beta 1,4-Galactosyltransferase (beta 4GalT)-IV Is Specific for GlcNAc 6-O-Sulfate

beta 4GalT-IV ACTS ON KERATAN SULFATE-RELATED GLYCANS AND A PRECURSOR GLYCAN OF 6-SULFOSIALYL-LEWIS X*

Akira SekoDagger , Naoshi Dohmae§, Koji Takio§, and Katsuko YamashitaDagger ||

From the Dagger  Department of Biochemistry, Sasaki Institute, Kanda-Surugadai 2-2, and Core Research for Evolutional Science and Technology (CREST) of the Japan Science and Technology Corporation, Kanda-Surugadai 2-3, Chiyoda-ku, Tokyo 101-0062, and § Biomolecular Characterization Division, the Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako, Saitama 351-0198, Japan

Received for publication, November 11, 2002, and in revised form, January 2, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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The Galbeta 1right-arrow4(SO<UP><SUB>3</SUB><SUP>−</SUP></UP>right-arrow6)GlcNAc moiety is present in various N-linked and O-linked glycans including keratan sulfate and 6-sulfosialyl-Lewis X, an L-selectin ligand. We previously found beta 1,4-galactosyltransferase (beta 4GalT) activity in human colonic mucosa, which prefers GlcNAc 6-O-sulfate (6SGN) as an acceptor to non-substituted GlcNAc (Seko, A., Hara-Kuge, S., Nagata, K., Yonezawa, S., and Yamashita, K. (1998) FEBS Lett. 440, 307-310). To identify the gene for this enzyme, we purified the enzyme from porcine colonic mucosa. The purified enzyme had the characteristic requirement of basic lipids for catalytic activity. Analysis of the partial amino acid sequence of the enzyme revealed that the purified beta 4GalT has a similar sequence to human beta 4GalT-IV. To confirm this result, we prepared cDNA for each of the seven beta 4GalTs cloned to date and examined substrate specificities using the membrane fractions derived from beta 4GalT-transfected COS-7 cells. When using several N-linked and O-linked glycans with or without 6SGN residues as acceptor substrates, only beta 4GalT-IV efficiently recognized 6SGN, keratan sulfate-related oligosaccharides, and Galbeta 1right-arrow3(SO<UP><SUB>3</SUB><SUP>−</SUP></UP>right-arrow6GlcNAcbeta 1right-arrow6) GalNAcalpha 1-O-pNP, a precursor for 6-sulfosialyl-Lewis X. These results suggested that beta 4GalT-IV is a 6SGN-specific beta 4GalT and may be involved in the biosynthesis of various glycoproteins carrying a 6-O-sulfated N-acetyllactosamine moiety.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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The Galbeta 1right-arrow4(SO<UP><SUB>3</SUB><SUP>−</SUP></UP>right-arrow6)GlcNAc structure has been found in N-linked and O-linked glycans of glycoproteins and is involved in various biological events (1, 2). The sulfated disaccharide unit has been believed to be biosynthesized by 6-O-sulfation of non-reducing terminal GlcNAc and, following beta 1,4-galactosylation, on the basis of the substrate specificities of GlcNAc 6-O-sulfotransferases (GlcNAc6STs)1 so far reported (2-11). Although the galactosylation step had long been unclear, we partially purified a GlcNAc 6-O-sulfate (6SGN)-specific beta 1,4-galactosyltransferase (beta 4GalT) from human colonic mucosa (12). The enzyme preferred 6SGN and 6SGN-containing oligosaccharides to non-sulfated GlcNAc residues as acceptor substrates, whereas bovine milk beta 4GalT-I showed the opposite specificity. The Neu5Acalpha 2right-arrow6GlcNAcbeta 1right-arrow sequence was a poor substrate for the 6SGN-specific beta 4GalT, suggesting that a sulfate residue at the C-6 of GlcNAc is essential for the acceptor recognition. This preference for 6SGN residues suggests that the enzyme is involved in the biosynthesis of keratan sulfate (KS), ((SO<UP><SUB>3</SUB><SUP>−</SUP></UP>right-arrow6)Galbeta 1right-arrow4(SO<UP><SUB>3</SUB><SUP>−</SUP></UP>right-arrow6)GlcNAcbeta 1right-arrow3)n (reviewed in Ref. 13), and 6-sulfosialyl-Lewis X, known as a L-selectin ligand (14-17). However, molecular cloning of the enzyme has not yet been performed.

It has been clarified so far (18-29) that seven beta 4GalT genes exist. All the beta 4GalTs are synthesized as type II membrane-bound proteins and reside in the Golgi apparatus (30). These seven enzymes have been characterized in terms of substrate specificity. beta 4GalT-I is abundant in bovine and human milk in a soluble form and is the first galactosyltransferase for which the corresponding cDNA has been isolated (18-21). beta 4GalT-I acts on non-reducing terminal GlcNAc as an acceptor, and whereas in the presence of alpha -lactalbumin, the enzyme prefers Glc as a lactose synthase to GlcNAc (31, 32). This enzyme is involved in the elongation of poly-N-acetyllactosamine repeats (33). beta 4GalT-II and -III act on GlcNAc residues in several glycoproteins and specific glycolipids, and beta 4GalT-II is affected by alpha -lactalbumin in a similar manner to beta 4GalT-I (22). beta 4GalT-IV acts on Lc3 (GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4Glcbeta 1right-arrowCer) (26) and Galbeta 1right-arrow3(GlcNAcbeta 1right-arrow6)GalNAc(core2); the latter galactosylation at the C-4 of GlcNAc leads to the formation of the sialyl-Lewis X structure (34). beta 4GalT-V has been shown to have strong activity for core2 and core6(GlcNAcbeta 1right-arrow6GalNAc) (35). The enzyme also recognized GlcNAcbeta 1right-arrow2(GlcNAcbeta 1right-arrow6)Manalpha 1right-arrow6 moieties in N-linked tetra-antennary glycans (23, 36) and was suggested to be involved in the biosynthesis of tumor-associated N-glycans in concert with beta N-acetylglucosaminyltransferase V (37). Lactosylceramide synthase has been purified from rat brain, and its molecular cloning was performed based on partial amino acid sequences (25). The enzyme is an orthologue of human beta 4GalT-VI (24, 27). beta 4GalT-VII is a beta -Xyl:beta 1,4GalT, equal to galactosyltransferase-I which is involved in the synthesis of the proximal sequence in various glycosaminoglycans, Galbeta 1right-arrow3Galbeta 1right-arrow 4Xylbeta 1right-arrowSer/Thr (28, 29). Although the substrate specificities of these seven beta 4GalTs have been extensively studied, it remained unclear which enzymes can act on 6SGN residues in KS, N-linked, and O-linked glycans, and whether or not 6SGN-specific beta 4GalT is encoded by a novel gene. This issue is important for studying the biological roles of 6-sulfo-N-acetyllactosamine-containing glycans including KS and 6-sulfosialyl-Lewis X.

