卵黄高磷蛋白磷酸肽调控生物矿化的功能位点和构效关系研究

Phosvitin is a highly phosphorylated protein from hen egg yolk with calcium-binding properties, can be used as calcium supplements or biomimetic materials for bone repair. Previous studies have shown that phosvitin has the effect of regulating mineralization in vitro mineralization model and can regulate the proliferation and differentiation of bone cells, its regulatory role is closely related to the degree of its phosphorylation. Based on this research, phosvitin phosphopeptide (PPP) used as the research object of this project, through the specific enzymolysis of phosvitin, PPP was separated and purified after applying the chromatographic and mass spectrometric methods. PPP with specific fragments were directional prepared through solid-phase synthesis, and its structural information was elucidated by using spectroscopy and nuclear magnetic resonance. Effect of the functional structure of directional prepared PPP on the regulation rules of mineralization was investigated through the collagen matrix model, the mineralization was characterized by various spectroscopic techniques, to identify the functional sites and the structure-activity relationship of PPP with the regulation of mineralization. In addition, PPP interacted with bone cells to regulate mineralization was verified through the molecular biology and cell biology techniques, and its signal pathway and the regulation approach were revealed. This project could provide a theoretical basis for the application of phosphopeptides as calcium supplement products and biomimetic materials for bone repair, and is of great significance to the deep processing of eggs.

卵黄高磷蛋白是鸡蛋蛋黄中的一种高度磷酸化的蛋白质，具有结合钙离子特性，可作为补钙产品或骨修复等仿生材料应用，前期研究表明卵黄高磷蛋白在体外矿化模型中具有调控矿化的效果且可调控骨细胞的生长分化，其调控作用与其磷酸化程度密切相关。在此基础上，本项目以卵黄高磷蛋白磷酸肽为研究对象，通过特异性酶解卵黄高磷蛋白，利用色谱和质谱法分离纯化并鉴定卵黄高磷蛋白磷酸肽，固相合成定向制备特异性片段的磷酸肽，利用光谱和核磁共振进行结构解析；通过胶原蛋白基质模型研究定向制备的磷酸肽功能结构对矿化的调控规律，利用多种光谱技术表征矿化物，探明卵黄高磷蛋白磷酸肽调控矿化的功能位点和构效关系。此外，利用分子生物学和细胞生物学技术，验证卵黄高磷蛋白磷酸肽与骨细胞交互调控矿化作用，揭示其调控矿化的信号通路及途径，为磷酸肽作为补钙产品及骨修复等仿生材料应用提供理论基础，对禽蛋精深加工具有十分重要的意义。

将卵黄高磷蛋白酶解后进行分离纯化及鉴定。以磷酸钙沉淀阻滞效果、钙螯合率以及肽钙螯合物得率为指标，分离鉴定得到一条分子量为1106.44 Da的9肽，序列为DEEENDQVK，其钙结合能力达到151.10±3.57 mg/g。研究九肽与钙离子的螯合机理的结果表明，随着钙离子的加入，肽发生折叠和修饰，其主要结合位点为羧基氧原子和氨基氮原子。. 选定DEEENDQVK（DK）和EDD-pSer-pSer（ES）两种活性肽为研究对象，进行骨细胞实验。两种多肽对MC3T3-E1细胞增殖没有显著影响。DK浓度在100 μg/mL及以上时可促进成骨细胞的ALP活力和矿化，且呈剂量依赖性。ES在50 μg/mL时抑制细胞ALP活力和矿化，在200 μg/mL及以上时表现出促进作用。转录组学分析表明，DK显著影响了具有促进骨化、骨发育、骨骼系统发育、生物矿化和调节细胞分化等功能的相关基因，这是通过调控核糖体和代谢途径实现的。而ES主要通过影响磷脂酰肌醇信号系统、肌醇磷酸代谢、粘合连接、细胞周期和Hedgehog信号通路实现这一功能。DK在200-300 μg/mL可有效抑制破骨细胞TRAP活性，而ES在200-500 μg/mL表现出显著抑制作用。破骨细胞中CTR、CTSK、MMP-9、TRAP、TRAF6的mRNA相对表达量被显著降低。通过共培养体系研究发现，两种肽显著促进了成骨细胞中ALP、BMP2、COL I和Runx2的mRNA相对表达量，上调了OPG/RANKL的比例，并能通过调节MAPK信号通路抑制破骨细胞分化。. 通过构建胶原蛋白基质矿化模型来验证筛选出的两条活性肽促进生物矿化的作用。ES和DK诱导羟基磷灰石在模型“D-周期”内的沉积，ES调控羟基磷灰石沿着c轴择优生长。ES相比于比DK，具有更好的调控矿化的作用。. 综上所述，DK、ES具有促进生物矿化的能力，揭示了其构效关系，阐明了其调控机制，本研究内容对生物矿化热点领域及禽蛋精深加工领域做出了重要贡献，卵黄高磷蛋白活性肽有望发展为补钙和骨修复等仿生材料产品。.