Sequential biosynthesis of sulfated and/or sialylated Lewis x determinants by transferases of the human bronchial mucosa

Sophie Degroote, Marie-Paule Ducourouble, Philippe Roussel, Geneviève Lamblin1

Unité INSERM n° 377, Place de Verdun, F-59045 Lille, France

Received on December 23, 1998; revised on April 9, 1999 ; accepted on April 17, 1999;

The structural determination of sulfated carbohydrate chains from a cystic fibrosis patient respiratory mucins has shown that sulfation may occur either on the C-3 of the terminal Gal, or on the C-6 of the GlcNAc residue of a terminal N-acetyllactosamine unit. The two enzymes responsible for the transfer of sulfate from PAPS to the C-3 of Gal or to the C-6 of GlcNAc residues have been characterized in human respiratory mucosa. These two enzymes, in conjunction with fucosyl- and sialyltransferases, allow the synthesis of different sulfated epitopes such as 3-sulfo Lewis x (with a 3-O-sulfated Gal), 6-sulfo Lewis x and 6-sulfo-sialyl Lewis x (with a 6-O-sulfated GlcNAc). In the present study, the sequential biosynthesis of these epitopes has been investigated using microsomal fractions from human respiratory mucosa incubated with radiolabeled nucleotide-sugars or PAPS, and oligosaccharide acceptors, mostly prepared from human respiratory mucins. The structures of the radiolabeled products have been determined by their coelution in HPAEC with known oligosaccharidic standards. In the biosynthesis of 6-O-sulfated carbohydrate chains by the human respiratory mucosa, the 6-O-sulfation of a terminal nonreducing GlcNAc residue precedes [beta]1-4-galactosylation, [alpha]2-3-sialylation (to generate 6-sulfo-sialyl-N-acetyllactosamine), and [alpha]1-3-fucosylation (to generate the 6-sulfo-sialyl Lewis x determinant). The 3-O-sulfation of a terminal N-acetyllactosamine may occur if this carbohydrate unit is not substituted. Once an N-acetyllactosamine unit is synthesized, [alpha]1-3-fucosylation of the GlcNAc residue to generate a Lewis x structure blocks any further substitution. Therefore, the present study defines the pathways for the biosynthesis of Lewis x, sialyl Lewis x, sulfo Lewis x, and 6-sulfo-sialyl Lewis x determinants in the human bronchial mucosa.

Key words: biosynthesis/bronchial mucins/cystic fibrosis/6-sulfo-sialyl Lewis x/transferases

INTRODUCTION

Human respiratory mucins consist of a broad family of high molecular weight and polydisperse O-glycosylproteins synthesized by specialized cells from the bronchial mucosa. Their peptide diversity stems from the expression of several genes (MUC2, 4, 5AC, 5B, 7, and 8) (Bobek et al., 1993; Jeffery and Li, 1997; Shankar et al., 1997; Desseyn et al., 1998) but the mucin heterogeneity is essentially due to post-translational phenomena, mostly O-glycosylation but also sulfation (Roussel and Lamblin, 1996). These phenomena lead to a remarkable diversity of carbohydrate chains, which allows the binding of inhaled microorganisms. These pathogens are then eliminated by the activity of the mucociliary system (Roussel and Lamblin, 1996).

Mucins are the most important compounds of the mucus layer: They are responsible for its rheological properties. Changes in the carbohydrate sequences of respiratory mucins could modify the mucus properties, leading to a nonefficient mucociliary clearance and to bacterial colonization and infection, as observed in cystic fibrosis (CF) and chronic bronchitis.

Cystic fibrosis is the most frequent autosomal recessive disease among Caucasians and is characterized in its most typical form by mucus hypersecretion and severe chronic lung infection by Pseudomonas aeruginosa. This disease is due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an apical membrane chloride channel (Riordan, 1993) that affects several other epithelial channels or transporters. [Delta]F508 is the most frequent mutation in CF patients and prevents CFTR from exiting the endoplasmic reticulum (Pind et al., 1994). It has been known for many years that there were abnormalities in glycosylation and sulfation of various glycoconjugates in CF. Several studies have shown that respiratory and salivary mucins secreted by CF patients as well as mucins synthesized by CF nasal epithelial cells were oversulfated (Roussel et al., 1975; Lamblin et al., 1977; Boat et al., 1977; Cheng et al., 1989; Carnoy et al., 1993). More recently, Zhang et al. (1995), using a model of human xenograft which eliminates the influence of infection and inflammation, have suggested that the increased sulfate content of CF mucins might be a primary defect. These abnormalities may be related to a defective acidification of the trans-Golgi/trans-Golgi network in CF cells, due to mislocation of mutated CFTR, and leading to modifications in the sulfation and glycosylation processes (Barasch et al., 1991; Barasch and Al-Awqati 1993; Dosanjh et al., 1994). However, Pasyk and Foskett (1997) have recently suggested that CFTR could modulate the amount of adenosine 3[prime]-phosphate 5[prime]-phosphosulfate (PAPS) in the Golgi apparatus and that mutated CFTR could be responsible for the accumulation of PAPS in the Golgi and for mucin hypersulfation.

In addition to oversulfation, recent studies on salivary and bronchial mucins from CF patients have shown an increased content of sialic acid (Barasch et al., 1991; Barasch and Al-Awqati, 1993; Carnoy et al., 1993; Dosanjh et al., 1994; Zhang et al., 1995; Pasyk and Foskett, 1997; Davril et al., 1999), and in the case of CF bronchial mucins, of sialyl Lewis x (sLex) determinants that might be related to the strong inflammatory response observed in this disease (Davril et al., 1999).

Such posttranslational modifications may lead to abnormal carbohydrate chains in respiratory mucins specifically recognized by Staphylococcus aureus or Pseudomonas aeruginosa that usually colonize the CF lung and are responsible for most of the morbidity and mortality of the disease (Høiby, 1988).

The structural determination of mucin carbohydrate chains from CF patients has shown the presence of various complex structures such as the sialyl Lewis x (sLex), the 3-sulfo Lewis x (3-sulfo Lex), and the 6-sulfo-sialyl Lewis x (6-sulfo-sLex) determinants (Lo-Guidice et al., 1994). The role of such determinants in the colonization of the CF airways by Pseudomonas aeruginosa has not yet been established. Moreover, the biosynthesis of these determinants remains controversial (Scudder et al., 1994; Crommie and Rosen, 1995).

