Heparan sulfate 2-O-sulfotransferase (HS2ST) transfers sulfate from PAPS to position 2 of hexuronic acid residues in heparan sulfate and heparin, but not to hexuronic acid residues neighboring GlcNSO3(6SO4) residues. HS2ST was purified from the detergent-solubilized extracts of cultured CHO cells and cloned. The enzymatic properties of HS2ST are as follows: The optimal pH is around 5.5. The enzyme activity is little affected by dithiothreitol (DTT) up to 10 mM. Protamine stimulates the activity to the maximum level at a concentration of 0.05 mg/mL.
Heparan sulfate 6-O-sulfotransferase (HS6ST) transfers sulfate from PAPS to position 6 of N-sulfoglucosamine and N-acetylglucosamine residues in heparan sulfate and heparin. HS6ST was purified from the conditioned-medium of cultured CHO cells and cloned. There are three isoforms, Hs6st-1, -2, -3 and at least 2 spliced forms of Hs6st-2, Hs6st-2S and Hs6st-2L, which are shorter and longer in size than the original form. The substrate specificities of these isoforms largely overlap, but the individual isoforms reveal characteristic preference for hexuronic acid residue neighboring the N-sulfoglucosamine. HS6ST-1 predominantly sulfates the IdoA-GlcNSO3 unit. On the other hand, HS6ST-2 transfers sulfate preferentially to the GlcA-GlcNSO3 unit and the IdoA(2SO4)-GlcNSO3 unit to produce IdoA(2SO4)-GlcNSO3(6SO4) unit in HS and heparin. The properties of HS6STs are as follows: the optimal pH is around 6.3. Protamine markedly activated the activity. Maximum stimulation by NaCl is around 175 mM. DTT, which has no effect on HS2ST, inhibits HS6ST activity to 19% of the control at a concentration of 10 mM. When CHO cells were cultured, more than 95% of HS2ST activity was retained in the cell layer, whereas more than 90% of HS6ST activity was secreted into the culture medium. HS2ST activity was rarely detected in mouse serum or human serum, while HS6ST activity was detected.
Heparan sulfate/heparin GlcNAc N-deacetylase/N-sulfotransferases (NDSTs) catalyzes the initial modification reaction of heparan sulfate and heparin. NDSTs have dual activities that are N-deacetylase and N-sulfotransferase. N-deacetylase activity removes N-acetyl group of GlcNAc residues and N-sulfotransferase activity transfers sulfate from PAPS to the resulting free amino group of glucosamine. Four isoforms of NDST-1, -2 , -3 and -4 are known. The enzymatic properties of these isoforms are markedly different in the ratio of N-sdeacetylase activity to N-sulfotransferase activity. |
| Category | Glycosyltransferases & related proteins |
| Protocol Name | Enzyme assay of sulfotransferases for heparan sulfate |
Authors
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Habuchi, Hiroko
*
Advanced Medical Research Center, Aichi Medical University
Kimata, Koji
Research Complex for the Medicine Frontiers, Aichi Medical University
*To whom correspondence should be addressed.
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| KeyWords |
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Reagents
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| ● |
[35 S]PAPS (10,000 cpm/pmol)(PerkinElmer, Waltham, MA, NEG010) |
| ● |
Chondroitin sulfate A (Seikagaku Corp., Tokyo, Japan) |
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Completely desulfated and N-resulfated heparin (CDSNS-heparin) or CDSNS-HS (Seikagaku Corp.) |
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N-desulfated heparin (see Notes 1) |
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Unsaturated heparan sulfate/heparin disaccharide mixture (H mix) (Seikagaku Corp.) |
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Heparitinase I (Seikagaku Corp.) |
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Heparitinase II (Seikagaku Corp.) |
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Heparinase (Seikagaku Corp.) |
| ● |
Ready Safe Scintillator (Beckman Coulter, Inc., Brea, CA) |
| ● |
pFLAG-CMV-2 vector (Sigma-Aldrich, St. Louis, MO) |
| ● |
FLAG M2 antibody-conjugated agarose (Sigma-Aldrich) |
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Instruments
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| ● |
YMC-Pack Polyamine II column (4.6 mm × 25 cm) (YMC Co., Ltd., Kyoto, Japan) |
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Fast Desalting Column HR 10/10 (GE Healthcare, Little Chalfont, UK) (see Notes 2) |
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Sepadex G-50 medium (Invitrogen/Life Technologies, Carlsbad, CA) |
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Liquid scintilation counter |
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| Methods |
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1. |
Enzyme assay of heparan sulfate 2-O-sulfotransferase (HS2ST), heparan sulfate 6-O-sulfotransferases (HS6STs) and heparan sulfate N-sulfotransferases (NDSTs) |
| 1) |
Prepare the recombinant enzymes. |
Comment 1
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| 2) |
Prepare the reaction mixture (50 μL) containing 2.5 μmol of imidazole-HCl, pH 5.6 (for HS2ST) or pH 6.3 (for HS6STs), 3.75 μg of protamine chloride, 25 nmol (as hexuronic acid) of CDSNS-heparin or other glycosaminoglycans, 50 pmol [35S]PAPS (about 5 × 105 cpm), 5 μL of 1 M NaCl in buffer A (10 mM Tris-HCl, pH 7.2, 0.1% Trton X-100, 10 mM MgCl2, 2 mM CaCl2, 20% (V/V) glycerol), and enzyme (Note 1-3). As to NDSTs, the reaction mixture (50 μL) contains 2.5 μmol of HEPES, pH 7.0, 0.1% Triton X-100, 0.5 μmol MgCl2, 0.05 μmol MnCl2, 25 μg of N-desulfated heparin, 50 pmol [35S]PAPS (about 5 × 105 cpm), and enzyme. |