In this study, we first purified 6SGN-specific beta 4GalT from porcine colonic mucosa and partially determined its amino acid sequence. We obtained a sequence similar to human beta 4GalT-IV. Moreover, we isolated cDNA for each of the seven beta 4GalTs cloned to date, and we introduced expression vectors containing these cDNAs individually into COS-7 cells and prepared membrane fractions to study their substrate specificities. As a result, we found that among the seven beta 4GalTs, beta 4GalT-IV is the only enzyme to efficiently act on KS-related oligosaccharides and GlcNAc-6-O-sulfated core2, a precursor of 6-sulfosialyl-Lewis X.

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EXPERIMENTAL PROCEDURES
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Materials-- UDP-[3H]galactose (1400 GBq/mmol) was purchased from PerkinElmer Life Sciences. PAPS, UDP-Gal, p-nitrophenyl(pNP)-beta -D-xylose (Xyl-O-pNP), and glucosylceramide (GlcCer) were purchased from Sigma. Galbeta 1right-arrow3(GlcNAcbeta 1right-arrow6)GalNAcalpha 1-O-pNP (core2-O-pNP) was obtained from Funakoshi Co., Ltd. (Tokyo, Japan). GlcNAcbeta 1right-arrow 2Manalpha 1right-arrow3(GlcNAcbeta 1right-arrow2Manalpha 1right-arrow6)Manbeta 1right-arrow4GlcNAcbeta 1right-arrow4GlcNAc (biGP) was prepared from egg yolk sialoglycopeptide (38) by hydrazinolysis re-N-acetylation (39), mild acid hydrolysis, and Streptococcus 6646K beta -galactosidase (40) (Seikagaku Corp., Tokyo, Japan) digestion. GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4Glc(GL) was prepared from lacto-N-tetraose (41) by digestion with Streptococcus 6646K beta -galactosidase. Ricinus communis agglutinin-I (RCA-I)-agarose(4 mg/ml gel) was purchased from Hohnen Oil Co. (Tokyo, Japan). Keratan sulfate (sodium salt and bovine cornea) was purchased from Seikagaku Corp. Galbeta 1right-arrow4(SO<UP><SUB>3</SUB><SUP>−</SUP></UP>right-arrow6)GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4(SO<UP><SUB>3</SUB><SUP>−</SUP></UP>right-arrow6)GlcNAc(L2L2) and Galbeta 1right-arrow4(SO<UP><SUB>3</SUB><SUP>−</SUP></UP>right-arrow6)GlcNAcbeta 1right-arrow3(SO<UP><SUB>3</SUB><SUP>−</SUP></UP>right-arrow6)Galbeta 1right-arrow4(SO<UP><SUB>3</SUB><SUP>−</SUP></UP>right-arrow6)GlcNAc(L2L4) were kindly provided by Seikagaku Corp. SO<UP><SUB>3</SUB><SUP>−</SUP></UP>right-arrow6GlcNAcbeta 1right-arrow 3Galbeta 1right-arrow4 (SO<UP><SUB>3</SUB><SUP>−</SUP></UP>right-arrow6)GlcNAc(agL2L2) and SO<UP><SUB>3</SUB><SUP>−</SUP></UP>right-arrow6GlcNAcbeta 1right-arrow3(SO<UP><SUB>3</SUB><SUP>−:</SUP></UP>right-arrow6)Galbeta 1right-arrow4(SO<UP><SUB>3</SUB><SUP>−</SUP></UP>right-arrow6)GlcNAc(agL2L4) were prepared by Streptococcus 6646K beta -galactosidase digestion from L2L2 and L2L4, respectively. GlcNAc 6-O-sulfate (6SGN), Neu5Acalpha 2right-arrow6GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4Glc(agLST-b), and SO<UP><SUB>3</SUB><SUP>−</SUP></UP>right-arrow6GlcNAcbeta 1right-arrow2Manalpha 1right-arrow3(Manalpha 1right-arrow6)Manbeta 1right-arrow4GlcNAcbeta 1right-arrow4(Fucalpha 1right-arrow6)GlcNAc(6S-biGP) were prepared as described previously (12). L-alpha -Phosphatidic acid, L-alpha -phosphatidylcholine, L-alpha -lysophosphatidylcholine, and L-alpha -phosphatidylethanolamine were from egg yolk lecithin; L-alpha -phosphatidylinositol was from soybean; L-alpha -phosphatidyl-L-serine and sphingomyelin were from bovine brain; ceramides were from bovine brain sphingomyelin; D-sphingosine was from bovine brain cerebrosides; N,N-dimethylsphingosine, N-acetyl-D-sphingosine, and N-stearoyl-D-sphingosine prepared from D-sphingosine, sphingosine 1-phosphate, and stearylamine were purchased from Sigma. Octylamine was purchased from Aldrich.

