Analysis Of Tissue-Nonspecific Alkaline Phosphatase With A Pro108Leu Mutation

Objectives: Hypophosphatasia is an inherited disease characterized by defective mineralization and reductions in the serum levels of alkaline phosphatase activity and caused by various missense mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene.Herasse M et al. have reported a missense mutation (Pro108Leu, P108L, according to standardized nomenclature, where the ATG initiator codon is numbered as 1) in a odonto-hypophosphatasia patient (J Med Genet., 2003).To define the molecular defects of TNSALP (P108L), we examined the biosynthesis and catalytic activity of this protein by expressing this mutant protein in mammalian cells to compare with those of the wild-type enzyme.

Methods: A plasmid encoding TNSALP (P108L) was created using site-directed mutagenesis. The wild type or TNSALP (P108L) was expressed in COS-1 cells transiently . Molecular size of expressed proteins was analyzed by an immunoblotting. Localization of TNSALP was examined with immunofluorescence, and alkaline phosphatase activity was determined using p-nitrophenylphosphate as a substrate.

Results: An immunofluorescence study showed the cell surface appearance of the mutant protein as in the case of the wild type. Nevertheless, TNSALP (P108L)  failed to exhibit the enzyme activity in contrast to the wild-type enzyme.

Conclusions: Since proline at position 108 of TNSALP is located very near the active site serine (110), it is suggested that the substitution of amino acid affected the structure and function of the catalytic site of the enzyme.