Comment 0
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| 4) |
Stop the reaction by heating at 100ºC for 1 min. |
Comment 0
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| 5) |
Add chondroitin sulfate A as carrier (0.1 μmol as GlcA) to the reaction mixture and vortex well. |
Comment 0
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| 6) |
Add 2.5 vol. of cold 95% ethanol/1.3% (w/v) potassium acetate/0.5 mM EDTA to the reaction mixture, and vortex well and then keep at −20ºC for 30 min. |
Comment 0
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| 7) |
Precipitate 35S-labeled polysaccharides by centrifugation at 10,000 × g for 30 min and discard the supernatant. |
Comment 0
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| 8) |
Add 70 μL of distilled in the precipitates and vortex well. |
Comment 1
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| 9) |
Inject 50 μL of the solution into a Fast Desalting column equilibrated with 0.2 M NH4HCO3 at a flow rate of 2 mL/min. |
Comment 1
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| 10) |
Collect the void volume to recover 35S-labeled products and mix with 3 mL of Scintillator, and determine the radioactivity by a liquid scintillation counter. |
Comment 0
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2. |
Determination of sulfated positions in the products |
| 1) |
Digest 35S-labeled products with a mixture of heparitinase I, heparitinase II and heparinase. |
Comment 1
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| 2) |
Stop the reaction by heating at 100ºC for 1 min. |
Comment 0
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| 3) |
Inject the digests together with 1 nmole of standard unsaturated disaccharides into a column of YMC-Pack Polyamine II. HPLC is performd* and the elution is fractionated into 0.6 mL portions to separate unsaturated disaccharide. |
Comment 1
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| 4) |
Each fraction is mixed with 3 mL of Ready Safe scintillator, and determine radioactivities of the fractions containing ∆HexA(2SO4)-GlcNSO3 for HS2ST and ∆HexA-GlcNSO3(6SO4) for HS6ST-1, -2, and -3, respectively. |
Comment 0
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| Notes |
- Chemically N-desulfated heparin is obtained from heparin (Sigma-Aldrich) by solvolysis with DMSO 6). Solvolysis with 5% (v/v) methanol in DMSO is performed at 50ºC for 1.5 h.
- Instead of a Fast Desalting column, a spin column can be utilized. The spin column (bed volume 0.9 mL) is prepared by packing Sephadex G-50 medium (GE Healthcare) suspended in 0.1 M NH4HCO3 into 1 mL syringe under centrifugation at 2,000 rpm for 4 min. A spin column is also commercially available 7). Samples (100 μL) are loaded to the top of the gel and centrifuged at 2,000 rpm for 4 min. [35S]Glycosaminoglycans are recovered in the flow through fractions.
- When crude extracts are used as enzyme, 1 μmole NaF and 0.1 μmole AMP are added to the reaction mixture to prevent degradation of PAPS.
- HS6ST isoforms but not HS2ST are inhibited by DTT. HS6ST-1 activity is decreased to 19% of control in 10 mM DTT.
- As to the sensitivity of this assay, crude extract from 1 × 105 fibroblasts (no transfection, endogenous activity) is enough to determine HS2ST and HS6ST activities.
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| Copyrights |
 Attribution-Non-Commercial Share Alike
This work is released underCreative Commons licenses
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| Date of registration：2014-07-31 09:28:45 |
- Habuchi, H., Habuchi, O., and Kimata, K. (1995) Purification and characterization of heparan sulfate 6-sulfotransferase from the culture medium of Chinese hamster ovary cells. J. Biol. Chem. 270, 4172–4179 [PMID : 7876170]
- Kobayashi, M., Habuchi, H., Habuchi, O., Saito, M., and Kimata, K. (1996) Purification and characterization of heparan sulfate 2-sulfotransferase from cultured Chinese hamster ovary cells. J. Biol. Chem. 271, 7645–7653 [PMID : 8631801]
- Kobayashi, M., Habuchi, H., Yoneda, M., Habuchi, O., and Kimata, K. (1997) Molecular cloning and expression of Chinese hamster ovary cell heparan-sulfate 2-sulfotransferase. J. Biol. Chem. 272, 13980–13985 [PMID : 9153262]
- Habuchi, H., Kobayashi, M., and Kimata, K. (1998) Molecular characterization and expression of heparan-sulfate 6-sulfotransferase. Complete cDNA cloning in human and partial cloning in Chinese hamster ovary cells. J. Biol. Chem. 273, 9208–9213 [PMID : 9535912]
- Aikawa, J., Grobe, K., Tsujimoto, M., Esko, J.D. (2001) Multiple isozymes of heparan sulfate/heparin GlcNAc N-deacetylase/GlcN N-sulfotransferase. Structure and activity of the fourth member, NDST4. J. Biol. Chem. 276, 5876–5882 [PMID : 11087757]
- Inoue, I., and Nagasawa, K. (1976) Selective N-desulfation of heparin with dimethyl sulfoxide containing water or methanol. Carbohydr. Res. 46, 87–95 [PMID : 1248016]
- Sueyoshi, T., Kakuta, Y., Pedersen, L.C., Wall, F.E., Perdersen, L.G., and Negishi, M. (1998) A role of Lys614 in the sulfotransferase activity of human heparan sulfate N-deacetylase/N-sulfotransferase. FEBS Letters 443, 211–214 [PMID : 9744796]
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