【作者】 张晓维；

【导师】 马美湖；

【作者基本信息】 华中农业大学 ， 食品科学， 2014， 博士

【摘要】 卵黄高磷蛋白是鸡蛋蛋黄中主要的磷蛋白,独特的氨基酸组成和高度磷酸化使其具有很多优良的物化和生物学特性,生物信息学分析推测卵黄高磷蛋白可能具有调控生物矿化的功能。本课题首先分离制备高纯度的卵黄高磷蛋白,对其结构进行表征,鉴定磷酸化位点并结合生物信息学分析,挖掘其潜在功能,最后研究卵黄高磷蛋白调控矿化的机制和活性位点。本课题为卵黄高磷蛋白在仿生矿化中的应用奠定基础。主要研究内容和结果如下：(1)建立一种条件温和、高效简单的分离卵黄高磷蛋白的新方法,将鸡蛋蛋黄依次经等质量蒸馏水和0.17mol/LNaCl稀释离心除去蛋黄浆质,获得蛋黄颗粒,用10%1.74mol/LNaCl溶解后,添加3%w/w PEG6000并调整溶液pH至4.0,离心后透析冻干,制备的卵黄高磷蛋白(Pvti)电泳鉴定纯度为99%,得率为47%。采用该方法分别从海兰褐和白莱航鸡蛋中分离制备卵黄高磷蛋白,HPLC测定纯度分别达93.14%和91.80%,得率分别为63.93%和58.89%。进一步分析表明,这两个品种中的卵黄高磷蛋白的含量、组成成分和二级构象均无明显差异,且与Sigma的一致。此外,使用离子交换层析法制备不含多价金属离子的卵黄高磷蛋白,最佳条件为：Pvti经Na2EDTA处理后,采用梯度增加NaCl浓度(0、0.35、0.5mol/L)的Tris-HCl缓冲液(0.05mol/L, pH7.5)在DEAE柱中洗脱,收集洗脱峰经透析冻干,得到纯化的不含多价金属离子的卵黄高磷蛋白(PVtf)。(2)开展卵黄高磷蛋白结构的系统分析,研究显示卵黄高磷蛋白的氨基酸组成含有高达30%的丝氨酸(Ser),富含酸性氨基酸(Asp. Glu)和碱性氨基酸(Arg、His),含有极少量的芳香族氨基酸(Tyr、Cys、 Trp)。使用红外光谱(FTIR)和圆二色谱(CD)分析卵黄高磷蛋白二级结构主要为无规卷曲和p-折叠结构,不含金属离子的卵黄高磷蛋白p-转角结构增多。LC-ESI-MS/MS (LTQ)生物质谱技术结合生物信息学预测分析表明卵黄高磷蛋白含有87个磷酸化位点(83个P-Ser和4个P-Thr)。利用生物信息学分析9个物种来源的11种卵黄高磷蛋白以及来源于人的5种磷酸化蛋白质,进行氨基酸组成、多序列比对和进化树分析,显示均具有高丝氨酸含量,且富含酸性氨基酸的特点。鸡卵黄高磷蛋白与牙本质磷蛋白(DPP)的结构及特性相似,进化关系较近,意味着卵黄高磷蛋白可能具有与牙本质磷蛋白相似的生物功能,即在生物矿化过程中发挥重要的调控作用。(3)利用钙离子浓度计(ISE)、等温滴定量热仪(ITC)、CD和荧光光谱方法,分别从化学、热力学以及结构信息方面,较为全面地研究了卵黄高磷蛋白与钙离子的相互作用。在中性和弱碱性条件下,卵黄高磷蛋白与钙离子相互作用存在高亲和性和低亲和性两类结合位点。高亲和性位点结合常数约为104mol-1,每摩尔卵黄高磷蛋白约结合30摩尔的钙离子,该结合为焓驱动反应,卵黄高磷蛋白的构象变化为无序和p-转角结构增加,而p-折叠结构减少,主要作用力为静电、氢键或范德华力；而低亲和性位点结合常数和结合位点数并不恒定,有大量的钙离子累积在饱和钙的分子或聚集区域的附近,该结合为熵驱动反应,卵黄高磷蛋白的p-折叠结构增加,主要作用力为疏水作用力；而在酸性条件下,卵黄高磷蛋白与钙离子相互作用为熵驱动的吸热反应,主要作用力为弱的疏水相互作用。(4)研究仿生矿化体系中卵黄高磷蛋白对磷酸钙矿物晶相转化的影响。FTIR、X射线衍射(XRD)、扫描电镜(SEM)和能谱分析(EDS)表征矿物的结构、组成以及表面微观形貌,结果表明在该体系中生成的磷酸钙矿物通过“溶解再结晶”途径从磷酸氢钙(DCPD)转变为羟基磷灰石(HAP)。卵黄高磷蛋白可以极大地促进晶相转化,使转化过程由6h缩短至0.5h以内。磷酸钙矿物的FTIR和XRD图谱显示出蛋白质特征峰,且矿化后滤液中蛋白含量降至原来的0.5%,表明卵黄高磷蛋白参与了磷酸钙矿物的形成,成为矿物的组成部分之一。不同添加顺序的实验结果显示,卵黄高磷蛋白在DCPD形成之前添加才能够发挥其促进作用,表明与游离钙离子的互作是其调控磷酸钙矿化的关键。作为对照的BSA亦对晶相转化具有促进作用,但其作用较弱且参与模式不同,BSA参与磷酸钙矿物晶相转化后期阶段,而卵黄高磷蛋白参与晶相转化起始阶段,卵黄高磷蛋白在磷酸钙矿化过程中的奇特作用与其高度的磷酸化密切相关。(5)使用0.1、0.2、0.3、0.4mol/L NaOH溶液处理卵黄高磷蛋白(Pv),经超滤分离得到不同去磷酸化的卵黄高磷蛋白：T1、T2、T3和T4,去磷酸化程度分别为2.98%、19.46%、43.39%和71.07%。并采用pH-stat体系研究了其对矿化的影响。卵黄高磷蛋白促进矿化的效果呈浓度剂量关系。通过CD、 FTIR、 XRD、 SEM和荧光光谱研究不同去磷酸化程度的卵黄高磷蛋白对矿化的影响,结果表明磷酸化在矿化反应中起到很重要的作用。去磷酸化后的卵黄高磷蛋白p-折叠结构增加,可以提供矿化模板。去磷酸化程度低的卵黄高磷蛋白由于无序结构减少,吸附矿物能力减弱,含磷量少,结合钙离子能力减弱,从而促进矿化效果减弱。但高浓度碱液处理卵黄高磷蛋白使其水解为了卵黄高磷蛋白磷酸肽,由于活性区域暴露,成核位点增加,显著的促进矿化。总之,促进矿化效果强弱顺序依次为T4>T3>卵黄高磷蛋白﹥T2﹥T1﹥Control。(6)通过碱液部分去磷酸化后再用胰蛋白酶酶解卵黄高磷蛋白,经DEAE离子交换色谱和Sepharose G-25凝胶过滤色谱分离纯化卵黄高磷蛋白磷酸肽(PPP)各组分,SDS-PAGE和CD分析显示PPPO和PPP1可能为卵黄高磷蛋白酶解的小分子片段(N端或C端),而PPP3和PPP4二级结构相似,只是PPP4折叠更多,且PPP3多含10kDa的小分子肽。利用pH-stat体系研究卵黄高磷蛋白磷酸肽各组分对矿化效果的影响,通过CD、 FTIR、 XRD、 SEM和荧光光谱研究发现PPPO、 PPP1、 PPP4促进效果并没有卵黄高磷蛋白强,原因为PPP0和PPP1是小分子肽,不参与矿化；同时,PPP4折叠太多,结构太紧凑,使得促进矿化能力减弱。而PPP3暴露了活性区域,因此促进效果最显著。促进矿化反应效果强弱顺序依次为：PPP3>卵黄高磷蛋白﹥PPP4﹥PPP1﹥Control﹥PPPO。经LTQ MS/MS鉴定可知促进矿化的活性区域为卵黄高磷蛋白核心区域中的D1165-R1258。更多还原

