Identification and Characterization of Three Novel beta 1,3-N-Acetylglucosaminyltransferases Structurally Related to the beta 1,3-Galactosyltransferase Family*

Norihiko ShiraishiDagger §, Ayumi NatsumeDagger §, Akira Togayachi||, Tetsuo EndoDagger , Tomohiro Akashima**, Yoji YamadaDagger , Nobuyuki ImaiDagger , Satoshi NakagawaDagger , Satoshi KoizumiDagger , Susumu SekineDagger , Hisashi Narimatsu, and Katsutoshi SasakiDagger DaggerDagger

From the Dagger  Tokyo Research Laboratories, Kyowa Hakko Kogyo Company, Limited, 3-6-6 Asahi-machi, Machida-shi, Tokyo 194-8533, the  Division of Cell Biology, Institute of Life Science, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577, the || Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, and the ** Laboratory of Animal Resources, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Aza-Yasaka, Abashiri-shi, Hokkaido 099-2422, Japan

Received for publication, June 2, 2000, and in revised form, September 28, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have isolated three types of cDNAs encoding novel beta 1,3-N-acetylglucosaminyltransferases (designated beta 3Gn-T2, -T3, and -T4) from human gastric mucosa and the neuroblastoma cell line SK-N-MC. These enzymes are predicted to be type 2 transmembrane proteins of 397, 372, and 378 amino acids, respectively. They share motifs conserved among members of the beta 1,3-galactosyltransferase family and a beta 1,3-N-acetylglucosaminyltransferase (designated beta 3Gn-T1), but show no structural similarity to another type of beta 1,3-N-acetylglucosaminyltransferase (iGnT). Each of the enzymes expressed by insect cells as a secreted protein fused to the FLAG peptide showed beta 1,3-N-acetylglucosaminyltransferase activity for type 2 oligosaccharides but not beta 1,3-galactosyltransferase activity. These enzymes exhibited different substrate specificity. Transfection of Namalwa KJM-1 cells with beta 3Gn-T2, -T3, or -T4 cDNA led to an increase in poly-N-acetyllactosamines recognized by an anti-i-antigen antibody or specific lectins. The expression profiles of these beta 3Gn-Ts were different among 35 human tissues. beta 3Gn-T2 was ubiquitously expressed, whereas expression of beta 3Gn-T3 and -T4 was relatively restricted. beta 3Gn-T3 was expressed in colon, jejunum, stomach, esophagus, placenta, and trachea. beta 3Gn-T4 was mainly expressed in brain. These results have revealed that several beta 1,3-N-acetylglucosaminyltransferases form a family with structural similarity to the beta 1,3-galactosyltransferase family. Considering the differences in substrate specificity and distribution, each beta 1,3-N-acetylglucosaminyltransferase may play different roles.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A family of human beta 1,3-galactosyltransferases (beta 3Gal-Ts)1 consisting of five members (beta 3Gal-T1, -T2, -T3, -T4, and -T5) was recently identified (1-4). The first beta 1,3-galactosyltransferase (beta 3Gal-T1), which catalyzes the formation of type 1 oligosaccharides, was isolated by us using an expression cloning approach (1). Expression patterns of beta 3Gal-T1 and type 1 oligosaccharides strongly suggested the existence of beta 3Gal-T1 homologs. For instance, type 1-derived oligosaccharides such as sialyl-Lea were known to be expressed in colon and pancreatic cancer cell lines, whereas expression of beta 3Gal-T1 was detected in brain, but not in cancer cells. Our early approach using Southern hybridization failed to detect the existence of beta 3Gal-T1 homologous genes. However, recent accumulation of nucleotide sequence information on human cDNAs and genes such as expressed sequence tags (ESTs) enabled us to search homologous genes that do not have high similarity as detected by hybridization, but show significant similarity. A homology search based on the nucleotide or amino acid sequence of beta 3Gal-T1 led to the isolation of beta 3Gal-T2, -T3, and -T4, indicating that beta 3Gal-Ts form a family (1-3).

beta 3Gal-T2 catalyzed a similar reaction, but showed different substrate specificity compared with beta 3Gal-T1. The activity of beta 3Gal-T3 has not been detected, whereas the corresponding mouse enzyme exhibits weak beta 3Gal-T activity for both GlcNAc and GalNAc (5). On the other hand, beta 3Gal-T4 transfers galactose to the GalNAc residue of asialo-GM2 or GM2 to catalyze the formation of asialo-GM1 or GM1, respectively (3). beta 3Gal-T4 may be a human homolog of rat GM1 and GD1 synthases (6) since these enzymes shows 79.4% identity at the amino acid level.

A PCR cloning approach using degenerate primers corresponding to conserved regions in the beta 3Gal-T family has enabled us to isolate a fifth member (beta 3Gal-T5) of this family, which catalyzes the synthesis of type 1 oligosaccharides and is the most probable candidate involved in the biosynthesis of a cancer-associated sugar antigen, sialyl-Lea, in gastrointestinal and pancreatic cancer cells (4).

Very interestingly, a beta 1,3-N-acetylglucosaminyltransferase (designated beta 3Gn-T1) has been recently isolated based on the structural similarity to the beta 3Gal-Ts (7). beta 3Gn-T1 shows significant overall similarity to beta 3Gal-Ts (15-19%) and shares motifs conserved among the beta 3Gal-Ts, but is structurally distinct from another type of beta 1,3-N-acetylglucosaminyltransferase (iGnT) that was isolated by expression cloning using an anti-i-antigen antibody (8). beta 3Gn-T1 exhibits beta 1,3-N-acetylglucosaminyltransferase activity instead of beta 1,3-galactosyltransferase activity. This result provides an exception that a glycosyltransferase structurally related to the beta 3Gal-T family uses distinct donor (GlcNAc versus Gal) and acceptor (Gal versus GlcNAc) substrates, maintaining the same linkage specificity (beta 1,3-linkage).

During the course of study to isolate beta 3Gal-T1 homologs, we have identified three additional types of putative members of the beta 3Gal-T family. In this study, we show additional examples that glycosyltransferases structurally related to the beta 3Gal-T family exhibit beta 1,3-N-acetylglucosaminyltransferase activity, but not beta 1,3-galactosyltransferase activity. These results indicate that beta 1,3-N-acetylglucosaminyltransferases (beta 3Gn-Ts) form a family having structural similarity to the beta 3Gal-T family. Alignment of primary sequences of all members of the beta 3Gn-T and beta 3Gal-T families revealed that the members are clustered into four subgroups, probably reflecting enzymatic activity and substrate specificity. Transfection experiments and in vitro enzymatic analysis have demonstrated that beta 3Gn-T2, -T3, and -T4 are able to catalyze the initiation and elongation of poly-N-acetyllactosamine sugar chains; however, they exhibit different substrate specificity. These results, taken together with the different distributions of these enzymes, indicate that beta 3Gn-T2, -T3, and -T4 each exert distinct roles in physiological and pathological processes.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Nomenclature of beta 1,3-Galactosyltransferases and beta 1,3-N-Acetylglucosaminyltransferases-- To simplify discussion, five members of the cloned human beta 3Gal-Ts will be called beta 3Gal-T1, -T2, -T3, -T4, and -T5 according to the designation of Kolbinger et al. (2), Clausen and co-workers (3), and Narimatsu and co-workers (4). Five types of beta 3Gn-Ts cloned to date will be referred to tentatively as follows. A beta 3Gn-T and newly isolated beta 3Gn-Ts in this study, which show structural similarity to the beta 3Gal-T family, will be called beta 3Gn-T1, -T2, -T3, and -T4. Another type of beta 3Gn-T, which was isolated by expression cloning using anti-i-antigen antibody (8) and showed no structural similarity to the beta 3Gal-T family, will be called iGnT according to Fukuda et al. (8).

Cell Lines-- Namalwa KJM-1, a subline of the human Burkitt lymphoma cell line Namalwa, was cultivated in serum-free RPMI 1640 medium as described (9, 10). Cell lines SK-N-MC and Colo205 were obtained from the American Type Culture Collection. These cell lines were cultured in RPMI 1640 medium containing 10% fetal calf serum. Sf9 and Sf21 insect cells were cultured at 27 °C in TNM-FH insect medium (Pharmingen) as described previously (11).

Preparation of cDNA Libraries and Single Strand cDNAs-- cDNA libraries of human gastric mucosa and human placenta were constructed as described previously (12). Single strand cDNAs were synthesized from total RNA prepared from the neuroblastoma cell line SK-N-MC.

