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Volume 270, Number 46, Issue of November 17, 1995 pp. 27544-27550
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of a Sulfotransferase from Human Airways Responsible for the 3-O-Sulfation of Terminal Galactose in N-Acetyllactosamine-containing Mucin Carbohydrate Chains (*)

(Received for publication, March 20, 1995; and in revised form, August 9, 1995)

Jean-Marc Lo-Guidice (1),  Jean-Marc Périni (1),  Jean-Jacques Lafitte (2),  Marie-Paule Ducourouble (1),  Philippe Roussel (1),  Geneviève Lamblin (1)(§)

From the  (1)Unité INSERM 377, 59045 Lille, France and the (2)Département de Pneumologie de l'Hôpital A. Calmette, 59045 Lille, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

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 HSO(3)-3Galbeta1-4GlcNAcbeta1-6GalNAc-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 Mn, and 10 mM AMP in a 30 mM 2-(N-morpholino)ethanesulfonic acid buffer at pH 6.1. The apparent K for methyl beta-galactoside and for adenosine 3`-phosphate 5`-phosphosulfate were observed at 0.69 times 10M and at 4 times 10M respectively. This sulfotransferase is different from that responsible for sulfatide synthesis.

INTRODUCTION

Human tracheobronchial mucus is an important component of the mucociliary system and constitutes the first line of mucosal defense against a variety of inhaled particles such as dust and microorganisms. Mucins are the main components of the mucus layer. They are responsible, to a large extent, for the rheological properties of the mucus, which are necessary for the efficiency of the mucociliary system. These high molecular mass glycoproteins consist of a broad family of molecules with different peptides, or apomucins, stemming from the expression of different genes(1) . This mucin diversity is increased by post-translational phenomena, mostly O-glycosylation, resulting in molecules that consist of about 70-80% carbohydrate by mass.

Structural studies have shown that respiratory mucins are sulfated either on galactose or on N-acetylglucosamine(2, 3, 4, 5, 6) , and it has been shown that respiratory epithelial cells in secondary culture were able to secrete sulfated mucins(7) . Strong negative charges confer important biological functions on sulfated carbohydrate chains. Thus, several studies have shown that sulfated sequences were involved in the recognition of microorganisms (8, 9, 10) or cell adhesion molecules (11, 12, 13, 14) .

It has been suspected for a long time that there are abnormalities in glycosylation and/or sulfation of respiratory mucins of patients suffering from cystic fibrosis (CF)(^1)(15, 16, 17) . More recently, abnormal sulfation of glycoproteins synthesized by CF nasal epithelial cells in culture have been reported(18) , and it has been suggested that these abnormalities could be related to abnormal alkalinization of the Golgi apparatus in CF cells(19) . Such modifications could lead to abnormal carbohydrate chains of respiratory mucins and be responsible for the persistent infection by Pseudomonas aeruginosa that usually colonizes the CF lung(20) .

Sulfotransferases are involved in the transfer of sulfate groups from adenosine 3`-phosphate 5`-phosphosulfate (PAPS) to specific substrate acceptors such as growing carbohydrate chains of glycoconjugates (21, 22, 23, 24, 25, 26, 27, 28, 29, 30) , serine residues of peptides(31) , steroids, and aromatic compounds(32, 33) . In order to study tracheobronchial mucin sulfation in CF, the present investigation was aimed at the characterization of sulfotransferase activities in human respiratory mucosa.

In this paper, 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. This enzyme is different from the galactosylceramide sulfotransferase.

EXPERIMENTAL PROCEDURES

Enzyme Preparation

Tissues were obtained from patients undergoing surgery for bronchial carcinoma. Specimens used in these studies were collected in macroscopically healthy areas of the bronchial tree. The tissues were immersed in Leibovitz L15 medium and immediately transported on ice to the laboratory and processed for mucosa isolation. Mucosa (2-3 cm^2) were cut into 1-mm^2 pieces, suspended in 50 mM Tris-HCl buffer, pH 7.4, containing 25 mM KCl, 250 mM saccharose, 5 mM beta-mercaptoethanol, 5 mM magnesium acetate, and disintegrated with a glass-Teflon homogenizer (1,400 rpm, 5 strokes). The mixture obtained was submitted to 16,000 times g centrifugation for 20 min at 4 °C. The supernatant was then submitted to 180,000 times g ultracentrifugation for 1 h at 10 °C. The resulting pellet containing microsomal fractions was stored at -80 °C until used.

