Analysis of mutated alkaline phosphatases associated with hypophosphatasia

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Hypophosphatasia is a congenital genetic disorder caused by mutations of tissue-nonspecific alkaline phosphatase (TNSALP) gene. In order to define the molecular defect of mutated TNSALP molecules, COS-1 cells were transfected with either wild-type or mutated TNSALP cDNA and transiently expressed TNSALP molecules in the cells were studied. We focused two mutated TNSALPs associated with severe form of hypophosphatasia in the present study.1. The TNSALP with an Ala162-Thr substitutionWhen synthesized in GOS-1 cells, only a fraction of newly synthesized TNSALP molecules underwent oligosaccharide processing and reached the cell surface as an active enzyme, while the remaining molecules were found to form a disulfide-bonded high molecular mass aggregate and to be arrested along the secretory pathway before it reached the Golgi apparatus.2.The TNSALP with a Gly3 17-Asp substitutionWhen expressed in COS-I cells, the cell surface appearance of this mutated molecule was totally blocked and the mutant protein formed a disulfide-bonded high molecular mass aggregate within the cell, presumably in the endoplasmic reticulum or a pre-Golgi compartment. Eventually the mutant protein was found to be degraded. The degradation of mutant protein was inhibited by lactacystin, a specific inhibitor of proteasome, Since both the TNSALP (Ala162-Thr) and the TNSALP(Gly3 17-Asp) were labeled with [3H]ethanolamine, the mutant proteins were modified with a glycosylphosphatidylinositol, through which TNSALP molecule is anchored to the cell membrane.Taken together, it is highly likely that missense mutataions found in patients with severe form of hypophosphatasia bring about a three-dimensional structural change of the protein, leading to the formation of an aggregate within the cells. As a consequense functional TNSALP molecules with alkaline phosphatase activity are greately decreased in number or totally absent from the cell surface.