A Family of Human beta 3-Galactosyltransferases
CHARACTERIZATION OF FOUR MEMBERS OF A UDP-GALACTOSE:beta -N-ACETYL-GLUCOSAMINE/beta -NACETYL-GALACTOSAMINE beta -1,3-GALACTOSYLTRANSFERASE FAMILY*

Margarida AmadoDagger §, Raquel AlmeidaDagger §, Fatima Carneiro§, Steven B. Levery, Eric H. Holmesparallel , Mitsuharu Nomoto**, Michael A. Hollingsworth**, Helle HassanDagger , Tilo SchwientekDagger , Peter A. NielsenDagger , Eric P. BennettDagger , and Henrik ClausenDagger Dagger Dagger

From the Dagger  School of Dentistry, University of Copenhagen, Nørre Allé 20, 2200 Copenhagen N, Denmark, § Institute of Molecular Pathology and Immunology of University of Porto, IPATIMUP, Rua Dr. R. Frias s/n, 4200 Porto, Portugal,  University of Georgia, Complex Carbohydrate Research Center, Athens, Georgia 30602, parallel  Department of Cell Surface Biochemistry, Northwest Hospital, Seattle, Washington 98125, and ** Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198 .

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

BLAST analysis of expressed sequence tags (ESTs) using the coding sequence of a human UDP-galactose:beta -N-acetyl-glucosamine beta -1,3-galactosyltransferase, designated beta 3Gal-T1, revealed no ESTs with identical sequences but a large number with similarity. Three different sets of overlapping ESTs with sequence similarities to beta 3Gal-T1 were compiled, and complete coding regions of these genes were obtained. Expression of two of these genes in the Baculo virus system showed that one represented a UDP-galactose:beta -N-acetyl-glucosamine beta -1,3-galactosyltransferase (beta 3Gal-T2) with similar kinetic properties as beta 3Gal-T1. Another gene represented a UDP-galactose:beta -N-acetyl-galactosamine beta -1,3-galactosyltransferase (beta 3Gal-T4) involved in GM1/GD1 ganglioside synthesis, and this gene was highly similar to a recently reported rat GD1 synthase (Miyazaki, H., Fukumoto, S., Okada, M., Hasegawa, T., and Furukawa, K. (1997) J. Biol. Chem. 272, 24794-24799). Northern analysis of mRNA from human organs with the four homologous cDNA revealed different expression patterns. beta 3Gal-T1 mRNA was expressed in brain, beta 3Gal-T2 was expressed in brain and heart, and beta 3Gal-T3 and -T4 were more widely expressed. The coding regions for each of the four genes were contained in single exons. beta 3Gal-T2, -T3, and -T4 were localized to 1q31, 3q25, and 6p21.3, respectively, by EST mapping. The results demonstrate the existence of a family of homologous beta 3-galactosyltransferase genes.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The data base of expressed sequence tags (ESTs)1 is now estimated to contain sequence information from more than half of human genes; it therefore provides a unique source for identifying novel members of homologous gene families by conserved sequence motifs (1). The identification of novel genes by sequence similarity was used recently to identify a large family of homologous UDP-galactose:beta -N-acetyl-glucosamine beta -1,4-galactosyltransferases (beta 4Gal-T). At least six novel genes were found of which four have been shown to represent functional beta 4Gal-Ts (2-5). Perhaps surprisingly, there are no sequence similarities between the beta -4-galactosyltransferases and a putative UDP-galactose:beta -N-acetyl-glucosamine beta -1,3-galactosyltransferase gene submitted to GenBank in 1996 and here designated beta 3Gal-T1. The disaccharides Galbeta 1-3GlcNAcbeta (type 1 chain) and Galbeta 1-4GlcNAcbeta (type 2 chain) are core structures in glycosphingolipids and glycoproteins, where they occur in linear or branched repeated structures (6, 7). The two core structures are differentially expressed in cells and organs (8) and synthesized by at least two independent galactosyltransferase activities (9, 10). However, a glycosyltransferase may transfer to either C-3 or C-4 of beta -GlcNAc (e.g. the alpha -3,4-fucosyltransferases (11)), suggesting that some enzymes may recognize common features of the two acceptor sites.

