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1、糖蛋白和糖肽的化学合成,Biosynthesis of N-glycoproteins,Synthesize of a lipid bound oligosaccharide as the precursors for all the various oligosaccharides expressed in N-glycoproteins Transfer precursor oligosaccharide from the lipid-bound form to an asparagine side chain of a polypeptide chain Trimming and pro
2、cessing of the oligosaccharide,N-Glycosylation in Eukaryotes,Step 1.1 Production of GlcNAc-P-P-Dolichol,Marquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79,Glycosylation mutants in Yeast, CHO cells (obtained by plant lectin resistance) and lymphoma cells missing Thy-1 glycoprotein (obtaine
3、d by antibody killing) Were useful in elucidating the pathway,CDG = Congenital Disorder of Glycosylation in Humans,Step 1.2 N-Glycan Precursor on the Cytosolic Leaflet of the Endoplasmic Reticulum (ER),Marquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79,Step 1.3 Biosynthesis of the N-Glyca
4、n Precursor on Lumenal Leaflet of ER,Marquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79,Step 1.4 Completion of Biosynthesis of N-Glycan Precursor on Lumenal Leaflet of ER,Marquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79,Step 2 Transfer Precursor Oligosaccharide to Protein,Oli
5、gosaccharyltransferase complex (OST) in the ER membrane transfers the dolichol N-glycan precursor to asparagine residues on nascently translated proteins,Yeast OST complex contains nine membrane-bound subunits,Target “sequon” for N-glycosylation Necessary but not sufficient Transfer co-translational
6、 before folding 2/3 of proteins have sequons 2/3 sequons actually occupied (some variably),ER,Golgi,Step 3.1 Initial Processing of N-Glycans in the ER and Golgi,Marquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79,Final products often show “microheterogeneity” at each N-Glycosylation site,S
7、tep 3.2 Completion of Processing of N-Glycans in the ER and Golgi,Marquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79,a-N-glycosidic asparagine,a-O-glycosidic serine,Lai-Xi Wang的化学生物学合成方法,Lai-Xi Wang的化学生物学合成方法,Lai-Xi Wang的化学生物学合成方法,寡糖结构分析,概述,自然界的糖：人体中常见的单糖分子,D-葡萄糖 Glc,D-半乳糖 Gal,D-甘露糖 Man,L
8、-岩藻糖 Fuc L-6-脱氧半乳糖,D-GlcNAc,D-GalNAc,D-ManNAc,D-木糖 Xyl,D-核糖,D-脱氧核糖,L-唾液酸 Neu5Ac,D-果糖 Fru,寡糖结构的要素: 各个单糖的化学结构（什么糖，吡喃还是呋喃） 糖苷键的链接顺序 糖苷键的立体构型,寡糖结构分析的技术手段: 质谱分析： 需要样品量少，但自身的结构信息少，谱图分析难度大 最易断裂处为糖苷键，特别是全甲基化修饰的寡糖 HPLC分析： 需要对照糖库分子的HPLC信息辅助 核磁分析： 能给出丰富的结构信息，但需要mg级的样品量和较高纯度 化学以及酶法修饰： 常作为必要的辅助手段，协助分析,寡糖结构分析中常用的
9、酶法和化学法修饰策略：,1）exoglycosidase配合质谱分析，以得到寡糖的结构 2）蛋白的N-glycan，使用PNGase，EndoF或者EndoH处理 3）蛋白的O-glycan，使用还原消除法处理 4）非还原端的荧光集团修饰（2-氨基吡啶等）：提高灵敏度 最常使用的耦合反应：还原氨化反应 5）寡糖酸性水解：唾液酸是最敏感的常见单糖，岩藻糖次之（质谱分析中，类似） 醋酸水溶液：唾液酸 HF水溶液：Fuc 0.5MHCl水溶液：全甲基化的Fuc 6）全甲基修饰 提高HPLC以及MS分析的灵敏度，更利于区分寡糖分析中的同分异构体 对照标准（甲基化）糖库，可提供寡糖中单糖的组分，以及单糖
10、连接顺序信息,Enzymes Useful in detecting Steps in N-glycan Biosynthesis,Complex-type glycans,核磁的结构分析： 核磁信息： H或者C的化学位移 氢谱： 糖环上多数的H，化学位移位于3.5-4.5之间 Fuc的6位甲基，位于1.1左右 唾液酸的3位次甲基（CH2），位于1.8-2.2之间 anomeric位置，氢原子b-H，一般位于4.5-5; a-H一般位于5-5.5之间 酰胺的甲基位于1.9左右 碳谱： 一般糖环上的碳原子一般位于60-80之间 anomeric的C,一般位于100左右， a构型的碳原子化学位移数
11、值常小于b构型 C-H，或者H-H的耦合相关（COSY,HSQC，HMBC） HMBC可以协助判断连接顺序 HSQC可以协助判断6位C和氢原子信号 耦合常数（phase-sensitive的COSY二维谱）（二面角越小，H-H耦合越弱） 多数糖，1,2的氢原子耦合常数为1-4（alpha），或者6-10hz（beta） 半乳糖的4位，和3位以及5位的耦合常数很小很弱 NOE的空间效应（分子量接近2000左右，常使用ROSY谱图） 可以协助判断连接顺序，以及糖苷键构型,J. Am. Chem. Soc., 2008, 130 (44), pp 1442014421,Carbohydrate Ch
12、emistry & Glycobiology,Importance of glycoanalysis,Pharmaceutical Concerns Regarding Carbohydrates Pharmacokinetics: Influence on receptor binding. Pharmacodynamics: Distribution. Clearance. Define product as “well-characterized”. Lot-to-lot variability. Definition of intellectual property Some Glyc
13、oconjugate Disease Associations immunity to infectious diseases, including HIV rheumatoid arthritis (altered composition of IgG and levels of the serum mannose-binding protein) prion diseases congenital disorders of glycosylation (CDGs) (rare, usually resulting in CNS impairment) oral pathologies cy
14、stic fibrosis heart pathologies cancer,A. Dell and H. Morris, Science291, 2001,Carbohydrate Analysis Offers Unique Challenges,Structural analysis of oligosaccharides is more challenging than that of oligonucleotides or proteins Synthesis is not “template driven”. This is because of the complexities
15、introduced by: Branching Linkages Anomericity Site-specific glycosylation. Microheterogeneity High Sensitivity is essential as the quantities of oligosaccharides are generally low (g-sub g),Workflow in glycoanalysis,Effective microisolation of glycoproteins (short LC microcolumns; lectin-based separ
16、ations; gels and blotting techniques) Sample treatment at microscale (microscale enzymatic and chemical cleavages; sample clean-up; possibly derivatization) Separation of glycan mixtures by CZE, CEC, or capillary LC Sensitive MS measurements to determine sequences, linkage forms, branching of glycan
17、s, sites of glycosylation, etc,PREREQUISITES FOR HIGH-SENSITIVITY MEASUREMENTS ON GLYCANS,Glycan analysis,This involves: Composition analysis sugars release for monosaccharide composition analysis Composition analysis by HPAE-PAD Composition analysis by GC-MS Glycan release from glycoprotein for further analysis: Chemical Enzymatic Glycomic analysis MS-based approaches Chromatography-based approaches Electrokinetically-driven approaches,
