title: Delayed transport of tissue-nonspecific alkaline phosphatase with missense mutations causing hypophosphatasia.
abstract: Hypophosphatasia is a rare genetic disease characterized by diminished bone and tooth mineralization due to deficient activity of tissue-nonspecific alkaline phosphatase (TNSALP). The disease is clinically heterogeneous due to different mutations in the TNSALP gene. In order to determine whether mutated TNSALP proteins may be sequestered, degraded, or subjected to delay in their transport to the cell membrane, we built a plasmid expressing a YFP-TNSALP fluorescent fusion protein allowing the observation of cellular localization in live cells by fluorescence confocal microscopy at different time points after transfection. We studied five mutants (c. 571G>A, c. 653T>C, c. 746G>T, c. 1363G>A and c. 1468A>T) exhibiting various levels of in vitro residual enzymatic activity. While the wild-type protein reached the membrane within the first 24h after transfection, the mutants reached the membrane with delays of 24, 48 or 72 h. For all of the tested mutations, accumulation of the mutated proteins, mainly in the Golgi apparatus, was observed. We concluded that reduced ALP activity of these TNSALP mutants results from structural disturbances and delay in membrane anchoring, and not from compromised catalytic activity.
authors.affiliation: Equipe Structure-Fonction et Génétique, EA 2493, CHU Paris Ile de France Ouest, Université de Versailles-Saint Quentin en Yvelines, France.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Brun-Heath
authors.forename: Anne-Sophie
authors.initials: AS
authors.lastname: Lia-Baldini
authors.forename: Stéphane
authors.initials: S
authors.lastname: Maillard
authors.forename: Agnès
authors.initials: A
authors.lastname: Taillandier
authors.forename: Boris
authors.initials: B
authors.lastname: Utsch
authors.forename: Mark E
authors.initials: ME
authors.lastname: Nunes
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Serre
authors.forename: Etienne
authors.initials: E
authors.lastname: Mornet
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D001693:Biological Transport, Active; D019556:COS Cells; D002462:Cell Membrane / Q000201:enzymology; D002522:Chlorocebus aethiops; D017931:DNA Primers / Q000235:genetics; D005260:Female; D006056:Golgi Apparatus / Q000201:enzymology; D006801:Humans; D007015:Hypophosphatemia, Familial / Q000201:enzymology* / Q000235:genetics*; D007223:Infant; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D020125:Mutation, Missense; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase