JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M107390200 on August 14, 2001

J. Biol. Chem., Vol. 276, Issue 42, 38588-38594, October 19, 2001
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
276/42/38588    most recent
M107390200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ikeda, N.
Right arrow Articles by Honke, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ikeda, N.
Right arrow Articles by Honke, K.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

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

Naoki IkedaDagger §, Hironobu EguchiDagger , Shoko Nishihara, Hisashi Narimatsu||, Reiji Kannagi**, Tatsuro IrimuraDagger Dagger , Mitsunori Ohta§, Hikaru Matsuda§, Naoyuki TaniguchiDagger , and Koichi HonkeDagger §§

From the Dagger  Department of Biochemistry and § Department of Surgery, Osaka University Medical School, Suita, Osaka 565-0871, Japan, the  Division of Cell Biology, Institute of Life Science, Soka University, Hachioji, Tokyo 192-8577, Japan, the || Institute of Molecular and Cell Biology, National Institute of Advanced Industrial Science and Technology, Central-2, 1-1-1 Umezono, Tsukuba 305-8568, Japan, the ** Program of Experimental Pathology, Aichi Cancer Center, Nagoya 464-8681, Japan, and the Dagger Dagger  Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

Received for publication, August 2, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta Gal-3-O-sulfotransferase GP3ST, hitherto known alpha 1,3/1,4-fucosyltransferases (FucT-III, IV, V, VI, VII, and IX) and arylsulfatase A. The pyridylaminated (PA) lacto-N-tetraose (Galbeta 1-3GlcNAcbeta 1-3Galbeta 1-4Glc) was first converted to 3'-sulfolacto-N-fucopentaose II (sulfo-3Galbeta 1-3(Fucalpha 1-4)GlcNAcbeta 1-3Galbeta 1-4Glc)-PA by sequential reactions with GP3ST and FucT-III. The 3'-sulfolacto-N-fucopentaose III (sulfo-3Galbeta 1-4(Fucalpha 1-3)GlcNAcbeta 1-3Galbeta 1-4Glc)-PA was then synthesized from lacto-N-neotetraose (Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4Glc)-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 alpha 1,3-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 GP3ST gene, indicating that GP3ST and alpha 2,3-sialyltransferase compete for the common Galbeta 1-4GlcNAc-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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (2-6).

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-beta Gal linkage is found in both N-glycans (8, 9) and O-glycans (10-17). Among these are the sulfo-3Galbeta 1-3(Fucalpha 1-4)GlcNAc-R (3'-sulfo-Lea) and sulfo-3Galbeta 1-4(Fucalpha 1-3)GlcNAc-R (3'-sulfo-Lex) structures (12, 14, 15, 17), which have been shown to be more potent ligands for both L- and E-selectin than the 3'-sialylated-Lea and -Lex determinants as evidenced by a binding assay using chemically synthesized oligosaccharides (14, 18, 19). The expression of the 3'-sulfo-Lea epitope decreases with increasing depth of invasion of human colon carcinomas (20), and human colon carcinoma cells expressing the 3'-sulfo-Lea epitope show a lower tumorigenicity in nude mice (21). On the other hand, the 3'-sulfo-Lea and/or -Lex determinants have been detected in cancer cells as well as in surrounding nonmalignant epithelia in human colon cancer tissues (22) and the 3'-sulfo-Lex 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 3'-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.

We recently reported on the cDNA cloning of a beta Gal 3-O-sulfotransferase (GP3ST)1 that acts on both type 1 (Galbeta 1-3GlcNAc-R) and type 2 (Galbeta 1-4GlcNAc-R) chains and is expressed in human colonic mucosa (23), based on its similarity to glycolipid 3-O-sulfotransferase (24). Its molecular cloning enabled us to develop a remodeling system of 3'-sulfated Lewis epitopes. The enzymatic degradation of these epitopes is also discussed.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 Ni2+ 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(+) (Invitrogen).

Mouse anti-Lea mAb MAB2108 (Chemicon, Temecula, CA), mouse anti-sialyl-Lea mAb ZY-CO9 (Zymed Laboratories Inc., South San Francisco, CA), mouse anti-Lex mAb P12 (Calbiochem), mouse anti-sialyl-Lex mAb KM93 (Calbiochem), mouse anti-3'-sulfo-Lea and 3'-sulfo-Lex mAb SU59 (29), and mouse anti-3'-sulfo-Lea mAb 91.9H (30, 31) were used for the detection of carbohydrate epitopes. Mouse IgG1 (Dako, Carpinteria, CA) was used as the negative control.

