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)
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)
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DOI：
10.1016/j.bone.2004.09.015
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发表时间：
2005-01-01
期刊：
影响因子：
4.1
通讯作者：
Gutierrez, GE
中科院分区：
文献类型：
--
作者：
Rowe, PSN;Garrett, IR;Gutierrez, GE
HYP 中基质细胞外磷酸糖蛋白 (MEPE) 和蛋白酶升高且 PHEX 缺陷。 PHEX 可防止 MEPE 的蛋白水解和耐蛋白酶 MEPE-ASARM 肽（一种矿化抑制剂（minhibin））的释放。因此，在 HYP 中，突变的 PHEX 可能有助于增加 ASARM 肽的释放。此外，MEPE 与 PHEX 的结合可能会调节正常受试者的这一过程。 PHEX-MEPE 非蛋白水解相互作用（直接或间接）的性质未知。我们的目的是确定 (1) PHEX 是否与 MEPE 特异性结合，(2) 结合是否涉及 ASARM 基序区域，以及 (3) 游离 ASARM 肽是否影响小鼠体内的矿化。使用表面等离子共振 (SPR) 测量 MEPE 和重组可溶性 PHEX (secPHEX) 之间的蛋白质相互作用。简而言之，将 secPHEX、MEPE 和对照蛋白 (IgG) 固定在 Biacore CM5 传感器芯片上，并在 Biacore 3000 高性能研究系统上进行 SPR 实验。然后以不同浓度注射纯 secPHEX，并测量与固定蛋白的相互作用。为了确定与 secPHEX 相互作用的 MEPE 序列，测量了 MEPE-ASARM 肽（磷酸化和非磷酸化）、对照肽和 MEPE 中区 RGD 肽对 secPHEX 与芯片固定的 MEPE 结合的抑制作用。通过小鼠后肢和颅盖的钙黄绿素荧光淬灭以及组织学桑德森染色来测定 ASARM 肽和依替膦酸介导的体内和体外矿化抑制。 secPHEX 和固定化 MEPE 之间会发生特异性、剂量依赖性和 Zn 依赖性蛋白质相互作用（EC50 为 553 nM）。合成 MEPE PO4-ASARM 肽可抑制 PHEX-MEPE 相互作用（K-Dapp = 15 uM 和 Bmax/inhib = 68%）。相反，对照和MEPE-RGD肽没有效果。相对于媒介物对照，皮下施用 ASARM 肽导致颅盖和后肢中的荧光显着猝灭，表明矿化受损。使用依替膦酸也获得了类似的结果。桑德森染色的颅骨还表明，ASARM 肽和依替膦酸组中未矿化的类骨质显着增加。我们得出结论，PHEX 和 MEPE 通过 MEPE 羧基末端 ASARM 基序形成非蛋白水解蛋白相互作用，并且 ASARM 肽抑制体内矿化。 PHEX 与 MEPE 和 ASARM 肽的结合可以解释为什么功能性成骨细胞表达的 PHEX 的丧失会导致 HYP 矿化缺陷。 (C) 2004 Elsevier Inc. 保留所有权利。
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-Dapp = 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. (C) 2004 Elsevier Inc. All rights reserved.