【作者】 周峻岗；

【导师】 王鹏； 祁庆生；

【作者基本信息】 山东大学 ， 微生物学， 2007， 博士

【摘要】 酿酒酵母（Saccharomyces cerevisiae）具有无毒、容易培养、遗传背景清晰和对外源蛋白有糖基化修饰等优点，是理想的外源蛋白表达宿主。酿酒酵母是第一个完成基因组测序的真核生物，酵母基因组数据库关于酵母基因组的详细注释（annotation）是研究糖基化酶的结构与功能之间的关系，寻找关键的结构域及阐明这些与膜相连的糖基化酶的拓扑学（topography）结构的重要工具。由于定位于酿酒酵母内质网和高尔基体的糖基化酶基因基本已被分离，有目的性的敲除这些酶将促进阐明糖基化与细胞功能的机制。在真核细胞中，分泌蛋白和定位蛋白经常被复杂的寡糖链修饰，糖蛋白中的寡糖在许多细胞识别过程中起着重要作用，如肿瘤转移、细胞粘附、病原体入侵和免疫反应等。另外，糖基化影响蛋白质的生物合成、折叠、抗原性、免疫原性及其在血浆中的半衰期。酿酒酵母蛋白N-糖基化和其他真核生物一样，首先发生在内质网中，N-连接的寡糖转移至新生肽的天冬酰胺上。转运到定位之前，N-糖链还经过高尔基体中进一步修饰加工至成熟的糖链结构。本课题主要研究了酵母高尔基体糖基化过程及N-糖链outer chain在细胞生命过程中的生物学功能。Mnn1p和Och1p是酿酒酵母高尔基体糖基化过程中的起始阶段的两个甘露糖基转移酶，Mnn1p参与N-糖链outer chain形成α1，3-甘露糖，Och1p则在核心糖链Man₈GlcNAc₂上加上一个α1，6-甘露糖，从而引发outer chain的α1，6-backbone的形成和N-糖链的过度甘露糖基化。利用Overlap extension PCR的方法，我们在体外构建了等位基因敲除DNA片段。通过敲除DNA片段上的同源序列与酵母基因组发生重组置换，敲除酵母N-糖基化过程中的甘露糖基转移酶基因MNN1和OCH1，构建了两株蛋白糖基化突变菌株：mnn1突变菌株和mnn1 och1突变菌株。为分析mnn1 och1突变菌株蛋白的糖基化形式，我们采用热柠檬酸抽提的方法，提取了酵母细胞壁的甘露糖蛋白；利用高甘露糖亲和柱concanavalin A-sepharose 4B亲和层析纯化抽提液中的甘露糖蛋白。利用糖酰胺酶PNGase F水解释放糖蛋白中的糖链；采用Shim-pack clc-NH2氨基柱Size-fractionation HPLC分析了2-氨基吡啶衍生后的糖链组份，结果表明，mnn1 och1突变菌株蛋白的糖链为单一组成，该组分的MALDI TOF／MS分子量鉴定为1794.66Da，与Man₈GlcNAc₂-PA的分子量相同。结果说明了MNN1和OCH1基因的敲除阻断了内质网核心糖链（ER core type glycan）在高尔基体中形成高聚合度甘露糖的outer chain，在mnn1 och1突变菌株中蛋白糖基化是单一的核心糖链结构，Man₈GlcNAc₂。糖蛋白不仅是细胞结构的重要组成部分，也是细胞生命活动的主要承担者之一。在mnn1 och1突变菌株中，蛋白糖基化在高尔基体阶段修饰受阻，通过突变菌株细胞形态及生化特征观察，发现高尔基体糖基化缺陷影响了细胞一些正常的活动；比较野生型和mnn1 och1突变菌株的生长曲线，发现突变菌株细胞生长速度明显减慢，菌体密度也不高；通过温度敏感性实验和台盼蓝染色（trypan blue dye staining）考察了高尔基体糖基化突变对细胞活力的影响。结果表明单个敲除基因MNN1并不影响细胞的生长表型，如果同时敲除MNN1和OCH1基因后，突变菌株的生长对温度变得敏感，这种温度敏感性的生长依赖于渗透压稳定剂。在mnn1 och1突变菌株中，细胞还表现一些细胞分裂的缺陷，而且渗透压稳定剂也不能抑制突变菌株细胞分裂缺陷。高尔基体糖基化突变影响了新生细胞壁的形成，导致子细胞不能正常地从母体细胞分离出来，细胞的不完全分裂造成了mnn1 och1突变菌株生长高度聚集，细胞堆积。另外，高尔基体糖基化缺陷也影响到分裂过程中细胞核的迁移，对mnn1 och1突变菌株细胞的细胞核DAPI染色发现一些芽孢没有细胞核。mnn1 och1突变使得N-糖链的完全丧失outer chain，因此减少了N-糖链的甘露糖磷酸化位点，这减弱细胞与细胞之间相同电荷的排斥作用，也破坏细胞表面的水化层，从而影响细胞的粘度，导致mnn1 och1细胞易堆积沉降。细胞凋亡（apoptosis）的生物学意义主要在于清除多余的、有害及衰老细胞，这一机制仍然保留在单细胞生物中。在哺乳动物和酵母中，蛋白整个糖基化过程缺陷，如ER糖基化起始阶段的基因突变或糖基化抑制剂，都诱导细胞的凋亡。本研究结果表明，N-连接糖链的不完整（高尔基体糖基化缺陷）足够引发酵母糖基化诱导的细胞凋亡。在trypan blue staining分析mnn1 och1突变菌细胞活力时发现，37℃培养时细胞大量死亡，显微观察发现部分死亡的细胞呈现与衰老细胞（ageing cell）相似的表征，如细胞表面疏松，皱褶。对mnn1 och1突变菌的凋亡形态学和生物化学特征进一步考察，结果显示，死亡的细胞中呈现细胞染色质浓缩（chromatin condensation），胞核碎化（nuclear fragmentation），磷脂酰丝氨酸外露（phosphatidylserine exposure）在细胞膜的外层，而细胞膜保持完整，这些细胞凋亡表型及生化的特征表明，mnn1 och1突变菌株细胞经历了程序性死亡过程（programme cell death）。活性氧自由基（reactive oxygen species，ROS）是酵母细胞凋亡关键的调控子，利用荧光探针二氢罗丹明123（dihydrorhodamine 123）检测mnn1 och1突变菌株细胞内的ROS水平，结果显示，在28℃和37℃培养的细胞中，ROS都出现不同程度的积累，说明高尔基体糖基化缺陷诱导的细胞凋亡是通过ROS途径进行调控。高尔基体糖基化缺陷可破坏酵母细胞壁的结构完整性，导致大量细胞坏死（necrosis），这一点通过mnn1 och1突变菌株增加了对50μg／ml的刚果红（Congo red）和细胞壁水解酶glucuronidase-lyticase敏感性以及1.0M的山梨醇（sorbitol）提高了细胞存活率中得到证实。人干扰素-β（HuIFN-β）是成纤维细胞受病毒感染或诱生剂诱导产生的细胞因子，广泛应用抗病毒，多发性硬化和肿瘤化疗。HuIFN-β是糖蛋白，在Asn⁸⁰上有一个N-糖基化位点。将HuIFN-β基因导入酿酒酵母分泌型表达载体pYFD18，构建重组载体pYFD18-HuIFN，通过电击转化法，将重组载体分别导入w3031A（wild type）菌株。mnn1突变菌株及mnn1 och1突变菌株，筛选转化子并表达外源蛋白，利用Western blot检测酵母细胞内和发酵液中的HuIFN-β的表达，结果显示，HuIFN-β蛋白在胞内积累，都没有在α-factor信号肽的引导下分泌到发酵液中。而且野生型和mnn1 och1突变菌株的Western blot杂交条带大小相同，由此证明，β-干扰素在酿酒酵母中表达时，由于对酵母细胞的毒性，没有经过分泌途径进入高尔基体进行糖基化修饰而分泌到胞外，从而造成HuIFN-β以α-factor信号肽融合蛋白的形式在细胞内积累。更多还原