3'-Sulfation and 3'-Sialylation of Lc4-PA and nLc4-PA-- 3'-Sulfo-Lc4-PA and 3'-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 spectrometerTM, Thermo Finnigan, San Jose, CA). The mass spectra were acquired by negative ion detection and 3'-sulfo-Lc4-PA and 3'-sulfo-nLc4-PA were identified at m/z 864.2 and 864.4, respectively.

3'-Sialyl-Lc4-PA and 3'-sialyl-nLc4-PA were synthesized by alpha 2,3-sialyltransferase from Lc4-PA and nLc4-PA, respectively. The reaction was carried out at 37 °C for 8 h in a solution comprised of 50 mM MOPS buffer (pH 7.4), 0.2% Triton X-100, 0.5 mg/ml bovine serum albumin, 2.5 mM CMP-NeuAc (Calbiochem), 1 mM Lc4-PA or nLc4-PA, and 0.6 µg of alpha 2,3-sialyltransferase (Calbiochem) in a final volume of 50 µl, and reaction products were isolated using HPLC as described above. 3'-Sialyl-nLc4-PA was identified at m/z 1075.3 by negative detection, using the quadrupole ion trap mass spectrometer.

alpha 1,3/1,4-Fucosylation of Lc4-PA, nLc4-PA, 3'-Sulfo-Lc4-PA, 3'-Sulfo-nLc4-PA, 3'-Sialyl-Lc4-PA, and 3'-Sialyl-nLc4-PA-- The standard incubation mixture contained the following components in a total volume of 10 µl: 50 mM MES buffer (pH 6.5), 25 mM MnCl2, 5 mM ATP, 10 mM L-fucose, 75 µM GDP-Fuc, 25 µM of each acceptor substrate except for 3'-sialyl-Lc4-PA, which was used at a concentration of 12.5 µM, and 5 µl of purified FucT-III. After incubation at 37 °C for 2 h, the reaction was terminated by boiling for 4 min. After the addition of 90 µl of water, the sample was centrifuged at 15,000 rpm for 5 min and 20 µl of the supernatant was injected onto a TSKgel ODS-80TM column (4.6 × 250 mm, Tosoh, Tokyo, Japan) equipped with a Shimazu LC-VP HPLC system (Kyoto, Japan) and eluted with a 20 mM ammonium acetate buffer (pH 4.0) at flow rate of 0.8 ml/min at 35 °C, and monitored with a fluorescence spectrophotometer (excitation, 320 nm; emission, 400 nm). The fractions containing 3'-sulfolacto-N-fucopentaose II-PA and 3'-sulfolacto-N-fucopentaose III-PA were pooled, dried, and determined by mass spectrometry as described above.

For assays of alpha 1,3-fucosyltranseferases (FucT-IV, -V, -VI, VII, and IX) toward the type 2 oligosaccharides, which were nLc4-PA, 3'-sulfo-nLc4-PA, and 3'-sialyl-nLc4-PA, enzyme reactions were performed in 20 µl of the following mixture: 50 mM MES buffer (pH 6.5), 25 mM MnCl2, 5 mM ATP, 10 mM L-fucose, 75 µM GDP-fucose, 25 µM each acceptor substrate and each enzyme source: FucT-IV, VII, and IX, 6 µl of the cell lysate (26); FucT-V, 1.8 µg (6 µl); FucT-VI, 240 ng (0.6 µl) for nLc4-PA and 3'-sialyl-nLc4-PA, 48 ng (0.12 µl) for 3'-sulfo-nLc4-PA. The other assay conditions were the same as described above.

