ANALYSIS OF LOCALIZATION OF MUTATED TISSUE NONSPECIFIC ALKALINE-PHOSPHATASE PROTEINS ASSOCIATED WITH NEONATAL HYPOPHOSPHATASIA USING GREEN FLUORESCENT PROTEIN CHIMERAS

Hypophosphatasia is associated with a defect of the tissue-nonspecific alkaline phosphatase (TNSALP) gene. The onset and clinical severity a re usually correlated in hypophosphatasia; patients with perinatal hyp ophosphatasia die approximately at the time of birth. In contrast, we describe a male neonatal patient with hypophosphatasia who had no resp iratory problems and survived. He was compound heterozygous for the co nversion of Phe to Leu at codon 310 (F310L) and the deletion of a nucl eotide T at 1735 (delT1735), causing the frame shift with the result o f the addition of 80 amino acids at the C-terminal of the protein. Bec ause the C-terminal portion of TNSALP is known to be important for TNS ALP to bind to the plasma membrane, the localization of wild-type and mutated TNSALP proteins was analyzed using green fluorescent protein c himeras. The expression vectors containing the complementary DNA of fu sion proteins consisting of signal peptide, green fluorescent protein, and wild-type or mutated TNSALP, caused by delT1735 or F310L mutation , were introduced transiently or stably in Saos-2 cells. The delT1735 mutant failed to localize at the cell surface membrane, whereas the wi ld-type and the F310L mutants were located in the plasma membrane and cytoplasm. The assay for enzymatic activity of TNSALP revealed that th e delT1735 mutant lost the activity and that the F310L mutant exhibite d an enzymatic activity level that was 72% of the normal level. The F3 10L mutation was also detected in another neonatal patient with relati vely mild (nonlethal) hypophosphatasia (reported in J Clin Endocrinol Metab, 81:4458-4461, 1996), suggesting that residual ALP activity of t he F310L mutant contributes to the less severe phenotype. The patient is unique, with respect to a discrepancy between onset and clinical se verity in hypophosphatasia.