In order to ultimately study the respective roles of CFTR abnormality and lung inflammation on the expression of the sialylated and/or sulfated determinants of CF mucins, we have analyzed the biosynthesis of these different Lex derivatives by microsomal fractions from human respiratory mucosa. The synthesized products were characterized by high performance anion exchange chromatography with pulsed amperometric (HPAEC-PAD) and radioactivity detection. The present work gives new information about the specificity of the various fucosyl-, sialyl-, and sulfotransferases involved in the biosynthesis of complex Lex structures.

RESULTS

6-O-sulfation of carbohydrate acceptors having a terminal nonreducing GlcNAc residue

As reported previously (Degroote et al., 1997), the GlcNAc-6-O-sulfotransferase from human respiratory mucosa is able to catalyze the transfer of a sulfate group onto the C-6 of GlcNAc[beta]1-O-Met (which leads to HO3S-6GlcNAc[beta]1-O-Met) and on the C-6 of the terminal GlcNAc residue of OS2 (which leads to oligosaccharide-alditol IVc-19) (Table I). This enzyme is not active anymore after the action of a [beta]1-4-galactosyltransferase from human respiratory mucosa which adds a Gal residue linked [beta]1-4 to this GlcNAc (Degroote et al., 1997). Thus, when OS1 (with a terminal Gal residue on the upper branch) was incubated with [35S]PAPS and bronchial microsomal fractions (which contained both GlcNAc-6-O- and Gal-3-O-sulfotransferase activities), the radiolabeled product which was obtained coeluted with oligosaccharide-alditol IVc-10 (Table II) which corresponded to OS1 with a sulfate group on the C-3 of the terminal Gal residue but there was no peak corresponding to oligosaccharide-alditol IVc-12 indicating that there was no direct sulfation of the GlcNAc residue of OS1 (Table I, Scheme 0).


Table I. GlcNAc-6-O-sulfotransferase assays with human bronchial microsomes
aRelative rates for each are expressed as a percentage of the incorporation with GlcNAc[beta]1-O-Met (17.27 pmol/mg of protein/min).


Table II. Gal-3-O-sulfotransferase assays with human bronchial microsomes
aRelative rates for each acceptor are expressed as a percentage of the incorporation with Gal[beta]1-O-Met (18.44 pmol/mg of protein/min).

Other substrates were assayed for 6-O-sulfation. No sulfation could be obtained with Fuc([alpha]1-3)GlcNAc[beta]1-O-Met, NeuAc([alpha]2-3)Gal([beta]1-4)GlcNAc, Gal([beta]1-4)[Fuc[alpha]1-3]GlcNAc, NeuAc([alpha]2-3)Gal([beta]1-4)[Fuc[alpha]1-3]GlcNAc, indicating that once the GlcNAc residue is substituted (by a Gal or a Fuc residue), GlcNAc-6-O-sulfation cannot occur anymore. The only peak which was observed on the HPAEC elution profile was the free [35S]sulfate peak, at 11 min 30 s using gradient I or at 45 min 24 s when gradient III was used. The activity of the GlcNAc-6-O-sulfotransferase on the different substrates which were tested is described in Table I.

These data indicate that, in mucin-type oligosaccharide biosynthesis, the GlcNAc-6-O-sulfotransferase must act on a nonsubstituted terminal GlcNAc residue.

3-O-Sulfation of carbohydrate acceptors having a terminal nonreducing Gal residue

As already mentioned, Gal[beta]1-O-Met and oligosaccharide-alditol OS1 with a terminal N-acetyllactosamine unit were sulfated by the Gal-3-O-sulfotransferase characterized by Lo-Guidice et al. (1995). However, this enzyme was not active on terminal Lex structures: when microsomal fractions were incubated with Gal([beta]1-4)[Fuc[alpha]1-3]GlcNAc-O-Met and [35S]PAPS, no Gal-3-O-sulfation could be observed (Table II). Fraction IIc (which contains in particular an oligosaccharide-alditol with a core type 2, a Lex determinant on the upper branch and a NeuAc([alpha]2-3)Gal unit on the lower branch) was also used as acceptor. No radiolabeled peak coeluted with IVc-2, which means that the bronchial Gal-3-O-sulfotransferase is not active on a Lex determinant (Table II). The activity of the Gal-3-O-sulfotransferase on the different substrates which were tested is described in Table II.

Fucosylation of different carbohydrate determinants by the [alpha]1-3-fucosyltransferase activity of the human bronchial mucosa

GlcNAc[beta]1-O-Met and HO3S-6GlcNAc[beta]1-O-Met were first used to test the [alpha]1-3-fucosyltransferase activity of the bronchial microsomal fractions (Table III). No radiolabeled peak could be observed on the HPAEC elution profile, indicating that the transfer of a Fuc residue from GDP-Fuc on the C-3 of a terminal GlcNAc (6-O-sulfated or not) was not possible. No peak corresponding to radiolabeled [3H]GDP-Fuc appeared on the elution profile; this compound was eluted with higher concentrations of sodium acetate. Similarly, no fucosylation of the oligosaccharide-alditols OS2 (with a terminal GlcNAc residue) and IVc-19 (with a terminal HO3S-6GlcNAc residue) could be obtained (Table III).

Table III. [alpha]1-3-fucosyltransferase assays with human bronchial microsomes
aRelative rates for each acceptor are expressed as a percentage of the incorporation with Gal([beta]1-4)BlcNAc (0.23 pmol/mg of protein/min).

When Gal([beta]1-4)GlcNAc was incubated with bronchial microsomal fractions and radiolabeled [3H]GDP-Fuc, the HPAEC elution profile showed a characteristic radiolabeled peak at 13 min 48 s (Figure 1a). In order to find out the position of the Fuc residue on this radiolabeled product, it was coinjected with a nonlabeled Gal([beta]1-4)[Fuc[alpha]1-3]GlcNAc, which was visualized by PAD. The neosynthesized fucosylated product coeluted exactly with Gal([beta]1-4)[Fuc[alpha]1-3]GlcNAc, indicating that a bronchial [alpha]1-3-fucosyltransferase activity was able to transfer a Fuc residue from [3H]GDP-Fuc onto the C-3 of the GlcNAc residue of a type 2 chain (Figure 1a). In our experimental conditions, the absence of any neosynthesized peak corresponding to Fuc([alpha]1-2)Gal([beta]1-4)GlcNAc suggested that [alpha]1-2-fucosyltransferases were absent or expressed at a very low level as compared to the [alpha]1-3-fucosyltransferase activity.