Acceptors Preparation

Neutral oligosaccharide-alditols (fraction Ic) were obtained by alkaline borohydride treatment of respiratory mucin glycopeptides obtained from a patient suffering from CF. They were fractionated by anion-exchange chromatography on a AG 1-X2 column (Bio-Rad) and then by gel-filtration chromatography on a Bio-Gel P4 column (Bio-Rad) according to Lamblin et al.(34) . Finally, they were purified by high performance liquid chromatography using a Lichrosorb-NH(2) column (Merck) according to Lamblin et al.(34) . Most of the peaks observed (20 peaks) were identified by 500 MHz ^1H NMR spectroscopy(34) .

2-O-Sulfated methyl alpha-galactoside, 3-O-sulfated methyl alpha-galactoside, and 4-O-sulfated methyl alpha-galactoside were generous gifts from J. F. G. Vliegenthart (University of Utrecht, The Netherlands).

6-O-Sulfated methyl alpha-galactoside was synthesized according to Van Kuik et al.(35) . Briefly, methyl alpha-galactoside (1 mmol) was dissolved in 5 ml of dry pyridine. After cooling to 5 °C, 73 µl of chlorosulfonic acid (1.1 mmol) in 300 µl of dry chloroform was added. The mixture was stirred for 30 min at 5 °C and then for 2 h at 25 °C. After addition of 2 ml of water, the solvent was evaporated to dryness. The sulfated methyl alpha-galactoside was purified on a silica column (2 times 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. The sulfation of methyl alpha-galactoside at C-6 was confirmed by the strong downfield shift for the H-6/H-6` protons (+0.473 and +0.413 ppm respectively) as well as the downfield shift for the H-5 proton (+0.254 ppm) as described by Contreras et al.(36

Sulfotransferase Assays

The incubation mixture (100 µl) for the sulfotransferase assays consisted of 50-100 µg of microsomal proteins, 0.5 µCi of [S]PAPS (DuPont NEN, 1.95-2.08 Ci/mmol) and 5 mM of methyl alpha/beta-galactoside or 700 µg of neutral oligosaccharide alditols Ic (about 0.5-1 µmol of carbohydrate chains) in a 30 mM MES/NaOH buffer, pH 6.1, containing 0.1% Triton X-100, 20 mM MnCl(2), 30 mM NaF, 10 mM AMP, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride (Sigma). After 1 h at 30 °C, the reaction was stopped by the addition of 300 µl of ice-cold methanol. The resulting mixture was kept overnight at 4 °C and the formed precipitate was eliminated by centrifugation for 20 min at 10,000 times g. The pellet was washed twice with ice-cold methanol and then centrifuged. Supernatants were pooled, evaporated to dryness, and then submitted to HPAEC.

When galactosylceramide was used either as the substrate acceptor or in competition with methyl beta-galactoside for sulfotransferase assays, it was dissolved in chloroform/methanol (2:1) and then added to the reaction tubes; before adding the other reagents, the solvent was evaporated under nitrogen(37) . Incubation was carried out under conditions already mentioned, except that two different concentrations of Triton X-100, 0.1 and 0.4%, were used. The reaction was stopped by the addition of 0.5 ml of methanol. The contents of the tube were thoroughly mixed, and chloroform and water were added to adjust the final composition of the solvent mixture to chloroform/methanol/water (2:1:0.8 (v/v/v)). After mixing and centrifugation, the chloroform phase, which is supposed to contain the S-labeled glycolipids, was counted for radioactivity; the upper aqueous phases were evaporated to dryness and then submitted to HPAEC.

Isolation of Labeled Products by HPAEC

Dry samples of sulfated galactosides or oligosaccharides were dissolved in water and directly injected onto a CarboPac PA-100 column (4 times 250 mm) for HPAEC (Dionex Corp., Sunnyvale, CA). The elution was monitored both by pulsed amperometric detection (PAD 2 model, Dionex Corp., Sunnyvale, CA) and by radioactivity detection on line (HPLC radioactivity detector LB 506 C-1, EG & G, Berthold, Wildbad, Germany).

