毕赤酵母N-糖基化改造的研究进展

毕赤酵母表达系统具有诸多优点,广泛用于生产重组蛋白.由于其糖基化途径与人不同,表达的糖蛋白为高甘露糖型,改变了糖蛋白的结构,影响其特性和功能,且具有免疫原性,限制了以毕赤酵母为表达系统大量生产重组蛋白的应用.在过去的20多年里,很多研究集中在毕赤酵母N-糖基化人源化改造研究上,希望产生类人的糖链结构,但进展缓慢.近期,毕赤酵母N-糖基化改造研究已经取得了重大进展,产生了类人的末端为唾液酸的糖链结构,为应用毕赤酵母表达系统大量生产重组蛋白铺平了道路.现对毕赤酵母N-糖基化的研究进展进行简述.     

重组糖蛋白激素类药物的质量控制和评价关注点:糖基化修饰的“宏观”和“微观”异质性

糖蛋白激素包括促卵泡激素、促黄体生成素、绒毛膜促性腺激素和促甲状腺激素,是由非共价结合的α亚基和β亚基组成的异源二聚体。糖基化复杂性和结构不均一性是糖蛋白激素类药物结构表征和质量控制的关注点。糖基化修饰位点、修饰类型、唾液酸含量与糖蛋白激素在机体的受体结合、信号转导、生物功能和清除速率密切相关。本文将梳理重组糖蛋白激素类药物的上市产品概况,解析该类药物糖基化修饰的“宏观”和“微观”复杂性,阐明结构异质性与生物功能的关系,梳理用于结构确证和糖链鉴定的分析技术,结合其质量控制难点提出研发路径和药学研究的相关考虑。     

用微载体培养重组哺乳动物细胞生产人免疫干扰素

真核细胞基因工程技术的应用大大增加了动物细胞培养的用处.哺乳动物细胞培养系统表达的人蛋白质酷似人体内合成的蛋白质,例如,可进行蛋白质的糖基化修饰,而许多重组原核细胞则没有这种转译后修饰功能.本文作者用携带编码人免疫干扰素(IFN-γ)的基因质粒转化中国仓鼠卵巢(CHO)细胞,使其能分泌糖基化的人     

解析重组糖蛋白激素的生物效价与质量控制

本文围绕重组糖蛋白激素的生物效价定义与来源,从糖基化修饰与生物活性之间重要关系为切入点,阐释建立糖基化分析方法和判断指标(如Z值)对于该类产品质量控制的重要性和可行性,对糖基化的一致性、糖谱(Z值)、蛋白含量等理化指标与生物效价的密切关系进行回顾解析,为今后此类生物类似药的研发评价提出意见和建议。     

通过糖基化工程提高治疗蛋白在体内的活性

由于活性低或消除快 ,治疗蛋白需频繁给药。而利用糖基化工程 ,可提高和延长一些蛋白的活性 ,以降低给药的频度。将新 N -连接的糖基化共有序列导入蛋白 ,产生一些糖基化类似物 ,并从中筛选出具额外 N -连接的糖基且有体外活性的物质。单个分子连接合适的共有序列 ,可生成重组人促红细胞生长素 (r Hu EPO )、肥胖蛋白 (leptin)和 Mpl配体的糖基化类似物。这些蛋白分属三种不同类型 ,r Hu EPO是 N -连接的糖蛋白 ,Mpl配体是 O-连接的糖蛋白 ,而 leptin则不含糖基 ,但均可大幅提高体内活性并延长作用时间。糖基化工程 r Hu EPO(darbop…     

N-乙酰葡萄糖胺糖基化修饰与糖尿病、阿尔茨海默病及心脏病的关系

蛋白质的O位N-乙酰葡萄糖胺(O-GlcNAc)糖基化作为一种区别于一般糖基化的翻译后修饰,会使蛋白质的功能发生多种改变。近年的研究发现,蛋白质O-GlcNAc糖基化修饰参与糖尿病、阿尔茨海默病与心脏病等多种疾病病理生理过程,因此对其研究具有积极意义。本文对蛋白质O-GlcNAc糖基化修饰与糖尿病、阿尔茨海默病和心脏病发生的关系作一综述。     