Isolation of Human beta Gn-T2, -T3, and -T4 cDNAs-- EST fragments encoding amino acid sequences similar to the human beta 3Gal-T1 sequence were retrieved from the EST division of the GenBankTM/EBI Data Bank using the FrameSearch algorithm (Compugen) (Table I). The human beta 3Gn-T2 cDNA fragment (600 bp) was isolated from a human gastric mucosa cDNA library by PCR using primers 5'-CCGGACAGATTTAAAGACTTTCTGC-3' and 5'-GTAGAGGCCAGAGTAAACAACTTCT-3'. The human beta 3Gn-T3 cDNA fragment (200 bp) was isolated from a human placenta cDNA library by PCR using primers 5'-CGTGGGGCAACTGATCCAAAACG-3' and 5'-ACCCAGGAAGACATCATCAATGGG-3'. The PCR conditions were 99 °C for 10 min; followed by 30 cycles of 95 °C for 1 min, 65 °C for 1 min, and 72 °C for 2 min; and 72 °C for 10 min of incubation. The beta 3Gn-T2 and -T3 cDNA fragments were cloned into pT7Blue (Novagen) and sequenced. Digoxigenin-labeled probes were prepared from the above-mentioned 600- and 200-bp fragments using a PCR DIG probe synthesis kit (Roche Molecular Biochemicals) and used to isolate the full-length cDNAs for beta 3Gn-T2 and -T3 by plaque hybridization. The full-length beta 3Gn-T2 cDNA (1.9 kb) was prepared from phage DNA by SalI and XbaI digestion and subcloned into pBluescript II SK(+) to yield pBS-beta 3Gn T2. A plasmid (pBS-beta 3Gn T3) containing the full-length beta 3Gn T3 cDNA in pBluescript SK(-) was recovered by in vivo excision. The full-length beta 3Gn T4 cDNA (1.3 kb) was amplified from single strand cDNAs derived from the neuroblastoma cell line SK-N-MC by PCR using primers 5'-CACAGCCTGAGACTCATCTCGCT-3' and 5'-AGGCATCAATTTCGCATCACGATAG-3' and was inserted into the pT7Blue-T vector to make pT7B-beta 3Gn T4.


                              
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Table I
List of the ESTs used to isolate beta 3Gn-T2, -T3, and -T4 cDNAs
GenBankTM/EBI accession numbers of EST clones are indicated according to the direction of the nucleotide sequences.

DNA Sequencing-- DNA sequences were determined by the dideoxynucleotide chain termination method using an ABI PRISMTM 377 DNA sequencer (Applied Biosystems, Inc.).

Construction of Plasmids for Expressing beta 3Gn T2, -T3, and -T4 in Animal Cells-- A beta 3Gn T2 cDNA fragment (prepared from pBS-beta 3Gn T2 by SalI and XbaI digestion, followed by blunting and addition of the SfiI linker (5'-CTTTAGAGCA-3' and 5'-CTCTAAAG-3')) was inserted between the SfiI sites of pAMo to yield pAMo-beta 3Gn T2. A beta 3Gn T3 cDNA fragment prepared from pBS-beta 3Gn T3 by HindIII and NotI digestion was inserted between the HindIII and NotI sites of pAMo to yield pAMo-beta 3Gn T3. A beta 3Gn T4 cDNA fragment (prepared from pT7B-beta 3Gn T4 by SmaI and HincII digestion, followed by addition of the SfiI linker) was inserted between the SfiI sites of pAMo to yield pAMo-beta 3Gn T4.

Expression of beta 3Gn T2, -T3, and -T4 in Namalwa KJM-1 Cells-- Namalwa KJM-1 cells were transfected with pAMo-beta 3Gn T2, pAMo-beta 3Gn T3, or pAMo-beta 3Gn T4 by electroporation as described (9, 10) and grown for 24 h. Stably transfected cells were selected by cultivation for >14 days in the presence of G418 (0.5 mg/ml).

Flow Cytometric Analysis-- Transfected Namalwa KJM-1 cells (5 × 106 cells) were incubated in 100 µl of phosphate-buffered saline for 60 min at 37 °C in the presence or absence of 20 milliunits of Clostridium perfringens neuraminidase (N2133, Sigma). These cells were stained with human anti-i-antigen serum (Den) (13), followed by fluorescein isothiocyanate-conjugated goat anti-human IgM, and were analyzed on a FACSCalibur apparatus (Becton Dickinson) as described (8). For lectin staining, cells were stained with 10 µg/ml fluorescein isothiocyanate-labeled Lycopersicon esculentum pokeweed mitogen (LEA) or agglutinin (PWM; both from EY Laboratories).

Construction and Purification of beta 3Gn-T Proteins Fused to the FLAG Peptide-- The putative catalytic domain of each beta 3Gn-T2, -T3, and -T4 was expressed as a secreted protein fused to the FLAG peptide in insect cells. A 1.1-kb DNA fragment encoding a COOH-terminal portion of beta 3Gn-T2 (amino acids 31-397) was amplified by PCR using primers 5'-CACGGATCCAGCCAAGAAAAAAATGGAAAAGGGGA-3' and 5'-ATCCGATAGCGGCCGCTTAGCATTTTAAATGAGCACTCTGCAAC-3', digested with BamHI and NotI, and inserted between the BamHI and NotI sites of pVL1393-F2 to yield pVL1393-F2G2. pVL1393-F2 is an expression vector derived from pVL1393 (Pharmingen) and contains a fragment encoding the signal peptide of human immunoglobulin kappa  (MHFQVQIFSFLLISASVIMSRG) and the FLAG peptide (DYKDDDDK). Joining in-frame a cDNA fragment with a unique BamHI site of pVL1393-F2 just downstream of the COOH terminus of the FLAG peptide enables the cDNA product to be secreted as a protein fused to the FLAG peptide. A 1.0-kb DNA fragment encoding a COOH-terminal portion of beta 3Gn-T3 (amino acids 38-372) was amplified by PCR using primers 5'-CGCGGATCCTCCCCACGGTCCGTGGACCAG-3' and 5'-ATAGTTTAGCGGCCGCGGAAGGGCTCAGCAGCGTCG-3', digested with BamHI and NotI, and inserted between the BamHI and NotI sites of pVL1393-F2 to yield pVL1393-F2G3. A 0.9-kb DNA fragment encoding a COOH-terminal portion of beta 3Gn-T4 (amino acids 56-378) was amplified by PCR using primers 5'-ATAAGATCTGCAGGAGACCCCACGGCCCACC-3' and 5'-ATAGTTATGCGGCCGCCTCAGGCTGTTGCCCAACCCAC-3', digested with BglII and NotI, inserted between the BamHI and NotI sites of pVL1393-F2 to yield pVL1393-F2G4. The PCR-amplified portions of pVL1393-F2G2, pVL1393-F2G3, and pVL1393-F2G4 were sequenced to confirm the absence of possible PCR errors.

Sf9 insect cells were cotransfected with BaculoGold viral DNA (Pharmingen) according to the manufacturer's instruction and each of plasmids pVL1393-F2G2, pVL1393-F2G3, and pVL1393-F2G4 and were incubated for 3 days at 27 °C to produce individual recombinant viruses. These viruses were amplified three times to reach titers of ~109 plaque-forming units/ml. Sf21 insect cells (4 × 107 cells; Pharmingen) were infected at a multiplicity of 10 and incubated in 30 ml of TNM-FH insect medium at 27 °C for 72 h to yield conditioned medium including recombinant beta 3Gn-T proteins fused to the FLAG peptide, which were readily purified by anti-FLAG M1 antibody resin (Sigma) according to the protocol of the manufacturer. Briefly, the culture medium (30 ml) was collected by centrifugation and added to NaCl (150 mM final concentration), NaN3 (0.1% final concentration), and M1 antibody resin (30 µl) to adsorb the recombinant beta 3Gn-T proteins on the resin. The resin was recovered by centrifugation and washed three times with buffer (1 ml) consisting of 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 1 mM CaCl2. The recombinant beta 3Gn-T proteins were eluted with buffer (90 µl) consisting of 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 2 mM EDTA, followed by the addition of CaCl2 (4 mM final concentration), and stored at 4 °C until use. The amount of the purified proteins was not enough for accurate quantification.

Silver Staining and Western Blot Analysis-- The enzymes purified above (3 µl) were subjected to SDS-polyacrylamide gel electrophoresis, followed by silver staining or Western blot analysis. Silver staining was performed using a silver staining kit (Wako Bioproducts). Proteins separated on 6% SDS-polyacrylamide gel were transferred to an polyvinylidene difluoride membrane (Immobilon, Millipore Corp.) in a Trans-Blot SD cell (Bio-Rad). The membrane was blocked with phosphate-buffered saline containing 5% skim milk at 4 °C overnight and then incubated with 10 µg/ml M2 antibody (Sigma). The membrane was stained with ECL Western blot detection reagents (Amersham Pharmacia Biotech) according to the manufacturer's instructions.

Glycosyltransferase Assays and Product Characterization-- The N-acetylglucosaminyltransferase activities of the purified proteins (15 µl) were assayed in 50 mM MOPS (pH 7.5), 5 mM MgCl2, 5 mM UDP-GlcNAc, and 10 mM unlabeled acceptors (a total volume of 40 µl). The following oligosaccharides were used as acceptors: lactose (Galbeta 1-4Glc), N-acetyllactosamine (Galbeta 1-4GlcNAc), lacto-N-neotetraose (LNnT; Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4Glc), p-lacto-N-neohexaose (p-LNnH; Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4Glc), and lacto-N-tetraose (LNT; Galbeta 1-3GlcNAcbeta 1-3Galbeta 1-4Glc). LNnT and LNT were purchased from Oxford Glycosystems. Lactose and p-LNnH were purchased from Sigma. Parallel reactions were done in the absence of UDP-GlcNAc to identify products. After incubation at 37 °C for the appropriate times (2 h for beta 3Gn-T2 and 16 h for beta 3Gn-T3 and -T4), the reactions were terminated by boiling. After centrifugation, the reaction mixtures were analyzed by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAE/PAD, Dionex Corp.). CarboPac PA10 was used as a column, and elution was performed with a gradient of 40-125 mM NaOH in 30 min at a flow rate of 1 ml/min. The structures of the reaction products derived from lactose and LNnT were confirmed by comparison of their retention times on HPLC with those of the standard oligosaccharides GlcNAcbeta 1-3Galbeta 1-4Glc and GlcNAcbeta 1-3Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4Glc, which were prepared from LNnT and p-LNnH by digestion with jack bean beta -galactosidase.