Purification of 6SGN-specific beta 4GalT from Porcine Colonic Mucosa-- The following procedures were performed at 4 °C. Six kg of porcine colon was purchased from Tokyo Shibaura Zohki Co. Ltd. (Tokyo, Japan). The mucus layer was scraped off the colon; 3 liters of PBS was added, and the mixture was homogenized with a Potter-Elvehjem type homogenizer and then centrifuged at 1,000 × g for 30 min. The extraction was performed once, and the two supernatant fractions were mixed and ultracentrifuged at 100,000 × g for 1 h. The precipitated microsomes were washed once with 0.5 M KCl and extracted twice with 1 liter of 20 mM HEPES-NaOH (pH 7.2), 1 M NaCl, 10 mM MnCl2, 1% (w/v) Triton X-100, 1 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride, and 10% (v/v) glycerol followed by ultracentrifugation. The extract was dialyzed against 10 mM HEPES-NaOH (pH 7.2), 5 mM MnCl2, 1 mM dithiothreitol, and 20% glycerol (buffer A), and applied on a UDP-hexanolamine-Sepharose column (7 µmol/ml of gel; 2 × 10 cm; equilibrated with 20 mM HEPES-NaOH (pH 7.2), 10 mM MnCl2, 0.1% (w/v) Triton X-100, 1 mM dithiothreitol, and 10% glycerol (buffer B)) (12). After washing with buffer B containing 0.15 M NaCl, the enzyme fractions were eluted with buffer B containing M NaCl and dialyzed against buffer A. Next, the dialysate was reapplied on a UDP-hexanolamine-Sepharose column (1.4 × 6.5 cm; equilibrated with buffer B) and eluted with buffer B containing 1 mM UDP. Each fraction was dialyzed against buffer A and assayed for beta 4GalT activity. The enzyme fractions were applied on an asialo-agalacto-ovomucin-Sepharose column (6.7 mg mucin/ml of gel; 1 × 7.5 cm; equilibrated with buffer B) (12). The enzyme was eluted with a linear gradient of NaCl (0-0.2 M) in buffer B and then dialyzed. The enzyme fractions were concentrated and used for biochemical analyses.

Cloning of the cDNAs Encoding beta 4GalTs-- cDNAs encoding beta 4GalT-II, -III, -IV, -V, and -VII were amplified from SuperScriptTM human testis cDNA library (Invitrogen) by PCR, and cDNAs encoding beta 4GalT-I and -VI were amplified by PCR from QUICK-CloneTM cDNA for human colon adenocarcinoma and mouse brain (Clontech, Palo Alto, CA), respectively. Oligonucleotide primers used for the PCRs were 5'-tttggatccTGTAGCCCACACC-3' (forward primer for beta 4GalT-I), 5'-tttgatatcAGGTCTCTTATCCGTG-3' (reverse primer for beta 4GalT-I), 5'-tttggatccGGATGAGCAGACTGCTG-3' (forward primer for beta 4GalT-II), 5'-tttgatatcGTCCATTAGTGTCAGCC-3' (reverse primer for beta 4GalT-II), 5'-tttggatccAGGATGTTGCGGAGGCT-3' (forward primer for beta 4GalT-III), 5'-tttgatatcGAGGAGTCAGTGTGAAC-3' (reverse primer for beta 4GalT-III), 5'-tttggatccACATGGGCTTCAACCTG-3' (forward primer for beta 4GalT-IV), 5'-tttgatatcATCCAGGGTCATGCACC-3' (reverse primer for beta 4GalT-IV), 5'-tttggatccTGGCTGCAGCATGC-3' (forward primer for beta 4GalT-V), 5'-tttgatatcTCAGTACTCGTTCAC-3' (reverse primer for beta 4GalT-V), 5'-tttggatccGGAAGATGTCTGTGCTC-3' (forward primer for beta 4GalT-VI), 5'-tttgatatcCAGCATTGTGGTCTA-3' (reverse primer for beta 4GalT-VI), 5'-tttggatccGCACGATGTTCCC-3' (forward primer for beta 4GalT-VII), and 5'-tttgatatcCAGCTCAGCTGAATG-3' (reverse primer for beta 4GalT-VII). Sequences in lowercase letters contain appropriate restriction sites. Amplified cDNAs were digested with BamHI and EcoRV and cloned between the respective sites of pcDNA3 (Invitrogen). The constructed plasmids were named pcDNA3-beta 4GalT-I, -II, -III, -IV, -V, -VI, and -VII, respectively, and these sequences were confirmed using an Applied Biosystems PRISM® 310 Genetic Analyzer (PE Biosystems).

Expression of beta 4GalTs in COS-7 Cells-- The plasmids (1 µg) were transfected into COS-7 cells on 35-mm dishes using Lipofectin Reagent (Invitrogen) according to the manufacturer's instructions. After 48 h, the cells were washed twice with phosphate-buffered saline, scraped off the dishes in 10 mM HEPES-NaOH (pH 7.2) and 0.25 M sucrose, and homogenized. The homogenates were ultracentrifuged at 100,000 × g for 1 h. The precipitated crude membranes were suspended in 20 mM HEPES-NaOH (pH 7.2) and kept at -80 °C until use.

