原核生物蛋白O-糖基化的研究进展

自从在原核生物中发现蛋白糖基化之后,越来越多的O-糖基化机制在不同种属的细菌中被发现。本文根据对O-寡糖基转移酶(O-oligosaccharide transferase,OTase)的依赖与否,将原核生物的O-糖基化分为OTase非依赖型和OTase依赖型,并分别对这两种糖基化机制进行了详细阐述。通过对不同的O-糖基化机制的深入了解,为以后更好地利用这些途径来合成工程化的目标糖蛋白奠定基础。     

植物蛋白质O-糖基化修饰的研究进展

糖基化是最主要的蛋白质翻译后修饰方式之一,主要有N-糖基化、O-糖基化和糖基磷脂酰肌醇锚定修饰三种类型。在植物细胞中, O-糖基化修饰广泛发生,它不仅参与蛋白质转录调节、信号转导,还与细胞壁合成等生物学过程紧密相关。在多种O-糖基化修饰类型中, O-N-乙酰氨基葡萄糖(O-GlcNAc)糖基化修饰结构独特、易于检测和表征,因此已经有许多相关技术实现了对其的表征。然而,其他类型O-糖基化修饰蛋白的结构和功能仍有待更全面的研究。该文综述了植物蛋白中不同类型O-糖基化修饰的相关研究进展,总结了植物O-糖基化修饰蛋白检测技术的优缺点,最后展望了这些技术在植物蛋白质O-糖基化修饰研究中的应用前景。     

蛋白分子O-糖基化的研究进展

蛋白分子的氧连接糖基化（O-糖基化）修饰是生物体内必不可少的转录后化学修饰之一,其作用方式类似磷酸化,并且两者之间相互作用,共同调节生物大分子的活性。O-糖基化修饰在生物体的转录、翻译、核运输、细胞骨架的形成以及调节细胞器的功能中发挥着重要的作用。通过影响细胞信号的传导,在细胞吞噬、炎性细胞的迁移以及细胞内大分子物质的循环中也起着重要作用。该文主要通过介绍蛋白分子O-糖基化修饰的基础理论以及。一糖基化修饰作用的几个方面,来简要阐述O-糖基化修饰在生物体内发挥的作用。     

O-糖基化位点预测及糖基化酶催化特点

O-糖链维持所连接蛋白质部分的空间构象。O-糖基化作为生物体内重要的生物过程,其起始步骤具有复杂的高度选择性,迄今为止还未发现固定的模式。人们通过比较已知O-糖基化部位周围的氨基酸序列,推测出O-糖基化位点的一些规律及其酶的催化特性。     

O-糖基化起始糖基转移酶的活性与结构研究

多肽：N-乙酰氨基半乳糖转移酶（ppGalNAc-T） 是催化N-乙酰氨基半乳糖（GalNAc）结合到蛋白质Ser或Thr上的糖基转移酶,是黏蛋白型O-糖基化修饰的起始糖基转移酶。ppGalNAc-T是一个酶家族,表达产物均为Ⅱ型膜蛋白。虽然氨基酸序列高度同源,但各成员具有独特的底物特异性和动力学特征。因此,ppGalNAc-T的底物作用机制是O-糖基化研究领域中的关键课题。近年来,通过利用定点突变及晶体结构解析技术,ppGalNAc-T中与底物相互作用的重要氨基酸残基以及由这些残基所形成的对底物结合起关键作用的空间构象逐渐被揭示,为了解ppGalNAc-T酶家族的底物作用机制及其蛋白结构与催化活性间的关系提供了理论依据。     

糖基化三氮唑白杨素衍生物的合成与抗缺氧活性研究

以叠氮糖、炔丙基溴和白杨素为原料,利用"Click chemistry"将糖基化三氮唑药效基团巧妙的引入白杨素分子结构中得到4种糖基化三氮唑白杨素衍生物,产物结构经1H NMR、IR、ESI-MS和元素分析确认。并通过小鼠常压密闭耐缺氧模型对4个目标化合物的药理活性进行评价,均能不同程度的延长小鼠的存活时间,其中化合物1b和4b的活性优于乙酰唑胺。     