Elution of sulfated methyl galactosides was performed at alkaline pH at a flow rate of 1 ml/min in 0.05 M NaOH, 0.2 M sodium acetate with a linear gradient of sodium acetate to 0.05 M NaOH, 0.3 M sodium acetate at 22 min, to 0.05 M NaOH, 0.95 M sodium acetate at 24 min, and followed by isocratic elution with 0.05 M NaOH, 0.95 M sodium acetate for 10 min (gradient I).

Elution of sulfated oligosaccharide-alditols was performed at alkaline pH at a flow rate of 1 ml/min in 0.1 M NaOH with a linear gradient of sodium acetate to 0.1 M NaOH, 0.05 M sodium acetate at 6 min, to 0.1 M NaOH, 0.07 M sodium acetate at 25 min, to 0.1 M NaOH, 0.32 M sodium acetate at 60 min, to 0.05 M NaOH, 0.95 M sodium acetate at 64 min and followed by isocratic elution with 0.05 M NaOH, 0.95 M sodium acetate for 10 min (gradient II). Fraction IIIc2 containing nine known sulfated oligosaccharides was used as a control(6) .

Protein determination

The protein content of microsomal fractions was measured by a Coomassie Brilliant Blue method as described by Périni et al.(38) .

RESULTS

Sulfation of Methyl Galactosides

Since it is difficult to obtain large amounts of neutral oligosaccharide alditols from human respiratory mucin, we first looked for sulfotransferase activity by examining the transfer of sulfate from [S]PAPS to methyl galactosides.

When microsomes were incubated with methyl alpha-galactoside, the HPAEC elution profile of the radiolabeled products obtained using gradient I showed mainly two peaks, a broad peak at 11 min 30 s corresponding to free [S]sulfate and a characteristic peak at 5 min 37 s (Fig. 1a), which was not visible when the enzyme preparation was incubated without substrate acceptor or when the incubation was performed after heating the microsomal preparation at 100 °C for 30 min. Thus, this peak seemed to correspond to a sulfated methyl alpha-galactoside. In order to define the position of the sulfate group on the galactose residue, we analyzed the HPAEC elution profile of different synthetic methyl alpha-galactosides bearing a sulfate group either on C-2, C-3, C-4, or C-6. The radiolabeled product synthesized from methyl alpha-galactoside co-eluted with 3-O-sulfated methyl alpha-galactoside (Fig. 1c). Methyl alpha-galactose 2-, 6-, and 4-sulfate were eluted at 7 min 34 s, 7 min 47 s, and 9 min 42 s, respectively. An identical peak eluted at 5 min 37 s was also obtained with gradient I, when methyl beta-galactoside was used as substrate acceptor indicating that the enzyme was able to transfer [S]sulfate to both methyl alpha- and beta-galactosides (Fig. 1b). The sulfotransferase activity was 17-fold higher for the beta anomer (18.44 pmol/mg of protein/min) than for the alpha anomer (1.08 pmol/mg of protein/min) under the same conditions of incubation (Fig. 1, a and b).

Figure 1: HPAEC elution profile of S-labeled products enzymatically obtained from methyl alpha-galactoside on CarboPac PA-100 column (4 times 250 mm) (a), S-labeled products enzymatically obtained from methyl beta-galactoside (b), and a mixture containing S-labeled products enzymatically obtained from methyl alpha-galactoside and different synthetic methyl alpha-galactosides bearing a sulfate group either on C-2, C-3, C-4, or C-6 (c). Elution was performed with gradient I described under ``Experimental Procedures.'' Peaks were detected by pulsed amperometric detection (dashed line) and by radioactivity detection (solid line).


Properties of the Galactose 3-O-Sulfotransferase

The effect of pH on the activity of respiratory mucosa 3-O-sulfotransferase was measured with MES buffer (pH 5.5-6.5) and with MOPS buffer (pH 6.0-7.9) (Fig. 2). The optimum condition for sulfation of methyl beta-galactoside was obtained with 30 mM MES buffer at pH 6.1.

