Molecular Cloning and Characterization of UDP-GlcNAc:Lactosylceramide beta 1,3-N-Acetylglucosaminyltransferase (beta 3Gn-T5), an Essential Enzyme for the Expression of HNK-1 and Lewis X Epitopes on Glycolipids*

Akira TogayachiDagger §, Tomohiro AkashimaDagger , Reiko OokuboDagger , Takashi KudoDagger , Shoko NishiharaDagger , Hiroko IwasakiDagger , Ayumi Natsume||, Hiroyuki Mio||, Jin-ichi Inokuchi**, Tatsuro Irimura§, Katsutoshi Sasaki||, and Hisashi NarimatsuDagger Dagger Dagger §§

From the Dagger  Division of Cell Biology, Institute of Life Science, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577, Japan, the § Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, Tokyo University, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, the  Laboratory of Animal Resources, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Aza-Yasaka, Abashiri, Hokkaido 099-2422, Japan, the ** Department of Biomembrane and Biofunctional Chemistry, Graduate school of Pharmaceutical Sciences, Hokkaido University, North-12, West-6, Kita-ku, Sapporo 060-0812, Japan, the || Tokyo Research Laboratories, Kyowa Hakko Kogyo Company Limited, 3-6-6 Asahi-machi, Machida, Tokyo 194-8582, Japan, and the Dagger Dagger  Laboratory of Gene Function Analysis, Institute of Molecular and Cell Biology (IMCB), National Institutes of Advanced Industrial Science and Technology (AIST), Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan

Received for publication, December 18, 2000, and in revised form, March 29, 2001

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A new member of the UDP-N-acetylglucosamine:beta -galactose beta 1,3-N-acetylglucosaminyltransferase (beta 3Gn-T) family having the beta 3Gn-T motifs was cloned from rat and human cDNA libraries and named beta 3Gn-T5 based on its position in a phylogenetic tree. We concluded that beta 3Gn-T5 is the most feasible candidate for lactotriaosylceramide (Lc3Cer) synthase, an important enzyme which plays a key role in the synthesis of lacto- or neolacto-series carbohydrate chains on glycolipids. beta 3Gn-T5 exhibited strong activity to transfer GlcNAc to glycolipid substrates, such as lactosylceramide (LacCer) and neolactotetraosylceramide (nLc4Cer; paragloboside), resulting in the synthesis of Lc3Cer and neolactopentaosylceramide (nLc5Cer), respectively. A marked decrease in LacCer and increase in nLc4Cer was detected in Namalwa cells stably expressing beta 3Gn-T5. This indicated that beta 3Gn-T5 exerted activity to synthesize Lc3Cer and decrease LacCer, followed by conversion to nLc4Cer via endogenous galactosylation. The following four findings further supported that beta 3Gn-T5 is Lc3Cer synthase. 1) The beta 3Gn-T5 transcript levels in various cells were consistent with the activity levels of Lc3Cer synthase in those cells. 2) The beta 3Gn-T5 transcript was presented in various tissues and cultured cells. 3) The beta 3Gn-T5 expression was up-regulated by stimulation with retinoic acid and down-regulated with 12-O-tetradecanoylphorbol-13-acetate in HL-60 cells. 4) The changes in beta 3Gn-T5 transcript levels during the rat brain development were determined. Points 2, 3, and 4 were consistent with the Lc3Cer synthase activity reported previously.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To date, three members of the human beta 1,3-N-acetylglucosaminyltransferase (beta 3Gn-T)1 family (beta 3Gn-T2, -T3, and -T4) (1, 2) and five members of the human beta 1,3-galactosyltransferase (beta 3Gal-T) family (beta 3Gal-T1, -T2, -T3, -T4, and -T5) have been identified (3-6). All of them share amino acid motifs (beta 3Gn-T motifs or beta 3Gal-T motifs) in three regions of the catalytic domain. The first, beta 3Gn-T, was cloned by an expression cloning method using an anti-i antibody (7). However, this enzyme is unique in that it does not have the beta 3Gn-T motifs although it transfers GlcNAc to Gal with an beta 1,3-linkage, resulting in the synthesis of polylactosamine chains. It was named iGn-T (7). Thereafter, beta 3Gn-T1 was isolated based on structural similarity with the beta 3Gal-T family (2). We previously reported three additional beta 3Gn-Ts, beta 3Gn-T2, -T3, and -T4, which are also structurally related to the beta 3Gn-T family (1). However, the cDNA sequence of beta 3Gn-T1 was recently corrected by Zhou et al. (see Ref. 2). The corrected sequence of beta 3Gn-T1 was identical to that of beta 3Gn-T2 which was isolated and reported by us (1). So, a total of four beta 3Gn-Ts, i.e. iGn-T, beta 3Gn-T2(-T1), -T3, and -T4, have been described to date. To avoid further confusion, we note this fact, but do not change the enzyme names used in this study. By transfection experiments and in vitro enzymatic analysis, these four beta 3Gn-Ts were found to exhibit beta 3Gn-T activity that catalyzes the synthesis of polylactosamine chains, but not beta 3Gal-T activity.

The monoclonal antibody HNK-1 reacts to a sulfoglucuronyl carbohydrate epitope, SO43-GlcAbeta 1-3Galbeta 1-4GlcNAc-R, which is expressed in several glycoproteins involved in neural cell recognition, and in two neolactoglycolipids, named sulfoglucuronylglycolipid (SGGL)-1 and -2 (SGGL-1, SO43-GlcAbeta 1-3Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4Glcbeta 1-1ceramide; and SGGL-2, SO43-GlcAbeta 1-3Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4Glcbeta 1-1ceramide) (8, 9). Neurobiologists have suggested in a series of studies that the HNK-1 epitope functions as a cell-cell interaction molecule during the development of the brain to complete the nervous system (10-14). The expression is developmentally and spatially regulated in the nervous system. In rat cerebellum, SGGLs are expressed in a biphasic manner, with the initial peak at around postnatal days (PD) 1-3, and the second peak at PD20 (15). In the adult rat cerebellum, the expression of SGGLs is kept constant (16, 17). Immunohistochemical studies localized SGGLs in specific cells during the development of the cerebellum, and suggested some biological functions for neuron guidance (14, 15, 17, 18). In rat cerebral cortex, the expression of SGGLs peaked at embryonic day (ED) 19 and then decreased until postnatal PD 5, and had almost completely disappeared by PD 20.