重组蛋白的糖基化及其影响因素

重组蛋白在生物体内的生物活性、免疫原性、体内代谢周期以及拮抗蛋白酶的水解等方面与其糖基化修饰密切相关。此文综述了重组蛋白的糖基化修饰受细胞内外环境多种因素的影响 ,对其生产工艺的改进具有指导意义。     

GM-CSF对红系造血的影响及其临床应用

人粒-巨噬细胞集落刺激因子(GM-CSF)为一分子量14500～35000KD的糖蛋白,其多肽链含127个氨基酸。不同来源的GM-CSF分子量因糖基化程度不同而异,但生物学活性相同。GM-CSF基因与IL-3、TL-4、IL-5基因密切连锁,定位于5号染色体长臂23～31位点,GM-CSF基因已被克隆,顺序已确定,目前已有重组产品生产。GM-CSF由活化T淋巴细胞、大颗粒细胞等多种细胞产生,除粒系与单核细胞系外,对红系及巨核系也有作用。由于重组产物、无血清培养技术、祖细胞纯化及低密度培养的成功,研究有很大进展。本文仅介绍其对红系造血的作用。     

蛋白质组学在口腔疾病研究中的应用

蛋白质组一词最早由澳大利亚学者Wilkins等［1］首次提出,是蛋白质和基因组两词的合并,意指“一种细胞、组织乃至一种生物的基因组所表达的全套蛋白质”,鉴定和分析蛋白质组的科学即为蛋白质组学［2］。蛋白质组学除了分离和识别蛋白质,它还利用蛋白质的表达水平以及翻译后的修饰（如磷酸化、羟基化、糖基化、乙酰化）相互之间的作用分析蛋白质的功能。与基因组不同,蛋白质组是动态的,一方面从整体上揭示生物体或细胞的蛋白质活动,另一方面也从机体或细胞的蛋白质水平发现和研究生命活动的生理和病理现象,更具整体性和可调节性。     

N-糖基化IL-24的表达及体外诱导肿瘤细胞凋亡的研究

目的构建IL-24基因的真核表达载体,在毕赤酵母GS115中高效表达,研究重组N-糖基化IL-24蛋白体外诱导肿瘤细胞凋亡的活性。方法借助过渡质粒α/pUC18,将IL-24基因插入到质粒pPIC9K的BamHⅠ和EcoRⅠ之间,构建重组质粒IL-24/pPIC9K,转化毕赤酵母GS115分泌表达,Tricine-SDS-PAGE和Western blot鉴定目的蛋白,ELISA检测蛋白表达量,糖苷酶PNGaseF分析IL-24糖基化形式和程度。MTT法和形态学分析重组IL-24诱导MCF-7乳腺癌细胞凋亡的活性。结果成功构建重组表达质粒IL-24/pPIC9K,IL-24在毕赤酵母最高表达量为(81.31±14.46)mg·L-1。约70%的IL-24发生了N-糖基化。重组IL-24诱导MCF-7乳腺癌细胞凋亡,对正常人肺成纤维细胞NHLF没有影响。N-糖基化IL-24对MCF-7抑制率约高于去糖基化IL-24。结论毕赤酵母分泌形式的表达和适度的糖基化修饰都有利于目的蛋白IL-24的生物学活性,为后续的研究提供基础。     

Development of fully functional proteins with novel glycosylation via enzymatic glycan trimming