糖基化对Notch信号传递系统的影响

Notch信号分子是多细胞生物发育过程中高度保守的一类十分重要的跨膜信 号受体糖蛋白家族.这一信号途径通过局部细胞间的相互作用而产生对多种不成熟细胞分化 的抑制信号, 精确调控细胞的分化潜能,在细胞发育、增殖、分化中起关键作用,参与造血 、T细胞发育、血管生成等重要生理过程.Notch受体分子上具有多种寡糖链,包括N-聚糖、O-岩藻糖聚糖、O-葡萄糖聚糖等,这些寡糖以及相关糖基转移酶对Notch受体-配体结合以及Notch信号传递功能有重要影响.本文就近年来有关Notch受体糖基化及其对Notch信号传递过程的研究进行综述.     

葡聚糖糖基化处理对绿豆蛋白抗氧化性影响研究

以绿豆分离蛋白（MBPI）和葡聚糖为原料,通过湿热法制备MBPI-Dextran共价复合物。本研究通过测定产物的还原力、DPPH自由基清除能力、超氧阴离子自由基、羟自由基清除能力共同评价糖基化反应对MBPI抗氧化能力的影响。结果表明：MBPI-Dextran共价复合物具有一定的抗氧化能力,特别是90℃条件下反应得到的产物,抗氧化能力较80℃产物显著提升,这与美拉德反应程度有关,较高温度下美拉德反应进程加快,促使更多具有抗氧化能力的物质生成。因此,糖基化改性后的绿豆蛋白在抗氧化能力研究上具有一定的应用价值。     

蛋白质糖基化修饰的研究方法及其应用

蛋白质糖基化是一种重要的翻译后修饰,它参与和调控生物体的许多生命活动。随着蛋白质组技术的不断发展,蛋白质糖基化研究越来越受到广泛的重视。本文介绍了蛋白质糖基化修饰的研究内容与方法,并综述了最近的研究进展。     

棉花纤维蛋白质3种提取及二维电泳方法的比较

高质量的蛋白样品制备是进行二维电泳的先决条件.棉花纤维中含有纤维素、多酚、多糖等严重干扰二维电泳的物质, 增加了蛋白提取和二维电泳的难度.分别采用3种提取植物组织蛋白的方法(水法、酚法和尿素法), 提取棉纤维总蛋白, 进而进行了二维电泳分析.在蛋白产量、蛋白纯度和电泳图谱等方面对3种方法进行了比较, 结果采用酚法提取的样品取得了较好的电泳图谱, 有望成为从棉纤维样品中提取总蛋白的可选方法.     

线虫surface coat proteins提取方法的建立与双向电泳分析

目的:建立一套适用于蛋白质双向电泳体系的线虫surface coat proteins(SCPs)样品制备技术,为今后研究线虫surfacecoat蛋白质组学及线虫病理生理学奠定基础.方法:以秀丽隐杆线虫(Caenorhabditis elegans)为研究材料,对比和分析不同的蛋白提取沉淀方法,进而采用SDS-PAGE电泳技术和双向电泳技术对所提蛋白进行评价.结果:通过35%乙醇结合TCA-丙酮沉淀法获得的质量较好的线虫SCPs,在12%的SDS-PAGE分析中该法提取的蛋白背景浅,蛋白条带多且清晰尖锐,含有丰富的蛋白信息量.通过双向电泳分析,可从提取的蛋白中鉴定出清晰蛋白点400多个.随机选择5个蛋白斑点,进行基质辅助激光解吸电离飞行时间质谱鉴定,鉴定得到高度匹配的已知线虫蛋白质2个.结论:所建立的方法可为今后研究线虫surface coat蛋白质组学及线虫病理生理学提供重要工具.     

