A bioassay for Lafora disease and laforin glucan phosphatase activity

Amanda R. Sherwood, Mary Beth Johnson, Antonio V. Delgado-Escueta, Matthew S. Gentry

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

Abstract

Objectives: 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. Design and 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.

Original languageEnglish
Pages (from-to)1869-1876
Number of pages8
JournalClinical Biochemistry
Volume46
Issue number18
DOIs
StatePublished - Dec 2013

Bibliographical note

Funding Information:
This work was supported by the National Institutes of Health Grants P20RR020171 and R01NS070899 and University of Kentucky College of Medicine startup funds to M.S.G. This publication was also supported by grant number TL1 RR033172 from the National Center for Research Resources (NCRR), funded by the Office of the Director, National Institutes of Health (NIH) and supported by the NIH Roadmap for Medical Research . The content is solely the responsibility of the authors and does not necessarily represent the official views of NCRR and NIH. We wish to thank Drs. Carol Beach and Martin Chow in the Molecular Basis of Human Disease COBRE Proteomics core and Protein Analytical Core, respectively, for technical assistance as well as members of the Gentry lab for fruitful discussions.

Funding Information:
The monoclonal antibody N84/37.1 was developed by and/or obtained from the UC Davis/NIH NeuroMab Facility, supported by NIH grant U24NS050606 and maintained by the Department of Neurobiology, Physiology and Behavior, College of Biological Sciences, University of California, Davis, CA 95616. This paper is subject to the NIH Public Access Policy. This work was carried out in accordance with the Uniform Requirements for Manuscripts Submitted to Biomedical Journals and the Code of Ethics of the World Medical Association (Declaration of Helsinki) for Experiments Involving Humans. All work involving human tissue has been cleared by the University of Kentucky College of Medicine Institution Review Board.

Keywords

  • Bioassay
  • Epilepsy
  • Glucan phosphatase
  • Lafora disease
  • Laforin
  • Malachite green

ASJC Scopus subject areas

  • Clinical Biochemistry

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