Carbohydrate chains on SGGLs are extended by stepwise reactions catalyzed by glycosyltransferases. Chou et al. (19-22) reported developmental changes in the activity of each enzyme in correlation with the SGGL expression in rat brain. It was demonstrated that Lc3Cer synthase, which catalyzes the transfer of GlcNAc to the Gal residue of lactosylceramide (LacCer; Galbeta 1-4Glcbeta 1-1Cer) with a beta 1-3-linkage resulting in the synthesis of Lc3Cer (GlcNAcbeta 1-3Galbeta 1-4Glcbeta 1-1Cer), is the key enzyme in the expression of SGGLs in the developing rat brain, because only the activity of this synthase was well correlated with the developmental change in SGGL expression.

The expression of the Lewis x (Lex; CD15) carbohydrate structure, alpha 1,3-fucosyl-N-acetyllactosamine, Galbeta 1-4(Fucalpha 1-3)GlcNAcbeta 1-R, is also regulated developmentally and region specifically in the brain (23-31). The Lex also functions as a cell-cell recognition molecule in the highly organized structures of the central nervous system (30, 32, 33). The Lex expression on GSLs in brain is also regulated by the Lc3Cer synthase which synthesizes a root structure of the GSLs carrying the Lex epitope (34). Thus, SGGLs and GSLs carrying the Lex epitope are of interest in terms of their biosynthetic regulation.

Lc3Cer synthase is an important enzyme with respect to hematopoietic cell differentiation (35). HL-60 cells, a human promyelocytic leukemic cell line, are capable of bidirectional differentiation into a monocytoid or granulocytoid lineage (35). The Lc3Cer synthase and GM3 synthase (lactosylceramide:alpha 2,3-sialyltransferase), which share the acceptor substrate lactosylceramide (LacCer); Galbeta 1-4Glcbeta 1-1Cer, are key enzymes in determining the biosynthetic flow of GSLs in the two different directions. During the monocytic differentiation of HL-60 cells induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment, ganglioside GM3 was markedly increased by up-regulation of GM3 synthase and down-regulation of Lc3Cer synthase. In contrast, during the granulocytic differentiation of HL-60 cells induced by all-trans-retinoic acid (RA), neolacto-series GSLs were markedly increased by up-regulation of Lc3Cer synthase.

Stults and Macher (36) concluded that mature myeloid cells exhibited beta 3Gn-T activity toward both acceptor substrates, leading to the expression of several types of neolacto-neutral GSLs in the cells, while lymphoid cells exhibited activity for nLc4Cer, but not LacCer. The lack of Lc3Cer synthesizing activity in lymphoid cells explains the absence of neolacto-neutral GSLs.

Lc3Cer synthase is the enzyme controlling the expression of neolacto-series GSLs and so plays important roles in many cells. In the present study, we cloned and characterized a fifth member of the beta 3Gn-T family (beta 3Gn-T5), and identified beta 3Gn-T5 as the most likely candidate for Lc3Cer synthase.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Tumor Cell Lines and Monoclonal Antibodies (MAbs)-- The tumor cell lines were cultured in RPMI 1640 medium (Life Technologies, Inc., Rockville, MD) supplemented with 10% fetal bovine serum.

The cell lines were donated or purchased from American Type Culture Collection (ATCC, Manassas, VA), Riken Cell Bank (RCB, Tsukuba, Japan), Japan Cell Research Bank (JCRB, Tokyo, Japan), Immunobiological laboratory (IBL, Fujioka, Japan), or Dai-Nippon Pharmaceutical Co., Ltd. (DNP, Osaka, Japan).

MAb 1B2 (anti-N-acetyllactosamine) which reacts to nLc4Cer and nLc6Cer (neolactohexaosylceramide) (IgM) (36, 37), were used for flow cytometry and immuno-TLC assays, respectively. The 1B2 hybridoma cells were cultured in RPMI 1640 medium (Life Technologies, Inc., Rockville, MD) supplemented with 15% fetal bovine serum. The supernatant of 1B2 hybridoma culture medium was used for experiments. The supernatant of 1B2 cells was kindly provided by Seikagaku Kogyo Co. Ltd (Tokyo, Japan).

Isolation of Rat and Human beta 3Gn-T5 cDNAs-- We constructed a rat shank cDNA library for a random sequencing project. During the sequencing, we found a rat cDNA encoding a partial sequence of beta 3Gn-T5. This novel sequence (738 bp) did not encode the full ORF. But it had the beta 3Gal-T (beta 3Gn-T) motifs which are shared by the known beta 3Gal-Ts and beta 3Gn-Ts. The cDNA library of Colo205 cells constructed in a previous study (6) was screened with the rat partial beta 3Gn-T5 cDNA as a probe to isolate the full-length human beta 3Gn-T5 cDNA which possessed a 3.7-kilobase pair insert DNA. We did not clone a rat full-length beta 3Gn-T5 in this study.

Quantitative Analysis of the Four beta 3Gn-T Transcripts in Human Tissues and Tumor Cell Lines by Competitive RT-PCR-- The principle behind the competitive RT-PCR method for quantification of transcripts was described in detail in our previous studies (6, 38). Total cellular RNAs of various human tissues were purchased from CLONTECH. Those of various tumor cell lines were extracted and purified in our laboratory. As to the measurement of the transcripts for three cloned beta 3Gn-T genes, beta 3Gn-T2, -T3, and -T4, the primers and the PCR conditions were also reported previously (1).

Regarding the beta 3Gn-T5 gene, a standard DNA plasmid was constructed by subcloning a full-length ORF cDNA into a pBluescript SK(-) vector. A competitor DNA plasmid carrying a small deletion (245 base pairs) within the ORF of the beta 3Gn-T5 cDNA was constructed as follows. The standard DNA plasmid was double digested with appropriate restriction endonucleases, MscI and BglII, and was blunt-ended with T4 DNA polymerase. This was followed by self-ligation of the deleted DNA plasmid. As to the measurement of the beta 3Gn-T5 transcripts by competitive RT-PCR, we used the following primer set: forward primer, 5'-TCTTATGACTGCTGATGATGACAT-3', and reverse primer, 5'-CTTTAGGATCTGTAGCATTCTTCC-3'.

Transfection Experiments to Express Each of the Four Human beta 3Gn-T Genes in Namalwa Cells-- Expression of glycosyltransferase genes subcloned in pAMo was described in detail in a series of previous papers (6, 7, 39-42). The construction of pAMo vector inserted with each of three cloned beta 3Gn-T genes, beta 3Gn-T2, -T3, and -T4, was also reported previously (1). Regarding the beta 3Gn-T5 gene, the beta 3Gn-T5 ORF fragment was excised from the standard beta 3Gn-T5 DNA in pBluescript SK(-), and then blunt-ended with T4 DNA polymerase. The blunt-ended fragment was flanked with SfiI linker and inserted into the SfiI site of pAMo vector. Each of the four beta 3Gn-T genes subcloned into a pAMo vector was transfected by electroporation into Namalwa cells. These cells were selected in the presence of geneticin (G418) (Life Technologies, Inc.) at a concentration of 0.8 mg/ml. Stable transfectant cells were obtained after 25 days exposure to geneticin. The levels of the transcripts expressed in the Namalwa transfectant cells were measured by means of competitive RT-PCR.

