Cloning, Expression, and Characterization of a Novel UDP-galactose:beta -N-Acetylglucosamine beta 1,3-Galactosyltransferase (beta 3Gal-T5) Responsible for Synthesis of Type 1 Chain in Colorectal and Pancreatic Epithelia and Tumor Cells Derived Therefrom*

Soichiro IsshikiDagger §, Akira TogayachiDagger parallel , Takashi KudoDagger , Shoko NishiharaDagger , Masahiko Watanabe§, Tetsuro Kubota§, Masaki Kitajima§, Norihiko Shiraishi**, Katsutoshi Sasaki**, Toshiwo Andohparallel , and Hisashi NarimatsuDagger Dagger Dagger

From the Dagger  Division of Cell Biology, Institute of Life Science, and parallel  Department of Bioengineering, Faculty of Engineering, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577, the § Department of Surgery, Keio University School of Medicine, Shinjuku-ku, Tokyo 160-8582, and ** Tokyo Research Laboratories, Kyowa Hakko Kogyo Company Limited, 3-6-6 Asahi-machi, Machida-shi, Tokyo 194-8533, Japan

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EXPERIMENTAL PROCEDURES
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The sialyl Lewis a antigen is a well known tumor marker, CA19-9, which is frequently elevated in the serum in gastrointestinal and pancreatic cancers. UDP-galactose:N-acetylglucosamine beta 1,3-galactosyltransferase(s) (beta 3Gal-Ts) are required for the synthesis of the sialyl Lewis a epitope. In the present study, a novel beta 3Gal-T, named beta 3Gal-T5, was isolated from a Colo205 cDNA library using a degenerate primer strategy based on the amino acid sequences of the four human beta 3Gal-T genes cloned to date. Transfection experiments demonstrated that HCT-15 cells transfected with the beta 3Gal-T5 gene expressed all the type 1 Lewis antigens. In gastrointestinal and pancreatic cancer cell lines, the amounts of beta 3Gal-T5 transcripts were quite well correlated with the amounts of the sialyl Lewis a antigens. The beta 1,3Gal-T activity toward agalacto-lacto-N-neotetraose was also well correlated with the amounts of beta 3Gal-T5 transcripts in a series of cultured cancer cells, and in Namalwa and HCT-15 cells transfected with the beta 3Gal-T5 gene. Thus, the beta 3Gal-T5 gene is the most probable candidate responsible for the synthesis of the type 1 Lewis antigens in gastrointestinal and pancreatic epithelia and tumor cells derived therefrom. In addition, beta 3Gal-T5 is a key enzyme that determines the amounts of the type 1 Lewis antigens including the sialyl Lewis a antigen.

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CA19-9 in serum is a well known tumor marker, which is frequently used for the clinical diagnosis of cancer, in particular, colorectal, pancreatic, and gastric cancers (1, 2). The 1116NS19-9 (19-9)1 monoclonal antibody detects a CA19-9 antigen, of which the antigenic epitope has been defined as the carbohydrate structure of sialyl Lewis a (sLea) (2-4). Besides its usefulness as a tumor marker, sLea antigen is known to be a ligand for selectins (5, 6). Clinical statistical analysis demonstrated that cancer patients who express abundant sLea antigens have a worse prognosis as to liver metastasis than patients who do not express sLea antigens (7, 8). Thus, it is of interest that sLea antigens may confer some metastatic capacity on cancer cells.

At least three glycosyltransferases are required for the synthesis of the sLea epitope. First, N-acetylglucosamine-beta 1,3-galactosyltransferase (beta 3Gal-T) transfers a galactose (Gal) to an N-acetylglucosamine (GlcNAc) with a beta 1,3-linkage, resulting in the synthesis of a type 1 chain, Galbeta 1,3GlcNAc, and then galactose-alpha 2,3-sialyltransferase (ST3Gal) transfers a sialic acid (SA) to the Gal residue of the type 1 chain with an alpha 2,3-linkage, resulting in sialyl-type 1 (sialyl Lewis c; sLec) chain, SAalpha 2,3Galbeta 1,3GlcNAc, synthesis. Finally, alpha 1,3/4-fucosyltransferase (Fuc-TIII, FUT3, Lewis enzyme) transfers a fucose (Fuc) to the GlcNAc residue of the sialyl-type 1 chain with an alpha 1,4-linkage to complete the synthesis of the structure, SAalpha 2,3Galbeta 1,3(Fucalpha 1,4)GlcNAc. In previous studies, we demonstrated that Fuc-TIII (FUT3) is the only enzyme determining the expression of sLea antigens in colorectal cancer (3, 4), and that ST3GalIV, one of the ST3Gals, mainly participates in the sLea synthesis in colorectal cancer (9).

Regarding beta 3Gal-Ts, we have reported for the first time the cloning of a beta 3Gal-T gene from human WM266-4 melanoma cells using an expression cloning method (10). The recent rapid growth of data bases of expressed sequence tags (ESTs) and the Human Genome Project enabled us to find novel genes homologous to the original one. Thus, three human beta 3Gal-T genes homologous to the original one were cloned very recently (11, 12). The four beta 1,3GalTs, including the original one, are named beta 3Gal-T1 to -T4 (12). Expression studies on the four human beta 3Gal-Ts demonstrated that two of them, beta 3Gal-T1 and T2, apparently transfer Gal to GlcNAc with a beta 1,3-linkage resulting in type 1 chain synthesis, but beta 3Gal-T4 transfers Gal to an N-acetylgalactosamine (GalNAc) residue, resulting in the synthesis of the type 3 chain, Galbeta 1,3GalNAc (12). The human beta 3Gal-T4 did not transfer Gal to a GlcNAc residue for the type 1 chain synthesis (12). The human beta 3Gal-T4 is likely to be the human homologue of the rat GM1/GD1 synthase (13), since the amino acid sequence of human beta 3Gal-T4 shows very high homology, 79.4%, to that of the rat GM1/GD1 synthase, and the human beta 3Gal-T4 apparently transfers Gal to the GalNAc residue of asialo-GM2 and GM2, resulting in the asialo-GM1 and GM1 synthesis, respectively (12). The activity of human beta 3Gal-T3 has not been detected toward any of the acceptor substrates used in their study (12). Three mouse beta 3Gal-T genes have been cloned and named mbeta 3GalT-I, mbeta 3GalT-II, and mbeta 3GalT-III, corresponding to human beta 3Gal-T1, beta 3Gal-T2, and beta 3Gal-T3, respectively (14). mbeta 3Gal-TII and mbeta 3GalT-III were found to exhibit the beta 3Gal-T activity toward both GlcNAc and GalNAc residues; however, they showed quite low activities for the type 1 chain synthesis, i.e. about 3% of the activity of mbeta 3GalT-I (14).