Recombinant glycoproteins present unique challenges to biopharmaceutical development, especially when efficacy is affected by glycosylation. In these cases, optimizing the protein's glycosylation is necessary, but difficult, since the glycan structures cannot be genetically encoded, and glycosylation in nonhuman cell lines can be very different from human glycosylation profiles. We are exploring a potential solution to this problem by designing enzymatic glycan optimization methods to produce proteins with useful glycan compositions. To demonstrate viability of this new approach to generating glycoprotein-based pharmaceuticals, the N-linked glycans of a model glycoprotein, ribonuclease B (RNase B), were modified using an α-mannosidase to produce a new glycoprotein with different glycan structures. The secondary structure of the native and modified glycoproteins was retained, as monitored using circular dichroism. An assay was also developed using an RNA substrate to verify that RNase B had indeed retained its function after being subjected to the necessary glycan modification conditions. This is the first study that verifies both activity and secondary structure of a glycoprotein after enzymatic glycan trimming for use in biopharmaceutical development methods. The evidence of preserved structure and function for a modified glycoprotein indicates that extracellular enzymatic modification methods could be implemented in producing designer glycoproteins. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:2581–2591, 2009     

GlycoFi's technology to control the glycosylation of recombinant therapeutic proteins

Importance of the field: Therapeutic properties of many glycoproteins strongly depend on the composition of their glycans. Most of the current approved glycoproteins are produced in mammalian cell lines, which yield mixture of different glycoforms close to the human one but not fully identical. Glyco-engineering is being developed as a method to control the composition of carbohydrates. Many alternative glycoprotein productions systems are actively investigated including new-engineered yeast strains, as developed by GlycoFi, a biotech company fully owned by Merck & Co. since 2006. Areas covered in this review: The objectives of this opinion paper is to present a comprehensive overview of the technological breakthrough developed by GlycoFi to produce recombinant human proteins with controlled glycosylation patterns in yeast, in comparison to other glyco-engineering technologies and to discuss the application to large-scale manufacturing of biologicals. What the reader will gain: Research papers and recent review articles on protein glycosylation and glyco-engineering, and in-depth search of the bibliography by the GlycoFi's research team, summary of recent meetings discussing the biosimilar topic were analyzed by the authors and will help the reader to gain insight in the field. Take home message: The glyco-engineering technology of the Pichia pastoris N-glycosylation pathway developed by GlycoFi allows producing human proteins with complex N-glycosylation modifications, which are similar to the ones performed in human. Moreover, more homogeneous glycosylation patterns are observed, as opposed to the large heterogeneity of glycan moieties that are found naturally in mammals or in other production systems such as Chinese hamster ovary and NS0 cell lines. These properties associated with the perspective to industrialize the manufacturing process of Pichia makes it a very promising expression system to produce large-scale batches of therapeutics at a lower cost.     

Effect of the alpha-glucosidase inhibitor N-hydroxyethyl-1-deoxynojirimycin (Bay m 1099) on the biosynthesis of liver secretory glycoproteins

The effect of the alpha-glucosidase inhibitor N-hydroxyethyl-1-deoxynojirimycin (Bay m 1099) on the glycosylation and secretion of alpha 1-antitrypsin (three complex type oligosaccharide chains) and of alpha 1-acid glycoprotein (six complex type oligosaccharide chains) was studied in rat hepatocyte primary cultures. In the presence of 4 mM Bay m 1099 the processing of high-mannose to complex type oligosaccharides was partially inhibited leading to the secretion of alpha 1-antitrypsin and alpha 1-acid glycoprotein carrying a mixture of both high-mannose and complex type oligosaccharides. The major part of alpha 1-antitrypsin secreted by Bay m 1099 treated cells still carried two complex type oligosaccharide chains, the majority of alpha 1-acid glycoprotein carried three to five. Despite its effects on protein glycosylation Bay m 1099 did not lead to pronounced changes in the synthesis or secretion of alpha 1-antitrypsin, alpha 1-acid glycoprotein or albumin. At concentrations of Bay m 1099 lower than 0.5 mM no inhibitory effect on oligosaccharide trimming could be observed. After removal of Bay m 1099 from hepatocytes its inhibitory effect on protein glycosylation was immediately reversible.     

