A Remodeling System of the 3′-Sulfo-Lewis a and 3′-Sulfo-Lewis x Epitopes*

It has been reported that the chemically synthesized 3′-sulfo-Lea and 3′-sulfo-Lex epitopes have a high potential as a ligand for selectins. To elucidate the physiological functions of 3′-sulfated Lewis epitopes, a remodeling system was developed using a combination of a βGal-3-O-sulfotransferase GP3ST, hitherto known α1,3/1,4-fucosyltransferases (FucT-III, IV, V, VI, VII, and IX) and arylsulfatase A. The pyridylaminated (PA) lacto-N-tetraose (Galβ1–3GlcNAcβ1–3Galβ1–4Glc) was first converted to 3′-sulfolacto-N-fucopentaose II (sulfo-3Galβ1–3(Fucα1–4)GlcNAcβ1–3Galβ1–4Glc)-PA by sequential reactions with GP3ST and FucT-III. The 3′-sulfolacto-N-fucopentaose III (sulfo-3Galβ1–4(Fucα1–3)GlcNAcβ1–3Galβ1–4Glc)-PA was then synthesized from lacto-N-neotetraose (Galβ1–4GlcNAcβ1–3Galβ1–4Glc)-PA by GP3ST and FucT-III, -IV, -V, -VI, -VII, or -IX in a similar manner. The substrate specificity for the 3′-sulfated acceptor of the α1,3-fucosyltransferases was considerably different from that for the non-substituted and 3′-sialylated varieties. When the GP3ST gene was introduced into A549 and Chinese hamster ovary cells expressing FucT-III, they began to express 3′-sulfo-Lea and 3′-sulfo-Lex epitopes, respectively, suggesting that GP3ST is responsible for their biosynthesis in vivo. The expression of the 3′-sialyl-Lex epitope on Chinese hamster ovary cells was attenuated by the introduction of GP3STgene, indicating that GP3ST and α2,3-sialyltransferase compete for the common Galβ1–4GlcNAc-R oligosaccharides. Last, arylsulfatase A, which is a lysosomal hydrolase that catalyzes the desulfation of 3-O-sulfogalactosyl residues in glycolipids, was found to hydrolyze the sulfate ester bond on the 3′-sulfo-Lex (type 2 chain) but not that on the 3′-sulfo-Lea (type 1 chain). The present remodeling system might be of potential use as a tool for the study of the physiological roles of 3′-sulfated Lewis epitopes, including interaction with selectins.

Sulfated glycoconjugates occur in a wide range of biological compounds, including glycoproteins, proteoglycans, glycolipids, and polysaccharides (for a review, see Ref. 1). The negative charge of the sulfate group is thought to serve as an adherent force in interactions with a variety of functional molecules, which include growth factors, cellular adhesion molecules, and extracellular matrix proteins (1). In fact, a considerable body of evidence has accumulated relative to the biological importance of sulfation of carbohydrate chains (2)(3)(4)(5)(6).
The sulfate group is attached to positions 3 and 6 of Gal, positions 3 and 6 of GlcNAc, and position 4 of GalNAc, in the case of N-linked or O-linked glycoproteins (1,7). The 3-sulfo-␤Gal linkage is found in both N-glycans (8,9) and O-glycans (10 -17). Among these are the sulfo-3Gal␤1-3(Fuc␣1-4)GlcNAc-R (3Јsulfo-Le a ) and sulfo-3Gal␤1-4(Fuc␣1-3)GlcNAc-R (3Ј-sulfo-Le x ) structures (12,14,15,17), which have been shown to be more potent ligands for both L-and E-selectin than the 3Ј-sialylated-Le a and -Le x determinants as evidenced by a binding assay using chemically synthesized oligosaccharides (14,18,19). The expression of the 3Ј-sulfo-Le a epitope decreases with increasing depth of invasion of human colon carcinomas (20), and human colon carcinoma cells expressing the 3Ј-sulfo-Le a epitope show a lower tumorigenicity in nude mice (21). On the other hand, the 3Ј-sulfo-Le a and/or -Le x determinants have been detected in cancer cells as well as in surrounding nonmalignant epithelia in human colon cancer tissues (22) and the 3Ј-sulfo-Le x epitope has been found to be a major carbohydrate motif in a human colon carcinoma cell line with a high metastatic tendency (15). These findings indicate that 3Ј-sulfated Lewis epitopes may serve as a relevant ligand for selectins in vivo and that their expression modulates tumor progression, in the case of human colon cancer. However, the lack of genetic tools for the remodeling of such epitopes has hampered the complete characterization of their biological functions.