【Abstract】 Phosvitin is the major phosphoprotein from hen egg yolk. Due to its unique amino acid composition and highly phosphorylation, phosvitin possess many excellent physical and biological properties. And phosvitin was speculated to possess regulation properties of biological mineralization through the bioinformatics analysis. In our research, highly purified phosvitin was firstly prepared, then characterized its structure, identified the phosphorylation sites, and excavated the potential function with combing the bioinformatics analysis, lastly investigated the regulation mechanism of phosvitin in the mineralization and clarified its active sites. This study will lay a foundation for the application of phosvitin in biomimetic mineralization. The main research contents and results are as follows:An attempt was made to develop a new protocol for preparing phosvitin with high efficiency and simplicity in a mild condition. Hen egg yolk sequentially diluted with equal mass of distilled water and0.17M NaCl, to obtain yolk granules after removing the yolk plasma by centrifugation. Granules was dissolved with10%1.74M NaCl, added3%w/w PEG6000and the solution was adjusted to pH4.0, desalted by dialysis after centrifugation and lyophilized. The purity of phosvitin (Pvti) was99%, which identified by SDS-PAGE, and yield was47%. Based on this protocol, phosvitin was prepared from egg yolk of Hy-Line Brown and White Leghorn, the purity examined by HPLC was93.14%and91.80%, respectively; and the yield was63.93%and58.89%, respectively. Further study showed that there had hardly significant between Hy-Line Brown and White Leghorn in the content, composition and secondary structure of phosvitin, they were all consistent with Sigma. Furthermore, Polyvalent metal-free phosvitin was purified through ion-exchange chromatography, the optimal condition was as follows: Pvti treated with Na2EDTA was eluted with an increasing gradient of NaCl (0,0.35and0.5mol/L) in Tris-HCl buffer (0.05mol/L, pH7.5) by DEAE column, the eluate was collected, polyvalent metal-free phosvitin (Pvtf) was obtained through dialysis against distilled water and lyophilized.(2) Systematic analysis the structure of phosvitin, the studies showed that amino acid composition of phosvitin contain up to30%Ser, rich in acidic amino acids (Asp, Glu) and basic amino acids (Arg, His), and contain a very small amount of the aromatic amino acids (Tyr, Cys and Trp). The conformation of phosvitin was mainly random coil and (3-sheet structure while metal-free phosvitin increased the β-turn content, by using Fourier transform infrared spectroscopy (FTIR) and circular dichroism (CD). Phosvitin possessed87phosphorylation sites (83P-Ser and4P-Thr), which were identified by LC-ESI-MS/MS (LTQ) and combined the prediction results by bioinformatics analysis. In addition, bioinformatics analyzed the11types of phosvitin from9different species and5types of phosphorylated protein from human through amino acid compositions, multiple sequence alignment and phylogenetic analysis, the results revealed that they all rich in acidic amino acids. Phosvitin and dentin phosphophoryn (DPP) have similar structure and properties, meanwhile the evolutionary relationships was close, this suggested phosvitin may have similar biological functional activity with DPP, can play an important regulation role in the process of bio mineralization.