GlycoFi's technology to control the glycosylation of recombinant therapeutic proteins

Importance of the field: Therapeutic properties of many glycoproteins strongly depend on the composition of their glycans. Most of the current approved glycoproteins are produced in mammalian cell lines, which yield mixture of different glycoforms close to the human one but not fully identical. Glyco-engineering is being developed as a method to control the composition of carbohydrates. Many alternative glycoprotein productions systems are actively investigated including new-engineered yeast strains, as developed by GlycoFi, a biotech company fully owned by Merck & Co. since 2006.

Areas covered in this review: The objectives of this opinion paper is to present a comprehensive overview of the technological breakthrough developed by GlycoFi to produce recombinant human proteins with controlled glycosylation patterns in yeast, in comparison to other glyco-engineering technologies and to discuss the application to large-scale manufacturing of biologicals.

What the reader will gain: Research papers and recent review articles on protein glycosylation and glyco-engineering, and in-depth search of the bibliography by the GlycoFi's research team, summary of recent meetings discussing the biosimilar topic were analyzed by the authors and will help the reader to gain insight in the field.

Take home message: The glyco-engineering technology of the Pichia pastoris N-glycosylation pathway developed by GlycoFi allows producing human proteins with complex N-glycosylation modifications, which are similar to the ones performed in human. Moreover, more homogeneous glycosylation patterns are observed, as opposed to the large heterogeneity of glycan moieties that are found naturally in mammals or in other production systems such as Chinese hamster ovary and NS0 cell lines. These properties associated with the perspective to industrialize the manufacturing process of Pichia makes it a very promising expression system to produce large-scale batches of therapeutics at a lower cost.     

Altered glycosylation of proteins in cancer: what is the potential for new anti-tumour strategies

It is becoming increasingly apparent that cell surface oligosaccharides play pivotal roles as recognition molecules in a range of cell communication and adhesion processes. Alterations in cellular glycosylation are also associated with diseases, including cancer, and may have functional significance. This paper gives an overview of the complex topic of cellular glycosylation mechanisms and reviews the well-documented alterations in cellular glycosylation of proteins in malignancy. One particular type of cancer-associated glycosylation change, the incomplete synthesis of O-linked glycans, is highlighted, and its possible functional significance in cancer cell metastatic mechanisms is discussed. The significance that cancer-associated changes in glycoprotein glycosylation may have in new approaches to anti-tumour therapies is explored.     

Increasing the sialylation of therapeutic glycoproteins: The potential of the sialic acid biosynthetic pathway

The number of therapeutic proteins has increased dramatically over the past years and most of the therapeutic proteins in the market today are glycoproteins. Usually, recombinant glycoproteins are produced in mammalian cell lines, such as Chinese-hamster-ovary-cells to obtain mammalian-type of glycosylation. The terminal monosaccharide of N-linked complex glycans is typically occupied by sialic acid. Presence of this sialic acid affects absorption, serum half-life, and clearance from the serum, as well as the physical, chemical and immunogenic properties of the respective glycoprotein. From a manufacturing perspective, the degree of sialylation is crucial since sialylation varies the function of the product. In addition, insufficient or inconsistent sialylation is also a major problem for the process consistency. Sialylation of over-expressed glycoproteins in all mammalian cell lines commonly used in biotechnology for the production of therapeutic glycoproteins is incomplete and there is a need for strategies leading to homogenous, naturally sialylated glycoproteins. This review will shortly summarize the biosynthesis of sialic acids and describe some recent strategies to increase or modify sialylation of specific therapeutic glycoproteins. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3499–3508, 2009     

Glycan mapping of glycotherapeutics

Glycosylation of proteins may, or may not, affect biological activity, either directly or indirectly. In addition, variability in glycosylation arises within the manufacturing procedure. Therefore, glycosylation would be an important parameter when assessing product quality. Regulatory authorities require biotechnological and biological products to be characterised, including determination of their biological activity, physicochemical and immunochemical properties, their purity and their impurity profiles. Here we outline how we can decide if and which types of glycan analysis and methodologies to use for glycoprotein therapeutics to answer the scientific questions as well as meeting regulatory requirements that already exist or are going to be developed/implemented.     