To further confirm the structure of the reaction product derived from lactose, it was digested with endo-beta -galactosidase or modified by beta 1,4-galactosyltransferase. To avoid the inhibitory effect of MOPS on endo-beta -galactosidase, an N-acetylglucosaminyltransferase assay was done without MOPS using lactose as an acceptor. The reaction was stopped by boiling, and the reaction mixture was recovered by centrifugation. The reaction mixture (15 µl) was added to acetate buffer (50 mM final concentration; pH 5.8) and incubated with 250 milliunits/ml Escherichia freundii endo-beta -galactosidase (Seikagaku Kogyo) (14) in a total volume of 41 µl at 37 °C for 16 h, followed by analysis with HPAE/PAD as described above. Alternatively, the reaction mixture (15 µl) was added to Tris-HCl (50 mM final concentration; pH 8.0) and incubated with 750 milliunits/ml bovine milk beta 1,4-galactosyltransferase (Sigma) in a total volume of 40 µl at 37 °C for 16 h, followed by analysis with HPAE/PAD as described above.

Endo-beta -galactosidase digestion of the product yielded two peaks that comigrated with the standard oligosaccharides GlcNAcbeta 1-3Galbeta and glucose at a 1:1 molar ratio. These results clearly indicated that the product derived from lactose was GlcNAcbeta 1-3Galbeta 1-4Glc.

Since the amount of the purified proteins was not enough for accurate quantification, enzymatic activity is defined as picomoles of acceptor substrate N-acetylglucosaminylated per ml of culture medium/h. The amounts of reaction products were determined from their absorbance intensities using individual standards.

Alternatively, the N-acetylglucosaminyltransferase activities of the purified proteins (15 µl) were assayed in 200 mM MOPS (pH 7.5), 20 mM MgCl2, 20 mM UDP-GlcNAc, and 50 µM pyridylaminated acceptors (total volume of 30 µl). As acceptors, the following pyridylaminated oligosaccharides were used: LNnT, lacto-N-fucosylpentaose (LNFP) III (Galbeta 1-4(Fucalpha 1-3)GlcNAcbeta 1-3Galbeta 1-4Glc), LNT, LNFP-II (Galbeta 1-3(Fucalpha 1-4)GlcNAcbeta 1-3Galbeta 1-4Glc), LNFP-V (Galbeta 1-3GlcNAcbeta 1-3Galbeta 1-4(Fucalpha 1-3)Glc), and lacto-N-difucosylhexaose II (Galbeta 1-3(Fucalpha 1-4)GlcNAcbeta 1-3Galbeta 1-4(Fucalpha 1-3)Glc). After incubation at 37 °C for the appropriate times (2 h for beta 3Gn-T2 and 15 h for beta 3Gn-T3 and -T4), the reactions were terminated by boiling and analyzed by HPLC as described previously, with exception that HPLC was performed at 50 °C with a flow rate of 0.5 ml/min (9, 10). Parallel reactions were done in the absence of UDP-GlcNAc to identify products and to check hydrolysis of substrate and product. The oligosaccharides were purchased from Oxford Glycosystems and pyridylaminated according to the method of Kondo et al. (15). The amounts of products were determined from their fluorescence intensities using pyridylaminated lactose as a standard.

The reaction product derived from pyridylaminated LNnT was identified by comparison of the retention time on HPLC with that of the pyridylaminated standard oligosaccharide GlcNAcbeta 1-3Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4Glc, which was prepared from pyridylaminated p-LNnH by digestion with jack bean beta -galactosidase. To further confirm the structure of the reaction product, the reaction product was modified by beta 1,4-galactosyltransferase to examine whether p-LNnH was produced or not. The reaction mixtures (20 µl) was incubated with 20 milliunits of bovine milk beta 1,4-galactosyltransferase in the presence or absence of UDP-Gal (20 mM) in a total volume of 30 µl at 37 °C for 15 h according to manufacturer's recommendations. The product further modified by beta 1,4-galactosyltransferase comigrated with pyridylaminated p-LNnH on HPLC. These results indicated that the product was pyridylaminated GlcNAcbeta 1-3Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4Glc. The galactosyltransferase activities of the purified proteins (15 µl) were assayed using pyridylaminated oligosaccharides (GlcNAcbeta 1-3Galbeta 1-4Glc and LNnT) as substrates as described previously (4).

Preparation and Fractionation of Blood Leukocytes-- Human polymorphonuclear leukocytes, monocyte-enriched population, and lymphocyte-enriched population were obtained as described previously (10).

Quantitative Analysis of the Three beta 3Gn-T Transcripts in Human Tumor Cell Lines and Human Tissues by Competitive RT-PCR-- The levels of the beta 3Gn-T2, -T3, and -T4 transcripts were measured by competitive RT-PCR as described in detail previously (4, 16). Competitor DNA plasmids carrying a small deletion within the respective cDNA were constructed by appropriate restriction endonuclease digestion as shown in Table II. For instance, a competitor DNA plasmid for measuring beta 3Gn-T2 transcripts was prepared by deleting the 227-bp Eco81I-PflMI fragment in beta 3Gn-T2 cDNA from the standard DNA plasmid pBS-beta 3Gn T2.


                              
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Table II
Oligonucleotide primers and conditions used for competitive RT-PCR analysis

Single strand cDNAs were synthesized with an oligo(dT) primer from 6 µg of DNase I-treated total RNA from human tissues (colon, jejunum, stomach body, stomach antrum, and esophagus) and cell lines (HL-60 and Colo205) as described previously (4). Single strand cDNAs from human leukocytes were prepared as described (10). In addition, single strand cDNAs were synthesized with an oligo(dT) primer from 1 µg of poly(A)+ RNAs from 35 human tissues (CLONTECH) using a SuperscriptTM pre-amplification system for first strand cDNA synthesis (Life Technologies, Inc.) according to manufacturer's instructions. After cDNA synthesis, the reaction mixture was diluted 50-fold with H2O and then stored at -80 °C until use.

Competitive RT-PCR was performed with AmpliTaq GoldTM (PerkinElmer Life Sciences). The annealing temperatures and specific primers used are listed in Table II. The amount of each of the beta 3Gn-T transcripts was normalized by the amount of beta -actin transcripts (4, 16).

Determination of Chromosomal Localization-- The chromosomal localizations of the beta 3Gn-T2, -T3, and -T4 genes were determined using 3'-EST mapping data (NCBI Protein Database). The chromosomal localization of the beta 3Gal-T1 gene was determined by PCR analysis using a series of genomic DNAs from hamster-human somatic hybrids (BIOSMAPTM Somatic Cell Hybrid PCRableTM DNAs, BIOS Laboratories) and specific primers (5'-TTCAGCCACCTAACAGTTGCCAGG-3' and 5'-ATACCTTCTTCGTGGCTTGGTGGAG-3'). The predicted fragment of 495 bp was amplified only when genomic DNA from a hybrid containing human chromosome 2 (hybrid 852) was used, indicating that this gene is located on chromosome 2.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification and Isolation of beta 3Gn-T2, -T3, and -T4-- A homology search in the EST division of the GenBankTM/EBI Data Bank using the FrameSearch algorithm revealed the existence of six types of cDNAs encoding proteins with low but significant similarity to beta 3Gal-T1, three of which have been reported recently to be beta 3Gal-T2, -T3, and -T4 (2, 3). Based on the nucleotide sequence of the ESTs shown in Table I, we prepared specific probes and isolated three types of full-length cDNAs encoding novel proteins (designated beta 3Gn-T2, -T3, and -T4) of 397, 372, and 378 amino acid residues, respectively, with structural similarity to beta 3Gal-T1. beta 3Gn-T2 and -T3 cDNAs were obtained from human gastric mucosa, and beta 3Gn-T4 cDNA was from the neuroblastoma cell line SK-N-MC.

A Kyte-Doolittle hydropathy analysis (17) revealed that beta 3Gn-T2, -T3, and -T4 show type 2 transmembrane topology typical of most glycosyltransferases. beta 3Gn-T2 is predicted to consist of an N-terminal cytoplasmic domain of 9 residues, a transmembrane segment of 19 residues, and a stem region and catalytic domain of 369 residues. beta 3Gn-T3 is predicted to consist of an N-terminal cytoplasmic domain of 11 residues, a transmembrane segment of 21 residues, and a stem region and catalytic domain of 340 residues. The predicted coding region of beta 3Gn-T4 has two potential initiation codons, both of which are in agreement with Kozak's rule (18). Therefore, it is predicted that beta 3Gn-T4 is composed of two different N-terminal cytoplasmic domains of 29 and 4 residues, a transmembrane segment of 20 residues, and 329 residues containing the stem region and catalytic domain (Fig. 1a).



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Fig. 1.   Comparison of novel beta 3Gn-Ts with beta 3Gn-T1 and the beta 3Gal-T family. A, multiple sequence analysis (ClustalX) of the beta 3Gn-T family (beta 3Gn-T1, -T2, -T3, and -T4) and the beta 3Gal-T family (beta 3Gal-T1, -T2, -T3, -T4, and -T5). Introduced gaps are shown by hyphens. The putative transmembrane domains are underlined. Asterisks indicate identical amino acids in all proteins. Conserved amino acids are shown by colons. Cysteine residues conserved in all the proteins or subgroups are shaded. The white arrows indicate cysteine residues conserved in all the proteins except for beta 3Gn-T1. The black arrow indicates the conserved possible N-glycosylation site. B, phylogenetic tree of the beta 3Gn-T and beta 3Gal-T families. The phylogenetic tree was produced with ClustalX presented in A using amino acid sequences of the predicted catalytic domains. The chromosomal localizations of the respective genes, except for the beta 3Gn-T1 gene, are indicated in parentheses (Refs. 4 and 49 and this study).