It has not been elucidated which beta 3Gal-T determines the expression of the sLea epitopes in gastrointestinal and pancreatic cancers. The tissue distributions of the four beta 3Gal-Ts were determined by Northern analysis (11, 12), it being found that neither beta 3Gal-T1 nor -T2 is expressed in the pancreas, which indicated that there may be unknown beta 3Gal-T(s) synthesizing the type 1 chain in the pancreas. They did not examine the expression of those beta 3Gal-Ts in the gastrointestinal tissues, such as colon and stomach, which frequently produce the sLea antigens when they become cancerous.

In this study, we first noticed that none of the four human beta 3Gal-Ts cloned to date, beta 3Gal-T1 to -T4, is responsible for the sLea expression in gastrointestinal and pancreatic cancers, and successfully cloned a novel beta 3Gal-T gene, named beta 3Gal-T5, from Colo205 cells. beta 3Gal-T5 is the most probable candidate participating in the synthesis of the sLea epitopes, i.e. CA19-9 antigens, in gastrointestinal and pancreatic cancer cells.

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Tumor Cell Lines and Monoclonal Antibodies-- Various tumor cell lines were cultured in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum. The monoclonal antibodies used in this study were as follows: 1116NS19-9 (19-9), which was used for the detection of tumor marker CA19-9, anti-sLea (IgG) (2); DU-PAN-2, anti-sLec (IgM) (4, 15); 7LE, anti-Lea (IgG) (16); and TT42, anti-Leb (IgM), of which the specificity against Leb was recently defined by us, and will be published elsewhere. 19-9 and TT42 were kind gifts from Fujirebio Inc. (Toyko, Japan), and Diagnostic Division, Otsuka Pharmaceutical Co. Ltd. (Tokushima, Japan), respectively. DU-PAN-2 and 7LE were purchased from Kyowa Medex Co., Ltd. (Tokyo, Japan), and Seikagaku-Kogyo Co., Ltd. (Tokyo, Japan), respectively.

Cloning of the Four Cloned beta 3Gal-T Genes from Various Human cDNA Libraries and Construction of Expression Plasmids-- The cDNA encoding beta 3Gal-T1 was cloned by the expression cloning method used in our previous study (10). We found three sequences homologous to that of beta 3Gal-T1 in the EST data bases. The full-length cDNAs encoding the other three homologous sequences were cloned from various human cDNA libraries using probes encoding the fragment sequences in the EST data base. They were identical to beta 3Gal-T2, -T3, and -T4, which were reported by Amado et al. (12), and Kolbinger et al. (11).

Thus, the four human cDNAs encoding the respective full-length open reading frames (ORFs) of beta 3Gal-T1, -T2, -T3, and -T4 were subcloned into the pAMo vector for expression in cultured cells (17, 18).

Construction of a cDNA Library from Colo205 Cells-- Total cellular RNA was isolated from Colo205 cells using the acid guanidium thiocyanate-phenol-chloroform method (19). Poly(A)+-rich-RNA was isolated with OligotexTM-dT30 (Super) (Roche, Tokyo, Japan). Complementary DNAs were synthesized with oligo(dT) primers from poly(A)+-rich-RNA using a Superscript Choice System for cDNA Synthesis (Life Technologies, Inc.). A cDNA library was constructed by inserting size-fractionated cDNAs (more than 1.5 kilobase pairs) into an expression vector, pAMo, using SfiI adaptors (17, 18). We obtained about 1 × 106 independent clones as a cDNA library and extracted plasmid DNAs from the library.

PCR for Cloning of a Fragment Encoding a Novel beta 3Gal-T-- On alignment of the amino acid sequences of the four cloned beta 3Gal-Ts, we found conserved amino acid sequences at three positions, and named them Motifs 1, 2, and 3 (Table I). The conserved amino acid sequences of Motifs 1, 2, and 3 were employed for the design of degenerate primer sequences, i.e. primers at-1 (5' primer for Motif 1), 5'-GCI AT(A/C/T) (A/C)GI CA(A/G) ACI TGG GG-3'; at-2 (3' primer for Motif 2), 5'-(A/G)TC (A/G)CT (A/G)TC IGT (C/T)TT CAT IAC (A/G)TA-3'; at-3 (5' primer for Motif 2), 5'-TA(C/T) GTI ATG AA(A/G) ACI GA(C/T) TCI GA(C/T)-3'; and at-4 (3' primer for Motif 3), 5'-(A/G)CA IA(A/G) ICC IAC (A/G)TA IAC (A/G)TC (C/T)TC-3', respectively.

                              
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Table I
Conserved amino acid motifs in the beta 3Gal-T family

The cDNAs of the Colo205 cDNA library described above were used as templates for PCR amplification to obtain a DNA fragment. Two PCRs were performed with two sets of degenerate primers, respectively, i.e. the first PCR was performed with primers at-1 and at-2, and the second PCR with primers at-3 and at-4. The amplified PCR products were inserted into a pBluescript SK (-) (pBS) vector (Stratagene, La Jolla, CA), and the DNA fragments obtained were sequenced by the dideoxynucleotide chain termination method using an ALF DNA sequencer (Amersham Pharmacia Biotech, Uppsala, Sweden). Two fragment DNAs contained novel nucleotide sequences, which, however, were homologous to the corresponding regions of the cloned beta 1,3GalT genes. On an additional PCR involving Colo205 cDNAs as templates using primers encompassing the two fragment sequences, both fragments were found to be encoded by one species of cDNA.

