Characterization of PHEX endopeptidase catalytic activity:: identification of parathyroid-hormone-related peptide107-139 as a substrate and osteocalcin, PPi and phosphate as inhibitors

Mutations in the PHEX gene (phosphate regulating gene with homologies to endopeptidases on the (X) over bar chromosome) are responsible for X-linked hypophosphataemia, and-studies in the Hyp mouse model of the human disease implicate the gene product in the regulation of renal phosphate (P-i) reabsorption and bone mineralization. Although the mechanism for PHEX action is unknown, structural homologies with members of the M13 family of endopeptidases suggest a function for PHEX protein in the activation or degradation of peptide factors involved in the control of renal P-i transport and matrix mineralization. To determine whether PHEX has endopeptidase activity, we generated a recombinant soluble, secreted form of human PHEX (secPHEX) and tested the activity of the purified protein with several peptide substrates, including a variety of bone-related peptides. We found that parathyroid-hormone-related peptide(107-139) is a substrate for secPHEX and that the enzyme cleaves at three positions within the peptide, all located at the N-terminus of aspartate residues. Furthermore, we show that osteocalcin, PPi and P-i, all of which are abundant in bone, are inhibitors of secPHEX activity. Inhibition of secPHEX activity by osteocalcin was abolished in the presence of Ca2+. We suggest that PHEX activity and mineralization may be controlled in vivo by PPi/P-i and Ca2+ and, in the latter case, the regulation requires the participation of osteocalcin.