Laforin is a glycogen phosphatase, deficiency of which leads to elevated phosphorylation of glycogen in vivo

Abstract

Lafora disease is a progressive myoclonus epilepsy with onset typically in the second decade of life and death within 10 years. Lafora bodies, deposits of abnormally branched, insoluble glycogen-like polymers, form 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 additionally contains a glycogen binding domain. The molecular basis for the formation of Lafora bodies is completely unknown. Glycogen, a branched polymer of glucose, contains a small amount of covalently linked phosphate whose origin and function are obscure. We report here that recombinant laforin is able to release this phosphate in vitro, in a time-dependent reaction with an apparent K(m) for glycogen of 4.5 mg/ml. Mutations of laforin that disable the glycogen binding domain also eliminate its ability to dephosphorylate glycogen. We have also analyzed glycogen from a mouse model of Lafora disease, Epm2a(-/-) mice, which develop Lafora bodies in several tissues. Glycogen isolated from these mice had a 40% increase in the covalent phosphate content in liver and a 4-fold elevation in muscle. We propose that excessive phosphorylation of glycogen leads to aberrant branching and Lafora body formation. This study provides a molecular link between an observed biochemical property of laforin and the phenotype of a mouse model of Lafora disease. The results also have important implications for glycogen metabolism generally.

Fig. 1.

Dephosphorylation of amylopectin and glycogen by laforin. The data shown are representative of at least five independent experiments. (A) Time-dependent release of phosphate from potato amylopectin (0.5 mg/ml) by purified, recombinant laforin (2.5 μg/ml). At the indicated times, the reaction was terminated by the addition of N-ethylmaleimide, and the inorganic phosphate was determined. (B) Concentration dependence of the rate of dephosphorylation of amylopectin by laforin. The reaction was for 15 min in the presence of 2.5 μg/ml laforin, at which time the reaction was close to linear. The apparent K_m for amylopectin was 1.5 mg/ml. (C) Time-dependent release of phosphate from rabbit skeletal muscle glycogen (5 mg/ml). Laforin was present at 25 μg/ml. (D) Concentration dependence of glycogen dephosphorylation by laforin. Laforin was present at 25 μg/ml, and the reaction time was 40 min. The apparent K_m for glycogen was 4.5 mg/ml. (E) Dephosphorylation of rabbit skeletal muscle glycogen in the presence of α-amylase and amyloglucosidase. Glycogen (5 mg/ml) dephosphorylation by laforin (25 μg/ml) was allowed to proceed until the reaction was essentially complete, at which time α-amylase (0.3 mg/ml) and amyloglucosidase (0.3 mg/ml) were added. Subsequent phosphate release was monitored. (F) Model for glycogen structure. Glycogen is believed to exist as a series of concentric shells of glucose residues, so that inner tiers would not be on the surface of the molecule. A full-size molecule would consist of 12 tiers.

Fig. 2.

Glycogen dephosphorylation requires the polysaccharide binding domain of laforin. WT laforin releases phosphate from glycogen (7.5 mg/ml). Mutation at the active site (C266S) essentially eliminated activity. Mutation in the carbohydrate binding domain (W32G), which is known to eliminate binding to polysaccharides, also abolished laforin's ability to dephosphorylate glycogen. The laforin proteins were present at 25 μg/ml, and the assay duration was 60 min.

Fig. 3.

Glycogen synthase and branching enzyme activity in tissues from WT and Epm2a^−/− mice. (A) Glycogen synthase activity in skeletal muscle of Epm2a^−/− mice (n = 4) and WT controls (n = 4). Empty bars indicate activity in the absence of the activator glucose-6-P, and filled bars indicate activity in the presence of glucose-6-P. (B) Branching enzyme activity in skeletal muscle Epm2a^−/− (n = 4) and WT (n = 4) mice. (C) Glycogen synthase activity in brain of Epm2a^−/− (n = 5) and WT (n = 4) mice. (D) Branching enzyme activity in brain of Epm2a^−/− (n = 5) and WT (n = 4) controls. No difference between genotypes was statistically significant. (E) Phosphorylation of GSK-3 in brain of WT and Epm2a^−/− mice (KO) analyzed by using phospho-specific antibodies. (F) (Upper) Glycogen synthase in muscle of WT and Epm2a^−/− mice (KO) analyzed with antiglycogen synthase antibodies. (Lower) Quantitation is shown. Also shown is the detection of laforin with an antilaforin antibody.

Fig. 4.

Phosphate content of glycogen from tissues of Epm2a^−/− and WT mice. Glycogen was isolated from liver or muscle of individual mice, and covalent phosphate content, expressed as mol phosphate/mol glucose, was determined. (A) Liver glycogen phosphate content of Epm2a^−/− (n = 4) and WT (n = 3) mice. The 40% increase in phosphate content in the Epm2a^−/− mice is significant with P = 0.03 (∗). (B) Skeletal muscle glycogen phosphate content of Epm2a^−/− (n = 8) and WT (n = 8) mice. Epm2a^−/− mice have 4-fold greater phosphate content, significant at P = 0.0001 (∗∗).