Cloning of Full-length cDNAs Encoding a Novel beta 3Gal-T-- The two DNA fragments obtained through the two PCRs, i.e. those with the Motif 1 and 2 primers, and the Motif 2 and 3 primers, respectively, were mixed and used as the probe for hybridization to isolate full-length cDNA clones. We screened the Colo205 cDNA library and isolated several distinct clones having inserts of different sizes. All inserts encoded the same sequence of one species of cDNA, this sequence being found to be homologous to those of the known four beta 3Gal-Ts. Thus, we named this novel gene the human beta 3Gal-T5 gene. After the cDNA sequences had been completed, we searched the data base of the Human Genome Project to determine whether the same sequence or homologous ones were registered or not. We found a genome sequence completely identical to the cDNA sequences in the data base, which was very recently registered (June 2nd, 1998). Its registration number is AF064860. By comparison between the cDNA sequences and the genome one, we determined the genomic organization of the beta 3Gal-T5 gene.

Identification of Alternatively Spliced Isoforms of beta 3Gal-T5 Transcripts and the Transcription Initiation Site-- By the 5'-rapid amplification of cDNA ends (5'-RACE) method using two primers, i.e. si-1 (5'-GAAAGGATTTAGACTGTACATGC-3'), this sequence being positioned close to the ATG codon in the ORF, and si-2 (5'-GTGAATTCCTCTTTCTCTGCTG-3'), we obtained five different types of amplified fragments, and subcloned them into a pBS (-) vector for sequencing. Thus, five isoforms, isoforms 1, 2, 3, 4, and 5, of beta 3Gal-T5 transcript were identified. Further RT-PCR experiments were performed to determine the abundance of each isoform of the beta 3Gal-T5 transcript expressed in Colo205 cells, for which the following primers were employed, primers si-1, si-2, si-3 (5'-TGAAAGGAACAAAATCCAATGAT-3'), and si-4 (5'-AGAACCCTGACTAATACACCTGGA-3').

Quantitative Analysis of the five beta 1,3GalT Transcripts in Human Tumor Cell Lines and Human Tissues by Competitive RT-PCR-- The principle of the competitive RT-PCR method was described in detail in our previous papers (9, 18). Competitor DNA plasmids each carrying a small deletion within the respective full-length ORF cDNA were constructed by appropriate restriction endonuclease digestion as shown in Table II. For instance, a competitor DNA plasmid of the beta 3Gal-T1 gene was prepared by deleting the 212-bp BanII-EcoRV fragment from the standard plasmid DNA containing the full-length cDNA of beta 3Gal-T1.

Total cellular RNA was isolated from various tumor cell lines and human tissues. Complementary DNAs were synthesized with an oligo(dT) primer from 6 µg of DNase I-treated total RNA in a 20-µl (total volume) reaction mixture using a SuperscriptTM Preamplification System for First Strand cDNA Synthesis (Life Technologies, Inc.). After cDNA synthesis, the reaction mixture was diluted 50-fold with H2O and then stored at -80 °C until use.

The competitive RT-PCR was performed with AmpliTaq GoldTM (Perkin Elmer) in a 50-µl (total volume) reaction mixture comprising 10 µl of standard plasmid DNA or sample cDNA, 10 µl of competitor DNA at the optimal concentration, which differs with the transcript, and 0.2 µM amounts of each primer of the gene-specific primer sets listed in Table II. The PCR buffer for the competitive RT-PCR comprised 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 µM of each dNTP, and 0.001% (w/v) gelatin. PCR was performed with a pre-PCR heat step at 95 °C for 11 min, followed by the optimal number of PCR cycles, each of which comprised 1 min at 95 °C, 1 min at the optimal annealing temperature (Table II), and 2 min at 72 °C. After the competitive RT-PCR, a 10-µl aliquot was electrophoresed in a 1% agarose gel and the bands were visualized by ethidium bromide staining. The intensities of the amplified fragments were quantified by scanning positive pictures using the public domain NIH Image program.2 Measurement of the beta -actin transcript in each sample was performed using the same competitive RT-PCR method as for the beta 3Gal-T transcripts. Each value for the beta 3Gal-T and beta -actin transcripts was plotted on the respective standard curve to obtain the actual amount of each transcript. The actual amount of each beta 3Gal-T transcript was divided by that of beta -actin for normalization.

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

Transfection Experiments to Express the Five Human beta 3Gal-T Genes in Namalwa (Burkitt Lymphoma) and HCT-15 Cells-- Each of the five beta 3Gal-T genes subcloned into the pAMo vector was stably transfected by the electroporation method into Namalwa or HCT-15 cells. These cells were selected in the presence of Geneticin (G418) (Life Technologies, Inc.) at a concentration of 0.8 mg/ml in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum. Stable transformant cells were obtained after 25 days of exposure to Geneticin. Cell homogenates of stable transformants expressing each of the beta 3Gal-T genes were subjected to assaying of beta 3Gal-T activity. The levels of the transcripts expressed in the transformant cells were measured by means of competitive RT-PCR to normalize the beta 3Gal-T activity. The stable transformants of HCT-15 cells were subjected to limiting dilution to obtain single transformant clones.

Flow Cytometry Analysis-- The expression of type 1 Lewis antigen epitopes, i.e. the Lea, Leb, sLec, and sLea epitopes, on the surface of the cultured tumor cells and the cells transfected with each of the beta 3Gal-T genes was examined by flow cytometry analysis using an Epics Elite (Coulter, Tokyo, Japan). The transfected cells (1 × 106) were incubated with a first antibody (10 µg/ml) for 1 h on ice, and then washed twice with PBS (pH 7.4) containing 1% BSA and 0.1% sodium azide, followed by incubation with fluorescein isothiocyanate-conjugated anti-mouse IgM or IgG (Bio-Rad). Then, the cells were washed again with PBS-BSA and finally subjected to flow cytometry analysis.

