人血清、唾液和H1N1流感病毒中的蛋白糖基化分析

发布时间：2018-01-01 19:32

本文关键词：人血清、唾液和H1N1流感病毒中的蛋白糖基化分析　出处：《西北大学》2011年博士论文　论文类型：学位论文

更多相关文章： 糖蛋白 糖基化 血清唾液 H1N1流感病毒

【摘要】：蛋白糖基化修饰是最重要、最普遍的蛋白质翻译后修饰之一。糖基化通过改变修饰蛋白的各种生物学特性、调节细胞间识别、调控、信号传导、免疫应答、细胞转化等而参与各种生物学过程。蛋白糖基化同样在流感病毒等微生物感染宿主过程中发挥重要作用,病毒蛋白糖基化影响病毒的宿主范围和毒力；蛋白糖基化程度及其糖链结构的异常变化常伴随于癌症及其他疾病的发生发展过程中,很多糖蛋白已用于人类相关疾病的诊断、分期和愈后评估。开发新的蛋白糖基化研究技术,并在糖组学水平开展各种生理病理条件下蛋白糖基化的改变分析研究,对于深入了解蛋白糖基化功能、寻找新的疾病相关生物标记物和探讨各种生理病理相关分子机制等都具有极其重要的意义。本实验中,第一部分研究内容：将磁性微粒与肼化学相结合,建立了一种简单快速地从生物样本中提取N-糖肽的新方法,并结合生物质谱检测方法,建立一套N-糖基化位点鉴定的新体系。通过对肼功能化磁性微粒制备方法的筛选及肼功能化磁粒提取N-糖肽的条件(封闭剂、磁粒用量、反应时间和温度等)的优化,肼功能化磁粒对N-糖蛋白的偶联容量和特异性均优于目前常用的酰肼树脂。通过对标准蛋白混合物中N-糖蛋白或N-糖肽的提取和N-糖基化位点的鉴定,证明了肼功能化磁粒提取N-糖肽新方法的可行性。第二部分研究内容：分别采用基于酰肼化学的N-糖蛋白提取、N-糖肽提取和唾液酸化N-糖肽提取三种提取方法,从健康志愿者血清和肝细胞癌患者血清中提取N-糖肽(已去糖基化),并结合生物质谱鉴定N-糖蛋白及其糖基化位点。同时,联合糖蛋白的非糖基化多肽质谱数据,利用emPAI无标记定量方法对两种血清中的N-糖蛋白进行定量分析。从肝癌患者血清和健康志愿者血清中共鉴定到169种非冗余N-糖肽,103种N-糖蛋白,包含183个特异N-糖基化位点。其中发生末端唾液酸化的糖蛋白和糖肽分别占44.7%和48.0%,新鉴定到的N-糖肽和糖蛋白分别为15%的和17.7%。所鉴定道的血清N-糖蛋白主要是细胞外蛋白细胞蛋白、细胞器蛋白、膜蛋白和高分子复合物组分蛋白；主要参与刺激反应、生物学调控、细胞过程、多细胞有机体过程、免疫过程、代谢过程和信号等生物学过程；具有结合、酶活性调节和酶催化等分子功能。在两种血清中共同鉴定到的糖蛋白有76种,在盯癌患者血清中单独鉴定到12种,健康志愿者血清中单独鉴定到15种。另外,在肝癌患者血清中有9种N-糖蛋白含量明显增加,7种明显减少。这些差异糖蛋白主要在防御反应,细胞过程,生物过程、细胞过程和发展过程的调控等方面发挥作用。此外,实验结果表明,改进的N-糖蛋白提取方法具有可以使糖蛋白鉴定结果更准确,可以借助emPAI方法进行糖蛋白定量,可以鉴定到一些无法通过N-糖肽提取方法鉴定的N-糖蛋白,与N-糖肽提取方法具有很好的互补性等优点。第三部分研究内容：采用酰肼化学和亲水亲和两种糖肽提取方法,对不同性别儿童、成年、老年三个年龄段(共6组)健康志愿者全唾液中的N-糖蛋白及其糖基化位点进行了鉴定、比较和和综合性分析。从各年龄段混合唾液中共鉴定到156个非冗余N-糖肽,包含164个特异N-糖基化位点,代表85个N-糖蛋白：其中新鉴定到的N-糖蛋白和N-糖肽分别占18%和24%,17种N-糖蛋白首次在唾液中发现。唾液中41.2%的N-糖蛋白和35.2%的N-糖肽同样存在于健康人血清中。唾液N-糖蛋白中,分子量在10-100KD之间的占78%,等电点小于7的占80.2%,说明唾液N-糖蛋白主要是中低分子量且呈酸性的蛋白质。唾液N-糖蛋白主要是细胞外蛋白、细胞蛋白及细胞器蛋白；主要参与细胞过程、刺激反应、生物调节、代谢过程和免疫反应等生物学过程；具有结合、酶催化和酶活性调节等分子功能。唾液N-糖蛋白的种类随着年龄增长均具有增加趋势,且男性的增加速度大于女性。成年男性、女性唾液中N-糖蛋白种类差异较大,而老年男女性唾液中N-糖蛋白差异较小。另外,也找到了一些年龄和性别相关唾液N-糖蛋白。年龄增长过程中增加的唾液糖蛋白主要在免疫,生物过程,酶活性抑制调节,血管内皮生长因子生产和生物过程负调控,细胞死亡和程序性死亡等方面发挥作用。其中免疫相关唾液糖蛋白的增加普遍发生于人年龄增长过程中,这些糖蛋白主要在补体通路途径中发挥作用。通过与血清糖蛋白的比较发现,41.2%的人全唾液糖蛋白同样存在于健康人血清中。另外,实验结果表明,酰肼方法提取到的N-糖肽和N-糖蛋白数量和提取特异性都明显优于亲水亲和方法,但两种方法在糖蛋白和糖肽提取中依然具有一定的互补性。该研究为性别和年龄相关人唾液糖蛋白质组研究奠定基础,同时也为年龄和性别相关人类疾病的发病机理研究提供重要科学数据。第四部分研究内容：应用一系列生物信息学工具,对H1N1流感病毒演化过程中糖基化位点改变的方式、规律和作用进行了全面分析和深入探讨。通过对H1N1流感病毒中的2770条血凝素(HA)全长序列和3235条神经氨酸酶(NA)的潜在N-糖基化位点预测,糖基化位点的进化和保守性进行分析,蛋白3D结构同源建模和计算机模拟蛋白糖基化,发现不同宿主体内的H1N1流感病毒经历不同的蛋白糖基化变化过程；人H1N1流感病毒进化过程中有两种不同病毒蛋白糖基化改变模式,一种是病毒进化前期较高频率的糖基化位点数量的增加,另一种则是病毒进化后期较低频率的糖基化位点位置的变更。病毒蛋白糖基化变化的可能功能有：屏蔽HA和NA上的抗原性位点,保护NA免受宿主蛋白酶酶切,稳定HA三聚体和NA四聚体的四级结构,调节HA的受体结合活性和NA的神经氨酸酶活性以及调节两种活性的平衡。虽然本文中的一些结论和假设还需要进一步实验数据的支持,这些结果可对流感病毒的蛋白糖基化研究和病毒疫苗生产起到一定的指导作用。
[Abstract]:Protein glycosylation is the most important, the most common post-translational modification of proteins. The glycosylation change through various biological characteristics of modified proteins, regulating cell recognition, signal transduction, regulation, immune response, cell transformation, and participate in a variety of biological processes. Protein glycosylation also plays an important role in microbial infection of influenza in the process of virus host, the host range and the virulence of the virus protein glycosylation of virus; abnormal protein glycosylation degree and sugar chain structure often associated with the occurrence and development of cancer and other diseases, many glycoproteins have been used for the diagnosis of human diseases, staging and prognostic evaluation of the development of new protein. Glycosylation of technology, and carry out the change of protein glycosylation in various physiological and pathological conditions of the level of sugar in the group, to deeply understand the functions of protein glycosylation, looking for The new disease related biomarkers and the study of various physiological and pathological molecular mechanisms are of great significance.