Separation of Glycolipids by Thin Layer Chromatography (TLC) and Immuno-TLC with Anti-nLc4Cer (Anti-paragloboside) MAb-- For glycolipid analysis, 1.0 × 108 of the Namalwa transfectant cells with each beta 3Gn-T gene were collected, washed twice with phosphate-buffered saline, and then lyophilized. Total glycolipids were extracted from the lyophilized cells. Crude glycolipids were extracted twice from the transfectant cells, first with chloroform/methanol (2:1, v/v), and then with chloroform/methanol/water (30:60:8, v/v/v). Samples dried with the N2 evaporator were dissolved in methanol, then subjected to mild alkaline treatment in 0.1 N KOH/methanol at 40 °C for 2 h, and neutralized with 1 N acetic acid. After the free fatty acids had been removed with n-hexane, the remaining fractions were dried with the N2 evaporator and subjected to Folch's partition. The lower neutral glycolipid fractions were dried with the N2 evaporator and subjected to immuno-TLC analysis. Neutral glycolipid equivalent to 1.0 × 107 cells was applied to each lane of TLC. Neutral glycolipids were separated by TLC (HPTLC Kieselgel 60, 5641; MERK, Germany) with mixtures of chloroform/methanol/water (60:35:8, v/v/v) and immuno-TLC analysis was performed with the antibody 1B2 as described (36).

Construction and Purification of beta 3Gn-T Proteins Fused with FLAG Peptide-- The putative catalytic domain of each of beta 3Gn-T2, -T3, -T4 had been expressed as a secreted protein fused with FLAG peptide in insect cells as described in a previous study (1). In the present study, a 1.1-kilobase pair DNA fragment encoding a COOH-terminal portion of beta 3Gn-T5 (amino acids 39 to 378) was amplified by PCR. The PCR was performed with Platinum Pfx DNA Polymerase (Life Technologies, Inc.), according to the supplier's manual. The 5' and 3' primer sequences were flanked with BamHI and XbaI sequences, respectively, to create the restriction sites. Those sequences were as follows: forward primer, 5'-CGGGATCCATTGTGAGCCATATGAAGTCATAT-3', and reverse primer, 5'-GCTCTAGATGACAGTGAAAACATACAACATTC-3'. The amplified fragment was first inserted between the BamHI and XbaI sites of the pBluescript SK(-) vector. Subsequently, the insert DNA was excised by BamHI and NotI, and was inserted between the BamHI and NotI sites of pVL1393-F2 to yield pVL1393-F2G5. pVL1393-F2 is an expression vector derived from pVL1393 (Pharmingen) and contains a fragment encoding the signal peptide of human immunoglobulin (MHFQVQIFSFLLISASVIMSRG) and FLAG peptide (DYKDDDDK).

We prepared recombinant viruses as described previously (1). Sf21 insect cells (Pharmingen) were infected with each individual recombinant virus at a multiplicity of infection of 10 and incubated at 27 °C for 72 h to yield conditioned media containing recombinant beta 3Gn-T proteins fused with FLAG peptide. Bacu3GnT proteins were readily purified by anti-FLAG M1 antibody resin (Sigma) and eluted with 50 mM TBS (50 mM Tris-HCl, pH 7.4, 150 mM NaCl), 4 mM CaCl2 buffer (pH 7.4). The recombinant proteins obtained in this system were named Bacu3GnT2, Bacu3GnT3, Bacu3GnT4, and Bacu3GnT5.

Bacu3GnT proteins separated by 10% SDS-polyacrylamide gel electrophoresis were transferred to an Immobilon PVDF membrane (Millipore, Bedford, MA). The membrane was probed with anti-FLAG monoclonal antibody, and stained with the ECL Western blotting detection reagents (Amersham Pharmacia Biotech). The intensity of positive bands on Western blotting was measured by densitometer (43) to determine the relative amounts of each Bacu3GnT protein.

Assaying of beta 3Gn-T Activity-- Two types of each recombinant beta 3Gn-T, the soluble enzyme produced in the baculoexpression system and the membrane-bound form expressed in the cell homogenates of Namalwa transfectant cells and other cultured cell lines, were used to determine the relative activities of each beta 3Gn-T toward various substrates. Each soluble Bacu3Gn-T purified was adjusted to the same amount as described above and used for the assay. The various cultured cells were solubilized in a 20 mM HEPES buffer (pH 7.2) containing 2% Triton X-100, and 20 µg of total protein in the cell homogenates was used for the enzyme reaction. Various oligosaccharides were fluorescently labeled with 2-aminobenzamide (2AB) or pyridylaminated (PA) (1, 44) and used for acceptors.

The beta 3Gn-T activity was assayed in a 20-µl reaction mixture containing 150 mM sodium cacodylate buffer (pH 7.2), 50 mM UDP-GlcNAc, 10 mM MnCl2, 0.4% Triton CF-54, 1 µM 2AB-acceptor or PA-acceptor substrate, and the enzyme source. After incubation at 37 °C for 16 h, the reaction was terminated by boiling for 3 min, and the mixture was diluted with 80 µl of water. After centrifugation at 15,000 rpm for 5 min, the supernatant was filtered using an Ultrafree-MC column (Millipore). A 10-µl aliquot of each supernatant was subjected to high performance liquid chromatography on a TSK-gel ODS-80TS QA column (4.6 × 300 mm; Tosoh, Tokyo, Japan). The reaction products were eluted with 20 mM ammonium acetate buffer (pH 4.0) containing 7% methanol at a flow rate of 1.0 ml/min at 50 °C and monitored with a fluorescence spectrophotometer, JASCO FP-920 (JASCO, Tokyo, Japan) (1, 44).

We conducted an assay of the incorporation of radioactive sugar into glycolipid acceptor substrates. The beta 3Gn-T activity for glycolipids was assayed in a 25-µl reaction mixture containing 150 mM sodium cacodylate buffer (pH 7.2), 480 µM UDP-GlcNAc, 175 nCi of UDP-[14C]GlcNAc (Amersham Pharmacia Biotech), 10 mM MnCl2, 0.4% Triton CF-54, 10 nmol of glycolipid acceptor substrate, and the enzyme source. After incubation at 37 °C for 16 h, the reaction was terminated by boiling for 3 min, and 200 µl of water containing 0.1 M KCl was added.