(3) Comprehensive study of the interaction of phosvitin with calcium ions from the chemical, thermodynamic and structural aspects, by using calcium ion selective electrode (ISE), isothermal titration calorimetry (ITC), CD and fluorescence spectroscopy, respectively. The results showed that, under neutral and alkaline conditions, there existed two classes of binding sites in the interaction between phosvitin and calcium: high affinity site and weak affinity site. The binding constant of high affinity is about104mol-1, and binding sites were nearly30mol of calcium per mole of phosvitin. This reaction was driven by enthalpy, the conformation of phosvitin increased random and (3-sheet structure, while decreased β-turn structure, the main interaction force was electrostatic, hydrogen bonds or Van der Waals. Meanwhile, the binding constant and the binding site of low affinity were not constant, since a large amount of calcium ions accumulated in the vicinity of the calcium saturation region or the molecule aggregation, the binding reaction was driven by entropy, the conformation of phosvitin increased β-sheet structure, and main interaction force was hydrophobic force. However, under acidic condition, the interaction between phosvitin and calcium was entropy-driven endothermic reaction, and the main interaction force was weak hydrophobic force.(4) Effect of phosvitin on the phase transformation of calcium phosphate mineral was investigated in the biomimetic system. The mineral structure, composition and surface morphology was characterized by FTIR, X-ray diffraction (XRD), scanning electron microscopy (SEM) and energy dispersive spectrometer (EDS). The results showed that the calcium phosphate mineral transformed from dicalcium phosphate dihydrate (DCPD) to hydroxyapatite (HAP), through the "dissolution-recrystallization" pathway in this system. Phosvitin significantly accelerated the phase transformation, since the transform process was shortened from6h to less than0.5h. Besides, The characteristic peaks of protein was occurred in the FTIR spectra and the XRD patterns of calcium phosphate mineral, and the phosvitin concentration in the filtrate during the mineralization was reduced to0.5%, indicated that phosvitin was involved in the phase transformation of calcium phosphate mineral and incorporated into the mineral. The results of the addition sequence of substrates on the phase transformation of calcium phosphate showed that, Phosvitin played its role only if adding phosvitin before DCPD formed, indicates that the interaction between phosvitin and the free calcium ions was the key to promote its regulation mineralization of calcium phosphate. BSA used as a control, also promoted the phase transformation. However, compared with phosvitin, its effect was weak and was involved in different modes. BSA involved in the later phase transformation stages of calcium phosphate mineral while phosvitin participate the initial phase transformation. The peculiar role of phosvitin in the calcium phosphate mineralization process was closely related to its degree of phosphorylation.