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 EffecteneTM 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 µg/ml G418, and/or 150 µg/ml Zeocin and harvested with PBS containing 1 mM EDTA. Fifty µl of cell suspensions (5-10 × 106 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 µl of fluorescein isothiocyanate-conjugated F(ab')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 3'-Sulfo-Lc4-PA, 3'-Sulfolacto-N-fucopentaose II-PA, 3'-Sulfo-nLc4-PA, and 3'-Sulfolacto-N-fucopentaose III-PA-- The reaction mixture contained the following components in a total volume of 50 µl: 0.5 M acetate/NaOH buffer (pH 5.0), 0.6 µM 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 µl 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 µl of cell suspension (1-5 × 107 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 µl 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(ab')2 fragment of goat anti-mouse immunoglobulins diluted 1:25 for 30 min on ice. Flow cytometry analysis was performed as described above.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Enzymatic Synthesis of 3'-Sulfo-Lea and 3'-Sulfo-Lex Structures in Vitro-- In a previous study (23), GP3ST was found to act on both lacto-N-tetraose and lacto-N-neotetraose, but not on lacto-N-fucopentaose II or lacto-N-fucopentaose III, suggesting that 3'-sulfation of the terminal Gal occurs prior to the alpha 3/4-fucosylation of the penultimate GlcNAc in the biosynthetic pathway of the 3'-sulfo-Lea and -Lex structures. Our knowledge is incomplete on the use of crude enzyme sources on the alpha 3/4-fucosylation of 3'-sulfated Galbeta 1-3/4GlcNAc-R oligosaccharides (32, 33). Therefore, we investigated biosynthesis of 3'-sulfo-Lea and -Lex structures extensively using recombinant GP3ST and alpha 1,3/1,4-fucosyltransferases. Since 3'-sialylation also occurs prior to the alpha 3/4-fucosylation in the synthetic pathway of 3'-sialyl-Lea and -Lex (34, 35), the effects of alpha 1,3/1,4-fucosyltransferases on non-substituted 3'-sialylated and 3'-sulfated Galbeta 1-3/4GlcNAc-R oligosaccharides were compared.

The alpha 1,4-fucosylation of the 3'-sulfated type 1 chain (Galbeta 1-3GlcNAc-R) was examined first. 3'-Sulfo-Lc4-PA was synthesized from Lc4-PA via catalysis by GP3ST (23). The resulting 3'-sulfo-Lc4-PA was then subjected to fucosylation by recombinant FucT-III, which is the sole alpha 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 3'-sulfolacto-N-fucopentaose II-PA (Fig. 1c). These results indicate that FucT-III has the capability to act on the 3'-sulfated type 1 chain and to synthesize the 3'-sulfo-Lea structure. The efficiency of FucT-III for non-substituted 3'-sialylated and 3'-sulfated acceptors was also compared (Table I). The result indicates that FucT-III prefers 3'-sulfo-Lc4-PA to the non-substituted Lc4-PA or 3'-sialyl-Lc4-PA.


View larger version (11K):
[in this window]
[in a new window]
  		Fig. 1.   Enzymatic synthesis of 3'-sulfo-Lea and 3'-sulfo-Lex structures in vitro. Lc4-PA (panels a and b) or nLc4-PA (panels d and e) were incubated with recombinant human FucT-III in the absence (panels a and d) or presence (panels b and e) of GDP-fucose. Reaction products were isolated by reverse-phase HPLC, with fluorescence monitoring as described under "Experimental Procedures." The arrows indicate the elution position of the products. The fractions containing the products were pooled, dried, and characterized by mass spectrometry. The determined m/z of the products corresponded with 3'-sulfo-lacto-N-fucopentaose II-PA (panel c) and 3'-sulfo-lacto-N-fucopentaose III-PA (panel f), respectively.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Substrate specificity of alpha 1.3/1.4-fucosyltransferase (FucT-III)

Thus far, six alpha 1,3-fucosyltransferase isozymes, FucT-III, -IV, -V, -VI, -VII, and -IX, are known (26, 36, 37). Since the substrate specificity of alpha 1,3-fucosyltransferases for 3'-sulfated acceptors has not been investigated, this was examined, compared with that for non-substituted and 3'-sialylated acceptors. When 3'-sulfo-nLc4-PA was incubated with FucT-III in the presence of GDP-Fuc, 3'-sulfolacto-N-fucopentaose III was produced (Fig. 1, e and f). As shown in Table I, FucT-III preferred the 3'-sulfated nLc4-PA to nLc4-PA or 3'-sialyl-nLc4-PA, and preferred the type 1 chain to the type 2 chain, as described previously (25). The alpha 3-fucosylation of 3'-sulfo-nLc4-PA was then examined with respect to the other alpha 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 3'-sialyl-nLc4-PA (FucT-VII) in Table II. The preference for the sulfated acceptor among the alpha 1,3-fucosyltransferases was considerably different from that for the sialylated or non-substituted acceptors. All the alpha 1,3-fucosyltransferases 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 3'-sulfated oligosaccharide was a better substrate than the non-substituted or 3'-sialylated oligosaccharide for FucT-III, -V, and -VI.

