糖组学研究策略及前沿技术研究进展

糖组学是继基因组学和蛋白质组学后的新兴研究领域,主要研究聚糖结构与功能.通过与蛋白质组数据库结合,糖捕捉法能系统鉴定糖蛋白和糖基化位点.糖微阵列技术可以对生物个体产生的全部蛋白聚糖结构进行鉴定与表征,提高了聚糖分析通量.而化学选择糖印迹技术简化了聚糖纯化步骤并提高了糖基化分析的灵敏度.双消化并串联柱法通过双酶消化双柱分离,在分析聚糖结构的同时也鉴定蛋白质的序列,并与蛋白质组学研究兼容.     

综述: 卵巢癌生物标志物的糖链谱研究进展

糖类抗原125(CA125)被认为是卵巢癌诊断的“金标准”,但在临床应用中普遍存在着特异性不高的问题．肿瘤形成和发展过程中常伴有糖基化修饰异常和糖链结构的改变,不同的肿瘤具有特异的异常糖链结构．近年来,借助凝集素芯片、多重质谱分析等糖蛋白组学和糖组学研究技术,发现不同来源CA125的O-糖链和N-糖链结构存在着明显的微观不均一性,以这些特征性糖链结构为标志物,可以显著提高CA125对卵巢癌的诊断特异性．在过去的10年,研究者们除对CA125糖链结构和糖基化模式做了深入的研究外,还利用糖组的研究方法,直接对来自卵巢癌患者血液、体液(腹水、囊泡液等)中糖蛋白的糖链做了精细的结构解析,结果显示,可有效鉴别卵巢癌患者和健康志愿者的特异性N-糖链结构,有可能成为灵敏度高和特异性好的卵巢癌生物标志物．卵巢癌生物标志物研究发展的总趋势是从传统的对蛋白质的定性和定量研究,逐步转向于对标志物糖基化修饰和特异性糖链结构的鉴定以及定量分析．本文从糖组学的视角,对卵巢癌标志物糖组学的研究现状及发展趋势进行了综述和展望．     

基于超滤膜辅助的糖蛋白全O-连接糖链的富集和MALDI-TOF/TOF质谱结构解析

哺乳动物中约有50%以上的蛋白质都发生了糖基化修饰.连接在丝氨酸或苏氨酸上的O-连接糖链是常见的蛋白质糖基化修饰方式之一,其主要功能是维持与其连接的蛋白质部分的空间构象,保护其免受蛋白酶水解及覆盖某些抗原决定簇.糖链结构的解析有助于更清楚地认识糖蛋白及其功能.本研究建立了一种基于超滤膜辅助(FASP)富集细胞、血清和尿液中糖蛋白全O-连接糖链的方法,根据糖蛋白与其糖链结构之间的分子质量差异,利用10 KD超滤膜富集蛋白质样品中由β消除反应释放的全O-连接糖链,将糖链甲基化修饰后再使用MALDI-TOF/TOF-MS进行解析,同时利用二级质谱进行结构确认.通过上述方法可从标准糖蛋白mucin、细胞、血清和尿液样本中分别鉴定到83、29、33和85种O-连接糖链结构,利用该方法可以从复杂样品中富集和解析糖蛋白全O-连接糖链,实现快速、高效、高通量地解析糖蛋白O-连接糖链的目的.     

蛋白质的糖组学研究进展

蛋白质的糖组学主要研究单个个体所包含的所有糖蛋白上聚糖的结构、功能等生物学作用。糖组学的出现使人类可以更深刻理解第三类生物信息大分子——聚糖在生命活动中的作用。目前,糖组学的发展仍处于初步阶段,基于DNA测序仪的荧光糖电泳、糖芯片等新技术的出现和质谱技术的应用对糖组学研究起到了很大的推动作用。     

糖蛋白质组学的信息学资源和方法

糖基化修饰是生物体内最常见、最重要的蛋白质翻译后修饰之一.哺乳动物体内超过50%的蛋白质都会发生糖基化修饰.糖蛋白广泛分布于各种组织的细胞膜表面,执行着重要的生物学功能.随着高通量、高灵敏度和高分辨率的蛋白质组学时代的来临,许多基于串级质谱技术解析糖链结构的生物数据库和分析软件也亦应运而生.本文综述了目前文献中最常用的糖类生物信息学资源,包括各种糖蛋白的数据库以及质谱解析糖类的相关工具和新技术、新方法.     