EXPERIMENTAL PROCEDURES
Materials-PAPS was purchased from Sigma; lacto-N-tetraose and lacto-N-neotetraose were purchased from Seikagaku Kogyo (Tokyo, Japan); L-fucose and GDP-Fuc from Nacalai Tesque (Kyoto, Japan). Lc4-PA and nLc4-PA were synthesized by the pyridylamination of lacto-N-tetraose and lacto-N-neotetraose using a GlycoTAG Reagent kit (Takara, Shiga, Japan) with an automated pyridylamination apparatus (GlycoTAG, Takara). FucT-III was isolated from a conditioned medium of CHO cells that had been transfected with pSec-FucT-III, which was constructed by recombination of the DNA fragments encoding the open reading frame portion of human FucT-III (25) into an expression vector pSecTagA (Invitrogen, Carlbad, CA), using Ni 2ϩ column chromatography. Human FucT-IV, -VII, and -IX were prepared as described previously (26). Human FucT-V and -VI were purchased from Calbiochem (San Diego, CA). Arylsulfatase A was purified from human placenta as described previously (27).
The GP3ST-expressing plasmid pcXN2-GP3ST was constructed via a recombination of the open reading frame portion of human GP3ST cDNA (23) into an expression vector pcXN2 (28). A lysate of the CHO cells transfected with pcXN2-GP3ST was used as a source of GP3ST. The FucT-III-expressing plasmid pcDNA-FucT-III was constructed by recombination of the open reading frame portions of human FucT-III (25) into an expression vector pcDNA3.1/Zeo(ϩ) (Invitrogen).
3Ј-Sulfation and 3Ј-Sialylation of Lc4-PA and nLc4-PA-3Ј-Sulfo-Lc4-PA and 3Ј-sulfo-nLc4-PA were synthesized by sulfation of Lc4-PA and nLc4-PA, respectively, using a recombinant GP3ST, and the resulting material was purified by anion exchange chromatography and subsequent reversed-phase HPLC as described previously (23). These substrates were characterized by NMR spectroscopy (23) and mass spectrometry using a quadrupole ion trap mass spectrometer fitted with an ESI source (LCQ ion trap mass spectrometer TM , Thermo Finnigan, San Jose, CA). The mass spectra were acquired by negative ion detection and 3Ј-sulfo-Lc4-PA and 3Ј-sulfo-nLc4-PA were identified at m/z 864.2 and 864.4, respectively.
Flow Cytometry Analysis of CHO and A549 Cells Stably Transfected with or without GP3ST and FucT-III Genes-CHO and A549 cells were transfected with linealized pcXN2-GP3ST and/or pcDNA-FucT-III genes using the Effectene TM Transfection Reagent (Qiagen, Hilden, Germany) according to the standard protocol for stable transfection and selected for clones stably expressing these genes, based on their resistance to G418 (Sigma) and/or Zeocin (Invitrogen) followed by measurement of the enzyme activities, as described above. The cloned cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 0.1 mg/ml streptomycin, 400 g/ml G418, and/or 150 g/ml Zeocin and harvested with PBS containing 1 mM EDTA. Fifty l of cell suspensions (5-10 ϫ 10 6 cells) were incubated with a primary antibody (SU59 diluted 1:5; P12 and KM93 diluted 1:25; MAB2108, ZY-CO9, 91.9H, and control immunogloblins at a dilution of 1:50) for 30 min on ice. Cells were then washed with 1 ml of PBS, resuspended in 100 l of fluorescein isothiocyanateconjugated F(abЈ) 2 fragment of goat anti-mouse immunoglobulins (Dako) diluted 1:25 and incubated for 30 min on ice. Flow cytometry analyses were performed using a FACScan instrument (Becton Dickinson, Frankin Lakes, NJ) operating with CELLQuest software.
