Outline Principle of Carbohydrate Analysis I Class of carbohydrate types of glycosylation II Why study carbohydrate BCMB 8020 III Approaches for carbohydrate analysis April 9th 2009 IV Sample paper Lianchun Wang 1 I Species of Carbohydrate Moieties 2 II Roles of Glycoprotein associated Carbohydrates 1 Quality Control folding deglycosylation reglycosylation glycosyltransferase 2 Solubility Peroxidase 3 Circulating half life Lutropin LH follicle stimulating hormone FSH 4 Cell cell interactions Glucose Glc Hexose Fucose Fuc Mannose Man unspecified Hex Xylose Xyl Galactose Gal Sialic acid unspecified Sia N acetylglucosamine GlcNAc Glucuronic acid GlcA N acetylgalactosamine GalNAc Iduronic acid IdoA N acetylhexosamine unspecified HexNAc Uronic acid unspecified HexA lymphocyte homing cell growth tumor metastasis 3 4 III Experimental approaches for carbohydrate analysis Significance Table 1 Primary structural features of a complex carbohydrate Fully understanding the biology of glycoconjugates requires in depth knowledge of the carbohydrate chains 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 Gal GlcNAc Man Fuc in ratio 2 4 3 1 D Gal D GlcNAc D Man L Fuc Manp Galf Positions of glycosidic linkages Sequence of monosaccharides including occurrence of branchpoints double or triple substitution of a monosaccharide Man 1 6 Man 1 3 Man 1 4 GlcNAc Nature number and location of appended noncarbohydrate groups phosphate sulfate acetate peptide lipid IdoA2SO3 Neu5Ac9OAc 5 III Experimental approaches for carbohydrate analysis Example Precise ring conformation of each monosaccharide Complete set of H C C H dihedral angles Orientation of monosaccharides with respect to each other Torsional angles around glycosidic bonds and or interatomic distances Flexibility of the spatial structure Dynamics parameters rotational correlation times order parameters Carbohydrate Analysis Offers Unique Challenges 1 Branched Table 2 Secondary structural features of a complex carbohydrate Conformational aspect 6 2 Synthesis is not template driven 3 Alternative linkage positions are possible 4 Alternative anomeric configurations are possible 5 Cell type specific glycosylation 6 Influence of environmental conditions Glucose NH3 pH For most carbohydrates the secondary and higher order structures in solution are not readily defined due to their inherent flexibility 7 Site specific glycosylation The secondary and higher order structure analysis of carbohydrates is not discussed in further detail in this lecture Instead the analytical methods used to determine composition and sequence of carbohydrates are discussed 8 Microheterogeneity 7 8 III General Considerations for Analyzing the Primary Structure of a Carbohydrate III General Considerations for Analyzing the Primary Structure of a Carbohydrate Information is expected to obtain through serial carbohydrate analysis Due to enormous structural diversity of naturally occurring glycans their structural analysis requires a flexible approach A Presence of carbohydrate moiety B Class of carbohydrate The choice of methodology and the final result expected are dictated by the amount and purity of carbohydrate material available C Quantity and monosaccharide composition D More detailed analysis linkage and sequence 9 IIIA Presence of Carbohydrates IIIA Presence of Carbohydrates Basic Strategies for the detection of carbohydrates in glycoconjugates Chemical Reaction Periodic acid Schiff PAS reaction Based on the susceptibility of sugars to periodate oxidation Sensitivity 5 10 ng of gycoprotein Radioactive labeling 10 SDS PAGE analysis of the S layer glycoprotein of G stearothermophilus NRS 2004 3a Lane 1 molecular mass standard lanes 2 4 S layer glycoprotein from continuous culture lanes 3 and 5 S layer glycoprotein from batch culture lanes 2 and 3 Coomassie blue staining lanes 4 and 5 periodic acid Schiff staining Steiner K et al J Bacteriol 2006 1 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 binding Lectins 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 digestion Carbohydrate degrading enzymes 11 12 IIIB Class of Carbohydrate G ly co o pr te Glycoproteins and Proteoglycans Proteoglycan in Glycoprotein carbohydrate moieties contain N glycan and O glycans Proteoglycan carbohydrate moieties contain glycosaminoglycan heparan sulfate chondroitin sulfate dematan sulfate keratan sulfate and hyaluronan A glycosylated protein typically presents diffused or multiple bands at electrophoresis gel due to heterogeneity of carbohydrate moiety Lysates from tumor cells were immunoprecipitated with antiMUC1 The precipitated MUC1 were left untreated or digested with N glycosidase and then immunoblotted Ramasamy S et al Molecular Cell 27 992 1004 2007 Carbohydrate staining PeriodateSchiff assay PAS Glucose Glc Hexose Fucose Fuc Mannose Man unspecified Hex Xylose Xyl Galactose Gal Sialic acid unspecified Sia N acetylglucosamine GlcNAc Glucuronic acid GlcA N acetylgalactosamine GalNAc Iduronic acid IdoA N acetylhexosamine unspecified HexNAc Uronic acid unspecified HexA Degrade carbohydrate by enzymes leading to mobility change 13 14 Presence of Carbohydrates Glycolipids Class of Carbohydrate Glycolipid glycosphingolipid an oligosaccharide attaches via glucose or galactose to the terminal primary hydroxyl group of the lipid moiety ceramide Fractionation by Thin layer Chromatography TLC Low abundant fractions need prepurification Carbohydrate staining PAS Degrade carbohydrate by enzymes or chemical method leading to mobility change Glucose Glc Hexose Fucose Fuc Mannose Man unspecified Hex Xylose Xyl Galactose Gal Sialic acid unspecified Sia N acetylglucosamine GlcNAc Glucuronic acid GlcA N acetylgalactosamine GalNAc Iduronic acid IdoA N acetylhexosamine unspecified HexNAc Uronic acid unspecified HexA Ocinol A1 NOR SBA NOR NOR galactose Fig 1 Characterization of NOR2