基于超滤膜辅助的糖蛋白全N-连接糖链的富集和质谱解析

糖基化作为一种常见的蛋白质翻译后修饰,对蛋白质的空间结构、生物功能等具有重要的影响.解析糖蛋白糖链结构有助于更清楚地认识糖蛋白及其功能.本研究建立了一种基于超滤膜富集血清中糖蛋白全N-连接糖链,并利用质谱技术对糖链结构进行分析的方法.根据糖蛋白及其糖链结构之间的分子质量差异,利用Millipore公司的10 ku超滤膜富集血清糖蛋白上酶解(PNGase F)释放的全N-连接糖链,并使用MALDI-TOF/TOF-MS解析糖链结构.通过该技术可以从血清中富集并鉴定到23种独特的N-连接的糖链结构,并且利用二级质谱进行了结构确认.该方法可以被用于从大量生物样本中富集糖蛋白全N-连接糖链,可以达到快速、高通量地解析糖蛋白N-连接糖链的目的.     

综述: 基于质谱技术的糖链结构解析研究进展

糖组学是继基因组学、蛋白质组学之后,又一门新兴的学科,其主要是研究糖分子的结构与功能．糖是一类比核酸、蛋白质更加独特的生物分子,它们不仅是生物体储存能量和释放能量的主要物质,更是生物体内的信息传递分子,并且在生理和病理过程中扮演着重要的角色,如细胞间的识别作用、炎症以及自身免疫疾病等．在结构上,糖类物质更为复杂,具有宏观不均一性(蛋白质上有多个糖基化位点)和微观不均一性(同一结合位点上可以连接不同的多糖),所以糖链的结构解析一直是糖组学研究的难题．相较于传统的分析方法,质谱法具有高灵敏度、高精度、高通量等优势,被认为是在糖链结构解析过程中重要的分析方法．本文综述了质谱、多级质谱、液相色谱-质谱、毛细管电泳-质谱等方法在糖组学中糖链结构解析的研究进展．     

胃癌相关糖蛋白糖链谱的研究进展

胃癌是一类高发病率和高死亡率的恶性肿瘤.研究表明,癌前感染与胃癌的发生发展过程始终伴随着蛋白糖基化的异常.例如在癌前感染阶段糖蛋白糖链发挥的作用:在感染阶段,幽门螺杆菌(Helicobacter pylori,H.pylori)吸附导致的唾液酸化路易斯X抗原的上调,增强了H.pylori的吸附作用使其在胃部定殖并诱发持续的炎症反应;在慢性胃炎和肠上皮化生阶段唾液酸化的Tn抗原表达上调.胃癌发生发展过程中涉及到了血清、组织、细胞中的蛋白糖基化的改变,如核心岩藻糖基化N-糖链表达的下调,β1,6-连接的N-乙酰葡糖胺分支型N-糖链的增加,以及细胞黏附分子糖基化的改变.本文综述了胃癌相关糖蛋白糖链研究的最新进展,阐述了糖基化在胃癌的发生发展中发挥的重要作用及其作为胃癌早期生物标志物与药物靶点的潜在临床应用价值.     

凝集素芯片技术检测糖蛋白方法的建立及初步应用

建立了凝集素芯片技术检测糖蛋白的方法,对实验条件进行了优化,应用凝集素芯片初步检测分析了Chang?蒺s liver正常肝细胞总蛋白中的糖蛋白糖链构成．将凝集素ConA、GNA固定于环氧化修饰的玻片表面,用Cy3标记标准糖蛋白RNaseB,利用凝集素识别特异糖链的原理建立凝集素芯片检测糖蛋白的方法．摸索出最佳封闭剂是含1% BSA的磷酸缓冲液,最佳孵育时间及温度为3 h和室温,最佳孵育缓冲液为含1% BSA和0.05% Tween-20的磷酸缓冲液,并用甘露糖抑制实验验证了凝集素芯片结合的特异性．用包含10种凝集素的芯片,成功解析了标准糖蛋白RNaseB、Fetuin的糖链构成,证实了凝集素芯片检测糖蛋白糖链的可行性．最后用凝集素芯片初步检测分析了Chang?蒺s liver正常肝细胞总蛋白中的糖蛋白糖链构成,发现 Chang's liver正常肝细胞总蛋白中的糖蛋白可能有多价 Sia或GlcNAc、terminalα-1,3 mannose、GalNAc、Galβ-1,4GlcNAc这些糖链结构的存在．蛋白质糖基化是一种重要的翻译后修饰,它在微生物感染、细胞分化、肿瘤转移、细胞癌变等生命活动中起着重要作用,因此近年来蛋白质的糖基化研究受到广泛的重视,但由于缺乏一种简便、快速、高通量的检测手段,蛋白质糖基化修饰的研究发展缓慢．凝集素芯片技术的出现实现了对糖蛋白的快速、准确、高通量的检测 分析．     

