(Received for publication, March 20, 1995; and in revised form, August 9, 1995)
From the
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 - or
-galactose 3-sulfate and three oligosaccharide alditols containing
the sequence
HSO
-3Gal
1-4GlcNAc
1-6GalNAc-itol were
identified by high performance anion-exchange chromatography.
Using
methyl -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
-galactoside
and for adenosine 3`-phosphate 5`-phosphosulfate were observed at 0.69
10
M and at 4
10
M respectively. This sulfotransferase is
different from that responsible for sulfatide synthesis.
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)()(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.
2-O-Sulfated
methyl -galactoside, 3-O-sulfated methyl
-galactoside, and 4-O-sulfated methyl
-galactoside
were generous gifts from J. F. G. Vliegenthart (University of Utrecht,
The Netherlands).
6-O-Sulfated methyl -galactoside was
synthesized according to Van Kuik et al.(35) .
Briefly, methyl
-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
-galactoside was purified on a silica column (2
7 cm), using a mixture of dichloromethane/methanol (5:3 (v/v))
as eluting system; its structure was verified by 400 MHz
H
NMR spectroscopy. The sulfation of methyl
-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
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 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.
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) .
When
microsomes were incubated with methyl -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
-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
-galactosides
bearing a sulfate group either on C-2, C-3, C-4, or C-6. The
radiolabeled product synthesized from methyl
-galactoside
co-eluted with 3-O-sulfated methyl
-galactoside (Fig. 1c). Methyl
-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
-galactoside was used as substrate acceptor indicating
that the enzyme was able to transfer [
S]sulfate
to both methyl
- and
-galactosides (Fig. 1b).
The sulfotransferase activity was 17-fold higher for the
anomer
(18.44 pmol/mg of protein/min) than for the
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
-galactoside on CarboPac PA-100 column (4
250 mm) (a),
S-labeled products enzymatically obtained
from methyl
-galactoside (b), and a mixture containing
S-labeled products enzymatically obtained from methyl
-galactoside and different synthetic methyl
-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).
Figure 2:
Effect of pH on the activity of human
respiratory mucosa sulfotransferase toward methyl -galactoside.
Incubations were performed at the indicated pH values under standard
assay conditions in MES (
) and in MOPS
(
).
The influence of divalent cations on the transfer of sulfate
group from PAPS to methyl -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 -galactoside.
Incubations were performed under standard assay conditions with
indicated amounts of Mn
(
), Mg
(
), Ca
(
), and EDTA
(
).
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 -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
10
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 -galactoside by human respiratory mucosa
sulfotransferase. Incubations were carried out under standard assay
conditions with indicated amounts of AMP (
), ATP (
), and NaF
(
).
Reducing agents such as
-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
-galactoside concentration is shown in Fig. 6a. The apparent K
for methyl
-galactoside was observed at 0.69
10
M. The effect of PAPS concentration on galactose
sulfotransferase is illustrated in Fig. 6b. The
apparent K
calculated for PAPS was 4
10
M.
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 -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
-galactoside (from 10 µM to
10 mM) and PAPS (from 0.44 µM to 22
µM).
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
H 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
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;
,
GlcNAc;
, 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 1-6 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 1-6 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 1-6
branch (IIIc2-25) (Fig. 7)(6) .
Peak C did not correspond to an already known sulfated oligosaccharide-alditol.
Figure 8:
Competition between methyl
-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 (
), B (
), C (
), and D (
). The
composition of the incubation mixture was the same as described under
standard assay conditions, except that different concentrations of
methyl
-galactoside (from 0.1 to 33 mM) were
used.
When microsomal fractions were incubated with 5
mM methyl -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
-galactoside (Fig. 9), although the sulfotransferase has a low affinity for
methyl
-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
-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.''
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 H 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 anomer was a much
better substrate than the
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 (
5
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 -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 -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-3Gal
1-4GlcNAc
1-6GalNAc-itol
sequence, indicating that the enzyme was able to transfer a sulfate
group to a terminal galactose residue involved in a Gal
1-4
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
-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
1-6 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 -D-galactosyl
residues in a
1-4 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.