title: A bioassay for Lafora disease and laforin glucan phosphatase activity.
abstract: OBJECTIVE
  Lafora 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.
  
  
  METHODS
  We 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.
  
  
  RESULTS
  We 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.
  
  
  CONCLUSIONS
  The 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.
authors.affiliation: Department of Molecular and Cellular Biochemistry, Center for Structural Biology, University of Kentucky, Lexington, KY 405036-0001, USA.
authors.forename: Amanda R
authors.initials: AR
authors.lastname: Sherwood
authors.forename: Mary Beth
authors.initials: MB
authors.lastname: Johnson
authors.forename: Antonio V
authors.initials: AV
authors.lastname: Delgado-Escueta
authors.forename: Matthew S
authors.initials: MS
authors.lastname: Gentry
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D054637:Dual-Specificity Phosphatases / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D047468:Immunoprecipitation; D020192:Lafora Disease / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018482:Muscle, Skeletal / Q000378:metabolism; D009596:Nitrophenols / Q000737:chemistry; D009943:Organophosphorus Compounds / Q000737:chemistry; D054558:Protein Tyrosine Phosphatases, Non-Receptor / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D011817:Rabbits; D012394:Rosaniline Dyes / Q000737:chemistry; D012867:Skin / Q000378:metabolism
chemical_list: D009596:Nitrophenols; D009943:Organophosphorus Compounds; D012394:Rosaniline Dyes; C005095:malachite green; C008644:nitrophenylphosphate; D054637:Dual-Specificity Phosphatases; C491989:Epm2a protein, mouse; D054558:Protein Tyrosine Phosphatases, Non-Receptor; C114990:EPM2A protein, human
keywords: BSA; Bioassay; CBM; CSF; DSP; DTT; Epilepsy; Glucan phosphatase; HEK; HRP; LB; LD; Lafora body; Lafora disease; Laforin; Malachite green; PAS; TBS; Tris-buffered saline; bovine serum albumin; carbohydrate binding module; cerebrospinal fluid; dithiothreitol; dual specificity phosphatase; horseradish peroxidase; human embryonic kidney; mRIPA; modified RIPA buffer; pNPP; para-nitrophenylphosphate; pcDNA3.1 FLAG; pcDNA3.1 with N-terminal FLAG tag; periodic acid Schiff