Figure 2: Effect of pH on the activity of human respiratory mucosa sulfotransferase toward methyl beta-galactoside. Incubations were performed at the indicated pH values under standard assay conditions in MES (bullet) and in MOPS (up triangle).


The influence of divalent cations on the transfer of sulfate group from PAPS to methyl beta-galactoside is illustrated on Fig. 3. The galactose 3-O-sulfotransferase activity was stimulated by Mg and particularly by Mn. A 20 mM concentration seemed to be optimal for this activity. A 4-fold increase in enzymatic activity was obtained with 20 mM Mn. The sulfotransferase acted to some extent in the absence of added cations, even in the presence of chelating agent such as EDTA in a range of 1-20 mM (Fig. 3). Ca had an inhibitory effect regardless of the concentration used.

Figure 3: Effect of divalent cations and EDTA on the transfer of sulfate group from PAPS to methyl beta-galactoside. Incubations were performed under standard assay conditions with indicated amounts of Mn (up triangle), Mg (circle), Ca (), and EDTA (bullet).


The presence of sodium fluoride in the incubation mixture had a stimulatory effect on sulfotransferase activity. The maximal activity was obtained at about 30 mM NaF concentration (Fig. 4). The effect of AMP and ATP on enzymatic sulfation of methyl beta-galactoside was also studied (Fig. 4). Both nucleotides at 1 mM concentration produced a 5-fold increase in sulfotransferase activity. For AMP, the stimulatory effect increased up to a 10 mM concentration. For ATP, the stimulatory effect decreased at a 5 mM concentration; the sulfotransferase activity was completely abolished at geq10 mM. The effect of NaF, AMP, and ATP on the liberation of [S]sulfate from [S]PAPS in the incubation mixture was also measured (data not shown); the amount of free [S]sulfate significantly decreased, 3-, 2-, and 9-fold when the microsomal fractions were incubated with 10 mM AMP, 10 mM ATP, and 30 mM NaF, respectively.

Figure 4: Effect of AMP, ATP, and NaF on enzymatic sulfation of methyl beta-galactoside by human respiratory mucosa sulfotransferase. Incubations were carried out under standard assay conditions with indicated amounts of AMP (up triangle), ATP (circle), and NaF ().


Reducing agents such as beta-mercaptoethanol had no effect on galactose 3-O-sulfotransferase activity (data not shown).

Optimal conditions were obtained with 0.1% Triton X-100, 30 mM NaF, 20 mM Mn, and 10 mM AMP in a 30 mM MES/NaOH buffer at pH 6.1. Under these conditions, the respiratory mucosa sulfotransferase activity increased linearly up to 90 min (Fig. 5a) and in a range of 20-220 µg of microsomal proteins (Fig. 5b). This activity was measured with different substrate acceptor or sulfate donor concentrations. The effect of methyl beta-galactoside concentration is shown in Fig. 6a. The apparent K(m) for methyl beta-galactoside was observed at 0.69 times 10M. The effect of PAPS concentration on galactose sulfotransferase is illustrated in Fig. 6b. The apparent K(m) calculated for PAPS was 4 times 10M.

Figure 5: Activity of human respiratory mucosa sulfotransferase as function of time (a) and of microsomal protein amount (b). The composition of the incubation mixture was the same as described under standard assay conditions, except the time of reaction (a) or the protein amount (b).


Figure 6: Effect of methyl beta-galactoside (a) and PAPS (b) concentrations on human respiratory mucosa sulfotransferase activity. Incubation mixtures were the same as described under ``Experimental Procedures,'' except for the concentrations of methyl beta-galactoside (from 10 µM to 10 mM) and PAPS (from 0.44 µM to 22 µM).


Sulfation of Neutral Oligosaccharide Alditols from Respiratory Mucins

Microsomal fractions extracted from human bronchial mucosa were analyzed for sulfation of neutral oligosaccharide alditols obtained from respiratory mucins.

The sulfotransferase activity was measured using a pool of neutral oligosaccharide-alditols (fraction Ic) isolated from the respiratory mucins of a CF patient, as substrate acceptors. Most of the carbohydrate chains contained in this fraction (20 chains) have already been identified(34) .