Western Blotting Analysis-- Cell pellets were solubilized in 20 mM HEPES buffer (pH 7.2) containing 2% Triton X-100 by brief sonication. Proteins separated on 6% SDS-polyacrylamide gel electrophoresis were transferred to an Immobilon PVDF membrane (Millipore, Bedford, MA) in a Transblot SD cell (Bio-Rad). The membrane was blocked with PBS containing 5% skim milk at 4 °C overnight and then incubated with 10 µg/ml 19-9. The membrane was stained according to the manual with the ECL Western blotting detection reagents (Amersham Pharmacia Biotech).

Assaying of beta 3Gal-T Activity-- Lacto-N-neotetraose (LNnT) was pyridylaminated as in the previous study (17). The pyridylaminated-LNnT (LNnT-PA) was digested with 20 milliunits/ml streptococcal beta -galactosidase (Seikagaku-Kogyo) to remove the galactose residue at the nonreducing end. Thus, agalacto-LNnT-PA was prepared, and used for assaying beta 3Gal-T activity. Namalwa and HCT-15 cells tranfected stably with each of the beta 3Gal-T genes, and various cultured cells were solubilized in 20 mM HEPES buffer (pH 7.2) containing 2% Triton X-100. The beta 1,3Gal-T activity was assayed in 14 mM HEPES buffer (pH7.4), 75 µM UDP-Gal, 11 µM MnCl2, 0.01% Triton X-100, and 25 µM acceptor substrate. After incubation at 37 °C for 2 h, the enzyme reactions were terminated by boiling for 3 min., followed by dilution with water. After centrifugation of the reaction mixtures at 15,000 rpm for 5 min., 10 µl of each supernatant was subjected to high performance liquid chromatography analysis on a TSK-gel ODS-80TS column (4.6 × 300 mm; Tosoh, Tokyo, Japan). The reaction products were eluted with 20 mM ammonium acetate buffer (pH 4.0) at the flow rate of 1.0 ml/min at 25 °C and monitored with a Jasco FP-920 fluorescence spectrophotometer (Jasco, Tokyo, Japan).

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Discrepancy between the Expression Levels of three beta 3Gal-T Transcripts, i.e. the beta 3Gal-T1, -T2, and -T3 Transcripts, and the Amounts of Type 1 Lewis Antigens Expressed in Various Tumor Cells-- Various tumor cell lines derived from different human tissues were examined as to the transcript levels of the four beta 3Gal-T genes that were cloned previously, and their expression levels were compared with the amounts of type 1 Lewis antigens, i.e. the sLea (19-9), Lea (7LE), and Leb (TT42) antigens, expressed in these cancer cells (Fig. 1). Flow cytometry analysis revealed that Colo205, Colo201, and SW1116 (colon cancer) cells, and Capan-2 (pancreatic cancer) cells expressed large amounts of type 1 Lewis antigens, these results being consistent with those of a previous study (20). All the above cell lines except for Capan-2 were strongly stained with the three antibodies, i.e. 19-9 (anti-sLea), 7LE (anti-Lea), and TT42 (anti-Leb) antibodies. Capan-2 cells were also strongly stained with 19-9, but not with 7LE or TT42. However, these four types of cells did not express beta 3Gal-T1 or beta 3Gal-T2 transcripts at all. The expression of beta 3Gal-T1 was abundantly detected in PC-1 (lung cancer) cells, and faintly detected in Jurkat (T cell leukemia) and PC-3 (prostatic cancer) cells. beta 3Gal-T2 was expressed in Namalwa and SK-N-MC (neuroblastoma) cells at intermediate levels, and faintly expressed in some cell lines from gastrointestinal cancers. beta 3Gal-T3 was abundantly expressed in some gastric cancer cells, i.e. KATO III and MKN45 cells, and PC-3 cells, and intermediately expressed in Capan-1 and Capan-2 (pancreatic cancer) cells, HCT-15 cells, and SK-N-MC and SK-N-SH (neuroblastoma) cells; however, it was not detected at all in Colo201 or Colo205 cells.


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Fig. 1.   Quantitative analysis of transcripts of beta 3Gal-T1, -T2, -T3, and -T4 in various human tumor cells by competitive RT-PCR, and flow cytometry analysis for type 1 Lewis antigens on these cells. A, for the quantitation of each transcript, each single-stranded cDNA was amplified together with 200 ag/µl of the respective competitor DNA. The human beta -actin transcripts were quantified with 200 fg/µl of the competitor DNA. The value for each beta 3Gal-T transcript was divided by that for the respective beta -actin transcript. B, the positive peaks observed on flow cytometry analysis are indicated as - to +++, depending on the intensity. NT, not tested.

We observed a significant discrepancy between the expression of the three beta 3Gal-Ts, i.e. beta 3Gal-T1, -T2, and -T3, and the expression of the type 1 Lewis antigens in these cell lines. In contrast, the expression of beta 3Gal-T4 appeared to be correlated with the type 1 Lewis antigen expression, i.e. the cells expressing type 1 Lewis antigens, i.e. Colo201, Colo205, SW1116, HT-29, and Capan-2 cells, also expressed substantial amounts of the beta 3Gal-T4 transcript.

beta 3Gal-T1, -T2, and -T3 could synthesize the type 1 chain (11, 12, 14); however, they were not correlated with the expression of type 1 Lewis antigens in the present study. The expression of beta 3Gal-T4 seemed to be correlated with the expression of type 1 Lewis antigens in some tumor cells; however, it could not synthesize the type 1 chain (12). From these results, we concluded that none of the four beta 3Gal-Ts is responsible for type 1 chain synthesis, resulting in sLea (CA19-9) antigen expression, in gastrointestinal and pancreatic cancer cells.

