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核糖体失活蛋白的结构生物学研究
中文摘要

本论文主要包括两个部分的内容,第一部分是关于几种Ⅰ型核糖体失活蛋白(八棱丝瓜蛋白1、南瓜蛋白、β、γ-天花粉蛋白)的结构生物学研究,第二部分初步地研究了整合素αMβ2 I-domain。 植物核糖体失活蛋白(RIPs)具有rRNA N-糖苷酶活性,能够抑制蛋白质的生物合成。它们具有多种生物功能,如抗肿瘤、抗病毒、抗真菌、诱导细胞凋亡、免疫抑制和抑制HIV整合酶等,这些发现激发了人们的研究兴趣。另外,核糖体失活蛋白在农业和生物医学研究中也具有重要的应用价值,如培育转基因农作物、研制免疫毒素等。因此核糖体失活蛋白已逐渐成为一个研究热点。 蛋白质的氨基酸顺序可以通过Edman降解、质谱法和cDNA测序获得,从高分辨率的晶体结构也可以获得蛋白质氨基酸序列的信息,但是在辨别Asp/Asn, Glu/Gln和Val/Thr氨基酸对时存在不可避免的困难。八棱丝瓜蛋白1是来源于八棱丝瓜种子的一种新型的核糖体失活蛋白,它除了具有rRNA N-糖苷酶活性外还可以诱导肿瘤细胞的凋亡和分化,从而抑制肿瘤细胞的增殖。八棱丝瓜蛋白1的晶体结构可以衍射到1.4 Å的分辨率,它的氨基酸顺序就是基于以下标准从高分辨率的晶体结构中推测的:1)高分辨率的晶体结构;2)不对称单元中两个分子的电子云密度图的比较;3)对某些难以区分的氨基酸对(Glu/Gln,Asp/Asn和Val/Thr)的化学环境的评估;4)与同源蛋白氨基酸顺序的比较。通过标准1和2,可以推断出66%的氨基酸顺序;再加上标准3,则可有86%的氨基酸顺序被推断出来,这也表明对某些氨基酸的化学环境的评估,对推断一些难以分辨的氨基酸对具有一定的有效性。总之,通过这4个标准利用X-射线测序的方法,能推断出94%的八棱丝瓜蛋白1的氨基酸顺序。从八棱丝瓜嫩叶中提取基因组DNA,以八棱丝瓜蛋白1的X-ray序列为基础,设计上下游引物,进行PCR扩增,从而测定八棱丝瓜蛋白1的氨基酸序列。通过比较八棱丝瓜蛋白1的X-ray顺序和用DNA方法测定的氨基酸顺序,证明超过90%的X-ray序列是正确的。在八棱丝瓜蛋白1的晶体结构中有两个N-乙酰基葡萄糖分别连接在77位和84位天冬酰胺上。Tyr70, Tyr110,Glu159和Arg162是八棱丝瓜蛋白1的活性位点。 南瓜蛋白是从南瓜瓜瓤(Cucurbita moschata)中提取的一种新型核糖体失活蛋白。除了具有rRNA N-糖苷酶活性外,南瓜蛋白还对鼠源和人源的三种癌细胞产生细胞毒性,但是对正常细胞的毒性较小。从南瓜的嫩叶中提取植物基因组DNA,基于N-端氨基酸顺序和C-端X-ray序列设计上下游引物,进行PCR扩增。南瓜蛋白全长的氨基酸顺序是通过N-端Edman降解,部分DNA测序获得,同时经高分辨率的晶体结构验证分析。南瓜蛋白的晶体结构可以衍射到1.04Å,目前还没有其它RIPs的晶体结构达到这样的分辨率。南瓜蛋白的晶体结构包含两个结构域:一个大的N-端结构域,由七个α-螺旋和八个β-折叠组成;一个小的C-端结构域,由三个α-螺旋和两个β-折叠构成。基于南瓜蛋白高分辨率的晶体结构,可以构建一个混合型寡糖链的模型GlcNAc₂Man₃Xyl,其中Ash225为糖基化位点。氨基酸Tyr70,,Tyr109,,Glu158和Arg161为南瓜蛋白的活性位点。 采用两步阳离子交换树脂洗脱技术,可以从葫芦科植物栝楼(Trichosanthes kirilowii Maxim)块根中,快速分离出α、β、γ-天花粉蛋白,α-天花粉蛋白就是先前报道的天花粉蛋白。纯化过的α、β、γ-天花粉蛋白用EMI-MS测定分子量分别为27167,27143和27262 Da。β-天花粉蛋白分别以氯化钾和聚乙二醇为沉淀剂均可长出单晶。前者(晶型A)在同步辐射光源上收集到一套1.6Å数据,晶体属于正交晶系P2₁2₁2₁空间群,后者(晶型B)在同步辐射光源上收集到一套1.2 Å数据,晶体属于单斜晶系P21空间群。目前β-天花粉蛋白两种晶型的模型构建和结构修正工作正在进行。γ-天花粉蛋白以硫酸铵为沉淀剂可以长出单晶,它在同步辐射光源上可以收集到一套2.2 Å数据,晶体属于六方晶系P622空间群,目前蛋白晶体的结构解析工作正在进行中。 αMβ2是整合素家族中的一员,是介导细胞和细胞外基质以及细胞间粘附作用的主要因子,可以识别、结合细胞外基质中相应的配体,为细胞黏附提供附着点。这些蛋白一般都是由α、β两条链经非共价键连接组成的异源二聚体。在α链的N末端包含了200个左右的氨基酸,被称为“I-domain”,它在识别许多蛋白配体以及非蛋白配体中起到关键作用,也是识别金属离子的主要位点。大量的、均一性好的蛋白对I-domain和其配体的结构研究是相当重要的。为了增加重组蛋白表达产量,可以对基因进行重新设计合成。本文借助DNAWorks在线程序,采用基因拼装法,合成了适合大肠杆菌表达菌株所偏爱的模板,提高了αMβ2 I-domain在大肠杆菌的表达量。另外我们采用不同咪唑浓度阶段洗脱的方法,简化了纯化步骤,使其只经过一步Ni柱纯化就能得到纯度达到90%的蛋白。由电喷雾质谱(ESI-MS)法测定出纯化蛋白的肽指纹图谱(PMF),经Mascort程序分析鉴定了目的蛋白。 关键词:核糖体失活蛋白(RIPs),晶体结构,X-ray序列,DNA测序,细胞毒性

英文摘要

This thesis is mainly composed of two parts: crystal structural study of some type 1 ribosome-inactivating proteins (luffaculin 1,cucurmosin, β- and γ-trichosanthin); the study on αMβ2 I-domain. Plant ribosome-inactivating proteins (RIPs) have rRNA N-glycosidase activity and can irreversibly block the protein biosynthesis. They have various biological functions, such as antitumor, antiviral,antifungal, apoptosis-inducing, immunosuppressive and HIV-1 integrase inhibitory activity. These findings inspire great interests of investigators. Moreover,RIPs