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)

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 (K(D(app)) = 15 uM and B(max/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.

Phosphorylated acidic serine–aspartate-rich MEPE-associated motif peptide from matrix extracellular phosphoglycoprotein inhibits phosphate regulating gene with homologies to endopeptidases on the X-chromosome enzyme activity

“…However, we have recently demonstrated that the Hyp phosphaturic phenotype is not corrected by transfer of MEPE-deficient mice on to the Hyp mouse background (Liu et al 2005b), indicating that MEPE is not the phosphaturic factor in this disorder. While these studies indicate that MEPE is not the proximate cause of hypophosphatemia in Hyp mice, it does not preclude an upstream effect of MEPE that is potentially mediated through MEPE binding to PHEX via the MEPE ASARM-motif or a role of MEPE in the mineralization defect in Hyp mice (Rowe et al 2005).…”

“…The reaction was followed by measuring the fluorescence from excitation at 320 nm and emission at 420 nm in a Synergy HT Multi-Detection Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA). Full-length insect expressed human MEPE was purified and expressed as described previously (Rowe et al , 2005. Synthetic peptides derived from MEPE sequence, including MEPEarginine glycine aspartate motif (RGD) peptide, phosphorylated ASARM (ASARM-PO 4 ), and non-phosphorylated ASARM peptide (Multiple Peptide Systems, Inc., San Diego, CA, USA) were used in the inhibition assay (Rowe et al 2005).…”

“…Full-length insect expressed human MEPE was purified and expressed as described previously (Rowe et al , 2005. Synthetic peptides derived from MEPE sequence, including MEPEarginine glycine aspartate motif (RGD) peptide, phosphorylated ASARM (ASARM-PO 4 ), and non-phosphorylated ASARM peptide (Multiple Peptide Systems, Inc., San Diego, CA, USA) were used in the inhibition assay (Rowe et al 2005). PHEX activities were expressed as an initial speed rate calculated by fitting the steepest linear slope over at least eight adjacent reading values.…”

“…To evaluate the functional significance of our previously reported MEPE and MEPE ASARM-peptide (phosphorylated and non-phosphorylated) interactions with PHEX (Rowe et al 2005), we examined if addition of recombinant MEPE or its ASARM peptide (phosphorylated and nonphosphorylated) inhibited PHEX enzymatic activity in vitro. For these studies, we used the previously reported soluble secreted recombinant PHEX enzyme and a synthetic fluorescent-quenched peptide that is specifically cleaved by PHEX (Campos et al 2003).…”

“…These findings suggest that its primary function is to regulate the mineralization process, although administration of recombinant MEPE is reported to induce phosphaturia in mice ). In addition, MEPE can be cleaved by cathepsin B to release a carboxy-terminal MEPE peptide (ASARM peptide) ) and the phosphorylated ASARM peptide inhibits mineralization in vivo and in vitro and binds to PHEX (Rowe et al , 2005, suggesting that MEPE also undergoes proteolytic processing.…”