Fig. 1A shows a multiple alignment of the amino acid sequences of beta 3Gn-T2, -T3, and -T4 as well as beta 3Gn-T1 and five members of the beta 3Gal-T family. beta 3Gn-T2, -T3, and -T4 show 19-24, 22-26, and 22-25% identities, respectively, to the beta 3Gal-T family (beta 3Gal-T1, -T2, -T3, -T5, and -T5), whereas they show 15, 18, and 15% identities to beta 3Gn-T1. beta 3Gn-T2, -T3, and -T4 show 40-45% identity one another. The sequence similarities are limited to the putative catalytic regions. Several sequence motifs conserved in the beta 3Gal-T family are also shared by beta 3Gn-T2, -T3, and -T4 as well as beta 3Gn-T1. Twenty-five amino acid residues located separately in the putative catalytic regions are identical among all the proteins. Three cysteine residues conserved in all members of the beta 3Gal-T family are also maintained in beta 3Gn-T2, -T3, and -T4, whereas two of these are not conserved in beta 3Gn-T1 (Fig. 1A, white arrows), indicating that beta 3Gn-T1 is relatively distinct from other members, especially in the context of the three-dimensional structure. There are five potential N-linked glycosylation sites in beta 3Gn-T2, three in beta 3Gn-T3, and three in beta 3Gal-T4. One site in a highly conserved motif is maintained among all the proteins (Fig. 1A, black arrow). The phylogenetic tree of these proteins generated using the amino acid sequences of the putative catalytic domains demonstrates that beta 3Gn-T2, -T3, and -T4 form a subgroup, indicating that they have similar enzymatic activity (Fig. 1B).

Production of Secreted Recombinant Proteins Fused to the FLAG Peptide-- To examine the enzymatic activities of beta 3Gn-T2, -T3, and -T4, we expressed the putative catalytic domain of each enzyme (amino acids 31-397 of beta 3Gn-T2, amino acids 38-372 of beta 3Gn-T3, and amino acids 56-378 of beta 3Gn-T4) as a secreted protein fused to the FLAG peptide in Sf21 insect cells. The FLAG-fused recombinant proteins were partially purified using anti-FLAG M1 antibody resin and analyzed by SDS-polyacrylamide gel electrophoresis, followed by silver staining (Fig. 2A) or Western blotting using anti-FLAG monoclonal antibody (Fig. 2B). Two major bands with apparent molecular masses of 45.5 and 48 kDa, broad bands of 42-45 kDa, and two major bands of 37.6 and 40 kDa were observed specifically for beta 3Gn-T2, -T3, and -T4, respectively. The FLAG-fused recombinant proteins for beta 3Gn-T2, -T3, and -T4 have predicted molecular masses of 43,674, 39,507, and 37,608 Da for the respective polypeptides, indicating glycosylation of the recombinant proteins produced by insect cells.



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Fig. 2.   SDS-polyacrylamide gel electrophoresis analysis of the secreted recombinant enzymes. The FLAG-fused secreted proteins for beta 3Gal-T2 (lanes 1), -T3 (lanes 2), and -T3 (lanes 3) were purified by anti-FLAG antibody resin and subjected to SDS-polyacrylamide gel electrophoresis, followed by silver staining (A) or Western blotting using anti-FLAG antibody M2 (B).

beta 3Gn-T2, -T3, and -T4 Are beta 1,3-N-Acetylglucosaminyltransferases-- The glycosyltransferase activities of the partially purified FLAG-fused recombinant proteins were examined. When lactose was used as an acceptor, beta 3Gn-T2, -T3, and -T4 showed a significant amount of N-acetylglucosaminyltransferase activity, whereas no activity was detected in a sample prepared from the conditioned medium of insect cells infected with empty vector virus. The structure of the product was estimated to be GlcNAcbeta 1-3Galbeta 1-4Glc by comparing the retention time on HPLC with that of the standard oligosaccharide (Fig. 3A). To further confirm the structure of the product, it was digested with endo-beta -galactosidase or modified by beta 1,4-galactosyltransferase. Digestion of the product by E. freundii endo-beta -galactosidase yielded two peaks comigrating with the standard oligosaccharides GlcNAcbeta 1-3Gal and glucose at a 1:1 molar ratio (Fig. 3, compare A and B). Modification of the product by bovine milk beta 1,4-galactosyltransferase yielded a peak comigrating with LNnT (Fig. 3, compare A and C). These results clearly indicated that the product was GlcNAcbeta 1-3Galbeta 1-4Glc.



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Fig. 3.   HPLC analysis of the reaction product generated from lactose by recombinant beta 3Gn-T2. A, the N-acetylglucosaminyltransferase activity of the purified beta 3Gn-T2 protein was assayed using lactose (Galbeta 1-4Glc) as an acceptor. The reaction mixture was analyzed using high-pH anion-exchange chromatography with pulsed amperometric detection. The peaks for substrate lactose and the generated product are labeled S and P, respectively. Arrow 1 indicates the elution position of the standard oligosaccharide GlcNAcbeta 1-3Galbeta 1-4Glc. Based on the elution position, the peak indicated by the asterisk seems to be GlcNAc, which may be a degradation product of UDP-GlcNAc. The peak with a retention time of 2-3 min may be glycerol, which appeared in the absence of UDP-GlcNAc. B, the reaction mixture described in A was analyzed after digestion with endo-beta -galactosidase. Arrows 2 and 3 indicate the elution positions of the standard oligosaccharides GlcNAcbeta 1-3Gal and glucose, respectively. C, the reaction mixture described in A was analyzed after galactosylation with beta 1,4-galactosyltransferase. Arrow 4 indicates the elution position of the standard oligosaccharide LNnT (Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4Glc). Based on the elution position, the peak indicated by the double asterisks seems to be Galbeta 1-4GlcNAc, which would be a galactosylation product of GlcNAc indicated by the asterisk in A.

On the other hand, beta 3Gn-T2, -T3, and -T4 showed no beta 1,3-galactosyltransferase activity for GlcNAcbeta 1-3Galbeta 1-4Glc or LNnT. Taken together, beta 3Gn-T2, -T3, and -T4 were demonstrated to be novel beta 1,3-N-acetylglucosaminyltransferases.

Substrate Specificity of beta 3Gn-T2, -T3, and -T4-- Analysis of the substrate specificity of beta 3Gn-T2, -T3, and -T4 revealed that these enzymes utilized common oligosaccharides as substrates, but the substrate preference was significantly different (Tables III and IV). beta 3Gn-T2 and -T4 showed more preferential activity for LNnT than for LNT, which is consistent with the nature of beta 3Gn-T1 and iGnT (7, 8). In contrast, beta 3Gn-T3 utilized LNT as a substrate comparable to LNnT. In common, fucosylation at the penultimate GlcNAc residue in LNnT or LNT yielded poor substrates (LNFP-III, LNFP-II, and lacto-N-difucosylhexaose II). LNFP-V, which is an LNT derivative fucosylated at the reducing terminal glucose residue, was a relatively good substrate for beta 3Gn-T2 compared with LNT.


                              
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Table III
Analysis of substrate specificity of beta 3Gn-T2, -T3, and -T4 using pyridylaminated oligosaccharides as substrates
Assay conditions were described under "Experimental Procedures." Activities are expressed as a percentage of the activity for pyridylaminated LNnT. The activities of beta 3Gn-T2, -T3, and -T4 for pyridylaminated LNnT were 330, 7.1, and 1.9 pmol/ml of medium/h, respectively. LNDFH-II, lacto-N-difucosylhexaose II.


                              
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Table IV
Analysis of substrate specificity of beta 3Gn-T2, -T3, and -T4 using unlabeled oligosaccharides as substrates
Assay conditions were described under "Experimental Procedures." Activities are expressed as a percentage of the activity for LNnT. The activities of beta 3Gn-T2, -T3, and -T4 for LNnT were 4900, 230, and 420 pmol/ml of medium/h, respectively. The activity of beta 3Gn-T1 was quoted from Ref. 7. LacNAc, N-acetyllactosamine; ND, not determined.

beta 3Gn-T2 transferred GlcNAc efficiently to both lactose and p-LNnH, as well as LNnT, whereas the relative activity N-acetyllactosamine was 21% compared with that of LNnT. beta 3Gn-T3 preferred lactose (235% relative activity) as a substrate, followed by LNnT (100%) and p-LNnH (45%), whereas it showed no activity for N-acetyllactosamine. beta 3Gn-T4 showed 33, 9, and 0% relative activities for lactose, N-acetyllactosamine, and p-LNnH, respectively, compared with LNnT (100%). beta 3Gn-T1 was reported to efficiently utilize N-acetyllactosamine as well as lactose (Table III), showing a significant difference in substrate specificity compared with beta 3Gn-T2, -T3, and -T4.

beta 3Gn-T2, -T3, and -T4 Each Direct Biosynthesis of Poly-N-acetyllactosamines in Namalwa KJM-1 Cells-- To examine the in vivo enzymatic activities of beta 3Gn-T2, -T3, and -T4, Namalwa KJM-1 cells were transfected with pAMo-beta 3Gn T2, pAMo-beta 3Gn T3, pAMo-beta 3Gn T4, or the empty vector pAMo and examined for changes in the surface expression of the poly-N-acetyllactosamine sugar chains by flow cytometric analyses using the anti-i-antigen antibody (Den) and specific lectins (LEA and PWM).