糖蛋白质组定量技术进展

蛋白质的糖基化是最重要和最普遍的蛋白质翻译后修饰之一,在生物体内起着极为重要的作用。糖蛋白质的量和（或）糖基化程度的改变以及糖链结构的改变等与许多疾病密切相关,因此定量糖蛋白质组研究已经成为一个新的热点。然而由于糖基化蛋白质所具有的独特特征,其定量面临严峻的挑战。糖蛋白质组学定量方法和技术的发展将为更好地研究糖基化蛋白质生物学功能起到重要作用。综述了基于生物质谱的糖蛋白质组定量研究的技术和方法,及其优缺点和未来的发展趋势。     

Endoplasmic reticulum-associated degradation of glycoproteins bearing Man5GlcNAc2 and Man9GlcNAc2 species in the MI8-5 CHO cell line.

Endoplasmic reticulum-associated degradation of newly synthesized glycoproteins has been demonstrated previously using various mammalian cell lines. Depending on the cell type, glycoproteins bearing Man9 glycans and glycoproteins bearing Man5 glycans can be efficiently degraded. A wide variety of variables can lead to defective synthesis of lipid-linked oligosaccharides and, therefore, in mammalian cells, species derived from Man9GlcNAc2 or Man5GlcNAc2 are often recovered on newly synthesized glycoproteins. The degradation of glycoproteins bearing these two species has not been studied. We used a Chinese hamster ovary cell line lacking Glc-P-Dol-dependent glucosyltransferase I to generate various proportions of Man5GlcNAc2 and Man9GlcNAc2 on newly synthesized glycoproteins. By studying the structure of the soluble oligomannosides produced by degradation of these glycoproteins, we demonstrated the presence of a higher proportion of soluble oligomannosides originating from truncated glycans, showing that glycoproteins bearing Man5GlcNAc2 glycans are degraded preferentially.     

Structural analysis of <Emphasis Type="Italic">N</Emphasis>-/<Emphasis Type="Italic">O</Emphasis>-glycans assembled on proteins in yeasts

Protein glycosylation, the most universal and diverse post-translational modification, can affect protein secretion, stability, and immunogenicity. The structures of glycans attached to proteins are quite diverse among different organisms and even within yeast species. In yeast, protein glycosylation plays key roles in the quality control of secretory proteins, and particularly in maintaining cell wall integrity. Moreover, in pathogenic yeasts, glycans assembled on cell-surface glycoproteins can mediate their interactions with host cells. Thus, a comprehensive understanding of protein glycosylation in various yeast species and defining glycan structure characteristics can provide useful information for their biotechnological and clinical implications. Yeast-specific glycans are a target for glyco-engineering; implementing human-type glycosylation pathways in yeast can aid the production of recombinant glycoproteins with therapeutic potential. The virulenceassociated glycans of pathogenic yeasts could be exploited as novel targets for antifungal agents. Nowadays, several glycomics techniques facilitate the generation of species-and strain-specific glycome profiles and the delineation of modified glycan structures in mutant and engineered yeast cells. Here, we present the protocols employed in our laboratory to investigate the N-and O-glycan chains released from purified glycoproteins or cell wall mannoproteins in several yeast species.     

Membrane biogenesis in embryonal carcinomas: glycoproteins destined for the cell surface are delayed in a pre-Golgi compartment

Embryonal carcinoma and early embryonic cells assemble a family of unusually large and complex carbohydrates. These glycans are highly branched, repeating copolymers of the sugars galactose and N-acetylglucosamine, referred to as polylactosamines, and are frequently decorated with fucose, sulfate, and sialic acid. We have previously shown that in teratocarcinoma cells these glycans are part of a large spectrum of glycans assembled on mannose cores derived from a common precursor glycan. Metabolic studies revealed a large excess of high-mannose glycans at a time when complex-type glycans cease to accumulate. The present studies demonstrate that these high-Man glycans are not degraded internally or secreted directly but are on glycoproteins destined for the cell surface. These unprocessed glycoproteins replace material lost during the extensive membrane turnover that occurs in these cells. Their export to the cell surface is delayed in a pre-Golgi compartment.     

