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UGA BCMB 8020 - carbo-analysis

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1Principle of Carbohydrate AnalysisBCMB 8020April 9th 2009Lianchun Wang2OutlineI. Class of carbohydrate (types of glycosylation)II. Why study carbohydrateIII. Approaches for carbohydrate analysisIV. Sample paper3I. Species of Carbohydrate Moieties= Glucose (Glc)= Mannose (Man)= Galactose (Gal)= N-acetylglucosamine (GlcNAc)= N-acetylgalactosamine (GalNAc)= N-acetylhexosamine, unspecified (HexNAc)= Fucose (Fuc)= Xylose (Xyl)= Sialic acid, unspecified (Sia)= Glucuronic acid (GlcA)= Iduronic acid (IdoA)= Uronic acid, unspecified (HexA)Hexose,unspecified (Hex)4II. Roles of Glycoprotein-associated Carbohydrates1. Quality Control/folding.(deglycosylation/reglycosylation)- glycosyltransferase2. Solubility-Peroxidase3. Circulating half-life-Lutropin (LH), follicle-stimulating hormone (FSH)4. Cell-cell interactions-lymphocyte homing, cell growth, tumor metastasis5SignificanceFully understanding the biology of glycoconjugatesrequires in-depth knowledge of the carbohydratechains6III. Experimental approaches for carbohydrateanalysisTable 1. Primary structural features of a complex carbohydrate General description Specific examples Qualitative and quantitative composition, i.e., nature and number of constituting monosaccharides including absolute configuration (D- or L-) and ring size (pyranose [p] or furanose [f]) Positions of glycosidic linkages Sequence of monosaccharides, including occurrence of branchpoints (double or triple substitution of a monosaccharide) Nature, number, and location of appended noncarbohydrate groups: phosphate, sulfate, acetate (peptide, lipid) Gal, GlcNAc, Man, Fuc in ratio 2:4:3:1 D-Gal, D-GlcNAc, D-Man, L-Fuc Manp, Galf , Manα(1–6)[Manα(1–3)]Manβ(1–4)GlcNAc IdoA2SO3, Neu5Ac9OAc 7III. Experimental approaches for carbohydrateanalysisTable 2. Secondary structural features of a complex carbohydrate Conformational aspect Example Precise ring conformation of each monosaccharide Orientation of monosaccharides with respect to each other Flexibility of the spatial structure Complete set of H-C-C′-H′ dihedral angles Torsional angles φ, ψ (ω) around glycosidic bonds and/or interatomic distances Dynamics parameters (rotational correlation times, order parameters) ! For most carbohydrates, the secondary and higher-order structures insolution are not readily defined, due to their inherent flexibility.! The secondary and higher-order structure analysis of carbohydrates isnot discussed in further detail in this lecture. Instead, the analyticalmethods used to determine composition and sequence of carbohydratesare discussed81. Branched2. Synthesis is not “template driven”3. Alternative linkage positions are possible4. Alternative anomeric configurations are possible5. Cell-type specific glycosylation6. Influence of environmental conditions[Glucose] [NH3] pH7. Site-specific glycosylation8. MicroheterogeneityCarbohydrate Analysis Offers Unique Challenges9! Due to enormous structural diversity of naturallyoccurring glycans, their structural analysis requires aflexible approach! The choice of methodology and the final resultexpected are dictated by the amount and purity ofcarbohydrate material availableIII. General Considerations for Analyzing thePrimary Structure of a Carbohydrate10Information is expected to obtain through serialcarbohydrate analysisA. Presence of carbohydrate moiety ?B. Class of carbohydrate ?C. Quantity and monosaccharide composition ?D. More detailed analysis: linkage and sequence ?III. General Considerations for Analyzing thePrimary Structure of a Carbohydrate11! Radioactive labeling1). Proteins metabolically labeled with radioactive sugar precursors. After purification, the presence of carbohydrate moiety can be detected by radioactivity.Sometimes, the ability to label proteins with specific radioactive precursors may suggest the presence of specific types of glycoprotein or carbohydrate structure.2). Label endogenous glycan by radioactive sugar nucleotides by glycosyltransferase ! Lectin bindingLectins are carbohydrate-binding proteins. Sensitivity: 5-10 ng. Based on the specificity, the lectin binding assay may also facilitate to determine the type of carbohydrate ! Enzyme digestionCarbohydrate degrading enzymesIIIA. Presence of Carbohydrates ?! Chemical ReactionPeriodic acid-Schiff (PAS)reaction: Based on thesusceptibility of sugars toperiodate oxidation. Sensitivity:5-10 ng of gycoproteinSDS-PAGE analysis of the S-layer glycoprotein of G. stearothermophilusNRS 2004/3a. Lane 1, molecular mass standard; lanes 2 & 4, S-layerglycoprotein from continuous culture; lanes 3 and 5, S-layer glycoproteinfrom batch culture; lanes 2 and 3, Coomassie blue staining; lanes 4 and 5,periodic acid-Schiff staining. Steiner K, et al. J. Bacteriol. 200612Basic Strategies for the detection of carbohydrates inglycoconjugatesIIIA. Presence of Carbohydrates ?13IIIB. Class of Carbohydrate ?= Glucose (Glc)= Mannose (Man)= Galactose (Gal)= N-acetylglucosamine (GlcNAc)= N-acetylgalactosamine (GalNAc)= N-acetylhexosamine, unspecified (HexNAc)= Fucose (Fuc)= Xylose (Xyl)= Sialic acid, unspecified (Sia)= Glucuronic acid (GlcA)= Iduronic acid (IdoA)= Uronic acid, unspecified (HexA)Hexose,unspecified (Hex)GlycoproteinProteoglycan14Glycoproteins and Proteoglycans ?! Glycoprotein carbohydrate moietiescontain N-glycan and O-glycans.! Proteoglycan carbohydratemoieties contain glycosaminoglycan(heparan sulfate, chondroitin sulfate,dematan sulfate, keratan sulfate andhyaluronan).! A glycosylated protein typicallypresents diffused or multiple bands atelectrophoresis gel due toheterogeneity of carbohydrate moiety. - Carbohydrate staining (Periodate-Schiff assay, PAS) - Degrade carbohydrate by enzymes,leading to mobility changeLysates from tumor cells were immunoprecipitated with anti-MUC1. The precipitated MUC1 were left untreated ordigested with N-glycosidase and then immunoblotted.Ramasamy S., et al. Molecular Cell 27, 992-1004 (2007).15Class of Carbohydrate ?= Glucose (Glc)= Mannose (Man)= Galactose (Gal)= N-acetylglucosamine (GlcNAc)= N-acetylgalactosamine (GalNAc)= N-acetylhexosamine, unspecified (HexNAc)= Fucose (Fuc)= Xylose (Xyl)= Sialic acid, unspecified (Sia)= Glucuronic acid (GlcA)= Iduronic acid (IdoA)= Uronic acid, unspecified (HexA)Hexose,unspecified (Hex)16Presence of Carbohydrates:GlycolipidsGlycolipid (glycosphingolipid): anoligosaccharide attaches viaglucose or galactose to theterminal primary


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