Background: The gene that encodes laforin, a dual-specificity phosphatase with a carbohydrate-binding module, is mutated in Lafora disease (LD). LD is an autosomal recessive, fatal progressive myoclonus epilepsy characterized by the intracellular buildup of insoluble, hyperphosphorylated glycogen-like particles, called Lafora bodies. Laforin dephosphorylates glycogen and other glucans in vitro, but the structural basis of its activity remains unknown. Recombinant human laforin when expressed in and purified from E. coli is largely insoluble and prone to aggregation and precipitation. Identification of a laforin ortholog that is more soluble and stable in vitro would circumvent this issue. Results: In this study, we cloned multiple laforin orthologs, established a purification scheme for each, and tested their solubility and stability. Gallus gallus (Gg) laforin is more stable in vitro than human laforin, Gg-laforin is largely monomeric, and it possesses carbohydrate binding and phosphatase activity similar to human laforin. Conclusions: Gg-laforin is more soluble and stable than human laforin in vitro, and possesses similar activity as a glucan phosphatase. Therefore, it can be used to model human laforin in structure-function studies. We have established a protocol for purifying recombinant Gg-laforin in sufficient quantity for crystallographic and other biophysical analyses, in order to better understand the function of laforin and define the molecular mechanisms of Lafora disease.
|State||Published - Apr 2 2014|
Bibliographical noteFunding Information:
We thank Dr. Carol Beach (University of Kentucky) for her assistance with the mass spectrometry as well as Dr. Martin Chow and the Center for Structural Biology (supported by NCRR grant P20RR0202171). This work was supported by NIH grant R01NS070899, an award from the Mizutani Foundation for Glycoscience, University of Kentucky College of Medicine startup funds to MSG, an American Heart Association Postdoctoral Award (12POST12030381) to VVD, and a Summer Research Grant to MKB from the Office of Undergraduate Research at the University of Kentucky.
- Carbohydrate-binding module
- Lafora disease
ASJC Scopus subject areas
- Molecular Biology