                              
View this table:
[in this window]
[in a new window]
 
Table II
Substrate specificity of alpha 1,3-fucosyltransferases for non-substituted, 3'-sialylated and 3'-sulfated acceptors

Reconstitution of 3'-Sulfo-Lex and 3'-Sulfo-Lea Epitopes on Living Cells-- To analyze biological roles of the 3'-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 3'-sulfated Lewis epitopes was examined by flow cytometry analysis using specific antibodies against the 3'-sulfated Lewis epitopes. The mAb SU59 recognizes both 3'-sulfo-Lea and 3'-sulfo-Lex epitopes (29), but mAb 91.9H recognizes only the 3'-sulfo-Lea 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 Lex (recognized by mAb P12), 3'-sialyl-Lex (recognized by mAb KM93), nor 3'-sulfo-Lex (recognized by mAb SU59). The CHO cells that had been transfected with only the FucT-III gene expressed Lex and 3'-sialyl-Lex (Fig. 2, panel c and g), but did not express 3'-sulfo-Lex (Fig. 2, panel k), indicating that CHO cells do not express the beta Gal-3-O-sulfotransferase. In addition, FucT-III-transfected CHO cells expressed neither Lea nor 3'-sialyl-Lea (data not shown), consistent with the previously reported observation that CHO cells express only the type 2 chain (38).


View larger version (22K):
[in this window]
[in a new window]
  		Fig. 2.   Expression of 3'-sulfo- and 3'-sialyl-Lex epitopes on GP3ST and FucT-III gene-transfected CHO cells. Parental CHO cells (panels a, e, and i) and CHO cells transfected with the 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-Lex mAb P12 (panels a-d, solid line), anti-sialyl-Lex mAb KM93 (panels e-h, solid line), and anti-3'-sulfo-Lea and 3'-sulfo-Lex 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 3'-sialyl-Lex is remarkably reduced, compared with those transfected only with the FucT-III gene (panels g and h).

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 3'-sulfo-Lex determinant but not the 3'-sulfo-Lea, which is in good agreement with the conclusion that CHO cells expresses only the type 2 chain. Furthermore, the expression of the 3'-sialyl-Lex 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 3'-sialyl-Lex epitope in vivo.

To analyze the specific molecules on which 3'-sulfo-Lex 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 3'-sulfo-Lex epitope was contained by several different proteins. These SU59-positive bands were nearly identical to the bands stained with anti-3'-sialyl-Lex antibody KM93 in CHO cells transfected with only the FucT-III gene (Fig. 3b, lane 3). In addition, the reactivity with anti-3'-sialyl-Lex 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 3'-sulfation and 3'-sialylation occur on common glycoproteins. 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 3'-sulfo-Lex epitope is mainly carried on N-linked glycoproteins in the CHO cells.


View larger version (45K):
[in this window]
[in a new window]
  		Fig. 3.   Expression of 3'-sulfo-Lex carrying glycoproteins in GP3ST and FucT-III gene-transfected CHO cells. a, the cell lysates of parental CHO cells (lane 1), CHO cells transfected with the GP3ST gene alone (lane 2), FucT-III gene alone (lane 3), and both GP3ST and FucT-III genes (clone 9, lane 4; clone 14, lane 5) were separated by SDS-PAGE, transferred to a nitrocellulose membrane and stained with mAb SU59. b, the same membrane as shown in panel a was stained with mAb KM93. c, lysates of CHO cells transfected with both GP3ST and FucT-III genes, corresponding to lanes 4 and 5 in panel a were separated by SDS-PAGE and transferred to a nitrocellulose membrane. After blocking, the membrane was incubated with N-glycanase F and then stained with mAb SU59.

To examine the ability of GP3ST to synthesize 3'-sulfo-Lea epitope in living cells, its gene was transfected into a human lung carcinoma cell line A549, which expresses Lea 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), indicating that GP3ST is also able to synthesize the 3'-sulfo-Lea epitope in vivo.


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 4.   Expression of 3'-sulfo-Lea epitope on GP3ST gene-transfected human lung cancer cells. Parental A549 cells (panel a) and GP3ST gene-transfected A549 cells (panel b) were examined by flow cytometry analysis using specific antibodies; anti-Lea mAb MAB2108 (solid line) and anti-sulfo-Lea mAb 91.9H (bold line). Mouse IgG1 was used as a negative control (dotted line).

