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Essentials of Glycobiology Lecture 29 May 18, 2004 Plant Glycans Marilynn Etzler Section of Molecular and Cellular Biology University of California Davis, CA 95616 e-mail: [email protected] LECTURE OUTLINE • Introduction • Classes of glycans in plants • Structure and biosynthesis of plant N-linked glycans • Other types of plant glycans • Plant cell wall • Plant glycosylation mutants • Molecular “farming” PLANTS Angiosperms FUNGI ANIMALS Vertebrates Gymnosperms Urochordates Insects Chordates Arthropods Mollusks Nematodes Brown algae Coelenterates Red algae Green algae Sponges Slime molds Unicellular PROTOZOA Yeasts EUKARYOTES Mosses Liverworts Multicellular Echinoderms Ferns Ancestral Prokaryotes Adapted from Figure 1-38, Molecular Biology of the Cell, 3rd ed., Garland Publishing, Inc. Comparison of Classes of Plant and Animal Glycans Major Glycan Classes in Plant Cells Glycolipids From Lecture 2 by Dr. Varki Cell wall glycans Major Classes of N-Glycans Found in Plants a4 b2 a3 High Mannose Complex = Man = Gal = GlcNAc = Fuc = Xylose = Sialic acid Pauci-mannose Hybrid Sialic Acid Has Recently Been Found in Plants Found in glycoproteins obtained from suspension-cultured cells from Arabidopsis thaliana, Nicotiana tabacum and Medicago sativa Evidence: Bound to Sambucus nigra and Maackia amurensis lectins Did not bind to these lectins if pretreated with a2-3,6 sialidase Sialic acids were released chemically, derivatized with DMB and analyzed by reverse phase chromatography, yielding a prominent peak of Neu5Gc and a smaller peak of Neu5Ac. Similar results were obtained with sialic acids released enzymatically. Analyses of DMB-SA derivatives were confirmed by MALDI-TOF Reference: Shah, M.M., K. Fujiyama, C.R. Flynn, and L. Joshi (2003) Nature Biotechnology 21: 1470 – 1471. Recognition and Processing of N-Glycans in the Plant Secretory Pathway Endoplasmic reticulum: Glucosidases I and II Calreticulin ER mannosidase a-mannosidase I Golgi: Processing in Golgi (continued) a-Man II GNT II GNT I b2-XylT a3-FucT b2 a4 a4 b3-GalT a4-FucT a3 J. Cell Science (2002) 115: 2423 Most of the volume of a typical plant cell is occupied by the vacuole(s) Processing in Vacuole or Enroute to Vacuole: Other Types of Plant Glycans Plant Glycolipids: Galactolipids – in chloroplast membranes Monogalactosyldiacylglycerol Digalactosyldiacylglycerol O-Diacylglycerol a6 b O-Diacylglycerol Sphingolipids – in plasma membrane Glucosylceramide ceramide Glycosylphosphatidylinositol anchors: Protein - Ethanol amine - PO4 a2 a6 a4 NH2 Phosphatidylinositol Protein - Ethanol amine - PO4 a2 a6 b4 a4 NH2 Phosphatidylinositol Types of O-Linked Glycans Found in Plants GlcNAc GalNAc Ser/Thr GlcNAc Gal Gal (Ara)1-4 Hydroxyproline (Glycosylation of hydroxyproline is unique to plants and Chlorophycean algae) Arabinogalactan proteins (carbohydrate usually > 90% by weight) ( b3 b3 b3 b3 b3 b3 ) - Hyp (many variations) Cell Wall Glycans Cellulose [ Glc b4 Glc]n Pectins: Homogalacturanan [GalU a4 GalU a4]n Rhamnogalacturonan I [GalU a2 L-Rha a4]n Rhamnogalacturonan II Figure 3 from Phytochem. 57: 929 (2001) Cell Wall Glycans (continued) Hemicelluloses: Xyloglucan: a2 b2 a6 b4 Galactomannan a6 b4 a6 b4 a6 n b4 a6 b4 a6 b4 n Plant Cell Wall Constitutes the extracellular matrix Alberts, et al., Molecular Biology of the Cell, Fig. 19-75 Model of Plant CellWall cellulose galactans homogalacturonan rhamnogalacturonan I calcium rhamnogalacturonan II xyloglucan arabinans Plasma membrane Adapted from Figure 2, Trends in Plant Science 9: 203 (2004) Plant Glycosyltransferases and Glycosidases Almost 800 glycosyltransferase and glycosidase-related genes have been found in the Arabidopsis genome. Comprises > 3.3% of its genes. By contrast, human genome has about 350 glycosyltransferase and glycosidase-related genes. Arabidopsis thaliana as a Model Plant System SOME ADVANTAGES: Complete genome sequenced Diploid Easily transformed Relatively rapid life cycle Many mutants available Plants small and thus take up little space Corn kernel Arabidopsis seed ARABIDOPSIS cgl MUTANT Identified by screening leaf extracts of EMS mutants with antiserum against complex glycans. DEFECT: Missing GNT I PHENOTYPE: No apparent effect on development and morphology of plants. No complex glycans. Accumulates Man5GlcNAc2 ARABIDOPSIS mur 1 MUTANT Identified by making acid hydrolysates of cell walls of EMS mutants and screening the alditol acetate derivatives by GLC. DEFECT: Deficient in an isoform of GDP-D-mannose-4,6-dehydratase. PHENOTYPE: Plants are dwarfed and have fragile cell walls. Deficient in fucose. b2 L-Gal a2 a3 L-Gal b2 a6 b4 a6 b4 a6 b4 a6 n b4 a6 b4 a6 b4 n ARABIDOPSIS SPY MUTANTS Originally identified in genetic screen for mutants with increased response to gibberellins from T- DNA mutants. Also from EMS mutants. DEFECT: Deficient in O-linked GlcNAc transferase activity. PHENOTYPE: A variety of alterations in growth and development. Proposed to be involved in various aspects of regulation of plant development.