糖链及其蛋白质糖基化

基因和蛋白质是生物统一性的重要标志,而糖链则是生物多样性最重要的标志分子。糖基化作为蛋白质翻译后重要的修饰方式,有其重要的生物学意义。本文综述了糖链的结构、功能及其蛋白质糖基化的类型、影响因素、表达系统等相关问题。     

植物糖生物学与糖链植物疫苗

植物糖生物学是研究植物与糖类互作机制、植物体内糖链与糖缀合物结构及生物学功能的科学,具体涉及糖信号、糖蛋白及其糖链功能、糖基转移酶及植物凝集素等研究方向。依据相关文献及实际研究经验,简要综述植物糖生物学的最新研究进展,其中重点介绍糖链植物疫苗并阐述其应用情况及作用机制。     

糖蛋白过敏原N-糖链与过敏的相关性研究

糖蛋白是一种含有寡糖链的蛋白质,糖链与蛋白质之间以共价键相连。N-糖蛋白为常见过敏原之一,主要来源于食物、吸入物、昆虫毒素等,能够引起过敏反应。N-糖蛋白过敏原的N-糖链结构影响过敏原与IgE的结合,影响抗原提呈细胞（APC）对过敏原的识别和提呈。本文在介绍与过敏相关的N-糖蛋白、常见N-糖蛋白过敏原的N-糖链结构及与过敏相关的糖基化酶的基础上,进一步分析过敏原N-糖链影响过敏的机制,为临床预防与治疗过敏性疾病提供新的思路。     

miR-10b对MCF10A细胞表达N-糖链 及O-糖链的影响

蛋白质糖基化是蛋白质翻译后修饰之一,对蛋白质功能有重要的调节作用,而异常糖基化在肿瘤的发生、发展以及癌细胞转移过程中起到关键作用.MiRNAs在癌症的发生发展过程中同样起到非常关键的作用,但其如何影响糖基化进而在肿瘤恶性转化过程中发挥生物学功能的研究甚少.本文将miR-10b在人正常乳腺上皮细胞MCF10A中过表达,利用糖类相关基因芯片系统筛选了发生显著变化的糖基转移酶;随后利用本实验室建立的N-糖链及O-糖链测定方法,分析糖链水平的表达差异;最后对关键糖基转移酶基因Fut8、MGAT3及OGT通过荧光定量PCR、蛋白质免疫印迹和凝集素免疫印迹进行了验证,为研究miR-10b在乳腺癌中的作用提供更多糖组学方面的理论基础.     

N-乙酰氨基葡萄糖苷内切酶的功能与机制

N-乙酰氨基葡萄糖苷内切酶（endo-beta-N-acetylglucosaminidase,ENGase）广泛分布于各种生物中,主要通过降解错误折叠的糖蛋白,参与细胞和生命的调控。ENGase也是糖链编辑的有效工具酶,可专一性水解游离寡糖链及糖肽或糖蛋白上核心五糖的N-乙酰氨基葡萄糖（GlcNAc）之间的β-1,4糖苷键。其水解产物是寡糖链和一个GlcNAc,或带有一个GlcNAc的糖肽或糖蛋白。本文对ENGase的发现、分布、蛋白质结构、酶学反应及生物学功能进行阐述,为ENGase的生物学研究提供思路,为糖生物学与糖组学的应用研究奠定基础。     

糖生物学

糖生物学是近年来迅速发展的一门新兴前沿分支学科,简单介绍了糖生物学的现状及其发展趋势和展望。     

免疫球蛋白糖链结构异常和自身免疫性疾病

免疫球蛋白在人体体液免疫中发挥巨大作用,而其均为糖蛋白.免疫球蛋白中与蛋白质相连的寡糖链结构及组成对其功能有很大影响,当寡糖链糖基化异常时,可导致一些自身免疫性疾病.从IgA肾病和类风湿性关节炎的结构基础、分子机制、酶学基础、临床意义等方面对这两类自身免疫性疾病的发病机制与糖基化异常之间的密切相关性予以详细论述,为这两类疾病在临床上建立一种特异、灵敏的血清学检测方法提供了理论基础,开辟了一条新途径.     

