moieties on the surface of starch by glucan water dikinase (GWD/R1) (8, 9). C6 phosphorylation triggers C3 phosphorylation by phosphoglucan water dikinase (PWD) (8,10,11). Recent data suggest that C6 phosphorylation fits within the unphosphorylated structure of the amylopectin helix, but C3 phosphorylation imposes significant steric effects and is predicted t...

Lafora Disease (LD) is a fatal neurodegenerative epileptic disorder that presents as a neurological deterioration with the accumulation of insoluble, intracellular, hyperphosphorylated carbohydrates called Lafora bodies (LBs). LD is caused by mutations in either the gene encoding laforin or malin. Laforin contains a dual specificity phosphatase domain and a carbohydrate-binding module, and is a member of the recently described family of glucan phosphatases. In the current study, we investigated the functional and physiological relevance of laforin dimerization. We purified recombinant human laforin and subjected the monomer and dimer fractions to denaturing gel electrophoresis, mass spectrometry, phosphatase assays, protein-protein interaction assays, and glucan binding assays. Our results demonstrate that laforin prevalently exists as a monomer with a small dimer fraction both in vitro and in vivo. Of mechanistic importance, laforin monomer and dimer possess equal phosphatase activity, and they both associate with malin and bind glucans to a similar extent. However, we found differences between the two states' ability to interact simultaneously with malin and carbohydrates. Furthermore, we tested other members of the glucan phosphatase family. Cumulatively, our data suggest that laforin monomer is the dominant form of the protein and that it contains phosphatase activity.

We initially demonstrated that the human phosphatase laforin and plant phosphatase Starch EXcess4 (SEX4) are glucan phosphatases. Mutations in the gene encoding laforin cause glycogen to become hyperphosphorylated, resulting in starch‐like glucans called Lafora bodies (LBs) and the fatal neurodegenerative disorder Lafora's disease. Plant starch contains covalently bound phosphate at the C6‐ and C3‐position of glucosyl residues. Reversible phosphorylation of starch outer glucans renders these glucans accessible to hydrolyzing enzymes. In the absence of starch phosphorylation or dephosphorylation, plants accumulate excessively large starch granules that they are unable to breakdown. SEX4 activity is a necessary step in the starch degradation cycle of glucan phosphorylation, hydrolysis, and dephosphorylation. We utilized X‐ray crystallography to determine the structure of SEX4 and this structure assisted in the identification of Like Sex Four2 (LSF2). Unlike SEX4, LSF2 lacks a carbohydrate‐binding module yet LSF2 binds starch. We found that SEX4 releases phosphate from the C3‐ and C6‐position, but prefers C6. Alternatively, LSF2 hydrolyzes phosphate from the C3‐position and plants lacking LSF2 have elevated C3‐bound phosphate. Lastly, we determined the crystal structure of LSF2 and utilize site‐directed mutagenesis with in vitro assays to define the structural elements of glucan phosphatases.

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