Since Den, LEA, and PWM are likely to recognize non-sialylated poly-N-acetyllactosamines more preferentially than sialylated ones, the transfected cells were treated with neuraminidase before staining. As shown in Fig. 4, expression of beta 3Gn-T2, -T3, or -T4 increased the levels of poly-N-acetyllactosamines recognized by Den, LEA, or PWM compared with the vector (pAMo) transfectant, consistent with in vitro enzymatic activity. In particular, expression of beta 3Gn-T3 or -T4 led to a remarkable increase in reactivity to Den, in contrast to the slight increase in the beta 3Gn-T2 transfectant. On the other hand, reactivity to LEA or PWM was increased in the beta 3Gn-T2 transfectant more clearly than in the other two transfectants. These results indicate that beta 3Gn-T2, -T3, and -T4 each are involved in the biosynthesis of poly-N-acetyllactosamine sugar chains in transfected cells.



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Fig. 4.   Flow cytometric analysis of Namalwa KJM-1 cells stably transfected with beta 3Gn-T2, -T3, or -T4 cDNA. Namalwa KJM-1 cells were stably transfected with plasmid pAMo-beta 3Gn T2, pAMo-beta 3Gn T3, or pAMo-beta 3Gn T4, which directs expression of beta 3Gn-T2, -T3, or -T4, respectively, or with the empty vector pAMo. These cells were stained with anti-i-antigen antiserum or poly-N-acetyllactosamine-recognizing lectins (LEA or PWM) and subjected to flow cytometric analysis as described under "Experimental Procedures" (thick lines). As controls, the transfectants were stained with phosphate-buffered saline (thin lines).

Expression Levels of the beta 3Gn-T2, -T3, and -T4 Transcripts-- The expression levels of the beta 3Gn-T2, -T3, and -T4 transcripts were examined by competitive RT-PCR. These genes were differentially expressed in human tissues and cells (Table V). beta 3Gn-T2 was ubiquitously expressed in the tissues and cells tested, but expression of beta 3Gn-T3 and -T4 was relatively restricted. beta 3Gn-T3 was expressed in colon, jejunum, stomach (body and antrum), esophagus, placenta, and trachea. beta 3Gn-T4 was mainly expressed in brain tissues such as whole brain, hippocampus, amygdala, cerebellum, and caudate nucleus, as well as in colon, esophagus, and kidney.


                              
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Table V
Quantitative analysis of transcripts of beta 3Gn-T2, -T3, and -T4 in various human tissues and cells by competitive RT-PCR

beta 1,3-N-Acetylglucosaminyltransferase activities were detected in several tissues, cells, and sera, some of which were characterized using partially purified enzymes (19-28). Poly-N-acetyllactosamines are known to serve as backbone oligosaccharides for presenting the sialyl-LeX and sialyl-Lea determinants, which function as selectin ligands in leukocytes and several cancer cells such as colon cancer cells (29-40). The human promyelocytic leukemia cell line HL-60 and the human colon adenocarcinoma cell line Colo205 are known to express beta 1,3-N-acetylglucosaminyltransferase activities as well as poly-N-acetyllactosamines presenting the sialyl-Lea and sialyl-LeX determinants. Therefore, it was of significant interest to examine the expression levels of beta 3Gn-T2, -T3, and -T4 in leukocytes and cancer cells such as HL-60 and Colo205. beta 3Gn-T2, but not beta 3Gn-T3 and -T4, was significantly expressed in HL-60 and Namalwa KJM-1 cells (Table V) as well as in human peripheral polymorphonuclear cells and lymphocytes (data not shown). On the other hand, beta 3Gn-T2 and -T3, but not beta 3Gn-T4, were highly expressed in the colon cancer cell line Colo205 (Table V).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, we identified three novel beta 1,3-N-acetylglucosaminyltransferases (beta 3Gn-T2, -T3, and -T4) that show structural similarity to beta 3Gn-T1 as well as the beta 3Gal-T family, including five members (beta 3Gal-T1, -T2, -T3, -T4, and T5), demonstrating the existence of a beta 3Gn-T family now consisting of four members (beta 3Gn-T1, -T2, -T3, and -T4). The existence of the multiple enzymes showing similar activity is a common feature of glycosyltransferases, which was demonstrated for Gal alpha 2,3-sialyltransferases I-VI (9, 41-47, 49-52), GalNAc alpha 2,6-sialyltransferases I-VI (53-58), NeuAc alpha 2,8-sialyltransferases I-V (59-69), alpha 1,3-fucosyltransferases III-VII and IX (10, 70-75, 77, 78)), core 2 beta 1,6-N-acetylglucosaminyltransferases 1-3 (79-82), large I beta 1,6-N-acetylglucosaminyltransferases (83), polypeptide N-acetylgalactosaminyltransferases (84-91), beta 1,4-galactosyltransferases 1-7 (92-100), and beta 1,3-galactosyltransferases 1-5 (1-4). Discovery of the beta 3Gn-T family in this study has clearly demonstrated a new feature of glycosyltransferases, that the beta 3Gn-T and beta 3Gal-T families show structural similarity despite of differences in both the transfer sugar (GlcNAc versus Gal) and the acceptor sugar (Gal versus GlcNAc).

We constructed the secreted recombinant proteins for beta 3Gn-T2, -T3, and -T4 fused to the FLAG peptide. Western blot analysis using anti-FLAG antibody revealed that the secreted enzymes were successfully produced by insect cells and were readily recovered by anti-FLAG M1 antibody resin. The FLAG-fused proteins adsorbed to the resin were eluted under mild conditions using buffer containing 2 mM EDTA. Since the eluted proteins showed activity comparative to that of the adsorbed proteins, it was confirmed that EDTA treatment did not damage the enzymes (data not shown). The molecular masses of the recovered proteins were equal to or larger than the predicted ones for their polypeptides, indicating some glycosylation and no significant degradation of the recovered proteins.

All of the recombinant proteins showed Gal beta 1,3-N-acetylgalactosaminyltransferase activity for common oligosaccharides, whereas their substrate preference was significantly different. Since the amount of the recombinant proteins used in this study was not enough for determination of the protein concentration, we could not precisely compare the relative activities of the enzymes. However, the relative activities of beta 3Gn-T2 for LNnT, lactose, Galbeta 1-4GlcNAc, and p-LNnH seemed to be higher than those of other enzymes (Tables III and IV). Considering the variety of acceptor substrates and the different reactivities of the transfected cells to anti-i-antigen antibody or PWM and LEA lectins, the higher activity of beta 3Gn-T2 for these oligosaccharides may reflect substrate specificity.

To date, beta 3Gn-T activities have been detected in several tissues, cells, and sera, some of which were characterized using partially purified enzymes (19-28). Based on the substrate specificity, beta 3Gn-T1, but not beta 3Gn-T2, -T3, and -T4, may correspond to a beta 3Gn-T partially purified from calf serum. beta 3Gn-T2 and -T4 showed more preferential activity for LNnT than for LNT, which was similar to the nature of the calf serum enzyme as well as beta 3Gn-T1 and iGnT (7, 8, 26); however, beta 3Gn-T2 and -T4 were distinguished from the calf serum enzyme and beta 3Gn-T1 by the activities for lactose (Galbeta 1-4Glc) and N-acetyllactosamine (Galbeta 1-4GlcNAc) (Table IV). On the other hand, beta 3Gn-T3 is quite unique since it showed activity for LNT comparable to LNnT. It has been reported that human colon cancer tissues and the colon cancer cell line Colo205 contain cancer-associated glycosphingolipids with dimeric Lea antigens (Galbeta 1-3(Fucalpha 1-4)GlcNAcbeta 1-3Galbeta 1-3(Fucalpha 1-4)GlcNAc) (101). Considering the substrate specificity and expression in colon tissues and Colo205 cells, beta 3Gn-T3 is likely to be the most probable candidate involved in the biosynthesis of the backbone structure of dimeric Lea (Galbeta 1-3GlcNAcbeta 1-3Galbeta 1-3GlcNAc). On the other hand, it is difficult to ascribe the beta 3Gn-T activities detected in crude samples to the isolated beta 3Gn-Ts (beta 3Gn-T1, -T2, -T3, and -T4 or iGnT) because of the following reasons: differences in experimental conditions such as substrates used, the possibility of the existence of multiple beta 3Gn-Ts in the crude samples, and the possibility of the existence of additional unidentified beta 3Gn-Ts.

Analysis of substrate specificity revealed that beta 3Gn-T2, -T3, and -T4 each could be involved in the initiation and elongation of poly-N-acetyllactosamine synthesis by itself, which was demonstrated by increased expression of poly-N-acetyllactosamines in the transfected cells. The different reactivities of the respective transfectants to the anti-i-antigen antibody or LEA and PWM lectins may reflect the preference of the antibody and lectins as well as substrate specificity of these enzymes. On the other hand, expression of two or more beta 3Gn-Ts in the same cell, which was clearly demonstrated for Colo205 cells, indicates that poly-N-acetyllactosamine sugar chains might be synthesized by the concerted action of multiple beta 3Gn-Ts. Since Namalwa KJM-1 cells express endogenous beta 3Gn-T2, at least two beta 3Gn-Ts may be involved in the biosynthesis of poly-N-acetyllactosamines in the beta 3Gn-T3 or beta 3Gn-T4 transfectants.

