Human recombinant endopeptidase PHEX has a strict S1' specificity for acidic residues and cleaves peptides derived from fibroblast growth factor-23 and matrix extracellular phosphoglycoprotein

Abstract

The PHEX gene (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) encodes a protein (PHEX) with structural homologies to members of the M13 family of zinc metallo-endopeptidases. Mutations in the PHEX gene are responsible for X-linked hypophosphataemia in humans. However, the mechanism by which loss of PHEX function results in the disease phenotype, and the endogenous PHEX substrate(s) remain unknown. In order to study PHEX substrate specificity, combinatorial fluorescent-quenched peptide libraries containing o -aminobenzoic acid (Abz) and 2,4-dinitrophenyl (Dnp) as the donor-acceptor pair were synthesized and tested as PHEX substrates. PHEX showed a strict requirement for acidic amino acid residues (aspartate or glutamate) in S(1)' subsite, with a strong preference for aspartate. Subsites S(2)', S(1) and S(2) exhibited less defined specificity requirements, but the presence of leucine, proline or glycine in P(2)', or valine, isoleucine or histidine in P(1) precluded hydrolysis of the substrate by the enzyme. The peptide Abz-GFSDYK(Dnp)-OH, which contains the most favourable residues in the P(2) to P(2)' positions, was hydrolysed by PHEX at the N-terminus of aspartate with a k(cat)/ K(m) of 167 mM(-1) x s(-1). In addition, using quenched fluorescence peptides derived from fibroblast growth factor-23 and matrix extracellular phosphoglycoprotein sequences flanked by Abz and N -(2,4-dinitrophenyl)ethylenediamine, we showed that these physiologically relevant proteins are potential PHEX substrates. Finally, our results clearly indicate that PHEX does not have neprilysin-like substrate specificity.

MEPE has the properties of an osteoblastic phosphatonin and minhibin

“…The physiological substrate for PHEX remains elusive although FGF23 and MEPE are possible candidates [11,12]. However, although small fluorogenic peptides of MEPE and FGF23 are reported to be cleaved by PHEX [12], full-length MEPE appears resistant to proteolysis [29,40].…”

“…However, although small fluorogenic peptides of MEPE and FGF23 are reported to be cleaved by PHEX [12], full-length MEPE appears resistant to proteolysis [29,40]. Also, the proteolysis of the full-length form of FGF23 by PHEX remains equivocal with one group reporting cleavage [11] and others no cleavage [29,40].…”

“…Recently, Campos et al [12] demonstrated that PHEX cleaves a synthetic fluorogenic MEPE peptide at the acidic residue distal to and adjacent to the MEPE ASARM peptide in a site, remarkably, that is in the same regional location as cathepsin B (Figs. 2 and 8A).…”

“…MEPE expression is markedly up-regulated in osteoblasts and bone from Hyp mice and is suppressed by PHEX and 1,25-dihydroxy vitamin-D 3 [2,3,29]. Moreover, recent evidence indicates that MEPE is a substrate or forms a complex with PHEX [12,29]. Recently we observed that PHEX inhibits the cathepsin B cleavage of MEPE through the C-terminal domain of PHEX [29].…”

Biological activity of FGF-23 fragments

“…This, in turn, suggests that further cleavage of FGF-23 in the carboxyl terminal domain needs to occur in order render the molecule inactive. Indeed, Campos and others have demonstrated the presence of PHEX cleavage sites at amino acid residue 183 and 215 [35]. Potential PHEX cleavage sites also exist at residues 186, 188 and 208, all of which may play a role in altering the bioactivity of the FGF-23.…”