DEFECTIVE INTRACELLULAR-TRANSPORT OF TISSUE-NONSPECIFIC ALKALINE-PHOSPHATASE WITH AN ALA(162)-]THR MUTATION ASSOCIATED WITH LETHAL HYPOPHOSPHATASIA

We have studied the biosynthesis and intracellular transport of tissue -nonspecific alkaline phosphatase (TNSALP) transiently expressed in CO S-1 cells. Mutations were introduced into TNSALP to examine the effect s of a single amino acid substitution on the activity and biosynthesis of TNSALP. The cells expressing wild-type TNSALP exhibited more than 200-fold higher alkaline phosphatase activity than untransfected ones, Pulse-chase experiments showed that TNSALP was synthesized as a 66-kD a endoglucosaminidase H (Endo H)-sensitive form and converted to EndoH -resistant forms with heterogenous molecular masses (similar to 80 kDa ), which finally appeared on the cell surface as judged by digestion w ith phosphatidylinositol-specific phospholipase C (PI-PLC), In contras t, a TNSALP with a Glu(218)-->Gly mutation exhibited no phosphatase ac tivity at all and the 66-kDa Endo H-sensitive form was the only molecu lar species throughout the chase in the transfected cells. In accordan ce with this finding, digestion with PI-PLC and immunofluorescence obs ervation confirmed that this mutant was never expressed on the cell su rface. Another mutant with a Ala(162)-->Thr substitution, which natura lly occurs in association with a lethal hypophosphatasia, exhibited a low activity and only a small fraction of the 66-kDa form acquired End o-H resistance and reached the cell surface, Since the wild-type and t he mutant TNSALPs were labeled with [H-3]ethanolamine, a component of glycosylphosphatidylinositol (GPI), it is unlikely that the impaired i ntracellular transport of the two mutants is due to a failure in their modification by GPI, Interestingly, the 66-kDa Endo H sensitive form of the TNSALP mutants but not that of the wild-type, was found to form an interchain disulfide-bonded high-molecular-mass aggregate within t he cells. These results suggest that impaired intracellular transport of the TNSALP (Ala(162)-->Thr) molecule caused by its aggregation is t he molecular basis for the lethal hypophosphatasia carrying this mutat ion.