Assessing the Biological Activity of the Glucan Phosphatase Laforin.
Ontology highlight
ABSTRACT:
SUBMITTER: Roma-Mateo C
PROVIDER: S-EPMC5339740 | biostudies-literature | 2016
REPOSITORIES: biostudies-literature
Publications
Assessing the Biological Activity of the Glucan Phosphatase Laforin.
Romá-Mateo Carlos C Raththagala Madushi M Gentry Mathew S MS Sanz Pascual P
Methods in molecular biology (Clifton, N.J.) 20160101
Glucan phosphatases are a recently discovered family of enzymes that dephosphorylate either starch or glycogen and are essential for proper starch metabolism in plants and glycogen metabolism in humans. Mutations in the gene encoding the only human glucan phosphatase, laforin, result in the fatal, neurodegenerative, epilepsy known as Lafora disease. Here, we describe phosphatase assays to assess both generic laforin phosphatase activity and laforin's unique glycogen phosphatase activity. ...[more]
Similar Datasets
Project description:Lafora disease is a rare yet invariably fatal form of progressive neurodegenerative epilepsy resulting from mutations in the phosphatase laforin. Several therapeutic options for Lafora disease patients are currently being explored, and these therapies would benefit from a biochemical means of assessing functional laforin activity following treatment. To date, only clinical outcomes such as decreases in seizure frequency and severity have been used to indicate success of epilepsy treatment. However, these qualitative measures exhibit variability and must be assessed over long periods of time. In this work, we detail a simple and sensitive bioassay that can be used for the detection of functional endogenous laforin from human and mouse tissue.We generated antibodies capable of detecting and immunoprecipitating endogenous laforin. Following laforin immunoprecipitation, laforin activity was assessed via phosphatase assays using para-nitrophenylphosphate (pNPP) and a malachite green-based assay specific for glucan phosphatase activity.We found that antibody binding to laforin does not impede laforin activity. Furthermore, the malachite green-based glucan phosphatase assay used in conjunction with a rabbit polyclonal laforin antibody was capable of detecting endogenous laforin activity from human and mouse tissues. Importantly, this assay discriminated between laforin activity and other phosphatases.The bioassay that we have developed utilizing laforin antibodies and an assay specific for glucan phosphatase activity could prove valuable in the rapid detection of functional laforin in patients to which novel Lafora disease therapies have been administered.
| S-EPMC3864130 | biostudies-literature
Project description:Laforin, encoded by a gene that is mutated in Lafora Disease (LD, OMIM 254780), is a modular protein composed of a carbohydrate-binding module and a dual-specificity phosphatase domain. Laforin is the founding member of the glucan-phosphatase family and regulates the levels of phosphate present in glycogen. Multiple reports have described the capability of laforin to form dimers, although the function of these dimers and their relationship with LD remains unclear. Recent evidence suggests that laforin dimerization depends on redox conditions, suggesting that disulfide bonds are involved in laforin dimerization. Using site-directed mutagenesis we constructed laforin mutants in which individual cysteine residues were replaced by serine and then tested the ability of each protein to dimerize using recombinant protein as well as a mammalian cell culture assay. Laforin-Cys329Ser was the only Cys/Ser mutant unable to form dimers in both assays. We also generated a laforin truncation lacking the last three amino acids, laforin-Cys329X, and this truncation also failed to dimerize. Interestingly, laforin-Cys329Ser and laforin-Cys329X were able to bind glucans, and maintained wild type phosphatase activity against both exogenous and biologically relevant substrates. Furthermore, laforin-Cys329Ser was fully capable of participating in the ubiquitination process driven by a laforin-malin complex. These results suggest that dimerization is not required for laforin phosphatase activity, glucan binding, or for the formation of a functional laforin-malin complex. Cumulatively, these results suggest that cysteine 329 is specifically involved in the dimerization process of laforin. Therefore, the C329S mutant constitutes a valuable tool to analyze the physiological implications of laforin's oligomerization.