Western Blotting of CHO Cells Transfected with or without GP3ST or FucT-III Genes-Parental CHO cells and CHO cells transfected with the GP3ST and/or FucT-III genes were suspended in 4 volumes of 10 mM Tris-HCl buffer (pH 7.4) containing 1% Triton X-100, 1 mM EDTA, and 0.1% protease inhibitor mixture for mammalian cell and tissue extracts (Wako, Osaka, Japan). After incubation on ice for 1 h, the solution was centrifuged at 15,000 rpm for 30 min and the supernatants were used as cell lysates. Protein concentration was assayed by means of a BCA protein assay kit (Pierce, Rockford, IL). The cell lysates were separated by SDS-PAGE on a 7.5% gel, transferred to a nitrocellulose transfer membrane (Schleicher & Schuell, Keene, NH), and stained with mAb SU59 diluted 1:5 and mAb KM93 diluted 1:20. In order to examine a susceptibility to an N-glycanase, a blotted membrane blocked with 3% bovine serum albumin was treated with 30 units of N-glycanase F (Roche Molecular Biochemicals, Basal, Switzerland) in 4 ml of PBS at 37°C for 24 h prior to incubation with SU59.
Desulfation of 3Ј-Sulfo-Lc4-PA, 3Ј-Sulfolacto-N-fucopentaose II-PA, 3Ј-Sulfo-nLc4-PA, and 3Ј-Sulfolacto-N-fucopentaose III-PA-The reaction mixture contained the following components in a total volume of 50 l: 0.5 M acetate/NaOH buffer (pH 5.0), 0.6 M of each substrate in the presence or absence of arylsulfatase A. After incubation at 37°C for 2 h, the reaction was terminated by boiling for 3 min. The sample was then centrifuged at 15,000 rpm for 5 min and 20 l of the supernatant was analyzed by HPLC as described above.
The doubly transfected CHO cells, which were stably expressing the GP3ST and FucT-III genes, were treated with arylsulfatase A. These cells were harvested with PBS containing 1 mM EDTA, and 150 l of cell suspension (1-5 ϫ 10 7 cells) was then incubated in 20 mM acetate-NaOH buffer (pH 5.0), 150 mM NaCl in the presence or absence of arylsulfatase A. After incubation at 37°C for 12 h by rotating, cells were washed three times with 1 ml of PBS and resuspended in 50 l with the primary antibody mAb SU59 diluted 1:5 for 30 min on ice. The cells were then washed and incubated with fluorescein isothiocyanate-conjugated F(abЈ) 2 fragment of goat anti-mouse immunoglobulins diluted 1:25 for 30 min on ice. Flow cytometry analysis was performed as described above.
The ␣1,4-fucosylation of the 3Ј-sulfated type 1 chain (Gal␤1-3GlcNAc-R) was examined first. 3Ј-Sulfo-Lc4-PA was synthesized from Lc4-PA via catalysis by GP3ST (23). The resulting 3Ј-sulfo-Lc4-PA was then subjected to fucosylation by recombinant FucT-III, which is the sole ␣1,4-fucosyltransferase (25). A strong product peak appeared, as shown by an arrow, in Fig. 1b in the presence of GDP-Fuc (Fig. 1b), whereas no peak was detected in the absence of the donor substrate (Fig. 1a). The m/z value of the material in the product peak was 1010.4, corresponding to that of 3Јsulfolacto-N-fucopentaose II-PA (Fig. 1c). These results indicate that FucT-III has the capability to act on the 3Ј-sulfated type 1 chain and to synthesize the 3Ј-sulfo-Le a structure. The efficiency of FucT-III for non-substituted 3Ј-sialylated and 3Ј-sulfated acceptors was also compared ( Table I). The result indicates that FucT-III prefers 3Ј-sulfo-Lc4-PA to the nonsubstituted Lc4-PA or 3Ј-sialyl-Lc4-PA.