Synthesis of Galbeta 1right-arrow3(SO<UP><SUB>3</SUB><SUP>−</SUP></UP>right-arrow6GlcNAcbeta 1right-arrow6)GalNAcalpha 1-O-pNP(6S-core2-O-pNP)-- The 6-O-sulfation of the GlcNAc residue in core2-O-pNP was performed by GlcNAc6ST-1 (42). Briefly, 2 ml of reaction mixture containing 0.1 M sodium cacodylate (pH 6.8), 10 mM MnCl2, 0.1% (w/v) digitonin, 0.1 M NaF, 2 mM Na2-ATP, 0.2 mM PAPS, 0.75 mM core2-O-pNP, and a membrane fraction prepared from GlcNAc6ST-1-expressing COS-7 cells (11) (the specific activity was 8 nmol/h when using 1 mM core2-O-pNP and 17 µM PAPS) was incubated at 37 °C for 24 h and subjected to paper electrophoresis. The product fractions were cut off and extracted with water. The extract was applied on a Sephadex G-25 column (1.4 × 68 cm, equilibrated and eluted with EtOH/water, 5:95 (v/v)). The sulfated oligosaccharide fractions were collected and concentrated. Finally, 150 nmol of 6S-core2-O-pNP was obtained.

Assay of beta 4GalT Activity-- Twenty µl of reaction mixture consisting of 50 mM HEPES-NaOH (pH 7.2), 10 mM MnCl2, 0.5% (w/v) Triton X-100, 250 µM UDP-Gal, 0.3 µM UDP-[3H]Gal (4.9 × 105 dpm), 0.5 mM acceptor substrate, and the membrane fraction approximately diluted was incubated at 37 °C for 1 h. In the cases of purified porcine 6SGN-specific beta 4GalT, 0.5%(w/v) Triton X-100 was replaced by 0.001% (w/v) D-sphingosine and 0.05% (w/v) Triton X-100. The 3H-labeled products derived from biGP, 6SGN, GL, Xyl-O-pNP, core2-O-pNP, 6S-core2-O-pNP, 6S-biGP, and KS were purified by paper electrophoresis (pyridine/acetic acid/water, 3:1:387, pH 5.4). The reaction mixtures in the case of agL2L2, agL2L4, and agLST-b were treated with 1 ml of 0.01 N HCl at 100 °C for 10 min, neutralized with 1 N NaOH, concentrated, and subjected to paper electrophoresis. The acid hydrolysis was necessary for the destruction of excess UDP-[3H]Gal, which moves to a similar position as 3H-galactosylated agL2L2 and agL2L4 and for removal of sialic acid residue in the case of agLST-b. After drying, in the cases of neutral substrates, the paper was further developed with a solvent system, pyridine/ethyl acetate/acetic acid/water (5:5:1:3). The paper was monitored for radioactivity with a radiochromatogram scanner, and the 3H-labeled products were extracted with water and applied on a RCA-I-agarose column (0.7 × 2.5 cm). Elution was started with 4 ml of 10 mM sodium phosphate (pH 7.0), 0.15 M NaCl, followed by the same buffer containing 10 mM lactose. The bound fractions (eluted with 10 mM lactose) or retarded fractions were collected and measured for radioactivity. In the case of GlcCer, the reaction mixtures were directly applied on a high performance TLC silica gel plate (Kieselgel 60 F254) (Merck) and developed with a solvent system (CHCl3/MeOH/0.2% CaCl2, 60:35:7). After drying, the plates were monitored for radioactivity, and the 3H-labeled products were extracted with CHCl3/MeOH (1:1) and counted. The membrane fraction derived from pcDNA3-transfected COS-7 cells (C-MF) was used as a control for intrinsic beta 4GalT activities. To calculate the amount of exogenous beta 4GalT activities, values of activities in C-MF were subtracted from those of apparent enzymatic activities obtained under the same conditions. Enzymatic activity values presented here were means of four independent experiments. Standard deviations were less than 5% in all cases. The concentration of KS was presented as moles of galactose.

Determination of Protein Concentrations-- The protein concentrations were estimated using the Bio-Rad Protein Assay dye reagent with bovine serum albumin as a standard.

Amino Acid Sequence Analysis-- Coomassie-stained bands in SDS-PAGE gels were excised and treated with 0.2 µg of Achromobacter protease I (a gift from Dr. Masaki, Ibaraki University) (43) at 37 °C for 12 h in 0.1 M Tris-HCl (pH 9.0) containing 0.1% SDS. Peptides generated were extracted from the gel and separated on columns of DEAE-5PW (2 × 20 mm; Tosoh, Tokyo) and Mightysil RP-18 (2 × 50 mm; Kanto Chemical, Tokyo) connected in series with a model 1100 (Hewlett-Packard) liquid chromatography system. Peptides were eluted at a flow rate of 0.1 ml/min using a linear gradient of 0-60% solvent B, where solvents A and B were 0.09% (v/v) aqueous trifluoroacetic acid and 0.075% (v/v) trifluoroacetic acid in 80%(v/v) acetonitrile, respectively. Selected peptides were subjected to Edman degradation using a model 477A automated protein sequencer (Applied Biosystems, Inc.) connected on-line to a model 120A PTH analyzer (PerkinElmer Life Sciences) and to a Reflex matrix-assisted laser desorption ionization time of flight mass spectrometer (Bruker-Franzen Analytik, Bremen, Germany) in linear mode using 2-mercaptobenzothiazole (44) as a matrix.

Northern Blot Analysis-- Human Multiple Tissue Northern blot membranes (Clontech, Palo Alto, CA) were used according to the manufacturer's instructions. The mRNA content in each lane of the Northern blot membrane was normalized to the mRNA expression level of beta -actin. 32P-Labeled probe was prepared from the cDNA fragment (excised from pcDNA3-beta 4GalT-IV by BamHI and EcoRV digestion) with a Random Primed DNA Labeling Kit (Roche Diagnostics) using [alpha -32P]dCTP (PerkinElmer Life Sciences) according to the manufacturer's instructions. The membranes were pre-hybridized in ExpressHyb Solution (Clontech) at 68 °C for 2 h and then hybridized with the 32P-labeled probe in the same solution at 68 °C for 16 h. The Northern blot membranes were washed in 2× SSC, 0.05% SDS at room temperature and then in 0.1× SSC, 0.1% SDS at 50 °C. The radioactivity was detected with FLA-2000 (Fuji Photo Film Co. Ltd., Tokyo).