are considered to have great potential in agricultural and biomedical applications, such as transgenic plants and immunotoxins. As a result, the studies of ribosome-inactivating proteins have been an active research field. Protein sequence can be obtained through Edman degradation, mass spectrometry, or cDNA sequencing. High resolution X-ray crystallography can also be used to derive protein sequence information, but faces the difficulty in distinguishing the Asp/Asn, Glu/Gln, and Val/Thr pairs. Luffaculin 1 is a new type 1 ribosome-inactivating protein (RIP) isolated from the seeds of Luffa acutangnla. Besides rRNA N-glycosidase activity, luffaculin 1 also demonstrates activities including inhibiting tumor cells' proliferation and inducing tumor cells' differentiation. The crystal structure of luffaculin 1 was determined at 1.4 Å resolution. Its amino-acid sequence was derived from this high resolution structure using the following criteria: 1) high resolution electron density; 2) comparison of electron density between two molecules that exist in the same crystal; 3) evaluation of the chemical environment of residues to break down the sequence assignment ambiguity in residue pairs Glu/Gln, Asp/Asn, and Val/Thr; 4) comparison with sequences of the homologous proteins. Using the criteria 1 and 2,66% of the residues can be assigned. By incorporating with criterion 3,86% of the residues were assigned, suggesting the effectiveness of chemical environment evaluation in breaking down residue ambiguity. In total, 94% of the luffaculin 1 sequence was assigned with high confidence using this improved X-ray sequencing strategy. Plant genomic DNA extracted from the tender leaves was amplified by PCR between primers based on the X-ray sequence of luffaculin 1. More than 90% X-ray sequence of luffaculin 1 was proved to be correct through comparing the X-ray sequence with the amino-acid sequence obtained by DNA sequencing. Two N-acetylglucosamine moieties, linked respectively to the residues Asn77 and Asn84, can be identified in the structure. Residues Tyr70, Tyr110, Glu159 and Arg162 define the active site of luffaculin 1 as an RNA N-glycosidase. Cucurmosin is a novel type 1 ribosome-inactivating protein isolated from the sarcocarp of Cucurbita moschata (pumpkin). Besides rRNA N-glycosidase activity, cucurmosin also exhibits strong cytotoxicities to three cancer cell lines of both human and murine origins, but low toxicity to normal cells. Plant genomic DNA extracted from the tender leaves was amplified by PCR between primers based on the N-terminal sequence and X-ray sequence of the C-terminal. The complete mature protein sequence was obtained from N-terminal protein sequencing and partial DNA sequencing, confirmed by high resolution crystal structure analysis. The crystal structure of cucurmosin has