【Abstract】 Since Saccharomyces cerevisiae has several advantages, such as safe, easy to cultivate, having glycosylation and a clear biochemical and genetic background, it has been one of the commonly used organisms for heterogenous protein expression. S. cerevisiae is the first sequenced genome eukaryotes, and the gene annotations in yeast genome database provide powerful information for structure-function studies, for identification of essential domains, and for elucidation of topography of membrane-bound glycosylation enzymes. This single-celled organism is also important in understanding cellular and molecular processes in eukaryotes. As some stages of glycosylation are highly conserved among eukaryotes, yeast glycosylation mutants can be used to isolate cDNAs encoding enzymes of these pathways from other species. The availability of genes encoding glycosylation enzymes in the ER and Golgi will be useful to identify their targeting mechanisms to these subcellular compartments. Depending on the proteins, glycans may contribute to their conformation, stability, and appropriate targeting. Furthermore, in multicellular eukaryotes, specific carbohydrate structures are known to participate in biological recognition processes. In eukaryotic cells, secreted and membrane proteins are frequently modified with complex glycan structures. The syntheses of these N-glycans, initiating in the endopiasmic reticulum （ER）, are catalyzed by the enzyme oligosaccharyltransferase complex （OST）, transfering the glycans from the lipid carrier （dolichol） to asparagine residues in the polypeptide chains. After the export of predominantly Man₈GlcNAc₂-containing glycoproteins to the Golgi, the core oligosaccharide may be hypermannosylated with up to 200 mannose residues. This work focused on the Golgi glycosylation pathway and the biofunction of the outer chains.The OCH1 gene encodes al, 6-mannosyltransferase functional in the initiating stage of mannose outer chain addition to the to the ER-form core oligosaccharide. Golgiα1, 3-mannosyltransferase （Mnn1p） is known to be responsible for the addition of the fourth mannose residue on N-linked chains, and it has been postulated to terminally mannosylate the core and outer chains on N-linked glycans as well. To get a mutant deficient in Golgi glycosylation, we deleted the two mannosyltransferase, Mnn1p and Och1p. The null disruptions of MNN1. 