N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to position 6 of GalNAc 4-sulfate residues of chondroitin sulfate and dermatan sulfate, and are involved in the biosynthesis of GlcA-GalNAc(4,6-bisSO4) and IdoA-GalNAc(4,6-bisSO4) units contained in chondroitin sulfate and dermatan sulfate. GalNAc4S-6ST is involved in the synthesis of chondroitin sulfate E in the bone marrow derived mast cells (BMMC) and contribute to packaging of mast cell-specific proteases in the granules of BMMC. In in vitro enzymeatic reaction, mammalian GalNAc4S-6ST transfers sulfate to position 6 of GalNAc(4SO4) residue located not only in the internal disaccharide units but also at the nonreducing terminal. A unique nonreducing terminal sequence, GalNAc (4SO4)-GlcA(2SO4)-GalNAc(6SO4), which is present in chondroitin sulfate A from whale cartilage, is converted by the sulfation with GalNAc4S-6ST to GalNAc(4,6-bisSO4)-GlcA(2SO4)-GalNAc(6SO4). Uronyl 2-O-sulfotransferase (2OST) transfers sulfate to position 2 of GlcA residues of chondroitin sulfate and position 2 of IdoA residues of dermatan sulfate. 2OST recognizes position of sulfate groups attached to the GalNAc residue adjacent to the targeting uronic acid; 2OST transfers sulfate to position 2 of GlcA residue located in GalNAc(4SO4)-GlcA-GalNAc(6SO4) in chondroitin sulfate, and to position 2 of IdoA residue located in IdoA-GalNAc(4SO4) in dermatan sulfate. |
| Category | Glycosyltransferases & related proteins |
| Protocol Name | Enzyme assay of sulfotransferases for chondroitin sulfate/dermatan sulfate |
Authors
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Habuchi, Osami
Advanced Medical Research Center, Aichi Medical University
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| KeyWords |
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Reagents
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[35S]PAPS (10,000 cpm/pmol) |
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Chondroitin sulfate A from whale cartilage (Seikagaku Corp., Tokyo, Japan) |
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Dermatan sulfate from pig skin (Seikagaku Corp.) |
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pFLAG-CMV-2 plasmid (Sigma-Aldrich, St. Louis, MO) |
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FLAG M2 antibody-conjugated agarose (Sigma-Aldrich) |
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Clearsol (Nacalai Tesque Inc., Kyoto, Japan) |
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Instruments
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Desalting column (1 × 10 cm) filled with Sephadex G-25 superfine |
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Partisil-10 SAX column (4.6 mm × 25 cm) (Whatman International Ltd., Kent, UK) |
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Liquid scintillation counter |
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| Methods |
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1. |
Enzyme assay of Sulfotransferases for chondroitin sulfate/dermatan sulfate |
| 1) |
Prepare the recombinant enzymes. |
Comment 1
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| 2) |
Prepare the acceptor substrate, chondroitin sulfate A and dermatan sulfate. |
Comment 1
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| 3) |
Prepare the donor substrate, [35S]PAPS. |
Comment 1
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| 4) |
Prepare the reaction mixture (50 μL). For GalNAc4S-6ST, the reaction mixtures contain 50 mM imidazole-HCl buffer (pH 6.8), 10 mM CaCl2, 20 mM reduced glutathione, 0.5 μmol/mL (as galactosamine) chondroitin sulfate A, 1 μM [35S]PAPS (about 5.0 × 105 cpm/50 μL). and 2 to 5 μL of the recombinant enzyme. For 2OST, 50 mM imidazole-HCl buffer (pH 6.8), 0.0052% protamine chloride, 0.5 μmol/mL (as galactosamine) chondroitin sulfate A, dermatan sulfate or desulfated dermatan, 1 μM [35S]PAPS (about 5.0 × 105 cpm/50 μL), and 2 to 5 μL of the recombinant enzyme. |
Comment 0
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| 6) |
Terminate the reaction by immersing the reaction tubes in a boiling water bath for 1 min. |
Comment 0
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Add 50 nmol CS-A as carrier and three volumes of ethanol containing 1% (w/v) potassium acetate. The mixtures are stirred well and placed on ice for 30 min. |
Comment 0
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Collect the precipitate with centrifugation at 10,000 rpm for 10 min and dissolve the precipitate in 70 μL water. |
Comment 0
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Inject 50 μL of the solution into a desalting column equilibrated with 0.1 M NH4HCO3 at the flow rate of 2 mL/min, and collect the void fractions. |
Comment 1
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Mix an aliquot of the [35S]glycosaminoglycan fraction with Clearsol and determine the radioactivity by a liquid scintillation counter. |
Comment 0
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Digest the [35S]glycosaminoglycan with chondroitinase ACII or chondroitinase ABC. |
Comment 1
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Inject the digests to Partisil-10 SAX column equilibrated with 5 mM KH2PO4 and separate digestion products. |
Comment 1
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| 13) |
For GalNAc4S-6ST, determine the radioactivity of GalNAc (4,6-bisSO4), ∆HexA-GalNAc(4, 6-bisSO4) and GalNAc (4,6-bisSO4)-GlcA(2SO4)-GalNAc(6SO4).
For 2OST, determine the radioactivity of ∆HexA(2SO4)-GalNAc(6SO4) when chondroitin sulfate is used as the acceptor, ∆HexA(2SO4)-GalNAc(4SO4) when dermatan sulfate is used as the acceptor, and ∆HexA(2SO4)-GalNAc when desulfated dermatan sulfate is used as the acceptor. |
Comment 1
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| Copyrights |
 Attribution-Non-Commercial Share Alike
This work is released underCreative Commons licenses
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| Date of registration：2014-07-29 16:23:25 |
- Ohtake, S., Ito, Y., Fukuta, M., and Habuchi, O. (2001) Human N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase cDNA is related to human B cell recombination activating gene-associated gene. J. Biol. Chem. 276, 43894–43900 [PMID : 11572857]
- Ohtake, S., Kimata, K., and Habuchi, O. (2003) A Unique Nonreducing Terminal Modification of Chondroitin Sulfate by N-Acetylgalactosamine 4-Sulfate 6-O-Sulfotransferase. J. Biol. Chem. 278, 38443–38452 [PMID : 12874280]
- Ohtake-Niimi, S., Kondo, S., Ito, T, Kakehi, S., Ohta, T., Habuchi, H., Kimata, K., and Habuchi, O. (2010) Mice Deficient in N-Acetylgalactosamine 4-Sulfate 6-O-Sulfotransferase are Unable to Synthesize Chondroitin/Dermatan Sulfate containing N-Acetylgalactosamine 4, 6-Bissulfate Residues and Exhibit Decreased Protease Activity in Bone Marrow-Derived Mast Cells. J. Biol. Chem. 285, 20793–20805 [PMID : 2043998]
- Kobayashi, M., Sugumaran, G., Liu, J., Shworak, N. W., Silbert, J. E., and Rosenberg, R. D. (1999) Molecular cloning and characterization of a human uronyl 2-sulfotransferase that sulfates iduronyl and glucuronyl residues in dermatan/chondroitin sulfate. J. Biol. Chem. 274, 10474–10480 [PMID : 10187838]
- Ohtake, S., Kimata, K., and Habuchi, O. (2005) Recognition of sulfation pattern of chondroitin sulfate by uronosyl 2-O-sulfotransferase. J. Biol. Chem. 280, 39115–39123 [PMID : 16192264]
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