基于构效关系揭示鸭蛋蛋清肽高效促钙吸收机制

Administration of safe and efficient calcium supplements is a crucial method to reverse the status of population calcium deficiency. The usually abandoned salted duck egg white and eggshell are economical sources of bio-calcium supplements and peptides. Duck egg white peptides (DPs), has been reported to have the high bio-activity of promoting calcium uptake as casein phosphopeptides in my laboratory. TRPV6 and Cav1.3 are the main routes of calcium transmembrane transport. In our preliminary experiment, TRPV6 has been clarified that it is the optimal target protein of DPs, but the precise mechanism are still unknown. Peptides, which is depolarized, has been considered to the effective stimulation of Cav1.3 calcium channel. A hypothesis is also proposed based on the peptide transport routes that DPs with acidic amino acid could be transported by PepT1 accompanied with calcium ions in my laboratories. On the one hand, the primary structures of the most important DPs will be identified and synthesized couple with the online mass spectrometry database and the structure-activity relationship, especially the structure of repeating Glu and Ser sequence and peptides with aromatic amino acid, will be researched in the cell model, in-vivo intestinal model and gene knock-out animal model, by the means of patch clamp technique, molecular biology, molecular docking technology and spectroscopy. On the other hand, the Cav1.3 calcium channel will be confirmed whether it is another calcium transport route by DPs and the interaction between DPs and TRPV6 will be the focused research. The underlying mechanism of promoting calcium uptake of DPs will be revealed in cell level, molecular level and animal level. The new research idea of the oligopeptides will be provided based on our current study.

补充安全高效补钙剂是解决当前国民普遍缺钙的重要手段，常被废弃的咸鸭蛋蛋清和蛋壳是经济的肽源和生物钙源，本室已发现鸭蛋蛋清肽(DPs)是具有可与酪蛋白磷酸肽媲美的促钙吸收小肽；TRPV6和Cav1.3 是钙在肠道跨膜吸收的重要钙离子通道，经预实验显示前者是DPs的重要作用靶标，但其作用机制尚不明了；去极化营养素肽适合激活Cav1.3通道。鉴于小肽有独立的转运系统，我们提出假说“富含酸性氨基酸的DPs小肽能携钙被PepT1转运”。我们拟通过细胞模型、在体肠道模型、基因敲除鼠模型，并借助于膜片钳技术、分子生物学手段、分子对接方法及波谱学等方法，鉴定并合成重要DPs，进而揭示含E簇、S簇和芳香族短肽的促钙吸收构效关系，考察Cav1.3 是否为 DPs的另一作用靶标，重点揭示DPs与TRPV6的作用机制。以期从细胞水平、分子水平及整体动物水平，阐明DPs的高效促钙吸收机制，为此类肽的研究提供新思路。

常被废弃的咸鸭蛋清是经济的促钙吸收肽源，本课题分别利用Caco-2细胞等体外实验和五种动物模型对鸭蛋蛋清肽(DPs)的促钙吸收、促骨合成及其机理进行了系统研究，主要研究结果如下：①DPs的促钙吸收方式为主动转运，当Ca2+浓度升高时，旁路转运被激活。且揭示了TRPV6和Cav1.3两种钙离子通道均为DPs的作用靶标；正常膳食和低钙膳食条件下，DPs均能有效激活TRPV6通路中的钙转运蛋白calbindinD9k，小肽在Ca2+与TRPV6间起到了桥梁作用。②鉴定出了7条有利于结合钙的肽段。构效关系研究显示：VSEE的碳端第一位的“E”是必须的，E簇对于促钙吸收作用有重要贡献，处于序列中间的“S”，若要发挥作用必须磷酸化。VHSS中“S”位于碳末端时，S簇能够促进钙的转运，且末端“S”磷酸化使钙转运量显著提高，而“SS”双磷酸化时，其钙转运量进一步提升。③低钙膳食模型、植酸抑制钙吸收模型及维甲酸模型三种动物模型均表明DPs具有较好的促钙吸收作用。在肉鸡模型中，VSEE+Ca能够促进小鸡肠道益生菌的生长；单独补充CaCO3会增加肠道内有害菌增殖；若CaCO3联合补充DPs则能显著抑制有害菌的增殖。④在体小肠单向灌流实验及分子对接实验结果表明，含芳香族氨基酸的肽段INSW和NFE能显著上调十二指肠中钙敏受体CaSR基因的表达量，此两肽段均能很好地进入CaSR蛋白的活性口袋，占据中心。⑤揭示了DPs不能携钙被小肽转运蛋白PepT1转运；代谢动力学实验表明VSEE能够直接被小肠吸收。⑥DPs和VSEE能通过改善去势大鼠的小肠屏障功能，显著上调Wnt/β-catenin信号通路相关蛋白表达量，促进前成骨细胞的增殖、分化和矿化，从而对雌激素水平降低引起的骨质疏松症起到明显的改善作用。综上，DPs/VSEE具有较好的促钙吸收及促骨合成活性，该研究成果对于高值化利用鸭蛋清资源具有重要意义。