Arabinogalactan proteins Pbio691 - Plant Cell Wall 11/05/2010 Laura Cristea

AGPs Plant primary cell wall Cellulose Hemicellulose Proteins

AGPs Overview 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)

AGPs Classification 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 glycoprotein Ala-poor, His-rich Extensin motif Intermediate between AGPs and extensins Repetitive consensus motif Ser-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

AGPs Biosynthesis Hydrophobic C-terminal – GPI 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 Buchanan, Gruissem, Jones Biochemistry & Molecular Biology of Plants

AGPs Prolyl hydroxylase Post-translational Type II integral membrane protein Affinity – > 4 Pro residues Atmosferic oxygen needed O of 4-OH from Hyp – oxygen Albersheim, P. et al. Plant Cell Walls (2010)

AGPs Hyp contiguity hypothesis 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.

AGPs Enzymes for degradation http://www.molbiol.saitama-u.ac.jp/bussitsu/research.html

AGPs O-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. Kieliszewski J. of Biol. Chem. Vol. 285, no. 32, 24575-24583 (2010)

Subcloning for gene expression Gene design (AP)51 IFNα2-(SP)20 Subcloning for gene expression Tobacco extensin signal sequence CaMV 35S Plant transformation vector pBI121 Agrobacterium LBA4404 transformation Tobacco suspension cells transformation Tobacco suspension cell culture Protein separation Protein biochemical analysis NMR structure determination

Gene design Gene design (AP)51 IFNα2-(SP)20 Tan, L. et al. (2003) Plant Physiology 132, 1362-1369 IFNα2-(Ser-Hyp)20 Note: IFNα2 sequence not detailed Xu, J. et al. (2007) Biotechnology & Bioengineering

Hydrophobic Interaction Chromatography Protein separation Protein biochemical analysis Tobacco cells media Hydrophobic Interaction Chromatography (HIC)

Cation & Size exclusion chromatography Hyp-arabinogalactan (Ala-Hyp)51-EGFP Cation & Size exclusion chromatography 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 AHP-1 tube 23 Tan, L. et al. (2004) The Journal of Biological Chemistry 279:13, 13156-13165

NMR spectroscopy One-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

A:B:C:D:E:F:G = 4:1:1:1:4:1:4 One dimensional 1H – AHP-1 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 Tan, L. et al. (2004) The Journal of Biological Chemistry 279:13, 13156-13165

HSQC & HMBC

INF Hyp-polysaccharide 1

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

INF Hyp-polysaccharide 1 Sugar ratio & configuration 1H NMR A:B:C:D:E:F:G = 6:2:2:1:5:1:4+2 A – Ara D – Hyp H-4 B – Ara E – Gal C – 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

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

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

Gal configurations 1H NMR HMBC Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583

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

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

1H NMR Ara linkages HMBC Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583

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

INF Hyp-polysaccharide 2

Gal:Ara:GlcUA:Rha – 10:5:4:1 Sugar composition 1H NMR Gal:Ara:GlcUA:Rha – 10:5:4:1 Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583

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

Side chains 1H NMR HMBC Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583

Primary structures IFN-Hyp polysaccharide 1 IFN-Hyp polysaccharide 2 Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583

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