Elsevier

Bone

Volume 36, Issue 1, January 2005, Pages 33-46
Bone

Surface plasmon resonance (SPR) confirms that MEPE binds to PHEX via the MEPE–ASARM motif: a model for impaired mineralization in X-linked rickets (HYP)

https://doi.org/10.1016/j.bone.2004.09.015Get rights and content

Abstract

Matrix Extracellular Phospho-glycoprotEin (MEPE) and proteases are elevated and PHEX is defective in HYP. PHEX prevents proteolysis of MEPE and release of a protease-resistant MEPE–ASARM peptide, an inhibitor of mineralization (minhibin). Thus, in HYP, mutated PHEX may contribute to increased ASARM peptide release. Moreover, binding of MEPE by PHEX may regulate this process in normal subjects. The nature of the PHEX–MEPE nonproteolytic interaction(s) (direct or indirect) is/are unknown. Our aims were to determine (1) whether PHEX binds specifically to MEPE, (2) whether the binding involves the ASARM motif region, and (3) whether free ASARM peptide affects mineralization in vivo in mice. Protein interactions between MEPE and recombinant soluble PHEX (secPHEX) were measured using surface plasmon resonance (SPR). Briefly, secPHEX, MEPE, and control protein (IgG) were immobilized on a Biacore CM5 sensor chip, and SPR experiments were performed on a Biacore 3000 high-performance research system. Pure secPHEX was then injected at different concentrations, and interactions with immobilized proteins were measured. To determine MEPE sequences interacting with secPHEX, the inhibitory effects of MEPE–ASARM peptides (phosphorylated and nonphosphorylated), control peptides, and MEPE midregion RGD peptides on secPHEX binding to chip-immobilized MEPE were measured. ASARM peptide and etidronate-mediated mineralization inhibition in vivo and in vitro were determined by quenched calcein fluorescence in hind limbs and calvariae in mice and by histological Sanderson stain. A specific, dose-dependent and Zn-dependent protein interaction between secPHEX and immobilized MEPE occurs (EC50 of 553 nM). Synthetic MEPE PO4-ASARM peptide inhibits the PHEX–MEPE interaction (KDapp = 15 uM and Bmax/inhib = 68%). In contrast, control and MEPE–RGD peptides had no effect. Subcutaneous administration of ASARM peptide resulted in marked quenching of fluorescence in calvariae and hind limbs relative to vehicle controls indicating impaired mineralization. Similar results were obtained with etidronate. Sanderson-stained calvariae also indicated a marked increase in unmineralized osteoid with ASARM peptide and etidronate groups. We conclude that PHEX and MEPE form a nonproteolytic protein interaction via the MEPE carboxy-terminal ASARM motif, and the ASARM peptide inhibits mineralization in vivo. The binding of MEPE and ASARM peptide by PHEX may explain why loss of functional osteoblast-expressed PHEX results in defective mineralization in HYP.

Introduction

Defects in two genes PHEX and FGF23 are primarily responsible for X-linked hypophosphatemic rickets (HYP) and autosomal dominant hypophosphatemic rickets (ADHR) [29], [92]. The molecular pathways(s) and the upstream factors impacting on mineralization, abnormal renal phosphate handling, and vitamin D metabolism remain unknown. However, evidence strongly suggests that PHEX and FGF23 pathways overlap and may well involve the direct or indirect regulation of extracellular matrix proteins (ECMP) from bone and teeth [62], [68], [71]. Matrix Extracellular Phospho-glycoprotEin (MEPE), an osteoblast and odontoblast expressed matrix protein, is a good downstream candidate ECMP factor whose expression or activity may be altered by PHEX and/or FGF23. MEPE was first cloned from a patient with oncogenic hypophosphatemic osteomalacia (OHO), a disease with many similarities to HYP and ADHR. The OHO tumor cloning was achieved by expression screening of an OHO tumor cDNA library with polyclonal antibodies that neutralized an OHO tumor-secreted renal phosphate uptake inhibiting factor(s) [69], [72]. MEPE is markedly up-regulated in Hyp osteoblasts and OHO tumors and is exclusively expressed in osteoblasts, osteocytes, and odontoblasts [2], [3], [25], [27], [31], [45], [60], [61], [69], [71], [72]. MEPE inhibits phosphate uptake and mineralization in vivo and in vitro. Phosphaturia in rodents can be induced via bolus administration or infusion of recombinant MEPE [14], [71]. The in vitro mineralization inhibition observed with MEPE is mediated by a short (2 kDa), protease-resistant, cathepsin B-released carboxy-terminal MEPE peptide (ASARM peptide) [69], [71]. This peptide likely also inhibits phosphate uptake. Recently, based on our published findings, we proposed a mineralization model that involved a nonproteolytic sequestration of MEPE by PHEX [68], [71]. Specifically, a reversible association of PHEX and MEPE was proposed to control release of a mineralization inhibitor ASARM peptide by transiently protecting MEPE from proteolysis. Also, in HYP, the reported massive up-regulation of MEPE, the excess protease expression, and the lack of functional PHEX should also collectively increase the levels of MEPE–ASARM peptide. This in turn was proposed to be responsible for the observed periosteocytic defects in mineralization [68], [71].

