糖基转移酶在去糖基化酶

在糖基化工程中,通过酶法对蛋白质进行糖基化和修饰和对天然糖蛋白去糖基化是研究糖蛋白结构与功能的重要手段。本文综述了近年来所纯化的主要的糖基化转移酶和去糖基化酶的性质和应用。     

糖蛋白的去糖基化方法研究进展

糖组学是继基因组学及蛋白质组学后的新兴研究领域,糖基化是非常重要的一种蛋白质翻译后修饰,在多种生物过程中扮演着重要角色,异常糖基化与一些疾病的发生发展密切相关,因此糖基化的研究已成为研究热点。去糖基化后能够影响糖蛋白的活性、分泌等物化性质,而且去糖基化也是糖基化研究的主要技术手段,因此综述了各种去糖基化的方法,各种方法的优缺点和适用范围及去糖基化后的结构检测方法等,为研究去糖基化对大分子物质的构效关系提供参考。     

蛋白质药物糖基化工程化改造研究进展

多种哺乳和非哺乳动物的蛋白质表达系统已成功用于重组糖蛋白药物的生产。糖基化对于生物药品的研究开发至关重要,对生物药品的药效、半衰期及抗原性等产生重要影响。糖基化工程的目的是生产组分明晰、结构均一的N-和O-连接的糖基化蛋白药物。N-糖基化改造的相关研究显示,利用哺乳动物和非哺乳动物表达系统可以表达均匀的N-聚糖重组糖蛋白。与N-糖基化改造相比, O-糖基化的改造研究尚处于起步阶段。首个糖基化工程单克隆抗体已在美国和日本获得上市批准。综述了重组蛋白表达系统的糖基化工程化改造的研究进展,包括蛋白质药物的 N-糖基化改造和O-糖基化改造的最新进展,以期为蛋白质药物的糖基化工程改造研究提供参考。     

N-糖基化位点鉴定方法和非经典N-糖基化序列

蛋白质的N-糖基化修饰是生物体调控蛋白质在组织和细胞中的定位、功能、活性、寿命和多样性的一种普遍的翻译后方式。N-糖基化位点是理解糖链功能的重要前提之一。应用新的糖蛋白、糖肽富集技术和质谱技术,科学家们在不同组织中完成了对N-糖基化位点的鉴定。此外,不同于经典三联子的N-糖基化序列的发现使人们对N-糖基化过程的认识向纵深发展。     

古菌蛋白质修饰研究进展

20世纪50年代中期,在古菌的表层(S-层)首次发现了糖蛋白;21世纪初又在空肠弯曲菌(Campylobacter jejuni)中发现了蛋白质N-糖基化修饰。由此,同行开始认识到,蛋白质的糖基化修饰广泛存在于古菌、细菌及真核生物三域中。近十年来,古菌蛋白质糖基化修饰的研究取得了进展,特别是古菌蛋白质N-糖基化修饰研究进展快速。但对古菌糖蛋白O-糖基化修饰和脂修饰的了解甚少。本文综述了古菌蛋白质糖基化修饰的研究进展。     

人肝癌细胞系的糖蛋白质组学研究

糖基化是最重要的蛋白质翻译后形式之一,糖基化蛋白的糖链部分影响着蛋白质的折叠和稳定性以及其生物学功能.许多恶性肿瘤组织与正常组织相比已显示出蛋白质糖基化的差异.采用蛋白质组学分析方法结合先进的糖蛋白荧光染色技术,研究了正常人肝细胞系(ChangLiver)和人肝癌细胞系(Hep3B)糖蛋白糖基化的差异.首先用细胞裂解法提取细胞总蛋白质,进行双向电泳(2-DE),然后用pro-QEmerald488糖蛋白荧光染料进行糖蛋白染色,得到两种细胞系糖基化蛋白表达谱,经2-DE分析软件Dymension分析2-DE图像,比较糖蛋白的糖基化程度,并对糖基化蛋白进行质谱鉴定.结果显示正常人肝细胞表达(74±2)个(n=3),而人肝癌细胞系表达(78±3)个糖蛋白(n=3).两者匹配的糖蛋白质点31个,Hep3B表达而ChangLiver不表达的糖蛋白质点47个,ChangLiver表达而Hep3B不表达的糖蛋白质点43个.两种细胞系糖基化程度存在明显差异,与正常人肝细胞相比,肝癌细胞发生糖基化改变的糖蛋白有25个,其中糖基化水平上调的有10个,下调的有15个,质谱鉴定出12个发生糖基化改变的糖蛋白.这些结果显示蛋白质糖基化改变可能在肝癌的发生和发展中起一定作用.     