| S-EPMC3724922 | biostudies-literature
Project description:Lafora disease (LD) is a rare, fatal neurodegenerative disorder characterized by the accumulation of glycogen-like inclusions in the cytoplasm of cells from most tissues of affected patients. One hundred years after the first description of these inclusions, the molecular bases underlying the processes involved in LD physiopathology are finally being elucidated. The main cause of the disease is related to the activity of two proteins, the dual-specificity phosphatase laforin and the E3-ubiquitin ligase malin, which form a functional complex. Laforin is unique in humans, as it is composed of a carbohydrate-binding module attached to a cysteine-based catalytic dual-specificity phosphatase domain. Laforin directly dephosphorylates glycogen, but other proteinaceous substrates, if they exist, have remained elusive. Recently, an emerging set of laforin-binding partners apart from malin have been described, suggestive of laforin roles unrelated to its catalytic activity. Further investigations based on different transgenic mouse models have shown that the laforin-malin complex is also involved in other cellular processes, such as response to endoplasmic reticulum stress and misfolded protein clearance by the lysosomal pathway. However, controversial data and some missing links still make it difficult to assess the concrete relationship between glycogen deregulation and neuronal damage leading to the fatal symptoms observed in LD patients, such as myoclonic seizures and epilepsy. Consequently, clinical treatments are far from being achieved. In the present review, we focus on the knowledge of laforin biology, not only as a glucan phosphatase, but also as an adaptor protein involved in several physiological pathways.
| S-EPMC3371293 | biostudies-literature
Project description:Lafora disease (LD) is a fatal autosomal recessive neurodegenerative disease. A hallmark of LD is cytoplasmic accumulation of insoluble glucans, called Lafora bodies (LBs). Mutations in the gene encoding the phosphatase laforin account for approximately 50% of LD cases, and this gene is conserved in all vertebrates. We recently demonstrated that laforin is the founding member of a unique class of phosphatases that dephosphorylate glucans.Herein, we identify laforin orthologs in a protist and two invertebrate genomes, and report that laforin is absent in the vast majority of protozoan genomes and it is lacking in all other invertebrate genomes sequenced to date. We biochemically characterized recombinant proteins from the sea anemone Nematostella vectensis and the amphioxus Branchiostoma floridae to demonstrate that they are laforin orthologs. We demonstrate that the laforin gene has a unique evolutionary lineage; it is conserved in all vertebrates, a subclass of protists that metabolize insoluble glucans resembling LBs, and two invertebrates. We analyzed the intron-exon boundaries of the laforin genes in each organism and determine, based on recently published reports describing rates of molecular evolution in Branchiostoma and Nematostella, that the conservation of laforin is linked to the molecular rate of evolution and the glucan metabolism of an organism.Our results alter the existing view of glucan phosphorylation/dephosphorylation and strongly suggest that glucan phosphorylation is a multi-Kingdom regulatory mechanism, encompassing at least some invertebrates. These results establish boundaries concerning which organisms contain laforin. Laforin is conserved in all vertebrates, it has been lost in the vast majority of lower organisms, and yet it is an ancient gene that is conserved in a subset of protists and invertebrates that have undergone slower rates of molecular evolution and/or metabolize a carbohydrate similar to LBs. Thus, the laforin gene holds a unique place in evolutionary biology and has yielded insights into glucan metabolism and the molecular etiology of Lafora disease.