The reaction mixture was centrifuged at 15,000 rpm for 5 min. Radioactive glycolipid products were separated from the free radioactive UDP-[14C]GlcNAc using a Sep-Pak Plus C18 Cartridge (Waters). For conditioning, a Sep-Pak Plus C18 Cartridge was washed with 10 ml of 100% methanol, and then washed twice with 10 ml of water. The supernatant of the reaction mixture was loaded on the equilibrated cartridge. Elution of the radioactive products was achieved using 5 ml of 100% methanol. Eluted products were dried with evaporator, and then the residues were dissolved in an adequate volume of 100% methanol. The dissolved residues were separated on a HPTLC plate with a solvent system of chloroform/methanol/0.2% CaCl2 (65:35:8, v/v/v). The radioactive intensities of the bands were measured with a BAS 2000 Imaging Analyzer (Fuji Film, Tokyo, Japan).

Differentiation of HL-60 Cells in in Vitro Culture-- Human myelogenous leukemia HL-60 cells were seeded at an initial density of 2 × 105 cells/ml and incubated with either 1 µM RA or 8 nM TPA (35). At various points over the course of the culture, the cells were harvested, and the total RNA in the cells was recovered for competitive RT-PCR.

Measurement of the Rat beta 3Gn-T5 Transcript in Developing Brain-- Brains of SD rats (Charles River Japan, Tokyo, Japan) were separated into cerebral cortex and cerebellum, and the total RNA recovered from each tissue was subjected to competitive RT-PCR assay. Total RNA was extracted from a mixture of 4-6 rat brains at each stage of development, and was used for cDNA synthesis.

The cDNA (738 bp; GenBankTM accession number AB045279) encoding the partial ORF of rat beta 3Gn-T5 was used as a standard, from which a competitor DNA was generated by deletion of a BsmBI fragment (185 bp). A primer set, a forward primer, 5'-CAAGATTTCACTGATTCTTTCCAC-3' and a reverse primer, 5'-GTCCTGTAGGTCTTGTGAGTGTCC-3', was used for the competitive RT-PCR assay. The competitive RT-PCR experiment was performed twice on each sample.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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A Novel cDNA Homologous to the Cloned beta 3Gal-Ts or beta 3Gn-Ts-- A novel cDNA sequence encoding beta 3Gn-T motifs was found during the random sequencing of a rat shank cDNA library. We cloned a full-length human cDNA homologous (AB045278) to the rat cDNA (AB045279), and named it beta 3Gn-T5. The amino acid sequence homology in the corresponding region between human and rat was 82.9%. This indicated that the two cDNAs are orthologous.

The human beta 3Gn-T5 cDNA contains a full-length ORF encoding a protein of 378 amino acids, as shown in Fig. 1. beta 3Gn-T5 is predicted to be a typical type II membrane protein consisting of a NH2-terminal cytoplasmic domain of 12 residues, a transmembrane segment of 20 residues, and a stem region and catalytic domain of 346 residues. beta 3Gn-T5 had the three motifs typical of members of the beta 3Gal-T and beta 3Gn-T families. On ClustalW analysis (Fig. 1), four cysteine residues were found to be conserved in the four beta 3Gn-Ts which indicates that some of these cysteines are essential for maintenance of the tertiary structure of beta 3Gn-Ts. In the second beta 3Gn-T motif, a triplet of aspartic acid residues, DDD, which may be a dication binding site as proposed in a crystallization study of other glycosyltransferases (45), was conserved. Four possible N-glycosylation sites were found in the primary sequence of beta 3Gn-T5. One of them was conserved in all beta 3Gn-Ts. The beta 3Gn-T5 gene was found to be localized to a draft genome sequence (GenBankTM accession number AC025833) and the 3.7-kilobase pair cDNA containing the ORF is composed of a single exon.


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Fig. 1.   ClustalW alignment for comparison of beta 3Gn-T5 with the other four beta 3Gn-Ts. Multiple sequence analysis (ClustalW) of the four members of the beta 3Gn-T family. Introduced gaps are shown with hyphens. The three beta 3Gn-T motifs are boxed. The putative transmembrane domains are underlined. Asterisks indicate the amino acids identical among all proteins. Conserved amino acids are shown by dots. Cysteine residues conserved in the four beta 3Gn-Ts, beta 3Gn-T2, -T3, -T4, and -T5, are indicated by open arrows. Possible N-glycosylation sites in the beta 3Gn-T5 sequence are double underlined. A possible N-glycosylation site conserved in all proteins is indicated by a closed arrow.

On a phylogenetic tree (Fig. 2), the four members of the beta 3Gn-T family apparently formed a cluster which is separated from beta 3Gal-T members. beta 3Gn-T5 is positioned in the beta 3Gn-T family branch, however, it is in an outer branch away from the cluster of the other members. Three enzymes, beta 3Gn-T2, -T3, and -T4, form a subfamily in the phylogenetic tree and beta 3Gal-T4 and iGn-T also form a subfamily. The divergence of beta 3Gn-T5 occurred earlier than that of any other beta 3Gn-T member.


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Fig. 2.   A phylogenetic tree of beta 3Gal-Ts and beta 3Gn-Ts. A phylogenetic tree of beta 3Gal-Ts and beta 3Gn-Ts was constructed by means of the neighbor joining method (53) based on the amino acid sequences. The branch length indicates the evolutionary distance between each member.

Quantitative Analysis of beta 3Gn-T5 Transcripts in Human Tissues and Various Cell Lines-- As summarized in Fig. 3 and Table I, this gene was expressed in almost all tissues and cell lines with very few exceptions, although the expression level was different depending on the tissue and cell line. The tissues expressing beta 3Gn-T5 at a considerably high level were lung, colon, placenta, testis, pituitary gland, and cerebellum. Brain, liver (very low), spleen, lymph node, and thymus expressed beta 3Gn-T5 at a low level. Colonic adenocarcinoma, Colo205, SW620, lung cancer cell lines, EBC-1, HAL8, LX-1, PC-7, and RERF-LC-MS, and stomach cancer cell lines, KATOIII, MKN7, and HSC43 expressed the beta 3Gn-T5 transcripts at a high level. All neuroblastoma cells examined, except for NAGAI and GOTO cells, expressed the beta 3Gn-T5 transcript at a relatively high level. On the other hand, all leukemic cells derived from lymphocytes, except for NALL-1 (lymphoblastic leukemia), expressed beta 3Gn-T5 almost at an undetectable level. U937 (monocyte-like) and HL-60 (promyelocytic leukemia) cells expressed it at a considerable level. HepG2 cells did not express it at all. The expression levels in the cultured cells reflected those in the original tissues derived therefrom.