糖链的生物质谱分析

糖链是重要的生物信息分子,在许多生理和病理过程中都发挥着独特作用。糖链结构非常复杂,具有微观不均一性,其分析和结构解析一直是糖生物学研究的瓶颈。质谱具有灵敏度高、可获得多种结构信息和适于分析混合物等优点,是糖链定性定量分析的一种理想手段。电喷雾电离质谱和基质辅助激光解析电离质谱两大生物质谱技术已被广泛应用于糖链的相对分子质量指纹谱分析、序列和连接方式测定及相对定量分析。对近年来以质谱为主要分析手段的糖链分析方法研究进展做一综述。     

红曲霉葡萄糖淀粉酶N-连接糖链的初步研究

 在加强了的碱性条件下对红曲霉葡萄糖淀粉酶进行β-消除反应,分离未发生消除的级分。对反应前后的样品进行糖组成及甲基化分析,证实了红曲霉葡萄糖淀粉酶上确有对β-消除有抵抗的含GlcNAc的糖链存在。     

细胞膜表面糖链结构与肿瘤转移的关系

连接在天冬酰胺上的N-糖链,连接在丝氨酸和苏氨酸上的O-糖链,连接在丝氨酸上的糖胺聚糖,连接在脂类物质上的糖脂等糖基化修饰在真核细胞表面普遍存在,并调节了细胞的各种功能,它们不仅参与生命活动中正常的生理生化活动,而且于疾病的发生发展密不可分。肿瘤细胞表面糖基异常化,如糖链在表达水平上的差异以及特殊糖链结构的出现,均与肿瘤细胞的侵袭和转移有密切关系。本综述主要介绍了肿瘤细胞糖基化的改变:一些主要的糖基化结构如β1,6分支和唾液酸的表达的增加会使某些肿瘤细胞的迁移能力增强。同时对引起这种变化的作用机制进行了介绍,提出一些潜在的抗肿瘤研究靶点,为肿瘤糖生物学的深入研究提供参考。     

Mass spectrometric approach for screening modifications of total serum N-glycome in human diseases: application to cirrhosis

Congenital and acquired modifications of glycosylation in diseases are a rapidly growing field that demonstrates the importance of glycosylation in human biology. Unfortunately, in clinical biochemistry, very few tests are available to explore oligosaccharide metabolism on a large scale. Such an assay needs to be of high throughput, rapid, and preferentially noninvasive. In the present study, we describe a method to analyze qualitative variations of N-glycosylation of human serum proteins. The method is based on direct release of N-linked oligosaccharides from patient serum samples, a single-step purification, and a matrix-assisted laser desorption ionization time of flight mass spectrometric analysis. A complementary structural study of the released oligosaccharides was achieved by enzymatic digestions, linkage analysis, and electrospray ionization ion trap mass spectrometry (ESI-IT-MS) of the permethylated N-glycome. A total of 26 oligosaccharide structures were individualized, their presence in human serum being the result of the combination of the biosynthesis and catabolic pathways. Application of the protocol to the serum of patients with cirrhosis demonstrates the ability of this assay to identify acquired modifications of glycosylation. Furthermore, we have analyzed the N-glycans and showed the increase in bisecting N-acetylglucosamine residue, core fucosylation, and the presence of an important population of neutral oligosaccharides. The study of total serum N-glycome modifications is a preliminary for the discovery of new noninvasive diagnostic or prognostic biomarkers resulting from the variations of the N-glycan metabolism during diseases.     

N-glycosylation at one rabies virus glycoprotein sequon influences N-glycan processing at a distant sequon on the same molecule