Scheme 1. Sequential biosynthesis of sulfated and/or sialylated Lewis x determinants in human respiratory mucosa. The enzymes involved in this biosynthesis are: 3sT, Gal-3-O-sulfotransferase; 6sT, GlcNAc-6-O-sulfotransferase; 4GT, [beta]1-4-galactosyltransferase; ST3, [alpha]2-3-sialyltransferase and FT, [alpha]1-3-fucosyltransferase from human respiratory mucosa.


Fig. 1. HPAEC elution profile on a CarboPac PA-100 column (4 - 250 mm) of a mixture containing [3H]-labeled products enzymatically synthesized from [3H]GDP-Fuc and Gal([beta]1-4)GlcNAc (a) or NeuAc([alpha]2-3)Gal([beta]1-4)GlcNAc (b), and unlabeled [alpha]1-3-fucosylated standards: Gal([beta]1-4)[Fuc[alpha]1-3]GlcNAc and NeuAc([alpha]2-3)Gal([beta]1-4)[Fuc[alpha]1-3]GlcNAc, respectively. Elution was performed with gradient III described under Materials and methods. Peaks were detected by pulsed amperometry (dashed line) and by radioactivity (solid line).

When NeuAc([alpha]2-3)Gal([beta]1-4)GlcNAc was incubated with microsomal fractions and radiolabeled [3H]GDP-Fuc, a characteristic radiolabeled peak was observed, which was eluted at 20 min 54 s (Figure 1b). When the radiolabeled products were coinjected with nonlabeled NeuAc([alpha]2-3)Gal[Fuc[alpha]1-3]GlcNAc, the characteristic peak at 20 min 54 s coeluted with this standard. This proved that the [alpha]1-3-fucosyltransferase activity was active on a [alpha]2-3-sialylated type 2 N-acetyllactosamine unit. Similarly, the [alpha]1-3-fucosyltransferase activity was tested on oligosaccharide-alditol IIIc1-26, which has an [alpha]2-3-sialylated type 2 unit on the upper branch (Table III). The HPAEC elution profile showed a characteristic radiolabeled peak at 33 min 54 s, which coeluted with oligosaccharide-alditol IIIc1-17 (Figure 2). This oligosaccharide has the same structure as IIIc1-26, with a Fuc residue ([alpha]1-3) linked to the internal GlcNAc residue (Table III). Altogether, these results indicate that the [alpha]1-3-fucosyltransferase activity from human bronchial mucosa is active on a type 2 N-acetyllactosamine unit, [alpha]2-3-sialylated or not, but is inactive on terminal GlcNAc or HO3S-6GlcNAc residues.


Fig. 2. HPAEC elution profile on a CarboPac PA-100 column (4 × 250 mm) of a mixture containing [3H]-labeled products enzymatically synthesized from [3H]GDP-Fuc and oligosaccharide-alditol IIIc1-26, and unlabeled [alpha]1-3-fucosylated standard oligosaccharide-alditol IIIc1-17. Elution was performed with gradient II described under Materials and methods. Peaks were detected by pulsed amperometry (dashed line) and by radioactivity (solid line).

Moreover, [beta]1-4-galactosylation was possible on terminal GlcNAc or HO3S-6GlcNAc residues, but not on Fuc([alpha]1-3)GlcNAc[beta]1-O-Met, indicating that the [alpha]1-3-fucosyltransferase activity from human bronchial mucosa was active after the action of the [beta]1-4-galactosyltransferase (data not shown).

In order to determine whether an internal HO3S-6GlcNAc residue in a type 2 N-acetyllactosamine unit may affect [alpha]1-3-fucosylation, the oligosaccharide-alditol IVc-12 which has a Gal([beta]1-4)[HO3S-6]GlcNAc structure on the upper branch (Table III) was incubated with microsomal fractions and [3H]GDP-Fuc (Table III). The elution profile showed a characteristic radiolabeled peak at 41 min 24 s which coeluted with oligosaccharide-alditol IVc-5 (Figure 3); IVc-5 has the same structure as IVc-12 but with a Fuc residue ([alpha]1-3) linked to the internal HO3S-6GlcNAc residue (Table III). No [alpha]1-2-fucosylation occurred on the terminal Gal residue, as proved by the absence of any radiolabeled oligosaccharide-alditol having the same retention time than IVc-8, the fucosylated derivative of IVc-5 with a Fuc residue linked ([alpha]1-2) to the terminal Gal residue.


Fig. 3. HPAEC elution profile on a CarboPac PA-100 column (4 × 250 mm) of a mixture containing [3H]-labeled products enzymatically synthesized from [3H]GDP-Fuc and oligosaccharide-alditol IVc-12, and unlabeled oligosaccharide-alditols from human respiratory mucins (fraction IVc). Elution was performed with gradient III described under Materials and methods. Peaks were detected by pulsed amperometry (dashed line) and by radioactivity (solid line).

These results suggest that, starting from a terminal GlcNAc residue, the enzymes responsible for the biosynthesis of a 6-sulfo Lex determinant act in the following order: (1) GlcNAc-6-O-sulfation, (2) [beta]1-4-galactosylation, and (3) [alpha]1-3-fucosylation.