Cloning and Sequence of a Novel cDNA Homologous to the Cloned beta 3Gal-Ts-- As described under "Experimental Procedures," we obtained two DNA fragments encoding novel sequences, which, however, are homologous to the corresponding regions of the four cloned beta 3Gal-T genes. The sequences of the two DNA fragments were found to be encoded by a single cDNA species. We named this gene beta 3Gal-T5. By use of the DNA fragments as probes, full-length cDNA clones were obtained from the Colo205 cDNA library. Complementary DNA sequencing analysis revealed that the beta 3Gal-T5 cDNA contains an ORF encoding a protein of 310 amino acids (Fig. 2). The position of the AUG start codon was assigned according to the Kozak consensus sequence (21). A hydropathy profile based on the Kyte and Doolittle method (22) indicated that the ORF encodes a type II membrane protein, which is a typical feature of glycosyltransferases (data not shown). The three motifs of amino acid sequences, Motifs 1, 2, and 3, which we employed for the design of degenerate primers in this study, were conserved in the sequence of beta 3Gal-T5. Four cysteine residues were conserved in the five beta 3Gal-Ts, which indicates that some of these cysteines are essential for maintenance of the tertiary structures of beta 3Gal-Ts. Fifty-four of the 310 amino acid residues of beta 3Gal-T5 were conserved in comparison with the sequences of the other four beta 3Gal-Ts. Three possible N-glycosylation sites were found in the primary sequence of beta 3Gal-T5.


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Fig. 2.   Nucleotide sequence of the beta 3Gal-T5 cDNA and the predicted amino acid sequence of beta 3Gal-T5. A putative transmembrane domain is boxed. Possible N-glycosylation sites are indicated by double underlines. Possible polyadenylation signals are single underlined. Exon-intron junctions are indicated. The four squares indicate cysteine residues conserved in the five beta 3Gal-Ts. The shaded amino acid letters indicate the three motifs conserved in the five beta 3Gal-Ts.

Genomic Structure of the beta 3Gal-T5 Gene and Alternatively Spliced Isoforms of Transcripts-- By comparison of the full-length cDNA sequence with the genome sequence, which has been registered in the Genome Project Database (registration no. AF064860), the chromosomal localization and the genomic structure of the beta 3Gal-T5 gene were determined (Fig. 3A). According to the description in GenBank, this gene is localized to human chromosome 21q22.3. The ORF of the beta 3Gal-T5 gene was found to be encoded by a single exon, as in the cases of the four cloned beta 3Gal-T genes. The A nucleotide of the translation initiation codon, ATG, was found to be the first nucleotide of exon 4 encoding the ORF.


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Fig. 3.   Genomic structure of the beta 3Gal-T5 gene. A, the four exons are shown as boxes, and introns as lines. The restriction sites in exon 2, XbaI, and in exon 3, BsmI, are indicated. ORF is indicated by a hatched box. B, structures of five isoforms of beta 3Gal-T5 transcripts. *, ratio means the abundance as a percentage of abundance of each isoform in Colo205 cells. C, RT-PCR analysis to determine abundance of the five isoforms of beta 3Gal-T5 transcripts. Molecular weight markers (100-bp ladders) are on the leftmost lane.

The sequence of the 5'-flanking region was extended by the 5'-RACE method using Colo205 transcripts. All subclones obtained with the 5'-RACE method had a common nucleotide sequence at the 5' end, i.e. all of them started at nucleotide position 85153 in the genome sequence AF064860 (Table III). Thus, five isoforms of beta 3Gal-T5 transcripts, isoforms 1, 2, 3, 4, and 5, were identified with the 5'-RACE method (Fig. 3B). All intron sequences at the exon-intron junctions complied with the acceptor and donor site sequences of splicing rule, i.e. the GT-AG rule (Table III). Each transcript of the five isoforms in Colo205 cells was quantified by determining the intensity of its band on a RT-PCR gel (Fig. 3C). As shown in Fig. 3A, exon 2 or 3 contains an XbaI or BsmI restriction site, respectively. The amplified bands on RT-PCR were digested with XbaI or BsmI to confirm exon 2 or 3, respectively. As shown in Fig. 3C, two bands were obtained on RT-PCR using the primer set, si-2 and si-1. The upper and lower bands correspond to isoform 1, consisting of exons 1, 3, and 4, and isoform 2, consisting of exons 1', 3, and 4, respectively, and both materials were digested by BsmI, but not by XbaI. The ratio of the band intensities of isoforms 1 and 2 was about 1 to 1. The other three isoforms, isoforms 3, 4, and 5, were not detected on this RT-PCR, which indicated that isoforms 3, 4, and 5 are minor transcripts in Colo205 cells. RT-PCR with primers si-4 and si-1 gave a single band for isoform 3, which was digested by both BsmI and XbaI. A band for isoform 4 was not detected on this RT-PCR, because the amount of isoform 4 transcripts may be very small. RT-PCR with primers si-2 and si-3 gave a single band for isoform 5. Isoform 1 and 2 were abundant among the five isoforms, and both isoforms amounted to approximately 50% of the total transcripts of the beta 3Gal-T5 gene, respectively. The other three isoforms, i.e. isoforms 3, 4, and 5, were only present in trace amounts.

                              
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Table III
Exon-intron junctions of the beta 3Gal-T5 gene

Correlation of the Expression Levels of the beta 3Gal-T5 Transcripts with the Amounts of Type 1 Lewis Antigens in Various Tumor Cells-- As can be seen in Fig. 4, the expression levels of beta 3Gal-T5 transcripts and the amounts of CA19-9 antigens in various cancer cells were determined by the competitive RT-PCR method and Western blot analysis, respectively. The results of flow cytometry analysis in Fig. 1 are well consistent with those of Western blot analysis in this section, i.e. the four types of cells, i.e. Colo205, Colo201, SW1116, and Capan-2 cells, that were stained strongly with 19-9 on flow cytometry also gave strong positive bands, which were smear ones with high molecular weights indicating they are mucins, with 19-9 on Western blotting (Fig. 4). These four cell lines also expressed abundant beta 3Gal-T5 transcripts (Fig. 4). The other cell lines, HT-29, WiDr, and Capan-1 cells, intermediately expressed the beta 3Gal-T5 gene.


