糖蛋白药物表达系统糖基化研究进展

随着基因重组技术的不断发展和蛋白糖基化机制研究的不断深入,糖蛋白药物对人类健康的重要作用越来越受到重视.迄今为止,哺乳动物表达载体仍然是最常见的药用蛋白生产系统.由于其存在成本高、产率低等问题,酵母和细菌表达系统也日益受到重视.受到理论和技术的限制,细菌表达系统的开发仍存在相当大的困难,而酵母表达系统的开发和应用已经取得了一系列突破性的成果.本文综述了国内外近年来改造不同表达系统N-糖基化途径用于生产人源糖蛋白的研究进展.     

真菌N-糖基化系统在异源表达中的研究进展

在近20年来,越来越多的真菌被开发成异源蛋白表达宿主,用来生产各种药用蛋白和酶类。随着对真菌异源表达系统的研究,人们也渐渐意识到真菌N-糖基化系统与高等动物的N-糖基化系统有着明显的区别,这也成为真菌生产高等动物源性糖蛋白的一个技术瓶颈。本文综述了真菌在异源表达糖蛋白工程中其N-糖基化系统的研究进展。包括N-糖基的检测技术和改造策略,并重点介绍了真菌N-糖基化系统与高等动物的N-糖基化系统的差异,以期为日后真菌N-糖基化系统动物源化甚至人源化改造提供参考。     

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。     

酵母表达人源化糖蛋白研究进展

与人体天然复杂型糖蛋白相比,使用酵母生产的药用蛋白带有高甘露糖型N-糖链。这一差异在临床应用中产生了许多不良影响。目前,可以通过消除酵母特有的内源糖基化反应,引入哺乳动物细胞中的一系列糖基转移酶及转运蛋白对酵母糖基化路径进行改造,从而使其表达出人源化的复杂型N-聚糖。本文介绍了酵母N-糖基化特点、糖基化不均一性,综述了近年来利用基因工程改造酵母N-糖基化路径获得特定的人源N-连接糖蛋白以及使用内切糖苷酶生产人源糖蛋白的研究进展,并且对存在的问题及今后的发展前景进行了讨论。     

酵母N-糖基化工程研究进展

酵母表达系统可用来生产具生物活性的重组糖蛋白,但其在N-糖基化过程中会生成高甘露糖型糖链。通过引入相关的甘露糖苷酶和糖基转移酶基因、切断酵母自身的高甘露糖链形成通道能够改变酵母宿主N-糖基化的类型。本对酵母N-糖基化工程的研究状况、最新进展及存在问题作简要阐述。     

CHO细胞表达糖蛋白的研究进展*

中国仓鼠卵巢细胞(Chinese hamster ovary cells,CHO)表达系统因具有较高密度培养、高表达和相对完整的蛋白质糖基化修饰系统等特点,成为生产糖蛋白广泛应用的宿主表达细胞之一。目前已产生不同的CHO细胞系和各种功能细胞株以满足对糖蛋白的大量生产和其他实验需求。近年来,随着基因工程、蛋白质工程、细胞工程和发酵调控等技术的发展应用,由CHO细胞生产糖蛋白的产量和糖基化修饰程度取得了突破。然而,随着生物制品市场对于糖蛋白的需求增加,如何获得大量、均质的糖蛋白也成为急需解决的问题。综述了不同工程CHO表达系统的研究、应用、糖基化修饰系统,以及影响外源糖蛋白在CHO系统表达和糖基化修饰的理化因素,结合文献总结并预测了未来CHO细胞表达系统研究的四个具有重大意义的研究方向,以期在未来可以改善由CHO细胞表达糖蛋白的产量和质量。     

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。     

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。     

酵母O-甘露糖转移酶的研究进展

随着糖蛋白类药物的需求量不断增加,酵母表达系统的人源化改造成为当务之急。其中,酵母蛋白的O-糖基化因O-糖链种类繁多、组分单一以及糖基化位点预测困难等因素,限制了酵母蛋白的O-糖基化人源化改造。从酵母蛋白的O-糖链结构、O-糖链发生过程及O-糖链的功能展开综述,重点介绍起始酵母蛋白O-糖基化过程的O-甘露糖转移酶家族(family of protein O-mannosyltransferases, PMTs)成员的研究现状,希望对酵母蛋白O-糖基化人源化改造研究提供参考。     

α-1,6-甘露糖转移酶基因敲除的毕赤酵母菌株构建及其用于融合蛋白HSA/GM-CSF 表达的研究

酵母对蛋白的糖基化修饰过程不同于哺乳动物,其特点为产生高甘露糖型糖基且易发生过度糖基化。本研究通过两步基因重组敲除目标基因的方法成功敲除了毕赤酵母中的α-1,6-甘露糖转移酶(och1p)基因,获得了och1敲除的菌株。以此为基础,构建了高效表达人血清白蛋白与粒细胞-巨噬细胞集落刺激因子融合蛋白(HSA/GM-CSF)的工程酵母,与野生型毕赤酵母表达的过度糖基化HSA/GM-CSF不同,och1敲除菌表达的该融合蛋白糖基化程度明显降低,这为该融合蛋白的开发提供了重要基础。och1敲除菌株的构建不仅提供了一个对糖蛋白进行低糖基化修饰的毕赤酵母表达系统,而且为进一步的酵母糖基工程改造提供了基础。     