In the present study, we found that beta 3Gal-T1 is one member of a beta 3-galactosyltransferase family. The human EST sequence data base was used to identify several novel members of a beta 3-galactosyltransferase gene family. The beta 3Gal-T1 gene was not found in the EST data base, but a large number of ESTs were identified that shared short sequence stretches with high similarity and had conserved cysteine residues. The full coding sequences of three of these genes were established; expression studies demonstrated that one gene encoded a new UDP-Gal: beta GlcNAc beta -1,3-galactosyltransferase (beta 3Gal-T2); and one gene encoded a UDP-Gal: beta GalNAc beta -1,3-galactosyltransferase gene (beta 3Gal-T4), with different acceptor substrate specificity.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Identification of beta 3Gal-T1 Homologues Genes

Data base searches were performed with the reported coding sequence of a human beta 3Gal-T1 sequence (GenBank accession number E07739) using the tBLASTn algorithm against the dbEST data base at The National Center for Biotechnology Information, U. S. A., as described previously (2). Overlapping sequences were merged (Fig. 1), and the Unigene data base was used to select cDNA clones with the longest inserts and chromosomal assignments. EST cDNA clones were obtained from Genome Systems Inc.

Cloning and Sequencing of the Full Coding Sequence of beta 3Gal-T2

Four partly overlapping ESTs with approximately 360 bp open reading frame with sequence similarity to the C-terminal sequence of beta 3Gal-T1 were identified (Fig. 1). A further 5' sequence was obtained by 5' rapid amplification of cDNA ends using human fetal brain Marathon-Ready cDNA (CLONTECH) in combination with antisense primers EBER405 (5'-GGTGCATATCCTCGCATTAGG), EBER409 (5'-GGTGCTAGACTTTCATTGCCCC), and EBER412 (5'-TTCTTTCCAAATGTTCCGAAGG) for 35 cycles at 95 °C, 45 s; 55 °C, 15 s; 68 °C, 3 min, using the Expand kit enzyme (Boehringer Mannheim). The rapid amplification of cDNA ends products were cloned into the BamHI site of pT7T3U19, and multiple clones were sequenced. The entire sequence was confirmed by sequencing genomic P1 clones. The composite sequence contained an open reading frame of 1266 bp encoding a putative protein with a type II domain structure (Fig. 2), and an overall sequence identity of approximately 42% to beta 3Gal-T1.

Cloning and Sequencing of the Full Coding Sequence of beta 3Gal-T3

Five partly overlapping ESTs with approximately 900 bp of open reading frame and sequence similarity to beta 3Gal-T1 were identified (Fig. 1). An additional 5' sequence was obtained by 5' rapid amplification of cDNA ends using primers EBER606 (5'-GCAGTTTGAATGCTCTCGAAGTGTG) and EBER612 (5'-AGCAGCAGGAGGCTCCATTTG) as described for beta 3Gal-T2 above. The composite sequence contained an open reading frame of 993 bp encoding a putative protein with a type II domain structure (Fig. 2) and an overall sequence identity of approximately 40% to beta 3Gal-T1 and 33% to -T2.

Cloning and Sequencing of the Full Coding Sequence of beta 3Gal-T4

Two overlapping EST clones with sequence similarity to the N-terminal sequence of beta 3Gal-T1 were identified (Fig. 1). Sequencing of the inserts revealed an open reading frame of 1134 bp potentially encoding a protein with a type II domain structure (Fig. 2) with an overall sequence identity of approximately 33% to beta 3Gal-T1.

