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。     

糖基转移酶和去糖基化酶

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。     

植物激素糖基化修饰研究进展

植物激素对植物的生长发育有重要的调节作用。由于激素的作用依赖于其浓度, 所以植物内源活性激素的水平必须受到严格控制, 而糖基化修饰被认为是调控激素活性水平的重要方式之一。随着植物激素糖基化修饰相关糖基转移酶基因不断被克隆与鉴定, 多种植物激素的糖基化修饰机制和功能作用逐渐被揭示。该文重点介绍了近年来植物生长素、细胞分裂素、脱落酸、油菜素内酯、水杨酸、茉莉酸等植物激素的糖基转移酶活性鉴定与功能研究进展。同时, 对植物激素糖基化修饰领域存在的问题和发展前景进行了讨论。     

人朊蛋白不同N-糖基化修饰突变体的构建及其在哺乳动物细胞中表达

构建并表达人朊蛋白N-糖基化修饰位点突变的真核表达载体,有助于进一步研究朊蛋白N-糖基化修饰的生物学功能。定点突变野生型人朊蛋白基因PRNP,将获得的突变体亚克隆至真核表达载体pcDNA3.1中,并在人宫颈癌细胞株HeLa中瞬时表达各种朊蛋白糖基化修饰位点突变体,利用免疫印迹和糖苷酶消化等糖蛋白分析方法鉴定表达产物的糖基化形式。经Western blot鉴定,野生型和突变型朊蛋白表达产物出现不同形式的泳动特征,分别出现特异性糖基化修饰的多个条带,单糖基化修饰的两条条带和无糖基化修饰的一条条带。经PNGase F糖苷酶消化,野生型和糖基化单点突变型表达产物均能被糖苷酶消化,其分子量下移,去糖基化突变型表达产物的分子条带位置不变。通过突变野生型人朊蛋白基因PRNP的N-糖基化修饰位点,获得单糖基化修饰和去N-糖基化修饰的6种人朊蛋白突变体,并能够在HeLa细胞株中瞬时表达单糖基化修饰和去N-糖基化修饰朊蛋白,为进一步研究朊蛋白的相关功能建立良好基础。     

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。     

糖基化修饰在嗜肺军团菌致病过程中的作用

糖基化修饰是由糖基转移酶催化的广泛存在于生物体内的一种蛋白质翻译后修饰,其与致病菌的毒力密切相关.嗜肺军团菌是一种胞内致病菌,通过其IVB型分泌系统向宿主细胞转运近300种效应蛋白,可介导多种蛋白质翻译后修饰,干扰宿主的细胞进程.其中,Lgt家族、SidI、SetA及LtpM等效应蛋白介导宿主蛋白糖基化修饰,影响宿主蛋...     

老兵又谱新传——新的糖基化方式、新的功能

在自然界中 ,糖类分子无所不在。虽然仍是那么一些单糖 ,但是在新的糖基转移酶作用下 ,以新的糖基化方式出现 ,就产生了新的功能。1.引子传统的概念是糖基转移酶定位在内质网和高尔基体中 ,它们的功能是合成糖苷键 ,在一组糖基转移酶的协同下 ,可以形成糖链。后来在哺乳动物精子的表面发现了半乳糖基转移酶 ,人们认识到糖基转移酶及其相关的糖链还可以参与细胞之间的识别和粘着。此后不仅在精子表面 ,而且在神经细胞表面也发现了一些糖基转移酶[1] 。在细胞质中发现Ｏ ＧｌｃＮＡｃ的糖基化及其对应的糖基转移酶和糖苷水解酶以后 ,极大地拓…     

天然产物的C-糖基化研究进展

自然界中的天然产物在其结构上存在各种修饰,C-糖基化为其中一种比较稀少的修饰方式.C-C糖苷键的形成由C-糖基转移酶负责催化.含C-糖基的化合物多数来自微生物,但高等植物也会有少量积累.综述了近些年来在天然产物C-糖基化方面的研究工作,并对其在药物开发方面的潜力进行了展望.     

植物小分子化合物的糖基化与糖基转移酶

文章介绍近年来植物体内亲脂性小分子化合物的糖基化和与其相关的糖基转移酶研究进展.     

N-糖基化修饰对热休克蛋白gp96免疫学功能的影响

文中旨在以N-糖基化位点突变的重组热休克蛋白gp96为对象,研究N-糖基化修饰对其免疫功能的影响。首先利用昆虫表达系统表达野生型和突变型gp96蛋白,并检测其糖基化水平。进一步通过体外和体内实验,利用流式细胞术和酶联免疫吸附试验 (Enzyme linked immunosorbent assay,ELISA) 检测小鼠CD8+IFN-γ+ T细胞亚群和IFN-γ的分泌,查明糖基化对gp96抗原呈递功能的影响,进一步用ATPase试剂盒检测gp96的ATPase活性。最后通过小鼠免疫实验探究糖基化对gp96疫苗佐剂功能和活化流感疫苗特异性T细胞的影响。结果显示,N-糖基化修饰位点突变后,重组gp96蛋白总含糖量下降了27.8%。与野生型重组蛋白相比,突变gp96的抗原呈递能力减弱,同时ATPase活性明显降低。同时与野生型重组gp96相比,突变gp96佐剂活化流感疫苗特异性T细胞水平也明显减少。这些结果表明,N-糖基化修饰参与调节gp96的ATPase活性和抗原呈递功能,进而影响其疫苗佐剂功能,为开发基于gp96的佐剂疫苗提供了依据。     

Bisecting GlcNAc Is a General Suppressor of Terminal Modification of N-glycan,

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Highlights

• •Loss of bisecting GlcNAc in N-glycan increases various terminal glycan modifications.
• •Glycosyltransferases commonly do not act well on glycans with bisecting GlcNAc.
• •Presence of bisecting GlcNAc alters overall conformation of N-glycan.
• •Bisecting GlcNAc serves as a general suppressor for terminal modification.

     

N-acetylglucosamine drives myelination by triggering oligodendrocyte precursor cell differentiation

Myelination plays an important role in cognitive development and in demyelinating diseases like multiple sclerosis (MS), where failure of remyelination promotes permanent neuro-axonal damage. Modification of cell surface receptors with branched N-glycans coordinates cell growth and differentiation by controlling glycoprotein clustering, signaling, and endocytosis. GlcNAc is a rate-limiting metabolite for N-glycan branching. Here we report that GlcNAc and N-glycan branching trigger oligodendrogenesis from precursor cells by inhibiting platelet-derived growth factor receptor-α cell endocytosis. Supplying oral GlcNAc to lactating mice drives primary myelination in newborn pups via secretion in breast milk, whereas genetically blocking N-glycan branching markedly inhibits primary myelination. In adult mice with toxin (cuprizone)-induced demyelination, oral GlcNAc prevents neuro-axonal damage by driving myelin repair. In MS patients, endogenous serum GlcNAc levels inversely correlated with imaging measures of demyelination and microstructural damage. Our data identify N-glycan branching and GlcNAc as critical regulators of primary myelination and myelin repair and suggest that oral GlcNAc may be neuroprotective in demyelinating diseases like MS.     

Inhibitory and Thermodynamic Studies on the Hydrolysis of Cyclodextrins Catalyzed by Taka-amylase A

The structures of N-glycans of total glycoproteins in royal jelly have been explored to clarify whether antigenic N-glycans occur in the famous health food. The structural feature of N-glycans linked to glycoproteins in royal jelly was first characterized by immunoblotting with an antiserum against plant complex type N-glycan and lectin-blotting with Con A and WGA. For the detail structural analysis of such N-glycans, the pyridylaminated (PA-) N-glycans were prepared from hydrazinolysates of total glycoproteins in royal jelly and each PA-sugar chain was purified by reverse-phase HPLC and size-fractionation HPLC. Each structure of the PA-sugar chains purified was identified by the combination of two-dimensional PA-sugar chain mapping, ESI-MS and MS/MS analyses, sequential exoglycosidase digestions, and 500 MHz ¹H-NMR spectrometry.

The immunoblotting and lectinblotting analyses preliminarily suggested the absence of antigenic N-glycan bearing β1-2 xylosyl and/or α1-3 fucosyl residue(s) and occurrence of β1-4GlcNAc residue in the insect glycoproteins.

The detailed structural analysis of N-glycans of total royal jelly glycoproteins revealed that the antigenic N-glycans do not occur but the typical high mannose-type structure (Man_9~4GlcNAc₂) occupies 71.6% of total N-glycan, biantennary-type structures (GlcNAc₂Man₃GlcNAc₂) 8.4%, and hybrid type structure (GlcNAc₁Man₄GlcNAc₂) 3.0%. Although the complete structures of the remaining 17% N-glycans; C4, (HexNAc₃Hex₃HexNAc₂: 3.0%), D2 (HexNAc₂Hex₅HexNAc₂: 4.5%), and D3 (HexNAc₃Hex₄HexNAc₂: 9.5%) are still obscure so far, ESI-MS analysis, exoglycosidase digestions by two kinds of β-N-acetylglucosaminidase, and WGA blotting suggested that these N-glycans might bear a β1-4 linkage N-acetylglucosaminyl residue.     

Profiling the Bisecting N-acetylglucosamine Modification in Amniotic Membrane via Mass Spectrometry

Bisecting N-acetylglucosamine(GlcNAc), a GlcNAc linked to the core β-mannose residue via a β1,4 linkage, is a special type of N-glycosylation that has been reported to be involved in various biological processes, such as cell adhesion and fetal development. This N-glycan structure is abundant in human trophoblasts, which is postulated to be resistant to natural killer cellmediated cytotoxicity, enabling a mother to nourish a fetus without rejection. In this study, we hypothesized that the human ...     

锌α2糖蛋白生物学功能

锌α2糖蛋白(zinc alpha-2 glycoprotein,ZAG)是一种42 kD可溶性蛋白,广泛存在于人体液中.自从ZAG被发现以来,有许多关于其结构及功能的报道.最初ZAG被认为与生殖及脂代谢有关,后又发现ZAG具有载体蛋白、核糖核酸酶的活性等功能.ZAG也参与免疫控制、细胞黏着、调节黑色素生成等.近年来,多项研究表明,ZAG与促进骨骼肌合成、抑制肿瘤细胞增殖、诊断早期癌症、抗糖尿病有关.本文从ZAG分子结构出发,对ZAG生物学功能的研究进展进行综述.     

蛋白质糖基化分析方法及其在蛋白质组学中的应用

作为一种普遍存在的翻译后修饰,糖基化对蛋白质的结构和功能有着重要影响。弄清糖基化发生发展的规律是理解蛋白质复杂多样的生物功能的一个重要前提。糖基化发生的特点决定了糖基化相关研究是对分析技术的一大挑战。作为蛋白质组学研究的重要组成部分,目前蛋白质糖基化研究的重点和难点主要集中于糖蛋白／糖肽的分离富集和糖蛋白的鉴定／糖基化位点的确定2个方面,相关技术已用于蛋白质组学水平的糖基化研究,但都还不够成熟。以生物质谱为核心、多学科交叉的蛋白质组学技术始终处于不断发展之中。基于糖基化发生规律的富集检测技术的发展、移动质子理论的提出及电子捕获裂解技术的应用必将极大地促进包括糖基化在内的翻译后修饰研究。蛋白质糖基化的研究有助于从基因组-蛋白组-糖组这样一个宏观的综合的水平观察分析生命现象,从而达到对生命现象更本质的认识。     

Biosynthesis of glycoproteins in Candida albicans: Solubilization and partial characterization of dolichol phosphate mannose synthase and protein mannosyl transferases

Incubation of a mixed membrane fraction isolated from C. albicans yeast cells with Nonidet P-40 at a detergent/protein ratio as low of 0.025 (0.016–0.019%, w/v) yielded a soluble fraction that catalyzed the transfer of mannose from GDP-[¹⁴C] Man into dolichol phosphate mannose and from this intermediate into mannoproteins. Over 95% of the sugar in mannoproteins was O-linked as judged from its release after -elimination. Mannose was identified as the sole product after this treatment. Transfer activity did not depend on exogenous lipid acceptor indicating that the latter was solubilized along with the mannosyl transferases. Synthesis of mannolipid and mannoproteins occurred at optima temperatures of 20 °C and 37 °C, respectively, and at a pH in the range of 7.5-9.5. Mannosyl transfer into the mannolipid was stimulated by Mg²⁺and inhibited by Ca²⁺and Mn²⁺whereas mannoprotein labeling was stimulated by Mn²⁺and to a lower extent by Mg²⁺. When measured as a function of substrate concentration, the synthesis of the mannolipid was a nearly linear function of GDP-Man concentration in the range of 5 to 32 M whereas protein mannosylation exhibited hyperbolic kinetics with saturation reached at about 10 M. The solubilized preparation was able to utilize an exogenous source of mannolipid as sugar donor for protein mannosylation. Dinucleotides and, to a higher extent trinucleotides, inhibited mannosyl transfer into the mannolipid and hence into mannoproteins.     

Structural Features of N-Glycans of Seaweed Glycoproteins: Predominant Occurrence of High-Mannose Type N-Glycans in Marine Plants

An ice nucleation-active strain of Erwinia uredovora shed vesicles when cultured at low temperature (l0?C). We isolated an ice nucleation-active vesicle fraction from the culture medium by ultrafiltration, ultracentrifugation, and gel filtration. Western blot analysis showed that this cell-free vesicle fraction contained an ice nucleation-active protein (InaU). The process of the InaU transport to a shed vesicle was examined by immunohistochemical analysis using electron microscopy. The examination showed the following successive processes: InaU molecules first assemble around the inner membrane, then the assembly enters a vesicle justforming on the surface of the outer membrane, and finally the vesicle, 100–400 nm in diameter, leaves the surface to be shed with InaU molecules occluded.