Recombinant Human C2GNT3/GCNT4 His-tag Protein

GCNT4 (Catalog # 11162-GT) recognizes core-1 O-glycan and convert it to core-2 type O-glycan.

1 μg/lane of Recombinant Human GCNT4 His-tag (Catalog # 11162-GT ) was resolved with SDS-PAGE under reducing conditions and visualized by silver staining, showing a band at 50-66 kDa.

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

Recombinant Human C2GNT3/GCNT4 His-tag Protein Summary

• Additional Information: Glucosaminyl (N-acetyl) Transferase 4 Core 2
• Details of Functionality: Measured by its ability to transfer GlcNAc from UDP-GlcNAc to B1-3 galactosyl-N-acetyl galactosamine. The specific activity is >75 pmol/min/μg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived human C2GNT3/GCNT4 protein
  Arg35-Ser453, with C-terminal 6-His tag
• Accession #: Q9P109.1
• N-terminal Sequence: Arg35
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 49.7 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: 50-66 kDa, under reducing conditions.

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Glycosyltransferase Activity Kit (Catalog # EA001)
  • Assay Buffer: 0.1 M MES, 10 mM DTT, 5 mM CaCl₂, pH 6.0
  • Recombinant Human C2GNT3/GCNT4 (rhGCNT4) (Catalog # 11162-GT)
  • UDP-GlcNAc (Sigma, Catalog # U4375), 50 mM stock in 50% ethanol
  • beta -1,3 Galactosyl-N-acetyl galactosamine (V-labs, Catalog # GN213), 20 mM stock in deionized water
  • 96-well Clear Plate (Catalog # DY990)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Prepare a standard curve from the 1 mM Phosphate Standard provided by the Glycosyltransferase Activity Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of Assay Buffer for a 100 µM stock.
  2. Continue standard curve by performing six additional one-half serial dilutions of the 100 µM Phosphate stock using Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
  3. Prepare a Reaction Mixture containing 0.4 mM UDP-GlcNAc, 1 mM beta -1,3 Galactosyl-N-acetyl galactosamine, and 4 µg/mL Coupling Phosphatase 1 in Assay Buffer.
  4. Dilute rhGCNT4 to 20 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 20 µg/mL rhGCNT4 into empty wells of the same plate as the curve. Include a Control containing 25 μL of Assay Buffer.
  7. Start the reactions by adding 25 µL of Reaction Mixture to all wells, excluding the standard curve and curve blank.
  8. Seal plate and incubate at 37 °C for 20 minutes.
  9. Add 30 µL of the Malachite Green Reagent A to all wells used, including standard curve. Mix briefly.
  10. Add 100 µL of deionized water to all wells used, including standard curve. Mix briefly.
  11. Add 30 µL of the Malachite Green Reagent B to all wells used, including standard curve. Mix briefly.
  12. Seal plate and incubate at room temperature for 20 minutes.
  13. Read plate at 620 nm (absorbance) in endpoint mode.
  14. 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:
  • rhGCNT4: 0.5 µg
  • Coupling Phosphatase 1: 0.1 µg
  • beta -1,3 Galactosyl-N-acetyl galactosamine: 0.5 mM
  • UDP-GlcNAc: 0.2 mM

Notes

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

Alternate Names for Recombinant Human C2GNT3/GCNT4 His-tag Protein

• beta-1,3-galactosyl-O-glycosyl-glycoproteinbeta-1,6-N-acetylglucosaminyltransferase 4
• C2GNT3
• core 2 beta-1,6-N-acetylglucosaminyltransferase 3
• Core 2-branching enzyme 3
• Core2-GlcNAc-transferase 3
• EC 2.4.1.102
• GCNT4
• glucosaminyl (N-acetyl) transferase 4, core 2
• glucosaminyl (N-acetyl) transferase 4, core 2(beta-1,6-N-acetylglucosaminyltransferase)
• Gm279
• Gm73

Background

Mucin-type O-glycans are initiated with an O-GalNAc attachment to a serine or threonine in a polypeptide. The O-GalNAc residues are subsequently extended by various glycosyltransferases resulting in different types of O-glycans. Most O-glycans contain the core 1 structure, Gal beta 1-3GalNAc. GCNT4 is a member of the glucosaminyl (N-acetyl) transferase family and it converts the core 1 O-glycan to core 2 O-glycan, Gal beta 1-3(GlcNAc beta 1-6)GalNAc, via the addition of a GlcNAc residue (1). Various ligand carbohydrates can be formed from core 2 branched oligosaccharides. For example, sialyl Le^x in mucin‑type glycoproteins of blood cells can be formed from core 2 branched oligosaccharides (2,3). GCNT4 does not have core 4 O-glycan or I-branching enzyme activity. GCNT4 has been shown to play a role in gastric cancers, where GCNT4 is downregulated and its overexpression prevents cancer cell growth (4, 5). The activity of the recombinant human GCNT-4 was determined using a phosphatase‑coupled glycosyltransferase assay (6).

1. Schwientek, T. et al. (2000) J Biol Chem. 275:11106.
2. Hemmerich, S. et al. (1995) J. Biol. Chem. 270:12035.
3. Wilkins, P.P. et al. (1996) J. Biol. Chem. 270:18732.
4. Sun, H. et al. (2020) Onco. Targets Ther. 25:8601.
5. Hu, W. et al. (2021) Bioengineered 12:11634.
6. Wu, Z.L. et al. (2011) Glycobiology 21:727.

Bioinformatics

• Uniprot:
  • Q9P109.1()