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• W2010595874 abstract "Lafora disease (progressive myoclonus epilepsy of Lafora type) is an autosomal recessive neurodegenerative disorder resulting from defects in the EPM2A gene. EPM2Aencodes a 331-amino acid protein containing a carboxyl-terminal phosphatase catalytic domain. We demonstrate that the EPM2Agene product also contains an amino-terminal carbohydrate binding domain (CBD) and that the CBD is critical for association with glycogen both in vitro and in vivo. The CBD domain localizes the phosphatase to specific subcellular compartments that correspond to the expression pattern of glycogen processing enzyme, glycogen synthase. Mutations in the CBD result in mis-localization of the phosphatase and thereby suggest that the CBD targets laforin to intracellular glycogen particles where it is likely to function. Thus naturally occurring mutations within the CBD of laforin likely result in progressive myoclonus epilepsy due to mis-localization of phosphatase expression. Lafora disease (progressive myoclonus epilepsy of Lafora type) is an autosomal recessive neurodegenerative disorder resulting from defects in the EPM2A gene. EPM2Aencodes a 331-amino acid protein containing a carboxyl-terminal phosphatase catalytic domain. We demonstrate that the EPM2Agene product also contains an amino-terminal carbohydrate binding domain (CBD) and that the CBD is critical for association with glycogen both in vitro and in vivo. The CBD domain localizes the phosphatase to specific subcellular compartments that correspond to the expression pattern of glycogen processing enzyme, glycogen synthase. Mutations in the CBD result in mis-localization of the phosphatase and thereby suggest that the CBD targets laforin to intracellular glycogen particles where it is likely to function. Thus naturally occurring mutations within the CBD of laforin likely result in progressive myoclonus epilepsy due to mis-localization of phosphatase expression. carbohydrate binding domain enhanced green fluorescence protein phosphate-buffered saline para-nitrophenylphosphate Lafora disease (OMIM 254780) is an autosomal recessive neurodegenerative disorder. It accounts for a subset of severe epilepsies with myoclonic, tonic seizures and progressive neurologic deterioration. The disease usually occurs between the ages of 7 and 20 and results in death within 10 years. Lafora disease is characterized by the accumulation of intraneuronal periodic acid-Schiff-positive cytoplasmic inclusion bodies (Lafora bodies), which contain 80–93% polyglucosan (1Minassian B.A. Pediatr. Neurol. 2001; 25: 21-29Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 2Serratosa J.M. Gardiner R.M. Lehesjoki A.E. Pennacchio L.A. Myers R.M. Adv. Neurol. 1999; 79: 383-398PubMed Google Scholar). Lafora bodies also develop in brain, liver, skin, kidney, skeletal, and cardiac muscle, and biopsy of axillary skin provides a reliable diagnosis for Lafora disease (3Busard B.L. Renier W.O. Gabreels F.J. Jaspar H.H. van Haelst U.J. Slooff J.L. Arch. Neurol. 1986; 43: 296-299Crossref PubMed Scopus (31) Google Scholar, 4Busard H.L. Gabreels-Festen A.A. Renier W.O. Gabreels F.J. Stadhouders A.M. Ann. Neurol. 1987; 21: 599-601Crossref PubMed Scopus (54) Google Scholar, 5Iannaccone S. Zucconi M. Quattrini A. Smirne S. J. Neurol. Neurosurg. Psychiatry. 1993; 56: 1339-1340Crossref PubMed Scopus (2) Google Scholar, 6Karimipour D. Lowe L. Blaivas M. Sachs D. Johnson T.M. J. Am. Acad. Dermatol. 1999; 41: 790-792Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). Minassian et al. (7Minassian B.A. Lee J.R. Herbrick J.A. Huizenga J. Soder S. Mungall A.J. Dunham I. Gardner R. Fong C.Y. Carpenter S. Jardim L. Satishchandra P. Andermann E. Snead III, O.C. Lopes-Cendes I. Tsui L.C. Delgado-Escueta A.V. Rouleau G.A. Scherer S.W. Nat. Genet. 1998; 20: 171-174Crossref PubMed Scopus (413) Google Scholar) and Serratosa et al. (8Serratosa J.M. Gomez-Garre P. Gallardo M.E. Anta B. de Bernabe D.B. Lindhout D. Augustijn P.B. Tassinari C.A. Malafosse R.M. Topcu M. Grid D. Dravet C. Berkovic S.F. de Cordoba S.R. Hum. Mol. Genet. 