Skip to main content
SpringerLink
Log in

Assessing the Biological Activity of the Glucan Phosphatase Laforin

  • Protocol
  • First Online:
Protein Tyrosine Phosphatases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1447))

Abstract

Glucan phosphatases are a recently discovered family of enzymes that dephosphorylate either starch or glycogen and are essential for proper starch metabolism in plants and glycogen metabolism in humans. Mutations in the gene encoding the only human glucan phosphatase, laforin, result in the fatal, neurodegenerative, epilepsy known as Lafora disease. Here, we describe phosphatase assays to assess both generic laforin phosphatase activity and laforin’s unique glycogen phosphatase activity.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Worby CA, Gentry MS, Dixon JE (2006) Laforin, a dual specificity phosphatase that dephosphorylates complex carbohydrates. J Biol Chem 281(41):30412–30418

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Gentry MS, Dowen RH III, Worby CA, Mattoo S, Ecker JR, Dixon JE (2007) The phosphatase laforin crosses evolutionary boundaries and links carbohydrate metabolism to neuronal disease. J Cell Biol 178(3):477–488

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Vilchez D, Ros S, Cifuentes D, Pujadas L, Valles J, Garcia-Fojeda B, Criado-Garcia O, Fernandez-Sanchez E, Medrano-Fernandez I, Dominguez J, Garcia-Rocha M, Soriano E, Rodriguez de Cordoba S, Guinovart JJ (2007) Mechanism suppressing glycogen synthesis in neurons and its demise in progressive myoclonus epilepsy. Nat Neurosci 10(11):1407–1413

    Article  CAS  PubMed  Google Scholar 

  4. Solaz-Fuster MC, Gimeno-Alcaniz JV, Ros S, Fernandez-Sanchez ME, Garcia-Fojeda B, Criado Garcia O, Vilchez D, Dominguez J, Garcia-Rocha M, Sanchez-Piris M, Aguado C, Knecht E, Serratosa J, Guinovart JJ, Sanz P, Rodriguez de Cordoba S (2008) Regulation of glycogen synthesis by the laforin-malin complex is modulated by the AMP-activated protein kinase pathway. Hum Mol Genet 17(5):667–678

    Article  CAS  PubMed  Google Scholar 

  5. Minassian BA, Ianzano L, Meloche M, Andermann E, Rouleau GA, Delgado-Escueta AV, Scherer SW (2000) Mutation spectrum and predicted function of laforin in Lafora’s progressive myoclonus epilepsy. Neurology 55(3):341–346

    Article  CAS  PubMed  Google Scholar 

  6. Wang J, Stuckey JA, Wishart MJ, Dixon JE (2002) A unique carbohydrate binding domain targets the lafora disease phosphatase to glycogen. J Biol Chem 277(4):2377–2380

    Article  CAS  PubMed  Google Scholar 

  7. Tonks NK (2006) Protein tyrosine phosphatases: from genes, to function, to disease. Nat Rev 7(11):833–846

    Article  CAS  Google Scholar 

  8. Ganesh S, Tsurutani N, Suzuki T, Ueda K, Agarwala KL, Osada H, Delgado-Escueta AV, Yamakawa K (2003) The Lafora disease gene product laforin interacts with HIRIP5, a phylogenetically conserved protein containing a NifU-like domain. Hum Mol Genet 12(18):2359–2368

    Article  CAS  PubMed  Google Scholar 

  9. Wang W, Roach PJ (2004) Glycogen and related polysaccharides inhibit the laforin dual-specificity protein phosphatase. Biochem Biophys Res Commun 325(3):726–730

    Article  CAS  PubMed  Google Scholar 

  10. Girard JM, Le KH, Lederer F (2006) Molecular characterization of laforin, a dual-specificity protein phosphatase implicated in Lafora disease. Biochimie 88(12):1961–1971

    Article  CAS  PubMed  Google Scholar 

  11. Tagliabracci VS, Turnbull J, Wang W, Girard JM, Zhao X, Skurat AV, Delgado-Escueta AV, Minassian BA, Depaoli-Roach AA, Roach PJ (2007) Laforin is a glycogen phosphatase, deficiency of which leads to elevated phosphorylation of glycogen in vivo. Proc Natl Acad Sci U S A 104(49):19262–19266

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Tagliabracci VS, Girard JM, Segvich D, Meyer C, Turnbull J, Zhao X, Minassian BA, Depaoli-Roach AA, Roach PJ (2008) Abnormal metabolism of glycogen phosphate as a cause for Lafora disease. J Biol Chem 283(49):33816–33825

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Raththagala M, Brewer MK, Parker MW, Sherwood AR, Wong BK, Hsu S, Bridges TM, Paasch BC, Hellman LM, Husodo S, Meekins DA, Taylor AO, Turner BD, Auger KD, Dukhande VV, Chakravarthy S, Sanz P, Woods VL Jr, Li S, Vander Kooi CW, Gentry MS (2015) Structural mechanism of laforin function in glycogen dephosphorylation and lafora disease. Mol Cell 57(2):261–272

    Article  CAS  PubMed  Google Scholar 

  14. Singh S, Ganesh S (2009) Lafora progressive myoclonus epilepsy: a meta-analysis of reported mutations in the first decade following the discovery of the EPM2A and NHLRC1 genes. Hum Mutat 30(5):715–723

