Analysis of recombinant Phex: an endopeptidase in search of a substrate

X-linked hypophosphatemia (XLH) is caused by inactivating mutations of Phex , a phosphate-regulating endopeptidase. Further advances in our knowledge o f the pathogenesis of XLH require identification of the biological function of Phex and its physiologically relevant substrates. We evaluated several potential substrates using mouse recombinant wild-type Phex proteins (rPhex -WT) and inactive mutant Phex proteins (rPhex-3'M) lacking the COOH-termina l catalytic domain as controls. By Western blot analysis, we demonstrated t hat Phex is a membrane-bound 100-kDa glycosylated monomer. Neither casein, a substrate for the related endopeptidase thermolysin, human stanniocalcin 1 (hSTC-1), an osteoblast-derived phosphate-regulating factor, nor FGF-23 p eptide (amino acid 172-186), comprising the region mutated in autosomal dom inant hypophosphatemia, was cleaved by rPhex-WT. In addition, membranes exp ressing rPhex-WT, rPhex-3'M, and the empty vector hydrolyzed parathyroid ho rmone-(1-34), indicating the lack of Phex-specific cleavage of parathyroid hormone. In contrast, rPhex-WT did display an EDTA-dependent cleavage of th e neutral endopeptidase substrate [Leu]enkephalin. Further studies with wil d-type and mutant rPhex proteins should permit the identification of physio logically relevant substrates involved in the pathogenesis of XLH.