| S-EPMC2714694 | biostudies-literature
Project description:Glucan phosphatases are central to the regulation of starch and glycogen metabolism. Plants contain two known glucan phosphatases, Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2), which dephosphorylate starch. Starch is water-insoluble and reversible phosphorylation solubilizes its outer surface allowing processive degradation. Vertebrates contain a single known glucan phosphatase, laforin, that dephosphorylates glycogen. In the absence of laforin, water-soluble glycogen becomes insoluble, leading to the neurodegenerative disorder Lafora Disease. Because of their essential role in starch and glycogen metabolism glucan phosphatases are of significant interest, yet a comparative analysis of their activities against diverse glucan substrates has not been established. We identify active site residues required for specific glucan dephosphorylation, defining a glucan phosphatase signature motif (C?AG?GR) in the active site loop. We further explore the basis for phosphate position-specific activity of these enzymes and determine that their diverse phosphate position-specific activity is governed by the phosphatase domain. In addition, we find key differences in glucan phosphatase activity toward soluble and insoluble polyglucan substrates, resulting from the participation of ancillary glucan-binding domains. Together, these data provide fundamental insights into the specific activity of glucan phosphatases against diverse polyglucan substrates.
| S-EPMC4645622 | biostudies-literature
Project description: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.
| S-EPMC3162602 | biostudies-literature
Project description:With the recent discovery of a unique class of dual-specificity phosphatases that dephosphorylate glucans, we report an in vitro assay tailored for the detection of phosphatase activity against phosphorylated glucans. We demonstrate that, in contrast to a general phosphatase assay using a synthetic substrate, only phosphatases that possess glucan phosphatase activity liberate phosphate from the phosphorylated glucan amylopectin using the described assay. This assay is simple and cost-effective, providing reproducible results that clearly establish the presence or absence of glucan phosphatase activity. The assay described will be a useful tool in characterizing emerging members of the glucan phosphatase family.
| S-EPMC3577954 | biostudies-literature
Project description:Living organisms utilize carbohydrates as essential energy storage molecules. Starch is the predominant carbohydrate storage molecule in plants while glycogen is utilized in animals. Starch is a water-insoluble polymer that requires the concerted activity of kinases and phosphatases to solubilize the outer surface of the glucan and mediate starch catabolism. All known plant genomes encode the glucan phosphatase Starch Excess4 (SEX4). SEX4 can dephosphorylate both the starch granule surface and soluble phosphoglucans and is necessary for processive starch metabolism. The physical basis for the function of SEX4 as a glucan phosphatase is currently unclear. Herein, we report the crystal structure of SEX4, containing phosphatase, carbohydrate-binding, and C-terminal domains. The three domains of SEX4 fold into a compact structure with extensive interdomain interactions. The C-terminal domain of SEX4 integrally folds into the core of the phosphatase domain and is essential for its stability. The phosphatase and carbohydrate-binding domains directly interact and position the phosphatase active site toward the carbohydrate-binding site in a single continuous pocket. Mutagenesis of the phosphatase domain residue F167, which forms the base of this pocket and bridges the two domains, selectively affects the ability of SEX4 to function as a glucan phosphatase. Together, these results reveal the unique tertiary architecture of SEX4 that provides the physical basis for its function as a glucan phosphatase.
| S-EPMC2932622 | biostudies-literature
Project description: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.
| S-EPMC2774450 | biostudies-literature
Project description:Laforin, encoded by the EPM2A gene, is a dual specificity protein phosphatase that has a functional glycogen-binding domain. Mutations in the EPM2A gene account for around half of the cases of Lafora disease, an autosomal recessive neurodegenerative disorder, characterized by progressive myoclonus epilepsy. The hallmark of the disease is the presence of Lafora bodies, which contain polyglucosan, a poorly branched form of glycogen, in neurons and other tissues. We examined the level of laforin protein in several mouse models in which muscle glycogen accumulation has been altered genetically. Mice with elevated muscle glycogen have increased laforin as judged by Western analysis. Mice completely lacking muscle glycogen or with 10% normal muscle glycogen had reduced laforin. Mice defective in the GAA gene encoding lysosomal alpha-glucosidase (acid maltase) overaccumulate glycogen in the lysosome but did not have elevated laforin. We propose, therefore, that laforin senses cytosolic glycogen accumulation which in turn determines the level of laforin protein.
| S-EPMC1850102 | biostudies-literature