Similarly, the oligosaccharide-alditol IVc-23 (which has a NeuAc([alpha]2-3)Gal([beta]1-4)[HO3S-6]GlcNAc structure on the upper branch and a NeuAc([alpha]2-3)Gal disaccharide on the lower branch) (Table III) was incubated with microsomal fractions and [3H]GDP-Fuc. The HPAEC elution profile of the neosynthesized product showed one radiolabeled peak (Figure 4), which had the same retention time as oligosaccharide-alditol IVc-14 (the fucosylated derivative of IVc-23 with a Fuc ([alpha]1-3) linked to the internal GlcNAc residue) (Table III). These results suggest that, in the biosynthesis of 6-sulfo-sLex, [alpha]1-3-fucosylation of the GlcNAc residue of a terminal Gal([beta]1-4)GlcNAc unit occurs after the actions of GlcNAc-6-O-sulfotransferase, [beta]1-4-galactosyltransferase and Gal-[alpha]2-3-sialylation. In fact, the [alpha]1-3-fucosyltransferase from human bronchial mucosa is also active (1) on the GlcNAc residue of a terminal Gal([beta]1-4)GlcNAc unit, (2) on the GlcNAc residue of a terminal Gal([beta]1-4)[HO3S-6]GlcNAc unit, and (3) on the GlcNAc residue of a terminal NeuAc([alpha]2-3)Gal([beta]1-4)GlcNAc unit.


Fig. 4. HPAEC elution profile on a CarboPac PA-100 column (4 × 250 mm) of a mixture containing [3H]-labeled products enzymatically synthesized from [3H]GDP-Fuc and IVc-23, and unlabeled oligosaccharide-alditols from human respiratory mucins (fraction IVc). Elution was performed with gradient III described under Materials and methods. Peaks were detected by pulsed amperometry (dashed line) and by radioactivity (solid line).

The influence of a sulfate group linked to the C-3 of the Gal residue of a N-acetyllactosamine unit on the [alpha]1-3-fucosyl transferase activity was also tested. The enzyme was active on oligosaccharide-alditol IVc-10, which has a Gal([beta]1-4)GlcNAc with a sulfate group on the C-3 of the terminal Gal residue on the upper branch (Table III) and led to the synthesis of oligosaccharide-alditol IVc-2 which has a terminal HO3S-3Gal([beta]1-4)[Fuc[alpha]1-3]GlcNAc structure on the upper branch (data not shown).

As the Gal-3-O-sulfotransferase from human bronchial mucosa is not active on terminal Lex structures, we can conclude that in the biosynthesis of the 3-sulfo Lex, Gal-3-O-sulfation occurs before GlcNAc-[alpha]1-3-fucosylation (Scheme 4). The relative activity of the [alpha]1-3-fucosyltransferase on the different substrates which were used is described in Table III.

Action of [alpha]2-3-sialyltransferase from human bronchial mucosa on oligosaccharidic acceptors

The [alpha]2-3-sialyltransferase activity was first tested with Gal([beta]1-4)GlcNAc. When microsomal fractions were incubated with this acceptor and radiolabeled [14C]CMP-NeuAc, the HPAEC elution profile, using gradient III, showed three radiolabeled peaks (Figure 5). Two of them were present when incubations were performed without any substrate; they were identified by coelution with nonlabeled standards whose retention times are known. The first radiolabeled peak, which was eluted at 21 min 4 s corresponded to free sialic acid. The one which was eluted at 47 min 30 s was identified as being [14C]CMP-NeuAc. The characteristic radiolabeled peak was eluted at 23 min 24 s and coeluted with the standard NeuAc([alpha]2-3)Gal([beta]1-4)GlcNAc. The [alpha]2-3-sialyltransferase activity was also tested on oligosaccharide-alditol OS1, which has a Gal([beta]1-4)GlcNAc unit on the upper branch (Table IV). When the radiolabeled products were fractionated by HPAEC using gradient III, three radiolabeled peaks were observed (Figure 6): two of them were not characteristic and corresponded to free sialic acid (retention time: 21 min 4 s), and to [14C]CMP-NeuAc (retention time: 47 min 30 s). A characteristic peak was eluted at 23 min 54 s. When the radiolabeled products were injected with the oligosaccharide-alditol IIIc1-26 (which has the same structure as OS1 with a NeuAc ([alpha]2-3) linked to the terminal Gal residue) (Table IV), the characteristic peak coeluted with IIIc1-26 (Figure 6); this proved that this [alpha]2-3-sialyltransferase was active on terminal Gal residue of OS1.


Fig. 5. HPAEC elution profile on a CarboPac PA-100 column (4 × 250 mm) of a mixture containing [14C]-labeled products enzymatically synthesized from [14C]CMP-NeuAc and oligosaccharide-alditol Gal([beta]1-4)GlcNAc, and unlabeled NeuAc([alpha]2-3)Gal([beta]1-4)GlcNAc. Elution was performed with gradient III described under Materials and methods. Peaks were detected by pulsed amperometry (dashed line) and by radioactivity (solid line).


Fig. 6. HPAEC elution profile on a CarboPac PA-100 column (4×250 mm) of a mixture containing [14C]-labeled products enzymatically synthesized from [14C]CMP-NeuAc and oligosaccharide-alditol OS1, and unlabeled oligosaccharide-alditol IIIc1-26. Elution was performed with gradient III described under Materials and methods. Peaks were detected by pulsed amperometry (dashed line) and by radioactivity (solid line).


Table IV. [alpha]2-3-sialylation assays with human bronchial microsomes
aRelative rates for each acceptor are expressed as a percentage of the incorporation with Gal(b1-4)GlcNAc (41.07 pmol/mg of protein/min).

In order to check if Gal-[alpha]2-3-sialylation was possible on a terminal Gal([beta]1-4)[HO3S-6]GlcNAc unit, the oligosaccharide-alditol IVc-12 (Table IV) was incubated with microsomal fractions and radiolabeled [14C]CMP-NeuAc. A neosynthesized radiolabeled peak coeluted with oligosaccharide-alditol IVc-23 (Figure 7), which has the same structure as IVc-12 with a NeuAc ([alpha]2-3) linked to the terminal Gal residue (Table IV). This result suggests that the [alpha]2-3-sialyltransferase is also active on a terminal Gal residue of a Gal([beta]1-4)[HO3S-6]GlcNAc unit.


Fig. 7. HPAEC elution profile on a CarboPac PA-100 column (4 × 250 mm) of a mixture containing [14C]-labeled products enzymatically synthesized from [14C]CMP-NeuAc and IVc-12 and unlabeled oligosaccharide-alditols from human respiratory mucins (fraction IVc). Elution was performed with gradient III described under Materials and methods. Peaks were detected by pulsed amperometry (dashed line) and by radioactivity (solid line).

