Home Protocols Protocol for α1-3,6 Galactosidase (P0731)

Protocol for α1-3,6 Galactosidase (P0731)

Introduction

Removal of terminal galactose from a glycoprotein containing tri- and tetra-antennary N-linked sugars.

With advances in transplantation and stem cell research, there has been a renewed interest in the study of glycoforms carrying the Galα1-3Gal epitope.

This motif is widely present in non-primate mammalian cells, while absent in Old World monkeys and humans (1). Naturally occurring high levels of anti-Gal antibodies cause xenotransplantations to fail within a few hours (2). This ability to ablate Gal-exposing cells has been exploited to develop safer human tissue grafts (3).

Specific glycosidases are required to characterize these kinds of systems. This application describes the use of an α1-3,6 Galactosidase from Xanthomonas manihotis (recombinant expressed in E.coli) to remove terminal galactose residues from the tri- and tetra- antennary N-glycoprotein Bovine Thyroglobulin (4).

Materials
α1-3,6-Galactosidase (NEB #P0731 ), Galactose standard (Sigma #G0750), Bovine Thyroglobulin (Calbiochem; #609310), 10X GlycoBuffer 1 (supplied with enzyme).


Protocol

  1. Preparation of Glycoprotein substrate: Dialyze 1 μl of a 10 mg/ml solution of Bovine Thyroglobulin in water against 100 volumes of G6 buffer, for 4 hours at 4°C. The dialyzed solution can be stored in aliquots of 100 μl.
  2. Set up the Degalactosylation reaction:
    Glycoprotein Substrate 10 mg/μl: 85 μl
    GlycoBuffer 1 (10X): 10 μl
    Xanthomonas α-1-3,6 galactosidase 5 μl: (20 units)
    Total volume: 100 μl
  3. Incubate at 37°C for 4 hours. Add 200 μl water followed by 600 μl methanol (1)*. Chill overnight at 4°C to precipitate proteins. After the overnight precipitation, spin the sample at 14 K rpm for 30 minutes, and reserve the supernatant.
  4. Concentrate supernatant to dryness with a Speed Vac set at medium heat (Savant; equipped with a high vacuum pump and finger trap immersed in a Dewar containing isopropanol and dry ice). Reconstitute with 400 μl Milli-Q water.
  5. De-ionize the sample from step 4 by gently rocking in 200 μl of prepared mixed bed ion exchange resin AGAG 501-X8 for 5 minutes (Bio-Rad; #142-6424). Collect the supernatant with a 1ml syringe using a 23 gauge needle. Note: before use, the resin must be converted to the acetate form by soaking in an equal volume of 1 M acetic acid followed by washing ten times with equal volumes of water.
  6. Remove the needle and load the entire sample (400 μl) from Step 5 to an activated Sep Pak cartridge (Waters; #WAT051910). Collect the entire flow through (400 μl). Wash the Sep-Pak 2 times with 400 μl of Milli-Q water and pool the washes with the flow through. Concentrate to 70 μl using a Speed Vac. Note: before use, the Sep-Paks are activated by washing two times with 400 μl methanol followed by 4 times with 400 μl Milli-Q water.
  7. Detect free galactose by HPAEC-PAD Chromatography using the following conditions: Column: CarboPac 20 with Amino Guard. Elution: 20mM NaOH isocratic for 12 minutes, 150mM regeneration for 10 minutes, flow rate: 0.5 μl/min. Detection: Pulse electrochemical, Au electrode, quadruple potential. Injection sample: 30 μl, with or without internal Galactose standard (30 nanograms).


References

  1. Koike, C. et al. (2002) The Journal of Biological Chemistry, 227, 10114-10120.
  2. Cooper, D.K., et al. (1993) Lancet, 342, 682-683.
  3. Hewitt, Z., et al. (2007) Stem Cells, 25, 10-18.
  4. Wong-Madden, T., et. al. (1995) Glycobiology, 5, 19-28.