The phylogenetic analysis using the amino acid sequences of the putative catalytic domains indicated that the members of the beta 3Gal-T and beta 3Gn-T families are clustered into the following four subgroups: beta 3Gal-T1, -T2, -T3, and -T5; beta 3Gn-T2, -T3, and -T4; beta 3Gal-T4; and beta 3Gn-T1 (Fig. 1b). Whereas beta 3Gal-T1, -T2, -T3, and -T5 catalyze the formation of the Galbeta 1-3GlcNAc structure, beta 3Gal-T4 forms the Galbeta 1-3GalNAc structure, indicating that enzymatic activity may reflect structural similarity. beta 3Gn-T1 was reported to have beta 3Gn-T activity (7); however, beta 3Gn-T2, -T3, and -T4 resemble the beta 3Gal-T family rather than beta 3Gn-T1. In addition, beta 3Gn-T1 is structurally distinct from the other members of the beta 3Gn-T and beta 3Gal-T families because it does not have 2 of the 3 cysteine residues conserved by all the other members. There was no direct relationship between the subgroups and chromosomal localizations of the genes (Fig. 1B). beta 3Gn-T1, -T2, -T3, and -T4 exhibited no structural similarity to another type of beta 3Gn-T (named iGnT) that was isolated by expression cloning using the anti-i-antigen antibody, suggesting that the in vivo substrate specificity of iGnT might be quite different from that of other beta 3Gn-Ts.

In this study, we isolated three types of novel beta 3Gn-T genes, which enabled us to discriminate the respective enzymes at the molecular level. Considering the enzymatic activities in vitro and in vivo as well as the expression patterns of the beta 3Gn-Ts, the respective enzymes are likely to play different roles. The poly-N-acetyllactosamine or GlcNAcbeta 1-3Gal structure appears in glycolipids, keratan sulfate proteoglycans, and human milk oligosaccharides, in addition to N- and O-glycans of glycoproteins. Therefore, the existence of multiple beta 3Gn-Ts is not strange. It remains to be determined which beta 3Gn-T makes which types of sugar chains. Poly-N-acetyllactosamines are known to be synthesized at various positions by the concerted action of several glycosyltransferases required for the elongation or formation of specific sugar branches preferred by elongation enzymes. For example, poly-N-acetyllactosamines are preferentially formed in the specific branch in complex type N-glycans, which are formed by beta 1,6-N-acetylglucosaminyltransferase V (102). In addition, core 2 beta 1,6-N-acetylglucosaminyltransferases 1 and 2 and large I beta 1,6-N-acetylglucosaminyltransferases are branching enzymes critical for elongation with poly-N-acetyllactosamines (48, 80, 81, 103-105). Discovery of the multiple beta 3Gn-Ts in addition to other multiple glycosyltransferases involved in the biosynthesis of poly-N-acetyllactosamines (e.g. beta 1,4-galactosyltransferases, beta 3Gal-Ts, beta 1,6-N-acetylglucosaminyltransferase V, core 2 beta 1,6-N-acetylglucosaminyltransferases, and large I beta 1,6-N-acetylglucosaminyltransferases) indicates that regulation of poly-N-acetyllactosamine synthesis may be more complex than previously recognized. Definitive determination of the enzymatic activities and expression patterns of the beta 3Gn-Ts as well as experiments using knockout mice may provide insight into their functions in physiological and pathological processes.

Recently, Amado et al. (76) have reported the existence of four additional members of the beta 3Gal-T family, although their enzymatic activities were not determined. Based on the characteristics of the primary structures and chromosomal localizations, three of them may correspond to beta 3Gal-T2, -T3, and -T4. Additional members of the beta 3Gn-T and beta 3Gal-T families remain to be investigated.


    ACKNOWLEDGEMENTS

We are grateful to Dr. Minoru Fukuda (Burnham Institute) for the generous gift of the anti-i-antigen antiserum. We thank Drs. Shoko Nishihara and Takashi Kudo and Hiroko Iwasaki (Soka University) for great help in and discussion of this study. We also thank Sachiko Kodama for excellent technical assistance, Reiko Koda for DNA sequencing, and Mayumi Ibonai and Dr. Atsuhiro Hasegawa for providing the synthetic oligonucleotides. We thank Kazumi Kurata-Miura for advice and suggestions throughout this work.


    Note Added in Proof

Recently, Zhou et al. (7) have corrected the nucleotide sequence and deduced amino acid sequence of the beta 1,3-N-acetylglucosaminyltransferase (beta 3Gn-T1) cDNA that they had previously published (see "Corrections" in Proc. Natl. Acad. Sci. U.S.A. (2000) 97, 11673-11975). This developed from an unfortunate cDNA clone substitution in their laboratory. The corrected sequence of beta 3Gn-T1 was identical to that of beta 3Gn-T2. Consequently, besides iGnT, there are three types of beta 3Gn-Ts described to date, not four. The sequence of beta 3Gn-T1 used in this paper is that of the corrected, substituted cDNA clone. To try to prevent further confusion, we point out this fact but do not change the enzyme names used in this paper.


    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.

The nucleotide sequences reported in this paper have been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession numbers AB049584 (beta 3Gn-T2), AB049585 (beta 3Gn-T3), and AB049586 (beta 3Gn-T4).

§ These authors contributed equally to this work and should be considered as first authors.

Dagger Dagger To whom correspondence should be addressed. Tel.: 81-427-25-2555; Fax: 81-427-26-8330; E-mail: ksasaki@kyowa.co.jp.