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Fig. 4.   Quantitative analysis of beta 3Gal-T5 transcripts by competitive RT-PCR, and Western blot analysis of CA19-9 antigens in various cancer cells. A, the expression levels of beta 3Gal-T5 transcripts in various cancer cells are shown as a bar chart. The results of actual gel electrophoresis after competitive RT-PCR are shown under the bars. B, Western blot analysis of various cancer cells with 19-9 (anti-sLea).

The Ability of Type 1 Chain Synthesis of beta 3Gal-T5 in Transfected Cells-- Namalwa cells do not possess the type 1 chain and lack alpha 1,3/4-fucosyltransferase (Fuc-TIII), which is the only enzyme capable of the synthesis of type 1 Lewis antigens, such as the Lea, Leb, and sLea antigens. Therefore, Namalwa cells transfected stably with the beta 3Gal-T5 gene were stained with DU-PAN-2 (anti-sLec), which recognizes the precursor structure, SAalpha 2,3Galbeta 1,3GlcNAc, of the sLea epitope (3, 4). As can be seen in Fig. 5, Namalwa cells transfected stably with the beta 3Gal-T5 gene gave positive peaks with DU-PAN-2. HCT-15 cells were chosen as the host cells for the transfection experiment with the beta 3Gal-T5 gene for the following reasons. First, they are cancer cells derived from colon tissue. Second, they are known to express substantial amounts of Fuc-TIII and ST3GalIV (data not shown), but not to express beta 3Gal-T5 at all (Fig. 4). A single transformant clone of HCT-15 cells, which had been transfected stably with the beta 3Gal-T5 gene, was obtained by the limiting dilution method and named HCT-3GT5H. Flow cytometry analysis of HCT-3GT5H cells apparently showed positive peaks with the antibodies against all type 1 Lewis antigens (Fig. 5). These results confirmed that beta 3Gal-T5 can synthesize the type 1 chain in transformant cells, i.e. not only in Namalwa cells but also in colon cancer (HCT-15) cells.


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Fig. 5.   Flow cytometry analysis of Namalwa cells and HCT-15 cells transfected stably with the beta 3Gal-T5 cDNA. The shaded peak in each panel represents the results of staining without the first antibody as a negative control. The results for mock-transfected Namalwa or HCT-15 cells are presented in the left panels. The results for Namalwa-3GT5 or HCT-3GT5H cells, which were stably transfected with the beta 3Gal-T5 gene, are presented in the right panels.

beta 3Gal-T Activity toward Agalacto-LNnT in Various Tumor Cells and HCT-3GT5 Cells-- Agalacto-LNnT-PA was used as an acceptor substrate to measure beta 3Gal-T activity, resulting in the synthesis of lacto-N-tetraose-PA (LNT-PA). The beta 3Gal-T activity, i.e. the LNT-PA synthesizing activity, in Namalwa cells transfected with the beta 3Gal-T5 gene, Namalwa-3GT5, was strongest among all samples of transfected cells and cultured cancer cells examined. Thus, the activity of Namalwa-3GT5 is expressed as 100% activity, and the beta 3Gal-T activities of the other samples relative to that of Namalwa-3GT5 cells are presented in Table IV. The amounts of transcripts for individual beta 3Gal-T genes were measured by competitive RT-PCR, and the relative amounts of individual transcripts normalized as to the amounts of beta -actin transcripts are shown in Table IV. The level of beta 3Gal-T activity synthesizing LNT-PA was quite parallel to the amounts of beta 3Gal-T5 transcripts, i.e. the cell homogenate of Colo205 showed the strongest activity among the cultured cancer cells, followed by those of SW1116 and Capan-2. This indicated that the LNT-PA synthesizing activity is mainly directed by beta 3Gal-T5. Mock-transfected HCT-15 (HCT-mock) cells did not exhibit any activity, but HCT-3GT5H cells exhibited strong activity almost equal to that of Colo205 cells. HCT-3GT5L cells, which expressed almost one-third of the amount of beta 3Gal-T5 transcripts in the HCT-3GT5H cells, showed one-third of the beta 3Gal-T activity of HCT-3GT5H cells. The cells expressing substantial amounts of transcripts for the other four beta 3Gal-T genes, i.e. the beta 3Gal-T1, -T2, -T3, and -T4 genes, did not exhibit LNT-PA synthesizing activity at all. Namalwa cells stably expressing beta 3Gal-T1, -T2, -T3 and -T4, which were named Namalwa-3GT1, -3GT2, -3GT3, and -3GT4 cells, respectively, also did not exhibit any LNT-PA synthesis activity.

                              
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Table IV
beta 1,3-Galactosyltransferase activity toward agalacto-LNnT

The above results confirmed that the beta 3Gal-T activity synthesizing LNT-PA is mainly directed by beta 3Gal-T5 in these cells, not by the other four beta 3Gal-Ts.

Tissue Distribution and Quantitative Measurement of the beta 3Gal-T5 Transcripts-- As can be seen in Fig. 6, beta 3Gal-T5 transcripts were substantially detected in the stomach, jejunum, colon, and pancreas, which are known to express sLea antigens frequently when they become cancerous. On the other hand, they were hardly detected in the lungs, liver, spleen, adrenal glands, and peripheral blood leukocytes, which rarely produce sLea antigens when they become malignant. This strongly suggested that beta 3Gal-T5 is responsible for the type 1 chain synthesis, resulting in the sLea antigen synthesis in gastrointestinal and other tissues.


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Fig. 6.   Quantitative analysis of beta 3Gal-T transcripts in various human tissues by competitive RT-PCR. For the quantitation of beta 3Gal-T5 transcripts, each single-stranded cDNA was amplified together with 200 ag/µl of the respective competitor DNA. The human beta -actin transcripts were quantified with 500 fg/µl of the competitor DNA. The amount of beta -actin differed with the tissue.

