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Research article

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

Marcelo CAMPOS, Constance COUTURE, Izaura Y. HIRATA, Maria A. JULIANO, Thomas P. LOISEL, Philippe CRINE, Luiz JULIANO, Guy BOILEAU, Adriana K. CARMONA
Biochemical Journal Jul 01, 2003, 373 (1) 271-279; DOI: 10.1042/bj20030287
Marcelo CAMPOS
Department of Biophysics, Universidade Federal de São Paulo, Escola Paulista de Medicina, Rua Três de Maio 100, 04044-020, São Paulo, Brazil
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Constance COUTURE
Department of Biochemistry, University of Montréal, C.P. 6128, Montréal, Quebec, Canada H3C 3J7
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Izaura Y. HIRATA
Department of Biophysics, Universidade Federal de São Paulo, Escola Paulista de Medicina, Rua Três de Maio 100, 04044-020, São Paulo, Brazil
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Maria A. JULIANO
Department of Biophysics, Universidade Federal de São Paulo, Escola Paulista de Medicina, Rua Três de Maio 100, 04044-020, São Paulo, Brazil
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Thomas P. LOISEL
BioMep Inc., 4631 Cumberland Ave. Montréal, Quebec, Canada H4B 2L5
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Philippe CRINE
Department of Biochemistry, University of Montréal, C.P. 6128, Montréal, Quebec, Canada H3C 3J7BioMep Inc., 4631 Cumberland Ave. Montréal, Quebec, Canada H4B 2L5
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Luiz JULIANO
Department of Biophysics, Universidade Federal de São Paulo, Escola Paulista de Medicina, Rua Três de Maio 100, 04044-020, São Paulo, Brazil
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Guy BOILEAU
Department of Biochemistry, University of Montréal, C.P. 6128, Montréal, Quebec, Canada H3C 3J7BioMep Inc., 4631 Cumberland Ave. Montréal, Quebec, Canada H4B 2L5
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Adriana K. CARMONA
Department of Biophysics, Universidade Federal de São Paulo, Escola Paulista de Medicina, Rua Três de Maio 100, 04044-020, São Paulo, Brazil
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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 S1´ subsite, with a strong preference for aspartate. Subsites S2´, S1 and S2 exhibited less defined specificity requirements, but the presence of leucine, proline or glycine in P2´, or valine, isoleucine or histidine in P1 precluded hydrolysis of the substrate by the enzyme. The peptide Abz-GFSDYK(Dnp)-OH, which contains the most favourable residues in the P2 to P2´ positions, was hydrolysed by PHEX at the N-terminus of aspartate with a kcat/Km 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.

  • combinatorial library
  • internally quenched fluorogenic substrate
  • M13 endopeptidase
  • PHEX substrate specificity

Footnotes

  • Abbreviations used: Abz, o-aminobenzoic acid; ADHR, autosomal dominant hypophosphataemic rickets; DMP1, dentin matrix protein 1; Dnp, 2,4-dinitrophenyl; DSPP, dentin sialophosphoprotein; ECE, endothelin-converting enzyme; EDDnp, N-(2,4-dinitrophenyl)ethylenediamine; FGF-23, fibroblast growth factor-23; IQFP, internally quenched fluorogenic peptide; LLC-PK1 cells, porcine renal epithelial proximal tubule cells; MALDI–TOF, matrix-assisted-laser-desorption ionization–time-of-flight; MEPE, matrix extracellular phosphoglycoprotein; NEP, neprilysin; OPN, osteopontin; PHEX, phosphate-regulating gene with homologies to endopeptidases on the X chromosome; PTHrP107–139, parathyroid hormone-related peptide residues 107–139; secPHEX, soluble secreted PHEX; TIO, tumour-induced osteomalacia; XLH, X-linked hypophosphataemia in humans.

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July 2003

Volume: 373 Issue: 1

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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
Marcelo CAMPOS, Constance COUTURE, Izaura Y. HIRATA, Maria A. JULIANO, Thomas P. LOISEL, Philippe CRINE, Luiz JULIANO, Guy BOILEAU, Adriana K. CARMONA
Biochemical Journal Jul 2003, 373 (1) 271-279; DOI: 10.1042/bj20030287
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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
Marcelo CAMPOS, Constance COUTURE, Izaura Y. HIRATA, Maria A. JULIANO, Thomas P. LOISEL, Philippe CRINE, Luiz JULIANO, Guy BOILEAU, Adriana K. CARMONA
Biochemical Journal Jul 2003, 373 (1) 271-279; DOI: 10.1042/bj20030287

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Keywords

combinatorial library
internally quenched fluorogenic substrate
M13 endopeptidase
PHEX substrate specificity

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