小麦地下茎双向电泳技术体系的优化

以东农冬麦1号为材料,对苗期地下茎处的蛋白提取方法、蛋白溶解、上样量、胶条的转移等方面进行试验,结果表明：在蛋白提取方面,TCA/丙酮法（T法）和尿素/硫脲法（N法）相比T法能减少低丰度蛋白的损失得到蛋白点数更多的图谱;在蛋白溶解方面,经过两次水化液溶解的蛋白纯度较高,在等电聚焦时能保持8000伏较高电压;上样量方面,10mg粗蛋白溶于两次水化液能得到清晰、分离效果好、蛋白点数较多的图像;胶条转移方面,先向胶面中加入400μl 0.3％普通琼脂糖溶液后,用200μl的电极缓冲液冲洗胶条的支撑膜会使胶条顺利转移到第二向胶面上且胶条与胶面间不会产生气泡。     

缺血后处理兔心肌蛋白质组学双向凝胶电泳分析的初步研究

目的:研究缺血后处理对缺血再灌注心肌保护的相关蛋白的变化。方法:将6只新西兰大白兔随机分为两组(每组3只):心肌缺血再灌注对照组(I/R组)和缺血后处理组(P组)。两组均接受左冠状动脉前降支阻断30min,开放再灌注180min。缺血后处理组,结扎LAD30min,然后灌注30s,阻断30s,重复4次,继而再灌注直至180min,分别取各组缺血区心肌进行二维凝胶电泳,利用ImageMaster2D软件分析实验结果。结果:P组和I/R组对比,有11个蛋白表达发生了显著变化,其中表达增强的有7个蛋白,表达降低的有4个蛋白。结论:这些差异表达的蛋白可能在缺血后处理对心肌缺血再灌注损伤的保护中发挥了作用。     

受白粉菌诱导大麦抗感等基因系蛋白质变化的双向电泳分析

分别对接种与否的大麦抗—感白粉病等基因系—叶期幼苗取材进行蛋白质双向电泳分析。结果表明,病原的侵入使抗—感两系在30Kd以下的低分子量区域的蛋白质发生了明显变化。接种48小时之后,抗病系在pH5.5、6.0、6.8及8.8附近出现了对照中所没有的蛋白质,而在pH6.0和8.8附近的蛋白质则较对照有减小的趋势;感病系在pH6.0附近蛋白质明显增多,在pH8.8处不仅在量上有大幅度提高,而且种类也有增加。结果还表明,抗—感系间在未接种的情况下双向电泳图谱也有差异,接种之后由于感病系在pH8.8处蛋白质的特异性合成,使抗—感两系间的差异缩小。     

建立和优化双向电泳分析柱花草根系蛋白谱的方法

本研究以柱花草根系为材料,比较了不同蛋白质提取方法和蛋白上样量等因素对双向电泳方法分析根系蛋白图谱的影响。结果表明,在苯酚法提取蛋白中,加入0.7mol·L-1NaCl和20%乙醇,并在蛋白沉淀过程中加入1/10倍体积5mol·L-1NaCl,能够有效去除组织样品中的非蛋白成分,结合使用pH4～7范围的IPG胶条,1mg根系蛋白可以在双向电泳图谱上分辨出较多蛋白点,图谱背景清晰,该体系适合柱花草根系蛋白质的双向电泳分析。     

松材线虫全蛋白的提取及其双向电泳体系优化

目的:一种适用于双向电泳体系的松材线虫全蛋白提取方法的建立及其双向电泳体系的优化.方法:以松材线虫为实验材料,比较2种不同的蛋白提取方法,并对双向电泳中的IPG胶条长度、IPG胶条最适pH范围、上样量等3个方面的条件进行优化.结果:采用TCA-丙酮法提取的蛋白质浓度较高,达到2.18μg/μl.使用pH5 ～8、24cm的IPG干胶条,上样量为120μg,经双向电泳分离可得到背景清晰、分辨率较高的2 - DE图谱,能检测到2 000个左右清晰的蛋白点,含有相对丰富的蛋白信息量.结论:该实验所建立的松材线虫提取方法和优化体系可以为今后松材线虫蛋白质组学的研究奠定技术基础.     