From planta to pharma with glycosylation in the toolbox

Plant-specific glycosylation has long been a major limitation to the extensive use of plant-made pharmaceuticals in human therapy. Our goal here is to highlight the progress recently made towards humanization of N-glycosylation in plants and to illustrate that plant-typical N- and O-glycosylation progressively emerge as additional advantages for using this promising expression system.     

细菌糖基化基本元件及其应用研究进展

近年来,细菌糖基化修饰系统的研究受到了越来越广泛的关注。已有文献报道了诸多细菌糖基化修饰系统,包括最具有代表性的空肠弯曲杆菌的N-糖基化修饰系统以及脑膜炎奈瑟菌的O-糖基修饰系统。本文在已有的研究基础上进行了系统的归纳总结,讨论对细菌蛋白质糖基化系统的理解,同时综述了细菌蛋白质糖基化应用方面的相关进展。     

Synthesis of N- and O-linked glycopeptides in oviduct membrane preparations

A hen oviduct membrane preparation that catalyzes both the N- and O-glycosylation of exogenous acceptor peptides was used to examine the possible involvement of lipid intermediates in enzymatic O-glycosylation. The results indicate that, under a variety of experimental conditions in which the dolichol-linked saccharides involved in N-glycosylation are readily observed, no lipid-linked intermediates for O-glycosylation could be detected. Whereas N-glycosylation is abolished by tunicamycin treatment and stimulated by dolichol phosphate addition, O-glycosylation is unaffected by such treatments. Further, the results of subcellular fractionation of oviduct membranes suggest that N-acetylgalactosaminyl:polypeptide transferase is localized primarily in membranes derived from the smooth endoplasmic reticulum and Golgi apparatus. This is in contrast to the subcellular site of N-glycosylation, which has previously been shown to be primarily the rough endoplasmic reticulum. These findings are discussed in relation to the function of dolichol phosphate in protein glycosylation.     

Structural requirements for additional N-linked carbohydrate on recombinant human erythropoietin

N-Linked glycosylation is a post-translational event whereby carbohydrates are added to secreted proteins at the consensus sequence Asn-Xaa-Ser/Thr, where Xaa is any amino acid except proline. Some consensus sequences in secreted proteins are not glycosylated, indicating that consensus sequences are necessary but not sufficient for glycosylation. In order to understand the structural rules for N-linked glycosylation, we introduced N-linked consensus sequences by site-directed mutagenesis into the polypeptide chain of the recombinant human erythropoietin molecule. Some regions of the polypeptide chain supported N-linked glycosylation more effectively than others. N-Linked glycosylation was inhibited by an adjacent proline suggesting that sequence context of a consensus sequence could affect glycosylation. One N-linked consensus sequence (Asn123-Thr125) introduced into a position close to the existing O-glycosylation site (Ser126) had an additional O-linked carbohydrate chain and not an additional N-linked carbohydrate chain suggesting that structural requirements in this region favored O-glycosylation over N-glycosylation. The presence of a consensus sequence on the protein surface of the folded molecule did not appear to be a prerequisite for oligosaccharide addition. However, it was noted that recombinant human erythropoietin analogs that were hyperglycosylated at sites that were normally buried had altered protein structures. This suggests that carbohydrate addition precedes polypeptide folding.     

Analysis of post-translational modification of mycobacterial proteins using a cassette expression system

A recombinant expression system was developed to analyse sequence determinants involved in O-glycosylation of proteins in mycobacteria. By expressing peptide sequences corresponding to known glycosylation sites within a chimeric lipoprotein construct, amino acids flanking modified threonine residues were found to have an important influence on glycosylation. The expression system was used to screen mycobacterial sequences selected using a neural network (NetOglyc) trained on eukaryotic O-glycoproteins. Evidence of glycosylation was obtained for eight of 11 proteins tested. The results suggest that sites involved in O-glycosylation of mycobacterial and eukaryotic proteins share similar structural features.     

Not just for Eukarya anymore: protein glycosylation in Bacteria and Archaea

Of the many post-translational modifications proteins can undergo, glycosylation is the most prevalent and the most diverse. Today, it is clear that both N-glycosylation and O-glycosylation, once believed to be restricted to eukaryotes, also transpire in Bacteria and Archaea. Indeed, prokaryotic glycoproteins rely on a wider variety of monosaccharide constituents than do those of eukaryotes. In recent years, substantial progress in describing the enzymes involved in bacterial and archaeal glycosylation pathways has been made. It is becoming clear that enhanced knowledge of bacterial glycosylation enzymes may be of therapeutic value, while the demonstrated ability to introduce bacterial glycosylation genes into Escherichia coli represents a major step forward in glyco-engineering. A better understanding of archaeal protein glycosylation provides insight into this post-translational modification across evolution as well as protein processing under extreme conditions. Here, we discuss new structural and biosynthetic findings related to prokaryotic protein glycosylation, until recently a neglected topic.     