Baculo Expression Constructs for beta 3-Galactosyltransferases

Constructs Lacking the Signal-anchor Sequence-- Expression constructs were designed to exclude the hydrophobic transmembrane segment and include a maximum amount of the putative stem region. (i) beta 3Gal-T1, encoding amino acid residues 21-326, was prepared by RT-PCR with mRNA from MKN-45 cell line using the primer pair EBER300FOR (5'-TGGTACTTGAGTATAACTCGC) and EBER304 (5'-TACCAACACCTATGGTCCCATTTC); (ii) beta 3Gal-T2, encoding amino acid residues 39-422 was prepared by RT-PCR with RNA from MKN45 gastric carcinoma cell line using the primer pair EBER400FOR (5'-ATGTTTTTGTTTTTCAATCATCATGAC) and EBER415 (5'-TCTAATGTAGTTTACGGTGGC) (Fig. 2); beta 3Gal-T3, encoding amino acid residues 33-331 was prepared by PCR with P1 and genomic DNA, as well as by RT-PCR with RNA from MKN45, using the primer pair EBER600FOR (5'-ATGTGGTACCTCAGCCTTCCC) and EBER614 (5'-GTTAATAATGGCATGTGGTGTTCC) (Fig. 2); and beta 3Gal-T4, encoding either residues 30-378 or 78-378 were prepared by RT-PCR with RNA from MKN45, using the primers EBER521 (5'-GAGGAGCTGCTGAGCCTCTCA) or EBER520 (5'-TGCACGGCTCCGGAGAACCCTG) in combination with EBER514 (5'-ACTCTCAGCTCTGAAGCC) (Fig. 2). The PCR products were cloned into the BamHI site of pAcGP67 (Pharmingen).

Full-length Coding Expression Constructs-- Expression constructs designed to encode the full open coding region were prepared by using primers that included the first potential initiation codon and genomic DNA as template (for beta 3Gal-T4 cDNA from EST H2O623, which contained the full coding sequence). The following primer pairs were used: (i) beta 3Gal-T1, EBER306 (5'-AGACAATGGCTTCAAAGGTCTC) and EBER 304; (ii) beta 3Gal-T2, EBER400FUL (5'-TACAACATGCTTCAGTGGAGGAG) and EBER415; (iii) beta 3Gal-T3, EBER600FUL (5'-TGACCATGGCCTCGGCTCTCTGGACTG) or EBER611 (5'-GTAGGATGTCACTGAGATCCC) (for second in-frame ATG) in combination with EBER614; and (iv) beta 3Gal-T4, EBER509 (5'-CCATGCAGCTCAGGCTCTTCC) and EBER514. The PCR products were cloned into the BamHI site of pVL1193 (Pharmingen). All constructs were fully sequenced.

Expression of beta 3-Galactosyltransferases in Sf9 Cells

The plasmids with pAcGP67 or pVL1193 were co-transfected with Baculo-GoldTM DNA (Pharmingen) as described previously (12). Recombinant Baculo virus were obtained after two successive amplifications in Sf9 cells grown in serum-containing medium. Controls included the pAcGP67-GalNAc-T3-sol (12). Standard assays were performed in 50 µl of total reaction mixtures containing 25 mM Tris (pH 7.5), 10 mM MnCl2, 0.25% Triton X-100, 100 µM UDP-[14C]Gal (2,300 cpm/nmol) (Amersham Pharmacia Biotech), and varying concentration of acceptor substrates (Sigma) (see Table I for structures). Reaction products were quantified by Dowex-1 chromatography. Assays with hen egg ovalbumin (Sigma) were performed with the standard reaction mixture modified to contain 200 µM UDP-Gal, 54 mM NaCl, and 1 mg of ovalbumin. The transfer of galactose was evaluated after separation by filtration through Whatman GF/C glass fiber filters. Constructs that encoded soluble secreted enzymes (lacking the signal-anchor sequence) were assayed with 5-20 µl of culture supernatant from infected cells, whereas the full-length enzymes were assayed with 1% Triton X-100 homogenates of cells. Assays used for assessment of Km of acceptor substrates and donor substrates were modified to include 500 µM UDP-[14C]Gal (2,300 cpm/nmol) or 100 mM GlcNAcbeta -benzyl. Assays with glycolipid acceptors were conducted as described previously (13) in reaction mixtures containing 2.5 µmol of HEPES buffer (pH 7.2), 1 µmol of MnCl2, 100 µg of TDOC (for beta 3Gal-T1 and -T2) or Triton CF-54 (for beta 3Gal-T4), 20 µg of acceptor glycolipid, 15 nmol of UDP-[14C]Gal (13,000 cpm/nmol), and enzyme in a total volume of 100 µl.

