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)
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
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2020, Molecular and Cellular EndocrinologyCitation 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).
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