Characterization of a Sulfotransferase from Human Airways Responsible for the 3-O-Sulfation of Terminal Galactose in N-Acetyllactosamine-containing Mucin Carbohydrate Chains

Jean-Marc Lo-Guidice,Jean-Marc Périni,Jean-Jacques Lafitte,Marie-Paule Ducourouble,Philippe Roussel,Geneviève Lamblin

doi:10.1074/jbc.270.46.27544

Jean-Marc Lo-Guidice, Jean-Marc Périni + Show 4 more

Open Access

https://doi.org/10.1074/jbc.270.46.27544

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Abstract

A galactose 3-O-sulfotransferase activity able to transfer a sulfate group from adenosine 3'-phosphate 5'-phosphosulfate to methyl galactosides or terminal N-acetyllactosamine-containing carbohydrate chains from human respiratory mucins was characterized in microsomal fractions prepared from human respiratory mucosa. The reaction products, methyl alpha- or beta-galactose 3-sulfate and three oligosaccharide alditols containing the sequence HSO3-3Gal beta 1-4GlcNAc beta 1-6GalNAc-itol were identified by high performance anion-exchange chromatography. Using methyl beta-galactoside as a substrate, the optimum activity was obtained with 0.1% Triton X-100, 30 mM NaF, 20 mM Mn2+, and 10 mM AMP in a 30 mM 2-(N-morpholino)ethanesulfonic acid buffer at pH 6.1. The apparent Km for methyl beta-galactoside and for adenosine 3'-phosphate 5'-phosphosulfate were observed at 0.69 x 10(-3) M and at 4 x 10(-6) M respectively. This sulfotransferase is different from that responsible for sulfatide synthesis.

Highlights

Using methyl ␤-galactoside as a substrate, the optimum activity was obtained with 0.1% Triton X-100, 30 mM NaF, 20 mM Mn2؉, and 10 mM AMP in a 30 mM 2-(Nmorpholino)ethanesulfonic acid buffer at pH 6.1
A galactose 3-O-sulfotransferase activity able to transfer a sulfate group from adenosine 3؅-phosphate 5؅-phosphosulfate to methyl galactosides or terminal N-acetyllactosamine-containing carbohydrate chains from human respiratory mucins was characterized in microsomal fractions prepared from human respiratory mucosa
We report, for the first time, the presence of sulfotransferase activity from human bronchial mucosa, which is able to transfer sulfate groups to C-3 of terminal galactose residues of neutral carbohydrate chains isolated from human respiratory mucins

Summary

EXPERIMENTAL PROCEDURES

Enzyme Preparation—Tissues were obtained from patients undergoing surgery for bronchial carcinoma. The sulfated methyl ␣-galactoside was purified on a silica column (2 ϫ 7 cm), using a mixture of dichloromethane/methanol (5:3 (v/v)) as eluting system; its structure was verified by 400 MHz 1H NMR spectroscopy. Sulfotransferase Assays—The incubation mixture (100 ␮l) for the sulfotransferase assays consisted of 50 –100 ␮g of microsomal proteins, 0.5 ␮Ci of [35S]PAPS (DuPont NEN, 1.95–2.08 Ci/mmol) and 5 mM of methyl ␣/␤-galactoside or 700 ␮g of neutral oligosaccharide alditols Ic (about 0.5–1 ␮mol of carbohydrate chains) in a 30 mM MES/NaOH buffer, pH 6.1, containing 0.1% Triton X-100, 20 mM MnCl2, 30 mM NaF, 10 mM AMP, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride (Sigma). When galactosylceramide was used either as the substrate acceptor or in competition with methyl ␤-galactoside for sulfotransferase assays, it was dissolved in chloroform/methanol (2:1) and added to the reaction tubes; before adding the other reagents, the solvent was evaporated under nitrogen [37]. Protein determination—The protein content of microsomal fractions was measured by a Coomassie Brilliant Blue method as described by Perini et al [38]

RESULTS

SCHEME I

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SCHEME III

DISCUSSION