0CH1 were carried out basing on an overlap extension PCR strategy. MNNI, OCHI was replaced by the S. cerevisiae URA3, HIS3, respectively. Transformants were confirmed by amplifying and sequencing the recombinant genomic region. Therefore, we generated two glycosylation mutants, mnn1 mutant and mnn1 och1 mutant. To characterize the N-glycosylation in the mnn1 och1 mutant, mannoproteins were obtained by hot citrate buffer extraction after the mnn1 och1 cells were crumbled. The extracted mannoprotein was precipitated by ethanol, and further purified by concanavalin A-sepharose 4B. The N-oligomannose saccharides were released from mannoprotein by PNGase F digestion, and then peptides and detergents were removed by passage through ion exchange columns. For desalting, glycans were applied to porous graphitic-carbon cartridge. 2-aminopyridine pyridylaminated sugars were profiled and purified by size fractionation HPLC with Shim-pack clc-NH2 column, and result showed dominantly a single peak. MALDI TOF/MS analysis of this peak revealed that its molecular weight was 1796.5 Da, which corresponds to the calculated mass of Man₈GlcNAc₂-PA. These results indicated that disruptions of MNN1 and OCH1 eliminated the hypermannosylation of the N-linked glycans, and glycoproteins were glycosylated with a single core type glycan, Man₈GlcNAc₂, in the mnn1 och1 mutant.N-glycosylation pathway involves the synthesis of lipid-linked oligosaccharide precursor and the subsequent processing events in the ER and the Golgi. It functions by modifying proteins with appropriate oligosaccharide structures, thus influencing their properties and bioactivities. N-glycan matures in Golgi apparatus and perturbations in Golgi N-glycosylation correlate with, and may result from, other malfunctions of the Golgi pathway. The mnn1 mutant yeast cells exhibit no observable change compared to the wild type strain at all temperature. The mnn1 och1 double mutant showed a slower growth rate and a thinner cell density. The Golgi glycosylation mutation also affected the cell viability; the mnn1 och1 mutant became temperature-sensitive and trypan blue dye staining showed more than 35% of cells died at nonpermissive temperature for 20h. However, these defects could be rescued in the presence of osmotic stabilizers. Additionally, the mnn1 och1 mutations impaired cell cytokinesis—most of the mother cells sporulated with two or three daughter cells. The double null mutant cells grew extremely clumped together. Even sonication could not disrupt cell clumps efficiently, indicating strong cell-cell interactions. DAPI staining revealed a nuclear migration defect in mnn1 och1 mutant cells; some of the buds were anucleate. The loss of mannosyl-phosphate accepting sites in mnn1 och1 also resulted in a loss of charge repulsion between cell surfaces and impairment of the surface