Laforin, a Dual Specificity Phosphatase Involved in Lafora Disease, Is Present Mainly as Monomeric Form with Full Phosphatase Activity

Abstract

Lafora Disease (LD) is a fatal neurodegenerative epileptic disorder that presents as a neurological deterioration with the accumulation of insoluble, intracellular, hyperphosphorylated carbohydrates called Lafora bodies (LBs). LD is caused by mutations in either the gene encoding laforin or malin. Laforin contains a dual specificity phosphatase domain and a carbohydrate-binding module, and is a member of the recently described family of glucan phosphatases. In the current study, we investigated the functional and physiological relevance of laforin dimerization. We purified recombinant human laforin and subjected the monomer and dimer fractions to denaturing gel electrophoresis, mass spectrometry, phosphatase assays, protein-protein interaction assays, and glucan binding assays. Our results demonstrate that laforin prevalently exists as a monomer with a small dimer fraction both in vitro and in vivo. Of mechanistic importance, laforin monomer and dimer possess equal phosphatase activity, and they both associate with malin and bind glucans to a similar extent. However, we found differences between the two states' ability to interact simultaneously with malin and carbohydrates. Furthermore, we tested other members of the glucan phosphatase family. Cumulatively, our data suggest that laforin monomer is the dominant form of the protein and that it contains phosphatase activity.

Dimeric Quaternary Structure of Human Laforin

“…3, A and B). This supports previous findings by Liu et al (45) and contradicts a recent report that laforin is monomeric based solely on size exclusion chromatography (44). Our structural analysis reveals that the laforin quaternary structure is stabilized by dimerization of its N-terminal CBM (Fig.…”

Structural Mechanism of Laforin Function in Glycogen Dephosphorylation and Lafora Disease

“…The physical and functional role of laforin oligomerization has been controversial (Dukhande et al, 2011; Koksal and Cingolani, 2011; Liu et al, 2006; Liu et al, 2009; Sanchez-Martin et al, 2013). In situ and in vivo experiments have demonstrated that laforin is able to both dimerize and multimerize, but different models have been proposed for the extent to which oligomers form and which domains contribute.…”

Structure of the Arabidopsis Glucan Phosphatase LIKE SEX FOUR2 Reveals a Unique Mechanism for Starch Dephosphorylation

“…Glucan binding assays were performed as previously described with the following modifications (Gentry et al, 2007;Dukhande et al, 2011). All proteins used in the glucan binding assays were purified as described above without cleavage of the His 6 tag to maintain the epitope for immunoblot analysis.…”