School of Dentistry and Department of Biochemistry, University of Western Ontario, London, Ontario, Canada.
PLoS One. 2010 Feb 19;5(2):e9330. doi: 10.1371/journal.pone.0009330.
In vitro studies have shown that the phosphoprotein osteopontin (OPN) inhibits the nucleation and growth of hydroxyapatite (HA) and other biominerals. In vivo, OPN is believed to prevent the calcification of soft tissues. However, the nature of the interaction between OPN and HA is not understood. In the computational part of the present study, we used molecular dynamics simulations to predict the adsorption of 19 peptides, each 16 amino acids long and collectively covering the entire sequence of OPN, to the {100} face of HA. This analysis showed that there is an inverse relationship between predicted strength of adsorption and peptide isoelectric point (P<0.0001). Analysis of the OPN sequence by PONDR (Predictor of Naturally Disordered Regions) indicated that OPN sequences predicted to adsorb well to HA are highly disordered. In the experimental part of the study, we synthesized phosphorylated and non-phosphorylated peptides corresponding to OPN sequences 65-80 (pSHDHMDDDDDDDDDGD) and 220-235 (pSHEpSTEQSDAIDpSAEK). In agreement with the PONDR analysis, these were shown by circular dichroism spectroscopy to be largely disordered. A constant-composition/seeded growth assay was used to assess the HA-inhibiting potencies of the synthetic peptides. The phosphorylated versions of OPN65-80 (IC(50) = 1.93 microg/ml) and OPN220-235 (IC(50) = 1.48 microg/ml) are potent inhibitors of HA growth, as is the nonphosphorylated version of OPN65-80 (IC(50) = 2.97 microg/ml); the nonphosphorylated version of OPN220-235 has no measurable inhibitory activity. These findings suggest that the adsorption of acidic proteins to Ca2+-rich crystal faces of biominerals is governed by electrostatics and is facilitated by conformational flexibility of the polypeptide chain.
体外研究表明，磷酸化蛋白骨桥蛋白（OPN）抑制羟磷灰石（HA）和其他生物矿物质的成核和生长。在体内，OPN 被认为可以防止软组织钙化。然而，OPN 与 HA 之间相互作用的性质尚不清楚。在本研究的计算部分，我们使用分子动力学模拟预测 19 个肽段（每个 16 个氨基酸长，总共覆盖 OPN 的整个序列）吸附到 HA 的{100}面上。该分析表明，预测的吸附强度与肽等电点（P<0.0001）之间存在反比关系。PONDR（天然无序区域预测器）对 OPN 序列的分析表明，预测与 HA 吸附良好的 OPN 序列高度无序。在研究的实验部分，我们合成了与 OPN 序列 65-80（pSHDHMDDDDDDDDDGD）和 220-235（pSHEpSTEQSDAIDpSAEK）相对应的磷酸化和非磷酸化肽。与 PONDR 分析一致，圆二色性光谱表明这些肽主要是无序的。恒组成/种子生长测定法用于评估合成肽对 HA 的抑制作用。OPN65-80 的磷酸化版本（IC（50）=1.93μg/ml）和 OPN220-235 的磷酸化版本（IC（50）=1.48μg/ml）是 HA 生长的有效抑制剂，非磷酸化的 OPN65-80 版本（IC（50）=2.97μg/ml）也是如此；OPN220-235 的非磷酸化版本没有可测量的抑制活性。这些发现表明，带负电荷的蛋白质吸附到富含 Ca2+的生物矿物质晶体面上是由静电控制的，并受到多肽链构象灵活性的促进。
