title: Function of mutant (G1144A) tissue-nonspecific ALP gene from hypophosphatasia.
abstract: Hypophosphatasia (HOPS) is a clinically heterogeneous heritable disorder characterized by defective skeletal mineralization, deficiency of tissue-nonspecific alkaline phosphatase (TNSALP) activity, and premature loss of deciduous teeth. The gene for TNSALP is located on chromosome 1p34-36.1 and consists of 12 exons and 11 introns. In our previous study, we found the novel point mutations (G1144A and T979C) from the genomic TNSALP gene of a patient with childhood HOPS. In this study, we have characterized the protein translated from the mutant G1144A gene. Wild-type and G1144A mutant-type TNSALP cDNA expression vector pcDNA3 have been constructed and transfected to COS-1 cells by lipofectin technique. After 48-h or 72-h transfection, cells were collected and homogenized using polytron homogenizer. After centrifugation at 10,000 g for 10 minutes, the supernatant was assayed. ALP activity was determined with 10 mM of p-nitrophenylphosphate as a substrate in 100 mM of 2-amino-2-methyl-1,3-propanediol-HCl buffer containing 5 mM of MgCl2. ALP activity of cells transfected with the mutant cDNA (G1144A) plasmid after 48-h or 72-h transfection exhibited 0.063 +/- 0.012 U/mg and 0.054 +/- 0.012 U/mg, respectively. As the enzymatic activity of the wild type was taken as 100%, the value of the mutant was estimated as 2.7% and 1.7%, respectively. These values were not significantly different from those found with mock-transfected cells, that is, 2.5% and 1.5%, respectively. This study indicated that the mutation (G1144A) produced the inactive ALP enzyme and would be a disease-causing mutation of the childhood-type HOPS.
authors.affiliation: Department of Hard Tissue Engineering, Graduate School, Tokyo Medical and Dental University, Japan.
authors.forename: Hisashi
authors.initials: H
authors.lastname: Watanabe
authors.forename: Masae
authors.initials: M
authors.lastname: Goseki-Sone
authors.forename: Hideo
authors.initials: H
authors.lastname: Orimo
authors.forename: Ryoko
authors.initials: R
authors.lastname: Hamatani
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Takinami
authors.forename: Isao
authors.initials: I
authors.lastname: Ishikawa
mesh_terms: D000328:Adult; D000469:Alkaline Phosphatase / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D002021:Buffers; D019556:COS Cells; D018076:DNA, Complementary; D004789:Enzyme Activation / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D007014:Hypophosphatasia / Q000201:enzymology* / Q000235:genetics*; D015636:Magnesium Chloride / Q000737:chemistry; D008297:Male; D009596:Nitrophenols / Q000378:metabolism; D009928:Organ Specificity; D009943:Organophosphorus Compounds / Q000378:metabolism; D017354:Point Mutation; D014162:Transfection
chemical_list: D002021:Buffers; D018076:DNA, Complementary; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D015636:Magnesium Chloride; C008644:nitrophenylphosphate; D000469:Alkaline Phosphatase