The [S] radiolabeled products that were obtained, were subsequently fractionated by HPAEC on a CarboPac PA-100 column eluted under conditions described under ``Experimental Procedures'' (gradient II). Five major radiolabeled peaks, A-E, were obtained (Fig. 7). Peak E was eluted at 44 min 32 s and was also observed when the incubation mixture did not contain substrate acceptors; it corresponded to free sulfate. The other peaks, A, B, C, and D eluted at 35 min 54 s, 36 min 30 s, 37 min 24 s, and 39 min 24 s, respectively, corresponded to synthesized products, and three of them (A, B, and D) had retention times identical to that of sulfated carbohydrate chains isolated from human respiratory mucins (fraction IIIc2) and which have already been described on the basis of methylation analysis and fast atom bombardment mass spectrometry in combination with ^1H NMR spectroscopy(6) .

Figure 7: HPAEC elution profile of a mixture containing S-labeled products enzymatically synthesized from neutral oligosaccharide-alditols (fraction Ic) and sulfated oligosaccharide-alditols (fraction IIIc2) isolated from human respiratory mucins on CarboPac PA-100 column (4 times 250 mm). Elution was performed with gradient II described under ``Experimental Procedures.'' Peaks were detected by pulsed amperometric detection (dashed line) and by radioactivity detection (solid line). , N-acetyl-D-galactosaminitol; , Gal; bullet, GlcNAc; box, Fuc.


When the radiolabeled products were injected simultaneously with fraction IIIc2, the major peaks A, B, and D co-eluted with sulfated oligosaccharides IIIc2-18, IIIc2-19, and IIIc2-25, respectively (Fig. 7)(6) .

Peak A corresponded to the sulfated derivative of the oligosaccharide alditol Ic-19 (34) with a sulfate group at C-3 of the Gal included in the beta1-6 branch (IIIc2-18) (Fig. 7)(6) .

The oligosaccharide Ic-19 represented 7.2% of the total neutral carbohydrate chains contained in fraction Ic.

Peak B corresponded to a sulfated derivative of the oligosaccharide alditol Ic-11 (34) with a sulfate group at C-3 of the Gal included in the beta1-6 branch (IIIc2-19) (Fig. 7)(6) .

The oligosaccharide acceptor Ic-11 was a major compound in the neutral fraction Ic (13% of the total carbohydrate chains) and its sulfated derivative (B) was the main sulfated product (Fig. 7).

Peak D corresponded to a sulfated derivative of the oligosaccharide alditol Ic-12(34) , which represented 2.2% of the neutral carbohydrate chains in fraction Ic, with a sulfate group at C-3 of the Gal included in the beta1-6 branch (IIIc2-25) (Fig. 7)(6) .

Peak C did not correspond to an already known sulfated oligosaccharide-alditol.

Competition Experiments

Considering that the average length of the oligosaccharide-alditols contained in fraction Ic was four to five sugars, the molar concentration of this fraction used in the competition experiments was roughly estimated as 5-10 mM. As shown on Fig. 8, beta-methyl galactoside had a significant inhibitory effect on the sulfation of mucin oligosaccharide-alditols. The sulfation of oligosaccharide A was 17-66% lower when the incubation medium was supplemented with 1-33 mM methyl beta-galactoside. Under the same conditions, the inhibition of sulfation went from 30 to 63% for oligosaccharide B, from 26 to 80% for compound C, and from 26 to 58% for oligosaccharide D. These data suggested that methyl beta-galactoside and mucin carbohydrate chains were sulfated by the same enzyme, which might have more affinity for the reduced mucin carbohydrate chains than for methyl beta-galactoside since high concentrations of beta-methyl galactoside did not completely inhibit the mucin carbohydrate chain sulfation (Fig. 8).

Figure 8: Competition between methyl beta-galactoside and neutral oligosaccharide-alditols (fraction Ic) for the galactose 3-O-sulfotransferase activity. Considering that the average length of the oligosaccharide-alditols contained in fraction Ic was four to five sugars, the molar concentration of this fraction used in the competition experiments was roughly estimated as 5-10 mM. These results show the residual sulfotransferase activity on oligosaccharides A (box), B (), C (up triangle), and D (bullet). The composition of the incubation mixture was the same as described under standard assay conditions, except that different concentrations of methyl beta-galactoside (from 0.1 to 33 mM) were used.