PHEX belongs to an M13 family of Zn metalloendopeptidases, and its physiological substrate remains elusive. Although small synthetic peptides of FGF23 and MEPE are PHEX substrates, a number of studies have failed to confirm cleavage of the full-length molecules [12], [27], [39]. Interestingly, we previously determined that PHEX protects MEPE from cathepsin B proteolysis [27]. More specifically, both full-length PHEX and/or a mutated PHEX protein that contains the COOH terminal extracellular domain (with zinc binding motif) prevent cathepsin B degradation of full-length MEPE in vitro. Moreover, this inhibition is not mediated through PHEX proteolysis of cathepsin B, and cathepsin B does not degrade PHEX [27]. However, our published experiments did not examine whether the observed in vitro PHEX-dependent protection of MEPE was direct or indirect. Indeed, PHEX potentially could either form a nonproteolytic complex with cathepsin B, MEPE, or both proteins. Others have also shown that PHEX activity is inhibited by a nonproteolytic association with another important bone matrix protein, osteocalcin [6]. The data presented in this study confirm that PHEX and MEPE do indeed form a specific, direct, Zn-dependent and nonproteolytic association. Moreover, the carboxy-terminal ASARM motif region of MEPE plays a key role in the MEPE–PHEX interaction. Finally, we confirm that the phosphorylated ASARM peptide quenches calcein bone fluorescence in vivo and increases the osteoid band in Sanderson-stained calvariae. This is consistent with the in vitro mineralization inhibition generated by MEPE, OPN, DMP-1, and statherin phosphorylated ASARM peptides or proteins [8], [9], [28], [44], [64], [68], [71], [76], [90]. The statherin ASARM peptide, for example, prevents ectopic mineralization of calcium and phosphate in supersaturated saliva and plays a key role in the mineralization dynamics of teeth [44], [64], [76]. The findings presented in this study are strongly supportive of the HYP mineralization ASARM model [68], [69], [71].

Section snippets

Expression of insect-expressed MEPE and soluble mammalian-expressed PHEX (secPHEX)

Expression and purification of full-length insect-expressed human MEPE were as described previously [71]. Briefly, insect S. frugiperda cells were infected with baculovirus containing the full-length human MEPE gene originally cloned into pBlueBac-4-5 (cDNA) and homologously recombined with Bac-N-Blue DNA™ to generate viral particles via transfection (Invitrogen kit). Infected cells were grown in a 10-l bioreactor for 48 h, and conditioned medium was concentrated (fivefold) and used as the

Specific Zn-dependent and dose-dependent direct binding of MEPE to PHEX

Fig. 1 shows a direct protein–protein Zn-dependent interaction between secPHEX and MEPE as monitored and plotted as an SPR sensorgram. A classic protein-association phase was followed by dissociation after the 6-min pulse of secPHEX. There were no significant signals generated between secPHEX and blank activated/blocked chip or control IgG protein. There was a very low-level barely detectable autologous interaction between injected secPHEX (analyte) and chip-immobilized secPHEX (ligand). The

Discussion

The primary defects in X-linked hypophosphatemic rickets (HYP) and autosomal dominant hypophosphatemic rickets (ADHR) are loss of function mutations in the PHEX gene (a Zn metalloendopeptidase) and activating mutations in FGF23, respectively [23], [29], [70], [73], [91], [92]. Oncogenic hypophosphatemic osteomalacia (OHO) is a rare tumor-induced disease and shares many pathophysiological features with HYP and ADHR. These include hypophosphatemia, defective/mineralization (osteomalacia/rickets),

Acknowledgments

The authors would like to acknowledge the very kind gift of pure secPHEX by Dr. Philippe Crine (Department of Biochemistry, University of Montréal and BIOMEP) and Dr. Guy Boileau (Department of Biochemistry, University of Montréal). We also acknowledge the generous financial support and awards to PSNR: Children's Cancer Research Center (CCRC) of the University of Texas Health Science Center at San Antonio (UTHSCSA), National Institutes of Health grant 1R03DE015900-01 (National Institute of

References (99)

  • S. Liu et al.