Multiple Sequence Alignment (ClustalW) of the Five Members of the Human beta 3Gal-T Family-- Multiple amino acid sequence alignment of the five beta 3Gal-Ts was constructed by ClustalW method (Fig. 7). Three conserved amino acid motifs, which were employed for design of the degenerate primers, and four conserved cysteine residues were indicated in Fig. 7. One possible N-glycosylation site was also conserved in all five hbeta 3Gal-Ts.


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Fig. 7.   Multiple sequence alignment (ClustalW) of the five human beta 3Gal-Ts. Multiple amino acid sequences of the five human beta 3Gal-Ts are shown. Introduced gaps are shown as hyphens. Three conserved motifs used for design of degenerate primers were shaded. Putative transmembrane domains are boxed, the four conserved cysteine residues are squared, and the conserved possible N-glycosylation site is double underlined. Asterisks indicate the amino acids conserved in the five beta 3Gal-Ts.

A phylogenetic tree of the five beta 3Gal-Ts was constructed by means of the neighbor-joining method based on the amino acid sequences (data not shown) (23). The position of beta 3Gal-T5 in the tree is closer to those of beta 3Gal-T1 and -T2, which can synthesize the type 1 chain as reported by us and others (10-12), than to those of beta 3Gal-T3 and -T4. These three enzymes, beta 3Gal-T1, -T2, and -T5, form a subfamily on the phylogenetic tree.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression of the sLea and sLec epitopes, which are mainly carried on the carbohydrate chains of mucins, is frequently elevated in gastrointestinal and pancreatic cancers. The sLea antigens are known to be some of the factors determining the prognoses of colorectal and gastric cancer patients (7, 8, 24). In this sense, it is very important to identify the beta 3GalT(s) responsible for type 1 chain synthesis, which results in the synthesis of the sLec and sLea epitopes in gastrointestinal and pancreatic cancers. The novel beta 3Gal-T, i.e. beta 3Gal-T5, isolated in the present study was demonstrated to be responsible for the type 1 chain (including the sLea and sLec antigen) synthesis in gastrointestinal and pancreatic cancers by the following evidence. 1) beta 3Gal-T5 could synthesize type 1 chains, leading to expression of Lea, Leb, sLec and sLea in transfected cells, i.e. in Namalwa-3GT5 and HCT-3GT5H cells. 2) beta 3Gal-T5 was expressed in cultured colon and pancreatic cancer cells expressing substantial amounts of type 1 chains, whereas the other three beta 3Gal-Ts, i.e. beta 3Gal-T1, -T2, and -T3, were not expressed in these cells. 3) The expression levels of beta 3Gal-T5 transcripts were well correlated with those of type 1 Lewis antigens in cultured cancer cells. 4) In the transfection experiment involving Namalwa cells, only beta 3Gal-T5 exhibited the LNT-PA synthesizing activity, whereas the other four beta 3Gal-Ts did not. 5) The expression level of beta 3Gal-T5 transcript in cultured cancer cells was well correlated with the LNT-PA synthesizing activity.

Kolbinger et al. demonstrated that beta 3Gal-T2 was able to synthesize type 1 chains in CHO cells through a transfection experiment (11). Amado et al. detected the beta 3Gal-T activities of beta 3Gal-T1 and -T2 (12). Hennet et al. demonstrated the beta 3Gal-T activities of three mouse beta 3Gal-Ts, i.e. mbeta 3GalT-I, -II and -III (14). In contrast, we could not detect LNT-PA synthesis activity in the Namalwa cells transfected stably with each of the beta 3Gal-T1, -T2, or -T3 genes. This was probably due to the amounts or structures of the enzymes used or the sensitivity of the assay system. In a previous study, we detected the activity of beta 3Gal-T1 using purified beta 3Gal-T1 that was expressed as a secreted form fused with the IgG binding domain of Staphylococcus aureus protein A (10). The other three groups, i.e. Hennet et al. (14), Kolbinger et al. (11), and Amado et al. (12), assayed beta 3Gal-T activity by measuring radioisotope incorporation using recombinant enzymes produced in soluble forms with a baculo-expression system or the S. aureus protein A fusion system, whereas we used cell homogenates as enzyme sources and a pyridylaminated acceptor substrate in the present study.

We apparently detected LNT-PA synthesizing activity in Namalwa-3GT5 cells and cultured cancer cells expressing beta 3Gal-T5, whereas Namalwa cells transfected with the other four beta 3Gal-T genes and cultured cells endogenously expressing beta 3Gal-T1 (PC-1), beta 3Gal-T2 (Namalwa), or beta 3Gal-T3 (MKN45) did not exhibit LNT-PA synthesizing activity at all. This indicated that beta 3Gal-T5 possesses the strongest activity as to type 1 chain synthesis among the four beta 3Gal-Ts, i.e. beta 3Gal-T1, -T2, -T3, and -T5. The beta 3Gal-T activity synthesizing LNT-PA in Colo205, SW1116, and Capan-2 cells decreased in accordance with the amount of the beta 3Gal-T5 transcript expressed in these cells (Table IV), and the activity was not detected in cells that did not express beta 3Gal-T5. These findings strongly indicated that the endogenous LNT-PA synthesizing activity in cell lines such as Colo205, SW1116, etc., is mainly directed by beta 3Gal-T5. Holmes reported that partially purified beta 3Gal-T(s) from Colo205 cells exhibit preferential activity toward lactotriaosylceramide (Lc3), GlcNAcbeta 1-3Galbeta 1-4Glcbeta 1-1Cer (25). Valli et al. found that the beta 3Gal-T activity in homogenates of human colorectal cancer cell lines is correlated with the expression levels of type 1 Lewis antigens (26). Their results are consistent with the beta 3Gal-T5 activity demonstrated in this study in these cultured cell lines. The beta 3Gal-T activity detected in their studies may be attributed to that of beta 3Gal-T5.

