Expression of the Mutant (1735T-DEL) Tissue-Nonspecific Alkaline Phosphatase Gene from Hypophosphatasia Patients

• DOI: 10.1359/jbmr.1998.13.12.1827
• Publication Date: 2006-04-26T23:51:59Z
• Authors: Masae Dr. Goseki-...; Hideo Orimo; Tadahiro Iimura; Hidetaka Miyazaki; Kimimitsu Oda; Hisanobu Shibata; Masaki Yanagishita; Yuzo Takagi; Hisashi Watanabe; Takashi Shimada; Shinichiro Oida
• Keywords: Male; 0301 basic medicine; 0303 health sciences; Molecular Sequence Data; Gene Expression; Hypophosphatasia; Infant; Alkaline Phosphatase; Pedigree; 03 medical and health sciences; Fluorescent Antibody Technique, Direct; Child, Preschool; Protein Biosynthesis; COS Cells; Animals; Humans; Electrophoresis, Polyacrylamide Gel; Female; Amino Acid Sequence; Child; Codon; Frameshift Mutation; Sequence Deletion

Abstract Hypophosphatasia (HOPS) is an inherited disorder characterized by defects in skeletal mineralization due to the deficiency of tissue-nonspecific alkaline phosphatase (TNSALP). To date, various mutations in the TNSALP gene have been identified. Especially, a deletion of T at position 1735 (1735T-del) located in exon 12 has been detected in three genetically unrelated Japanese patients, which seems to be one of the hot spots among the causative mutations in Japanese HOPS patients. 1735T-del causes a frame shift downstream from codon 503 (Leu), and consequently the normal termination codon at 508 is eliminated. Since a new inframe termination codon appears at codon 588 in the mutant DNA, the resultant protein is expected to have 80 additional amino acids. Expression of the mutant TNSALP gene using COS-1 cells demonstrated that the protein translated from the mutant 1735T-del had undetectable ALP activity, and its molecule size was larger than normal, as expected. Interestingly, an immunoprecipitation study of patients' sera using antibody against TNSALP revealed an abnormal protein which corresponded in size to the mutated TNSALP expressed by COS-1 cells, suggesting that the abnormal TNSALP is made by HOPS patients. The detection of TNSALP in cells transfected with 1735T-del using an immunofluorescent method exhibited only a faint signal on the cell surface, but an intense intracellular fluorescence after permeabilization.