(5) Different dephosphorylation degrees of phosvitin were accomplished by treating phosvitin (Pv) with0.1,0.2,0.3,0.4mol/L NaOH solution. T1, T2, T3and T4were obtained through ultrafiltration, the dephosphorylation degree was2.98%,19.46%,43.39%,71.07%, respectively. Their effect on the mineralization were studied by using a pH-stat system. Phosvitin promote mineralization depended on concentration-dose relationship. Effect of different dephosphorylation degrees of phosvitin on the mineralization was studied by CD, FTIR, XRD, SEM, fluorescence spectroscopy. The results showed that phosphorylation was very important in the mineralization reaction. In addition, the dephosphorylation phosvitin increased its P-sheet structure, providing the template of mineralization. Due to its random structure reduced, low dephosphorylated levels of phosvitin decreased its adsorption capacity on mineral, decreased the calcium binding capacity as less phosphorus, and thereby weakened the effect of promotion on the mineralization. However, phosvitin could hydrolyzed to phosvitin peptides by high concentration of alkaline solution, the active regions were exposed, thus increased the nucleation sites, and significantly promoted mineralization. In summary, efficiency of promoting mineralization order was T4﹥T3﹥Pv﹥T2﹥T1﹥Control.(6) Phosvitin was partially dephosphorylated by alkaline and then digested with trypsin. The phosvitin phosphopeptides (PPP) fractions were separated and purified through DEAE ion-exchange chromatography and Sepharose G-25gel filtration chromatography. SDS-PAGE and CD analysis showed that PPPO and PPP1were possible the small molecule fragments (N-or C-terminal) after enzymatic hydrolysis, while PPP3and PPP4had similar secondary structure, but PPP4had more compact and β-sheet structure, while PPP3contained10kDa peptides. Effect of phosvitin phosphopeptides fractions on the mineralization by means of FTIR, XRD, SEM, fluorescence spectroscopy in pH-stat system. The results showed that, as compared with Pv, PPPO, PPP1and PPP4decreased its promoting efficiency, because PPPO and PPP1were small peptides, and not involved in the mineralization; meanwhile, PPP4owed too much β-sheet structure resulted in more compact, thus weakened the promotion effect. PPP3exposed its active region, thus the promoted effect was the most significant. The mineralization reaction rate was as follows: PPP3﹥Pv﹥PPP4﹥PPP1﹥Control﹥PPPO. The active region of promotion mineralization was D1165-R1258in the core region of phosvitin by LTQ MS/MS identification.更多还原