Recombinant Human HS3ST4 Protein, CF

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

Recombinant Human HS3ST4 Protein, CF Summary

• Details of Functionality: Measured by its ability to transfer sulfate from PAPS to heparan sulfate. The specific activity is >100 pmol/min/μg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived human Heparan Sulfate 3-O-Sulfotransferase 4/HS3ST4 protein
  Gly184-Lys456 with an N-terminal 6-His tag
• Accession #: NP_006031
• N-terminal Sequence: HHHHHHGGAV
• Protein/Peptide Type: Recombinant Enzymes
• Gene: HS3ST4
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 32 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: 35-43 kDa, 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, NaCl and Glycerol.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.
• Assay Procedure:
  • Universal Sulfotransferase Activity Kit (Catalog # EA003)
  • 10X Assay Buffer (supplied in kit): 500 mM Tris, 150 mM MgCl2, pH 7.5
  • Recombinant human HS3ST4 (rhHS3ST4) (Catalog # 6085-ST)
  • Donor Substrate: PAPS (3'-Phosphoadenosine-5'-Phosphosulfate) (Catalog # ES019)
  • Acceptor Substrate: Heparan Sulfate (Celsus Labs, Catalog # HO-3105), 50 mg/mL stock in deionized water
  • 96-well Clear Plate (Catalog # DY990)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Prepare 1X Assay Buffer by diluting 10X stock 10 fold with deionized water.
  2. Dilute 1 mM Phosphate Standard by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of 1X Assay Buffer for a 100 µM stock. This is the first point of the standard curve.
  3. Continue standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock in 1X Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
  4. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of 1X Assay Buffer.
  5. Prepare a reaction mixture containing 0.4 mM PAPS and 4 mg/mL Heparin Sulfate in 1X Assay Buffer.
  6. Dilute Coupling Phosphatase 3 (supplied in kit) to 50 µg/mL in 1X Assay Buffer.
  7. Dilute rhHS3ST4 to 20 µg/mL in 1X Assay Buffer.
  8. Load 15 µL of the 20 µg/mL rhHS3ST4 into empty wells of the same plate as the curve. Include a Negative Control containing 15 µL of 1X Assay Buffer and an Enzyme Only Control containing 15 µL of the 20 µg/mL rhHS3ST4.
  9. Add 10 µL of 50 µg/mL Coupling Phosphatase 3 to wells containing enzyme and Negative Control, excluding the standard curve and Enzyme Only Control.
  10. Add 25 µL of reaction mixture to enzyme and Negative Control, excluding the standard curve and Enzyme Only Control.
  11. Add 35 µL of 1X Assay Buffer to Enzyme Only Controls. Note: All wells now have a volume of 50 µL.
  12. Seal plate and incubate at 37 °C for 30 minutes.
  13. Add 30 µL of the Malachite Green Reagent A to all wells.
  14. Add 100 µL of deionized water to all wells. Mix briefly.
  15. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
  16. Read plate at 620 nm (absorbance) in endpoint mode.
  17. 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 Negative Control and Enzyme Only Control.

  Per Reaction:
  • rhHS3ST4: 0.3 µg
  • Coupling Phosphatase 3: 0.5 µg
  • Heparan Sulfate: 100 µg
  • PAPS: 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 HS3ST4 Protein, CF

• 30ST4
• 3OST4 heparan sulfate 3-O-sulfotransferase-4
• 3-OST-4
• EC 2.8.2
• EC 2.8.2.23
• h3-OST-4
• heparan sulfate (glucosamine) 3-O-sulfotransferase 4
• Heparan sulfate 3-O-sulfotransferase 4
• Heparan sulfate D-glucosaminyl 3-O-sulfotransferase 4
• heparan sulfate glucosamine 3-O-sulfotransferase 4
• HS3ST4

Background

Heparan sulfate is a highly sulfated polysaccharide that can be found on the cell surface and within the extracellular matrix. It is typically covalently attached to the protein core of proteoglycans, such as syndecans and glypicans. Heparin, on the other hand, can be considered as a highly sulfated version of heparan sulfate that is predominantly found in mast cells. Both heparin and heparan sulfate contain disaccharide repeats of uronic acid and N‑acetylglucosamine and are modified by the same sulfotransferases (1, 2). The uronic acid residues can be sulfated at the 2-O position by heparan sulfate 2‑O sulfotransferase (HS2ST). The N‑acetylglucosamine residues can be sulfated at the N, 3-O, and 6-O positions by N‑deacetylase/N‑sulfotransferases (NDSTs), heparan sulfate 3‑O sulfotransferases (HS3STs) and heparan sulfate 6-O sulfotransferases (HS6STs) respectively. There are seven HS3STs in the human genome (3, 4). HS3ST4 and HS3ST2 are brain specific and may participate in HS-dependent neurobiologic events (5). HS3ST4 can generate tetrasulfated heparan sulfate disaccharide, the most highly sulfated sugar found in biological samples (6, 7), and may have a role in assisting HSV-1 entry and spread (8). HS3ST4 is a Golgi resident type II membrane protein and has the longest proline rich stem region among all HS3STs (3, 5). The activity of the recombinant human CHST1 is measured using a PAP-specific phosphatase-coupled sulfotransferase assay (9).

1. Bernfield, M. et al. (1999) Annu. Rev. Biochem. 68:729.
2. Esko, J.D. and Selleck, S.B. (2002) Annu. Rev. Biochem. 71:435.
3. Shworak, N.W. et al. (1999) J. Biol. Chem. 274:5170.
4. Xu, D. et al. (2005) Biochem. J. 386:451.
5. Lawrence, R. et al. (2007) Matrix Biol. 26:442.
6. Mochizuki, H. et al. (2003) J. Biol. Chem. 278:26780.
7. Wu, Z.L. et al. (2004) J. Biol. Chem. 279:1861.
8. Tiwari,V. et al. (2005) Biochem. Biophys. Res. Commun. 338:930.
9. Prather, B. et al. (2012) Anal. Biochem. 423:86.

Publications for Heparan Sulfate 3-O-Sulfotransferase 4/HS3ST4 (6085-ST)(1)

Publication using 6085-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)

Bioinformatics

• Gene Symbol: HS3ST4
• Uniprot:
  • NP_006031()