    RESULTS AND DISCUSSION
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Purification of 6SGN-specific beta 4GalT from Porcine Colonic Mucosa and Its Basic Lipid Requirement for Activity-- We showed previously (12) that a 6SGN-specific beta 4GalT exists in human colonic mucosa. To purify this enzyme and to isolate a corresponding cDNA based on partial amino acid sequences, we used porcine colonic mucosa as an enzyme source. Porcine colonic mucosa also contained 6SGN-specific beta 4GalT activity. When the enzyme was purified 2,300-fold by a second UDP-hexanolamine-Sepharose column chromatography (Table I), the enzymatic activity disappeared (Fig. 1A). Because such a loss of enzymatic activity has been reported for a glucuronyltransferase, which requires sphingomyelin for activity in a highly purified state (45), we added various lipid compounds to the enzyme reaction mixtures. As shown in Table II, most of the phospholipids and ceramides had no ability to restore the enzymatic activity, whereas the activity appeared in the presence of D-sphingosine and N,N-dimethylsphingosine. Stearylamine could also restore the activity, whereas octylamine could not, suggesting that basic, lipidous compounds with long hydrophobic chains are essential at least for the in vitro enzymatic activity. The minimum concentrations of D-sphingosine, N,N-dimethylsphingosine, and stearylamine giving the maximal activity were ~10, 20, and 10 µM, respectively (Fig. 2).

                              
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Table I
Purification of 6SGN-specific beta 4GaIT from porcine colonic mucosa


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Fig. 1.   A, second UDP-hexanolamine-Sepharose column chromatography of the 1 M NaCl-eluted fractions on first UDP-hexanolamine-Sepharose column chromatography. Each 30-ml fraction was collected. The column was washed with buffer B followed by buffer B containing 1 mM UDP (arrowheads). 6SGN-specific beta 4GalT activities () were assayed in the absence (upper) or presence (lower) of 0.001% D-sphingosine as described under "Experimental Procedures." The fractions indicated by the bar were collected. B, asialo-agalacto-ovomucin-Sepharose column chromatography of the enzyme fractions obtained in A. Each 12-ml fraction was collected. The column was washed with buffer B followed by a linear gradient of 0-0.2 M NaCl (- - -) in buffer B. The enzymatic activities were assayed in the presence of 0.001% D-sphingosine. The fractions indicated by the bar were collected and used for biochemical analysis.

                              
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Table II
Effects of various lipids on purified 6SGN-specific beta 4GaIT activity


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Fig. 2.   Effects of D-sphingosine (), N,N-dimethylsphingosine (open circle ), and stearylamine (black-triangle) on the purified 6SGN-specific beta 4GalT. Indicated concentrations of the lipids were added to the enzyme reaction mixtures. The concentration of Triton X-100 was 0.05% (w/v) in these assays.

After asialo-agalacto-ovomucin-Sepharose chromatography, 6SGN-specific beta 4GalT was purified 24,000-fold (Fig. 1B and Table I). We tried further purification by various chromatographic methods but were unsuccessful. The final enzyme fractions contained two major (45kDa and 42kDa) and two minor (59kDa and 52kDa) proteins as determined by SDS-PAGE analysis (Fig. 3). Each protein band was digested with a lysine-specific protease, and the peptide fragments were separated by reversed phase high pressure liquid chromatography, and their amino acid sequence was analyzed. One or two peptide fragments from each band were sequenced (Table III), and the sequences were compared with known protein sequences using the BLAST search system. The band a contained one sequence that is equal to that of human polypeptide:N-acetylgalactosaminyltransferase-2 (46). The band b contained two sequences that are very similar to that of human N-acetylglucosaminyltransferase-I (47, 48). Because the two enzymes utilize UDP-sugars as donor substrates, they may be co-purified with 6SGN-specific beta 4GalT by UDP-hexanolamine-Sepharose column chromatographies. On the other hand, the bands c and d contained a common peptide sequence, which is similar to that of human beta 4GalT-IV (24, 26). This result suggested that the purified 6SGN-specific beta 4GalT corresponds to beta 4GalT-IV.


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Fig. 3.   SDS-PAGE analysis of the enzyme fractions obtained by asialo-agalacto-ovomucin-Sepharose column chromatography. The enzyme fractions (10 µg as protein) were subjected to SDS-PAGE (10% gel) under reducing conditions and visualized with Coomassie Brilliant Blue.

                              
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Table III
Amino acid sequences of peptide fragments derived from the bands a-d in Fig. 3
X in analyzed sequence indicates the amino acid (aa) residues that could not be determined. The underlined amino acid residues in corresponding sequence (human) indicate discrepancies from analyzed sequence (porcine).

Expression of Seven beta 4GalTs in COS-7 Cells and Their Substrate Specificities-- To confirm that beta 4GalT-IV is 6SGN-specific, and the only 6SGN-specific beta 4GalT among the seven beta 4GalTs so far identified, we prepared expression vectors containing each beta 4GalT cDNA and analyzed substrate specificities using the membrane fractions derived from vector-transfected COS-7 cells. Seven beta 4GalT genes have been identified within the data bases provided by the human genome project to date (18-29). The membrane fraction derived from vacant pcDNA3-transfected COS-7 cells (C-MF) was used as a control for intrinsic beta 4GalT activities. C-MF had weak beta 4GalT activity; the specific activities using biGP, 6SGN, agL2L2, Xyl-O-pNP, GL, core2-O-pNP, GlcCer, and 6S-core2-O-pNP as acceptor substrates were 0.20, 0.34, 0.43, 0.20, 0.12, 0.47, 0, and 0.16 nmol/min/mg of protein, respectively. To calculate the amount of exogenous beta 4GalT activities, values derived from C-MF were subtracted from those of the apparent enzymatic activities obtained under the same conditions.