hydration layer, causing cells to aggregate.Apoptosis functions to clear unused or potentially harmful cells remained in the unicellular organism. Here, for the first time, we showed that the accidental cell death and programmed cell death induced with defect in the outer sugar chain by blocking the Golgi N-glycosylation elongation in S. cerevisiae. Microscopic visualization of trypan blue stained mnn1 och1 cells at 37℃showed that the surface of some dead cells displayed a loose and wrinkled appearance, one of the characteristics of ageing cells. To test whether the mnn1 och1 mutation induces apoptosis at nonpermissive temperature, we examined the morphological and biochemical features of apoptosis. Results showed that the mnn1 och1 cells displayed chromatin condensation and nuclear fragmentation. PS was also exposed on the outer surface of the plasma membrane when cells were still metabolically active and able to exclude the vital dye PI. These evidences indicated that the mnn1 och1 mutant underwent a program cell death. The production of reactive oxygen species in the mnn1 och1 mutant was also detected by Dihydrorhodamine 123. As the ROS have been shown to be a regulator of inducing apoptosis in yeast, the program cell death of mnn1 och1 mutant was probably due to the accumulation of ROS. The mnn1 och1 mutant also underwent a necrotic cell death caused by the cell wall defects. The phenomenon was verified by the increase of cell survival in the presence of an osmotic stabilizer, increase of susceptibility to glucuronidase digestion and sensitivity to the Congo red. Therefore, our study provides a new insight into the correlation between glycosylation with the cell death in yeast.Beta Human interferon （HuIFN-β） is a glycoprotein, secreted by fibroblasts in response to viral infection or exposure to double-stranded RNA. It has an antiviral activity, and has also been used in chemotherapy of certain types of tumors and therapy of multiple sclerosis. To study the HuIFN-βexpression in different strains, the human interferon-βgene was inserted in the Hind III clone site of the secretion-expression plasmid YFD18, and the recombinant plasmid, pYFD18-HuIFN was constructed. The recombinant plasmid was then transformed into strain W303-1A, mnn1 mutant and mnn1 och1 respectively by electroporation. Western blot was applied to analyse the HuIFN-βexpression. However, results showed the HuIFN-βremained intracellular, and the alpha-factor secretion signal had not led the HuIFN-βto secrete into fermentation liquid. Probably, the expression of the HuIFN-βwas very toxic for S. cerevisiae, which affected the HuIFN-βexport to Golgi modification and secretion pathway. Therefore, the HuIFN-βaccumulated intracellularly as a fusion protein with alpha-factor signal peptide.更多还原