Published, JBC Papers in Press, October 19, 2000, DOI 10.1074/jbc.M004800200


    ABBREVIATIONS

The abbreviations used are: beta 3Gal-Ts, UDP-galactose:beta -N-acetylglucosamine beta 1,3-galactosyltransferases; EST, expressed sequence tag; GM1, Galbeta 1-3GalNAcbeta 1-4(NeuAcalpha 2-3)Galbeta 1-4Glc-Cer; GM2, GalNAcbeta 1-4(NeuAcalpha 2-3)Galbeta 1-4Glc-Cer; GD1, Galbeta 1-3GalNAcbeta 1-4(NeuAcalpha 2-8NeuAcalpha 2-3)Galbeta 1-4Glc-Cer; PCR, polymerase chain reaction; beta 3Gn-T, UDP-GlcNAc:beta -galactose beta 1,3-N-acetylglucosaminyltransferase; bp, base pair(s); kb, kilobase pair(s); LEA, L. esculentum agglutinin; PWM, pokeweed mitogen; MOPS, 4-morpholinepropanesulfonic acid; LNnT, lacto-N-neotetraose; p-LNnH, p-lacto-N-neohexaose; LNT, lacto-N-tetraose; LNFP, lacto-N-fucosylpentaose; HPLC, high performance liquid chromatography; RT-PCR, reverse transcription-polymerase chain reaction.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Sasaki, K., Sasaki, E., Kawashima, K., Hanai, N., Nishi, T., and Hasegawa, M. (July 5, 1994) Japanese Patent 0618759 A 940705
2. Kolbinger, F., Streiff, M. B., and Katopodis, A. G. (1998) J. Biol. Chem. 273, 433-440[Abstract/Free Full Text]
3. Amado, K., Almeida, R., Carneiro, F., Leverly, S. B., Holmes, E. H., Nomoto, M., Hollingsworth, M. A., Hassan, H., Schwientek, T., Nielsen, P. A., Bennett, E. P., and Clausen, H. (1998) J. Biol. Chem. 273, 12770-12778[Abstract/Free Full Text]
4. Isshiki, S., Togayachi, A., Kudo, T., Nishihara, S., Watanabe, M., Kubota, T., Kitajima, M., Shiraishi, N., Sasaki, K., Andoh, T., and Narimatsu, H. (1999) J. Biol. Chem. 274, 12499-12507[Abstract/Free Full Text]
5. Hennet, T., Dinter, A., Kuhnert, P., Mattu, T. S., Rudd, M. P., and Berger, E. G. (1998) J. Biol. Chem. 273, 58-65[Abstract/Free Full Text]
6. Miyazaki, H., Fukumoto, S., Okada, M., Hasegawa, T., Furukawa, K., and Furukawa, K. (1997) J. Biol. Chem. 272, 24794-24799[Abstract/Free Full Text]
7. Zhou, D., Dinter, A., Gutierrez Gallego, R., Kamerling, J. P., Vliegenthart, J. F., Berger, E. G., and Hennet, T. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 406-411[Abstract/Free Full Text]
8. Sasaki, K., Kurata-Miura, K., Ujita, M., Angata, K., Nakagawa, S., Sekine, S., Nishi, T., and Fukuda, M. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 14294-14299[Abstract/Free Full Text]
9. Sasaki, K., Watanabe, E., Kawashima, K., Sekine, S., Dohi, T., Oshima, M., Hanai, N., Nishi, T., and Hasegawa, M. (1993) J. Biol. Chem. 268, 22782-22787[Abstract/Free Full Text]
10. Sasaki, K., Kurata, K., Funayama, K., Nagata, M., Watanabe, E., Ohta, S., Hanai, N., and Nishi, T. (1994) J. Biol. Chem. 269, 14730-14737[Abstract/Free Full Text]
11. Shinkai, A., Shinoda, K., Sasaki, K., Morishita, Y., Nishi, T., Matsuda, Y., Takahashi, I., and Anazawa, H. (1997) Protein Expression Purif. 10, 379-385[CrossRef][Medline] [Order article via Infotrieve]
12. Ikehara, Y., Kojima, N., Kurosawa, N., Kudo, T., Kono, M., Nishihara, S., Issiki, S., Morozumi, K., Itzkowitz, S., Tsuda, T., Nishimura, S. I., Tsuji, S., and Narimatsu, H. (1999) Glycobiology 9, 1213-1224[Abstract/Free Full Text]
13. Feizi, T., Childs, R. A., Watanabe, K., and Hakomori, S.-I. (1979) J. Exp. Med. 149, 975-980[Abstract]
14. Fukuda, M. N. (1981) J. Biol. Chem. 256, 3900-3905[Abstract/Free Full Text]
15. Kondo, A., Suzuki, J., Kuraya, N., Hase, S., Kato, I., and Ikenaka, T. (1990) Agric. Biol. Chem. 54, 2169-2170[Medline] [Order article via Infotrieve]
16. Kudo, T., Ikehara, Y., Togayachi, A., Morozumi, K., Watanabe, M., Nakamura, M., Nishihara, S., and Narimatsu, H. (1998) Lab. Invest. 78, 797-811[Medline] [Order article via Infotrieve]
17. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132[Medline] [Order article via Infotrieve]
18. Kozak, M. (1989) J. Cell Biol. 108, 229-241[Abstract]
19. Holmes, E. H. (1988) Arch. Biochem. Biophys. 260, 461-468[Medline] [Order article via Infotrieve]
20. Piller, F., and Cartron, J. P. (1983) J. Biol. Chem. 258, 12293-12299[Abstract/Free Full Text]
21. Holmes, E. H., Hakomori, S., and Ostrander, G. K. (1987) J. Biol. Chem. 262, 15649-15658[Abstract/Free Full Text]
22. van den Eijnden, D. H., Koenderman, A. H., and Schiphorst, W. E. (1988) J. Biol. Chem. 263, 12461-12471[Abstract/Free Full Text]
23. Holmes, E. H., and Levery, S. B. (1989) Arch. Biochem. Biophys. 274, 14-25[Medline] [Order article via Infotrieve]
24. Basu, M., Khan, F. A., Das, K. K., and Zhang, B. J. (1991) Carbohydr. Res. 209, 261-277[CrossRef][Medline] [Order article via Infotrieve]
25. Gu, J., Nishikawa, A., Fujii, S., Gasa, S., and Taniguchi, N. (1992) J. Biol. Chem. 267, 2994-2999[Abstract/Free Full Text]
26. Kawashima, H., Sueyoshi, S., Li, H., Yamamoto, K., and Osawa, T. (1990) Glycoconjugate J. 7, 323-334[Medline] [Order article via Infotrieve]
27. Stults, C. L., and Macher, B. A. (1993) Arch. Biochem. Biophys. 303, 125-133[CrossRef][Medline] [Order article via Infotrieve]
28. Tsuji, Y., Urashima, T., and Matsuzawa, T. (1996) Biochim. Biophys. Acta 1289, 115-121[Medline] [Order article via Infotrieve]
29. Fukuda, M., Spooncer, E., Oates, J. E., Dell, A., and Klock, J. C. (1984) J. Biol. Chem. 259, 10925-10935[Abstract/Free Full Text]
30. Lowe, J. B., Stoolman, L. M., Nair, R. P., Larsen, R. D., Berhend, T. L., and Marks, R. M. (1990) Cell 63, 475-484[Medline] [Order article via Infotrieve]
31. Phillips, M. L., Nudelman, E., Gaeta, F. C. A., Perez, M., Singhai, A. K., Hakomori, S.-I., and Paulson, J. C. (1990) Science 250, 1130-1132[Medline] [Order article via Infotrieve]
32. Walz, G., Aruffo, A., Kolanus, W., Bevilacqua, M., and Seed, B. (1990) Science 250, 1132-1135[Medline] [Order article via Infotrieve]
33. Lowe, B. J. (1994) in Molecular Glycobiology (Fukuda, M. , and Hindsgaul, O., eds) , pp. 163-205, Oxford University Press, Oxford
34. Fukushima, K., Hirota, M., Terasaki, P. I., Wakisaka, A., Togashi, H., Chia, D., Suyama, N., Fukushi, Y., Nudelman, E., and Hakomori, S. (1984) Cancer Res. 44, 5279-5285[Abstract]
35. Nakamori, S., Kameyama, M., Imaoka, S., Furukawa, H., Ishikawa, O., Sasaki, Y., Kabuto, T., Iwanaga, T., Matsushita, Y., and Irimura, T. (1993) Cancer Res. 53, 3632-3637[Abstract]
36. Sawada, R., Tsuboi, S., and Fukuda, M. (1994) J. Biol. Chem. 269, 1425-1431[Abstract/Free Full Text]
37. Carlsson, S. R., Sasaki, H., and Fukuda, M. (1986) J. Biol. Chem. 261, 12787-12795[Abstract/Free Full Text]
38. Maemura, K., and Fukuda, M. (1992) J. Biol. Chem. 267, 24379-24386[Abstract/Free Full Text]
39. Lee, N., Wang, W. C., and Fukuda, M. (1990) J. Biol. Chem. 265, 20476-20487[Abstract/Free Full Text]
40. Wilkins, P. P., McEver, R. P., and Cummings, R. D. (1996) J. Biol. Chem. 271, 18732-18742[Abstract/Free Full Text]
41. Gillespie, W., Kelm, S., and Paulson, J. C. (1992) J. Biol. Chem. 267, 21004-21010[Abstract/Free Full Text]
42. Kitagawa, H., and Paulson, J. C. (1994) J. Biol. Chem. 269, 17872-17878[Abstract/Free Full Text]
43. Lee, Y. C., Kojima, N., Wada, E., Kurosawa, N., Nakaoka, T., Hamamoto, T., and Tsuji, S. (1994) J. Biol. Chem. 269, 10028-10033[Abstract/Free Full Text]
44. Kim, Y. J., Kim, K. S., Kim, S. H., Kim, C. H., Ko, J. H., Choe, I. S., Tsuji, S., and Lee, Y. C. (1996) Biochem. Biophys. Res. Commun. 228, 324-327[CrossRef][Medline] [Order article via Infotrieve]
45. Kitagawa, H., and Paulson, J. C. (1993) Biochem. Biophys. Res. Commun. 194, 375-382[CrossRef][Medline] [Order article via Infotrieve]
46. Wen, D. X., Livingston, B. D., Medzihradszky, K. F., Kelm, S., Burlingame, A. L., and Paulson, J. C. (1992) J. Biol. Chem. 267, 21011-21019[Abstract/Free Full Text]
47. Kitagawa, H., and Paulson, J. C. (1994) J. Biol. Chem. 269, 1394-1401[Abstract/Free Full Text]
48. Brockhausen, I. (1999) Biochim. Biophys. Acta 1473, 67-95[Medline] [Order article via Infotrieve]
49. Kono, M., Takashima, S., Liu, H., Inoue, M., Kojima, N., Lee, Y. C., Hamamoto, T., and Tsuji, S. (1998) Biochem. Biophys. Res. Commun. 253, 170-175[CrossRef][Medline] [Order article via Infotrieve]
50. Ishii, A., Ohta, M., Watanabe, Y., Matsuda, K., Ishiyama, K., Sakoe, K., Nakamura, M., Inokuchi, J., Sanai, Y., and Saito, M. (1998) J. Biol. Chem. 273, 31652-31655[Abstract/Free Full Text]
51. Fukumoto, S., Miyazaki, H., Goto, G., Urano, T., Furukawa, K., and Furukawa, K. (1999) J. Biol. Chem. 274, 9271-9276[Abstract/Free Full Text]
