Recombinant Human GALNT10 Protein, CF

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

Recombinant Human GALNT10 Protein, CF Summary

• Details of Functionality: Measured by its ability to transfer GalNAc from UDP-GalNAc to peptide MUC5AC-3/13 from AnaSpec, Inc. The specific activity is >180 pmol/min/μg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human Polypeptide GalNac Transferase 10/GALNT10 protein
  Leu71-Asn603, with a C-terminal 6-His tag
• Accession #: Q86SR1
• N-terminal Sequence: Leu71
• Protein/Peptide Type: Recombinant Enzymes
• Gene: GALNT10
• Purity: >80%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Endotoxin Note: <0.10 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 62 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: 60-68 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: >80%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• 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 10/GALNT10 (rhGALNT10) (Catalog # 7575-GT)
  • UDP-GalNAc (Sigma, Catalog # U5252), 10 mM stock in deionized water
  • MUC5AC-3/13 peptide (AnaSpec Inc, Catalog # 61332), 2 mM in deionized water
  • 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. Dilute Coupling Phosphatase I to 10.67 ng/µL in Assay Buffer.
  4. Prepare reaction mixture by combining 26 µL of 10 mM UDP-GalNAc, 39 µL of 2 mM MUC5AC-3/13 peptide, and 195 µL of 10.67 ng/µL Coupling Phosphatase I (sufficient to test 10 wells).
  5. Dilute rhGALNT10 to 6 ng/µL in Assay Buffer.
  6. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
  7. Load 25 µL of the 6 ng/µL rhGALNT10 into the plate. Include a Control containing 25 µL of Assay Buffer.
  8. Add 25 µL of reaction mixture to the wells, excluding the standard curve and curve blank.
  9. Cover the plate with parafilm or a plate sealer and incubate at 37 ºC for 20 minutes.
  10. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
  11. Add 100 µL of deionized water to all wells. Mix briefly.
  12. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
  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:
  • rhGALNT-10: 0.15 µg
  • Coupling Phosphatase I: 0.2 µg
  • MUC5AC-3/13 peptide: 0.15 mM
  • UDP-GalNAc: 0.5 mM

Notes

Coomassie is a registered trademark of Imperial Chemical Industries Ltd.

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

Alternate Names for Recombinant Human GALNT10 Protein, CF

• DKFZp586H0623
• EC 2.4.1.41
• FLJ00205
• FLJ11715
• GalNAc transferase 10
• GALNACT10
• GalNAc-T10
• GALNT10
• Polypeptide GalNAc transferase 10
• polypeptide N-acetylgalactosaminyltransferase 10
• PPGALNACT10
• pp-GaNTase 10
• PPGANTASE10
• Protein-UDP acetylgalactosaminyltransferase 10
• UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 10
• UDP-N-acetyl-alpha-D-galactosamine:polypeptideN-acetylgalactosaminyltransferase 10 (GalNAc-T10)

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). GALNT10 exhibits a single large preference for Ser/Thr‑O‑GalNAc at the +1 (C‑terminal) position relative to the Ser or Thr acceptor site (7) and is able to glycosylate substrates of tri‑ and even tetraglycosylated peptides, which may complete mucin domain assembly; therefore it is classified as the late transferase (6). Human GALNT10 is found in the small intestine, stomach, pancreas, ovary, thyroid gland and spleen (8). The enzymatic activity of recombinant human GALNT10 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. Perrine, C.L. et al. (2009) J. Biol. Chem. 284:20387.
8. Cheng, L. et al. (2002) FEBS Lett. 531:115.
9. Wu, Z.L. et al. (2011) Glycobiology 21:727.

Bioinformatics

• Gene Symbol: GALNT10
• Uniprot:
  • Q86SR1(Human)