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Essentials of Glycobiology

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: meetzler@ucdavis.edu. LECTURE OUTLINE. Introduction Classes of glycans in plants

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Essentials of Glycobiology

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  1. 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: meetzler@ucdavis.edu

  2. 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”

  3. PLANTS FUNGI ANIMALS Angiosperms Vertebrates Gymnosperms Urochordates Insects Chordates Echinoderms Ferns Mosses Liverworts Arthropods Multicellular Mollusks Nematodes Brown algae Coelenterates EUKARYOTES Green algae Red algae Sponges Slime molds Yeasts Unicellular PROTOZOA Ancestral Prokaryotes Adapted from Figure 1-38, Molecular Biology of the Cell, 3rd ed., Garland Publishing, Inc.

  4. Major Glycan Classes in Plant Cells Cell wall glycans Glycolipids Comparison of Classes of Plant and Animal Glycans From Lecture 2 by Dr. Varki

  5. Major Classes of N-Glycans Found in Plants a4 b2 a3 = Man = Gal = GlcNAc = Fuc = Xylose = Sialic acid High Mannose Complex Pauci-mannose Hybrid

  6. 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.

  7. Recognition and Processing of N-Glycans in the Plant Secretory Pathway Endoplasmic reticulum: Glucosidases I and II Calreticulin ER mannosidase -mannosidase I Golgi:

  8.  4 Processing in Golgi (continued)  4 -Man II GNT II GNT I 2-XylT 3-FucT 2 3 3-GalT 4-FucT

  9. J. Cell Science (2002) 115: 2423 Most of the volume of a typical plant cell is occupied by the vacuole(s)

  10. Processing in Vacuole or Enroute to Vacuole:

  11. 2 2 6 6 4 4 NH2 NH2 Other Types of Plant Glycans Plant Glycolipids: Galactolipids – in chloroplast membranes Monogalactosyldiacylglycerol O-Diacylglycerol O-Diacylglycerol Digalactosyldiacylglycerol a6 b Sphingolipids – in plasma membrane Glucosylceramide ceramide Glycosylphosphatidylinositol anchors: Protein - Ethanol amine - PO4 Protein - Ethanol amine - PO4 4 Phosphatidylinositol Phosphatidylinositol

  12. GlcNAc GalNAc GlcNAc Gal (Ara)1-4 Gal Ser/Thr Hydroxyproline (Glycosylation of hydroxyproline is unique to plants and Chlorophycean algae) Arabinogalactan proteins (carbohydrate usually > 90% by weight) ) ( 3 3 3 3 3 3 - Hyp (many variations) Types of O-Linked Glycans Found in Plants

  13. Cell Wall Glycans Cellulose [ Glc 4 Glc]n Pectins: [GalU 4 GalU 4]n Homogalacturanan [GalU 2 L-Rha 4]n Rhamnogalacturonan I Rhamnogalacturonan II Figure 3 from Phytochem. 57: 929 (2001)

  14. 2 2 6 6 6 6 6 6 4 4 4 4 4 4 n n Cell Wall Glycans (continued) Hemicelluloses: Xyloglucan: Galactomannan

  15. Plant Cell Wall Constitutes the extracellular matrix Alberts, et al., Molecular Biology of the Cell, Fig. 19-75

  16. cellulose galactans homogalacturonan rhamnogalacturonan I calcium rhamnogalacturonan II xyloglucan arabinans Plasma membrane Model of Plant CellWall Adapted from Figure 2, Trends in Plant Science 9: 203 (2004)

  17. 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 glyco- sidase-related genes.

  18. 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

  19. 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

  20. L-Gal 2 2 3 L-Gal 2 6 6 6 6 6 6 4 4 4 4 4 4 n n 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.

  21. 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.

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