1999; 8: 345-352Crossref PubMed Scopus (197) Google Scholar) independently identified the gene mutated in Lafora disease to be present on chromosome 6q24. This chromosome localization distinguishes Lafora disease from the progressive myoclonus epilepsy of Unverricht-Lundborg type (21q22.3). Positional cloning of theEPM2A (epilepsy of progressivemyoclonus Type 2) gene revealed an encoded protein product of 331 amino acids containing a dual specificity protein phosphatase catalytic active site motif, HCXXGXXRS/T (9Denu J.M. Stuckey J.A. Saper M.A. Dixon J.E. Cell. 1996; 87: 361-364Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar, 10Ganesh S. Agarwala K.L. Ueda K. Akagi T. Shoda K. Usui T. Hashikawa T. Osada H. Delgado-Escueta A.V. Yamakawa K. Hum. Mol. Genet. 2000; 9: 2251-2261Crossref PubMed Scopus (131) Google Scholar). A total of 30 different disease-related mutations have been described in the EPM2A gene (11Gomez-Garre P. Sanz Y. Rodriguez De Cordoba S.R. Serratosa J.M. Eur. J. Hum. Genet. 2000; 8: 946-954Crossref PubMed Scopus (49) Google Scholar, 12Minassian B.A. Ianzano L. Meloche M. Andermann E. Rouleau G.A. Delgado-Escueta A.V. Scherer S.W. Neurology. 2000; 55: 341-346Crossref PubMed Scopus (114) Google Scholar), of which 12 cause missense mutations (Fig. 1A). In this study, we show that the NH2 terminus of laforin contains a carbohydrate binding domain that targets the phosphatase to glycogen where it is likely to function. Mutations within the CBD1 abolish the binding of laforin to glycogen, and this is likely to be the cause of some forms of progressive myoclonus epilepsy. Laforin was amplified from a human muscle cDNA library (CLONTECH) by PCR and cloned into BamHI and HindIII sites of the bacteria expression plasmid pET21a (Novagen) to produce a recombinant protein with a COOH-terminal 6-histidine tag. The cDNA of laforin was also cloned into a mammalian expression vector pcDNA3.1NF (13Taylor G.S. Maehama T. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8910-8915Crossref PubMed Scopus (279) Google Scholar) by PCR. Fusion proteins expressed from vector pcDNA3.1NF contain an NH2-terminal M2-FLAG epitope of 8 amino acid residues. To produce a fusion protein with a COOH-terminal enhanced green fluorescence protein (EGFP), the cDNA sequence of laforin was excised from pET21a-Laf with NheI and HindIII and ligated to the same sites of pEGFP-N1 (CLONTECH). All site-directed mutations were confirmed by nucleotide sequencing. The cDNA of human glycogen synthase was amplified from the human muscle cDNA library (CLONTECH) and contained a COOH-terminal Myc epitope of 10 amino acid residues. Recombinant proteins were expressed in Escherichia coli BL21 (DE3) Codonplus cells (Stratagene). The expressed proteins were purified using Ni2+-agarose (Qiagen) as described previously (14Maehama T. Taylor G.S. Slama J.T. Dixon J.E. Anal. Biochem. 2000; 279: 248-250Crossref PubMed Scopus (99) Google Scholar). HEK 293 cells and COS1 cells were grown in modified Eagle's medium supplemented with fetal bovine serum (10% v/v), penicillin (100 units/ml), streptomycin (100 mg/ml), and l-glutamine (2 mm) (Invitrogen). Transfections with the cDNA constructs were carried out using FuGENE 6 (Roche Diagnostics). For in vitroexperiments, 0.5 μg of purified recombinant protein was incubated in 1 ml of 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.1% (v/v) 2-mercaptoethanol, 0.1 mg/ml bovine serum albumin containing 10 mg/ml glycogen (Roche Diagnostics) at 4 °C for 30 min. After centrifugation at 100,000 × g for 90 min, the supernatant and the pellet fractions were collected and subjected to Western blot analysis using anti-polyhistidine antibody His probe (H-15) (Santa Cruz Biotechnology). For in vivo experiments, HEK 293 cells were transiently transfected and after 24 h, and the cells were washed with cold PBS three times and harvested in hypotonic buffer consisting of 20 mm Tris-HCl, pH 7.5, 10 mm NaCl, 200 mm sucrose, 1 mmphenylmethylsulfonyl fluoride, 1 mm EDTA, 1 μg/ml of aprotinin, leupeptin, and pepstatin. The cells were then lysed on ice using Dounce homogenizer, and the cell lysates were cleared by centrifugation at 10,000 × g for 10 min. After incubating at 30 °C for 30 min in the presence or absence of 5 units/ml of α-amylase (Sigma), the cell lysates were ultracentrifuged at 100,000 × g for 90 min, and the supernatant and pellet fractions were collected and subjected to Western blot analysis using anti-FLAG M2 antibody (Sigma). 