    Regulation of FGF23 expression but not degradation by Phex

    J. Biol. Chem.

    (2003)
  • D. Miao et al.

    Cartilage abnormalities are associated with abnormal Phex expression and with altered matrix protein and MMP-9 localization in Hyp mice

    Bone

    (2004)
  • J.R. Morgan et al.

    Eps15 homology domain-NPF motif interactions regulate clathrin coat assembly during synaptic vesicle recycling

    J. Biol. Chem.

    (2003)
  • J.R. Morgan et al.

    Uncoating of clathrin-coated vesicles in presynaptic terminals: roles for Hsc70 and auxilin

    Neuron

    (2001)
  • A.E. Nelson et al.

    Phosphate wasting in oncogenic osteomalacia: PHEX is normal and the tumor-derived factor has unique properties

    Bone

    (2001)
  • A.E. Nelson et al.

    Characteristics of tumor cell bioactivity in oncogenic osteomalacia

    Mol. Cell. Endocrinol.

    (1996)
  • D.N. Petersen et al.

    Identification of osteoblast/osteocyte factor 45 (OF45), a bone-specific cDNA encoding an RGD-containing protein that is highly expressed in osteoblasts and osteocytes

    J. Biol. Chem.

    (2000)
  • P.A. Raj et al.

    Salivary statherin. Dependence on sequence, charge, hydrogen bonding potency, and helical conformation for adsorption to hydroxyapatite and inhibition of mineralization

    J. Biol. Chem.

    (1992)
  • N.D. Rawlings et al.

    Evolutionary families of metallopeptidases

  • P.S.N. Rowe et al.

    MEPE, a new gene expressed in bone-marrow and tumours causing osteomalacia

    Genomics

    (2000)
  • P.S.N. Rowe et al.

    MEPE has the properties of an osteoblastic phosphatonin and minhibin

    Bone

    (2004)
  • P.S.N. Rowe et al.

    Candidate 56 and 58 kDa protein(s) responsible for mediating the renal defects in oncogenic hypophosphataemic osteomalacia

    Bone

    (1996)
  • M. Szczepanska-Konkel et al.

    Phosphonocarboxylic acids as specific inhibitors of Na+-dependent transport of phosphate across renal brush border membrane

    J. Biol. Chem.

    (1986)
  • P.H. Tartaix et al.

    Dentin matrix protein-1: in vitro effects on hydroxyapatite formation provide insights into in vivo functions

    J. Biol. Chem.

    (2004)
  • S. Toyosawa et al.

    Expression of dentin matrix protein 1 in tumors causing oncogenic osteomalacia

    Mod. Pathol.

    (2004)
  • K.E. White et al.

    Autosomal-dominant hypophosphatemic rickets (ADHR) mutations stabilize FGF-23

    Kidney Int.

    (2001)
  • T. Yamamoto et al.

    Abnormal response of osteoblasts from Hyp mice to 1,25-dihydroxyvitamin D3

    Bone

    (1992)
  • T. Yamashita et al.

    Identification of a novel fibroblast growth factor, FGF-23, preferentially expressed in the ventrolateral thalamic nucleus of the brain

    Biochem. Biophys. Res. Commun.

    (2000)
  • X. Bai et al.

    Partial rescue of the Hyp phenotype by osteoblast-targeted PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) expression

    Mol. Endocrinol.

    (2002)
  • L. Bianchetti et al.

    M13 endopeptidases: new conserved motifs correlated with structure, and simultaneous phylogenetic occurrence of PHEX and the bony fish

    Proteins

    (2002)
  • G. Boileau et al.

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

    Biochem. J.

    (2001)
  • J.P. Bonjour et al.

    Action of 1,25-dihydroxyvitamin D3 and a diphosphonate on calcium metabolism in rats

    Am. J. Physiol.