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Fig. 3.   Quantitative analysis of transcripts of beta 3Gn-Ts in various human tissues by competitive RT-PCR. The transcript levels of beta 3Gn-T5 in 36 kinds of human tissue were determined by competitive RT-PCR. The single stranded cDNA for the beta 3Gn-T5 transcript was amplified together with 200 ag/µl of the respective competitor DNA. The human beta -actin transcripts in the respective tissues were also quantified with 200 fg/µl of the beta -actin competitor DNA. The value for the beta 3Gn-T5 transcript divided by that for the respective beta -actin transcript is shown as a bar chart.

                              
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Table I
Quantitative analysis of beta 3Gn-T transcripts in various human cell lines by competitive RT-PCR

Relative Activities of Four Recombinant beta 3Gn-Ts, Which Were Produced as Truncated Forms of Fusion Protein with the FLAG Peptide in a Baculoexpression System, Toward Oligosaccharides-- The recombinant enzymes of four beta 3Gn-Ts, beta 3Gn-T2, -T3, -T4, and -T5, produced as fusion proteins with a FLAG peptide in the baculoexpression system were named Bacu3GnT2, -3GnT3, -3GnT4, and -3GnT5, respectively. The amounts of each enzyme were made equal for assaying the beta 3Gn-T activity.

We observed no galactosyltransferase activity of the four beta 3Gn-Ts toward LNnT-PA and agalacto-LNnT-PA (data not shown). The beta 3Gn-T activity of Bacu3GnT2 toward LNnT-PA was strongest among all combinations of the enzyme and the substrate. Thus, the activity of Bacu3GnT2 toward LNnT-PA is presented as 100%, and all other activities are given as relative values.

Bacu3GnT2 exhibited relative activity toward the following pyridylaminated substrates: LNnT-PA, 100%; LNT-PA, 3.3%; LNFP-III-PA, 4.2%; and LNFP-V-PA, 4%; and exhibited no activity toward LNFP-II-PA or LNDFH-II-PA. Bacu3GnT3 exhibited relative activity toward LNnT-PA, 1.0%; LNT-PA, 0.8%; and LNFP-V-PA, 0.8%, and exhibited no activity toward LNFP-II-PA, LNFP-III-PA, or LNDFH-II-PA. Bacu3GnT4 exhibited relative activity toward only LNFP-V-PA, 0.2%, and exhibited no activity toward LNnT-PA, LNT-PA, LNFP-II-PA, LNFP-III-PA, or LNDFH-II-PA. Bacu3GnT5 exhibited relative activity toward pyridylaminated substrates, LNnT-PA, 18.2%; LNT-PA, 1.0%; LNFP-III-PA, 0.9%; and LNFP-V-PA, 0.9%. It exhibited no activity toward LNFP-II-PA or LNDFH-II-PA.

The beta 3Gn-T activity of Bacu3GnT5 toward LNnT-PA was almost one-fifth the activity of Bacu3GnT2. The three Bacu3GnTs, -3GnT2, -3GnT3, and -3GnT5, exhibited weak activity for LNT-PA and LNFP-V-PA. Bacu3GnT2 and -3GnT5 also exhibited weak activity for LNFP-III-PA. LNFP-II-PA and LNDFH-II-PA could not be utilized as acceptor substrate for any Bacu3GnT examined. Bacu3GnT4 activity was almost undetectable for all substrates except a very faint activity for LNFP-V-PA. In a previous study, we confirmed that Bacu3GnT3 and Bacu3GnT4 apparently exhibited detectable levels of beta 3Gn-T activity toward LNnT-PA and LNT-PA because we used an excess of recombinant enzyme for the reaction (1).

The oligosaccharide substrates having the polylactosamine structures, repeats of units of lactosamine (Galbeta 1-4GlcNAc; LN), were labeled with 2AB and used as acceptor substrates (44). The LNnT-2AB oligosaccharide was used as a control. 2LN, 3LN, 4LN, and 5LN in Table II indicate that each oligosaccharide has 2-, 3-, 4-, or 5-repeating lactosamine (LN) units, respectively. The beta 3Gn-T activity toward LNnT-2AB of Bacu3GnT2 was again the strongest among all combinations of enzyme and substrate, therefore its activity is expressed as 100%, and the activities of the other combinations are expressed relative to this value in Table II. Bacu3GnT2 transferred a GlcNAc with almost the same level of activity to all polylactosamine substrates regardless of the number of LN units. Bacu3GnT3 exhibited low, but apparently positive activity for all lengths of polylactosamine substrate. The activity of Bacu3GnT4 was again hardly detected with the amount of recombinant protein used in the present study. Interestingly, Bacu3GnT5 preferred the shorter substrates, i.e. 2LN-2AB and 3LN-2AB. The activities of Bacu3GnT5 for the longer polylactosamine chains, 4LN-2AB and 5LN-2AB, were almost one-tenth of those for the shorter chains.

                              
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Table II
Specific activity of recombinant beta 3Gn-Ts expressed in the baculo system toward polylactosamine chains

Relative Activities of Four beta 3Gn-Ts, Which Were Produced in Two Different Types of Recombinant Enzymes, Toward Glycolipid Substrates-- To elucidate the relative activities toward glycolipid acceptors, two recombinant beta 3Gn-Ts were compared. First, the homogenates of Namalwa cells stably expressing each beta 3Gn-T gene were used as a source of recombinant enzyme.

Leading to measurement of beta 3Gn-T activity in Namalwa transfectant cells, we determined the transcript level of each beta 3Gn-T gene expressed in each Namalwa transfectant cell. The wild-type Namalwa cells expressed substantial amounts of beta 3Gn-T2 endogenously, 3.3 units, but not the other beta 3Gn-Ts (beta 3Gn-T3, -T4, and -T5). The amounts of transcript in cells transfected with the beta 3Gn-T2, beta 3Gn-T3, beta 3Gn-T4, or beta 3Gn-T5 gene, were 21.2, 15.4, 24.4, and 10.0 units, respectively. The amount of each transcript in cells mock-transfected with the pAMo vector alone was just as much as that in Namalwa wild cells. We used HL-60 cells as a control for this experiment. HL-60 expressed substantial amounts of two beta 3Gn-T transcripts, i.e. beta 3Gn-T2; 5.3 units and beta 3Gn-T5; 0.9 units, but possessed no transcript of beta 3Gn-T3 and beta 3Gn-T4.

As seen in Fig. 4, the homogenates of Namalwa-3GnT5 cells exhibited strong activity for the synthesis of both Lc3Cer and nLc5Cer. The homogenates of HL-60 cells also showed positive activities toward both acceptors, LacCer and nLc4Cer. Relative activities were obtained by measurement of the positive bands in Fig. 4. The activity of Namalwa-3GnT5 toward LacCer is presented as 100%, and all other activities are given as relative values. Namalwa-3GnT5 exhibited strong activity toward LacCer resulting in the synthesis of Lc3Cer (100%), however, the other three beta 3Gn-Ts showed no activity toward LacCer. The wild-type Namalwa cells and the mock-transfected Namalwa cells showed faint activity, 2.6 and 2.5%, respectively, for the synthesis of nLc5Cer. The other transfectant cells did not show an increase in either activity, although the transcript for the transfected gene was apparently overexpressed in the cells. Their activities were 3.2, 2.8, and 2.4%, respectively. In contrast, Namalwa-3GnT5 exhibited strong activity toward nLc4Cer. The relative activity of Namalwa-3GnT5 for nLc5Cer synthesis was almost the same, 105.2%, as that for Lc3Cer synthesis.