Thus far, six ␣1,3-fucosyltransferase isozymes, FucT-III, -IV, -V, -VI, -VII, and -IX, are known (26,36,37). Since the substrate specificity of ␣1,3-fucosyltransferases for 3Ј-sulfated acceptors has not been investigated, this was examined, compared with that for non-substituted and 3Ј-sialylated acceptors. When 3Ј-sulfo-nLc4-PA was incubated with FucT-III in the presence of GDP-Fuc, 3Ј-sulfolacto-N-fucopentaose III was produced (Fig. 1, e and f). As shown in Table I, FucT-III preferred the 3Ј-sulfated nLc4-PA to nLc4-PA or 3Ј-sialyl-nLc4-PA, and preferred the type 1 chain to the type 2 chain, as described previously (25). The ␣3-fucosylation of 3Ј-sulfo-nLc4-PA was then examined with respect to the other ␣1,3-fucosyltransferases. Since the sources and specific activities of the fucosyltransferases used were different, the activities toward individual acceptors are expressed relative to those toward nLc4-PA (FucT-III, -IV, -V, -VI, and -IX) or 3Ј-sialyl-nLc4-PA (FucT-VII) in Table II. The preference for the sulfated acceptor among the ␣1,3-fucosyltransferases was considerably different from that for the sialylated or non-substituted acceptors. All the ␣1,3fucosyltransferases acted on the sulfated acceptor unlike the sialylated one, although the extent of relative reaction efficiency was varied, depending on the specific enzyme. It was noted that the 3Ј-sulfated oligosaccharide was a better substrate than the non-substituted or 3Ј-sialylated oligosaccharide for FucT-III, -V, and -VI.
Reconstitution of 3Ј-Sulfo-Le x and 3Ј-Sulfo-Le a Epitopes on Living Cells-To analyze biological roles of the 3Ј-sulfated Lewis epitopes, information on the expression of these epitopes on the living cell surface is required. Therefore, the GP3ST and FucT-III genes, which had been inserted into the expression vectors, were transfected into CHO cells and the expression of 3Ј-sulfated Lewis epitopes was examined by flow cytometry analysis using specific antibodies against the 3Ј-sulfated Lewis epitopes. The mAb SU59 recognizes both 3Ј-sulfo-Le a and 3Јsulfo-Le x epitopes (29), but mAb 91.9H recognizes only the 3Ј-sulfo-Le a epitope (30,31).
The parent CHO cells (Fig. 2, panels a, e, and i) and CHO cells transfected with the GP3ST gene alone (Fig. 2, panel b, f, and j) expressed neither Le x (recognized by mAb P12), 3Ј-sialyl-Le x (recognized by mAb KM93), nor 3Ј-sulfo-Le x (recognized by mAb SU59). The CHO cells that had been transfected with only the FucT-III gene expressed Le x and 3Ј-sialyl-Le x (Fig. 2, panel  c and g), but did not express 3Ј-sulfo-Le x (Fig. 2, panel k), indicating that CHO cells do not express the ␤Gal-3-O-sulfotransferase. In addition, FucT-III-transfected CHO cells expressed neither Le a nor 3Ј-sialyl-Le a (data not shown), consistent with the previously reported observation that CHO cells express only the type 2 chain (38).
CHO cells transfected with both the GP3ST and FucT-III genes were SU59-positive (Fig. 2, panel l) but 91.9H-negative (data not shown), indicating that the cells express the 3Ј-sulfo-Le x determinant but not the 3Ј-sulfo-Le a , which is in good agreement with the conclusion that CHO cells expresses only the type 2 chain. Furthermore, the expression of the 3Ј-sialyl-Le x epitope on both gene-transfected cells was remarkably reduced, compared with that on only the FucT-III gene-transfected cells (Fig. 2, panels g and h). This finding indicates that the expression of GP3ST interferes with the biosynthesis of 3Ј-sialyl-Le x epitope in vivo.
To analyze the specific molecules on which 3Ј-sulfo-Le x epitope is carried, glycoproteins were extracted from CHO cells transfected with the GP3ST and FucT-III genes and examined by Western blotting. As shown in Fig. 3a, several protein bands with a relatively high molecular weight were specifically stained with mAb SU59 (lanes 4 and 5), indicating that the 3Ј-sulfo-Le x epitope was contained by several different proteins. These SU59-positive bands were nearly identical to the bands stained with anti-3Ј-sialyl-Le x antibody KM93 in CHO cells transfected with only the FucT-III gene (Fig. 3b, lane 3). In addition, the reactivity with anti-3Ј-sialyl-Le x antibody was reduced in CHO cells that had been transfected with both the GP3ST and FucT-III genes (Fig. 3b, lanes 4 and 5), consistent with the flow cytometry results. These observations suggest that 3Ј-sulfation and 3Ј-sialylation occur on common glycopro-teins. In addition, most SU59-positive bands in Fig. 3a, lanes 4 and 5, disappeared after treatment with N-glycanase (Fig. 3c,  lanes 1 and 2). When glycolipids were extracted from the doubly transfected CHO cells and analyzed by thin-layer chromatography immunostaining, no SU59-positive band could be detected (data not shown). These findings indicate that 3Јsulfo-Le x epitope is mainly carried on N-linked glycoproteins in the CHO cells.
To examine the ability of GP3ST to synthesize 3Ј-sulfo-Le a epitope in living cells, its gene was transfected into a human lung carcinoma cell line A549, which expresses Le a antigen but does not react with mAb 91.9H (Fig. 4a). After the introduction of the GP3ST gene, the cells became 91.9H positive (Fig. 4b),   The values represent the percentage of the activity, compared with that for nLc4-PA, except for FucT-VII in which relative activities to that for 3Ј-sialyl-nLc4-PA are shown. ␣1,3-Fucosyltransferase activities for nLc4-PA of FucT-III, -IV, -V, -VI, and -IX were 3.6, 64.9, 75.1, 6.0, and 144.9 pmol/min/ml, respectively. ␣1,3-Fucosyltransferase activity for 3Ј-sialyl nLc4-PA of FucT-VII was 593.0 pmol/min/ml. GP3ST gene (panels b, f, and j), the FucT-III gene (panels c, g, and k), and both genes (panels d, h, and l) were examined by flow cytometry analysis using specific antibodies; anti-Le x mAb P12 (panels a-d, solid line), anti-sialyl-Le x mAb KM93 (panels e-h, solid line), and anti-3Ј-sulfo-Le a and 3Ј-sulfo-Le x mAb SU59 (panels i-l, solid line). Mouse IgG1 was used as a negative control (dotted line). Note that only the CHO cells transfected with both GP3ST and FucT-III genes are SU59-positive (panel l), while the expression of 3Ј-sialyl-Le x is remarkably reduced, compared with those transfected only with the FucT-III gene (panels g and h).

FIG. 2. Expression of 3-sulfo-and 3-sialyl-Le x epitopes on GP3ST and FucT-III gene-transfected CHO cells. Parental CHO cells (panels a, e, and i) and CHO cells transfected with the
indicating that GP3ST is also able to synthesize the 3Ј-sulfo-Le a epitope in vivo.
