A bioassay for Lafora disease and laforin glucan phosphatase activity |
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Authors: | Amanda R Sherwood Mary Beth Johnson Antonio V Delgado-Escueta Matthew S Gentry |
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Institution: | 1. Department of Molecular and Cellular Biochemistry, Center for Structural Biology, University of Kentucky, Lexington, KY 405036-0001, USA;2. Department of Neurology, University of California Los Angeles, Los Angeles CA, 90095-1769, USA |
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Abstract: | ObjectivesLafora disease is a rare yet invariably fatal form of progressive neurodegenerative epilepsy resulting from mutations in the phosphatase laforin. Several therapeutic options for Lafora disease patients are currently being explored, and these therapies would benefit from a biochemical means of assessing functional laforin activity following treatment. To date, only clinical outcomes such as decreases in seizure frequency and severity have been used to indicate success of epilepsy treatment. However, these qualitative measures exhibit variability and must be assessed over long periods of time. In this work, we detail a simple and sensitive bioassay that can be used for the detection of functional endogenous laforin from human and mouse tissue.Design and methodsWe generated antibodies capable of detecting and immunoprecipitating endogenous laforin. Following laforin immunoprecipitation, laforin activity was assessed via phosphatase assays using para-nitrophenylphosphate (pNPP) and a malachite green-based assay specific for glucan phosphatase activity.ResultsWe found that antibody binding to laforin does not impede laforin activity. Furthermore, the malachite green-based glucan phosphatase assay used in conjunction with a rabbit polyclonal laforin antibody was capable of detecting endogenous laforin activity from human and mouse tissues. Importantly, this assay discriminated between laforin activity and other phosphatases.ConclusionsThe bioassay that we have developed utilizing laforin antibodies and an assay specific for glucan phosphatase activity could prove valuable in the rapid detection of functional laforin in patients to which novel Lafora disease therapies have been administered. |
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Keywords: | BSA bovine serum albumin CBM carbohydrate binding module CSF cerebrospinal fluid DSP dual specificity phosphatase DTT dithiothreitol HRP horseradish peroxidase HEK human embryonic kidney LB Lafora body LD Lafora disease mRIPA modified RIPA buffer pcDNA3 1 FLAG pcDNA3 1 with N-terminal FLAG tag PAS periodic acid Schiff pNPP para-nitrophenylphosphate TBS Tris-buffered saline |
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