    (1975)
  • A.L. Boskey

    Osteopontin and related phosphorylated sialoproteins: effects on mineralization

    Ann. N. Y. Acad. Sci.

    (1995)
  • A.L. Boskey et al.

    Osteopontin deficiency increases mineral content and mineral crystallinity in mouse bone

    Calcif. Tissue Int.

    (2002)
  • Q. Cai et al.

    Brief report: inhibition of renal phosphate transport by a tumor product in a patient with oncogenic osteomalacia

    N. Engl. J. Med.

    (1994)
  • M. Campos et al.

    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

    Biochem. J.

    (2003)
  • S.M. De Beur et al.

    Tumors associated with oncogenic osteomalacia express genes important in bone and mineral metabolism

    J. Bone Miner. Res.

    (2002)
  • H. Dobbie et al.

    Infusion of the bone-derived protein MEPE causes phosphaturia in rats (abstract)

    J. Am. Soc. Nephrol.

    (2003)
  • K. Donath et al.

    A method for the study of undecalcified bones and teeth with attached soft tissues. The Sage-Schliff (sawing and grinding) technique

    J. Oral. Pathol.

    (1982)
  • M.K. Drezner

    Tumor-induced osteomalacia

    Rev. Endocr. Metab. Disord.

    (2001)
  • S.G. Dubois et al.

    Role of abnormal neutral endopeptidase-like activities in Hyp mouse bone cells in renal phosphate transport

    Am. J. Physiol.: Cell Physiol.

    (2002)
  • B. Ecarot et al.

    Defective bone formation by Hyp mouse bone cells transplanted into normal mice: evidence in favor of an intrinsic osteoblast defect

    J. Bone Miner. Res.

    (1992)
  • B. Ecarot et al.

    Effect of 1,25-dihydroxyvitamin D3 treatment on bone formation by transplanted cells from normal and X-linked hypophosphatemic mice

    J. Bone Miner. Res.

    (1995)
  • F.H. Ecarot et al.

    Effect of dietary phosphate deprivation and supplementation of recipient mice on bone formation by transplanted cells from normal and X-linked hypophosphataemic mice

    J. Bone Miner. Res.

    (1992)
  • N.S. Fedarko et al.

    Three small integrin-binding ligand N-linked glycoproteins (SIBLINGs) bind and activate specific matrix metalloproteinases

    FASEB J.

    (2004)
  • F. Francis et al.

    Genomic organisation of the human PEX gene mutated in X-linked dominant hypophosphataemic rickets

    Genet. Res.

    (1997)
  • I.R. Garrett et al.

    Selective inhibitors of the osteoblast proteasome stimulate bone formation in vivo and in vitro

    J. Clin. Invest.

    (2003)
  • R. Guo et al.

    Analysis of recombinant Phex: an endopeptidase in search of a substrate

    Am. J. Physiol.: Endocrinol. Metab.

    (2001)
  • HYP-consortium et al.

    A gene (PEX) with homologies to endopeptidases is mutated in patients with X-linked hypophosphatemic rickets. The HYP Consortium

    Nat. Genet.

    (1995)
  • Cited by (129)

    • FGF23 and bone disease

      2021, Fibroblast Growth Factor 23
    • Active sites of human MEPE-ASARM regulating bone matrix mineralization

      2020, Molecular and Cellular Endocrinology
      Citation Excerpt :

      The anti-mineralization activity of synthetic nonphosphorylated ASARM (MEPE-nASARM) peptide (hereinafter, “peptide” was abbreviated) was initially uncovered in mouse 2T3 osteoblastic cell cultures (Rowe et al., 2004). Thereafter, three phosphorylated Ser (pSer) residues in ASARM (MEPE-pASARM) were shown to be necessary for its anti-mineralization effect (e.g., increased osteoid in mouse bones) (Rowe et al., 2005), mineralization defects in mouse bone marrow cell (Liu et al., 2007), MC3T3-E1 cell (Addison et al., 2008), and mouse chondrocyte/cartilage (Staines et al., 2012) cultures, and inhibition of hydroxyapatite crystal formation (Boskey et al., 2010). Similar results were obtained in the synthetic pASARM of SPP1 (SPP1-pASARM) in vitro (Addison et al., 2010).

    • Phosphatonins

      2020, Marcus and Feldman’s Osteoporosis
    • Osteocytes

      2020, Marcus and Feldman’s Osteoporosis
    View all citing articles on Scopus
    View full text