Importance of phosphorylation for osteopontin regulation of biomineralization
Importance of phosphorylation for osteopontin regulation of biomineralization
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DOI：
10.1007/s00223-004-1288-1
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发表时间：
2005-07-01
影响因子：
4.2
通讯作者：
Boskey, AL
中科院分区：
文献类型：
--
作者：
Gericke, A;Qin, C;Boskey, AL
先前的体外和体内研究表明，骨桥蛋白 (OPN) 是羟基磷灰石 (HA) 和其他生物矿物质形成和生长的抑制剂。本研究测试了以下假设：OPN 与 HA 的相互作用是由蛋白质磷酸化的程度决定的，并且这种相互作用调节矿化过程。先前报道的骨 OPN 抑制明胶-凝胶系统中 HA 的形成和 HA 种子的生长。连接转谷氨酰胺酶的 OPN 聚合物也具有类似的效果。重组、非磷酸化 OPN 和化学去磷酸化 OPN 对该系统中 HA 的形成或生长没有影响。相比之下，高度磷酸化的牛奶 OPN (mOPN) 则促进 HA 的形成。 mOPN 稳定了无定形磷酸钙（牛奶的非结晶成分）向 HA 的转化，而骨 OPN 对这种转化的影响较小。 OPN 和骨钙素的混合物已知在体外形成复合物，出乎意料地促进了 HA 的形成。为了检验由磷酸化引起的蛋白质构象的微小变化导致观察到的 OPN 与 HA 相互作用能力差异的假设，通过衰减全反射 (ATR) 红外 (IR) 光谱测定了存在和不存在结晶 HA 时骨 OPN 和 mOPN 的构象。两种蛋白质均表现出主要为随机卷曲结构，该结构不受添加 Ca2+ 的影响。与 HA 的结合不会改变骨 OPN 的二级结构，但会诱导 mOPN 中 β-折叠的小幅增加（几个百分点）。这些数据综合表明 OPN 磷酸化是调节 OPN 介导的矿化过程的重要因素。
Previous in vitro and in vivo studies demonstrated that osteopontin (OPN) is an inhibitor of the formation and growth of hydroxyapatite (HA) and other biominerals. The present study tests the hypotheses that the interaction of OPN with HA is determined by the extent of protein phosphorylation and that this interaction regulates the mineralization process. Bone OPN as previously reported inhibited HA formation and HA-seeded growth in a gelatin-gel system. A transglutaminase-linked OPN polymer had similar effects. Recombinant, nonphosphorylated OPN and chemically dephosphorylated OPN, had no effect on HA formation or growth in this system. In contrast, highly phosphorylated milk OPN (mOPN) promoted HA formation. The mOPN stabilized the conversion of amorphous calcium phosphate (a noncrystalline constituent of milk) to HA, whereas bone OPN had a lesser effect on this conversion. Mixtures of OPN and osteocalcin known to form a complex in vitro, unexpectedly promoted HA formation. To test the hypothesis that small alterations in protein conformation caused by phosphorylation account for the differences in the observed ability of OPN to interact with HA, the conformation of bone OPN and mOPN in the presence and absence of crystalline HA was determined by attenuated total reflection (ATR) infrared (IR) spectroscopy. Both proteins exhibited a predominantly random coil structure, which was unaffected by the addition of Ca2+. Binding to HA did not alter the secondary structure of bone OPN, but induced a small increase of beta-sheet (few percent) in mOPN. These data taken together suggest that the phosphorylation of OPN is an important factor in regulating the OPN-mediated mineralization process.