When microsomal fractions were incubated with 5 mM methyl beta-galactoside and galactosylceramide, in a range of 2.5-500 µg, under the conditions described under ``Experimental Procedures,'' galactosylceramide (up to 500 µg) did not affect the sulfation of methyl beta-galactoside (Fig. 9), although the sulfotransferase has a low affinity for methyl beta-galactoside. Moreover, when galactosylceramide was used as the only substrate acceptor for the sulfotransferase, [S]sulfate incorporation into the glycolipid could not be detected. After incubation, as described under ``Experimental Procedures,'' all of the radioactivity was found in the upper aqueous phase and corresponded to [S]PAPS and its degradation derivatives (data not shown). No radioactivity was found in the lower chloroform phase. Our results point out that the galactose 3-O-sulfotransferase activity characterized in the present work is different from the sulfotransferase involved in sulfatide synthesis.

Figure 9: Competition between methyl beta-galactoside (5 mM) and galactosylceramide in a range of 2.5-500 µg for human respiratory mucosa sulfotransferase activity. The composition of the incubation mixture was the same as described under ``Experimental Procedures.''


DISCUSSION

In the present work we have characterized a microsomal sulfotransferase activity from the human respiratory mucosa, which was able to transfer sulfate groups from PAPS to C-3 of terminal galactose residues of carbohydrate chains from human airways mucins. As we have shown in a previous paper(6) , HPAEC was a suitable and reliable method to separate with high resolution and to identify sulfated carbohydrates. The structure of enzymatically radiolabeled products was deduced by comparison of their elution profile with that of well known compounds. Previous structural studies have proposed that carbohydrate chains, isolated from respiratory mucins of CF patients, could be sulfated on the C-6 of N-acetylglucosamine residues or on galactose. The data concerning the sulfation of galactose in these mucins are controversial. Using methylation and enzymatic degradation, Mawhinney et al.(2, 4, 5) have found that galactose was sulfated either on C-4 or C-6. Using ^1H NMR, methylation, and fast atom bombardment mass spectrometry, Lamblin et al.(3) and Lo-Guidice et al.(6) have observed the sulfation of galactose only on C-3 among the structures, which have been determined so far. These different data suggest that the sulfation of human respiratory mucin involves several sulfotransferases, and the present work reports the characterization of a galactose 3-O-sulfotransferase activity in human respiratory mucosa.

Since it was able to sulfate methyl galactosides, the enzyme present in human respiratory mucosa microsomal preparations seemed to act on unsubstituted terminal galactose; the beta anomer was a much better substrate than the alpha anomer.

The presence of free [S]sulfate in the reaction mixture is an indication of the PAPS degradation by hydrolases present in our microsomal preparations. These hydrolytic activities seem to be similar to those found in several tissue extracts(39, 40, 41) . Inclusion of NaF and AMP or ATP in sulfotransferase assays effectively protects the PAPS from degradation, allowing a reduction in the free [S]sulfate liberation in the reaction mixture and a stimulation of the sulfotransferase activity. Concerning ATP, the stimulatory effect was only observed at low concentration (leq5 mM); at higher concentration, ATP had an inhibitory effect. ATP, which may be considered as a structural analog of PAPS, has already been reported to inhibit sulfotransferase activities(42) ; at high concentrations, the impact on PAPS degradation would be lower than the inhibitory effect on the binding of PAPS to the sulfotransferase.

The mucosal galactose 3-O-sulfotransferase activity did not absolutely require the presence of divalent cations since it acted to some extent without added cations and was not inhibited by EDTA but was stimulated significantly by Mg and particularly by Mn at a 20 mM concentration.

The transfer of the sulfate group from PAPS to methyl beta-galactoside was optimal with 0.1% Triton X-100, 30 mM NaF, 20 mM Mn, and 10 mM AMP in a 30 mM MES buffer at pH 6.1.

The enzyme responsible for the synthesis of sulfatides also transfers the sulfate group of PAPS onto the C-3 of the galactose residue of galactosylceramides(21) . Nevertheless, this galactosylceramide sulfotransferase activity is different from that described in this paper since our enzyme preparation obtained from respiratory mucosa did not catalyze the sulfation of galactosylceramide (nor did the addition of galactosylceramide to the enzymatic assay affect the sulfation of methyl beta-galactoside).

