Recombinant Human HS3ST1 Protein, CF

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

Recombinant Human HS3ST1 Protein, CF Summary

• Details of Functionality: Measured by its ability to transfer sulfate from PAPS to heparan sulfate. The specific activity is >30 pmol/min/μg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived human Heparan Sulfate 3-O-Sulfotransferase 1/HS3ST1 protein
  Arg21-His307, with an N-terminal 6-His tag
• Accession #: O14792
• N-terminal Sequence: His
• Protein/Peptide Type: Recombinant Enzymes
• Gene: HS3ST1
• Purity: >90%, 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: 35 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: 38-50 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-HCl and NaCl.
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by silver stain
• 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 Heparan Sulfate3-O-Sulfotransferase 1/HS3ST1 (rhHS3ST1) (Catalog # 5968-ST)
  • Donor Substrate: PAP³²S (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 combining 10X stock and diluting 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 PAP³²S 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 rhHS3ST1 to 100 µg/mL in 1X Assay Buffer.
  8. Load 15 µL of the 100 µg/mL rhHS3ST1 into empty wells of the same plate as the curve. Include a control containing 15 µL of Assay Buffer.
  9. Add 10 µL of 50 µg/mL Coupling Phosphatase 3 to wells containing enzyme and control, excluding the standard curve.
  10. Add 25 µL of reaction mixture to the wells, excluding the standard curve.
  11. Seal plate and incubate at 37 ºC for 20 minutes.
  12. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
  13. Add 100 µL of deionized water to all wells. Mix briefly.
  14. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
  15. Read plate at 620 nm (absorbance) in endpoint mode.
  16. 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:
  • rhHS3ST1: 1.5 µg
  • Coupling Phosphatase 3: 0.5 µg
  • Heparan Sulfate: 100 µg
  • PAP³²S: 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 HS3ST1 Protein, CF

• 3OST
• 3OST1
• 3-OST-1
• 3OST1Heparan sulfate 3-O-sulfotransferase 1
• EC 2.8.2
• EC 2.8.2.23
• h3-OST-1
• heparan sulfate (glucosamine) 3-O-sulfotransferase 1
• Heparan sulfate D-glucosaminyl 3-O-sulfotransferase 1
• heparan sulfate glucosamine 3-O-sulfotransferase 1
• heparin-glucosamine 3-O-sulfotransferase
• HS3ST1

Background

Heparan sulfate is a highly sulfated polysaccharide that can be found on cell surface and within 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 detached from the protein core and 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 2‑O position by heparan sulfate 2‑O sulfotransferase (HS2ST). The N‑acetylglucosamine residues can be sulfated at 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). HS3ST1 is a rate-limiting enzyme for generating an antithrombin-binding pentasaccharide epitope on heparan sulfate and heparin (5, 6). Unlike other sulfotransferases that have signal-anchor domains and are type II membrane integral proteins in Golgi apparatus, HS3ST1 lacks a transmembrane domain and is likely to be an intraluminal enzyme (7, 8). The enzyme activity of the recombinant HS3ST1 is measured using a phosphatase-coupled 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. Rosenberg, R.D. et al. (1997) J. Clin. Invest. 100:S67.
6. Esko, J.D and Lindahl, U. (2001) J. Clin. Invest. 108:169.
7. Shworak, N.W. et al. (1997) J. Biol. Chem. 272:28008.
8. Liu, J. et al. (1999) J. Biol. Chem. 274:5158.
9. Prather, B. et al. (2012) Anal.Biochem.423:86

Publications for Heparan Sulfate 3-O-Sulfotransferase 1/HS3ST1 (5968-ST)(2)

Publications using 5968-ST

• J Deng, C Koutras, J Donnelier, M Alshehri, M Fotouhi, M Girard, S Casha, PS McPherson, SM Robbins, JEA Braun Neurons Export Extracellular Vesicles Enriched in Cysteine String Protein and Misfolded Protein Cargo Sci Rep, 2017-04-19;7(1):956. 2017-04-19 [PMID: 28424476] (Bioassay, Human)
• 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: HS3ST1
• Uniprot:
  • O14792()