蛋白质糖基化表达体系及应用

糖基化对治疗性蛋白质的溶解度、稳定性、半衰期、活性等具有重要的影响。选择合适的表达载体对蛋白质进行合适的糖基化修饰,可以大大提高治疗效果和降低毒副作用。该文主要介绍糖链对糖蛋白性质的影响,各种糖蛋白表达载体的优势和不足,并简要探讨糖基化工程在生物制药中的应用。     

酵母糖基化改造工程研究进展

酵母真核表达系统是常用的安全性较高的外源蛋白表达系统。酵母细胞内存在翻译后糖基化修饰过程,对其糖基化修饰系统进行改造可用于生产人源糖蛋白。研究表明,可以通过基因工程手段消除酵母特有的内源糖基化反应、引入哺乳动物细胞表达系统中糖基化类型等方法对酵母糖基化路径进行改造。近年来许多研究通过对酵母菌株糖基化位点突变、基因缺失等方法对酵母糖基化系统进行改造,探究糖基化修饰对蛋白质功能的影响,这为利用酵母生产治疗性蛋白和新型糖基化疫苗提供了新的思路。本综述将对近年来酵母糖基化改造成果及研究进展进行综述。     

构建大肠埃希菌底盘细胞合成N-糖基化蛋白的研究进展

N-糖基化是自然界中主要的翻译后修饰之一,对蛋白质结构和功能的影响十分重要。随着糖工程领域的快速发展,在大肠埃希菌(Escherichia coli)中完成治疗性蛋白的N-糖基化修饰变得更加普遍。利用基因编辑技术对大肠埃希菌基因组进行编辑,使大肠埃希菌获得新的性状和生产能力,可以提高目标糖蛋白的产量。本文综述了通过基因编辑技术改造大肠埃希菌基因组来构建大肠埃希菌底盘细胞,及在此基础上优化N-糖基化效率以提高N-糖基化蛋白产量的研究进展,为构建具有N-糖基化修饰功能的工程菌株提供依据,为更好地进行糖蛋白生产,及进一步高效开发“糖蛋白工厂”提供策略。     

粘液糖蛋白去糖基化作用的研究进展

由于粘液糖蛋白中的寡糖侧链对其蛋白质多肽链的水解具有保护作用,为了研究粘液糖蛋白多肽链的结构与功能,必须进行去糖基化作用的处理。处理的方法主要有酶法和化学法。到目前为止,以碘酸三氯甲烷(TFMSA)预处理,再以高碘酸盐氧化,然后在碱性条件下进行β-消去的方法最为理想。     

Effect of glycosylation on the biochemical properties of <Emphasis Type="Italic">β</Emphasis>-xylosidases from <Emphasis Type="Italic">Aspergillus versicolor</Emphasis>

Aspergillus versicolor grown on xylan or xylose produces two β-xylosidases with differences in biochemical properties and degree of glycosylation. We investigated the alterations in the biochemical properties of these β-xylosidases after deglycosylation with Endo-H or PNGase F. After deglycosylation, both enzymes migrated faster in PAGE or SDS-PAGE exhibiting the same R_f. Temperature optimum of xylan-induced and xylose-induced β-xylosidases was 45°C and 40°C, respectively, and 35°C after deglycosylation. The xylan-induced enzyme was more active at acidic pH. After deglycosylation, both enzymes had the same pH optimum of 6.0. Thermal resistance at 55°C showed half-life of 15 min and 9 min for xylose- and xylan-induced enzymes, respectively. After deglycosylation, both enzymes exhibited half-lives of 7.5 min. Native enzymes exhibited different responses to ions, while deglycosylated enzymes exhibited identical responses. Limited proteolysis yielded similar polypeptide profiles for the deglycosylated enzymes, suggesting a common polypeptide core with differential glycosylation apparently responsible for their biochemical and biophysical differences.     

Processing of a class I-restricted epitope from tyrosinase requires peptide N-glycanase and the cooperative action of endoplasmic reticulum aminopeptidase 1 and cytosolic proteases

Although multiple components of the class I MHC processing pathway have been elucidated, the participation of nonproteasomal cytosolic enzymes has been largely unexplored. In this study, we provide evidence for multiple cytosolic mechanisms in the generation of an HLA-A*0201-associated epitope from tyrosinase. This epitope is presented in two isoforms containing either Asn or Asp, depending on the structure of the tyrosinase precursor. We show that deamidation of Asn to Asp is dependent on glycosylation in the endoplasmic reticulum (ER), and subsequent deglycosylation by peptide-N-glycanase in the cytosol. Epitope precursors with N-terminal extensions undergo a similar process. This is linked to an inability of ER aminopeptidase 1 to efficiently remove N-terminal residues, necessitating processing by nonproteasomal peptidases in the cytosol. Our work demonstrates that processing of this tyrosinase epitope involves recycling between the ER and cytosol, and an obligatory interplay between enzymes involved in proteolysis and glycosylation/deglycosylation located in both compartments.     

