Chondroitin 4-sulfotransferase (C4ST) and chondroitin 6-sulfotransferase transfer sulfate from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to position 4 and 6, respectively, of GalNAc residues of chondroitin, and are involved in the biosynthesis of chondroitin sulfate A and C. Three isoforms of C4ST, C4ST-1, C4ST-2 and C4ST-3, and two isoforms of C6ST, C6ST-1 and C6ST-2, are known. C4ST-1 and C6ST-1 are able to sulfate dermatan, but these enzymes differ from each other in the recognition of uronic acid residues adjacent to the targeted GalNAc residue; C4ST-1 transfers sulfate preferably to GalNAc residues adjacent to the reducing side of the GlcA residue but C6ST-1 does not show any requirement for the presence of GlcA residues. Dermatan 4-sulfotransferase (D4ST) preferentially transfers sulfate to position 4 of GalNAc residues of dermatan, and is involved in the biosynthesis of dermatan sulfate. In C4ST-1 deficient mice, multiple skeletal abnormalities are observed and the morphology of growth plates is disturbed. Deficient in C6ST-1 in human causes spondyloepiphyseal dysplasia with severe skeletal abnormality. Deficient in D4ST-1 in human causes musculocontractural Ehlers-Danlos syndrome. |
| Category | Glycosyltransferases & related proteins |
| Protocol Name | Enzyme assay of sulfotransferases for chondroitin/dermatan |
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 from squid skin |
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pFLAG-CMV-2 plasmid (Sigma-Aldrich, St. Louis, MO) |
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pcDNA3.1 (Invitrogen/Life Technologies, Carlsbad, CA) |
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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 (Invitrogen/Life Technologies) |
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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/dermatan |
| 1) |
Prepare the recombinant enzymes. |
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 C4ST and C6ST, the reaction mixtures contain 50 mM imidazole-HCl buffer (pH 6.8), 0.0025% protamine chloride, 2 mM dithiothreitol, 0.5 μmol/mL (as galactosamine) chondroitin, 1 μM [35S]PAPS (about 5.0 × 105 cpm/50 μL), and the recombinant enzyme. For D4ST, the reaction mixtures contain 50 mM imidazole-HCl buffer (pH 6.8), 2 mM dithiothreitol, 5.0 × 105 cpm [35S]PAPS , 2 μM PAPS, 0.05% protamine chloride, 50 μg desulfated dermatan sulfate, and the recombinant enzyme. |
Comment 0
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| 5) |
Incubate at 37˚C for 20 min (C4ST and C6ST) or 2 h (D4ST). |
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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| 8) |
Collect the precipitate with centrifugation at 10,000 rpm for 10 min. |
Comment 0
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| 9) |
Dissolve the precipitate in 70 μL water and 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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| 10) |
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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| 11) |
Digest the [35S]glycosaminoglycan with chondroitinase ACII or chondroitinase ABC. |
Comment 1
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| 12) |
Inject the digests to Partisil-10 SAX column equilibrated with 5 mM KH2PO4 and separate ∆HexA-GalNAc(4SO4) and ∆HexA-GalNAc(6SO4). |
Comment 1
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| 13) |
Determine the radioactivity of ∆HexA-GalNAc(4SO4) for C4ST and D4ST, ∆HexA-GalNAc(6SO4) for C6ST. |
Comment 0
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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 16:22:41 |
- Habuchi, O., Matsui, Y., Kotoya, Y., Aoyama, Y., Yasuda, Y., and Noda, M. (1993) Purification of chondroitin 6-sulfotransferase secreted from cultured chick embryo chondrocytes. J. Biol. Chem. 268, 21968–21974 [PMID : 8408053]
- Fukuta, M., Uchimura, K., Nakashima, K., Kato, M., Kimata, K., Shinomura, T., and Habuchi, O. (1995) Molecular cloning and expression of chick chondrocyte chondroitin 6-sulfotransferase. J. Biol. Chem. 270, 18575–18580 [PMID : 7629189]
- Yamauchi, S., Mita, S., Matsubara, T., Fukuta, M., Habuchi, H., Kimata, K., and Habuchi, O. (2000) Molecular cloning and expression of chondroitin 4-sulfotransferase. J. Biol. Chem. 275, 8975–8981 [PMID : 10722746]
- Yamada, T., Ohtake, S., Sato, M., and Habuchi, O. (2004) Chondroitin 4-sulphotransferase-1 and chondroitin 6-sulphotransferase-1 are affected differently by uronic acid residues neighboring the acceptor GalNAc residues. Biochem. J. 384, 567–575 [PMID : 15324304]
- Evers, M. R., Xia, G., Kang, H. G., Schachner, M., Baenziger, J. U. (2001) Molecular cloning and characterization of a dermatan-specific N-acetylgalactosamine 4-O-sulfotransferase. J. Biol. Chem. 276, 36344–36353 [PMID : 11470797]
- Anno, K., Kawai, Y.. and Seno, N. (1964) Isolation of chondroitin from squid skin. Bioichim. Biophys. Acta. 83, 348–349 [PMID : 14236709]
- Habuchi, O., and Miyata, K. (1980) Stimulation of glycosaminoglycan sulfotransferase from chick embryo cartilage by basic proteins and polyamines. Bioichim. Biophys. Acta. 616, 208–217 [PMID : 6938246]
- Nagasawa, K., Inoue Y, and Tokuyasu, T. (1979) An improved method for the preparation of chondroitin by solvolytic desulfation of chondroitin sulfates. J. Biochem. 86, 1323–1329 [PMID : 521436]
- Derfert, D.M., and Conrad H.E. (1985) Preparation and high-performance liquid chromatography of 3'-phosphoadenosine-5'-phospho[35S]sulfate with a predetermined specific activity. Anal. Biochem. 148, 303–310 [PMID : 4061812]
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