title: Glycogen and related polysaccharides inhibit the laforin dual-specificity protein phosphatase.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Center for Diabetes Research, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Roach
mesh_terms: D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry*; D054637:Dual-Specificity Phosphatases; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000737:chemistry; D006003:Glycogen / Q000737:chemistry*; D051379:Mice; D011134:Polysaccharides / Q000737:chemistry; D011485:Protein Binding; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry*; D054558:Protein Tyrosine Phosphatases, Non-Receptor; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D002352:Carrier Proteins; C483189:EPM2AIP1 protein, human; D004791:Enzyme Inhibitors; D011134:Polysaccharides; D006003:Glycogen; D054637:Dual-Specificity Phosphatases; C491989:Epm2a protein, mouse; D017027:Protein Tyrosine Phosphatases; D054558:Protein Tyrosine Phosphatases, Non-Receptor