A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation

Characterization of glycoproteins using mass spectrometry ranges from determination of carbohydrate-protein linkages to the full characterization of all glycan structures attached to each glycosylation site. In a novel approach to identify N-glycosylation sites in complex biological samples, we performed an enrichment of glycosylated peptides through hydrophilic interaction liquid chromatography (HILIC) followed by partial deglycosylation using a combination of endo-beta-N-acetylglucosaminidases (EC 3.2.1.96). After hydrolysis with these enzymes, a single N-acetylglucosamine (GlcNAc) residue remains linked to the asparagine residue. The removal of the major part of the glycan simplifies the MS/MS fragment ion spectra of glycopeptides, while the remaining GlcNAc residue enables unambiguous assignment of the glycosylation site together with the amino acid sequence. We first tested our approach on a mixture of known glycoproteins, and subsequently the method was applied to samples of human plasma obtained by lectin chromatography followed by 1D gel-electrophoresis for determination of 62 glycosylation sites in 37 glycoproteins.     

The effect of individual N-glycans on enzyme activity

In a series of investigations, N-glycosylation has proven to be a key determinant of enzyme secretion, activity, binding affinity and substrate specificity, enabling a protein to fine-tune its activity. In the majority of cases elimination of all putative N-glycosylation sites of an enzyme results in significantly reduced protein secretion levels, while removal of individual N-glycosylation sites often leads to the expression of active enzymes showing markedly reduced catalytic activity, with the decreased activity often commensurate with the number of glycosylation sites available, and the fully deglycosylated enzymes showing only minimal activity relative to their glycosylated counterparts. On the other hand, several cases have also recently emerged where deglycosylation of an enzyme results in significantly increased catalytic activity, binding affinity and altered substrate specificity, highlighting the very unique and diverse roles that individual N-glycans play in regulating enzyme function.     

Automated measurement of site‐specific N‐glycosylation occupancy with SWATH‐MS

Asparagine‐linked glycosylation is a common post‐translational modification of proteins catalyzed by oligosaccharyltransferase that is important in regulating many aspects of protein function. Analysis of protein glycosylation, including glycoproteomic measurement of the site‐specific extent of glycosylation, remains challenging. Here, we developed methods combining enzymatic deglycosylation and protease digestion with SWATH‐MS to enable automated measurement of site‐specific occupancy at many glycosylation sites. Deglycosylation with peptide‐endoglycosidase H, leaving a remnant N‐acetylglucosamine on asparagines previously carrying high‐mannose glycans, followed by trypsin digestion allowed robust automated measurement of occupancy at many sites. Combining deglycosylation with the more general peptide‐N‐glycosidase F enzyme with AspN protease digest allowed robust automated differentiation of nonglycosylated and deglycosylated forms of a given glycosylation site. Ratiometric analysis of deglycosylated peptides and the total intensities of all peptides from the corresponding proteins allowed relative quantification of site‐specific glycosylation occupancy between yeast strains with various isoforms of oligosaccharyltransferase. This approach also allowed robust measurement of glycosylation sites in human salivary glycoproteins. This method for automated relative quantification of site‐specific glycosylation occupancy will be a useful tool for research with model systems and clinical samples.     

细胞外基质金属蛋白酶诱导分子(CD147)在胰腺癌细胞及胰腺星状细胞中的表达(英文)

目的:探讨细胞外基质金属蛋白酶诱导分子(CD147)在胰腺癌细胞(Panc-1)及胰腺星状细胞(PSCs)的表达。方法:应用QRT-PCR,免疫细胞化学和免疫印迹分析方法检测Panc-1和PSCs细胞中EMMRPIN的表达,应用脱糖基化试剂N-glycosidase F及Endoglycosidase H鉴定CD147糖基化形式。结果:CD147在Panc-1和PSCs细胞质膜及细胞质中高表达,通过脱糖基化法首次鉴定出胰腺癌细胞及胰腺星状细胞中CD147不同的糖基化修饰。结论:CD147的糖基化修饰具有细胞特异性,可能与细胞恶性程度相关。     

Production and secretion of a biologically active Closterium sex pheromone by Saccharomyces cerevisiae

The cDNA fragment coding for the Closterium sex pheromone (protoplast-release-inducing protein inducer, PR-IP inducer) was inserted into a Saccharomyces cerevisiae–Escherichia coli shuttle vector, under the control of the glucose-repressible alcohol dehydrogenase (ADH2) promoter of yeast. The yeast cells transformed by this construct produced and secreted recombinant PR-IP inducer into the surrounding culture medium. The release of PR-IP from mt⁺ cells was induced by the recombinant pheromone, whereas amino- and carboxy-terminal truncated forms did not show any biological activity. High levels of asparagine-linked glycosylation in the recombinant pheromone were also confirmed after the treatment by deglycosylation enzymes.     