In this experiment, the first part of the research: the combination of magnetic particles and chemical hydrazine, established a simple and rapid method for isolation of N- peptide from a biological sample, combined with biological mass spectrometry detection method, a new system to establish a set of N- glycosylation sites. Through the screening and identification of hydrazide functionized magnetic hydrazide functionized magnetic particle preparation method of particle N- extraction conditions (glycopeptide sealant, magnetic particle dosage, reaction temperature and time) of the optimization, hydrazide functionized magnetic microspheres on N- glycoprotein coupling capacity and specificity were better than the commonly used hydrazide resin. Through the extraction and identification of N- glycosylation sites the standard protein mixture of glycoprotein N- or N- peptide, proved the feasibility of hydrazine functionalized magnetic particle detection method of N- peptide.
The second part of the research were extracted using hydrazide chemistry N- glycoprotein based on three kinds of extraction methods of N- extraction and sialyl N- glycopeptide glycopeptide glycopeptide N- extracted from serum serum samples from healthy volunteers and in patients with hepatocellular carcinoma (has deglycosylation), and combined with the biomass identification of spectrum N- glycoprotein glycosylation site at the same time, non glycosylated peptide data combined with glycoprotein, using emPAI label free quantitative method for quantitative analysis of two kinds of serum N- glycoprotein. From the sera of HCC patients and healthy volunteers serum identified in total of 169 non redundant N- peptide, 103 N- glycoprotein, 183 specific glycosylation sites containing N- which terminal sialylation of the glycoproteins and glycopeptides accounted for 44.7% and 48%, the new identified N- glycopeptides and glycoproteins were 15% and 17.7%. identified road serum N- glycoprotein is mainly extracellular egg The white blood cell protein, cell protein, membrane protein and polymer composite component protein; mainly involved in stimulus response, biological regulation, cells, multicellular organisms, immune process, metabolic process and signal biological process; binding, enzyme regulator activity and catalytic activity of molecular function. Two serum glycoproteins in common to identify the 76 species at in serum of cancer patients and identified separately to 12 healthy volunteers, serum alone to identify 15 species. In addition, in the serum of patients with hepatocellular carcinoma 9 N- glycoprotein content increased 7, significantly reduced. These differences mainly in the glycoprotein defense reaction, cellular process, biological process that play a role in the cell process and the development process of regulation. In addition, the experimental results show that the improved N- glycoprotein extraction method has more accurate identification results can make the glycoprotein, can use emPAI By quantitation of glycoprotein, we can identify some N- glycoproteins that can not be identified by N- glycopeptide extraction method, and have good complementarity with N- glycopeptide extraction methods.
