本文關(guān)鍵詞：人血清、唾液和H1N1流感病毒中的蛋白糖基化分析　出處：《西北大學(xué)》2011年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 糖蛋白 糖基化 血清 唾液 H1N1流感病毒

【摘要】：蛋白糖基化修飾是最重要、最普遍的蛋白質(zhì)翻譯后修飾之一。糖基化通過改變修飾蛋白的各種生物學(xué)特性、調(diào)節(jié)細(xì)胞間識別、調(diào)控、信號傳導(dǎo)、免疫應(yīng)答、細(xì)胞轉(zhuǎn)化等而參與各種生物學(xué)過程。蛋白糖基化同樣在流感病毒等微生物感染宿主過程中發(fā)揮重要作用,病毒蛋白糖基化影響病毒的宿主范圍和毒力；蛋白糖基化程度及其糖鏈結(jié)構(gòu)的異常變化常伴隨于癌癥及其他疾病的發(fā)生發(fā)展過程中,很多糖蛋白已用于人類相關(guān)疾病的診斷、分期和愈后評估。開發(fā)新的蛋白糖基化研究技術(shù),并在糖組學(xué)水平開展各種生理病理?xiàng)l件下蛋白糖基化的改變分析研究,對于深入了解蛋白糖基化功能、尋找新的疾病相關(guān)生物標(biāo)記物和探討各種生理病理相關(guān)分子機(jī)制等都具有極其重要的意義。 本實(shí)驗(yàn)中,第一部分研究內(nèi)容：將磁性微粒與肼化學(xué)相結(jié)合,建立了一種簡單快速地從生物樣本中提取N-糖肽的新方法,并結(jié)合生物質(zhì)譜檢測方法,建立一套N-糖基化位點(diǎn)鑒定的新體系。通過對肼功能化磁性微粒制備方法的篩選及肼功能化磁粒提取N-糖肽的條件(封閉劑、磁粒用量、反應(yīng)時(shí)間和溫度等)的優(yōu)化,肼功能化磁粒對N-糖蛋白的偶聯(lián)容量和特異性均優(yōu)于目前常用的酰肼樹脂。通過對標(biāo)準(zhǔn)蛋白混合物中N-糖蛋白或N-糖肽的提取和N-糖基化位點(diǎn)的鑒定,證明了肼功能化磁粒提取N-糖肽新方法的可行性。 第二部分研究內(nèi)容：分別采用基于酰肼化學(xué)的N-糖蛋白提取、N-糖肽提取和唾液酸化N-糖肽提取三種提取方法,從健康志愿者血清和肝細(xì)胞癌患者血清中提取N-糖肽(已去糖基化),并結(jié)合生物質(zhì)譜鑒定N-糖蛋白及其糖基化位點(diǎn)。同時(shí),聯(lián)合糖蛋白的非糖基化多肽質(zhì)譜數(shù)據(jù),利用emPAI無標(biāo)記定量方法對兩種血清中的N-糖蛋白進(jìn)行定量分析。從肝癌患者血清和健康志愿者血清中共鑒定到169種非冗余N-糖肽,103種N-糖蛋白,包含183個(gè)特異N-糖基化位點(diǎn)。其中發(fā)生末端唾液酸化的糖蛋白和糖肽分別占44.7%和48.0%,新鑒定到的N-糖肽和糖蛋白分別為15%的和17.7%。所鑒定道的血清N-糖蛋白主要是細(xì)胞外蛋白細(xì)胞蛋白、細(xì)胞器蛋白、膜蛋白和高分子復(fù)合物組分蛋白；主要參與刺激反應(yīng)、生物學(xué)調(diào)控、細(xì)胞過程、多細(xì)胞有機(jī)體過程、免疫過程、代謝過程和信號等生物學(xué)過程；具有結(jié)合、酶活性調(diào)節(jié)和酶催化等分子功能。在兩種血清中共同鑒定到的糖蛋白有76種,在盯癌患者血清中單獨(dú)鑒定到12種,健康志愿者血清中單獨(dú)鑒定到15種。另外,在肝癌患者血清中有9種N-糖蛋白含量明顯增加,7種明顯減少。這些差異糖蛋白主要在防御反應(yīng),細(xì)胞過程,生物過程、細(xì)胞過程和發(fā)展過程的調(diào)控等方面發(fā)揮作用。此外,實(shí)驗(yàn)結(jié)果表明,改進(jìn)的N-糖蛋白提取方法具有可以使糖蛋白鑒定結(jié)果更準(zhǔn)確,可以借助emPAI方法進(jìn)行糖蛋白定量,可以鑒定到一些無法通過N-糖肽提取方法鑒定的N-糖蛋白,與N-糖肽提取方法具有很好的互補(bǔ)性等優(yōu)點(diǎn)。 第三部分研究內(nèi)容：采用酰肼化學(xué)和親水親和兩種糖肽提取方法,對不同性別兒童、成年、老年三個(gè)年齡段(共6組)健康志愿者全唾液中的N-糖蛋白及其糖基化位點(diǎn)進(jìn)行了鑒定、比較和和綜合性分析。