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Fig. 4.   Assaying of Lc3Cer and nLc5Cer synthesizing activities for the acceptors, LacCer and nLc4Cer, respectively, in the homogenates of Namalwa cells stably expressing each beta 3Gn-T gene. The homogenates of Namalwa wild-type cells, Namalwa mock-transfected cells, and Namalwa cells stably expressing each beta 3Gn-T gene were used as enzyme sources. The Lc3Cer synthesizing activity (left panel) and the nLc5Cer synthesizing activity (right panel) were determined from [14C]GlcNAc incorporation into LacCer (left panel) and nLc4Cer (right panel). Following the enzyme reaction, reaction products were separated by HPTLC and the radioactivity in the positive bands was measured by BAS 2000.

Second, we measured the relative activities of the four Bacu3GnTs, three of which, Bacu3Gn-T2, -T3, and -T4, were the same recombinant enzymes used in the previous experiments (1) toward glycolipid acceptors. Bacu3GnT5 was prepared in this study. As summarized in Table III, the activity of Bacu3GnT5 toward LacCer is presented as 100%. Bacu3GnT5 exhibited very strong activities toward both LacCer and nLc4Cer in comparison with the other enzymes. Interestingly, Bacu3GnT5 preferred nLc4Cer over LacCer as an acceptor, and transferred GlcNAc to nLc4Cer 4.67-fold more efficiently than to LacCer. Bacu3GnT5 did not utilize galactosylceramide (GalCer) as an acceptor. Bacu3GnT2 exhibited considerable activity toward nLc4Cer, 21.8%, but not toward LacCer. Bacu3GnT3 showed very faint activity toward nLc4Cer, 0.8%. Again, Bacu3GnT4 did not show any activity toward the glycolipid acceptors.

                              
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Table III
Specific activity of recombinant beta 3Gn-Ts expressed in the baculo system toward glycolipid acceptors

Immuno-TLC Analysis of Glycoshingolipids (GSLs) Extracted from Namalwa Cells Transfected with Each beta 3Gn-T Gene-- As seen from the orcinol staining results of neutral GSLs (Fig. 5A), among multiple bands of GSLs, the band intensity of LacCer of Namalwa-3GnT5 apparently decreased as compared with that of the other transfectants. By immunostaining using the 1B2 mAb (Fig. 5B), positive bands were detected in all transfectants including the wild-type and mock transfectant cells. However, the nLc4Cer band of Namalwa-3GnT5 cells showed a very strong intensity as compared with that of the other transfectants. The positive band faintly detected below that of nLc4Cer of Namalwa-3GnT5 cells corresponded to nLc6Cer. It is known that mAb 1B2 reacts with both nLc4cer and nLc6Cer (37). The above results are interpreted as follows. The overexpressed beta 3Gn-T5 in the cells consumed the substrate, LacCer, and thereafter, the product Lc3Cer was galactosylated by (an) endogenous beta 1,4-galactosyltransferase(s) to produce nLc4Cer, because Namalwa cells are known to endogenously possess an excess amount of beta 4Gal-T1. Some of the nLc4Cer produced in the cells was further converted to nLc5Cer by the overexpressed beta 3Gn-T5 and again galactosylated to produce nLc6Cer by endogenous beta 4Gal-T(s).


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Fig. 5.   Orcinol staining and HPTLC immunostaining of neutral glycolipids extracted from Namalwa cells and transfectants. A, neutral glycolipids were visualized by orcinol staining. Total glycolipids were extracted from lyophilized cells, equivalent to 1.0 × 108 cells. Standard glycolipids, GlcCer, LacCer, Lc3Cer, and nLc4Cer (from top to bottom), are presented in the left lane. B, the neutral GSL bands were immunostained with mAb 1B2 (anti-nLc4Cer and anti-nLc6Cer).

Correlation of Lc3Cer Synthesizing Activity with the Amount of beta 3Gn-T5 Transcript in Various Cultured Tumor Cells-- The Lc3Cer synthesizing activity was highest in KATOIII cells among various cultured cancer cells examined in this study (Table IV). Thus, the activity of KATOIII was set as 100%, and the values for Lc3Cer synthesizing activity of the other cells were calculated relative to this (see Table IV). The level of Lc3Cer synthesizing activity was almost correlated with the amount of beta 3Gn-T5 transcript (the mathematical correlation coefficient is 0.83), but not with the levels of the other beta 3Gn-T transcripts (the mathematical correlation coefficients of other beta 3Gn-Ts are all under 0.45). KATOIII cells possessed the most beta 3Gn-T5, followed by Colo205, EBC-1, LS180, and AOI cells in decreasing order both for activity and the amount of transcript. However, SK-N-SH cells are an exception which exhibited low activity, despite the relatively high expression of beta 3Gn-T5 transcripts. We do not know why the Lc3Cer synthesizing activity of SK-N-SH cells conflicted with the amount of beta 3Gn-T5 transcript. Some cancer cells, HepG2, Jurkat, Daudi, Ramos, and GOTO cells, did not express the beta 3Gn-T5 gene at all, and exhibited no activity of Lc3Cer synthase.

                              
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Table IV
Correlation of Lc3Cer synthase activity with the transcript level of beta 3Gn-T5 in various tumor cells

On the other hand, Colo205 cells, exhibiting weaker activity than KATOIII cells, expressed larger amounts of beta 3Gn-T2 transcript than KATOIII cells. HepG2 cells expressed considerable amounts of beta 3Gn-T2 and -T3 transcripts, but they exhibited no activity of Lc3Cer synthase. beta 3Gn-T4 was expressed at a very low or undetectable level in all cell lines examined. The above results indicated that the Lc3Cer synthesizing activity is mainly directed by beta 3Gn-T5 in these cell lines.

Expression of the Human beta 3Gn-T5 Transcripts during Differentiation in the Human Promyelocytic Cell Line HL-60-- As seen in Fig. 6, the expression level of the beta 3Gn-T5 transcript during the granulocytic differentiation by RA treatment apparently increased in comparison to that of the wild-type HL-60 cell. On the other hand, it markedly declined during the monocytic differentiation induced by TPA treatment.


