• Purity

  >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie? Blue stain at 5 μg per lane

• Endotoxin Level

  <1.0 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to hydrolyze 4-methylumbelliferyl-N-acetyl-beta -D-glucosaminide (4-MU-GlcNAc) The specific activity is >3500 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  E. coli-derived Gln22-Lys737, with an N-terminal Met and 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Met

• Predicted Molecular Mass

  83 kDa

• SDS-PAGE

  66-76 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 50 mM MES, 100 mM NaCl, pH 5.5

• Recombinant B. thetaiotaomicron O-GlcNAcase/OGA (rBtOGA) (Catalog # 6779-GH)

• Substrate: 4-Methylumbelliferyl-N-acetyl-beta -D-glucosaminide (Sigma, Catalog # M2133), 50 mM stock in DMSO

• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)

• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rBtOGA to 2 ng/μL in Assay Buffer.

2. Dilute Substrate to 2 mM in Assay Buffer.

3. Load into a plate 50 μL of 2 ng/μL rBtOGA, and start the reaction by adding 50 μL of 2 mM Substrate. For Substrate Blanks, load 50 μL of Assay Buffer and 50 μL of 2 mM Substrate.

4. Read plate at excitation and emission wavelengths of 365 nm and 445 nm, respectively, in kinetic mode for 5 minutes.

5. Calculate specific activity:

     *Adjusted for Substrate Blank
     **Derived using calibration standard 4-Methylumbelliferone (4-MU) (Sigma, Catalog # M1381).

Per Well:

• rBtOGA: 0.1 μg

• Substrate: 1 mM

Background: O-GlcNAcase/OGA

The addition of the monosaccharide beta -N-acetyl-D-glucosamine to serine and threonine residues in proteins (O?GlcNAc glycosylation) is a dynamic, intracellular, post?translational modification that shares features with phosphorylation (1). Almost all major classes of intracellular proteins are modified with O?GlcNAc glycosylation. O?GlcNAc is known to regulate gene transcription, act as an energy sensor to desensitize insulin response, and coordinate phosphorylation to control protein activity (2, 3, 4, 5). In humans, O?GlcNAc is introduced by a single O?linked N?acetylglucosamine transferase, OGT, and removed by a single glycosidase, OGA. Both OGT and OGA are cytosolic. Enzymes with high sequence homology to human OGA have been found in human pathogens and symbionts (6, 7, 8), where these enzymes are proposed to metabolize O?GlcNAc in human proteins. OGA from the human gut symbiont   Bacteroides thetaiotaomicron and its human counterpart are very similar in structure and function, and both enzymes operate via an unusual 'substrate-assisted' catalytic mechanism (8, 9). Recombinant   B. thetaiotaomicron OGA can be used as an enzymatic tool to investigate O?GlcNAc glycosylation.

• References:

1. Wells, L. et al. (2001) Science 291:2376.

2. Wells, L. et al. (2003) Cell. Mol. Life Sci. 60:222.

3. Yang, X. et al. (2008) Nature 451:964-9.

4. Love, D.C.and Hanover, J.A. (2005). Sci. STKE 312:1.

5. Hart, G. W. et al. (2011)  Annu. Rev. Biochem. in press.

6. Martinez-Fleites, C. (2008) Nat. Struct. Mol. Biol. 15:764.

7. Rao, F. V. et al. (2006) The EMBO J. 25:1569.

8. Dennis, R.J. et al. (2006) Nat. Struct. Mol. Biol. 13:365.

9. He, Y. et al. (2008) Carbohydr. Res. 344:627.

• Entrez Gene IDs:

  10724 (Human); 76055 (Mouse); 154968 (Rat); 1074035 (B. thetaiotaomicron)

• Alternate Names:

  bifunctional protein NCOAT; FLJ11229; GH84; HEXC; HEXC3; Hexosaminidase B; hyaluronidase in meningioma; KIAA0679; MEA5FLJ23355; meningioma expressed antigen 5 (hyaluronidase); Meningioma-expressed antigen 5; MGEA5; NCOAT; Nuclear cytoplasmic O-GlcNAcase and acetyltransferase; OGA; OGlcNAcase; O-GlcNAcase