Modulation of the CD95-induced apoptosis: the role of CD95 N-glycosylation

Protein modifications of death receptor pathways play a central role in the regulation of apoptosis. It has been demonstrated that O-glycosylation of TRAIL-receptor (R) is essential for sensitivity and resistance towards TRAIL-mediated apoptosis. In this study we ask whether and how glycosylation of CD95 (Fas/APO-1), another death receptor, influences DISC formation and procaspase-8 activation at the CD95 DISC and thereby the onset of apoptosis. We concentrated on N-glycostructure since O-glycosylation of CD95 was not found. We applied different approaches to analyze the role of CD95 N-glycosylation on the signal transduction: in silico modeling of CD95 DISC, generation of CD95 glycosylation mutants (at N136 and N118), modulation of N-glycosylation by deoxymannojirimycin (DMM) and sialidase from Vibrio cholerae (VCN). We demonstrate that N-deglycosylation of CD95 does not block DISC formation and results only in the reduction of the procaspase-8 activation at the DISC. These findings are important for the better understanding of CD95 apoptosis regulation and reveal differences between apoptotic signaling pathways of the TRAIL and CD95 systems.     

Protein O-glycosylation in fungi: diverse structures and multiple functions

Protein glycosylation is essential for eukaryotic cells from yeasts to humans. When compared to N-glycosylation, O-glycosylation is variable in sugar components and the mode of linkages connecting the sugars. In fungi, secretory proteins are commonly mannosylated by protein O-mannosyltransferase (PMT) in the endoplasmic reticulum, and subsequently glycosylated by several glycosyltransferases in the Golgi apparatus to form glycoproteins with diverse O-glycan structures. Protein O-glycosylation has roles in modulating the function of secretory proteins by enhancing the stability and solubility of the proteins, by affording protection from protease degradation, and by acting as a sorting determinant in yeasts. In filamentous fungi, protein O-glycosylation contributes to proper maintenance of fungal morphology, hyphal development, and differentiation. This review describes recent studies of the structure and function of protein O-glycosylation in industrially and medically important fungi.     

Glyco-engineering of biotherapeutic proteins in plants

Many therapeutic glycoproteins have been successfully generated in plants. Plants have advantages regarding practical and economic concerns, and safety of protein production over other existing systems. However, plants are not ideal expression systems for the production of biopharmaceutical proteins, due to the fact that they are incapable of the authentic human N-glycosylation process. The majority of therapeutic proteins are glycoproteins which harbor N-glycans, which are often essential for their stability, folding, and biological activity. Thus, several glyco-engineering strategies have emerged for the tailor-making of N-glycosylation in plants, including glycoprotein subcellular targeting, the inhibition of plant specific glycosyltranferases, or the addition of human specific glycosyltransferases. This article focuses on plant N-glycosylation structure, glycosylation variation in plant cell, plant expression system of glycoproteins, and impact of glycosylation on immunological function. Furthermore, plant glyco-engineering techniques currently being developed to overcome the limitations of plant expression systems in the production of therapeutic glycoproteins will be discussed in this review.     

Systematic mutagenesis of potential glycosylation sites of lysosomal acid lipase

Lysosomal acid lipase (LAL; EC 3.1.1.13) is a key enzyme in the intracellular lipid metabolism. It hydrolyzes exogenous triglycerides and cholesterol esters taken up by various cell types. LAL has six potential N-glycosylation sites and one potential O-glycosylation site. Elimination of each of the six Asn-(X)-Ser/Thr sites by site-directed mutagenesis and expression in baculovirus-infected Spodoptera frugiperda cells resulted in two single-mutant enzymes without lipolytic activities (N134Q and N246Q) and four mutants with preserved activities. The two inactive mutants were not detectable on immunoblot analysis, indicating that they were not secreted. Six double mutants in all possible combinations except for the two inactive single mutants were produced and expressed. Double mutants in combination with the N9 glycosylation site showed reduced activities as compared to the other mutants or the wild-type enzyme. Kinetic data of LAL glycosylation mutants indicate that substrate affinity of N9Q was not changed, but k (cat) of N9 mutants was reduced distinctly compared to the wild-type enzyme. Peanut agglutinin lectin did not recognize LAL, demonstrating that the protein has no core1 structure (Galbeta 1-3 GalNAc) of O-glycosylation. These data indicate that at least two of the six N-glycosylation sites are used in native lipase. N134 and N246 were found to be essential for LAL activity. We conclude that glycosylation plays an important role in the formation of functional LAL.