Essentials of Glycobiology Lecture 29 May 18, 2004 Plant Glycans Marilynn Etzler Section of Molecular and Cellular Biology University of California Davis,

Essentials of Glycobiology

Lecture 29

May 18, 2004

Plant Glycans

Marilynn EtzlerSection of Molecular and Cellular BiologyUniversity of CaliforniaDavis, CA 95616e-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 FUNGI ANIMALS

Urochordates

Yeasts

Red algae Green algae

Brown algae

Vertebrates

Chordates

MossesLiverworts

Ferns

GymnospermsAngiosperms

PR

OT

OZ

OA

EU

KA

RY

OT

ES

Mu

ltic

ellu

lar

Un

icel

lula

r

Ancestral Prokaryotes

Arthropods

Insects

Slime moldsSponges

Coelenterates

MollusksNematodes

Echinoderms

Adapted from Figure 1-38, Molecular Biology of the Cell, 3rd ed., Garland Publishing, Inc.

Comparison of Classes of Plant and Animal Glycans

From Lecture 2 by Dr. Varki

Cell wall glycans

Glycolipids

MajorGlycan

Classes inPlantCells

Major Classes of N-Glycans Found in Plants

High Mannose

3

2

4

Complex

Pauci-mannoseHybrid= Sialic acid

= Man

= Gal

= GlcNAc

= Fuc

= Xylose

Sialic Acid Has Recently Been Found in Plants

Evidence:

Found in glycoproteins obtained from suspension-cultured cells from Arabidopsis thaliana, Nicotiana tabacum and Medicago sativa

Bound to Sambucus nigra and Maackia amurensis lectinsDid not bind to these lectins if pretreated with 2-3,6 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 IICalreticulinER mannosidase

-mannosidase I

Golgi:

Processing in Golgi (continued)

GNT I-Man IIGNT II

2-XylT3-FucT

3 2

3-GalT4-FucT 4

4

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

O-Diacylglycerol6

Sphingolipids – in plasma membraneGlucosylceramide ceramide

Glycosylphosphatidylinositol anchors:

462

NH2

Protein - Ethanol amine - PO4

Phosphatidylinositol

4 462

NH2

Protein - Ethanol amine - PO4

Phosphatidylinositol

Types of O-Linked Glycans Found in Plants

GlcNAc

Ser/Thr

GalNAc Gal GlcNAc Gal (Ara)1-4

Hydroxyproline

(Glycosylation of hydroxyprolineis unique to plants and Chlorophyceanalgae)

Arabinogalactan proteins (carbohydrate usually > 90% by weight)

3 33 333)(

(many variations)

- Hyp

Cell Wall Glycans

Cellulose [ Glc 4 Glc]n

Pectins:

Homogalacturanan [GalU 4 GalU 4]n

Rhamnogalacturonan I [GalU 2 L-Rha 4]n

Rhamnogalacturonan II

Figure 3 from Phytochem. 57: 929 (2001)

Xyloglucan:

4 44 4 44

2

6 6 6 6 6 6

2

nn

Hemicelluloses:

Cell Wall Glycans (continued)

Galactomannan

Plant Cell Wall

Alberts, et al., Molecular Biology of the Cell, Fig. 19-75

Constitutes the extracellular matrix

cellulose

galactans

homogalacturonan

rhamnogalacturonan I

calcium

rhamnogalacturonan II

xyloglucan

arabinans

Plasma membrane

Adapted from Figure 2, Trends in Plant Science 9: 203 (2004)

Model of Plant CellWall

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.

Arabidopsis thaliana as a Model Plant System

SOME ADVANTAGES:

Complete genome sequencedDiploidEasily transformedRelatively rapid life cycleMany mutants availablePlants small and thus take up little space

Arabidopsis seed

Corn kernel

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

DEFECT: Deficient in an isoform of GDP-D-mannose-4,6-dehydratase.

PHENOTYPE: Plants are dwarfed and have fragile cell walls. Deficient in fucose.

Identified by making acid hydrolysates of cell walls of EMS mutants and screening the alditol acetate derivatives by GLC.

3 2

L-Gal

4 44 4 44

2

6 6 6 6 6 6

2

nn

L-Gal

ARABIDOPSIS SPY MUTANTS

DEFECT: Deficient in O-linked GlcNAc transferase activity.

PHENOTYPE: A variety of alterations in growth and development.

Originally identified in genetic screen for mutants with increased response to gibberellins from T- DNA mutants. Also from EMS mutants.

Proposed to be involved in various aspects of regulation of plant development.