长效卵泡刺激素的分子设计与新药开发

卵泡刺激素(FSH)是一种促进女性卵泡发育和成熟、男性精子正常发生的重要糖蛋白激素。常用FSH药物半衰期相对较短,体内代谢和消除迅速,患者一般需要每天1次或2次皮下注射,才能维持FSH血药浓度在卵巢刺激的阈值水平以上。长效FSH的开发有利于增强生物活性和患者的依从性,减缓患者的精神压力和用药不当。目前用于长效FSH分子设计的技术手段包括在FSH亚基末端融合绒毛膜促性腺激素β亚基的羧端肽(CTP),或引进新的糖基化位点序列,或创建由CTP或糖基化连接序列连接而成的FSHβ和α亚基单链融合蛋白,或创建FSH与IgG1的Fc片段的融合蛋白等。基于各种重组FSH分子优良的药动学和药效学特性,体内半衰期长、生物活性高的新型FSH药物一定会在将来临床应用中发挥重要作用。     

Influence of chronic ethanol consumption on toxic effects of 1,2-dichloroethane: glycolipoprotein retention and impairment of dolichol concentration in rat liver microsomes and Golgi apparatus

Our previous investigations demonstrated that 1,2-dichloroethane (DCE) and chronic ethanol treatment separately are able to impair glycoprotein metabolism and secretion, and reduce dolichol concentration in liver membranes. The purpose of this study was to investigate whether chronic ethanol consumption can induce potentiation of rat liver damage due to DCE haloalkane used in several chemical processes and in agriculture. Rats were given 36% of their total energy as ethanol in the Lieber-DeCarli liquid diet for 8 weeks (CH group). The pair-fed control group received an isocaloric amount of dextrine-maltose (PF group). "In vitro" experiments: the DCE (6.5 mM) treatment of isolated hepatocytes from CH rats enhanced glycoprotein retention and further reduced glycoprotein secretion and 14C-glucosamine incorporation compared to the hepatocytes from CH or from PF and DCE treated rats. "In vivo" experiments: a marked decrease of dolichol concentration in microsomes (in which dolichyl phosphate is rate-limiting for the initial glycosylation of protein) and in Golgi membranes (in which total dolichol is very important for membrane permeability, fluidity and vesicle fusion) was observed in CH rats acutely treated with 628 mg/kg bw of DCE (CH+DCE) compared with CH or PF+DCE treated rats. These data suggest that chronic ethanol consumption increases DCE liver toxicity by affecting protein glycosylation processes and impairing glycolipoprotein secretion, with a concomitant retention at the level of the Golgi apparatus.     

Role of Glycosylation in Conformational Stability, Activity, Macromolecular Interaction and Immunogenicity of Recombinant Human Factor VIII

Factor VIII (FVIII) is a multi-domain glycoprotein that is an essential cofactor in the blood coagulation cascade. Its deficiency or dysfunction causes hemophilia A, a bleeding disorder. Replacement using exogenous recombinant human factor VIII (rFVIII) is the first line of therapy for hemophilia A. The role of glycosylation on the activity, stability, protein–lipid interaction, and immunogenicity of FVIII is not known. In order to investigate the role of glycosylation, a deglycosylated form of FVIII was generated by enzymatic cleavage of carbohydrate chains. The biochemical properties of fully glycosylated and completely deglycosylated forms of rFVIII (degly rFVIII) were compared using enzyme-linked immunosorbent assay, size exclusion chromatography, and clotting activity studies. The biological activity of degly FVIII decreased in comparison to the fully glycosylated protein. The ability of degly rFVIII to interact with phosphatidylserine containing membranes was partly impaired. Data suggested that glycosylation significantly influences the stability and the biologically relevant macromolecular interactions of FVIII. The effect of glycosylation on immunogenicity was investigated in a murine model of hemophilia A. Studies showed that deletion of glycosylation did not increase immunogenicity.