EXPRESSION OF THE MUTANT (1735T-DEL) TISSUE-NONSPECIFIC ALKALINE-PHOSPHATASE GENE FROM HYPOPHOSPHATASIA PATIENTS

Hypophosphatasia (HOPS) is an inherited disorder characterized bg defe cts in skeletal mineralization due to the deficiency of tissue-nonspec ific alkaline phosphatase (TNSALP), To date, various mutations in the TNSALP gene have been identified. Especially, a deletion of T at posit ion 1735 (1735T-del) located in exon 12 has been detected in three gen etically 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 conseque ntly the normal termination codon at 508 is eliminated. Since a new in frame termination codon appears at codon 588 in the mutant DNA, the re sultant protein is expected to have 80 additional amino acids. Express ion of the mutant TNSALP gene using COS-1 cells demonstrated that the protein translated from the mutant 1735T-del had undetectable ALP acti vity, and its molecule size was larger than normal, as expected, Inter estingly, an inmunoprecipitation study of patients' sera using antibod y against TNSALP revealed an abnormal protein which corresponded in si ze 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 exh ibited only a faint signal on the cell surface, but an intense intrace llular fluorescence after permeabilization.