The specific activities of beta 4GalT-I, -II, -III, -IV, -V, -VI, and -VII were 6.6 (using biGP as an acceptor), 3.4 (core2-O-pNP), 2.6 (core2-O-pNP), 1.5 (6SGN), 1.5 (GlcCer), 0.32 (GlcCer), and 3.7 nmol/min/mg of protein (Xyl-O-pNP), respectively. The relative activities of the seven beta 4GalTs for eight acceptor substrates are summarized in Table IV. Among the seven beta 4GalTs, only beta 4GalT-IV recognized 6SGN as a good acceptor in comparison with biGP and GL, and this profile of beta 4GalT-IV was equal to the substrate specificity of human colonic 6SGN-specific beta 4GalT previously reported by us (12). The linkage position of [3H]Gal in [3H]Galbeta 1right-arrowagL2L2 and ([3H]Galbeta 1right-arrow)6S-core2-O-pNP synthesized by beta 4GalT-IV was confirmed to be the C-4 of GlcNAc by RCA-I-agarose affinity chromatography (Fig. 4), which binds to Galbeta 1right-arrow4GlcNAc (49). [3H]Galbeta 1right-arrow agL2L2 (Fig. 4B, solid line) and ([3H]Galbeta 1right-arrow)6S-core2-O-pNP (Fig. 4C, solid line) bound to the column weaker than ([3H]Galbeta 1right-arrow)core2-O-pNP (Fig. 4A). To examine whether the weak binding is caused by the presence of 6-O-sulfate, the sulfated products were treated with mild methanolysis (0.05 N HCl/MeOH, 25 °C, 4 h) (50) and applied onto the lectin column. The digests bound to the column and were eluted with 10 mM lactose (Fig. 4, B and C, dotted lines), but the digests flowed through the column by digestion with Galbeta 1right-arrow4GlcNAc-specific diplococcal beta -galactosidase (data not shown), showing that [3H]Gal is attached to the C-4 of GlcNAc in the sulfated products. 6SGN was a poor substrate at best for the other beta 4GalTs (Table IV) even at the lower concentrations, 0.1 or 0.2 mM (data not shown), excluding the possibility that the absence or low level of activity was due to the inhibitory effect of a high concentration of 6SGN; such as has been found in several beta 4GalTs (34, 51). These results indicated that beta 4GalT-IV is the only 6SGN-specific enzyme among the seven beta 4GalTs, consistent with the amino acid sequence of the purified porcine 6SGN-specific beta 4GalT as described above.

                              
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Table IV
Substrate specificities of seven beta 4GalTs
The enzymatic activities were the means of four independent experiments (SD, less than 5%). The relative values are taken from the value of biGP (I), core2-O-pNP (II and III), 6SGN (IV), GlcCer(V and VI), or Xyl-O-pNP (VII) as 100.


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Fig. 4.   RCA-I-agarose column chromatography of the reaction products, [3H]Galright-arrowcore2-O-pNP (A), [3H]Galright-arrowagL2L2 (B, solid line), and ([3H]Galright-arrow)6S-core2-O-pNP (C, solid line). The reaction products purified by paper electrophoresis and paper chromatography were applied on a RCA-I-agarose column and eluted as described under "Experimental Procedures." [3H]Galright-arrowagL2L2 and ([3H]Galright-arrow)6S-core2-O-pNP were treated with mild methanolysis and applied on a RCA-I column (dotted lines in B and C, respectively). V0, void volume.

beta 4GalT-III prefers O-linked type core2-O-pNP to N-linked type biGP, whereas beta 4GalT-I and -II recognize both biGP and core2-O-pNP as good acceptors. It has been shown that beta 4GalT-V acts on core2 (35) and a specific branching structure of N-linked glycans (36). As shown in Table IV, beta 4GalT-V could act on GlcCer, similar to beta 4GalT-VI. Recently, Lee et al. (52) reported that beta 4GalT-VI-deficient CHO cells have an ability to synthesize lactosylceramides, and they suggested the existence of a lactosylceramide synthase other than beta 4GalT-VI. beta 4GalT-V may be the second lactosylceramide synthase.

beta 4GalT-IV recognizes several 6SGN-containing oligosaccharides as good acceptors (Table V). KS-related oligosaccharides, agL2L2 and agL2L4, were good substrates for beta 4GalT-IV. AgL2L2 was a poor substrate at best for the other beta 4GalTs (Table IV). KS is also a substrate for beta 4GalT-IV with a specific activity of 0.045 nmol/min/mg of protein at 1 mM KS. To expose GlcNAc residues at the non-reducing termini of KS chains, we treated KS by mild acid hydrolysis to remove sialic acid and Streptococcus 6646K beta -galactosidase digestion. The enzymatic activity for the asialo-agalacto-KS was 3.3-fold higher than that for intact KS. This result suggests that beta 4GalT-IV can also act on KS long chains. Because agLST-b was a poor substrate for beta 4GalT-IV, sialic acid cannot replace sulfate at the C-6 of GlcNAc. This substrate selectivity is the same as for human colonic 6SGN-specific beta 4GalT (12). beta 4GalT-IV also efficiently acts on 6S-biGP and 6S-core2-O-pNP; the 6SGN moiety is present in N-linked and O-linked glycans in various glycoproteins (1). The 6-sulfosialyl-Lewis X on core2 glycans, which is synthesized from 6S-core2 by beta 1right-arrow4-galactosylation, alpha 2right-arrow3-sialylation, and alpha 1right-arrow3-fucosylation, functions as an L-selectin ligand moiety in the early step of lymphocyte homing in lymph nodes (14-17). beta 4GalT-IV is the only enzyme recognizing 6S-core2-O-pNP as a good substrate among the seven beta 4GalTs (Table IV), suggesting that beta 4GalT-IV is involved in the synthesis of 6-sulfosialyl-Lewis X. 