24 h after transfection, HEK 293 cells were washed with ice-cold PBS three times and harvested in ice-cold lysis buffer consisting of 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Triton X-100, 0.1% 2-mercaptoethanol, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, leupeptin, and pepstatin. The cells were lysed at 4 °C by constant agitation for 30 min, and then the cell lysates were cleared by centrifugation at 10,000 × g for 10 min. The supernatants were subjected to binding with prewashed anti-FLAG M2 affinity resin (Sigma) at 4 °C for 4 h by constant rotation (using 25 μl of resin slurry/106 cells). Then the resins were pelleted by centrifugation at 500 × g for 1 min and washed three times with 1 ml of lysis buffer without 1% Triton X-100. The resulting resins were subjected to phosphatase activity assay and Western blot analysis. The samples were analyzed on 12% SDS-polyacrylamide gels and transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore). The membranes were probed with appropriate first antibodies and horseradish peroxidase-conjugated second antibody. Para-nitrophenylphosphate (pNPP) assays were carried out at 30 °C as described previously (15Taylor G.S. Liu Y. Baskerville C. Charbonneau H. J. Biol. Chem. 1997; 272: 24054-24063Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 16Taylor G.S. Dixon J.E. Anal. Biochem. 2001; 295: 122-126Crossref PubMed Scopus (28) Google Scholar), using 0.1 μg of recombinant protein. COS1 cells were transfected with cDNA constructs of laforin-EGFP fusion proteins. After 24 h, the cells were washed with ice-cold PBS three times and fixed in 4% paraformaldehyde at room temperature for 10 min. For COS1 cells co-transfected with pEGFP-Laf and pcDNA4/Myc-GS, after fixation with 4% paraformaldehyde, the cells were permeablized in methanol at −20 °C for 5 min. Then the cells were washed three times with PBS and blocked in 3% bovine serum albumin for 30 min at room temperature, followed by incubating with mouse anti-Myc antibody (Santa Cruz Biotechnology) and Texas red anti-mouse IgG (Vector Laboratories) at room temperature for 1 h, respectively. The Myc-tagged glycogen synthase proteins were visualized as red fluorescence under fluorescence microscope, while the laforin EGFP fusion proteins were visualized as green fluorescence. We cloned the laforin cDNA from a human muscle library (CLONTECH). The sequence of the coding region was identical to that published by Ganesh et al. (10Ganesh S. Agarwala K.L. Ueda K. Akagi T. Shoda K. Usui T. Hashikawa T. Osada H. Delgado-Escueta A.V. Yamakawa K. Hum. Mol. Genet. 2000; 9: 2251-2261Crossref PubMed Scopus (131) Google Scholar). Analysis of the protein conserved domain data base (CDD; www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) revealed that laforin contained a putative starch binding domain (CBD-4), encompassing the NH2-terminal 116 amino acids (Fig.1B). CBD-4 is found in a variety of glycosylhydrolases from bacteria and fungi (17Svensson B. Jespersen H. Sierks M.R. MacGregor E.A. Biochem. J. 1989; 264: 309-311Crossref PubMed Scopus (177) Google Scholar, 18Svensson B. Plant Mol. Biol. 1994; 25: 141-157Crossref PubMed Scopus (407) Google Scholar, 19Sauer J. Sigurskjold B.W. Christensen U. Frandsen T.P. Mirgorodskaya E. Harrison M. Roepstorff P. Svensson B. Biochim. Biophys. Acta. 2000; 1543: 275-293Crossref PubMed Scopus (176) Google Scholar), where the function of CBD-4 is to bind polysaccharide substrates prior to cleavage. To investigate the function of the CBD of laforin, several point mutations were created at the CBD-4 invariant residues (Fig.1B) and their effects on both phosphatase activity and carbohydrate binding examined. Trp32 was mutated to Gly (W32G), since this is a mutation that is also found in Lafora disease. In addition, we mutated the invariant Lys87 to Ala (K87A) and prepared the double mutant (W32G/K87A). We expressed both wild type and mutant laforin proteins in HEK 293 cells. The expressed proteins were immunoprecipitated and the phosphatase activities determined usingpNPP. All three CBD mutant proteins, W32G, K87A and W32G/K87A, showed about 50% of the wild type phosphatase activity