Lysosomal metabolism of glycoproteins

The lysosomal catabolism of glycoproteins is part of the normal turnover of cellular constituents and the cellular homeostasis of glycosylation. Glycoproteins are delivered to lysosomes for catabolism either by endocytosis from outside the cell or by autophagy within the cell. Once inside the lysosome, glycoproteins are broken down by a combination of proteases and glycosidases, with the characteristic properties of soluble lysosomal hydrolases. The proteases consist of a mixture of endopeptidases and exopeptidases, which act in concert to produce a mixture of amino acids and dipeptides, which are transported across the lysosomal membrane into the cytosol by a combination of diffusion and carrier-mediated transport. Although the glycans of all mature glycoproteins are probably degraded in lysosomes, the breakdown of N-linked glycans has been studied most intensively. The catabolic pathways for high-mannose, hybrid, and complex glycans have been established. They are bidirectional with concurrent sequential removal of monosaccharides from the nonreducing end by exoglycosidases and proteolysis and digestion of the carbohydrate-polypeptide linkage at the reducing end. The process is initiated by the removal of any core and peripheral fucose, which is a prerequisite for the action of the peptide N-glycanase aspartylglucosaminidase, which hydrolyzes the glycan-peptide bond. This enzyme also requires free alpha carboxyl and amino groups on the asparagine residue, implying extensive prior proteolysis. The catabolism of O-linked glycans has not been studied so intensively, but many lysosomal glycosidases appear to act on the same linkages whether they are in N- or O-linked glycans, glycosaminoglycans, or glycolipids. The monosaccharides liberated during the breakdown of N- and O-linked glycans are transported across the lysosomal membrane into the cytosol by a combination of diffusion and carrier-mediated transport. Defects in these pathways lead to lysosomal storage diseases. The structures of some of the oligosaccharides that accumulate in these diseases are not digestion intermediates in the lysosomal catabolic pathways but correspond to intermediates in the biosynthetic pathway for N-linked glycans, suggesting another route of delivery of glycans to the lysosome. Incorrectly folded or glycosylated proteins that are rejected by the quality control mechanism are broken down in the ER and cytoplasm and the end product of the cytosolic degradation of N-glycans is delivered to the lysosomes. This route is enhanced in cells actively secreting glycoproteins or producing increased amounts of aberrant glycoproteins. Thus interaction between the lysosome and proteasome is important for the regulation of the biosynthesis and distribution of N-linked glycoproteins. Another example of the extralysosomal function of lysosomal enzymes is the release of lysosomal proteases into the cytosol to initiate the lysosomal pathway of apoptosis.     

Fermentation Monitoring and Control: A Perspective

Abstract

One of the fastest growing fields in the pharmaceutical industry is the market for therapeutic glycoproteins. Today, these molecules play a major role in the treatment of various diseases, and include several protein classes, i.e., clotting factors, hormones, cytokines, antisera, enzymes, enzyme inhibitors, Ig-Fc-Fusion proteins, and monoclonal antibodies. Optimal glycosylation is critical for therapeutic glycoproteins, as glycans can influence their yield, immunogenicity and efficacy, which impact the costs and success of such treatments. While several mammalian cell expression systems currently used can produce therapeutic glycoproteins that are mostly decorated with human-like glycans, they can differ from human glycans by presenting two structures at the terminal and therefore most exposed position. First, natural human N-glycans are lacking the terminal Gal? 1–3Gal (alpha-Gal) modification;     

Oligosaccharide analysis and molecular modeling of soluble forms of glycoproteins belonging to the Ly-6, scavenger receptor, and immunoglobulin superfamilies expressed in Chinese hamster ovary cells