小麦苗期地下茎蛋白质双向电泳技术体系的优化

以东农冬麦1号为材料,对苗期地下茎处的蛋白提取方法、蛋白溶解、上样量、胶条的转移等方面进行了试验.结果表明:在蛋白提取方面,TCA/丙酮法(T法)和尿素/硫脲法(N法)相比T法能减少低丰度蛋白的损失得到蛋白点数更多的图谱.在蛋白溶解方面,经过两次水化液溶解的蛋纯度较高,在等电聚焦时能保持8000伏较高电压.上样量方面,10mg粗蛋白溶于两次水化液能得到清晰、分离效果好、蛋白点数较多的图像.胶条转移方面,先向胶面中加入400μl0.3%普通琼脂糖溶液后,用200μl的电极缓冲液冲洗胶条支撑膜会使胶条顺利转移到第二向胶面上且胶条与胶面间不会产生气泡.     

蓝色温和凝胶双向电泳用于分析嗜盐菌质膜蛋白复合物

为了揭示细胞对盐胁迫渗透适应的分子机制,以新鉴定的中度嗜盐芽孢杆菌Bacillussp.I121为实验材料,分析了该嗜盐菌质膜上的盐胁迫响应蛋白.为此,通过蓝色温和凝胶双向电泳(BN/SDS-PAGE)对纯化的质膜组分进行了差异蛋白质组学研究.经MALDI-TOF/TOF质谱分析,鉴定了8个盐胁迫响应蛋白.盐胁迫诱导上调表达的蛋白质包括ABC型转运蛋白、3-磷酸甘油透性酶、嘧啶核苷转运蛋白和甲酸脱氢酶,下调表达的蛋白质包括琥珀酸脱氢酶(succinate dehydrogenase)铁硫亚基、黄素蛋白亚基、细胞色素b556亚基以及分子伴侣DnaJ的同源蛋白;酶活力测定结果表明胁迫条件下上述蛋白质的活性变化与表达量变化相一致.这些蛋白质中绝大多数属于高度疏水的跨膜蛋白,主要负责物质跨膜运输及能量代谢.上述结果表明,中度嗜盐菌Bacillus sp.I121可通过加快跨膜物质运输,同时抑制TCA循环完成盐胁迫条件下相容性溶质脯氨酸和四氢嘧啶的合成与积累.也进一步证明,蓝色温和凝胶双向电泳不仅可用于线粒体、叶绿体中蛋白质复合物的分析,也同样适用于细胞质膜上高度疏水蛋白复合物的比较研究.     

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling^1,2, while LRRK2 is implicated in the pathogenesis of Parkinson''s disease^3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with ³²P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing ³²P-orthophosphate. The ³²P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (³²P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.     

