Arabinogalactan proteins

Pbio691 - Plant Cell Wall11/05/2010

Laura Cristea

AGPs

Cellulose

Hemicellulose

Proteins

Plant primary cell wall

AGPsOverview

HRGPs Lowest protein content among HRGPs (1-10%) Highest sugar content among HRGPs (90-99%) Complex glycosylation modules Protein backbone O-glycosylated Ara, Gal, Rha, GlcUA, Fuc Soluble or GPI-anchored Associated with plasma membrane, cell wall Regulation, signaling, growth and development Cell-cell interaction, pathogen defense Substrate for pollen growth, wound-induced (gum arabic)

AGPsClassification based on

structure Classical Hyp-rich AGPs (A)

Classical AGPs with Lys-rich domain (B)

AG peptide (12 aa) (C)

Nonclassical AGPs with Asn-rich domain (D)

Proteins with two AGP and two fasciclin domain (E)

Proteins with two AGP and one fasciclin domain (F)

Proteins with one AGP and one fasciclin domain (G)

Albersheim, P. et al. Plant Cell Walls (2010)

AGPs

Gum arabic

Ala-poor, His-rich

Extensin motif

Intermediate between AGPs and extensins

Repetitive consensus motifSer-Hyp-Hyp-Hyp-Thr-Leu-Ser-Hyp-Ser-Hyp-Thr-Hyp-Thr-Hyp-Hyp-Leu-Gly-Pro-His

Sugar composition resembles the AGPs with arabinose and galactose as major ones

Gum arabic glycoprotein

AGPs

Secretory pathway Hydrophobic C-terminal – GPI Ala, Thr, Ser, Pro, Hyp rich ER – protein backbone Prolyl hydroxylase – not all Pro Golgi – Hyp-O-glycosylation –not all Hyp Glycosyltransferases Hyp glycosylation hypothesis

beta glucosyl Yariv reagent – identification problems

Biosynthesis

Buchanan, Gruissem, Jones Biochemistry & Molecular Biology of Plants

AGPsProlyl hydroxylase

Albersheim, P. et al. Plant Cell Walls (2010)

Post-translational Type II integral membrane protein Affinity – > 4 Pro residues Atmosferic oxygen needed O of 4-OH from Hyp – oxygen

AGPs

Contiguous Hyp residues are arabinosylated (extensins)

Noncontiguous Hyp are not glycosylated or just have one arabinose

Clustered Hyp residues are linked to a galactose backbone with arabinose and galactose as major components in the side chains; other types of sugar might be present

Conclusion: the glycosylation pattern of the AGP protein backbone is determined by the amino acid sequence.

Hyp contiguity hypothesis

AGPsEnzymes for degradation

http://www.molbiol.saitama-u.ac.jp/bussitsu/research.html

AGPsO-glycosylation in general

Wilson, Iain BH (2002) Curr. Opinion in Structural Biology 12, 569-577

Plant O-Hydroxyproline Arabinogalactans Are Composed of

Repeating Trigalactosyl Subunits with Short Bifurcated Side Chains

Li Tan, Peter Varnai, Derek T.A. Lamport, Chunhua Yuan, Jianfeng Xu, Feng Qiu,

Marcia J. KieliszewskiJ. of Biol. Chem. Vol. 285, no. 32, 24575-24583 (2010)

Gene design(AP)51

IFNα2-(SP)20Subcloning for gene expressionTobacco extensin signal sequence

CaMV 35S Plant transformation vector pBI121

Agrobacterium LBA4404 transformation

Tobacco suspension cells

transformation

Protein separation

Tobacco suspension cell

culture

NMR structure determination

Protein biochemicalanalysis

Gene design

IFNα2-(Ser-Hyp)20 Note: IFNα2 sequence not detailed

Tan, L. et al. (2003) Plant Physiology 132, 1362-1369

Xu, J. et al. (2007) Biotechnology & Bioengineering

Gene design(AP)51

IFNα2-(SP)20

Protein separation

Hydrophobic Interaction

Chromatography

(HIC)

Tobacco cells media

Protein biochemicalanalysis

Hyp-arabinogalactan

INFα2-(Ser-Hyp)20

Isolation & purification NaOH hydrolysis (108°C, 18 h) Separation on size-exclusion chromatography (Superdex-Peptide column) Hyp analysis - colorimetric Monosaccharide analysis

Total sugar content – colorimetric (anthrone method) Neutral sugar content – gas chromatography (alditol acetates method)

Nuclear Magnetic Resonance (NMR) INF Hyp-polysaccharide 1 AHP-1

(Ala-Hyp)51-EGFP Cation & Size exclusion

chromatography

AHP-1tube 23

Tan, L. et al. (2004) The Journal of Biological Chemistry 279:13, 13156-13165

NMR spectroscopyOne-dimensional 1H

Two-dimensional 1H homonuclear COrrelation SpectroscopY (COSY) TOtal Correlation SpectroscopY (TOCSY) ROtating Frame NOE SpectroscopY (ROESY) Nuclear Overhausser Effect SpectroscopY (NOESY)

Two-dimensional 13C, 1H

Heteronuclear Single Quantum Coherence (HSQC) Heteronuclear Multiple Bond Coherence (HMBC)

Two dimensional 13C, 1H heteronuclear HSQC-TOCSY Two dimensional 13C, 1H HSQC—NOESY

NMRPipe NMRView

Standard: 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS)

Chemical shifts of 1H & 13C

Gane, A.M. et al. (1995) Carbohydrate Research 277, 67-85

1H NMR, HSQC, HSMB from a previous paper

One dimensional 1H – AHP-1

Tan, L. et al. (2004) The Journal of Biological Chemistry 279:13, 13156-13165

A:B:C:D:E:F:G = 4:1:1:1:4:1:4

A – Ara D – Hyp H-4

B – Ara E – Gal

C – Rha F – Gal

G – Gal & GlcA

HSQC & HMBC

INF Hyp-polysaccharide 1

Sugar ratio & configuration

Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583

Sugar ratio & configuration

A:B:C:D:E:F:G = 6:2:2:1:5:1:4+2A – Ara D – Hyp H-4B – Ara E – GalC – Rha F – Gal linked to Hyp G – Gal + GlcUA

INF Hyp-polysaccharide 1

Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583

1H NMR

Hyp-Gal linkage

Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583

HSQC

TOCSY

Hyp-Gal linkage

Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583

HMBC

Gal configurations

HMBC

Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583

1H NMR

Rha residues

Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583

TOCSY

HSQC

Rha – GlcUAGlcUA - Gal

Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583

HMBC

Ara linkages

HMBC

Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583

1H NMR

Ara linkages

Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583

HSQC

HMBC

INF Hyp-polysaccharide 2

Sugar composition

Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583

Gal:Ara:GlcUA:Rha – 10:5:4:1

1H NMR

Gal linkage & backbone

Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583

HMBC

Side chains

Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583

HMBC

1H NMR

Primary structures

Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583

IFN-Hyp polysaccharide 2IFN-Hyp polysaccharide 1

Conclusions

Complete structure elucidation by NMR INF Hyp-polysaccharide 1 has six-residue galactan chain with 2 beta

1,3 linked by a beta 1,6 linkage INF Hyp-polysaccharide has four side chains Repetitive trisaccharide with two six-residue bifurcated side chains Six-residue side chain – identical with gum arabic side chain (no ter 5-

Ara) Glycosylation is not determined by the non-glycosylated sequence or

type of peptide Incomplete glycosylation

Molecular Modeling