Recombinant Human CHST4 Protein, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human CHST4 Protein, CF Summary

• Details of Functionality: Measured by its ability to transfer sulfate from PAPS to N-acetyl-D-glucosamine. The specific activity is >150 pmol/min/μg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived human Carbohydrate Sulfotransferase 4/CHST4 protein
  His29-His386, with an N-terminal 6-His tag
• Accession #: NP_005760
• N-terminal Sequence: His
• Protein/Peptide Type: Recombinant Enzymes
• Gene: CHST4
• Purity: >85%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 43 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 45 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol.
• Purity: >85%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Assay Buffer (1X Phosphatase Buffer 3): 50 mM Tris, 15 mM MgCl₂, pH 7.5
  • Recombinant Human Carbohydrate Sulfotransferase 4/CHST4 (rhCHST4) (Catalog # 5357-ST)
  • 3'-Phosphoadenosine-5'-phosphosulfate (PAPS) (Catalog # ES019)
  • N-acetyl-alpha -D-glucosamine (GlcNAc) (Calbiochem, Catalog # 1079), 1 M stock in deionized water
  • Universal Sulfotransferase Activity Kit (Catalog # EA003)
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute 1 mM Phosphate Standard by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of Assay Buffer for a 100 µM stock. This is the first point of the standard curve.
  2. Continue standard curve by performing six one‑half serial dilutions of the 100 µM Phosphate stock in Assay Buffer. The standard curve has a range of 0.078 to 5.0 nmol per well.
  3. Prepare a reaction mixture containing 0.4 mM PAPS, 20 mM GlcNAc and 20 μg/mL Coupling Phosphatase 3 in Assay Buffer.
  4. Dilute rhCHST4 to 10 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 10 µg/mL rhCHST4 into the plate. Include a Control containing 25 µL of Assay Buffer.
  7. Add 25 µL of reaction mixture to the wells, excluding the standard curve.
  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:

  Specific Activity (pmol/min/µg) = (Phosphate released* (nmol) x (1000 pmol/nmol)) / (Incubation time (min) x amount of enzyme (µg))

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

  Per Reaction:
  • rhCHST4: 0.25 µg
  • Coupling Phosphatase 3: 0.500 μg
  • PAPS: 0.2 mM
  • GlcNAc: 10 mM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human CHST4 Protein, CF

• carbohydrate (N-acetylglucosamine 6-O) sulfotransferase 4
• Carbohydrate Sulfotransferase 4
• CHST4
• EC 2.8.2
• EC 2.8.2.-
• EC 2.8.2.17
• EC 2.8.2.21
• galactose/N-acetylglucosamine/N-acetylgalactosamine 6-O-sulfotransferase 3
• Galactose/N-acetylglucosamine/N-acetylglucosamine 6-O-sulfotransferase 3
• GlcNAc6ST2
• GlcNAc6ST-2
• gn6st-2
• GST3
• GST-3
• HECGLCNAC6ST
• HEC-GlcNAc6ST
• HEC-GLCNAC-6-ST
• High endothelial cells N-acetylglucosamine 6-O-sulfotransferase
• L-selectin ligand sulfotransferase
• LSST
• LSSTcarbohydrate sulfotransferase 4
• N-acetylglucosamine 6-O-sulfotransferase 2

Background

The CHST family is comprised of 14 enzymes in human. All members of this family are Golgi-localized type II membrane proteins (1). Only the luminal and enzymatic domain is expressed in each of our recombinant CHST proteins. These enzymes transfer sulfate (i.e., sulfonate) onto the 6-O or 4-O positions of GalNAc, Gal and GlcNAc residues on glycoproteins, proteoglycans and glycolipids (2). This sulfation often creates specific epitopes that can be recognized by extracellular matrix proteins, cell surface receptors and viruses (3). CHST4, also known as high endothelial cells N-acetylglucosamine 6-O-sulfotransferase (HEC-GlcNAc6ST) or L‑selectin ligand sulfotransferases (LSST), catalyzes the transfer of sulfate to position 6 of non-reducing GlcNAc residues within mucin-associated glycans that ultimately serve as L‑selectin ligands (4). It has a catalytic preference for core 2-branched mucin-type O‑glycans, but also has activity toward core 3 type of O‑glycan (5). Human CHST4 shares 72% amino acid sequence identity with the mouse ortholog.The enzymatic activity of the recombinant human CHST4 is measured using a phosphatase-coupled assay (6).

1. deGraffenried, D. and Bertozzi, C.R. (2003) J. Biol. Chem. 278:40282.
2. Hemmerich, S. and Rosen, S. (2000) Glycobiology 10:849.
3. Bowman, K. G. and Bertozzi, C. R. (1999) Chem. Biol. 5:447.
4. Bistrup, A. et al. (1999) J. Cell Biol. 145:899.
5. Uchimura, K. et al. (2002) J. Biol. Chem. 277:3979.
6. Prather, B. et al. (2012) Anal. Biochem. 423:86.

Publications for Carbohydrate Sulfotransferase 4/CHST4 (5357-ST)(2)

Publications using 5357-ST

• Wu ZL, Prather B, Ethen CM Detection of specific glycosaminoglycans and glycan epitopes by in vitro sulfation using recombinant sulfotransferases. Glycobiology, 2010-12-17;21(5):625-33. 2010-12-17 [PMID: 21169395] (Enzyme Assay, Bovine, Porcine)
• Wu ZL, Ethen CM, Larson S, Prather B, Jiang W A versatile polyacrylamide gel electrophoresis based sulfotransferase assay. BMC Biotechnol., 2010-02-10;10(0):11. 2010-02-10 [PMID: 20146816] (Enzyme Assay)

Bioinformatics

• Gene Symbol: CHST4
• Uniprot:
  • NP_005760()