Preparation of Arabinogalactan Glycoproteins from Plant Tissue

[Principle] Disrupted plant tissues release soluble AGPs that can be precipitated as their Yariv complex. This procedure yields mainly classical AGPs; these comprise the bulk of cell surface AGPs. Extraction with CaCl₂ rather than the more usual NaCl has two advantages:

1. It results in Ca²⁺ tightly bound by the glucuronic acid residues (Lamport and Varnai, 2013) at > pH 4.5 thus enhancing AGP solubility after its release from the insoluble Yariv complex.
   
2. It removes pectin as insoluble calcium pectate crosslinked by intermolecular Ca²⁺ bridges while AGPs with intramolecular Ca²⁺ remain soluble.
   

Materials and Reagents

1. Tobacco BY-2 cells or other plant tissue
   
2. Liquid nitrogen
   
3. CaCl₂ (2% w/v)
   
4. Distilled water
   
5. Na metabisulphite (Sigma-Aldrich, catalog number: S-1256 )
   
6. Dialysis tubing 12 kDa MW cutoff (3.2 cm flat width) (Sigma-Aldrich, catalog number: D-0530 )
   
7. Superose-6, 10/300 GL (GE Healthcare, catalog number: 17-5172-01 )
   
8. Hydroxyproline
   
9. Glucuronic acid
   
10. Gum arabic (Sigma-Aldrich, catalog number: G-9752 )
    
11. NaCl
    
12. NaOH
    
13. Yariv reagent (Biosupplies Australia Pty, catalog number: 100-2 ) (see Recipes)
    

Equipment

1. Blender/coffee mill
   
2. Minifuge centrifuge
   
3. 2 ml Sarstedt tube (with screw cap)
   
4. Spectrophotometer or microplate reader
   
5. Mass spectroscope
   
6. Microfuge
   
7. Fine-tip pipette
   
8. Fine tip sonic probe
   
9. Block heater
   

Procedure

1. Freeze ~10 to 100 g plant tissue in liquid nitrogen.
   
2. Pulverise frozen fresh tissue to a fine powder in a cold blender/coffee mill.
   
3. Stir tissue in 2% w/v CaCl₂ for 2-3 h at RT (2 ml for each gram of tissue).
   
4. Centrifuge 30 minutes at ~10,000 x g (e.g. minifuge at RT)。
   
5. Assay 10, 50 and 100 μl aliquots to estimate total AGP in extract (see Notes).
   
6. Add a slight excess (see Notes) of Yariv reagent to the remaining extract.
   
7. Allow to precipitate at least 1 h or overnight at RT.
   
8. Collect precipitate by low speed centrifugation (10 min at 2,000 x g).
   
9. Resuspend precipitate in 1.5 ml distilled water.
   
10. Transfer to 2 ml Sarstedt tube (with screw cap).
    
11. Add ~25 mg Na metabisulphite (final conc. 70 mM) to reduce the diazo linkage (top up with H₂O to exlude oxygen which otherwise results in the formation of elemental sulfur).
    
12. Cap tube tightly and heat at ~50 °C until decolourised (5-20 min).
    
13. Transfer to small (~5 ml) dialysis bag; stir overnight at RT in 500 ml distilled H₂O and change H₂O three times.
    
14. Freeze dialysate in liquid nitrogen, lyophilise, then and weigh the product. (AGP yields vary from 30-300 μg AGP/g fresh weight depending on tissue source) (see Notes).
    
15. Validate classical AGPs by size and composition:
    
    1. Gel filtration on Superose-6 (Lamport, et al., 2006).
       
    2. Hydroxyproline content (Kivirikko and Liesmaa, 1959).
       
    3. Uronic acid content (Blumenkrantz and Asboe-Hansen, 1973).
       
    4. Bound calcium via colourimetry (Gindler and King, 1972) or ICPMS (inductively coupled plasma mass spectroscopy) (Lamport and Varnai, 2013).
       
    5. Amino acid and sugar analyses.
       

Notes

1. Arabinogalactan protein assay via Yariv reagent.
   All steps at RT.
   1. Add test samples to Eppendorf microfuge tubes.
      
   2. Make up to ~500 μl in 1% CaCl₂.
      
   3. Use 10 and 20 μg gum arabic as AGP "standards" as follows:
      Add 10 μl gum arabic (1 mg/ml) to 500 μl 1% CaCl₂
      Add 20 μl gum arabic (1 mg/ml) to 500 μl 1% CaCl₂
      
   4. Use 500 μl 1% CaCl₂ as a reagent blank.
      
   5. Then to each tube:
      Add 200 μl β-D-Galactosyl-Yariv reagent (1 mg/ml in 2% CaCl₂)
      Or use β-D-Glucosyl-Yariv reagent – choice depends on availability.
      