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Fig. 6.   Change of beta 3Gn-T5 transcript levels during differentiation of HL-60 cells by induction with RA or TPA. The expression level of the beta 3Gn-T5 transcript in HL-60 cells was quantified by competitive RT-PCR. The value was divided by that for the respective beta -actin transcript. Non-treated HL-60 cells (open circle ) are indicated at 0 days. HL-60 cells were cultured in the presence of 1 µM RA (black-square) or 8 nM TPA () for up to 4 or 2 days, respectively.

Change of the Transcript Level of beta 3Gn-T5 in the Developing Rat Brain-- As shown in Fig. 7, in the rat cerebral cortex, the expression level of the beta 3Gn-T5 transcript peaked at around ED19, and had almost completely disappeared by PD14. The adult cerebral cortex expressed no beta 3Gn-T5 transcript. In contrast, cerebellum showed biphasic changes in the transcript level. The level of beta 3Gn-T5 transcript decreased from ED19 to PD3, and then increased until PD8. Subsequently, the level again decreased till PD14. In the adult cerebellum, the expression level was comparatively high.


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Fig. 7.   Change of beta 3Gn-T5 transcript levels during rat brain development. Total RNA was extracted from a mixture of 5-7 rat brains at each developmentally stage, fetal (ED19), neonatal (PD3, PD8, and PD14), and adult, and subjected to the measurement of beta 3Gn-T5 transcript by competitive RT-PCR. The value for the transcript in cerebellum () and cerebral cortex (black-square) was divided by that for the respective rat beta -actin transcript for normalization. The competitive RT-PCR experiments were performed twice on each sample. The values of two experiments are expressed as the mean ± S.D.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Lc3Cer synthase plays a key role in the control of carbohydrate synthesis in GSLs during cell differentiation and development. As demonstrated in the present study, beta 3Gn-T2, -T3, and -T4 are not the Lc3Cer synthase. We did not examine activities of the recombinant enzymes of iGn-T. However, the transcript levels of iGn-T in various tumor cell lines were not correlated with the Lc3Cer and nLc5Cer synthesizing activities in the respective cell lysates (data not shown). So, we can rule out iGn-T as a candidate for the Lc3Cer synthase. We concluded that beta 3Gn-T5 is the most feasible candidate for the following reasons. 1) Bacu3GnT5 and the homogenates of Namalwa-3GnT5 cells exhibited strong activity to synthesize Lc3Cer in vitro. 2) LacCer was consumed to be converted to neolacto-series GSLs in the Namalwa-3GnT5 cells. 3) The transcript levels of beta 3Gn-T5 in various tissues and cell lines were consistent with the Lc3Cer synthesizing activity as reported previously. 4) The expression level of the beta 3Gn-T5 transcript in various cultured cancer cells was well correlated with the Lc3Cer synthesizing activity. 5) The changes in the beta 3Gn-T5 transcript level during HL-60 differentiation and during rat brain development were consistent with those of the Lc3Cer synthesizing activity as reported by others (15, 20, 35, 46).

The truncated enzyme expressed in the insect cells preferred nLc4Cer which has a longer carbohydrate chain than LacCer, while the membrane-bound form exhibited almost the same activity toward both substrates. The difference in activity between the two forms may be related to structural difference or the presence of detergent in the reaction mixture. Glycosyltransferases are the Golgi enzymes bound to the Golgi membrane by the transmembrane domain. We assume that the truncated form, which is soluble due to the absence of transmembrane domain, may easily access GSLs with a long carbohydrate chain, which are more hydrophilic than GSLs with shorter carbohydrate chains. The membrane-bound form expressed in Namalwa cells probably has more physiological activity than the truncated form. In the reaction mixture for beta 3Gn-T assay, the membrane-bound form probably exists as a micellar penetrating the Golgi membrane. It is likely that the membrane-bound enzyme rather than the truncated soluble form interacts with glycolipid substrates.

Lc3Cer synthase has been detected in many tissues, including the developing rat brain (14, 15, 20, 46), hematopoietic cells (35, 36, 47-49), and colorectal tissues (50-52). In all tissues examined, the Lc3Cer synthase was identified to be a key enzyme in the extension of the carbohydrate chain on neolacto-series GSLs.

The homogenates of Namalwa-3GnT5 cells exhibited strong activity for both GSL substrates, LacCer and nLc4Cer, as well as toward LNnT-2AB and the two shorter polylactosamine chains, 2LN-2AB and 3LN-2AB. This indicated that beta 3Gn-T5 effectively recognizes a polylactosamine structure within two units of lactosamine. The marked reduction in the beta 3Gn-T5 activity for the longer polylactosamine chain would suggest that GSLs with a long polylactosamine chain, such as nLc6Cer, are not good substrates for beta 3Gn-T5. In previous studies (15, 20, 46), the activity of beta 3Gn-T toward LacCer and nLc4Cer was measured using tissue homogenates of rat brain during development. Both activities showed not only very similar profiles of change during the development, but almost the same level. This may be consistent with the present results showing that the two activities are directed by a single enzyme, beta 3Gn-T5. The wild-type and mock-transfected Namalwa cells showed weak beta 3Gn-T activity for nLc4Cer, but no activity for LacCer. We could not identify which enzyme directs this activity in the wild-type Namalwa cells. The activity may be directed by endogenous beta 3Gn-T2 or there may be some unknown beta 3Gn-T(s) in the Namalwa cells.

The Lc3Cer synthase is a key enzyme in the expression of a series of neolactoglycolipids, i.e. nLc4Cer and its derivatives. In particular, the expression level of two SGGLs, SGGL-1 and SGGL-2, carrying the HNK-1 epitope is determined by the Lc3Cer synthase (15, 19-22, 46). The change in the level of beta 3Gn-T5 transcript in developing rat brain almost paralleled that in Lc3Cer synthesizing activity reported previously (15, 20, 46). This strongly indicated that beta 3Gn-T5 is the Lc3Cer synthase. To confirm this, we will examine whether or not beta 3Gn-T5 is co-localized with HNK-1 on SGGLs and CD15 on neolacto-series GSLs in a future study.

Lc3Cer synthase is also an important enzyme in hematopoietic cell differentiation. The changes in the level of beta 3Gn-T5 transcript during HL-60 differentiation shown in Fig. 6 are consistent with the results of Nakamura et al. (35). Stults et al. (36) reported that lymphoid cell lines lack Lc3Cer synthesizing activity, but possess nLc5Cer synthesizing activity, whereas myeloid cell lines express both activities. Almost all lymphoid cell lines we examined in this study showed very low or undetectable levels of beta 3Gn-T5 transcript, while HL-60 cells (promyelocytic leukemia) and U937 cells (monocyte-like) expressed substantial amounts of beta 3Gn-T5. This again supported that beta 3Gn-T5 is responsible for the synthesis of Lc3Cer in hematopoietic cells.