Western blot analysis revealed that the amounts of beta 3Gal-T5 transcripts were also well correlated with those of sLea antigens on proteins, probably on mucins in colorectal and pancreatic cancer cells. This means that beta 3Gal-T5 utilizes carbohydrate chains on proteins as acceptor substrates. In the future, the substrate specificities of beta 3Gal-T5, as well as the other beta 3Gal-Ts, should be examined by employing substrates as analogous as possible to physiological carbohydrate structures.

beta 3Gal-T5 was demonstrated to be physiologically expressed in a set of gastrointestinal and other tissues. The substantial amounts of beta 3Gal-T5 transcripts are expressed in stomach, jejunum, colon, and pancreas, strongly suggesting that beta 3Gal-T5 is responsible for expressing sLea antigens when those tissues become cancerous.

We previously determined the expression levels of 12 glycosyltransferase genes, i.e. those of five alpha 1,3-fucosyltransferases (Fuc-TIII, -TIV, -TV, -TVI, and -TVII), four ST3Gals (ST3Gal I, -II,- III, and -IV), one ST6Gal (ST6Gal I), beta 4Gal-T1, and core2-GlcNAc transferase, in colorectal cancer tissues in order to correlate them with the amounts of the sLex and sLea antigens (9). Although Fuc-TIII and ST3Gal IV are essentially required for the sLea synthesis in colorectal cancers, no single enzyme among the 12 was correlated with the amounts of sLea antigens. Therefore, we conjectured in the previous study that the combinatorial up-regulated expression of multiple enzymes determines the amounts of the antigens. However, it is a noteworthy finding in the present study that the expression levels of beta 3Gal-T5 were well correlated with the amounts of sLea antigens and the other type 1 Lewis antigens in the cultured cancer cells. This means that beta 3Gal-T5 is a key enzyme determining the expression levels of type 1 Lewis antigens including sLea antigens in these cells. The above, together with the results of the present study, indicated that beta 3Gal-T5 and ST3Gal IV are responsible for sLec synthesis, and Fuc-TIII is further required for sLea synthesis in colorectal cancers in addition to beta 3Gal-T5 and ST3Gal IV.

On the other hand, the type 2 chain, Galbeta 1,4GlcNAc, was found to be expressed in all cell lines examined in the present study, since beta 4Gal-T1 is a ubiquitous enzyme, and was substantially expressed in all cell lines (data not shown).

It is of interest to determine whether or not the up-regulation of the beta 3Gal-T5 gene expression determines the levels of sLea antigens in native cancer tissues. If this is the case, transcriptional regulation of the beta 3Gal-T5 gene will be an attractive subject in the future. In this study, we determined the transcription initiation site of the beta 3Gal-T5 gene in Colo205 cells. The nucleotide sequence in the upstream region was examined for the binding sites of transcription factors using the TFSEARCH (transcription factor search) program,3 based on the data bases deposited by Heinemeyer et al. (27). We searched the 1-kilobase pair upstream region from the transcription initiation site of the beta 3Gal-T5 gene, but found no TATA box. Within 150 bp upstream of the transcription initiation site, two CdxA sites, an AP-1 site, and a myeloid zinc finger 1 protein, MZF1, site were found. AP-1 and MZF1 have been reported to be potential targets of neoplastic transformation (28, 29). CdxA, known as a chicken homeobox-containing gene related to caudal in Drosophila, was previously shown to be expressed in the endoderm-derived gut epithelium during early embryogenesis (30). In the future, we will examine whether or not these transcription factors function in regulation of the beta 3Gal-T5 gene.

Finally, the results of the present study strongly indicate that beta 3Gal-T5 is the most probable candidate responsible for the sLea antigen synthesis in gastrointestinal and pancreatic cancer cells. In the future, it will be interesting to determine whether or not expression of the beta 3Gal-T5 gene changes some characteristics of cancer cells, especially those related to malignancy.

    ACKNOWLEDGEMENTS

We thank Dr. Nobuyuki Imai, Dr. Kyoko Takeuchi, Dr. Satoshi Nakagawa, Sachiko Kodama, Hiromi Inagaki, Reiko Koda, and Hiromi Ohara of Kyowa Hakko Kogyo Co. for cloning and sequencing of the four beta 3Gal-T genes, beta 3Gal-T1, -T2, -T3, and -T4. We also thank Dr. Satoshi Ito, of Fujirebio Inc., and Dr. Tetsuya Tachikawa, of Diagnostic Division, Otsuka Pharmaceutical Co. Ltd. for the kind gifts of the 19-9 and TT42 antibodies, respectively.

    FOOTNOTES

* This work was supported in part by Grant-in-aid for Scientific Research on Priority Areas 10178104, from the Ministry of Education, Science, and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number AB020337.

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

Dagger Dagger To whom correspondence and reprint requests should be addressed: Div. of Cell Biology, Inst. of Life Science, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577, Japan. Tel.: 81-426-91-9466; Fax: 81-426-91-9315; E-mail: hisashi{at}scc1.t.soka.ac.jp.

2 The NIH Image program was developed at the National Institutes of Health and is available through the Internet by anonymous FTP from zippy.nimh.nih.gov or on a floppy disk from the National Technical Information Service, Springfield, VA (part no. PB95-500195GEI).

3 The TFSEARCH program was developed by Y. Akiyama and is available via the World Wide Web (http://www.rwcp.or.jp/ lab/pdappl/papia.html).

    ABBREVIATIONS

The abbreviations used are: 19-9, 1116NS19-9; sLea, sialyl Lewis a; sLex, sialyl Lewis x; Lea, Lewis a; Leb, Lewis b; PCR, polymerase chain reaction; RT-PCR, reverse transcription-polymerase chain reaction; Fuc, fucose; SA, sialic acid; ORF, open reading frame; 5'-RACE, 5'-rapid amplification of cDNA ends; EST, expressed sequence tags; PBS, phosphate-buffered saline; BSA, bovine serum albumin; bp, base pair(s); beta 3Gal-T, UDP-galactose:beta -N-acetylglucosamine:beta 1,3-galactosyltransferase; LNT, lacto-N-tetraose; LNnT, lacto-N-neotetraose.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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