Differential in vitro and in vivo glycosylation of human erythropoietin expressed in adenovirally transduced mouse mammary epithelial cells

The expression of human erythropoietin in the mammary gland is an attractive approach to diminish its current production cost. Previous attempts to produce erythropoietin in the milk of transgenic animals resulted in very low expression levels and in a detrimental effect in the health of the founder animals. Here, we show that the direct transduction of the mouse mammary gland with an adenoviral vector carrying the cDNA of erythropoietin promotes its expression in milk at a level as high as 3.5 mg/ml. The recombinant erythropoietin derived from mouse milk showed a different migration and distribution after SDS-PAGE electrophoresis as well as a low in vivo hematopoietic activity. Enzymatic deglycosylation showed that these molecular weight disparities are in part due to differential glycosylation compared to with its counterpart produced in CHO and HC11 cell lines. The difference between in vivo and in vitro glycosylation of human erythropoietin expressed in adenovirally transduced mammary epithelial cells suggests that key enzymes in the glycosylation pathway may be insufficient during lactation. Thus, the direct transduction of the mammary epithelium seems to be a powerful tool to express toxic proteins in milk at levels high enough for their physical, chemical and biological characterization before undertaking the generation of a transgenic mammal.     

Two-dimensional electrophoresis of recombinant human erythropoietin: a future method for the European Pharmacopoeia?

Quality assurance of recombinant protein drugs concerning identity and purity represents a difficult task, in particular, when post-translational modifications lead to a heterogeneous mixture of biomolecules. We chose Neorecormon (rh-EPO, Roche) for our studies to demonstrate the efficiency of two-dimensional electrophoresis (2-DE) to analyse post-translationally modified recombinant drugs. More than 40 protein spots in the range from isoelectric point (pI) 3.5-4.5 and 32-45 kDa could be separated. Enzymatic deglycosylation revealed that the heterogeneity of the protein pattern is mainly caused by variations in glycosylation. In comparison to the separately performed isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as requested by the European Pharmacopoeia, we see a great synergy to use 2-DE for the analysis of rh-EPO. A by far higher resolution can be achieved, allowing an improved differentiation of the various rh-EPO glycoforms. Sequential deglycosylation of sialic acids, N-glycosides and the O-glycoside lead to significant shifts both in apparent relative molecular mass and pI. Comparing the 2-DE patterns of rh-EPO before and after deglycosylation allows on the one hand valuable information to be gained on the glycosylation of the recombinant protein and shows on the other hand how significantly the 2-DE protein pattern can be influenced by the glycosylation. As the equipment for the performance of 2-DE has improved significantly over the last decade, we see 2-DE as a reliable method, which should be approved for the routine quality assurance of recombinant drugs and also recommended for the European Pharmacopoeia.     

Glycosylation of purified buffalo heart galectin-1 plays crucial role in maintaining its structural and functional integrity

A buffalo heart galectin-1 purified by gel filtration chromatography revealed the presence of 3.55% carbohydrate content, thus it is the first mammalian heart galectin found to be glycosylated in nature and emphasizes the need to perform deglycosylation studies. Physicochemical comparative analysis between the properties of the native and deglycosylated proteins was carried out to understand the significance of glycosylation. The deglycosylated protein exhibited lesser thermal and pH stability compared to the native galectin. When exposed to thiol blocking reagents, denaturants, and detergents, remarkable differences were observed in the properties of the native and deglycosylated protein. Compared to the native glycosylated protein, the deglycosylated galectin showed enhanced fluorescence quenching when exposed to various agents. CD and FTIR analysis showed that deglycosylation of the purified galectin and its exposure to different chemicals resulted in significant deviations from regular secondary structure of the protein, thus emphasizing the significance of glycosylation for maintaining the active conformation of the protein. The remarkable differences observed in the properties of the native and deglycosylated galectin add an important dimension to the significance of protein glycosylation and its associated biological and clinical relevance.