Recombinant Human GALNT4 Protein, CF

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

Recombinant Human GALNT4 Protein, CF Summary

• Details of Functionality: Measured by its ability to transfer GalNAc from UDP-GalNAc to peptide EA2 from AnaSpec, Inc. The specific activity is >200 pmol/min/μg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human Polypeptide GalNac Transferase 4/GALNT4 protein
  Ala36-Lys578, with C-terminal 6-His tag
• Accession #: Q8N4A0
• N-terminal Sequence: Ala36
• Protein/Peptide Type: Recombinant Enzymes
• Gene: GALNT4
• Purity: >90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <0.10 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 64 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: 57-62 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 and NaCl.
• Purity: >90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: 25 mM Tris, 5 mM MnCl₂ (supplied in kit), 2.5 mM CaCl₂, pH 7.5
  • Recombinant Human Polypeptide GalNac Transferase 4/GALNT4 (rhGALNT4) (Catalog # 7528-GT)
  • UDP-GalNAc (Sigma, Catalog # U5252), 10 mM stock in deionized water
  • EA2 peptide (AnaSpec Inc, Catalog # 63841), 5 mM in 5 mM Tris, pH 7.0
  • Glycosyltransferase Activity Kit (Catalog # EA001)
  • 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 the 1 mM Phosphate Standard to 360 µL of Assay Buffer for a 100 µM stock.
  2. Prepare 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 nmol per well.
  3. Prepare a reaction mixture composed of 1 mM UDP-GalNAc, 0.5 mM EA2 peptide, and 4 ng/µL Coupling Phosphatase I in Assay Buffer.
  4. Dilute rhGALNT4 to 8 ng/µL 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 8 ng/µL rhGALNT4 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 and curve blank.
  8. Cover the plate with parafilm or 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:

  • rhGALNT4: 0.2 µg
  • Coupling Phosphatase I: 0.1 µg
  • EA2 peptide: 0.25 mM
  • UDP-GalNAc: 0.5 mM

Notes

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

Alternate Names for Recombinant Human GALNT4 Protein, CF

• EC 2.4.1.41
• FLJ93314
• GALNACT4
• GalNAc-T4
• GALNT4
• Polypeptide GalNAc transferase 4
• polypeptide N-acetylgalactosaminyltransferase 4
• pp-GaNTase 4
• Protein-UDP acetylgalactosaminyltransferase 4
• UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 4
• UDP-N-acetyl-alpha-D-galactosamine:polypeptideN-acetylgalactosaminyltransferase 4 (GalNAc-T4)

Background

O-glycosylation is a ubiquitous post-translational modification present in secreted and membrane-bound proteins. Polypeptide N-acetylgalactosaminyltransferases (GALNTs) calalyze the initial step for O-glycosylation by transferring GalNAc to Thr or Ser residues (GalNAc alpha 1-O-Ser/Thr) in the Golgi compartment. Structurally, the GALNTs consist of an N-terminal catalytic domain tethered by a short linker to a C-terminal ricin-like lectin domain containing three potential carbohydrate-binding sites (1, 2). Twenty distinct GALNT isoforms have been detected in humans. These isoforms display both unique and overlapping substrate specificities (3, 4, 5) with no known universal consensus glycosylation sequence. Glycosylation of mucins results from the successive, often hierarchical, action of several specific GALNTs (6). Human GALNT4 is widely expressed with particularly high expression in the immune system (7). The enzymatic specificity of GALNT4 depends on the selective binding between the C-terminal lectin domain and the existing O-GalNAc modified residues present within the substrate polypeptide (8). Along with GALNT3, GALNT4 is classified as an intermediate transferase that glycosylates mucin polypeptides that have been previously mono- or di-glycosylated by other transferases (6). The enzymatic activity of recombinant human GALNT4 was determined using a phosphatase-coupled assay (9).

1. Gerken, T.A. et al. (2011) J. Biol. Chem. 286:14493.
2. Ten Hagen, K.G. et al. (2003) Glycobiology 13:1R.
3. Hagen, F.K. et al. (1997) J. Biol. Chem. 272:13843.
4. Gerken, T.A. et al. (2006) J. Biol. Chem. 281:32403.
5. Wandall, H.H. et al. (1997) J. Biol. Chem. 272:23503.
6. Pratt, M.R. et al. (2004) Chem. Biol. 11:1009.
7. Bennett, E.P. et al. (1998) J. Biol. Chem. 273:30472.
8. Hassan, H. et al. (2000) J. Biol. Chem. 275:38197.
9. Wu, Z.L. et al. (2011) Glycobiology 21:727.

Bioinformatics

• Gene Symbol: GALNT4
• Uniprot:
  • Q8N4A0(Human)