toward pNPP (Fig. 2A). As expected, the active site mutant (C266S) had no phosphatase activity. These results suggested that mutations in the CBD had a limited effect on protein phosphatase activity of laforin. Cells overexpressing laforin with the mutations in the CBD of laforin were then examined by subcellular fractionation. The glycogen-microsomal complexes were sedimented from cytosol by ultracentrifugation. Approximately 50% of the wild type laforin was associated with the pelleted glycogen-microsomal complexes (Fig.2B). Treatment of the glycogen-microsomal suspension with α-amylase is known to lead to digestion of the glycogen particles (20Fong N.M. Jensen T.C. Shah A.S. Parekh N.N. Saltiel A.R. Brady M.J. J. Biol. Chem. 2000; 275: 35034-35039Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). As shown in Fig. 2B, α-amylase treatment released laforin from the glycogen complexes, suggesting specific association of laforin with the intracellular glycogen complexes. The catalytically inactive phosphatase mutant (C266S) was also released upon treatment with α-amylase, indicating that phosphatase activity is not required for the glycogen association. Interestingly, the CBD mutant proteins, W32G and W32G/K87A, could not be detected in the glycogen-microsomal complexes, suggesting that they are not associated with the intracellular glycogen complexes. Only a very small fraction of K87A protein was present in the glycogen-microsomal complexes, and after α-amylase treatment all of the K87A protein was released into the cytosol. These results indicate that both the W32G and K87A mutations abolish the association of laforin with glycogen complexes. Many proteins associate with intracellular glycogen complexes via interactions with glycogen binding proteins. For instance, the catalytic subunit of protein phosphatase 1 is associated with glycogen only when it binds to the glycogen-targeting regulatory subunits (21Newgard C.B. Brady M.J. O'Doherty R.M. Saltiel A.R. Diabetes. 2000; 49: 1967-1977Crossref PubMed Scopus (152) Google Scholar). To determine whether laforin interacts with an intermediate glycogen-binding protein or directly binds to glycogen, we carried out an in vitro glycogen binding experiment using protein-free glycogen and recombinant wild type and mutant laforin proteins. A His-tagged recombinant laforin was expressed in bacteria and purified on Ni2+-agarose column. To determine whether the recombinant laforin was properly folded, the phosphatase activity was analyzed. The specific activity of the recombinant laforin towardpNPP was 1.34 × 10−5 mol/mg/min at the optimum pH of 5.0. Under the same conditions, the CBD mutant protein W32G/K87A showed 75% activity of the wild type enzyme, while the C266S mutant was inactive. We were unable to purify the W32G mutant protein, because it was present in insoluble bacteria pellet. We incubated the purified recombinant proteins with glycogen at 4 °C for 30 min and then precipitated the glycogen particles by ultracentrifugation. Both wild type enzyme and the C266S mutant protein co-sedimented with glycogen, while the CBD mutant protein W32G/K87A remained in the supernatant (Fig. 2C). Wild type laforin protein did not exhibit any tendency to aggregate, excluding this a reason for its presence in the glycogen pellet (Fig. 2D). Thus our results suggest that laforin associates directly with glycogen, and mutations that disrupt the CBD of laforin abrogate the glycogen binding. Based on our glycogen binding studies of laforin CBD domain, we developed a functional model by threading its amino acid sequence onto the crystal structure of cyclodextrin glycosyltransferase (protein data bank number: 2DIJ) using program O (22Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). Our model predicts that the two invariant Trp residues in the CBD of laforin (Fig.3, W32 and W99) directly interact with the polysaccharide in a manner similar to the two Trp residues (Trp616 and Trp662) in cyclodextrin glycosyltransferase (Fig. 1B, CDGT). The invariant residue Lys87 is also predicted to directly interact with the carbohydrate via several hydrogen bonds. Lafora disease-related mutation of Trp32 to glycine (W32G) would disrupt the polysaccharide binding pocket and also potentially