Hydrolysis of the Sulfate Ester Bond on the 3'-Sulfo-Lex 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 3'-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 3'-sulfo-nLc4-PA and 3'-sulfolacto-N-fucopentaose III-PA, while 3'-sulfo-Lc4-PA and 3'-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 3'-sulfo-Lex 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-3Galbeta 1-4GlcNAc-R oligosaccharides irrespective of whether the penultimate GlcNAc residue is alpha 3-fucosylated.


View larger version (16K):
[in this window]
[in a new window]
  		Fig. 5.   Enzymatic degradation of 3'-sulfo-Lea and 3'-sulfo-Lex structure in vitro. 3'-Sulfo-Lc4-PA (panel a), 3'-sulfolacto-N-fucopentaose II-PA (panel b), 3'-sulfo-nLc4-PA (panel c), and 3'-sulfolacto-N-fucopentaose III-PA (panel d) were incubated in the absence (-) or presence (+) of arylsulfatase A (ASA) and separated on a reverse-phase HPLC system as described under "Experimental Procedures." Arrows indicate the positions of desulfated products. Note that only type 2 chain carbohydrates (panels c and d) are desulfated.


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 6.   Effect of treatment with arylsulfatase A on the expression of 3'-sulfo-Lex epitope on GP3ST and FucT-III gene-transfected CHO cells. Both GP3ST and FucT-III gene-transfected CHO cells were incubated in 20 mM acetate-NaOH buffer (pH 5.0) and 150 mM NaCl in the presence (bold line) or absence (solid line) of arylsulfatase A (ASA), and then subjected to flow cytometry analysis with mAb SU59.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We report herein, the enzymatic synthesis and degradation of 3'-sulfo-Lea and -Lex epitopes in vitro and in vivo. Previous studies have suggested that 3'-sulfation of the nonreducing terminal Gal occurs prior to the alpha 3/4-fucosylation of the penultimate GlcNAc in the biosynthetic pathway of 3'-sulfo-Lea and -Lex structures (23, 32, 33), as 3'-sialylation occurs before the 3/4-fucosylation in the synthetic pathway of the 3'-sialyl-Lea and -Lex (34, 35). In the present study, the biosynthesis of the 3'-sulfo-Lea and -Lex structures was comprehensively investigated by sequential reactions with GP3ST and all the currently known alpha 1,3/1,4-fucosyltransferases.

Since FucT-III is the sole alpha 1,4-fucosyltransferase (25), it was employed to synthesize the 3'-sulfo-Lea structure. As expected, FucT-III catalyzed the alpha 1,4-fucosylation of 3'-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 3'-sulfo-Lea epitope, indicating that GP3ST is involved in the biosynthesis of this epitope in vivo.

Thus far, six alpha 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 Galbeta 1-4GlcNAc-R and 3'-sialyl-Galbeta 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 3'-sialylated oligosaccharide is a poor one. FucT-V and FucT-VI are active on both neutral and 3'-sialylated substrates. FucT-VII acts on only the 3'-sialylated type 2 chain whereas FucT-IX is active only on the neutral one. Therefore, the issue of which fucosyltransferases act on the 3'-sulfated type 2 chain is of interest. Prior to this study, we anticipated a result similar to that for the 3'-sialylated acceptors. Unexpectedly, all the alpha 1,3-fucosyltransferases acted on the 3'-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 alpha 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 3'-sulfo-Lex 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 3'-sialyl-Lex epitope on FucT-III-transfected CHO cells was inhibited by the introduction of the GP3ST gene. This result suggests that GP3ST and alpha 2,3-sialyltransferase are located in the same compartment of the Golgi apparatus and compete for the Galbeta 1-4GlcNAc-R oligosaccharide on the common oligosaccharides in CHO cells. This finding was verified by Western blotting analysis, where the protein bands with 3'-sialyl-Lex were found to be nearly identical to those with 3'-sulfated Lex 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 Lex and 3'-sialyl-Lex epitopes there. A similar regulation may occur in terms of the expression of Lea and 3'-sialyl-Lea epitopes. Mutual interference by glycosyltransferases and carbohydrate-modifying enzymes in the biosynthesis of carbohydrate chains occurs under various situations (40, 41).