                              
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Table V
Relative values of the enzymatic activities of beta 4GalT-IV for various acceptor substrates
The enzymatic activities were the means of four independent experiments (SD, less than 5%). The relative values are taken the value at 0.5 mM 6SGN as 100.

Results of kinetic analysis of beta 4GalT-IV for several glycans with or without 6-O-sulfation at GlcNAc are summarized in Table VI. AgL2L4 and 6S-core2-O-pNP had an inhibitory effect on beta 4GalT-IV activities at high concentrations (Fig. 5), so that kinetic constants were calculated using only the data obtained with lower concentrations of these substrates. The Km value for 6SGN was lower than that for GlcNAc, and the Vmax/Km value for 6SGN was 1900-fold higher than that for GlcNAc. Similarly, the Vmax/Km values for 6S-biGP and 6S-core2-O-pNP were much higher than those for biGP and core2-O-pNP, respectively. These results indicate that 6-O-sulfation of GlcNAc residues is important for efficient catalytic activity of beta 4GalT-IV.

                              
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Table VI
Kinetic analysis of beta 4GalT-IV for several oligosaccharides containing 6-O-sulfated or non-substituted GlcNAc


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Fig. 5.   Enzymatic activities at various concentrations of acceptor substrates. The activities were measured as under "Experimental Procedures." Acceptor substrates used were 6SGN (), core2-O-pNP (open circle ), 6S-core2-O-pNP (black-triangle), and agL2L4 (triangle ).

Expression of beta 4GalT-IV in Various Human Tissues-- Lo et al. (24) and Schwientek et al. (26) reported the expression profile of beta 4GalT-IV in human adult and fetal tissues, but they had not examined its expression in colon or lymph node, where 6SGN-specific beta 4GalT should be expressed. To clarify the expression pattern more extensively, we analyzed it using commercial Northern blot membranes. As shown in Fig. 6, a 2.4-kb transcript of beta 4GalT-IV was expressed ubiquitously. Relatively high expression levels of the enzyme were observed in kidney, placenta, lymph node, prostate, stomach, thyroid, tongue, and trachea. Recently, it has been shown that the 6-sulfosialyl-Lewis X determinant is present in colonic mucosa (53); in colon, beta 4GalT-IV was also moderately expressed (Fig. 7).


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Fig. 6.   Northern blot analysis of beta 4GalT-IV transcript. The amount of poly(A)+ RNA was normalized to the mRNA expression levels of beta -actin. The blots were hybridized with a 32P-labeled cDNA probe specific for human beta 4GalT-IV. On the left, the migration position standard is indicated.


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Fig. 7.   Proposed schemes for biosynthesis of (A) keratan sulfate and (B) 6-sulfosialyl-Lewis X structure.

General Discussion-- In this study, we showed the following. (i) Porcine 6SGN-specific beta 4GalT was purified and identified as beta 4GalT-IV. (ii) The substrate specificities of the seven beta 4GalTs so far cloned were investigated, and beta 4GalT-IV is the only 6SGN-specific beta 4GalT among them and is involved in the synthesis of several 6-O-sulfated N-acetyllactosamine glycans. (iii) Porcine beta 4GalT-IV requires basic lipidous compounds for the catalytic activity at least in a highly purified state. It has been reported that sphingosine increases several times the enzymatic activities of alpha 1right-arrow3-fucosyltransferase, GM2:beta 1right-arrow3GalT, GM3:beta 1right-arrow4-N-acetylgalactosaminyltransferase (54), and glycoprotein sulfotransferase (55) and decreases that of beta 1right-arrow3-glucuronyltransferase (56). However, to our knowledge, this is the first demonstration that basic lipidous compounds are essential for the catalytic activity of glycosyltransferases. Sphingosine is an intermediate of the metabolism of sphingolipids and a precursor for sphingosine 1-phosphate, a second messenger molecule (reviewed in Refs. 57-59); however, it has been believed that sphingosine is present at quite low levels in living cells. The molecular aspect of the requirement is unclear, but one explanation is that in the process of purification, a certain basic compound, which binds to the enzyme in the crude extract, is detached from the enzyme protein. We are in the process of resolving this issue by analyzing biochemically the binding of recombinant beta 4GalT-IV to various basic compounds.

KS is poly-6-O-sulfated poly-N-acetyllactosamine (reviewed in Ref. 13). Several glyco- and sulfotransferases involved in KS synthesis have been reported. Five GlcNAc6ST genes have so far been identified, and all the enzymes can act on non-reducing terminal GlcNAc but not on internal GlcNAc such as Galbeta 1right-arrow4GlcNAc. Recently, Akama et al. (60) showed that GlcNAc6ST-5 (CGn6ST) is involved in KS synthesis. Fukuta et al. (61) identified a Gal6ST, which prefers an internal Gal residue in the di-N-acetyllactosamine chain (62), suggesting that 6-O-sulfation of Gal is a later step in KS synthesis. Among beta 3GnTs so far cloned, beta 3GnT-2 has strong enzymatic activity for p-lacto-N-neohexaose (63), suggesting that the enzyme may be a candidate involved in the elongation of the KS backbone structure, although there is no evidence that beta 3GnT-2 has beta 3GnT activity for KS elongation. In this study, we showed that among the seven beta 4GalTs, only beta 4GalT-IV acts on KS-related glycans, agL2L2 and agL2L4. From these results, it is proposed that KS is synthesized by the sequential actions of four enzymes, GlcNAc6ST-5, beta 4GalT-IV, beta 3GnT, and then Gal6ST (Fig. 7A).