Rabies glycoprotein (RGP(WT)) contains N-glycosylation sequons at Asn(37), Asn(247), and Asn(319), although Asn(37) is not efficiently glycosylated. To examine N-glycan processing at Asn(247) and Asn(319), full-length glycosylation mutants, RGP(-2-) and RGP(--3), were expressed, and Endo H sensitivity was compared. When the Asn(247) sequon is present alone in RGP(-2-), 90% of its N-glycans are high-mannose type, whereas only 35% of the N-glycans at Asn(319) in RGP(--3) are high-mannose. When both sequons are present in RGP(-23), 87% of the N-glycans are of complex type. The differing patterns of Endo H sensitivity at sequons present individually or together suggests that glycosylation of one sequon affects glycosylation at another, distant sequon. To explore this further, we constructed soluble forms of RGP: RGP(WT)T441His and RGP(--3)T441His. Tryptic glycopeptides from these purified secreted proteins were isolated by HPLC and characterized by a 3D oligosaccharide mapping technique. RGP(WT)T441His had fucosylated, bi- and triantennary complex type glycans at Asn(247) and Asn(319). However, Asn(247) had half as many neutral glycans, more monosialylated glycans, and fewer disialylated glycans when compared with Asn(319). Moreover, when comparing the N-glycans at Asn(319) on RGP(--3)T441His and RGP(WT)T441His, the former had 30% more neutral, 28% more monosialylated, and 33% fewer disialylated glycans. This suggests that the N-glycan at Asn(247) allows additional N-glycan processing to occur at Asn(319), yielding more heavily sialylated bi- and triantennary forms. The mechanism(s) by which glycosylation at one sequon influences N-glycan processing at a distant sequon on the same glycoprotein remains to be determined.     

植物糖生物学研究进展

自1988年糖生物学概念提出以来, 国内外科学家在动物、微生物领域取得了大量的研究成果, 但植物糖生物学的研究进展较慢, 目前少见系统的专著或综述。该文围绕植物正常生长时糖信号、逆境时糖信号、糖蛋白及其糖链、重要糖基转移酶及植物凝集素等植物糖生物学的主要问题, 全面阐述植物糖生物学的各个研究分支, 并介绍各领域的最新研究进展。提出了植物糖生物学的概念, 并将其定义为研究植物与糖类互作机制及植物体内糖(糖链与糖分子)结构及生物学功能的科学。     

植物糖生物学研究进展

自1988年糖生物学概念提出以来,国内外科学家在动物、微生物领域取得了大量的研究成果,但植物糖生物学的研究进展较慢,目前少见系统的专著或综述。该文围绕植物正常生长时糖信号、逆境时糖信号、糖蛋白及其糖链、重要糖基转移酶及植物凝集素等植物糖生物学的主要问题,全面阐述植物糖生物学的各个研究分支,并介绍各领域的最新研究进展。提出了植物糖生物学的概念,并将其定义为研究植物与糖类互作机制及植物体内糖（糖链与糖分子）结构及生物学功能的科学。     

Impact of Fc N-glycan sialylation on IgG structure

ABSTRACT

Human IgG antibodies containing terminal alpha 2,6-linked sialic acid on their Fc N-glycans have been shown to reduce antibody-dependent cell-mediated cytotoxicity and possess anti-inflammatory properties. Although terminal sialylation on complex N-glycans can happen via either an alpha 2,3-linkage or an alpha 2,6-linkage, sialic acids on human serum IgG Fc are almost exclusively alpha 2,6-linked. Recombinant IgGs expressed in Chinese hamster ovary (CHO) cells, however, have sialic acids through alpha 2,3-linkages because of the lack of the alpha 2,6-sialyltransferase gene. The impact of different sialylation linkages to the structure of IgG has not been determined. In this work, we investigated the impact of different types of sialylation to the conformational stability of IgG through hydrogen/deuterium exchange (HDX) and limited proteolysis experiments. When human-derived and CHO-expressed IgG1 were analyzed by HDX, sialic acid-containing glycans were found to destabilize the CH2 domain in CHO-expressed IgG, but not human-derived IgG. When structural isomers of sialylated glycans were chromatographically resolved and identified in the limited proteolysis experiment, we found that only alpha 2,3-linked sialic acid on the 6-arm (the major sialylated glycans in CHO-expressed IgG1) destabilizes the CH2 domain, presumably because of the steric effect that decreases the glycan-CH2 domain interaction. The alpha 2,6-linked sialic acid on the 3-arm (the major sialylated glycan in human-derived IgG), and the alpha 2,3-linked sialic acid on the 3-arm, do not have this destabilizing effect.     