During the preparation of this article, the molecular cloning of another beta 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 3'-sulfo-Lex epitope in the thyroid, although only the sulfo-3Galbeta 1-4GlcNAc-R structure without alpha 3-fucose was found on human thyroglobulin (44). Gal3ST-3 may also be involved in the biosynthesis of 3'-sulfo-Lex 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 3'-sulfo-Lea and 3'-sulfo-Lex 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 beta Gal in glycolipids prompted us to examine the issue of whether it is able to desulfate the 3'-sulfo-Lea and -Lex structures. As a result, arylsulfatase A was found to hydrolyze the sulfate ester bond on 3'-sulfo-Lex but not on 3'-sulfo-Lea in vitro. This suggests that arylsulfatase A can be used as a tool for the dissection of functions between the 3'-sulfated-Lea and -Lex epitopes. On the other hand, arylsulfatase A acted only weakly on the 3'-sulfo-Lex 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 3'-sulfo-Lea epitope in a collective manner; 2) GP3ST and FucT-III, IV, V, VI, VII, and IX are involved in the biosynthesis of the 3'-sulfo-Lex epitope; 3) GP3ST and alpha 2,3-sialyltransferase compete for the common Galbeta 1-4GlcNAc-R oligosaccharides in vivo; 4) arylsulfatase A hydrolyzes the sulfate ester bond on 3'-sulfo-Lex but not on the 3'-sulfo-Lea. In the future, the present remodeling system of the 3'-sulfated Lewis epitopes may provide a useful tool for the study on their biological roles including their interaction with selectins.

    ACKNOWLEDGEMENTS

We thank Yoshie Tawara and Emmanuel S. Palacpac for technical assistance.

    FOOTNOTES

* This work was supported by a grant-in-aid for Scientific Research on Priority Area No. 10178104 from the Ministry of Education, Science and Culture, Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§§ To whom correspondence should be addressed: Dept. Biochemistry, Osaka University Medical School, Room B1, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: 81-6-6879-3421; Fax: 81-6-6879-3429; E-mail: khonke@biochem.med.osaka-u.ac.jp.