On the other hand, 6-sulfosialyl-Lewis X is synthesized by sequential reactions of GlcNAc6ST, beta 4GalT, alpha 2,3-sialyltransferase, and alpha 1,3-fucosyltransferase. It has been shown that in lymph nodes, this structure is attached on beta 1,6-branching GlcNAc in O-linked core2 (14) or on elongated core1 (64) and that the GlcNAc 6-O-sulfation is performed by GlcNAc6ST-2. However, Uchimura et al. (10) showed that GlcNAc6ST-1 is responsible for the synthesis of 6-sulfosialyl-Lewis X. Next, galactosylation may be catalyzed by beta 4GalT-IV as indicated from our results that only beta 4GalT-IV efficiently acts on 6S-core2-O-pNP among the seven beta 4GalTs. Ujita et al. (34) reported that among beta 4GalT-I to -V, beta 4GalT-IV efficiently acts on non-sulfated core2-O-pNP. Here we showed that 6S-core2-O-pNP is a better substrate for beta 4GalT-IV than core2-O-pNP. From these results, a biosynthetic scheme for 6-sulfosialyl-Lewis X is proposed as shown in Fig. 7B. beta 4GalT-IV is extensively expressed in human tissues as shown in Fig. 6, and there seems to be no relationship between expression profiles of 6-sulfosialyl-Lewis X and beta 4GalT-IV. This indicates that beta 4GalT-IV is not a rate-limiting enzyme for biosynthesis of 6-sulfosialyl-Lewis X and that the preceding 6-O-sulfation of GlcNAc may be a rate-limiting step. The next subject of study should be whether 6-sulfosialyl-Lewis X is synthesized in cultured cells defective in beta 4GalT-IV expression.

beta 4GalT-IV is expressed in various human tissues including lymph node as shown in Fig. 6. This result indicates that the 6-O-sulfated N-acetyllactosamine structure occurs extensively. In fact, this sulfated glycan moiety has been shown to be present in various glycoproteins including ovomucin (65), glycoproteins derived from several culture cells (66), thyroglobulin (67), zona pellucida glycoproteins (68, 69), gp120 of human immunodeficiency virus type 1 (70), respiratory mucins (71, 72), carcinoembryonic antigen (73), hyosophorin (74), and oviducal mucins (75). 6SGN-specific beta 4GalT-IV identified in this study may be useful to elucidate the biological roles of these sulfated moieties.

    FOOTNOTES

* 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.

Present address: High Throughput Factory, RIKEN Harima Institute, 1-1-1, Kouto, Mikazukichou, Sayo-gun, Hyogo 679-5148, Japan.

|| To whom correspondence should be addressed: Dept. of Biochemistry, Sasaki Institute, Kanda-Surugadai 2-2, Chiyoda-ku, Tokyo 101-0062, Japan. Tel.: 81-3-3294-3286; Fax: 81-3-3294-2656; E-mail: yamashita@sasaki.or.jp.

Published, JBC Papers in Press, January 2, 2003, DOI 10/.1074/jbc.M211480200

    ABBREVIATIONS

The abbreviations used are: GlcNAc6ST, GlcNAc 6-O-sulfotransferase; KS, keratan sulfate; agL2L2, SO<UP><SUB>3</SUB><SUP>−</SUP></UP>right-arrow6GlcNAcbeta 1right-arrow3Galbeta 1right-arrow 4(SO<UP><SUB>3</SUB><SUP>−</SUP></UP>right-arrow6)GlcNAc; agL2L4, SO<UP><SUB>3</SUB><SUP>−</SUP></UP>right-arrow6GlcNAcbeta 1right-arrow3(SO<UP><SUB>3</SUB><SUP>−</SUP></UP>right-arrow6)Galbeta 1right-arrow 4(SO<UP><SUB>3</SUB><SUP>−</SUP></UP>right-arrow6)GlcNAc; agLST-b, Neu5Acalpha 2right-arrow6GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4Glc; beta 4GalT, beta 1,4-galactosyltransferase; biGP, GlcNAcbeta 1right-arrow2Manalpha 1right-arrow 3(GlcNAcbeta 1right-arrow2Manalpha 1right-arrow6)Manbeta 1right-arrow4GlcNAcbeta 1right-arrow4GlcNAc; core 2, Galbeta 1right-arrow3(GlcNAcbeta 1right-arrow6)GalNAcalpha 1right-arrow; core 3, GlcNAcbeta 1right-arrow 3GalNAcalpha 1right-arrow; GL, GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4Glc; GlcCer, glucosylceramide; L2L2, Galbeta 1right-arrow4(SO<UP><SUB>3</SUB><SUP>−</SUP></UP>right-arrow6)GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4(SO<UP><SUB>3</SUB><SUP>−</SUP></UP>right-arrow6)GlcNAc; L2L4, Galbeta 1right-arrow4(SO<UP><SUB>3</SUB><SUP>−</SUP></UP>right-arrow6)GlcNAcbeta 1right-arrow3(SO<UP><SUB>3</SUB><SUP>−</SUP></UP>right-arrow6)Galbeta 1right-arrow4(SO<UP><SUB>3</SUB><SUP>−</SUP></UP>right-arrow6)GlcNAc; LST-b, Galbeta 1right-arrow3(Neu5Acalpha 2right-arrow6)GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4Glc; Man, mannose; Neu5Ac, N-acetylneuraminic acid; pNP, p-nitrophenyl; RCA-I, R. communis agglutinin-I; 6S-biGP, SO<UP><SUB>3</SUB><SUP>−</SUP></UP>right-arrow6GlcNAcbeta 1right-arrow 2Manalpha 1right-arrow3(Manalpha 1right-arrow6)Manbeta 1right-arrow4GlcNAcbeta 1right-arrow4(Fucalpha 1right-arrow6)GlcNAc; 6S-core2, Galbeta 1right-arrow3(SO<UP><SUB>3</SUB><SUP>−</SUP></UP>right-arrow6GlcNAcbeta 1right-arrow6)GalNAcalpha 1right-arrow; 6SGN, GlcNAc 6-O-sulfate; PAPS, adenosine 3'-phosphate 5'-phosphosulfate; C-MF, the membrane fraction derived from pcDNA3-transfected COS-7 cells.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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