Characterization of the Products Formed with beta 3Gal-T2 and -T4

Terminal glycosylation of Lc3Cer (43) with beta 3Gal-T2 was performed in a reaction mixture consisting of 1 milliunit of beta 3Gal-T2 (specific activity determined with beta GlcNAc-benzyl), 150 µg of Lc3Cer, 25 mM Tris (pH 7.4), 10 mM MnCl2, 50 µg of taurodeoxycholate, and 0.5 µmol of UDP-Gal in a final volume of 100 µl. The secreted form of beta 3Gal-T2 was partially purified by sequential DEAE and S-Sepharose chromatographies from serum-free medium as described previously (14). Terminal glycosylation of GM2 (Sigma) was performed with 50 µl of a 1:1 suspension of a membrane fraction prepared from High FiveTM (Invitrogen) cells infected with the full coding expression construct of beta 3Gal-T4. Briefly, cells were harvested 2 days postinfection, lysed, and homogenized in 1.0% CF54, 150 mM cacodylate (pH 6.5), 10 mM MnCl2, and 2 mM EDTA. The extract was pelleted by low speed centrifugation (3,000 rpm for 10 min), and the supernatant from this was pelleted again by high speed centrifugation (30,000 rpm for 30 min). Most of the enzyme activity was retained in the high speed-pelleted fraction and used as enzyme source. The reaction mixture included 250 µg of GM2, 150 mM cacodylate (pH 6.5), 10 mM MnCl2, 0.3% Triton CF-54, and 5 mM UDP-Gal in a final volume of 200 µl. The glycosylations were monitored by high performance TLC, and the products were purified on octadecyl silica cartridges (Bakerbond, J. T. Baker Inc.) and deuterium-exchanged as described previously (2). One-dimensional 1H NMR spectroscopy of the product of beta 3Gal-T2 was performed on a Bruker AMX-500 spectrometer (temperature, 308 K; spectral width, 5000 Hz acquired over 16,000 data points; relaxation delay, 2 s; solvent suppression by presaturation pulse). One-dimensional 1H NMR spectroscopy of the product of beta 3Gal-T4 was performed on a Varian Unity-INOVA 600 MHz spectrometer (temperature, 308 K; spectral width, 6000 Hz acquired over 16,000 data points; relaxation delay, 1.5 s; solvent suppression by presaturation pulse).

Northern Analysis

The human multiple tissue northern blot was obtained from CLONTECH and used once for the experiments shown. The soluble expression constructs were used as probes. Probes were random-primed-labeled using [alpha -32P]dCTP (Amersham) and an oligo labeling kit (Amersham). The blots were probed overnight at 42 °C as described previously (12), washed 2× 10 min at RT with 2×SSC (1× SSC = 0.15 M NaCl and 0.015 M sodium citrate), 1% Na4P2O2; 2× 20 min at 65 °C with 0.2×SSC, 1% SDS, 1% Na4P2O2; and once 10 min with 0.2 x SSC at RT.

Genomic Cloning and Characterization of the Organization of beta 3Gal-T1, -T2, and -T3

P1 genomic clones were obtained for beta 3Gal-T2 and -T3 by screening a human foreskin P1 library (DuPont Merck Pharmaceutical Co. Human Foreskin Fibroblast P1 Library) using the primer pairs EBER400 (5'-ACCAGACCTCTACCCAAGTGAGCG)/EBER402 (5'-CAGCTCGAATAAGAGACTCGC) or EBER603 (5'-GTGATAGAACGCGTGAACTGG)/EBER604 (5'-CCCCAAGTAACTCTAATGGCC). Three P1 clones each for beta 3Gal-T2 and -T3 were obtained from Genome Systems (Fig. 1). DNA from P1 phages were prepared as recommended by Genome Systems.