Quantitative Proteomics by Metabolic Labeling of Model Organisms

In the biological sciences, model organisms have been used for many decades and have enabled the gathering of a large proportion of our present day knowledge of basic biological processes and their derailments in disease. Although in many of these studies using model organisms, the focus has primarily been on genetics and genomics approaches, it is important that methods become available to extend this to the relevant protein level. Mass spectrometry-based proteomics is increasingly becoming the standard to comprehensively analyze proteomes. An important transition has been made recently by moving from charting static proteomes to monitoring their dynamics by simultaneously quantifying multiple proteins obtained from differently treated samples. Especially the labeling with stable isotopes has proved an effective means to accurately determine differential expression levels of proteins. Among these, metabolic incorporation of stable isotopes in vivo in whole organisms is one of the favored strategies. In this perspective, we will focus on methodologies to stable isotope label a variety of model organisms in vivo, ranging from relatively simple organisms such as bacteria and yeast to Caenorhabditis elegans, Drosophila, and Arabidopsis up to mammals such as rats and mice. We also summarize how this has opened up ways to investigate biological processes at the protein level in health and disease, revealing conservation and variation across the evolutionary tree of life.Well before the genomics era, the foundation for our current understanding of genetics was largely established by biological research performed using model organisms. Early genetics discoveries such as the chromosome theory of heredity and bacterial conjugation were first described in the fruit fly Drosophila melanogaster (1) and the bacterium Escherichia coli (2), respectively. Apart from these organisms, most of the current knowledge of development, evolution, and genetics originates from other classical model organisms including the bakers'' yeast Saccharomyces cerevisiae, the nematode Caenorhabditis elegans, and the mouse Mus musculus. Nowadays, they hold a primary position in the analysis of biological, disease, and pharmaceutical processes in modern biology and probably claim an even more promising position in future biological research. With increasing numbers of completed genome annotations, however, the focus is also shifting somewhat from these classical model organisms toward organisms that have unique genetic properties, are economically interesting, or are more directly related to human disease such as puffer fish, rice, and Plasmodium, respectively. Consequently, the definition of a model organism has broadened over the past decade, and today model organisms are found in nearly all branches of the “tree of life,” providing extensive means to further investigate conservation or diversification of biological principles through evolution (3). This has gained momentum tremendously by the completion of genome sequencing efforts in hundreds of organisms. In relatively simple organisms (bacteria and yeast), this has allowed the systematic investigation of multiple basic biological processes conserved through evolution (e.g. apoptosis (4) and vacuolar transport (5)). Higher organisms are highly useful for the study of complex traits, which is facilitated by large collections of mutant strains (6, 7). This is of particular relevance where model systems of human physiology, either in a healthy or diseased state, are studied. Fruit flies and C. elegans, the “classical” model organisms, have been used as models for a variety of diseases (8) but also for natural processes like aging (9, 10), sleep (11, 12), and olfaction (13). Mouse and rat models have been a long-standing model for human biology (14), especially in cancer (15). Particularly the availability of strains engineered to represent human diseases has increased our understanding of pathological processes tremendously (16).So far, the focus has primarily been on genetic and genomic aspects of these processes and disorders, but with the maturation of proteomics techniques, ways to study these at the protein level in a meaningful way are coming within reach. Over the last decade, proteomics research has experienced significant advances and has evolved into an indispensable technology to investigate the proteomic composition of biological samples. Proteomics has shifted from the analysis of small sets of proteins toward the comprehensive investigation of a much larger number of proteins expressed in a cell, tissue, or organism (17). Nowadays, a typical proteomics experiment is peptide-centric and starts with the enzymatic digestion of a protein mixture followed by fractionation using one or more chromatographic steps to reduce sample complexity (18, 19) as illustrated in Fig.1. Peptides are fragmented in the mass spectrometer as they elute, and subsequent matching of fragmentation profiles against a protein database leads to peptide and protein identification. When performed at a large scale, this can be used for the identification of thousands of proteins in cells or subcellular structures (20–23). Although such qualitative approaches are fruitful in providing information on proteins present in cells or tissues, they largely ignore the dynamics of protein expression when different conditions are to be investigated. This is highly relevant because in general only the proteins that differ between biological states (e.g. healthy/diseased) are likely to be of primary interest. Because mass spectrometry is not inherently quantitative, it is beneficial to add an internal standard as a reference for the peptide of interest. For large scale experiments, often all proteins or peptides in one sample are modified with a stable isotope-coded mass label. After mixing the labeled sample with an unmodified sample, the intensity ratio between the modified peptide and the unlabeled peptide accurately reflects the change in expression level.Open in a separate windowFig. 1.Qualitative proteomics work flow. Proteins are extracted, digested, and separated by strong cation exchange. Each strong cation exchange fraction is then analyzed by nano-LC-MS/MS. Peptide fragment spectra are used in a database search to identify the peptide sequence and the corresponding protein.Various approaches have been developed for the incorporation of stable isotopes into proteins that can be divided into in vivo and in vitro methods. In the former, isotope-enriched compounds (salts or amino acids) are added to the growth media that can be metabolized by the cell and incorporated into proteins. In vitro labeling can be established using chemical derivatization of proteins or peptides after protein extraction. The choice for either approach depends on the biological system under investigation, but there are a few considerations that should be taken into account because of their impact on the experimental work flow. In