Kinetic characterization of hypophosphatasia mutations with physiological substrates

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dc.contributor.authorDi Mauro, S.
dc.contributor.authorManes, T.
dc.contributor.authorHessle, L.
dc.contributor.authorKozlenkov, A.
dc.contributor.authorPizauro, J. M.
dc.contributor.authorHoylaerts, M. F.
dc.contributor.authorMillan, J. L.
dc.contributor.institutionBurnham Inst
dc.contributor.institutionUniversidade Estadual Paulista (Unesp)
dc.contributor.institutionUniv Leuven
dc.contributor.institutionUmea Univ
dc.date.accessioned2014-05-20T15:28:06Z
dc.date.available2014-05-20T15:28:06Z
dc.date.issued2002-08-01
dc.description.abstractWe have analyzed 16 missense mutations of the tissue-nonspecific AP (TNAP) gene found in patients with hypophosphatasia. These mutations span the phenotypic spectrum of the disease, from the lethal perinatal/infantile forms to the less severe adult and odontohypophosphatasia. Site-directed mutagenesis was used to introduce a sequence tag into the TNAP cDNA and eliminate the glycosylphosphatidylinositol (GPI)-anchor recognition sequence to produce a secreted epitope-tagged TNAP (setTNAP). The properties of GPI-anchored TNAP (gpiTNAP) and setTNAP were found comparable. After introducing each single hypophosphatasia mutation, the setTNAP and mutant TNAP cDNAs were expressed in COS-1 cells and the recombinant flagged enzymes were affinity purified. We characterized the kinetic behavior, inhibition, and heat stability properties of each mutant using the artificial substrate p-nitrophenylphosphate (pNPP) at pH 9.8. We also determined the ability of the mutants to metabolize two natural substrates of TNAP, that is, pyridoxal-5'-phosphate (PLP) and inorganic pyrophosphate (PPi), at physiological pH. Six of the mutant enzymes were completely devoid of catalytic activity (R54C, R54P, A94T, R206W, G317D, and V365I), and 10 others (A16V, A115V, A160T, A162T, E174K, E174G, D277A, E281K, D361V, and G439R) showed various levels of residual activity. The A160T substitution was found to decrease the catalytic efficiency of the mutant enzyme toward pNPP to retain normal activity toward PPi and to display increased activity toward PLP. The A162T substitution caused a considerable reduction in the pNPPase, PPiase, and PLPase activities of the mutant enzyme. The D277A mutant was found to maintain high catalytic efficiency toward pNPP as substrate but not against PLP or PPi. Three mutations ( E174G, E174K, and E281K) were found to retain normal or slightly subnormal catalytic efficiency toward pNPP and PPi but not against PLP. Because abnormalities in PLP metabolism have been shown to cause epileptic seizures in mice null for the TNAP gene, these kinetic data help explain the variable expressivity of epileptic seizures in hypophosphatasia patients.en
dc.description.affiliationBurnham Inst, La Jolla, CA 92037 USA
dc.description.affiliationUNESP, Fac Ciências Agrarias & Vet, São Paulo, Brazil
dc.description.affiliationUniv Leuven, Ctr Mol & Vasc Biol, Louvain, Belgium
dc.description.affiliationUmea Univ, Dept Med Genet, Umea, Sweden
dc.description.affiliationUnespUNESP, Fac Ciências Agrarias & Vet, São Paulo, Brazil
dc.format.extent1383-1391
dc.identifierhttp://dx.doi.org/10.1359/jbmr.2002.17.8.1383
dc.identifier.citationJournal of Bone and Mineral Research. Washington: Amer Soc Bone & Mineral Res, v. 17, n. 8, p. 1383-1391, 2002.
dc.identifier.doi10.1359/jbmr.2002.17.8.1383
dc.identifier.fileWOS000177048900006.pdf
dc.identifier.issn0884-0431
dc.identifier.urihttp://hdl.handle.net/11449/37990
dc.identifier.wosWOS:000177048900006
dc.language.isoeng
dc.publisherAmer Soc Bone & Mineral Res
dc.relation.ispartofJournal of Bone and Mineral Research
dc.relation.ispartofjcr6.314
dc.relation.ispartofsjr2,808
dc.rights.accessRightsAcesso aberto
dc.sourceWeb of Science
dc.subjectgenetic diseasept
dc.subjectmissense mutationspt
dc.subjectcatalytic efficiencypt
dc.subjectnatural substratespt
dc.subjectalkaline phosphatasept
dc.titleKinetic characterization of hypophosphatasia mutations with physiological substratesen
dc.typeArtigo
dcterms.licensehttp://olabout.wiley.com/WileyCDA/Section/id-406071.html
dcterms.rightsHolderAmer Soc Bone & Mineral Res

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