Most cell surface molecules are glycoproteins consisting of linear arrays of globular domains containing stretches of amino acid sequence with similarities to regions in other proteins. These conserved regions form the basis for the classification of proteins into superfamilies. Recombinant soluble forms of six leukocyte antigens belonging to the Ly-6 (CD59), scavenger receptor (CD5), and immunoglobulin (CD2, CD48, CD4, and Thy-1) superfamilies were expressed in the same Chinese hamster ovary cell line, thus providing an opportunity to examine the extent to which N-linked oligosaccharide processing might vary in a superfamily-, domain-, or protein-dependent manner in a given cell. While we found no evidence for superfamily-specific modifications of the glycans, marked differences were seen in the types of oligosaccharides attached to individual proteins within a given superfamily. The relative importance of local protein surface properties versus the overall tertiary structure of the molecules in directing this protein-specific variation was examined in the context of molecular models. These were constructed using the 3D structures of the proteins, glycan data from this study, and an oligosaccharide structural database. The results indicated that both the overall organization of the domains and the local protein structure can have a large bearing on site-specific glycan modification of cells in stasis. This level of control ensures that the surface of a single cell will display a diverse repertoire of glycans and precludes the presentation of multiple copies of a single oligosaccharide on the cell surface. The glycans invariably shield large regions of the protein surfaces although, for the glycoproteins examined here, these did not hinder the known active sites of the molecules. The models also indicated that sugars are likely to play a role in the packing of the native cell surface glycoproteins and to limit nonspecific protein-protein interactions. In addition, glycans located close to the cell membrane are likely to affect crucially the orientation of the glycoproteins to which they are attached.     

Sialic acids in T cell development and function

Virtually all cell surface proteins and many cell membrane lipids are glycosylated, creating a cell surface glycocalyx. The glycan chains attached to cell surface glycoproteins and glycolipids are complex structures with specific additions that determine functions of the glycans in cell–cell communication and cell sensing of the environment. One type of specific modification of cell surface glycans is decoration of glycan termini by sialic acids. On T cells, these terminal sialic acid residues are involved in almost every aspect of T cell fate and function, from cell maturation, differentiation, and migration to cell survival and cell death. The roles that sialylated glycans play in T cell development and function, including binding to specific sialic acid-binding lectins, are reviewed here.     

Glycan reductive isotope labeling for quantitative glycomics

Many diseases and disorders are characterized by quantitative and/or qualitative changes in complex carbohydrates. Mass spectrometry methods show promise in monitoring and detecting these important biological changes. Here we report a new glycomics method, termed glycan reductive isotope labeling (GRIL), where free glycans are derivatized by reductive amination with the differentially coded stable isotope tags [¹²C₆]aniline and [¹³C₆]aniline. These dual-labeled aniline-tagged glycans can be recovered by reverse-phase chromatography and can be quantified based on ultraviolet (UV) absorbance and relative ion abundances. Unlike previously reported isotopically coded reagents for glycans, GRIL does not contain deuterium, which can be chromatographically resolved. Our method shows no chromatographic resolution of differentially labeled glycans. Mixtures of differentially tagged glycans can be directly compared and quantified using mass spectrometric techniques. We demonstrate the use of GRIL to determine relative differences in glycan amount and composition. We analyze free glycans and glycans enzymatically or chemically released from a variety of standard glycoproteins, as well as human and mouse serum glycoproteins, using this method. This technique allows linear relative quantitation of glycans over a 10-fold concentration range and can accurately quantify sub-picomole levels of released glycans, providing a needed advancement in the field of glycomics.     

Identification of N-glycosylated proteins from the central nervous system of Drosophila melanogaster

Although the function of many glycoproteins in the nervous system of fruit flies is well understood, information about the glycosylation profile and glycan attachment sites for such proteins is scarce. In order to fill this gap and to facilitate the analysis of N-linked glycosylation in the nervous system, we have performed an extensive survey of membrane-associated glycoproteins and their N-glycosylation sites isolated from the adult Drosophila brain. Following subcellular fractionation and trypsin digestion, we used different lectin affinity chromatography steps to isolate N-glycosylated glycopeptides. We identified a total of 205 glycoproteins carrying N-linked glycans and revealed their 307 N-glycan attachment sites. The size of the resulting dataset furthermore allowed the statistical characterization of amino acid distribution around the N-linked glycosylation sites. Glycan profiles were analyzed separately for glycopeptides that were strongly and weakly bound to Concanavalin A (Con A), or that failed to bind Concanavalin A, but did bind to wheat germ agglutinin (WGA). High- or paucimannosidic glycans dominated each of the profiles, although the wheat germ agglutinin-bound glycan population was enriched in more extensively processed structures. A sialylated glycan structure was unambiguously detected in the wheat germ agglutinin-bound fraction. Despite the large amount of starting material, insufficient amount of glycopeptides was retained by the Wisteria floribunda (WFA) and Sambucus nigra columns to allow glycan or glycoprotein identification, providing further evidence that the vast majority of glycoproteins in the adult Drosophila brain carry primarily high-mannose, paucimannose, and hybrid glycans. The obtained results should facilitate future genetic and molecular approaches addressing the role of N-glycosylation in the central nervous system (CNS) of Drosophila.