A Trypanosoma brucei β3 glycosyltransferase superfamily gene encodes a β1-6 GlcNAc-transferase mediating N-glycan and GPI anchor modification

The parasite Trypanosoma brucei exists in both a bloodstream form (BSF) and a procyclic form (PCF), which exhibit large carbohydrate extensions on the N-linked glycans and glycosylphosphatidylinositol (GPI) anchors, respectively. The parasite''s glycoconjugate repertoire suggests at least 38 glycosyltransferase (GT) activities, 16 of which are currently uncharacterized. Here, we probe the function(s) of the uncharacterized GT67 glycosyltransferase family and a β3 glycosyltransferase (β3GT) superfamily gene, TbGT10. A BSF-null mutant, created by applying the diCre/loxP method in T. brucei for the first time, showed a fitness cost but was viable in vitro and in vivo and could differentiate into the PCF, demonstrating nonessentiality of TbGT10. The absence of TbGT10 impaired the elaboration of N-glycans and GPI anchor side chains in BSF and PCF parasites, respectively. Glycosylation defects included reduced BSF glycoprotein binding to the lectin ricin and monoclonal antibodies mAb139 and mAbCB1. The latter bind a carbohydrate epitope present on lysosomal glycoprotein p67 that we show here consists of (-6Galβ1-4GlcNAcβ1-)_≥4 poly-N-acetyllactosamine repeats. Methylation linkage analysis of Pronase-digested glycopeptides isolated from BSF wild-type and TbGT10 null parasites showed a reduction in 6-O-substituted- and 3,6-di-O-substituted-Gal residues. These data define TbGT10 as a UDP-GlcNAc:βGal β1-6 GlcNAc-transferase. The dual role of TbGT10 in BSF N-glycan and PCF GPI-glycan elaboration is notable, and the β1-6 specificity of a β3GT superfamily gene product is unprecedented. The similar activities of trypanosome TbGT10 and higher-eukaryote I-branching enzyme (EC 2.4.1.150), which belong to glycosyltransferase families GT67 and GT14, respectively, in elaborating N-linked glycans, are a novel example of convergent evolution.     

Small scale affinity purification and high sensitivity reversed phase nanoLC-MS N-glycan characterization of mAbs and fusion proteins

N-glycosylation is a complex post-translational modification with potential effects on the efficacy and safety of therapeutic proteins and known influence on the effector function of biopharmaceutical monoclonal antibodies (mAbs). Comprehensive characterization of N-glycosylation is therefore important in biopharmaceutical development. In early development, e.g. during pool or clone selection, however, only minute protein amounts of multiple samples are available for analytics. High sensitivity and high throughput methods are thus needed. An approach based on 96-well plate sample preparation and nanoLC-MS of 2- anthranilic acid or 2-aminobenzoic acid (AA) labeled N-glycans for the characterization of biopharmaceuticals in early development is reported here. With this approach, 192 samples can be processed simultaneously from complex matrices (e.g., cell culture supernatant) to purified 2-AA glycans, which are then analyzed by reversed phase nanoLC-MS. Attomolar sensitivity has been achieved by use of nanoelectrospray ionization, resulting in detailed glycan maps of mAbs and fusion proteins that are exemplarily shown in this work. Reproducibility, robustness and linearity of the approach are demonstrated, making use in a routine manner during pool or clone selection possible. Other potential fields of application, such as glycan biomarker discovery from serum samples, are also presented.     

Small scale affinity purification and high sensitivity reversed phase nanoLC-MS N-glycan characterization of mAbs and fusion proteins

N-glycosylation is a complex post-translational modification with potential effects on the efficacy and safety of therapeutic proteins and known influence on the effector function of biopharmaceutical monoclonal antibodies (mAbs). Comprehensive characterization of N-glycosylation is therefore important in biopharmaceutical development. In early development, e.g. during pool or clone selection, however, only minute protein amounts of multiple samples are available for analytics. High sensitivity and high throughput methods are thus needed. An approach based on 96-well plate sample preparation and nanoLC-MS of 2- anthranilic acid or 2-aminobenzoic acid (AA) labeled N-glycans for the characterization of biopharmaceuticals in early development is reported here. With this approach, 192 samples can be processed simultaneously from complex matrices (e.g., cell culture supernatant) to purified 2-AA glycans, which are then analyzed by reversed phase nanoLC-MS. Attomolar sensitivity has been achieved by use of nanoelectrospray ionization, resulting in detailed glycan maps of mAbs and fusion proteins that are exemplarily shown in this work. Reproducibility, robustness and linearity of the approach are demonstrated, making use in a routine manner during pool or clone selection possible. Other potential fields of application, such as glycan biomarker discovery from serum samples, are also presented.