Home Protocols Endo-α-N-Acetylgalactosaminidase Application Note 1

Endo-α-N-Acetylgalactosaminidase Application Note 1

For use with O-Glycosidase (P0733) and α2-3,6,8 Neuraminidase (P0720). 

Introduction

Enzymatic deglycosylation of a protein containing Core 1 O-glycans. Endo-α-N-Acetylgalactosaminidase (O-glycosidase) removes O-glycans from Bovine fetuin.

For many cancers, such as colon, ovary, uterus and bladder (mucosas), tumor progression and its poor prognosis strongly correlates with alterations in the patterns of mucin O-glycosylation. For instance, β3Gn-T6 (the enzyme responsible for Core 3 O-glycan synthesis), is abundant in normal colon tissue while its expression is strongly downregulated in adenocarcinoma (1,2,3). As a result, mucin glycosylation switches from common Core 3 O-glycan structures to short Core 1 structures. These Core 1 structures, T and Tn, are hallmark epitopes of cell malignancy (4).

The study of glycan aberrations in cancer opens new avenues in the development of novel therapies. In this regard, the endo-O-glycosidase is proving useful to characterize and quantitate a variety of commonly found O-glycan forms. 

This application describes the use of a recombinant Endo-α-N-Acetylgalactosaminidase that can cleave core 1 O-linked disaccharides (5) and immature Tn core (GalNAc). Conditions have been determined that allow this O-glycosidase to be used under the same denaturing conditions used for PNGase F digestion. 

Materials
Endo-α-N-Acetylgalactosaminidase (NEB #P0733); Fetuin (NEB #P6042); G7 buffer (NEB #B0701S); Denaturing buffer (NEB #B0701S); 10X NP-40 (NEB #B0701S); α2-3,6,8 Neuraminidase (NEB #P0720S); Core 1 disaccharide (Galβ1,3GalNAc; Accurate Chemical #BCR20/06).

Protocol

  1. Preparation of substrate: Dissolve 10 mg of Fetuin in 1 ml water.
  2. A) Denaturing Reaction Conditions:

    Denaturation Reaction:
    Fetuin (10 mg/ml) in water: 18 μl
    10X Denaturing Buffer: 2 μl
    Heat 95°C for 5 min.; Chill on ice and spin.

    Digestion of substrate:
    Denaturing Reaction: 20 μl
    10X NP-40: 4 μl
    10X G7 Buffer: 4 μl
    Milli-Q Water: 18 μl
    α2-3,6,8 Neuraminidase (50 u/μl): 2 μl
    Endo-α-N-Acetylgalactosaminidase (40,000 u/μl): 2 μl

    B) Non-Denaturing Reaction Conditions:
    Fetuin (10 mg/ml) in water: 18 μl

    Digestion of substrate:
    10X G7 Buffer: 4 μl
    Milli-Q Water: 24 μl
    α2-3,6,8 Neuraminidase (50 u/μl): 2 μl
    Endo-α -N-Acetylgalactosaminidase (40,000 u/μl): 2 μl
  3. Incubate at 37°C for 1 to 4 hours. After incubation, add 1 μl of 4M KCl followed by 150 μl of methanol. Chill overnight at 4°C to precipitate proteins. After the overnight precipitation, spin the sample at 14,000 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 (Bio-Rad; #142-6424) for 5 minutes. Collect the supernatant with a 1 ml 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 sugars by HPAEC-PAD Chromatography using the following conditions:
    Column: CarboPac 20 with Amino Guard.
    Elution: 40 mM NaOH isocratic for 12 minutes, 150 mM 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 Galβ1,3GalNAc standard (30 nanograms).

References

  1. Iwai, T. et al. (2005) Proceedings of the National Academy of Sciences U.S.A., 102, 4572-7.
  2. Iwai, T. et al. (2002) Journal of Biological Chemistry, 277, 12808-9.
  3. Capon, C. et al. (2001) Biochemical Journal, 358, 657-64.
  4. Springer, G.F. et al. (1997) Journal of Molecular Medicine, 75, 594-602.
  5. Koutsioulis, D. et al. (2008) Glycobiology, 18, 799-805.