Published, JBC Papers in Press, August 14, 2001, DOI 10.1074/jbc.M107390200

    ABBREVIATIONS

The abbreviations used are: GP3ST, beta Gal-3-O-sulfotransferase; CHO cells, Chinese hamster ovary cells; MES, 2-(N-morpholino)ethanesulfonic acid; mAb, monoclonal antibody; PA, 2-aminopyridine; PAPS, adenosine 3'-phosphate,5'-phosphosulfate; Lc4, lacto-N-tetraose, Galbeta 1-3GlcNAcbeta 1-3Galbeta 1-4Glc; nLc4, lacto-N-neotetraose, Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4Glc; lacto-N-fucopentaose II, Galbeta 1-3(Fucalpha 1-4)GlcNAcbeta 1-3Galbeta 1-4Glc; lacto-N-fucopentaose III, Galbeta 1-4(Fucalpha 1-3)GlcNAcbeta 1-3Galbeta 1-4Glc; Lea, Lewis a antigen, Galbeta 1-3(Fucalpha 1-4)GlcNAc-R; Lex, Lewis x antigen, Galbeta 1-4(Fucalpha 1-3)GlcNAc-R; MOPS, 4-morpholinepropanesulfonic acid; HPLC, high performance liquid chromatography; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Brockhausen, I., and William, K. (1997) Trends Glycosci. Glycotechnol. 9, 379-398
2. Humphries, D. E., Wong, G. W., Friend, D. S., Gurish, M. F., Qiu, W. T., Huang, C., Sharpe, A. H., and Stevens, R. L. (1999) Nature 400, 769-772[CrossRef][Medline] [Order article via Infotrieve]
3. Forsberg, E., Pejler, G., Ringvall, M., Lunderius, C., Tomasini-Johansson, B., Kushe-Gullberg, M., Eriksson, I., Ledin, J., Hellman, L., and Kjellen, L. (1999) Nature 400, 773-775[CrossRef][Medline] [Order article via Infotrieve]
4. Bullock, S. L., Fletcher, J. M., Beddington, R. S. P., and Wilson, V. A. (1998) Genes Dev. 12, 1894-1906[Abstract/Free Full Text]
5. Tangemann, K., Bistrup, A., Hemmerich, S., and Rosen, S. D. (1999) J. Exp. Med. 190, 935-42[Abstract/Free Full Text]
6. Akama, T. O., Nishida, K., Nakayama, J., Watanabe, H., Ozaki, K., Nakamura, T., Dota, A., Kawasaki, S., Inoue, Y., Maeda, N., Yamamoto, S., Fujiwara, T., Thonar, E. J., Shimomura, Y., Kinoshita, S., Tanigami, A., and Fukuda, M. N. (2000) Nat. Genet. 26, 237-241[CrossRef][Medline] [Order article via Infotrieve]
7. Kudo, M., Kitajima, K., Inoue, S., Shiokawa, K., Morris, H. R., Dell, A., and Inoue, Y. (1996) J. Biol. Chem. 271, 32667-32677[Abstract/Free Full Text]
8. Kato, Y., and Spiro, R. (1989) J. Biol. Chem. 264, 3364-3371[Abstract/Free Full Text]
9. de Waard, P., Koorevaar, A., Kamerling, J., and Vliegenhart, J. (1991) J. Biol. Chem. 319, 209-216
10. Mawhinney, T., Adelstein, E., Morris, D., Mawhinney, A., and Barbergo, G. (1987) J. Biol. Chem. 262, 2994-3001[Abstract/Free Full Text]
11. Strecker, G., Wieruszeski, J., Martel, C., and Montreuil, J. (1987) Glycoconj. J. 4, 329-337
12. Capon, C., Leory, Y., Wieruszeski, J.-M., Ricart, G., Strecker, G., Montreuil, J., and Fournet, B. (1989) Eur. J. Biochem. 182, 139-152[Medline] [Order article via Infotrieve]
13. Lamblin, G., Rahmoune, H., Wieruszeski, J., Lhermitte, M., Strecker, G., and Roussel, P. (1991) Biochem. J. 275, 199-206
14. Yuen, C. T., Lawson, A. M., Chai, W., Larkin, M., Stoll, M. S., Stuart, A. C., Sullivan, F. X., Ahern, T. J., and Feizi, T. (1992) Biochemistry 31, 9126-9131[CrossRef][Medline] [Order article via Infotrieve]
15. Capon, C., Wieruszeski, J. M., Lemoine, J., Byrd, J. C., Leffler, H., and Kim, Y. S. (1997) J. Biol. Chem. 272, 31957-31968[Abstract/Free Full Text]
16. Hokke, C., Damm, J., Penninkhof, B., Aitken, R., Kamerling, J., and Vliegenhart, J. (1994) Eur. J. Biochem. 221, 491-512[Medline] [Order article via Infotrieve]
17. Lo-Guidice, J., Perini, J., Lafitte, J., Ducourouble, M., Roussel, P., and Lamblin, G. (1995) J. Biol. Chem. 270, 27544-27550[Abstract/Free Full Text]
18. Green, P. J., Tamatani, T., Watanabe, T., Miyasaka, M., Hasegawa, A., Kiso, M., Yuen, C. T., Stoll, M. S., and Feizi, T. (1992) Biochem. Biophys. Res. Commun. 188, 244-251[CrossRef][Medline] [Order article via Infotrieve]
19. Yuen, C. T., Bezouska, K., O'Brien, J., Stoll, M., Lemoine, R., Lubineau, A., Kiso, M., Hasegawa, A., Bockovich, N. J., Nicolaou, K. C., and Feizi, T. (1994) J. Biol. Chem. 269, 1595-1598[Abstract/Free Full Text]
20. Yamachika, T., Nakanishi, H., Inada, K., Kitoh, K., Kato, T., Irimura, T., and Tatematsu, M. (1997) Virch. Arch. 431, 25-30