When microsomal fractions were incubated with radiolabeled [14C]CMP-NeuAc and Gal([beta]1-4)[Fuc[alpha]1-3]GlcNAc, no characteristic radiolabeled peak was observed, the enzyme was not active on this substrate. The [alpha]2-3-sialyltransferase activity was neither active on oligosaccharide-alditol IVc-5 (Table IV) nor on Gal[beta]1-O-Met. The relative activity of the [alpha]2-3-sialyltransferase on the different substrates which were used is indicated in Table IV.

These results suggest that Gal-[alpha]2-3-sialylation occurs after GlcNAc-6-O-sulfation but before GlcNAc-[alpha]1-3-fucosylation. Indeed, once there was a Fuc residue ([alpha]1-3) linked to the internal GlcNAc residue, the [alpha]2-3-sialyltransferase was not active anymore (Scheme 7).

DISCUSSION

The structural determination of oligosaccharide-alditols from a CF patient respiratory mucins has shown the presence of different sulfated epitopes, such as terminal 3-sulfo Lex, 6-sulfo Lex, and 6-sulfo-sLex structures (Lo-Guidice et al., 1994). We report here the sequential biosynthesis of these structures in human respiratory mucosa.

Our analysis concerning the biosynthesis of 6-sulfo-sLex argues that GlcNAc-6-O-sulfation is the first event, followed by [beta]-galactosylation, [alpha]2-3-sialylation and [alpha]1-3-fucosylation (Scheme 7).

As reported before, the GlcNAc-6-O-sulfotransferase from human bronchial mucosa is only active on terminal GlcNAc residues (Degroote et al., 1997). These results are in agreement with those obtained by Spiro et al. (1996) who characterized a GlcNAc-6-O-sulfotransferase from rat liver. Recently, a human GlcNAc-6-O-sulfotransferase was cloned by Uchimura et al. (1998b), based on the sequence homology to the cloned cDNA of mouse GlcNAc-6-O-sulfotransferase (Uchimura et al., 1998a). This enzyme was active on terminal GlcNAc residues and involved in the biosynthesis of NeuAc([alpha]2-3)Gal([beta]1-4)-[HO3S-6]GlcNAc and Gal([beta]1-4)[Fuc[alpha]1-3][HO3S-6]GlcNAc. This enzyme is strongly expressed in cerebrum, cerebellum, eye, pancreas, and lung of adult mice. Another report by Bowman et al. (1998) dealing with the identification of a GlcNAc-6-O-sulfotransferase activity specific for lymphoid tissue indicates the requirement for a substrate with a terminal GlcNAc residue, suggesting that, as found for the GlcNAc-6-O-sulfotransferase described here, sulfation precedes further biosynthetic assembly of 6-sulfo-sLex.

The Gal-3-O-sulfotransferase from human respiratory mucosa was active on terminal Gal residues of Gal([beta]1-4)GlcNAc disaccharide from oligosaccharide-alditols carbohydrate chains (Lo-Guidice et al., 1995). Our results showed that this enzyme was inactive if the internal GlcNAc residue was [alpha]1-3-fucosylated. Thus, in the biosynthesis of 3-sulfo Lex, an [alpha]1-3-fucosyltransferase acts after the action of the Gal-3-O-sulfotransferase (Scheme 7). These results are in good agreement with those of Chandrasekaran et al. (1997) who characterized two groups of Gal-3-O-sulfotransferases which were both inactive on terminal Lex structures.

An [alpha]2-3-sialyltransferase activity was also present in the microsomal fractions, adding N-acetylneuraminic acid on the terminal Gal residue of Gal([beta]1-4)GlcNAc or Gal([beta]1-4)[HO3S-6]GlcNAc disaccharide (Table IV). Similar results were obtained by Spiro et al. (1996) concerning an [alpha]2-3-sialyltransferase from rat liver. Moreover, the [alpha]2-3-sialyltransferase from human bronchial mucosa was inactive on Lex structures (with or without a sulfate group on the C-6 of the GlcNAc residue). Among the human [alpha]2-3-sialyltransferases already cloned, ST3Gal III and ST3Gal IV use the disaccharide sequence Gal([beta]1-4)GlcNAc as an acceptor to form NeuAc([alpha]2-3)Gal([beta]1-4)GlcNAc (Kitagawa and Paulson, 1994). These two enzymes are expressed in the human bronchial mucosa (unpublished observations).

Concerning the [alpha]1-3-fucosyltransferase activity which was also present in the microsomal fractions, it was active on Gal([beta]1-4)GlcNAc, Gal([beta]1-4)[HO3S-6]GlcNAc, NeuAc([alpha]2-3)Gal([beta]1-4)GlcNAc, NeuAc([alpha]2-3)Gal([beta]1-4)[HO3S-6]GlcNAc and on HO3S-3Gal([beta]1-4)GlcNAc structures (Table III). Recent studies about the acceptor specificity of three human [alpha]1-3/(4) fucosyltransferases (FucT-III, FucT-IV and FucT-V) (Kukowska-Latallo et al., 1990; Kumar et al., 1991; Weston et al., 1992a) have shown that they have slightly different, but mainly overlapping substrate specificities (De Vries et al., 1995; Chandrasekaran et al., 1996a). FucT-III is mostly active on type 1 but also on type 2 units, sialylated or not. Concerning FucT-IV, neutral and GlcNAc-6-O-sulfated type 2 chains were good substrates whereas [alpha]2-3-sialylated type 1 and 2 structures were poor acceptors. FucT-V was active on a broad variety of neutral and acidic substrates. The two last human [alpha]1-3-fucosyltransferases that have been cloned, FucT-VI (Weston et al., 1992b) and FucT-VII (Sasaki et al., 1995), can both be involved in the biosynthesis of sLex epitopes (De Vries et al., 1996; Britten et al., 1998). However, a recent study of the expression of [alpha]1-3-fucosyltransferases in human bronchial epithelial cells in secondary culture has shown that only FucT-III and FucT-IV were expressed in these cells (Emery et al., 1997). FucT-V, FucT-VI, and FucT-VII have not been detected by reverse transcriptase polymerase chain reaction (RT-PCR) in human bronchial epithelial cells in secondary culture (unpublished observations). Thus, our results suggest that the [alpha]1-3-fucosyltransferases involved in the biosynthesis of sLex and 6-sulfo-sLex epitopes by human bronchial mucosa are FucT-III and FucT-IV (Davril et al., 1999).

The absence of [alpha]1-2-fucosyltransferase activity was quite unexpected because the two cloned enzymes FucT-I and FucT-II (Larsen et al., 1990; Kelly et al., 1995) mRNAs were detected in human bronchial epithelial cells in secondary culture (Emery et al., 1997). When IVc-12 (which has a Gal([beta]1-4)[HO3S-6]GlcNAc on the upper branch) was incubated with microsomal fractions and [3H]GDP-Fuc, the only neosynthesized product coeluted with IVc-5, the [alpha]1-3-fucosylated derivative (Table III). HPAEC-PAD is a suitable method to separate oligosaccharides, even those with an identical molecular weight but with different linkages (Lo-Guidice et al., 1994). The only difference between oligosaccharide-alditols IVc-5 and IVc-8 is the Fuc linkage, [alpha](1-3) and [alpha](1-2), respectively, and they have different retention times: 41 min 24 s and 43 min 36 s, respectively (Figure 3). According to Chandrasekaran et al. (1996b), FucT-I and FucT-II were found to be active on terminal Gal([beta]1-4)GlcNAc but a substituent on GlcNAc decreased or abolished the acceptor ability. This could explain the absence of [alpha]1-2-fucosyltransferase activity on IVc-12, which could be a better substrate for the [alpha]1-3-fucosyltransferase from human bronchial mucosa. The absence of [alpha]1-2-fucosyltransferase activity on Gal([beta]1-4)GlcNAc may correspond to a competition between the [alpha]1-2- and the [alpha]1-3-fucosyltransferases, in favor of the [alpha]1-3-fucosylation. However, it is quite possible that, as for the Gal-3-O-sulfotransferase and the [alpha]2-3-sialyltransferase, the [alpha]1-2-fucosyltransferase activity from human bronchial mucosa is inactive on terminal Lex structures.

No [alpha]2-6-sialylation was observed on terminal Gal residues of the different substrates which were tested. [alpha]2-6-Sialylation of terminal Gal residue may occur very rarely in human respiratory mucins. More than 150 oligosaccharides have been described so far in human respiratory mucins, and only three of them have a [alpha]2-6-sialylated terminal Gal residue (Breg et al., 1987; Mawhinney et al., 1992; Lo-Guidice et al., 1994).

In this study, we did not test for the formation of the sulfated and/or sialylated Lea derivatives. Indeed, in our previous work on structural determination of oligosaccharides from mucins secreted by patients with cystic fibrosis or chronic bronchitis, all the patients chosen were Secretor and Lewis positive. So the presence of both H and Lewis epitopes on type 1 chain leads to the Leb structure or Ley on a type 2 chains. In the bronchial mucins most of the chains are type 2 chains and express Lex and Ley epitopes. So we were more interested in the biosynthesis of sulfated and/or sialylated Lex derivatives. Moreover, we do not have any standard which contain the sulfated and/or sialylated derivatives of the Lea structure.

Our finding in the ordering of [alpha]2-3-sialylation versus [alpha]1-3-fucosylation is in good agreement with previous studies (Natsuka et al., 1994; Crommie and Rosen, 1995): [alpha]2-3-sialylation precedes [alpha]1-3-fucosylation in sLex and 6-sulfo-sLex biosynthesis. Moreover, the [alpha]2-3-sialyltransferases, as well as the Gal-3-O-sulfotransferase and the [alpha]1-2-fucosyltransferases from human respiratory mucosa, were not active on a terminal Lex structure.

Altogether, these results suggest that 6-O-sulfation of the GlcNAc residue is the first event in the biosynthesis of 6-sulfated chains leading to the 6-sulfo-sLex in human respiratory mucosa and that it precedes both galactosylation and [alpha]2-3-sialylation. [alpha]1-3-Fucosylation of the GlcNAc residue is the last event. Similarly, the Gal-3-O-sulfotransferase active on terminal Gal, in N-acetyllactosamine-containing mucin carbohydrate chains, cannot act if the C-3 position of the GlcNAc residue is fucosylated.

The activity of the GlcNAc-6-O-sulfotransferase in respiratory mucins biosynthesis leads to the synthesis of carbohydrate chains having the same structure as those of GlyCAM-1, one of the best ligand for L-selectin. GlyCAM-1 contains both 6-sulfo-sLex and 6[prime]-sulfo-sLex structures, but recent studies have shown that 6-sulfo-sLex (found in mucin carbohydrate chains) was the best ligand for L-selectin (better than 6[prime]-sulfo-sLex and 6-6[prime]-bis-sulfo-sLex) (Galustian et al., 1997). Crottet et al. (1996) have shown that human respiratory mucins containing such oligosaccharides were bound by L-selectin and thus could interact with leukocytes.

In CF, two types of events may influence the biosynthesis of mucin carbohydrate chains: (1) oversulfation which has been reported to be linked to the primary defect (Zhang et al., 1995) and (2) hypersialylation and sLex hyperexpression which are related to the inflammatory response (Davril et al., 1999).

In the future, it will be important to investigate the influence of cytokines on the activity of various sulfo-, sialyl-, and fucosyltransferases and to find out if the overexpression of the resulting carbohydrate determinants may favor the airway colonization by Pseudomonas aeruginosa, the major problem faced by the patients suffering from this disease.

MATERIALS AND METHODS

Preparation of microsomes from the human respiratory mucosa

Fragments of bronchial mucosa from patients undergoing surgery for bronchial carcinoma were collected in macroscopically healthy areas of the bronchial tree. Microsomal fractions were prepared from a pool of bronchial mucosa fragments from different patients as previously described and stored at -80°C until used (Lo-Guidice et al., 1995).

Acceptors and/or standards

The different compounds used for acceptor specificity studies were from the following sources: GlcNAc[beta]1-O-Met, Gal[beta]1-O-Met, and Gal([beta]1-4)GlcNAc were from Sigma; Fuc([alpha]1-3)GlcNAc[beta]1-O-Met, Gal([beta]1-4)[Fuc[alpha]1-3]GlcNAc, Gal([beta]1-4)[Fuc[alpha]1-3]GlcNAc[beta]1-O-Met, NeuAc([alpha]2-3)Gal([beta]1-4)GlcNAc ([alpha]2-3sialyl-N-acetyllactosamine), HO3S-3Gal([beta]1-4)[Fuc[alpha]1-3]GlcNAc[beta]1-O-Met, and NeuAc([alpha]2-3)Gal([beta]1-4)[Fuc[alpha]1-3]GlcNAc (sLex) were from Toronto Research Chemicals Inc.; HO3S-6GlcNAc[beta]1-O-Met was synthesized according to the protocol described by Van Kuik et al. (1987). The oligosaccharide-alditols OS1 and OS2 from Collocalia mucins, whose structures are described in Table I, were prepared as described previously (Strecker et al., 1992).

Ten oligosaccharide-alditols from human respiratory mucins, whose structures were determined by Lo-Guidice et al. (1994), were also used as acceptors (IIIc1-26, IVc-5, IVc-10, IVc-12, IVc-19, IVc-23) and/or standards (IIIc1-17, IIIc1-26, IVc-2, IVc-5, IVc-8, IVc-10, IVc-12, IVc-14, IVc-19, IVc-23). Briefly, these oligosaccharide-alditols were prepared by alkaline borohydride treatment of mucin glycopeptides, fractionated by anion-exchange chromatography on an AG 1-X2 column (Bio-Rad) according to acidity and then by gel-filtration on a Bio-Gel P4 column (Bio-Rad) according to size (Lamblin et al., 1991). The pools of acidic oligosaccharide-alditols IIIc1 and IVc were further fractionated by HPAEC, using a CarboPac PA-100 column (Dionex Corp.) and a PAD 2-pulsed amperometric detector (Dionex Corp.). The structure of 24 sialylated and/or sulfated oligosaccharide-alditols were determined by high resolution 1H NMR in combination with fast atom bombardment-mass spectrometry (Lo-Guidice et al., 1994). The oligosaccharides used in the present study are shown in Tables I-IV. Most of them have a core type 2.

A fraction of monosialyl oligosaccharide-alditols from a CF patient respiratory mucins was also used as acceptors (fraction IIc) (Breg et al., 1987). This fraction contains 24 oligosaccharide-alditols whose structure was determined by 1H NMR. One of them is particularly interesting for this study: it has a core type 2, a Lex structure on the upper branch, and a NeuAc([alpha]2-3)Gal disaccharide on the lower branch (Table II).

GlcNAc-6-O-Sulfotransferase and Gal-3-O-sulfotransferase assays

The GlcNAc-6-O-sulfotransferase assays were performed as described previously (Degroote et al., 1997). The incubation mixture contained 50-100 µg of microsomal proteins, 0.5 µCi [35S]PAPS (NEN Life Science Products, 2.25-2.50 Ci/mmol), 5 mM of one of the following substrates: GlcNAc[beta]1-O-Met, Gal([beta]1-4)[Fuc[alpha]1-3]GlcNAc, Fuc([alpha]1-3)GlcNAc[beta]1-O-Met, NeuAc([alpha]2-3)Gal([beta]1-4)GlcNAc, NeuAc([alpha]2-3)Gal([beta]1-4)[Fuc[alpha]1-3]GlcNAc, or 100 µg of one of the oligosaccharide-alditols, OS1 or OS2 (Table I), in a Mops/NaOH buffer, pH 6.7, containing 0.1 % (w/v) Triton X-100, 20 mM MnCl2, 30 mM NaF, 5 mM AMP, 1 mM 4-(2-aminoethyl)benzene sulfonylfluoride (AEBSF).

For the Gal-3-O-sulfotransferase assays, the incubation mixture contained 50-100 µg of microsomal proteins, 0.5 µCi [35S]PAPS (NEN Life Science Products, 2.25-2.50 Ci/mmol), 5 mM of Gal[beta]1-O-Met, Gal([beta]1-4)[Fuc[alpha]1-3]GlcNAc[beta]1-O-Met, or 100 µg of OS1 or 700 µg of fraction IIc (Table II), in a Mes/NaOH buffer, pH 6.1, containing 0.1 % (w/v) Triton X-100, 20 mM MnCl2, 30 mM NaF, 10 mM AMP, 1 mM AEBSF.

After incubation for 1 h at 30°C, the reactions were stopped by addition of ice-cold methanol. The mixtures were kept overnight at 4°C and the resulting precipitates were eliminated by centrifugation at 10,000 × g for 20 min. The pellets were washed twice with ice-cold methanol and centrifuged. The supernatants were pooled, evaporated to dryness, and then directly submitted to HPAEC-PAD.

The standards used for the identification of the 6-O-sulfated neosynthesized products were HO3S-6GlcNAc[beta]1-O-Met, IVc-12 (which has the same structure as OS1 but with a sulfate group on the internal GlcNAc residue), IVc-19 (which corresponds to OS2 with a sulfate group on the terminal GlcNAc residue) (Table I).

For the identification of the 3-O-sulfated neosynthesized products, the standards used were HO3S-3Gal[beta]1-O-Met, HO3S-3Gal([beta]1-4)[Fuc[alpha]1-3]GlcNAc[beta]1-O-Met, IVc-10 (which has the same structure as OS1 with a terminal HO3S-3Gal) and IVc-2 (which has a core type 2 and a 3-sulfo Lex on the upper branch) (Table II).

[alpha]1-3-Fucosyltransferase assays

The reaction mixture for the fucosyltransferase assays was performed according to De Vries et al. (1995): 50-100 µg of microsomal proteins were incubated with 0.3 µCi [3H]GDP-Fuc (Amersham Life Science Products, 61 Ci/mmol), 5 mM of one of the following substrates: GlcNAc[beta]1-O-Met, HO3S-6GlcNAc[beta]1-O-Met, Gal([beta]1-4)GlcNAc, NeuAc([alpha]2-3)Gal([beta]1-4)GlcNAc, or 100 µg of one of the following oligosaccharide-alditols: OS2, IIIc1-26, IVc-10, IVc-12, IVc-19, or IVc-23 (Table III), in a Mops/NaOH buffer, pH 7.5, containing 20 mM MnCl2, 0.1 % Triton X-100, 100 mM NaCl, 4 mM ATP, 1 mM AEBSF. Incubations were performed for 2 h at 30°C and stopped as described for the sulfotransferase assays. The neosynthesized products were then studied by HPAEC-PAD. The [alpha]1-3-fucosylated standards used were: Fuc([alpha]1-3)GlcNAc[beta]1-O-Met, Gal([beta]1-4)[Fuc[alpha]1-3]GlcNAc, NeuAc([alpha]2-3)Gal([beta]1-4)[Fuc[alpha]1-3]GlcNAc, oligosaccharide-alditol IIIc1-17 (which is the [alpha]1-3-fucosylated derivative of IIIc1-26), IVc-2 (the [alpha]1-3-fucosylated derivative of IVc-10), IVc-5 (the [alpha]1-3-fucosylated derivative of IVc-12), and IVc-14 (the [alpha]1-3-fucosylated derivative of IVc-23) (Table III).

[alpha]2-3-Sialyltransferase assays

Reactions were performed according to the slightly modified method of Majuri et al. (1994), in a Tris/acetate buffer, pH 6.7, containing 50-100 µg of microsomal proteins, 0.5 µCi of [14C]CMP-NeuAc (Amersham Life Science Products 0.294 Ci/mmol), 0.1 % Triton X-100, 1 mM AEBSF, 5 mM of one of the following substrates: Gal[beta]1-O-Met, Gal([beta]1-4)GlcNAc, Gal([beta]1-4)[Fuc[alpha]1-3]GlcNAc, or 100 µg of one of the following oligosaccharide-alditols, OS1, IVc-12, IVc-5 (Table IV). Incubations were performed 5 h at 30°C and stopped as described for the sulfotransferase assays. The neosynthesized products were then studied by HPAEC-PAD. The standards used for the identification of the [alpha]2-3-sialylated neosynthesized products were NeuAc([alpha]2-3)Gal([beta]1-4)GlcNAc, NeuAc([alpha]2-3)Gal([beta]1-4)[Fuc[alpha]1-3]GlcNAc, the oligosaccharide alditols IIIc1-26 (which is the [alpha]2-3-sialylated derivative of OS1), IVc-23 (the [alpha]2-3-sialylated derivative of IVc-12), and IVc-14 (the [alpha]2-3-sialylated derivative of IVc-5) (Table IV).

Characterization of radiolabeled products by HPAEC-PAD

Dry samples of sulfated, fucosylated, or sialylated radiolabeled products were dissolved in water and directly injected onto a CarboPac PA-100 column (4 × 250 mm) for HPAEC (Dionex Corp.). The elution of neosynthesized products was monitored both by pulsed amperometric detection (PAD 2 model, Dionex Corp.) and by radioactivity on line (high performance liquid chromatography radioactivity detector LB 506 C-1, EG & G, Berthold).

Elution of sulfated GlcNAc[beta]1-O-Met was performed at alkaline pH with 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 the fucosylated radiolabeled oligosaccharide-alditol synthesized from IIIc1-26 was performed at alkaline pH at a flow rate of 1 ml/min in 0.1 M NaOH for 10 min followed by a linear gradient of sodium acetate to 0.1 M NaOH/0.1 M sodium acetate at 75 min, and to 0.1 M NaOH/0.4 M sodium acetate at 80 min (gradient II).

Elution of the radiolabeled products synthesized from all other substrates was performed at alkaline pH at a flow rate of 1 ml/min in 0.1 M NaOH for 10 min followed by a linear gradient of sodium acetate to 0.1 M NaOH/0.07 M sodium acetate at 16 min, to 0.1 M NaOH/0.1 M sodium acetate at 30 min and to 0.1 M NaOH/0.45 M sodium acetate at 80 min (gradient III).

The activity of the different enzymes was expressed as picomoles of radiolabeled nucleotide-sugar transferred/min/mg of protein. Relative rates for each acceptor are expressed as a percentage of the incorporation of [35S] in GlcNAc[beta]1-O-Met for the GlcNAc-6-O-sulfotransferase (Table I), and in Gal[beta]1-O-Met for the Gal-3-O-sulfotransferase (Table II). For the fucosyl- and sialyltransferase assays, relative rates for each acceptor are expressed as a percentage of the incorporation with Gal([beta]1-4)GlcNAc (Tables III, IV).

ACKNOWLEDGMENTS

We are indebted to Prof. J.J.Lafitte for kindly providing human respiratory mucosa. This work was supported by INSERM and by the Association Française de Lutte contre la Mucoviscidose (AFLM).

ABBREVIATIONS

CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; HPAEC, high performance anion exchange chromatography; HPAEC-PAD, high performance anion exchange chromatography with pulsed amperometric detection; AEBSF, 4-(2-aminoethyl)benzenesulfonylfluoride; [14C]CMP-NeuAc, cytidine 5[prime]-monophospho[14C]sialic acid; [3H]GDP-Fuc, guanosine 5[prime]-diphospho-[beta]-L-[5,6-3H]fucose; Mes, 2-(N-morpholino)ethanesulfonic acid; Mops, 3-(N-morpholino)propanesulfonic acid; PAPS, adenosine 3[prime]-phosphate 5[prime]-phosphosulfate; Lex, Lewis x (Gal([beta]1-4)[Fuc[alpha]1-3]GlcNAc[beta]1); sLex, sialyl Lewis x (NeuAc([alpha]2-3)Gal([beta]1-4)[Fuc[alpha]1-3]GlcNAc[beta]1); 6-sulfo Lex, Lewis x with 6-O-sulfation of the GlcNAc residue; 6-sulfo-sLex, sialyl Lewis x with 6-O-sulfation of the GlcNAc residue; 6[prime]-sulfo-sLex, sialyl Lewis x with 6-O-sulfation of the Gal residue; 3-sulfo Lex, Lewis x with 3-O-sulfation of the Gal residue; ST3Gal, [alpha]2-3-sialyltransferase; FucT, fucosyltransferase; RT-PCR, reverse transcriptase polymerase chain reaction.

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