Hydrolysis of the Sulfate Ester Bond on the 3Ј-Sulfo-Le x Structure-Since arylsulfatase A catalyzes the desulfation of 3-O-sulfogalactosyl containing glycolipids (39), the issue of whether the sulfatase is capable of desulfating 3-O-sulfogalactosyl residues on 3Ј-sulfo-Lewis epitopes would be of interest. As shown by the arrows in Fig. 5, c and d, peaks corresponding to desulfated products were detected on treatment with arylsulfatase A for 3Ј-sulfo-nLc4-PA and 3Ј-sulfolacto-N-fucopentaose III-PA, while 3Ј-sulfo-Lc4-PA and 3Ј-sulfolacto-N-fucopentaose II-PA did not undergo desulfation (Fig. 5, a and b). This indicates that arylsulfatase A acts on the type 2 chain but not on the type 1 chain. Furthermore, the intensity of the 3Ј-sulfo-Le x epitope for the doubly transfected CHO cells was slightly but significantly reduced by treatment with arylsulfatase A, as evidenced by flow cytometry (Fig. 6). These findings indicate that arylsulfatase A desulfates 3-O-sulfogalactosyl residues on sulfate-3Gal␤1-4GlcNAc-R oligosaccharides irrespective of whether the penultimate GlcNAc residue is ␣3-fucosylated. DISCUSSION We report herein, the enzymatic synthesis and degradation of 3Ј-sulfo-Le a and -Le x epitopes in vitro and in vivo. Previous studies have suggested that 3Ј-sulfation of the nonreducing terminal Gal occurs prior to the ␣3/4-fucosylation of the penultimate GlcNAc in the biosynthetic pathway of 3Ј-sulfo-Le a and -Le x structures (23,32,33), as 3Ј-sialylation occurs before the 3/4-fucosylation in the synthetic pathway of the 3Ј-sialyl-Le a and -Le x (34,35). In the present study, the biosynthesis of the 3Ј-sulfo-Le a and -Le x structures was comprehensively investigated by sequential reactions with GP3ST and all the currently known ␣1,3/1,4-fucosyltransferases.
Since FucT-III is the sole ␣1,4-fucosyltransferase (25), it was employed to synthesize the 3Ј-sulfo-Le a structure. As expected, FucT-III catalyzed the ␣1,4-fucosylation of 3Ј-sulfo-Lc4-PA in vitro. Furthermore, introduction of the GP3ST gene into human lung cancer cells that produce the type 1 chain led to the expression of the 3Ј-sulfo-Le a epitope, indicating that GP3ST is involved in the biosynthesis of this epitope in vivo.
Thus far, six ␣1,3-fucosyltransferase isozymes, FucT-III, IV, V, VI, VII, and IX, are known (26,36,37). The preference of these fucosyltransferases toward acceptor substrates Gal␤1-4GlcNAc-R and 3Ј-sialyl-Gal␤1-4GlcNAc-R varies considerably. FucT-III is largely active on type 1 but also on type 2 chains, whether they are sialylated or not. Concerning FucT-IV, the neutral type 2 chain is a good substrate while the 3Ј-sialylated oligosaccharide is a poor one. FucT-V and FucT-VI are active on both neutral and 3Ј-sialylated substrates. FucT-VII acts on only the 3Ј-sialylated type 2 chain whereas FucT-IX is active only on the neutral one. Therefore, the issue of which fucosyltransferases act on the 3Ј-sulfated type 2 chain is of interest. Prior to this study, we anticipated a result similar to that for the 3Ј-sialylated acceptors. Unexpectedly, all the ␣1,3fucosyltransferases acted on the 3Ј-sulfated acceptor and no correlation was found for the relative activity for the sulfated substrate of individual fucosyltransferases with that for the sialylated one. These findings suggest that the mechanism by which ␣1,3-fucosyltransferases recognize the sulfate group or the sialic acid attached to the terminal Gal residue of type 2 chain involves, not only anionic charge, but other factors, depending on the isozymes. The similarity in substrate specificity for non-substituted, sialylated, and sulfated acceptors of FucT-III, -V, and -VI may reflect the homology in their primary structures (37).
