Structural Mechanism of Laforin Function in Glycogen Dephosphorylation and Lafora Disease

Abstract

SUMMARY Glycogen is the major mammalian glucose storage cache and is critical for energy homeostasis. Glycogen synthesis in neurons must be tightly controlled, due to neuronal sensitivity to perturbations in glycogen metabolism. Lafora disease (LD) is a fatal, congenital, neurodegenerative epilepsy. Mutations in the gene encoding the glycogen phosphatase laforin result in hyperphosphorylated glycogen that forms water-insoluble inclusions called Lafora bodies (LBs). LBs induce neuronal apoptosis and are the causative agent of LD. The mechanism of glycogen dephosphorylation by laforin and dysfunction in LD is unknown. We report the crystal structure of laforin bound to phosphoglucan product, revealing its unique integrated tertiary and quaternary structure. Structure-guided mutagenesis combined with biophysical and biochemical analyses reveal the basis for normal function of laforin in glycogen metabolism. Analyses of LD patient mutations define the mechanism by which subsets of mutations disrupt laforin function. These data provide fundamental insights connecting glycogen metabolism to neurodegenerative disease.

Unique carbohydrate binding platforms employed by the glucan phosphatases

“…A laforin ortholog is conserved in all vertebrate genomes, but it has been lost in the vast majority of invertebrate genomes and lower organisms (including yeast, fly and worm genomes), and laforin-like orthologs are conserved in a subset of protists and invertebrates [44]. The vertebrate laforin orthologs are 66% identical at the amino acid level, while laforin orthologs from humans to C. merolae are only 25% identical [24, 44, 47]. The invertebrates and protists that contain laforin-like orthologs are organisms with genomes that are predicted to have undergone slower rates of molecular evolution and/or organisms that metabolize a carbohydrate similar to a Lafora body [44, 48].…”

“…To define the specificity of glucan phosphatases, we and others have employed a radiolabelling assay that utilizes the specific action of the glucan dikinases [11-13, 37, 46, 47]. Two independent paths are followed to generate radiolabelled starch at either the C6 or C3 position.…”

“…LSF2 differs in that it exclusively dephosphorylates the C3 position [13, 37]. Insights from recent structures as well as structure-guided mutagenesis demonstrated that the DSP domain is responsible for engagement and orientation of the glucan to achieve position-specific specificity [11, 37, 46, 47]. These studies also allowed for the identification of a glucan phosphatase catalytic site (PTP-loop) consensus motif of Cys, hydrophilic (ζ), Ala, Gly, long chain aliphatic (Ψ), Gly, Arg (GζAGΨGR) [11].…”

Regulatory Mechanisms and Novel Therapeutic Targeting Strategies for Protein Tyrosine Phosphatases

“…199–202 The three-dimensional structure, along with complementary biophysical data, suggests that laforin can exist as an antiparallel dimer, which is mediated by residues in its phosphatase domain. 203 Importantly, it was shown that the Lafora disease associated mutation F321S can interrupt dimerization of laforin and reduces its phosphatase activity toward glycogen. Myotubularin related protein 2 (MTMR2), a phosphoinositide lipid phosphatase, can also form as a homodimer (Figure 10C), which is important for its function inside the cell.…”

Recombinant Human Laforin Expressed in Insect Cells: Expression, Purification, and Biochemical Characterizations

“…Very recently, crystal structure of nearly full-length Laforin was determined and it provided the functional mechanism of Laforin and the structural basis of disease. 16 Although crystal structure of Laforin was determined using the E. coli expressed recombinant Laforin, I-Laforin can be more preferable over B-Laforin for future biochemical studies because I-Laforin exhibited enhanced substrate specificity in our studies. …”