    Article  CAS  PubMed  Google Scholar 

  15. Gottlin EB, Xu X, Epstein DM, Burke SP, Eckstein JW, Ballou DP, Dixon JE (1996) Kinetic analysis of the catalytic domain of human cdc25B. J Biol Chem 271(44):27445–27449

    Article  CAS  PubMed  Google Scholar 

  16. Lanzetta PA, Alvarez LJ, Reinach PS, Candia OA (1979) An improved assay for nanomole amounts of inorganic phosphate. Anal Biochem 100(1):95–97

    Article  CAS  PubMed  Google Scholar 

  17. Harder KW, Owen P, Wong LK, Aebersold R, Clark-Lewis I, Jirik FR (1994) Characterization and kinetic analysis of the intracellular domain of human protein tyrosine phosphatase beta (HPTP beta) using synthetic phosphopeptides. Biochem J 298(Pt 2):395–401

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. McCain DF, Zhang ZY (2002) Assays for protein-tyrosine phosphatases. Methods Enzymol 345:507–518

    Article  PubMed  Google Scholar 

  19. Sherwood AR, Johnson MB, Delgado-Escueta AV, Gentry MS (2013) A bioassay for Lafora disease and laforin glucan phosphatase activity. Clin Biochem 46(18):1869–1876

    Article  CAS  PubMed  Google Scholar 

  20. Sanchez-Martin P, Raththagala M, Bridges TM, Husodo S, Gentry MS, Sanz P, Roma-Mateo C (2013) Dimerization of the glucan phosphatase laforin requires the participation of cysteine 329. PLoS One 8(7):e69523

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Huyer G, Liu S, Kelly J, Moffat J, Payette P, Kennedy B, Tsaprailis G, Gresser MJ, Ramachandran C (1997) Mechanism of inhibition of protein-tyrosine phosphatases by vanadate and pervanadate. J Biol Chem 272(2):843–851

    Article  CAS  PubMed  Google Scholar 

  22. Itaya K, Ui M (1966) A new micromethod for the colorimetric determination of inorganic phosphate. Clin Chim Acta 14(3):361–366

    Article  CAS  PubMed  Google Scholar 

  23. Meekins DA, Guo HF, Husodo S, Paasch BC, Bridges TM, Santelia D, Kotting O, Vander Kooi CW, Gentry MS (2013) Structure of the Arabidopsis glucan phosphatase like sex four2 reveals a unique mechanism for starch dephosphorylation. Plant Cell 25(6):2302–2314

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Meekins DA, Raththagala M, Husodo S, White CJ, Guo HF, Kotting O, Vander Kooi CW, Gentry MS (2014) Phosphoglucan-bound structure of starch phosphatase Starch Excess4 reveals the mechanism for C6 specificity. Proc Natl Acad Sci U S A 111(20):7272–7277

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Sherwood AR, Paasch BC, Worby CA, Gentry MS (2013) A malachite green-based assay to assess glucan phosphatase activity. Anal Biochem 435(1):54–56

    Article  CAS  PubMed  Google Scholar 

  26. Walton KM, Dixon JE (1993) Protein tyrosine phosphatases. Annu Rev Biochem 62:101–120

    Article  CAS  PubMed  Google Scholar 

  27. Dukhande VV, Rogers DM, Roma-Mateo C, Donderis J, Marina A, Taylor AO, Sanz P, Gentry MS (2011) Laforin, a dual specificity phosphatase involved in lafora disease, is present mainly as monomeric form with full phosphatase activity. PLoS One 6(8):e24040

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. DePaoli-Roach AA, Contreras CJ, Segvich DM, Heiss C, Ishihara M, Azadi P, Roach PJ (2015) Glycogen phosphomonoester distribution in mouse models of the progressive myoclonic epilepsy, Lafora disease. J Biol Chem 290(2):841–850

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This study was supported by grants from the Spanish Ministry of Education and Science SAF2014-54604-C3-1-R (P.S.), Generalitat Valenciana (PrometeoII/2014/029) (P.S.), NIH Grants R01NS070899 (M.S.G.), P20GM103486 (M.S.G.), Mizutani Foundation for Glycoscience Award (M.S.G.), and NSF Grant IIA-1355438 (M.S.G.). C.R.-M. was supported by the Saving Lives at Birth Consortium.

Author information

Authors and Affiliations

  1. Department of Physiology, School of Medicine and Dentistry, University of Valencia and FIHCUV-INCLIVA, Valencia, Spain

    Carlos Romá-Mateo

  2. Department of Molecular and Cellular Biochemistry, Center for Structural Biology, University of Kentucky, Lexington, KY, USA

    Madushi Raththagala & Mathew S. Gentry

  3. Instituto de Biomedicina de Valencia, CSIC, Valencia, Spain

    Pascual Sanz

  4. Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Jaime Roig 11, 46010, Valencia, Spain

    Carlos Romá-Mateo & Pascual Sanz

Authors
  1. Carlos Romá-Mateo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Madushi Raththagala
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mathew S. Gentry
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Pascual Sanz
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Pascual Sanz .

Editor information

Editors and Affiliations

  1. BioCruces Health Research Institute, Barakaldo, Bizkaia, Spain

    Rafael Pulido

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Romá-Mateo, C., Raththagala, M., Gentry, M.S., Sanz, P. (2016). Assessing the Biological Activity of the Glucan Phosphatase Laforin. In: Pulido, R. (eds) Protein Tyrosine Phosphatases. Methods in Molecular Biology, vol 1447. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3746-2_7

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-3746-2_7

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3744-8

  • Online ISBN: 978-1-4939-3746-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation