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Arabinogalactan proteins

Arabinogalactan proteins. Pbio691 - Plant Cell Wall 11/05/2010 Laura Cristea. AGPs. Plant primary cell wall. Cellulose Hemicellulose Proteins . AGPs. Overview. HRGPs Lowest protein content among HRGPs (1-10%) Highest sugar content among HRGPs (90-99%)

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Arabinogalactan proteins

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

  2. AGPs Plant primary cell wall • Cellulose • Hemicellulose • Proteins

  3. AGPs Overview • 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)

  4. 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)

  5. AGPs Gum arabic glycoprotein • Gum arabic • 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

  6. AGPs Biosynthesis • 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

  7. 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)

  8. 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.

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

  10. AGPs O-glycosylation in general Wilson, Iain BH (2002) Curr. Opinion in Structural Biology 12, 569-577

  11. 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)

  12. 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

  13. Gene design (AP)51 IFNα2-(SP)20 Gene design 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

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

  15. 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

  16. NMR spectroscopy One-dimensional 1H Two-dimensional 1H homonuclear • COrrelation SpectroscopY (COSY) • TOtal Correlation SpectroscopY (TOCSY) • ROtating Frame NOESpectroscopY (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)

  17. Chemical shifts of 1H & 13C Gane, A.M. et al. (1995) Carbohydrate Research 277, 67-85

  18. 1H NMR, HSQC, HSMB from a previous paper

  19. 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

  20. HSQC & HMBC

  21. INF Hyp-polysaccharide 1

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

  23. 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

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

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

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

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

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

  29. 1H NMR Ara linkages HMBC Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32, 24575-24583

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

  31. INF Hyp-polysaccharide 2

  32. 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

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

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

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

  36. 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

  37. Molecular Modeling

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