52. Okajima, T., Fukumoto, S., Miyazaki, H., Ishida, H., Kiso, M., Furukawa, K., Urano, T., and Furukawa, K. (1999) J. Biol. Chem. 274, 11479-11486[Abstract/Free Full Text]
53. Kurosawa, N., Hamamoto, T., Lee, Y. C., Nakaoka, T., Kojima, N., and Tsuji, S. (1994) J. Biol. Chem. 269, 1402-1409[Abstract/Free Full Text]
54. Kurosawa, N., Inoue, M., Yoshida, Y., and Tsuji, S. (1996) J. Biol. Chem. 271, 15109-15116[Abstract/Free Full Text]
55. Sjoberg, E. R., Kitagawa, H., Glushka, J., van Halbeek, H., and Paulson, J. C. (1996) J. Biol. Chem. 271, 7450-7459[Abstract/Free Full Text]
56. Lee, Y. C., Kaufmann, M., Kitazume-Kawaguchi, S., Kono, M., Takashima, S., Kurosawa, N., Liu, H., Pircher, H., and Tsuji, S. (1999) J. Biol. Chem. 274, 11958-11967[Abstract/Free Full Text]
57. Okajima, T., Fukumoto, S., Ito, H., Kiso, M., Hirabayashi, Y., Urano, T., Furukawa, K., and Furukawa, K. (1999) J. Biol. Chem. 274, 30557-30562[Abstract/Free Full Text]
58. Okajima, T., Chen, H.-H., Ito, H., Kiso, M., Tai, T., Furukawa, K., Urano, T., and Furukawa, K. (2000) J. Biol. Chem. 275, 6717-6723[Abstract/Free Full Text]
59. Sasaki, K., Kurata, K., Kojima, N., Kurosawa, N., Ohta, S., Hanai, N., Tsuji, S., and Nishi, T. (1994) J. Biol. Chem. 269, 15950-15956[Abstract/Free Full Text]
60. Nara, K., Watanabe, Y., Maruyama, K., Kasahara, K., Nagai, Y., and Sanai, Y. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7952-7956[Abstract]
61. Haraguchi, M., Yamashiro, S., Yamamoto, A., Furukawa, K., Takamiya, K., Lloyd, K. O., Shiku, H., and Furukawa, K. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10455-10459[Abstract/Free Full Text]
62. Nakayama, J., Fukuda, M. N., Hirabayashi, Y., Kanamori, A., Sasaki, K., Nishi, T., and Fukuda, M. (1996) J. Biol. Chem. 271, 3684-3691[Abstract/Free Full Text]
63. Livingston, B. D., and Pauson, J. C. (1993) J. Biol. Chem. 268, 11504-11507[Abstract/Free Full Text]
64. Eckhardt, M., Mühlenhoff, M., Bethe, A., Koopman, J., Frosch, M., and Gerardy-Schahn, R. (1995) Nature 373, 715-718[CrossRef][Medline] [Order article via Infotrieve]
65. Yoshida, Y., Kojima, N., Kurosawa, N., Hamamoto, T., and Tsuji, S. (1995) J. Biol. Chem. 270, 14628-14633[Abstract/Free Full Text]
66. Nakayama, J., Fukuda, M. N., Fredette, B., Ranscht, B., and Fukuda, M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7031-7035[Abstract]
67. Scheidegger, E. P., Sternberg, L. R., Roth, J., and Lowe, J. B. (1995) J. Biol. Chem. 270, 22685-22688[Abstract/Free Full Text]
68. Kojima, N., Yoshida, Y., and Tsuji, S. (1995) FEBS Lett. 373, 119-122[CrossRef][Medline] [Order article via Infotrieve]
69. Kono, M., Yoshida, Y., Kojima, N., and Tsuji, S. (1996) J. Biol. Chem. 271, 29366-29371[Abstract/Free Full Text]
70. Kukowska-Latallo, J. F., Larsen, R. D., Nair, R. P., and Lowe, J. B. (1990) Genes Dev. 4, 1288-1303[Abstract]
71. Lowe, J. B., Kukowska-Latallo, J. F., Nair, R. P., Larsen, R. D., Marks, R. M., Macher, B. A., Kelly, R. J., and Ernst, L. K. (1991) J. Biol. Chem. 266, 17467-17477[Abstract/Free Full Text]
72. Goelz, S. E., Hession, C., Goff, D., Griffiths, B., Tizard, R., Newman, B., Chi-Rosso, G., and Lobb, R. (1990) Cell 63, 1349-1356[Medline] [Order article via Infotrieve]
73. Weston, B. W., Nair, R. P., Larsen, R. D., and Lowe, J. B. (1992) J. Biol. Chem. 267, 4152-4160[Abstract/Free Full Text]
74. Weston, B. W., Smith, P. L., Kelly, R. J., and Lowe, J. B. (1992) J. Biol. Chem. 267, 24575-24584[Abstract/Free Full Text]
75. Nishihara, S., Nakazato, M., Kudo, T., Kimura, H., Ando, T., and Narimatsu, H. (1993) Biochem. Biophys. Res. Commun. 190, 42-46[CrossRef][Medline] [Order article via Infotrieve]
76. Amado, M., Almeida, R., Schwientek, T., and Clausen, H. (1999) Biochim. Biophys. Acta 1473, 35-53[Medline] [Order article via Infotrieve]
77. Natsuka, S., Gersten, K. M., Zenita, K., Kannagi, R., and Lowe, J. B. (1994) J. Biol. Chem. 269, 16789-16794[Abstract/Free Full Text]
78. 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]
79. Bierhuizen, M. F., and Fukuda, M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9326-9330[Abstract]
80. Yeh, J.-C., Ong, E., and Fukuda, M. (1999) J. Biol. Chem. 274, 3215-3221[Abstract/Free Full Text]
81. Schwientek, T., Nomoto, M., Levery, S. B., Merkx, G., van Kessel, A. G., Bennett, E. P., Hollingsworth, M. A., and Clausen, H. (1999) J. Biol. Chem. 274, 4504-4512[Abstract/Free Full Text]
82. Schwientek, T., Yeh, J.-C., Levery, S. B., Keck, B., Merkx, G., van Kessel, A. G., Fukuda, M., and Clausen, H. (2000) J. Biol. Chem. 275, 11106-11113[Abstract/Free Full Text]
83. Bierhuizen, M. F. A., Mattei, M.-G., and Fukuda, M. (1993) Genes Dev. 7, 468-478[Abstract]
84. Bennett, E. P., Hassan, H., and Clausen, H. (1996) J. Biol. Chem. 271, 17006-17012[Abstract/Free Full Text]
85. Bennett, E. P., Hassan, H., Hollingsworth, M. A., and Clausen, H. (1999) FEBS Lett. 460, 226-230[CrossRef][Medline] [Order article via Infotrieve]
86. Bennett, E. P., Hassan, H., Mandel, U., Hollingsworth, M. A., Akisawa, N., Ikematsu, Y., Merkx, G., van Kessel, A. G., Olofsson, S., and Clausen, H. (1999) J. Biol. Chem. 274, 25362-25370[Abstract/Free Full Text]
87. Bennett, E. P., Hassan, H., Mandel, U., Mirgorodskaya, E., Roepstorff, P., Burchell, J., Taylor-Papadimitriou, J., Hollingsworth, M. A., Merkx, G., van Kessel, A. G., Eiberg, H., Steffensen, R., and Clausen, H. (1998) J. Biol. Chem. 273, 30472-30481[Abstract/Free Full Text]
88. Homa, F. L., Hollander, T., Lehman, D. J., Thomsen, D. R., and Elhammer, A. P. (1993) J. Biol. Chem. 268, 12609-12616[Abstract/Free Full Text]
89. Ten Hagen, K. G., Hagen, F. K., Balys, M. M., Beres, T. M., Van Wuyckhuyse, B., and Tabak, L. A. (1998) J. Biol. Chem. 273, 27749-27754[Abstract/Free Full Text]
90. White, T., Bennett, E. P., Takio, K., Sorensen, T., Bonding, N., and Clausen, H. (1995) J. Biol. Chem. 270, 24156-24165[Abstract/Free Full Text]
91. White, K. E., Lorenz, B., Evans, W. E., Meitinger, T., Strom, T. M., and Econs, M. J. (2000) Gene (Amst.) 246, 347-356[CrossRef][Medline] [Order article via Infotrieve]
92. Narimatsu, H., Sinha, S., Brew, K., Okayama, H., and Qasba, P. K. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 4720-4724[Abstract]
93. Shaper, N. L., Hollis, G. F., Douglas, J. G., Kirsch, I. R., and Shaper, J. H. (1988) J. Biol. Chem. 263, 10420-10428[Abstract/Free Full Text]
94. Masri, K. A., Appert, H. E., and Fukuda, M. N. (1988) Biochem. Biophys. Res. Commun. 157, 657-663[Medline] [Order article via Infotrieve]
95. Almeida, R., Amado, M., David, L., Levery, S. B., Holmes, E. H., Merkx, G., van Kessel, A. G., Hassan, H., Bennett, E. P., and Clausen, H. (1997) J. Biol. Chem. 272, 31979-31992[Abstract/Free Full Text]
96. Sato, T., Furukawa, K., Bakker, H., van den Eijnden, D. H., and Van Die, I. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 472-477[Abstract/Free Full Text]
97. Nomura, T., Takizawa, M., Aoki, J., Arai, H., Inoue, K., Wakisaka, E., Yoshizuka, N., Imokawa, G., Dohmae, N., Takio, K., Hattori, M., and Matsuo, N. (1998) J. Biol. Chem. 273, 13570-13577[Abstract/Free Full Text]
98. Schwientek, T., Almeida, R., Levery, S. B., Holmes, E., Bennett, E. P., and Clausen, H. (1998) J. Biol. Chem. 273, 29331-29340[Abstract/Free Full Text]
99. Okajima, T., Yoshida, K., Kondo, T., and Furukawa, K. (1999) J. Biol. Chem. 274, 22915-22918[Abstract/Free Full Text]
100. Almeida, R., Levery, S. B., Mandel, U., Kresse, H., Schwientek, T., Bennett, E. P., and Clausen, H. (1999) J. Biol. Chem. 274, 26165-26171[Abstract/Free Full Text]
101. Stroud, M. R., Levery, S. B., Nudelman, E. D., Salyan, M. E., Towell, J. A., Roberts, C. E., Watanabe, M., and Hakomori, S. (1991) J. Biol. Chem. 266, 8439-8446[Abstract/Free Full Text]
102. Dennis, J. W., Granovsky, M., and Warren, C. E. (1999) Biochim. Biophys. Acta 1473, 21-34[Medline] [Order article via Infotrieve]
103. Ujita, M., McAuliffe, J., Suzuki, M., Hindsgaul, O., Clausen, H., Fukuda, M. N., and Fukuda, M. (1999) J. Biol. Chem. 274, 9296-9304[Abstract/Free Full Text]
104. Ujita, M., McAuliffe, J., Hindsgaul, O., Sasaki, K., Fukuda, M. N., and Fukuda, M. (1999) J. Biol. Chem. 274, 16717-16726[Abstract/Free Full Text]
105. Ujita, M., McAuliffe, J., Schwientek, T., Almeida, R., Hindsgaul, O., Clausen, H., and Fukuda, M. (1998) J. Biol. Chem. 273, 34843-34849[Abstract/Free Full Text]


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