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

2. AGPs
Plant primary cell wall
Cellulose
Hemicellulose
Proteins

3. 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)

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 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 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

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

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

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, (2010)

12. 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

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

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

15. 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,

16. 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)

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. 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,

20. HSQC & HMBC

21. INF Hyp-polysaccharide 1

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

23. 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,

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

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

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

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

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

31. 1H NMR
Ara linkages
HMBC
Tan, L. et al. (2010) The Journal of Biological Chemistry 285:32,

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

34. INF Hyp-polysaccharide 2

35. 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,

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

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

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

39. 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

40. Molecular Modeling