Enzyme assay of sulfotransferases for chondroitin/dermatan

[³⁵S]PAPS (PerkinElmer NEG010, PerkinElmer, Waltham MA)

Chondroitin from squid skin or desulfated chondroitin

Desulfated dermatan

CS-A (Sigma-Aldrich, St. Louis, MO)

pFLAG-CMV-2 plasmid (Sigma-Aldrich, St. Louis, MO)

pcDNA3.1 (Thermo Fisher Scientific, Tokyo)

COS-7 cells

FLAG M2 antibody-conjugated agarose (Sigma-Aldrich)

Clearsol (Nacalai Tesque, Inc., Kyoto, Japan)

Chondroitinase ACII (Commercial chondroitinase ACII is presently unavailable and can be replaced by chondroitinase AC from Flavobacterium heparinum (Sigma-Aldrich))

Chondroitinase ABC (Sigma-Aldrich)

Desalting column (1 × 10 cm) filled with Sephadex G-25 superfine (Cytiva, Tokyo)

HPLC system

Partisil-10 SAX column (4.6 mm × 25 cm) (Sigma-Aldrich)

Liquid scintillation counter

Prepare the recombinant enzymes (Note 1).

Prepare the acceptor substrate (Note 2).

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, [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 (Note 3).

Incubate at 37˚C for 20 min (C4ST and C6ST) or 2 h (D4ST).

Terminate the reaction by immersing the reaction tubes in a boiling water bath for 1 min.

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.

Collect the precipitate with centrifugation at 10,000 rpm for 10 min.

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 (Note 4).

Mix an aliquot of the [³⁵S]glycosaminoglycan fraction with Clearsol and determine the radioactivity by a liquid scintillation counter.

Digest the [³⁵S]glycosaminoglycan with chondroitinase ACII or chondroitinase ABC (Note 5).

Inject the digests to Partisil-10 SAX column equilibrated with 5 mM KH₂PO₄ and separate ∆4,5Hexuronic acid (∆HexA)-GalNAc(4SO₄) and ∆HexA-GalNAc(6SO₄) (Note 6).

The recombinant human C4ST-1 and C6ST-1 are expressed in COS-7 cells as a fusion protein with a FLAG peptide (4). From the transfected cells, C4ST-1 and C6ST-1 are extracted with 1.5 ml/10-cm dish 10 mM Tris-HCl (pH 7.2), 10 mM MgCl₂, 2 mM CaCl₂, 0.5% Triton X-100, and 20% glycerol for 30 min on a rotatory shaker at 4°C. The extracts are centrifuged at 10,000 × g for 10 min. The cellular extracts from ten 10-cm dishes are applied to an anti-FLAG mAb-conjugated agarose column (0.5 mL) (Sigma-Aldrich) equilibrated with buffer A (10 mM Tris-HCl (pH 7.2), 20 mM MgCl₂, 2 mM CaCl₂, 10 mM 2-mercaptoethanol, 0.1% Triton X-100, and 20% glycerol) containing 50 mM NaCl for purification of C4ST-1 or with buffer A containing 150 mM NaCl for purification of C6ST-1. The absorbed materials are eluted with 1.5 mL of buffer A containing 118 μg FLAG peptide (Sigma-Aldrich). The recombinant human D4ST-1 is expressed in CHO/Tag cells transfected with pcDNA3.1 containing the ORF of D4ST-1(5). From the transfected cells, D4ST-1 is extracted with 200 μL 20 mM HEPES buffer (pH 7.4), 5 mM MgCl₂, 175 mM KCl, 2% Triton X-100, and protease inhibitors (23 millitrypsin inhibitor units of aprotinin and 4 μg each of leupeptin, antipain, pepstatin, and chymostatin) per 100-mm diameter culture plate. The homogenate is mixed by rotation for 1 h and sedimented at 12,000 × g for 20 min. The supernatant is designated as the cell extract. The culture medium is pooled and sedimented at 12,000 × g for 20 min. The culture supernatant is adjusted to a final concentration of 20 mM HEPES (pH 7.4), and protease inhibitors are added as noted above.

Chondroitin is obtained from the skin of squid, Todarodes pacificus as described (14,15). Briefly, a crude polysaccharide fraction is obtained from the acetone-dried squid skin by digestion with pronase, deproteinization with 5% trichloroacetic acid and precipitation with 3 volumes of ethanol containing 1% (w/v) potassium acetate. The crude polysaccharide is applied to the DEAE-cellulose column equilibrated with 0.02 M Tris-HCl (pH 7.2), and washed with the same buffer. The column is eluted with 0.4 M NaCl in 0.02 M Tris-HCl (pH 7.2). The fractions containing uronic acid are pooled, dialyzed against distilled water, and precipitated with 2-volumes of ethanol containing 1% (w/v) potassium acetate. Chemically desulfated chondroitin and dermatan are obtained from CS-A from whale cartilage (Seikagaku Corp., Tokyo, Japan) and dermatan sulfate from pig skin (Seikagaku Corp.), respectively, by solvolysis with DMSO (16). Solvolysis with 90% (v/v) DMSO is performed at 100˚C for 60 min.

Enzyme activities of C6ST and C4ST are detectable by using 5 to 10 μL of the affinity-purified preparations.

The desalting column (1.0 x 10 cm) is prepared by packing Sephadex G-25 superfine suspended in 0.1 M NaCl at a flow rate of 6 mL/min. This column is equilibrated with 0.1 M NH₄HCO₃ and run at a flow rate of 2 mL/min. As a column packed with the same material, HiTrap® Desalting Columns (1.6 x 2.5 cm, GE17-1408-01, Cytiva 17-1408-01) is commercially available. Instead of the desalting column, a spin column can be utilized. The spin column (bed volume 0.9 mL) is made by packing Sephadex G-50 medium suspended in 0.1 M NH₄HCO₃ into 1 mL syringe under centrifugation at 2000 rpm for 4 min. Samples (100 μL) are loaded to the top of the gel and centrifuged at 2000 rpm for 4 min. [³⁵S]glycosaminoglycans are recovered in the flow through fractions. Alternatively, dissolve the precipitates in 140 μL water and separate from [³⁵S]PAPS and degradation products by Bio-Spin 6 column (Bio-Rad Laboratories, Hercules, CA) (5).

Digestion with chondroitinase ACII (for C6ST and C4ST) or chondroitinase ABC (for D4ST) under the standard conditions is carried out for 4 h at 37°C in the reaction mixture containing, in a final volume of 25 μL, ³⁵S-labeled glycosaminoglycans, 50 mM Tris-acetate buffer (pH 7.5), 0.1 mg/m of bovine serum albumin and 30 milli units of chondroitinase ACII or chondroitinase ABC (17).

The Partisil-10 SAX column is developed with 5 mM KH₂PO₄ for 10 min followed by a linear gradient from 5 to 500 mM KH₂PO₄. A column temperature is 40°C. Fractions (0.5 mL) are collected at a flow rate of 1 mL/min. Elution time of the standard disaccharides varies with the lot of the column; typically, ∆HexA-GalNAc(6SO₄) and ∆HexA-GalNAc(4SO₄) are eluted at 25.5 and 26.5 min, respectively (18).