   6. Mix well and leave for at least 30 min at room temperature.
      
   7. Spin 10 min at ~15,000 x g in microfuge.
      
   8. With CARE use fine-tip pipette to remove & discard supernate.
      
   9. Wash pellet twice with 1 ml 2% CaCl₂.
      
   10. Add 1 ml 20 mM NaOH.
       
   11. Shake vigorously to dissolve pellet or sonicate 10-20 sec with fine tip probe or in a sonic bath. If solution is turbid, spin at ~15,000 x g to clarify.
       
   12. Read A₄₅₇ nm against reagent blank within an hour or so. A₄₅₇ avoids phenolic interference when eluting Yariv from intact BY-2 cells; for general assays read at 440 nm.
       
   13. Plot "standard curve" and calculate unknowns as μg AGP/tube.
       Note: Gum arabic quantification is only an approximation as each AGP binds different amounts of Yariv.
       As a general guide, however, a given weight of Yariv reagent will precipitate the same weight of AGP. So for AGP isolation a 10% excess of Yariv reagent generally suffices to precipitate all the AGP.
       
2. AGP cellular distribution (background information):
   Classical AGPs are essential glycoproteins distributed in three cell surface compartments: bound to the outer surface of the plasma membrane by a GPI anchor; soluble in the periplasm; and "bound" or trapped in the wall matrix.
   Thus AGP cellular distribution T = M + S + W (Lamport et al., 2006)
   M = AGPs bound to plasma membrane
   S = Soluble AGPs released by cell disruption
   W = AGPs bound to cell wall
   
   Table 1. Total of AGPs in tobacco BY-2 cells. In tobacco BY-2 cells, T = 600 μg AGPs g fresh weight.
   

   BY-2 cells (data in 6)	T Total	M Membrane bound	S Soluble periplasmic	W Wall bound
   Salt-adapted*	600	60	354	186
   Control	600	210	282	108

   * AGPs upregulated by high salt appear in the growth medium.
   

Recipes

1. Preparation of Yariv reagent (1,3,5-tri-(p-β-D-galactosyloxyphenylazo)-2,4,6-trihydroxybenzene) or the β-D-glucosyl derivative
   Dissolve 100 mg β-D-galactosyl Yariv in 100 ml 2% w/v CaCl₂
   

Acknowledgments

This protocol is adapted from Popper (2011), Lamport and Varnai (2013) and Lamport et al. (2006).

References

1. Blumenkrantz, N. and Asboe-Hansen, G. (1973). New method for quantitative determination of uronic acids. Anal Biochem 54(2): 484-489.
   
2. Darjania, L., Ichise, N., Ichikawa, S., Okamoto, T., Okuyama, H. and Thompson Jr, G. A. (2002). Dynamic turnover of arabinogalactan proteins in cultured Arabidopsis cells. Plant Physiol Biochem 40(1): 69-79.
   
3. Gindler, E. and King, J. (1972). Rapid colorimetric determination of calcium in biologic fluids with methylthymol blue. Am J Clin Pathol 58(4):376-82.
   
4. Kivirikko, K. I. and Liesmaa, M. A. (1959). A Colorimetric method for determination of hydroxyproline in tissue hydrolysates. Scandinavian J Clin Lab 11(2):128-133.
   
5. Lamport, D. T. and Varnai, P. (2013). Periplasmic arabinogalactan glycoproteins act as a calcium capacitor that regulates plant growth and development. New Phytol 197(1): 58-64.
   
6. Lamport, D. T., Kieliszewski, M. J. and Showalter, A. M. (2006). Salt stress upregulates periplasmic arabinogalactan proteins: using salt stress to analyse AGP function. New Phytol 169(3): 479-492.
   
7. Popper, Z. A. (2011). Extraction and Detection of Arabinogalactan Proteins in The Plant Cell Wall - Methods and Protocols, edited by John M. Walker. Humana Press, New York, pp.245-254.
   
8. Qi, W., Fong, C. and Lamport, D. T. (1991). Gum arabic glycoprotein is a twisted hairy rope: a new model based on o-galactosylhydroxyproline as the polysaccharide attachment site. Plant Physiol 96(3): 848-855.
   
9. Zhao, Z. D., Tan, L., Showalter, A. M., Lamport, D. T. and Kieliszewski, M. J. (2002). Tomato LeAGP-1 arabinogalactan-protein purified from transgenic tobacco corroborates the Hyp contiguity hypothesis. Plant J 31(4): 431-444.