The third part of the research: using hydrazide chemistry and hydrophilic affinity of two kinds of extraction methods of glycopeptide, adult children, gender, old age three (6 groups) healthy volunteers N- glycoprotein and its glycosylation sites in whole saliva were identified, compared and analyzed. From the age of mixed saliva the identification of 156 nonredundant N- peptide, 164 specific glycosylation sites including N-, on behalf of the 85 N- glycoprotein which identified new N- glycoprotein and N- peptide accounted for 18% and 24%, 17 for the first time N- glycoprotein found in saliva. The saliva of 41.2% N- and 35.2% N- glycopeptide as glycoprotein in a healthy human serum. The salivary glycoprotein of N-, molecular weight between 10-100KD accounted for 78% and the isoelectric point of less than 7 accounted for 80.2%, that saliva N- glycoprotein is mainly low molecular weight and acidic protein. The salivary glycoprotein of N- mainly fine Extracellular protein, cell protein and organelle protein; mainly involved in cellular processes, stimulus response, biological regulation, metabolism and immune responses such as biological process; binding, enzyme catalysis and enzyme activity regulation. The molecular function of N- glycoprotein sialic species with age has increased, and the increase speed is higher than the female male. Adult males, N- glycoprotein species differences of the female in saliva is large, and old men and women in the saliva of N- glycoprotein were smaller. In addition, also found some age and sex age. Salivary glycoprotein N- in the process of increasing salivary glycoproteins mainly in immune, biological process, enzyme inhibition, vascular endothelial growth factor production and biological processes play a role in the negative regulation of cell death and programmed cell death. The increase of immune associated N-glycoproteins occurs in people age increase Long process, mainly these glycoproteins play a role in the complement pathway. By comparing with the serum glycoprotein found 41.2% people total salivary glycoproteins also exist in human serum. In addition, the experimental results show that the N- peptide and N- glycoprotein and the number of extraction of specific hydrazine method are much better than the hydrophilic affinity method, but the two methods in the extraction of glycoproteins and glycopeptides still are complementary. This study laid the foundation for the sex and age of human salivary sugar proteomics research, but also provide important scientific data for the study of pathogenesis of age and gender related human diseases.
The fourth part of the research: using a series of bioinformatics tools, the evolution of potential glycosylation sites to change the way of the H1N1 influenza virus, rule and function are analyzed and discussed. Through 2770 of the H1N1 influenza virus hemagglutinin (HA) sequence and 3235 neuraminidase (NA) to predict potential N- glycosylation sites, and evolutionary conserved glycosylation site analysis, protein 3D structure homology modeling and computer simulation of protein glycosylation, found in different hosts of influenza virus H1N1 protein through different sugar residues change process; H1N1 influenza virus evolution has two different viral protein glycosylation change the mode, one is to increase the number of glycosylation sites of virus evolution early high frequency, the other is a glycosylation site location of virus evolution late low frequency changes. The virus. May function based sugar: the antigenic sites shield HA and NA, protect NA from host protease digestion, four stable structure of HA trimer and NA four dimer, neuraminidase activity regulation of HA receptor binding activity and NA and regulating the balance of two kinds of activity. Although the number of conclusions and assumptions need to be further supported by experimental data, these results may be protein glycosylation research and the production of viral vaccines against influenza virus to play a guiding role.

【学位授予单位】：西北大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R341

【引证文献】