從各年齡段混合唾液中共鑒定到156個(gè)非冗余N-糖肽,包含164個(gè)特異N-糖基化位點(diǎn),代表85個(gè)N-糖蛋白：其中新鑒定到的N-糖蛋白和N-糖肽分別占18%和24%,17種N-糖蛋白首次在唾液中發(fā)現(xiàn)。唾液中41.2%的N-糖蛋白和35.2%的N-糖肽同樣存在于健康人血清中。唾液N-糖蛋白中,分子量在10-100KD之間的占78%,等電點(diǎn)小于7的占80.2%,說明唾液N-糖蛋白主要是中低分子量且呈酸性的蛋白質(zhì)。唾液N-糖蛋白主要是細(xì)胞外蛋白、細(xì)胞蛋白及細(xì)胞器蛋白；主要參與細(xì)胞過程、刺激反應(yīng)、生物調(diào)節(jié)、代謝過程和免疫反應(yīng)等生物學(xué)過程；具有結(jié)合、酶催化和酶活性調(diào)節(jié)等分子功能。唾液N-糖蛋白的種類隨著年齡增長均具有增加趨勢,且男性的增加速度大于女性。成年男性、女性唾液中N-糖蛋白種類差異較大,而老年男女性唾液中N-糖蛋白差異較小。另外,也找到了一些年齡和性別相關(guān)唾液N-糖蛋白。年齡增長過程中增加的唾液糖蛋白主要在免疫,生物過程,酶活性抑制調(diào)節(jié),血管內(nèi)皮生長因子生產(chǎn)和生物過程負(fù)調(diào)控,細(xì)胞死亡和程序性死亡等方面發(fā)揮作用。其中免疫相關(guān)唾液糖蛋白的增加普遍發(fā)生于人年齡增長過程中,這些糖蛋白主要在補(bǔ)體通路途徑中發(fā)揮作用。通過與血清糖蛋白的比較發(fā)現(xiàn),41.2%的人全唾液糖蛋白同樣存在于健康人血清中。另外,實(shí)驗(yàn)結(jié)果表明,酰肼方法提取到的N-糖肽和N-糖蛋白數(shù)量和提取特異性都明顯優(yōu)于親水親和方法,但兩種方法在糖蛋白和糖肽提取中依然具有一定的互補(bǔ)性。該研究為性別和年齡相關(guān)人唾液糖蛋白質(zhì)組研究奠定基礎(chǔ),同時(shí)也為年齡和性別相關(guān)人類疾病的發(fā)病機(jī)理研究提供重要科學(xué)數(shù)據(jù)。 第四部分研究內(nèi)容：應(yīng)用一系列生物信息學(xué)工具,對H1N1流感病毒演化過程中糖基化位點(diǎn)改變的方式、規(guī)律和作用進(jìn)行了全面分析和深入探討。通過對H1N1流感病毒中的2770條血凝素(HA)全長序列和3235條神經(jīng)氨酸酶(NA)的潛在N-糖基化位點(diǎn)預(yù)測,糖基化位點(diǎn)的進(jìn)化和保守性進(jìn)行分析,蛋白3D結(jié)構(gòu)同源建模和計(jì)算機(jī)模擬蛋白糖基化,發(fā)現(xiàn)不同宿主體內(nèi)的H1N1流感病毒經(jīng)歷不同的蛋白糖基化變化過程；人H1N1流感病毒進(jìn)化過程中有兩種不同病毒蛋白糖基化改變模式,一種是病毒進(jìn)化前期較高頻率的糖基化位點(diǎn)數(shù)量的增加,另一種則是病毒進(jìn)化后期較低頻率的糖基化位點(diǎn)位置的變更。病毒蛋白糖基化變化的可能功能有：屏蔽HA和NA上的抗原性位點(diǎn),保護(hù)NA免受宿主蛋白酶酶切,穩(wěn)定HA三聚體和NA四聚體的四級結(jié)構(gòu),調(diào)節(jié)HA的受體結(jié)合活性和NA的神經(jīng)氨酸酶活性以及調(diào)節(jié)兩種活性的平衡。雖然本文中的一些結(jié)論和假設(shè)還需要進(jìn)一步實(shí)驗(yàn)數(shù)據(jù)的支持,這些結(jié)果可對流感病毒的蛋白糖基化研究和病毒疫苗生產(chǎn)起到一定的指導(dǎo)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.

【學(xué)位授予單位】：西北大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R341

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 王秦哲;肝癌細(xì)胞的N-糖蛋白質(zhì)鑒定及糖基化位點(diǎn)分析[D];西北大學(xué);2012年

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本文編號：1365901