• Purity

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

• Endotoxin Level

  <0.1 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to transfer GalNAc from UDP-GalNAc to d-Glucuronic Acid. The specific activity is >700 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com

• Source

  Mouse myeloma cell line, NS0-derived Asn43-Met330, with an N-terminal 8-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  His

• Predicted Molecular Mass

  34 kDa

• SDS-PAGE

  35-40 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 25 mM HEPES, 10 mM MnCl₂, 10 mM CaCl₂, pH 7.5 (supplied in kit)

• Recombinant Mouse Exostosin‑like 2/EXTL2 (rmEXTL2) (Catalog # 2536-GT)

• UDP-GalNAc (Sigma, Catalog # U5252), 10 mM stock in deionized water

• D-Glucuronic Acid Sodium Salt Monohydrate (Sigma, Catalog # G8645), 0.2 M stock in deionized water

• Glycosyltransferase Activity Kit (Catalog # )

• 96-well Clear Plate (Costar, Catalog # 92592)

• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute 1 mM Phosphate Standard provided by the Glycosyltransferase Kit by adding 40 µL of standard to 360 µL Assay Buffer. This is the first point of the standard curve.

2. Perform six additional one-half serial dilutions in Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.

3. Prepare reaction mixture containing 2 mM UDP-GalNAc, 30 mM D-Glucuronic Acid, and 4 µg/mL Coupling Phosphatase I in Assay Buffer.

4. Dilute rmEXTL2 to 2.5 µg/mL in Assay Buffer.

5. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.

6. Load 25 µL of the 2.5 µg/mL rmEXTL2 into the plate. Include a Substrate Blank containing 25 µL of Assay Buffer.

7. Add 25 µL of reaction mixture (step 3) to the wells, excluding the standard curve and curve blank.

8. Cover the plate with a plate sealer and incubate at 37 °C for 20 minutes.

9. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.

10. Add 100 µL of deionized water to all wells. Mix briefly.

11. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.

12. Read plate at 620 nm (absorbance) in endpoint mode.

13. Calculate specific activity:

     *Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control.

Per Reaction:

• rmEXTL2: 0.0625 µg

• Coupling Phosphatase I: 0.1 µg

• UDP-GalNAc: 1 mM

• D-Glucuronic Acid: 15 mM

Background: Exostosin-like 2/EXTL2

Exostosin (EXT) family of glycosyltransferases are involved in heparan sulfate (HS) and heparin biosynthesis. Mutations of these enzymes are the causes of hereditary multiple exostoses, a rare medical condition in which multiple bony spurs or lumps (also known as exostoses) develop on the bones of a child. Five members of this family have been cloned to date: EXT1, EXT2, EXTL1, EXTL2, and EXTL3. EXT1 and EXT2 are bifunctional enzymes with both beta ‑1,4‑glucuronyltransferase and alpha ‑1,4‑N‑acetylglucosaminyltransferase activities. EXT1 and EXT2 together function as a polymerase for HS synthesis by addition of alternating beta -1,4-GlcA and alpha ‑1,4‑GlcNAc residues on an HS‑protein linkage tetrasaccharide (1). EXTL2 has no beta ‑1,4‑glucuronyltransferase activity but has alpha ‑1,4‑N‑acetylhexosaminyltransferase activity that can transfer either GlcNAc or GalNAc residues (2). Recently, it was reported that that EXTL2 together with a xylose kinase can terminate the polymerization of HS chains by transferring a GlcNAc residue to a phosphorylated HS‑protein linkage tetrasaccharide (3). Given that chondroitin sulfate shares the same linkage tetrasaccharide for biosynthesis, this may be a general mechanism for modulating glycosaminoglycan synthesis. The enzyme activity of recombinant mouse EXTL2 is measured using a phosphatase-coupled assay (4).

• References:

1. Busse, M. et al. (2007) J. Biol. Chem. 282:32802.

2. Kitagawa, H. et al. (1999) J. Biol. Chem. 274:13933.

3. Nadanaka, S. et al. (2013) J. Biol. Chem. 288:9321.

4. Wu, Z.L. et al. (2011) Glycobiology 21:727.

• Entrez Gene IDs:

  2135 (Human); 58193 (Mouse)

• Alternate Names:

  Alpha-1,4-N-acetylhexosaminyltransferase EXTL2; alpha-1,4-N-acteylhexosaminyltransferase; Alpha-GalNAcT EXTL2; EC 2.4.1.223; exostoses (multiple)-like 2; Exostosin like 2; Exostosin-like 2; EXTL2; EXTR2; EXT-related protein 2; Glucuronyl-galactosyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase; processed exostosin-like 2