Human recombinant endopeptidase PHEX has a strict S-1 ' specificity for acidic residues and cleaves peptides derived from fibroblast growth factor-23 and matrix extracellular phosphoglycoprotein

Human recombinant endopeptidase PHEX has a strict S-1 ' specificity for acidic residues and cleaves peptides derived from fibroblast growth factor-23 and matrix extracellular phosphoglycoprotein

Author Campos, Marcelo Autor UNIFESP Google Scholar
Couture, Constance Google Scholar
Hirata, Izaura Y. Autor UNIFESP Google Scholar
Juliano, Maria Aparecida Autor UNIFESP Google Scholar
Loisel, Thomas P. Google Scholar
Crine, Philippe Google Scholar
Juliano, Luiz Autor UNIFESP Google Scholar
Boileau, Guy Google Scholar
Carmona, Adriana Karaoglanovic Autor UNIFESP Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
Univ Montreal
BioMep Inc
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, 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) . 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.
Keywords combinatorial library
internally quenched fluorogenic substrate
M13 endopeptidase
PHEX substrate specificity
Language English
Date 2003-07-01
Published in Biochemical Journal. London: Portland Press, v. 373, p. 271-279, 2003.
ISSN 0264-6021 (Sherpa/Romeo, impact factor)
Publisher Portland Press
Extent 271-279
Origin http://dx.doi.org/10.1042/BJ20030287
Access rights Open access Open Access
Type Article
Web of Science ID WOS:000184060200028
URI http://repositorio.unifesp.br/handle/11600/27296

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