Characterization of PHEX endopeptidase catalytic activity: identification of parathyroid-hormone-related peptide(107-139) as a substrate and osteocalcin, PPi and phosphate as inhibitors

Mutations in the PHEX gene (phosphate regulating gene with homologies to en dopeptidases on the (X) over bar chromosome) are responsible for X-linked h ypophosphataemia, and-studies in the Hyp mouse model of the human disease i mplicate the gene product in the regulation of renal phosphate (P-i) reabso rption and bone mineralization. Although the mechanism for PHEX action is u nknown, structural homologies with members of the M13 family of endopeptida ses 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 ge nerated a recombinant soluble, secreted form of human PHEX (secPHEX) and te sted the activity of the purified protein with several peptide substrates, including a variety of bone-related peptides. We found that parathyroid-hor mone-related peptide(107-139) is a substrate for secPHEX and that the enzym e cleaves at three positions within the peptide, all located at the N-termi nus 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 controlle d in vivo by PPi/P-i and Ca2+ and, in the latter case, the regulation requi res the participation of osteocalcin.