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.

Figure 1. Laforin monomer is abundant compared with its dimer form.

(A) The chromatogram is of Hs-laforin-HIS₆ purified using a Superdex-75 column and contains two distinct peaks, peak A and peak B. This is a representative of 6 purifications. (B) Fractions from peak B (65–78) were collected, concentrated, and re-loaded onto a Superdex-75 column. The chromatograms are representatives of 4 experiments. (C) Proteins from these peak A (41–43) and B (65–78) were collected, separated using denaturing gel electrophoresis, and stained with Coomassie. (D) The gel bands of monomeric and dimeric peaks were excised, trypsin digested, and subjected to mass spectrometric identification (MS/MS).

Figure 2. Laforin monomer and dimer have equal phosphatase activity.

(A) Specific activity of laforin monomer (peak B) and dimer (peak A) fractions obtained by size exclusion chromatography (Figure 1A) against pNPP. The activities are compared based on total protein content. (B) A representative immunoblotting image of varying concentration of laforin monomer and dimer fractions detected using anti-HIS₆ monoclonal antibody. (C) Specific activity for laforin monomer and dimer fractions against pNPP. The activities are compared based on total laforin content from the blot in panel B. (D) Phosphate release measured by malachite green assays using amylopectin as a substrate for laforin monomer and dimer fractions. Normalization of the activity was carried out for laforin content. All values are means ± SEM (**p<0.001) analyzed by independent sample ‘t’ test.

Figure 3. Phosphatase activity of monomeric and dimeric forms of SEX4 and Cm-laforin.

(A) Specific activity of SEX4 monomer and dimer fractions obtained by size exclusion chromatography (Figure S1) against pNPP. The activities are compared based on total SEX4 content (Figure S2). (B) Phosphate release measured by malachite green assays using amylopectin as a substrate for SEX4 monomer and dimer fractions. The activities are compared based on total SEX4 content. (C) Specific activity for Cm-laforin monomer and dimer fractions obtained by size exclusion chromatography (Figure S1) against pNPP. The activities are compared based on total Cm-laforin content (Figure S2). (D) Phosphate release measured by malachite green assays using amylopectin as a substrate for Cm-laforin monomer and dimer fractions. The activities are compared based on total Cm-laforin content. All values are means ± SEM (*p<0.05, **p<0.001) analyzed by one-way ANOVA.

Figure 4. Reducing agents prevent laforin oligomer formation.

(A) Gel-filtration analysis on Superdex 200 10/300 GL column of human laforin stored in the presence or absence of reducing agents (10 mM DTT). A laforin sample stored at −20°C in the absence of DTT showed an elution profile (non-reducing peak; squares) corresponding to an apparent molecular weight higher than 2,000 kDa. A laforin sample stored in the presence of 10 mM DTT (reducing peak; line) showed an elution profile corresponding to an apparent molecular weight of approximately 37 kDa. Calibration of the column with size standards is indicated; ordinates indicate the natural logarithm (Ln) of molecular weight (Mr) in kDa. (B) The non-reducing peak of purified laforin was treated or not with different amounts of DTT before analysis by non-reducing gel electrophoresis and immunoblotting using anti-laforin antibodies. The position of the monomeric, dimeric and multimeric forms of laforin is indicated. (C) Phosphatase activity of the non-reducing and reducing peaks of laforin was measured in the presence or absence of 10 mM DTT in the reaction mixture. All values are means ± SEM (**p<0.01; n: 3) analyzed by independent sample ‘t’ test. (D) Cell extracts from HEK293 cells transfected with plasmid pCMVmyc-laforin were analyzed by non-reducing gel electrophoresis. When indicated, samples were treated with different amounts of DTT before loading them into the electrophoresis gel. The position of the monomeric, dimeric and multimeric forms of myc-laforin is indicated.

Figure 5. Dimerization of laforin does not affect its ability to bind glucans.

(A) Equal amounts of monomeric (0.5 µg peak B fraction, Figure 1A) and dimeric (1 µg peak A fraction, Figure 1A) laforin were incubated with amylopectin and glucan-binding assay was performed as described in Methods. A representative image of the I (input), P (pellet), and S (supernatant) fractions analyzed by Western blotting is presented. (B) The OMFP phosphatase activity of GST-laforin fusion protein purified from bacteria was measured in the presence of different amounts of glycogen in the reaction mixture. We assigned the maximal phosphatase activity in the absence of glycogen as 100% and then compared activity in the presence of glycogen to this maximal amount. (C) Phosphatase activity of GST-laforin and GST-VHR in the absence or presence of glycogen (0.5 mg/ml) in the reaction mixture. As in Figure B, we assigned maximal phosphatase activities as 100% and compared activities in the presence of glycogen to the untreated samples (control). Values are means ± SEM of three independent experiments (*p<0.05) analyzed by independent sample ‘t’ test.

Figure 6. Dimerization of laforin does not affect its ability to associate with malin.

(A) Equal amount of monomeric and dimeric laforin used in section A of Figure 6, were mixed with FLAG-malin and immunoprecipitation was carried out as described in Methods. A representative image showing detection of monomeric and dimeric laforin in samples immunoprecipitated using anti-Flag agarose beads is presented. (B) Phosphatase activity of monomeric and dimeric laforin in the presence of malin was determined using amylopectin as substrate. (C) Representative image demonstrating the presence in the I (input), P (pellet), and S (supernatant) fractions from the glucan-binding assay of laforin monomer (0.5 µg) and dimer (1.0 µg), that had been previously mixed with Hs-malin-HIS₆ (1.0 µg). The membrane was blotted with α-laforin antibody.