title: Characterization of the mutant (A115V) tissue-nonspecific alkaline phosphatase gene from adult-type 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. To date, various mutations in the TNSALP gene have been identified. Especially, A115V located in exon 5 has been detected in a Japanese patient with severe periodontitis and adult-type HOPS. In this study, we have characterized the protein translated from the mutant A115V gene. Wild-type and A115V mutant-type TNSALP cDNA expression vector pcDNA3 have been constructed and transfected to COS-1 cells by lipofectin technique. After 48-h transfection, the cells were subjected to assay ALP activity. In order to identify possible dominant effect of the mutation, we performed co-transfections of wild-type and mutated cDNA, and evaluated the residual activities of each mutation. Detection of TNSALP synthesized by COS-1 cells transfected with the wild- or the mutated-type was also performed by using an immunofluorescent method. ALP activity of cell transfected with the mutant cDNA (A115V) plasmid after 48-h transfection exhibited 0.399+/-0.021 U/mg. As the enzymatic activity of the wild type was taken as 100%, the value of the mutant was estimated as 16.9%. When co-transfected this mutant showed no inhibition of the wild-type enzyme. TNSALP in COS-1 cells with transfected with the mutant exhibited strong fluorescence at the surface of cells as wild-type. This study indicated that the mutant (A115V) TNSALP gene produced the defective ALP enzyme and it could be recessively transmitted and be a disease-causing mutation of the adult-type hypophosphatasia.
authors.affiliation: Department of Hard Tissue Engineering (Periodontology), Graduate School, Tokyo Medical and Dental University, Tokyo, Japan. wantanabe.peri@tmd.ac.jp
authors.forename: Hisashi
authors.initials: H
authors.lastname: Watanabe
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Takinami
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: Isao
authors.initials: I
authors.lastname: Ishikawa
mesh_terms: D000409:Alanine / Q000235:genetics* / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007014:Hypophosphatasia / Q000201:enzymology* / Q000235:genetics* / Q000473:pathology; D007150:Immunohistochemistry; D008875:Middle Aged; D009154:Mutation / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D012333:RNA, Messenger; D000469:Alkaline Phosphatase; D000409:Alanine