Delayed transport of tissue-nonspecific alkaline phosphatase with missense mutations causing hypophosphatasia

• Published in: European journal of medical genetics Vol. 50; no. 5; pp. 367 - 378
• Main Authors: Brun-Heath, Isabelle, Lia-Baldini, Anne-Sophie, Maillard, Stéphane, Taillandier, Agnès, Utsch, Boris, Nunes, Mark E, Serre, Jean-Louis, Mornet, Etienne
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier Masson SAS 01-09-2007; Elsevier
• Subjects: Alkaline Phosphatase - chemistry; Alkaline Phosphatase - genetics; Alkaline Phosphatase - metabolism; Animals; Base Sequence; Biological and medical sciences; Biological Transport, Active; Cell Membrane - enzymology; Cercopithecus aethiops; COS Cells; DNA Primers - genetics; Female; Fundamental and applied biological sciences. Psychology; General aspects. Genetic counseling; Genetics of eukaryotes. Biological and molecular evolution; Golgi Apparatus - enzymology; Humans; Hypophosphatasia; Hypophosphatemia, Familial - enzymology; Hypophosphatemia, Familial - genetics; Infant; Medical Education; Medical genetics; Medical sciences; Membrane anchoring; Microscopy, Fluorescence; Mineralization; Missense mutation; Models, Molecular; Molecular and cellular biology; Mutation, Missense; Phenotype–genotype correlation; Plasmids - genetics; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Transfection; Membrane anchoring; Missense mutation; Mineralization; Hypophosphatasia; Phenotype–genotype correlation; Human; Alkaline phosphatase; Correlation; Typing; Enzyme; Phosphoric monoester hydrolases; Metabolic diseases; Phenotype-genotype correlation; Esterases; Enzymopathy; Delay; Genetic disease; Tissue; Phenotype; Hydrolases; Genetics; Transport
• ISSN: 1769-7212; 1878-0849
• DOI: 10.1016/j.ejmg.2007.06.005

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 24 h 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.