1. ESSENTIALS OF GLYCOBIOLOGY Lecture 30 May 18, 2004 Marilynn Etzler Section of Molecular and Cellular Biology University of California Davis, CA 95616 (meetzler@ucdavis.edu) FREE GLYCANS AND THEIR ROLES AS SIGNALING MOLECULES

2. LECTURE OUTLINE Background Oligosaccharide signals trigger the initiation of the plant defense response Nod factor signals initiate the nitrogen-fixing Rhizobium-legume symbiosis Chitin oligosaccharide signals in plant defense and early animal development Other oligosaccharide signals in early plant and animal development Pattern recognition receptors and innate immunity

3. Background: Potential of oligosaccharides as signals: 1) Variety of linkages between monomers enables a large number of conformational variations. Portion of Table shown in Lecture 1: Macromolecule Protein Nucleic Acid Carbohydrate Building block Amino acids Nucleotides Hexoses Possible variations in a trimer 6 1,056 to 27,648 2) Many hydroxyls available for modification.

4. Basic elements of signaling system: Signal Receptor Transduction mechanism Response

5. Cell wall Pathogen Elicitor Plasma membrane Early Plant Responses Changes in ion fluxes Oxidative burst  H 2 O 2 and O 2  Activation of early defense-related genes Phytoalexin production Structural changes in cell walls Plant Defense Responses: Oligosaccharide Signals Trigger the Initiation of the Plant Defense Response

6. 33 66 66 66 66 33 Isolated from cell walls Elicits phytoalexin production in soybean seedlings Structure confirmed by chemical synthesis Hepta-  -glucoside The first oligosaccharide signal was identified in the fungus, Phytophthora megasperma, a fungal pathogen of soybean.

7. Relative Activities of Oligo-  -Glucosides Relative Elicitor Activity Relative Binding Activity 33 66 66 66 66 33 = reduced glucose 33 66 66 66 66 33 66 66 66 66 33 33 33 1000 270 93 1.2 1.3

8. Nod factor signals initiate the nitrogen-fixing Rhizobium-legume symbiosis

9. GENERIC STRUCTURE OF NOD FACTORS R 1 = H, Methyl R 2 = C16:2, C16:3 C18:1, C18:3, C18:4 C20:3, C20:4 R 3 = H, Cb R 4 = H, Cb R 5 = H, Ac R 6 = H, Ac, SO 4 Fuc AcFuc MeFuc R 7 = H Glycerol n = 1 - 4

10. Chitin oligosaccharide signals in plant defense and early animal development Elicit alkalinization of medium of tomato cell cultures Biological activity (nanomolar) 44 44 44 44 44 44 44 44 44 44 0.1 50 100,000 >1,000,000

11. Evidence for chitin oligosaccharide signaling in early animal development: In Xenopus laevis the developmentally regulated protein, DG42, is homologous to Nod C, the enzyme that synthesizes the chitin backbone of the Nod factors in rhizobia. DG42 is only expressed between the gastrula and neurulaation stages. DG42 can direct the synthesis of chitin oligosaccharides in vitro. Chitin oligosaccharides can be synthesized by extracts of gastrulation stage embryos of cyprinid fishes (zebra fish and carp). Microinjection of fertilized eggs with antibodies against DG42 leads to severe defects in trunk and tail development.

12. Oligogalacturonides - isolated from plant cell walls = Galacturonic acid Serve as signals in both defense and in plant development 44 44 44 44 44 44 44 44 44 DEFENSE: Usually need DP of 10-14 to elicit phytoalexin accumulation. DEVELOPMENT: Different DP elicit different responses. DP = degree of polymerization Other oligosaccharide signals in early plant and animal development

13. 44 44 44 22 66 66 66 22 Xyloglucan nonasaccharide: Concentrations of 10 -8 M inhibit auxin-induced elongation of stem fragments. Hyaluronan fragments: 44 33 33 33 44 n Activate antigen presenting cells and other proinflammatory responses, signal cell motility and adhesion

14. Innate immunity Dependent on proteins and phagocytic cells that recognize conserved features of pathogens that are absent in the host. Found in vertebrates, invertebrates and plants. Pattern recognition receptors Recognize pathogen-associated immunostimulants Peptidoglycan cell wall and flagella of bacteria Examples of repeating patterns that often occur on pathogen surfaces Lipopolysaccharide on Gram-negative bacteria Teichoic acids on Gram-positive bacteria Chitin, glucan and other polysaccharides in cell walls of fungi Such repeating patterns called PAMPS (Pathogen-Associated Molecular Patterns)

15. Pathogen associated immunostimulants Pattern recognition receptor Macrophage plasma mem- brane Phagosome Lysosome Acid hydrolases Lysozyme Actin rearrangement Transcription of target genes When signal binds to the receptor, it causes the rearrangement of actin filaments as well as the transcription of new genes. The pathogen is endocytosed

16. Pattern recognition receptors Distinguish self from conserved microbial structures. Drosophila – identified Toll receptors Mammals – identified Toll-like receptors (TLRs) Membrane Leucine rich repeat (LRR) domain Indirectly associated with binding PAMPs TIR domain Interacts with adaptor protein May be part of receptor complex

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

18. Animals Insects Plants Pathogen Perception and Defense Systems Legumes Mammals Last common ancestor may have used a related TLR for pathogen recognition