The fact that GP3ST and FucT-III were collaboratively able to synthesize the 3Ј-sulfo-Le x epitope in vivo was verified by flow cytometry analysis, where only CHO cells transfected with both GP3ST and FucT-III genes reacted with mAb SU59. Flow cytometry analysis also revealed that the robust expression of 3Ј-sialyl-Le x epitope on FucT-III-transfected CHO cells was inhibited by the introduction of the GP3ST gene. This result suggests that GP3ST and ␣2,3-sialyltransferase are located in the same compartment of the Golgi apparatus and compete for the Gal␤1-4GlcNAc-R oligosaccharide on the common oligosaccharides in CHO cells. This finding was verified by Western blotting analysis, where the protein bands with 3Ј-sialyl-Le x were found to be nearly identical to those with 3Ј-sulfated Le x and their signals were attenuated in the cells expressing GP3ST. Since the GP3ST gene is expressed in various human tissues (23), GP3ST may regulate the expression of Le x and 3Ј-sialyl-Le x epitopes there. A similar regulation may occur in terms of the expression of Le a and 3Ј-sialyl-Le a epitopes. Mutual interference by glycosyltransferases and carbohydratemodifying enzymes in the biosynthesis of carbohydrate chains occurs under various situations (40, 41).
During the preparation of this article, the molecular cloning of another ␤Gal 3-O-sulfotransferase (Gal3ST-3, GAL3ST2), which acts on only the type 2 chain and is expressed in confined tissues such as thyroid and brain, was independently reported by two groups (42,43). This sulfotransferase may synthesize the 3Ј-sulfo-Le x epitope in the thyroid, although only the sulfo-3Gal␤1-4GlcNAc-R structure without ␣3-fucose was found on human thyroglobulin (44). Gal3ST-3 may also be involved in the biosynthesis of 3Ј-sulfo-Le x epitope on the N-glycans of human thyrotropin in the anterior pituitary gland (45), where the sulfotransferase gene is expressed (44). In contrast, GP3ST is expressed in various tissues, including colon epithelia (23), and may be responsible for the biosynthesis of both 3Ј-sulfo-Le a and 3Ј-sulfo-Le x epitopes in these tissues.
Arylsulfatase A is a lysosomal hydrolase that catalyzes the desulfation of 3-O-sulfogalactosyl-containing glycolipids (39). The fact that arylsulfatase A hydrolyses the sulfate ester attached to position 3 of the nonreducing terminal ␤Gal in glycolipids prompted us to examine the issue of whether it is able to desulfate the 3Ј-sulfo-Le a and -Le x structures. As a result, arylsulfatase A was found to hydrolyze the sulfate ester bond on 3Ј-sulfo-Le x but not on 3Ј-sulfo-Le a in vitro. This suggests that arylsulfatase A can be used as a tool for the dissection of functions between the 3Ј-sulfated-Le a and -Le x epitopes. On the other hand, arylsulfatase A acted only weakly on the 3Ј-sulfo-Le x structure attached to proteins, suggesting that it may degrade this structure after digestion of the peptide portion in vivo. This study is the first report which conclusively shows that arylsulfatase A hydrolyzes physiological endogenous substrates other than sulfoglycolipids.
In conclusion, the present study demonstrates that: 1) GP3ST and FucT-III catalyze the synthesis of the 3Ј-sulfo-Le a epitope in a collective manner; 2) GP3ST and FucT-III, IV, V, VI, VII, and IX are involved in the biosynthesis of the 3Ј-sulfo-Le x epitope; 3) GP3ST and ␣2,3-sialyltransferase compete for the common Gal␤1-4GlcNAc-R oligosaccharides in vivo; 4) ar- ylsulfatase A hydrolyzes the sulfate ester bond on 3Ј-sulfo-Le x but not on the 3Ј-sulfo-Le a . In the future, the present remodeling system of the 3Ј-sulfated Lewis epitopes may provide a useful tool for the study on their biological roles including their interaction with selectins.