When using a mixture of various neutral oligosaccharide alditols from human respiratory mucins, four carbohydrate chains could be sulfated. Three of the four sulfated products (A, B, and D) were identified and had in common the HSO(3)-3Galbeta1-4GlcNAcbeta1-6GalNAc-itol sequence, indicating that the enzyme was able to transfer a sulfate group to a terminal galactose residue involved in a Galbeta1-4 GlcNAc sequence. The main products A and B corresponded to the sulfated derivatives of the main potential acceptors contained in the neutral oligosaccharide mixture. The fourth sulfated product, present in peak C, was not co-eluted with a known sulfated carbohydrate chain and thus cannot be identified on the basis of its HPAEC retention time. However one might deduce that it was sulfated on a galactose residue since its sulfation was significantly affected by the presence of methyl beta-galactoside in the incubation mixture. In fraction Ic, four other neutral chains, Ic-10a, Ic-17, Ic-15.2a, and Ic-15.1b, representing, respectively, 5.3, 2.6, 1.8, and 0.6% of the total carbohydrate chains, contained a N-acetyllactosamine unit in the beta1-6 branch and could have been substrate for galactose 3-O-sulfotransferase, especially Ic-10a. However, to date, such sulfated carbohydrate chains have not been described in human respiratory mucins. There might be two explanations for that: (i) these sulfated chains exist but we have not yet been able to isolate them and (ii) they do not exist in the respiratory mucins that were studied.

Some properties of human respiratory mucosa sulfotransferase described in this paper are quite similar to those of the thyroid sulfotransferase responsible for the N-glycan sulfation of thyroglobulin(29) , including the 3-O-sulfation of terminal beta-D-galactosyl residues in a beta1-4 linkage to GlcNAc. Even if there is a difference in optimal pH between these two enzymes, it can not be anticipated that these two activities are different. One may also wonder what is the relationship between the present 3-O-sulfotransferase and the sulfotransferase activity recently described in human colorectal adenoma cell, and which is able to incorporate sulfate on the galactose residue of the O-glycan core 1(43) . Other sulfotransferase activities acting on mucin carbohydrate chains have previously been studied in rat salivary(44) , intestine(45) , and stomach(46) . They are able to transfer sulfate to C-6 of GlcNAc.

In conclusion, this is the first description of galactose sulfotransferase activity from human respiratory mucosa capable of acting on human bronchial mucin carbohydrate chains. This enzyme might act in the Golgi apparatus and add sulfate on the carbon 3 of a galactose residue belonging to an N-acetyllactosamine unit.

In the future, it will be necessary to determine the activity of this enzyme in CF tissues. Barasch et al.(19) have suggested that alkalinization of the trans-Golgi of CF cells due to defective chloride conductance was responsible for alterations in glycosyl and/or sulfotransferase activities and for the oversulfation of mucins in CF. Our results suggest that the present galactose 3-O-sulfotransferase cannot be stimulated by alkaline pH and that the oversulfation of CF respiratory glycoproteins cannot be explained by a simple effect of pH on this enzyme. However, there are obviously other sulfotransferases involved in mucin biosynthesis, and CF mucin oversulfation might be due to alterations of these other sulfotransferase activities or to a disregulation of some glycosyltransferase(s) acting before the sulfotransferases, leading to the formation of more substrates for these enzymes.

FOOTNOTES

*
This investigation was supported by the Association Française de Lutte contre la Mucoviscidose. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Unité INSERM 377, place de Verdun, 59045 Lille, France. Tel.: 33-20-29-88-62; Fax: 33-20-53-85-62.

(^1)
The abbreviations used are: CF, cystic fibrosis; MES, 2-(N-morpholino)ethanesulfonic acid; MOPS, 3-(N-morpholino)propanesulfonic acid; PAPS, adenosine 3`-phosphate 5`-phosphosulfate; HPAEC, high performance anion-exchange chromatography.

ACKNOWLEDGEMENTS

We thank Dr. Yves Plancke for running the NMR spectra and Dr. Reuben Ramphal for helpful discussion.

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