Holmes (50) reported the beta 3Gn-T activities for the synthesis of GSL in the cells homogenates of a human colon cancer cell line, SW403. The SW403 cell homogenates showed almost the same level of beta 3Gn-T activity toward two GSL acceptors, LacCer and nLc4Cer. This could be interpreted to mean that both activities were directed by a single enzyme, beta 3Gn-T5, as in the case of rat brain. In the present study, we demonstrated that recombinant beta 3Gn-T5 effectively catalyzes the biosynthesis of both Lc3Cer and nLc5Cer at almost the same efficiency. There is some controversy (52) over whether a single enzyme, beta 3Gn-T5, synthesizes both Lc3Cer and nLc5Cer in colon tissue. Based on the Basu study (52), there appear to be unknown beta 3Gn-Ts in colon tissue, which differentially catalyze the synthesis of Lc3Cer or nLc5Cer. Another controversy with the present study relates to the report by Holmes et al. (51) in which significantly high activity of Lc3Cer synthase was detected in colonic adenocarcinoma tissues of patients and cell lines derived therefrom, but in contrast, the activity was undetectable in normal colonic epithelial cells. In the present study, all colonic adenocarcinoma cell lines expressed substantial amounts of beta 3Gn-T5 transcript. This is consistent with the results of Holmes et al. (51). However, normal colon tissue also expressed substantial amounts of the transcript. We will examine whether or not the beta 3Gn-T transcripts are markedly up-regulated in the colorectal cancer tissues of patients.

Regarding the other beta 3Gn-Ts examined in this study, beta 3Gn-T2 was most active toward polylactosamine acceptors, and it effectively extended the polylactosamine chain even on the longer chain acceptors. Thus, beta 3Gn-T2 is the most probable candidate for the enzyme which functions to extend the polylactosamine chain. beta 3Gn-T5 is involved only in the synthesis of short polylactosamine chains or initiation of polylactosamine synthesis. beta 3Gn-T3 and beta 3Gn-T4 exhibited very little activity, almost undetectable, toward all substrates examined. They apparently showed positive beta 3Gn-T activity in a previous study (1), because excess amounts of enzyme were used for the assay. The native acceptor substrates for beta 3Gn-T3 and -T4 may be different from the Gal residue acceptor, and some unknown native acceptors may exist for beta 3Gn-T3 and -T4.

The beta 3Gn-T (beta 3Gal-T) family is very large with 10 members, 1Gn-T, beta 3Gal-T1-5, and beta 3Gn-T2-5, having been cloned and analyzed to date. The possibility therefore exists that some as yet unidentified beta 3Gn-T(s) exhibits Lc3Cer synthase activity.

The results of the present study strongly indicated that beta 3Gn-T5 is the most feasible candidate for Lc3Cer synthase. In the future, we will assess the biological functions of this synthase, the key enzyme determining the expression of biologically functional epitopes on GSLs.

    ACKNOWLEDGEMENT

We thank Kazuya Kabayama, Graduate School of Pharmaceutical Sciences, Hokkaido University, for help with immuno-TLC analysis. We also thank Dr. Satoshi Nakagawa and Sachiko Yokokawa, Kyowa Hakko Co. Ltd., for homology search and excellent technical assistance, respectively.

    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 sequence(s) reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with the accession numbers AB045278 for human beta 3Gn-T5 and AB045279 for rat beta 3Gn-T5.

§§ To whom all correspondence should be addressed: Laboratory of Gene Function Analysis, Institute of Molecular and Cell Biology (IMCB), National Institutes of Advanced Industrial Science and Technology (AIST), Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan. Tel.: 81-298-61-3200; Fax: 81-298-61-3201; E-mail: h.narimatsu@aist.go.jp.

Published, JBC Papers in Press, March 3, 2001, DOI 10.1074/jbc.M011369200

    ABBREVIATIONS

The abbreviations used are: beta 3Gn-T, UDP-GlcNAc:beta -galactose beta 1,3-N-acetylglucosaminyltransferase; beta 3Gal-T, UDP-galactose:beta -N-acetylglucosamine beta 1,3-galactosyltransferase; UDP-GlcNAc, uridine diphosphate-N-acetylglucosamine; mAb, monoclonal antibody; bp, base pair(s); PCR, polymerase chain reaction; ORF, open reading frame; HPTLC, high performance thin layer chromatography; SGGL, sulfoglucuronylglycolipid; GSL, glycosphingolipid; Lea, Lewis a; Leb, Lewis b; Lex, Lewis x; SSEA-1, stage-specific embryonal antigen-1; LacCer, lactosylceramide (Galbeta 1-4Glcbeta 1-1Cer); Lc3Cer, lactotriaosylceramide (GlcNAcbeta 1-3Galbeta 1-4Glcbeta 1-1Cer); nLc4Cer, neolactotetraosylceramide (paragloboside: Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4Glcbeta 1-1Cer); nLc5Cer, GlcNAcbeta 1-3Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4Glcbeta 1-1Cer; nLc6Cer, Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4Glcbeta 1-1Cer; PA, pyridylaminated; 2AB, 2-aminobenzamide; LNnT, lacto-N-neotetraose (Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4Glc); LNT, lacto-N-tetraose (Galbeta 1-3GlcNAcbeta 1-3Galbeta 1-4Glc); LNFP-II, lacto-N-fucopentaose-II (Galbeta 1-3(Fucalpha 1-4)GlcNAcbeta 1-3Galbeta 1-4Glc); LNFP-III, lacto-N-fucopentaose-III (Galbeta 1-4(Fucalpha 1-3)GlcNAcbeta 1-3Galbeta 1-4Glc); LNFP-V, lacto-N-fucopentaose-V (Galbeta 1-3GlcNAcbeta 1-3Galbeta 1-4(Fucalpha 1-3)Glc); LNDFH-II, lacto-N-difucohexaose-II (Galbeta 1-3(Fucalpha 1-4)GlcNAcbeta 1-3Galbeta 1-4(Fucalpha 1-3)Glc); LN, N-acetyllactosamine (Galbeta 1-4GlcNAc); 2LN, Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4GlcNAc; 3LN, (Galbeta 1-4GlcNAcbeta 1-3)2Galbeta 1-4GlcNAc; 4LN, (Galbeta 1-4GlcNAcbeta 1-3)3Galbeta 1-4GlcNAc; 5LN, (Galbeta 1-4GlcNAcbeta 1-3)4Galbeta 1-4GlcNAc; RA, retinoic acid; TPA, 12-O-tetradecanoylphorbol-13-acetate; RT-PCR, reverse transcriptase-polymerase chain reaction; GM3 synthase, lactosylceramide:alpha 2,3-sialyltransferase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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