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Department of Glycobiology Research areas: Enzymes involved in formation and modification of complex saccharides a) glycosyl transferases b) transglycosylases a) glycosyl transferases (Dr. Mucha) N-linked glycans Mammals Plants N-acetylglucosamine Galactose Xylose (ß1,2) Mannose Fucose (a1,6) Fucose (a1,3) Sialic acid Molecular cloning and mutagenesis 1. RT-PCR 2. Inverse PCR in mutagenesis Mutation Site His EK Rb Gnt I Catalytic Domain Heterologous Expression - Insect Cells GT I mutant pVTB acHis BaculoG old Recombinat ion Virus Propagation Recombin ant Virus DNA Cells Transfection and Virus Packaging AcMNPV virus Protein Purification Purification of the GT mutants by Ni2+ -NTA column Imidazole Concentration 20mM Conditioned medium 40mM 80mM 250mM 0,2 0,18 0,16 0,14 0,12 0,1 0,08 0,06 0,04 0,02 0 Rb Gnt I Wt R269 Fraction 34 31 28 25 22 19 16 13 10 7 4 H187R269 1 Elution OD280 Elution Analysis Western blotting: heterologous expression of GT mutants in insect cells Sf 21 Biomolecular Interactions Analysis (BIA) of GT Basic Principle • A interacting molecule (ligand) is bound to the sensor surface.(e.g. a peptide, substrate,..) • Another (analyte) is passed over the surface and binds to it. b) Transglycosylases Xyloglucan endotransglycosylase/hydrolase, XET/XTH An enzyme involved in cleavage and re-formation of glycosidic bonds in xyloglucan (XG). XG molecules are tethering the cellulose microfibrils (CM) in primary plant cell walls. Action of XET is essential for plant cell wall growth: it enables the sliding of CM during expansion and incorporation of newly arriving XG molecules into the cell wall structure. XG Enzyme Bond cleavage Development of assay methods for XET: •Viscometric •Radiometric •Fluorimetric •Colorimetric •Chromatographic (HPLC) HO 2 (slow) XGOS (labeled) (fast) Hydrolysis Product XG (labeled) Transglycosylation CM XE XET XG T XET CM Scheme depicting the release of XET from the cell walls by transglycosylation to labeled XGOs* XET XG CM * XGOS* * XET * * CM * XG * XET * CM XE T * XET XG CM Fluorescent detection of XETs after IEF separation