unfold the region immediately adjacent to the binding pocket. Our K87A mutation would also affect the carbohydrate binding by disrupting hydrogen bonds between the protein and carbohydrate. Other mutations found in patients with myoclonus epilepsy are also predicted to alter the ability of the protein to bind to glycogen. For example, F84L would affect the positioning of Trp85 in the polysaccharide binding pocket, while F88L would disturb the structural integrity of the polysaccharide binding site by altering the positioning of Lys87. To study the subcellular localization of laforin, the full-length protein was fused to the NH2 terminus of EGFP. The laforin protein was localized to punctate cytoplasmic structures in transfected COS1 cells (Fig. 4,A and B). Glycogen synthase is known to localize to intracellular glycogen particles (23Brady M.J. Nairn A.C. Saltiel A.R. J. Biol. Chem. 1997; 272: 29698-29703Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 24Brady M.J. Kartha P.M. Aysola A.A. Saltiel A.R. J. Biol. Chem. 1999; 274: 27497-27504Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 25Fernandez-Novell J.M. Bellido D. Vilaro S. Guinovart J.J. Biochem. J. 1997; 321: 227-231Crossref PubMed Scopus (66) Google Scholar, 26Ferrer J.C. Baque S. Guinovart J.J. FEBS Lett. 1997; 415: 249-252Crossref PubMed Scopus (49) Google Scholar) and was used as a control for laforin expression. Expression of cDNA constructs pEGFP-Laf and pcDNA4/Myc-GS showed that laforin and glycogen synthase co-localize in the same punctate structures within the cytoplasm of transfected COS1 cells (Fig. 4A; laforin was shown ingreen fluorescence; glycogen synthase was shown inred fluorescence.). The subcellular distribution of the phosphatase-inactive laforin mutant proteins (C266S and D234A) showed the same punctate pattern as the wild type enzyme (Fig. 4B). In contrast, the CBD mutant laforins (W32G, K87A, W32G/K87A) distributed evenly throughout the cytoplasm and did not exhibit a punctate expression pattern (Fig. 4B), suggesting that the CBD domain is responsible for the specific subcellular localization of laforin. Taking together, our subcellular localization studies support the concept that laforin is targeted to intracellular glycogen particles by its CBD domain and mutations that disrupt carbohydrate binding abolish laforin targeting. What is the in vivo substrate of laforin? Although there currently is no answer to this question, the clinical and genetic features of Lafora disease distinguish it from other well known glycogen storage diseases (27Wolfsdorf J.I. Holm I.A. Weinstein D.A. Endocrinol. Metab. Clin. 1999; 28: 801-823Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Our work clearly demonstrated that laforin is targeted directly to glycogen. It would not be surprising that the substrate would also be directly involved in glycogen metabolism. The substrate likely contributes to the relative complexity of glycogen metabolism in vertebrates, since we have been unable to find laforin orthologues in worms or flies. Efforts are currently in progress to identify candidate phosphoprotein substrates for the laforin phosphatase. In summary, our studies indicate that the laforin phosphatase contains an NH2-terminal CBD that targets the protein to glycogen and that mutations in CBD, including the W32G mutation found in Lafora disease, lead to mis-targeting of laforin. The characteristic histology of Lafora disease is an intraneuronal accumulation of Lafora bodies. The targeting of laforin to intracellular glycogen complexes suggests that it may act on proteins important in glycogen metabolism, which are also co-localized to similar sites within the cell. Mutations that disrupt the CBD of laforin would attenuate its localization to glycogen particles, where the substrate of laforin may reside. This provides an explanation for why some patients with mutations only in the CBD, but not in the phosphatase domain, develop Lafora disease. We thank Gregory S. Taylor for his technical assistance." @default.
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• W2010595874 title "A Unique Carbohydrate Binding Domain Targets the Lafora Disease Phosphatase to Glycogen" @default.
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