title: Biophysical characterization of laforin-carbohydrate interaction.
abstract: Laforin is a human dual-specificity phosphatase (DSP) involved in glycogen metabolism regulation containing a carbohydrate-binding module (CBM). Mutations in the gene coding for laforin are responsible for the development of Lafora disease, a progressive fatal myoclonus epilepsy with early onset, characterized by the intracellular deposition of abnormally branched, hyperphosphorylated insoluble glycogen-like polymers, called Lafora bodies. Despite the known importance of the CBM domain of laforin in the regulation of glycogen metabolism, the molecular mechanism of laforin-glycogen interaction is still poorly understood. Recently, the structure of laforin with bound maltohexaose was determined and despite the importance of such breakthrough, some molecular interaction details remained missing. We herein report a thorough biophysical characterization of laforin-carbohydrate interaction using soluble glycans. We demonstrated an increased preference of laforin for the interaction with glycans with higher order of polymerization and confirmed the importance of tryptophan residues for glycan interaction. Moreover, and in line with what has been described for other CBMs and lectins, our results confirmed that laforin-glycan interactions occur with a favourable enthalpic contribution counter-balanced by an unfavourable entropic contribution. The analysis of laforin-glycan interaction through the glycan side by saturation transfer difference (STD)-NMR has shown that the CBM-binding site can accommodate between 5 and 6 sugar units, which is in line with the recently obtained crystal structure of laforin. Overall, the work in the present study complements the structural characterization of laforin and sheds light on the molecular mechanism of laforin-glycan interaction, which is a pivotal requisite to understand the physiological and pathological roles of laforin.
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, U.K. Coimbra Chemistry Center, University of Coimbra, 3004-535 Coimbra, Portugal Department of Life Sciences, Faculty of Science and Technology, University of Coimbra, 3000-393 Coimbra, Portugal.
authors.forename: David M
authors.initials: DM
authors.lastname: Dias
authors.affiliation: Biocant-Biotechnology Innovation Center, Parque Tecnológico de Cantanhede, Núcleo 4, Lote 8-3060-197 Cantanhede, Portugal.
authors.forename: Joana
authors.initials: J
authors.lastname: Furtado
authors.affiliation: Coimbra Chemistry Center, University of Coimbra, 3004-535 Coimbra, Portugal.
authors.forename: Emeric
authors.initials: E
authors.lastname: Wasielewski
authors.affiliation: Biocant-Biotechnology Innovation Center, Parque Tecnológico de Cantanhede, Núcleo 4, Lote 8-3060-197 Cantanhede, Portugal.
authors.forename: Rui
authors.initials: R
authors.lastname: Cruz
authors.affiliation: Applied Photophysics Ltd., 21 Mole Business Park, Leatherhead, Surrey KT22 7BA, U.K.
authors.forename: Bernard
authors.initials: B
authors.lastname: Costello
authors.affiliation: Applied Photophysics Ltd., 21 Mole Business Park, Leatherhead, Surrey KT22 7BA, U.K.
authors.forename: Lindsay
authors.initials: L
authors.lastname: Cole
authors.affiliation: CNC-Center for Neuroscience and Cell Biology of Coimbra, University of Coimbra, 3004-517 Coimbra, Portugal.
authors.forename: Tiago Q
authors.initials: TQ
authors.lastname: Faria
authors.affiliation: Nanotemper Technologies GmbH, Flößergasse 4, 81369 Munich, Germany.
authors.forename: Philipp
authors.initials: P
authors.lastname: Baaske
authors.affiliation: Coimbra Chemistry Center, University of Coimbra, 3004-535 Coimbra, Portugal CNC-Center for Neuroscience and Cell Biology of Coimbra, University of Coimbra, 3004-517 Coimbra, Portugal Chemistry Department, Faculty of Science and Technology, University of Coimbra, 3004-535 Coimbra, Portugal.
authors.forename: Rui M M
authors.initials: RM
authors.lastname: Brito
authors.affiliation: College of Life Sciences, University of Dundee, Division of Biological Chemistry and Drug Discovery, James Black Centre, Dow Street, Dundee, DD1 5EH, U.K.
authors.forename: Alessio
authors.initials: A
authors.lastname: Ciulli
authors.affiliation: Biocant-Biotechnology Innovation Center, Parque Tecnológico de Cantanhede, Núcleo 4, Lote 8-3060-197 Cantanhede, Portugal CNC-Center for Neuroscience and Cell Biology of Coimbra, University of Coimbra, 3004-517 Coimbra, Portugal.
authors.forename: Isaura
authors.initials: I
authors.lastname: Simões
authors.affiliation: IBMC-Institute for Molecular and Cell Biology, Rua do Campo Alegre, 823, 4150-180, Porto, Portugal.
authors.forename: Sandra
authors.initials: S
authors.lastname: Macedo-Ribeiro
authors.affiliation: Biocant-Biotechnology Innovation Center, Parque Tecnológico de Cantanhede, Núcleo 4, Lote 8-3060-197 Cantanhede, Portugal CNC-Center for Neuroscience and Cell Biology of Coimbra, University of Coimbra, 3004-517 Coimbra, Portugal.
authors.forename: Carlos
authors.initials: C
authors.lastname: Faro
authors.affiliation: Coimbra Chemistry Center, University of Coimbra, 3004-535 Coimbra, Portugal Department of Life Sciences, Faculty of Science and Technology, University of Coimbra, 3000-393 Coimbra, Portugal CNC-Center for Neuroscience and Cell Biology of Coimbra, University of Coimbra, 3004-517 Coimbra, Portugal geraldes@ci.uc.pt pedro.castanheira@biocant.pt.
authors.forename: Carlos F G C
authors.initials: CF
authors.lastname: Geraldes
authors.affiliation: Biocant-Biotechnology Innovation Center, Parque Tecnológico de Cantanhede, Núcleo 4, Lote 8-3060-197 Cantanhede, Portugal geraldes@ci.uc.pt pedro.castanheira@biocant.pt.
authors.forename: Pedro
authors.initials: P
authors.lastname: Castanheira
mesh_terms: D001665:Binding Sites; D006003:Glycogen / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D020192:Lafora Disease / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D054558:Protein Tyrosine Phosphatases, Non-Receptor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D011134:Polysaccharides; D006003:Glycogen; D054558:Protein Tyrosine Phosphatases, Non-Receptor; C114990:EPM2A protein, human
keywords: Lafora disease; carbohydrate-binding module; glycogen; laforin; protein–carbohydrate interaction