Kinetic characterization of hypophosphatasia mutations with physiological substrates
Autor(a) principal: | |
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Data de Publicação: | 2002 |
Outros Autores: | , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UNESP |
Texto Completo: | http://dx.doi.org/10.1359/jbmr.2002.17.8.1383 http://hdl.handle.net/11449/37990 |
Resumo: | We 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. |
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Kinetic characterization of hypophosphatasia mutations with physiological substratesgenetic diseasemissense mutationscatalytic efficiencynatural substratesalkaline phosphataseWe 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.Burnham Inst, La Jolla, CA 92037 USAUNESP, Fac Ciências Agrarias & Vet, São Paulo, BrazilUniv Leuven, Ctr Mol & Vasc Biol, Louvain, BelgiumUmea Univ, Dept Med Genet, Umea, SwedenUNESP, Fac Ciências Agrarias & Vet, São Paulo, BrazilAmer Soc Bone & Mineral ResBurnham InstUniversidade Estadual Paulista (Unesp)Univ LeuvenUmea UnivDi Mauro, S.Manes, T.Hessle, L.Kozlenkov, A.Pizauro, J. M.Hoylaerts, M. F.Millan, J. L.2014-05-20T15:28:06Z2014-05-20T15:28:06Z2002-08-01info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/article1383-1391application/pdfhttp://dx.doi.org/10.1359/jbmr.2002.17.8.1383Journal of Bone and Mineral Research. Washington: Amer Soc Bone & Mineral Res, v. 17, n. 8, p. 1383-1391, 2002.0884-0431http://hdl.handle.net/11449/3799010.1359/jbmr.2002.17.8.1383WOS:000177048900006WOS000177048900006.pdfWeb of Sciencereponame:Repositório Institucional da UNESPinstname:Universidade Estadual Paulista (UNESP)instacron:UNESPengJournal of Bone and Mineral Research6.3142,808info:eu-repo/semantics/openAccess2023-11-05T06:13:23Zoai:repositorio.unesp.br:11449/37990Repositório InstitucionalPUBhttp://repositorio.unesp.br/oai/requestopendoar:29462024-08-05T16:59:49.720214Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)false |
dc.title.none.fl_str_mv |
Kinetic characterization of hypophosphatasia mutations with physiological substrates |
title |
Kinetic characterization of hypophosphatasia mutations with physiological substrates |
spellingShingle |
Kinetic characterization of hypophosphatasia mutations with physiological substrates Di Mauro, S. genetic disease missense mutations catalytic efficiency natural substrates alkaline phosphatase |
title_short |
Kinetic characterization of hypophosphatasia mutations with physiological substrates |
title_full |
Kinetic characterization of hypophosphatasia mutations with physiological substrates |
title_fullStr |
Kinetic characterization of hypophosphatasia mutations with physiological substrates |
title_full_unstemmed |
Kinetic characterization of hypophosphatasia mutations with physiological substrates |
title_sort |
Kinetic characterization of hypophosphatasia mutations with physiological substrates |
author |
Di Mauro, S. |
author_facet |
Di Mauro, S. Manes, T. Hessle, L. Kozlenkov, A. Pizauro, J. M. Hoylaerts, M. F. Millan, J. L. |
author_role |
author |
author2 |
Manes, T. Hessle, L. Kozlenkov, A. Pizauro, J. M. Hoylaerts, M. F. Millan, J. L. |
author2_role |
author author author author author author |
dc.contributor.none.fl_str_mv |
Burnham Inst Universidade Estadual Paulista (Unesp) Univ Leuven Umea Univ |
dc.contributor.author.fl_str_mv |
Di Mauro, S. Manes, T. Hessle, L. Kozlenkov, A. Pizauro, J. M. Hoylaerts, M. F. Millan, J. L. |
dc.subject.por.fl_str_mv |
genetic disease missense mutations catalytic efficiency natural substrates alkaline phosphatase |
topic |
genetic disease missense mutations catalytic efficiency natural substrates alkaline phosphatase |
description |
We 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. |
publishDate |
2002 |
dc.date.none.fl_str_mv |
2002-08-01 2014-05-20T15:28:06Z 2014-05-20T15:28:06Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://dx.doi.org/10.1359/jbmr.2002.17.8.1383 Journal of Bone and Mineral Research. Washington: Amer Soc Bone & Mineral Res, v. 17, n. 8, p. 1383-1391, 2002. 0884-0431 http://hdl.handle.net/11449/37990 10.1359/jbmr.2002.17.8.1383 WOS:000177048900006 WOS000177048900006.pdf |
url |
http://dx.doi.org/10.1359/jbmr.2002.17.8.1383 http://hdl.handle.net/11449/37990 |
identifier_str_mv |
Journal of Bone and Mineral Research. Washington: Amer Soc Bone & Mineral Res, v. 17, n. 8, p. 1383-1391, 2002. 0884-0431 10.1359/jbmr.2002.17.8.1383 WOS:000177048900006 WOS000177048900006.pdf |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
Journal of Bone and Mineral Research 6.314 2,808 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
1383-1391 application/pdf |
dc.publisher.none.fl_str_mv |
Amer Soc Bone & Mineral Res |
publisher.none.fl_str_mv |
Amer Soc Bone & Mineral Res |
dc.source.none.fl_str_mv |
Web of Science reponame:Repositório Institucional da UNESP instname:Universidade Estadual Paulista (UNESP) instacron:UNESP |
instname_str |
Universidade Estadual Paulista (UNESP) |
instacron_str |
UNESP |
institution |
UNESP |
reponame_str |
Repositório Institucional da UNESP |
collection |
Repositório Institucional da UNESP |
repository.name.fl_str_mv |
Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP) |
repository.mail.fl_str_mv |
|
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1808128733509320704 |