Laforin, a dual specificity phosphatase that dephosphorylates complex carbohydrates

Abstract

Laforin is the only phosphatase in the animal kingdom that contains a carbohydrate-binding module. Mutations in the gene encoding laforin result in Lafora disease, a fatal autosomal recessive neurodegenerative disorder, which is diagnosed by the presence of intracellular deposits of insoluble complex carbohydrates known as Lafora bodies. We demonstrate that laforin interacts with proteins known to be involved in glycogen metabolism and rule out several of these proteins as potential substrates. Surprisingly, we find that laforin displays robust phosphatase activity against a phosphorylated complex carbohydrate. Furthermore, this activity is unique to laforin, since several other phosphatases are unable to dephosphorylate polysaccharides. Finally, fusing the carbohydrate-binding module of laforin to the dual specific phosphatase VHR does not result in the ability of this phosphatase to dephosphorylate polysaccharides. Therefore, we hypothesize that laforin is unique in its ability to utilize a phosphorylated complex carbohydrate as a substrate and that this function may be necessary for the maintenance of normal cellular glycogen.

Figure 1

GSK3β is not a substrate of laforin in vivo or in vitro. (A) HEK293 cells were cotransfected with FLAG-tagged laforin and HA-tagged GSK3β. Western analysis probed with α-FLAG HRP of the FLAG IP is shown in the left panel, while Western analysis probed with α-HA HRP of the FLAG and HA IPs is shown in the right panel. (B) WT and C/S FLAG-tagged laforin were co-transfected along with HA-tagged GSK3β into CHO-IR cells. Western analysis of WCL probed with α-FLAG demonstrates the expression level of laforin (top panel). Western analysis of α-HA immunoprecipitates using an antibody directed against PSer9 of GSK3β is shown in the bottom panel. C/S laforin is consistently expressed at a higher level than WT laforin in all cell types analyzed. (C) WT His-tagged laforin was expressed in and purified from bacteria. HA-tagged GSK3β was immunoprecipitated from CHO-IR cells treated with insulin as described in Materials and methods. Laforin and GSK3β were allowed to react in the presence or absence of tungstate (T) in standard phosphatase assays followed by Western analysis of the samples using α-PSer9 (top panel) or α-HA to assess equal loading (bottom panel). Samples were run in duplicate. (D) HA-tagged GSK3β was immunoprecipitated from transiently transfected HEK239 cells. Increasing amounts of bacterially expressed laforin were allowed to react with immunoprecipitated GSK3β in the absence (top panel) or presence (bottom panel) of tungstate. Western analysis of the samples was performed using α-PTyr (4G10).

Figure 2

Laforin interacts with proteins involved in glycogen metabolism. (A) CHO-IR cells were transfected with WT FLAG-tagged laforin (lane 1), C/S FLAG-tagged laforin (lane 2) or empty vector (lane 3). Laforin was immunoprecipitated using α-FLAG resin and endogenous GS was immunoprecipitated using α-GS. Western analyses of the FLAG IPs using α-GS and α-FLAG are shown in the left panels (* denotes a nonspecific band) while Western analyses of the GS IPs are shown in the right panels. (B) HEK293 cells were co-transfected with WT FLAG-tagged laforin and myc-tagged PTG family members. Whole cell lysates (WCLs) were immunoblotted with α-myc (PTG, G_L, R6) or immunoprecipitated using α-myc (G_M) followed by immunoblotting with α-myc (left panels) to ascertain the expression levels of the PTG family members. The remainder of the WCLs was immunoprecipitated using α-FLAG resin and immunoblotted with α-myc or α-FLAG (right panels).

Figure 3

Laforin dephosphorylates amylopectin. WT and C/S His-tagged laforin were expressed in and purified from bacteria. Standard malachite green assays were performed containing 100 ng of enzyme and 45 μg of amylopectin or glycogen as described in Materials and Methods. Phosphate release was calculated from the change of A₆₂₀. Error bars represent the standard error of the mean (SEM).

Figure 4

Laforin is unique in its ability to dephosphorylate amylopectin. (A) All enzymes were subjected to standard pNPP assays as described in Materials and Methods. Phosphatase activity was calculated from the change of absorbance at 410nm. (B) Malachite green assays were performed as described in Materials and Methods. The numbers positioned above the bars represent the ratio of phosphate release (malachite green assay) to phosphatase activity (pNPP assay). Phosphate release was calculated from the change of A₆₂₀. Error bars represent the SEM.

Figure 5

VHR containing a CBM is not able to dephosphorylate amylopectin. (A) Amino acids 1–162 of laforin containing laforin’s CBM were fused in frame to amino acids 35–185 of VHR followed by a 6His tag. The alignment of laforin with that of VHR is shown at the fusion point. (B) Phosphatase assays utilizing laforin and CBM-VHR were performed using pNPP as a substrate (left graph) or amylopectin as a substrate (right graph). Error bars represent the SEM.