been determined at 1.04 Å,a resolution which has never been achieved for any RIP. The structure contains two domains: a large N-terminal domain composed of seven α-helices and eight β-strands,and a smaller C-terminal domain consisting of three α-helices and two β-strands. High resolution structural determination established a glycosylation pattern of GlcNAc₂Man₃Xy1. Asn225 was identified as a glycosylation site. Residues Tyr70,Tyr109,Glu158 and Arg161 define the active site of cucurmosin as an RNA N-glycosidase. α-, β- and γ-trichosanthin, isolated from the root tuber of Trichosanthe kirilowii Maxim by two-step cation exchange chromatography, are isoforms of trichosanthin. α-trichosanthin was the major protein corresponding closely to the previously reported trichosanthin (TCS). The exact molecular weight of α-,β- and γ-trichosanthin is 27167,27143 and 27262 Da, respectively, by ESI-MS. β-trichosanthin could be crystallized by the vapor-diffusion method using potassium chloride or polyethylene glycol as a precipitant. X-ray data have been collected to 1.6 and 1.2 Å resolution for the former (form A) and the latter (form B) crystals, respectively, using a synchrotron source. The form A and form B crystals belong to the orthorhombic space group P2₁2₁2₁ and monoclinic space group P2₁, respectively. Final model rebuilding and refinement of β-trichosanthin,two crystal forms are in progress. γ-trichosanthin has been crystallized by the vapor-diffusion method using ammonium sulphate as a precipitant. X-ray data has been collected to 2.2 Å using a synchrotron source and the crystals belong to hexagonal space group P622. The resolution of γ-trichosanthin is in progress. αMβ2 is a member of the integrins which mediate cell adhesion and migraton by binding to extracellular matrix or counter-receptors on other cells via their adhesion site. These proteins consist of two noncovalently associated subunits, an α chain and a β chain. The α chain contains a 200 amino acid insert near the N-terminus, termed the "I-domain" that is key role in substrate recognition. αMβ2 I-domain also contains a metal-ion-dependent adhesion site. A large amount and homogeneous protein is very important to the structural study of αMβ2 I-domain and it's receptor. The DNA sequence could be designed and synthesized again to enhance the yield of recombinant protein. As described in chapter five,codon optimized DNA sequences of αMβ2 I-domain was assembled by PCR from primers designed by DNAWorks. The recombinant protein was expressed in E.coli, purified by a single step affinity chromatography with Ni-NTA to a purity of 90%,and was characterized by peptide mass fingerprinting. Key words: ribosome-inactivating proteins (RIPs),crystal structure, X-ray sequence, DNA sequencing, cytotoxicity

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