21. Vavasseur, F., Dole, K., Yang, J., Matta, K. L., Myerscough, N., Cornfield, A., Paraskeva, C., and Brockhausen, I. (1994) Eur. J. Biochem. 222, 415-424[Medline] [Order article via Infotrieve]
22. Izawa, M., Kumamoto, K., Mitsuoka, C., Kanamori, A., Ohmori, K., Ishida, H., Nakamura, S., Kurata-Miura, K., Sasaki, K., Nishi, T., and Kannagi, R. (2000) Cancer Res. 60, 1410-1416[Abstract/Free Full Text]
23. Honke, K., Tsuda, M., Koyota, S., Wada, Y., Iida-Tanaka, N., Ishizuka, I., Nakayama, J., and Taniguchi, N. (2001) J. Biol. Chem. 276, 267-274[Abstract/Free Full Text]
24. Honke, K., Tsuda, M., Hirahara, Y., Ishii, A., Makita, A., and Wada, Y. (1997) J. Biol. Chem. 272, 4864-4868[Abstract/Free Full Text]
25. Kukowska-Latallo, J. F., Larsen, R. D., Nair, R. P., and Low, J. B. (1990) Genes Dev. 4, 1288-1303[Abstract/Free Full Text]
26. Nishihara, S., Iwasaki, H., Kaneko, M., Tawada, A., Ito, M., and Narimatsu, H. (1999) FEBS Lett. 462, 289-294[CrossRef][Medline] [Order article via Infotrieve]
27. Fujii, T., Kobayashi, T., Honke, K., Gasa, S., Ishikawa, M., Shimizu, T., and Makita, A. (1992) Biochim. Biophys. Acta 1122, 93-98[CrossRef][Medline] [Order article via Infotrieve]
28. Niwa, H., Yamamura, K., and Miyazaki, J. (1991) Gene (Amst.) 108, 193-199[CrossRef][Medline] [Order article via Infotrieve]
29. Mitsuoka, C., Sawada-Kasugai, M., Ando-Furui, K., Isawa, M., Nakanishi, H., Nakamura, S., Ishida, H., Kiso, M., and Kannagi, R. (1998) J. Biol. Chem. 273, 11225-11233[Abstract/Free Full Text]
30. Tsuiji, H., Hong, J. C., Kim, Y. S., Ikehara, Y., Narimatsu, H., and Irimura, T. (1998) Biochem. Biophys. Res. Commun. 253, 374-381[CrossRef][Medline] [Order article via Infotrieve]
31. Loveless, R. W., Yuen, C. T., Tsuiji, H., Irimura, T., and Feizi, T. (1998) Glycobiology 8, 1237-1242[Abstract/Free Full Text]
32. Chandrasekaran, E. V., Jain, R. K., and Matta, K. L. (1992) J. Biol. Chem. 267, 23806-23814[Abstract/Free Full Text]
33. Degroote, S., Ducourouble, M.-P., Roussel, P., and Lamblin, G. (1999) Glycobiology 9, 1199-1211[Abstract/Free Full Text]
34. Hansson, G. C., and Zopf, D. (1985) J. Biol. Chem. 260, 9388-9392[Abstract/Free Full Text]
35. Holmes, E. H., Ostrander, G. K., and Hakomori, S. (1986) J. Biol. Chem. 261, 3737-3743[Abstract/Free Full Text]
36. Kudo, T., Ikehara, Y., Togayachi, A., Kaneko, M., Hiraga, T., Sasaki, K., and Narimatsu, H. (1998) J. Biol. Chem. 273, 26729-26738[Abstract/Free Full Text]
37. Kaneko, M., Kudo, T., Iwasaki, H., Ikehara, Y., Nishihara, S., Nakagawa, S., Sasaki, K., Shiina, T., Inoko, H., Saitou, N., and Narimatsu, H. (1999) FEBS Lett. 452, 237-242[CrossRef][Medline] [Order article via Infotrieve]
38. Salvini, R., Bardoni, A., Valli, M., and Trinchera, M. (2001) J. Biol. Chem. 276, 3564-3573[Abstract/Free Full Text]
39. von Figura, K., Gieselmann, V., and Jaeken, J. (2001) in The Metabolic and Molecular Bases of Inherited Disease (Scriver, C. R. , Beaudet, A. L. , Sly, W. S. , and Valle, D., eds), 8th Ed. , pp. 3695-3724, McGraw Hill, New York
40. Tanemura, M., Miyagawa, S., Koyota, S., Koma, M., Matsuda, H., Tsuji, S., Shirakura, R., and Taniguchi, N. (1998) J. Biol. Chem. 273, 16421-16425[Abstract/Free Full Text]
41. Aubert, M., Panicot, L., Crotte, C., Gibier, P., Lombardo, D., Sadoulet, M. O., and Mas, E. (2000) Cancer Res. 60, 1449-1456[Abstract/Free Full Text]
42. Suzuki, A., Hiraoka, N., Suzuki, M., Angata, K., Misra, A. K., McAuliffe, J., Hindsgaul, O., and Fukuda, M. (2001) J. Biol. Chem. 276, 24388-24395[Abstract/Free Full Text]
43. El-Fasakhany, F. M., Uchimura, K., Kannagi, R., and Muramatsu, T. (2001) J. Biol. Chem. 276, 26988-26994[Abstract/Free Full Text]
44. Spiro, R. G., and Bhoyroo, V. D. (1988) J. Biol. Chem. 263, 14351-14358[Abstract/Free Full Text]
45. Hiyama, J., Weisshaar, G., and Renwick, A. G. (1992) Glycobiology 2, 401-409[Abstract/Free Full Text]

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?



This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
276/42/38588    most recent
M107390200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ikeda, N.
Right arrow Articles by Honke, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ikeda, N.
Right arrow Articles by Honke, K.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg