Recombinant Human Active Heparanase/HPSE Protein, CF

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Summary
Reactivity HuSpecies Glossary
Applications Enzyme Activity
Format
Carrier-Free

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Recombinant Human Active Heparanase/HPSE Protein, CF Summary

Details of Functionality
Measured by its ability to release biotinylated heparan sulfate from Recombinant Human Syndecan‑4 (Catalog # 2918-SD). <60 ng of Recombinant Human Active Heparanase/HPSE is required for the release of 50% of heparan sulfate from 2 μg of Recombinant Human Syndecan-4, as measured under the described conditions.
Source
Chinese Hamster Ovary cell line, CHO-derived human Heparanase/HPSE protein
Gln36-Ile543 with N-terminal 6-His tag
The linker peptide from Ser110-Gln157 is removed by protease treatment
Accession #
N-terminal Sequence
His & K158
Protein/Peptide Type
Recombinant Enzymes
Gene
HPSE
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

Theoretical MW
43 kDa & 9 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
50-60 kDa & 7-9 kDa, reducing conditions
Publications
Read Publications using
7570-GH in the following applications:

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 E64.
Purity
>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Assay Procedure
  • Labeling Buffer: 25 mM MES, 0.5% (v/v) Triton® X-100, 2.5 mM MgCl2, 2.5 mM MnCl2, 1.25 mM CaCl2, 0.75 mg/mL BSA, pH 7.0
  • Assay Buffer: 0.1 M NaOAc, pH 4.0
  • Gel Running Buffer: 40 mM Tris, 1 mM EDTA, adjust to pH 8.0 with acetic acid
  • Recombinant Human Active Heparanase/HPSE (rhHPSE) (Catalog # 7570-GH)
  • Recombinant Human Heparan Sulfate 3-O-Sulfotransferase 1/HS3ST1 (rhHS3ST1) (Catalog # 5968-ST)
  • 8% SDS-PAGE (approximately 15 cm x 20 cm, 20 lanes per gel)
  • Recombinant Human Syndecan-4 (rhSyndecan-4) (Catalog # 2918-SD)
  • PAP35S (prepared in-house using the PAPS Synthesis Kit (Catalog # EA005), ~1 μM and ~2 x 106 cpm/μL)
  • Gel loading buffer: 0.15 M Tris, 20.8 mM SDS, 1.15 M Glycine, 174 µM Bromophenol Blue, 30% Glycerol
  • Blotting paper (Fisher Sci., Catalog # 05-714-4)
  • Gel dryer
  • Glogos® II autorad markers (Stratagene, Cat. # 420202) or equiv.
  • Blue sensitive medical X-ray film
  • X-ray film cassette
  • Film developer (Konica SRX-101A Medical Film Processor) or equiv.
  • Liquid scintillation counter (Beckman Coulter, Model # LS5000TD) or equiv.
  • Liquid scintillation fluid (Beckman Coulter, Catalog # 141349) or equiv.
  1. Mix rhSyndecan4, rhHS3ST1 and PAP35S at concentrations of 0.133 mg/mL, 0.067 mg/mL, and 0.067 μM respectively in Labeling Buffer.
  2. Incubate the mixture at room temperature overnight (some precipitation may occur).
  3. Dilute rhHPSE to 66.7, 6.67, 3.34, 1.67, 0.667, 0.334, 0.0667, and 0.00667 µg/mL in Assay Buffer.
  4. Combine 15 µL of rhHPSE at each dilution with 15 µL of radiolabeled reaction mix. Include a control containing 15 µL Assay Buffer and 15 µL reaction mix. Incubate at 37 °C for 20 min.
  5. Add 15 µL gel loading buffer to each reaction. Mix.
  6. Load 30 µL of each rxn per lane on a gel. Leave empty lanes between samples. Run at 200 V for 35 min.
  7. Transfer gel onto blotting paper and dry with gel dryer for 1 hr or until fully dry.
  8. Affix two autorad markers to the blotting paper next to the dried gel.
  9. In a darkroom expose dried gel to X-ray film by enclosing overnight in a cassette. Develop the following day.
  10. Using the dried gel, begin marking regions to be cut out for scintillation counting. Mark a horizontal line across the top of the entire gel just under the bottom of the wells.
  11. Using the developed film as an overlay, mark a second line just above the loading dye migration which should correspond to a region between substrate and cleaved product. Note: It will be easiest to use the highest enzyme containing lanes to identify the product. For the control, identify the empty region where the product would appear.
  12. Draw a third line just below where the labeled product migrated (ignore any free sulfate which will appear in equivalent quantity in all lanes and will have migrated the furthest).
  13. The area between the top two lines is considered to contain the labeled starting material and between the bottom two lines is the compact cleavage product resulting from the reaction.
  14. Mark vertical lines distinguishing one lane (reaction condition) from another.
  15. Cut each region (two per lane) and place each into a separate liquid scintillation vial.
  16. Add 5 mL liquid scintillation fluid to each vial and count the vials for 35S.
  17. Determine the amount of rhHPSE required for 50% cleavage by plotting % cleavage vs. ng of rhHPSE with 4-PL fitting.
Per Reaction:
  • Syndecan-4: 2 μg
  • rhHPSE: 1000, 100, 50, 25, 10, 5, 1 and 0.1 ng

Notes

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

Alternate Names for Recombinant Human Active Heparanase/HPSE Protein, CF

  • EC 3.2
  • Endo-glucoronidase
  • HEP
  • Heparanase
  • Heparanase-1
  • HPA1
  • HPAheparanase exon 9 and 10 deletion
  • HPR1
  • HPSE
  • HPSE1heparanase-1
  • HSE1
  • HSE1heparanase exon10-deletion

Background

Heparanase (HPSE) selectively cleaves heparan sulfate at specific sites on heparan sulfate proteoglycans (HSPGs) (1, 2, 3, 4). The enzyme is synthesized as an inactive 65 kDa proenzyme that is secreted via the Golgi apparatus and associates with the cell membrane through interaction with HSPGs (5). It is then endocytosed and transferred to lysosomes (6) where cathepsin L activates it by removing an internal inhibitory peptide, forming a heterodimer composed of an 8 kDa and a 50 kDa subunit (7, 8). Under certain stimuli, the active enzyme is transferred back to the cell surface, where it participates in extracellular matrix degradation and remodeling (9). HPSE facilitates cell migration associated with metastasis, wound healing and inflammation (10). An increase in its activity is associated with an increase in VEGF activity, which further enhances angiogenesis (11). HPSE also enhances shedding of syndecans and increases endothelial invasion and angiogenesis in myelomas (12). It acts as a procoagulant by increasing the generation of activation factor X in the presence of tissue factor and activation factor VII (13). In addition, it increases cell adhesion to the extracellular matrix (ECM), independent of its enzymatic activity (14). HPSE is highly expressed in placenta and spleen and weakly expressed in lymph node, thymus, peripheral blood leukocytes, bone marrow, endothelial cells, fetal liver and tumor tissues (15). The enzyme activity of recombinant human HPSE was assayed using 35S-labeled recombinant syndecan 4 (16).
  1. Vlodavsky, I. et al. (1999) Nat. Med. 5:793.
  2. Hulett, M.D. et al. (1999) Nat. Med. 5:803.
  3. Gong, F. et al. (2003) J. Biol. Chem. 278:35152.
  4. Peterson, S.B. and Liu, J. (2010) J. Biol. Chem. 285:14504.
  5. Nadav L. et al. (2002) J. Cell Sci. 115:2179.
  6. Gingis-Velitski, S. et al. (2004) J. Biol. Chem. 279:44084.
  7. Abboud-Jarrous, G. et al. (2008) J. Biol. Chem. 283:18167.
  8. Zetser, A. et al. (2004) J. Cell Sci. 117:2249.
  9. Zcharia E. et al. (2001) J. Mammary Gland Biol. Neoplasia 6:311.
  10. Fux, L. et al. (2009) Trends Biochem. Sci. 34:511.
  11. Cohen-Kaplan, V. et al. (2008) Int. J. Cancer 123:2566.
  12. Purushothaman, A. et al. (2010) Blood 115:2449.
  13. Nadir, Y. et al. (2010) Haematologica 95:1927.
  14. Goldshmidt, O. et al. (2003) FASEB J. 17:1015.
  15. Kussie, P.H. et al. (1999) Biochem. Biophys. Res. Commun. 261:183.
  16. Ethen, C.M. et al. http://www.rndsystems.com/resources/images/21388.pdf

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7570-GH
Species: Hu
Applications: Enzyme Activity

Publications for Heparanase/HPSE (7570-GH)(5)

We have publications tested in 1 confirmed species: Human.

We have publications tested in 3 applications: Bioassay, Click Chemistry, WB.


Filter By Application
Bioassay
(3)
Click Chemistry
(1)
WB
(1)
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Human
(3)
All Species
Showing Publications 1 - 5 of 5.
Publications using 7570-GH Applications Species
Wu Z, Person A, Anderson M, Burroughs B, Tatge T, Khatri K, Zou Y, Wang L, Geders T, Zaia J, Sackstein R Imaging specific cellular glycan structures using glycosyltransferases via click chemistry. Glycobiology, 2017;0(0):. 2017 [PMID: 29186441] (Click Chemistry, Human) Click Chemistry Human
N Poupard, P Badarou, F Fasani, H Groult, N Bridiau, F Sannier, S Bordenave-, C Kieda, JM Piot, C Grillon, I Fruitier-A, T Maugard Assessment of Heparanase-Mediated Angiogenesis Using Microvascular Endothelial Cells: Identification of ?-Carrageenan Derivative as a Potent Anti Angiogenic Agent Mar Drugs, 2017;15(5):. 2017 [PMID: 28486399] (Bioassay) Bioassay
N Poupard, H Groult, J Bodin, N Bridiau, S Bordenave-, F Sannier, JM Piot, I Fruitier-A, T Maugard Production of heparin and ?-carrageenan anti-heparanase derivatives using a combination of physicochemical depolymerization and glycol splitting Carbohydr Polym, 2017;166(0):156-165. 2017 [PMID: 28385219] (Bioassay) Bioassay
Nataliya Bohdan Heparanase Activates Antithrombin through the Binding to Its Heparin Binding Site PLoS ONE, 2016;11(6):e0157834. 2016 [PMID: 27322195] (WB,, Human) WB Human
Tinholt M, Stavik B, Louch W, Carlson C, Sletten M, Ruf W, Skretting G, Sandset P, Iversen N Syndecan-3 and TFPI colocalize on the surface of endothelial-, smooth muscle-, and cancer cells. PLoS ONE, 2015;10(1):e0117404. 2015 [PMID: 25617766] (Bioassay, Human) Bioassay Human

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Bioinformatics

Gene Symbol HPSE
Entrez
Uniprot