The chromosomal localization of beta 3Gal-T2, -T3, and -T4 were determined using 3'EST mapping data (National Center for Biotechnology Information). No ESTs corresponding to the available sequence of beta 3Gal-T1 was found; thus, the localization of this gene was not determined.

    RESULTS
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Introduction
Procedures
Results
Discussion
References

Identification and Cloning of Human beta 3Gal-T2, beta 3Gal-T3, and beta 3Gal-T4-- The search and cloning strategy outlined in Fig. 1 produced three novel genes with significant sequence similarity to beta 3Gal-T1 (Fig. 2). Additionally, three genes with less similarity were identified (not shown). Multiple sequence alignment of the three beta 3-galactosyltransferases as well as a homologous Drosophila gene designated Brainiac (15) (GenBank accession number U41449) is shown in Fig. 2. The sequence similarities between the four human genes are limited to the central regions; there were no significant similarities in the N-terminal regions. Several sequence motifs in the putative catalytic domains are conserved between all the sequences. In the high similarity region, beta 3Gal-T2 is most similar to beta 3Gal-T1, beta 3Gal-T3 was the second most similar, and the least similar is beta 3Gal-T4. At least three cysteine residues align within all the human genes; an additional two align within beta 3Gal-T1, -T2, and -T3 (Fig. 2). Although Brainiac has most of the sequence motifs conserved among the human genes, none of the conserved cysteines in the human genes were found in Brainiac. There are two potential N-linked glycosylation sites in beta 3Gal-T1, five in beta 3Gal-T2 and -T3, and one in beta 3Gal-T4, respectively. The glycosylation sites are mainly in the N-terminal region, to the carboxyl side of the putative transmembrane region. Interestingly, one site occurs in the region of high sequence similarity and is conserved among the four human genes and Brainiac.


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Fig. 1.   Strategy for identification and cloning of beta 3Gal-T2, -T3, and -T4. Identified ESTs are indicated by their GenBank accession numbers, with available sequence lengths in parenthesis. RACE, rapid amplification of cDNA ends. DMPC-HFF, DuPont Merck Pharmaceutical Co. Human Foreskin Fibroblast.


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Fig. 2.   Multiple sequence analysis (ClustalW) of human beta 3Gal-T1, -T2, -T3, and -T4 and the homologous Drosophila gene designated as Brainiac. Introduced gaps are shown as hyphens, and aligned identical residues are indicated by boxing, black for all sequences, dark gray for four sequences, and light gray three sequences. The hydrophobic segments representing putative transmembrane domains are underlined with a single line (Kyte and Doolittle (38), window of 8). Three cysteine residues conserved between the four human genes are indicated by a *, and a conserved putative N-linked glycosylation site in all genes is indicated by an arrow.

The predicted coding region of beta 3Gal-T2 has two potential initiation codons, both of which are in agreement with Kozak's rule (16). The coding sequence thus depicts a type II transmembrane glycoprotein with two different N-terminal cytoplasmic domains of 24 or 11 residues and a transmembrane segment of 19 residues flanked by charged residues, and 379 residues contain the stem region and catalytic domain (Fig. 2). beta 3Gal-T2 differs significantly from the other genes by having an extended putative stem region (approximately 50 residues) which is hydrophilic, as is found in most glycosyltransferase genes (Figs. 2 and 3). The 3'-UTR with one polyadenylation signal at position 2497 (1228 bp 3'-UTR) was found in all the identified EST clones. The 3'ESTs (WI-13433) were linked to chromosome 1q31 between microsatellite markers D1S461 and D1S412 at 208-213 centimorgan.


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Fig. 3.   Kyte and Doolittle (38) (window of 8) hydropathy plot of human beta 3Gal-T1, -T2, -T3, and -T4. The position of conserved sequence motifs as shown in Fig. 2 is indicated with dotted lines. TM indicates the putative transmembrane region. the arrow indicates the position of the putative hydrophobic stem region of beta 3Gal-T4.

The predicted coding region of beta 3Gal-T3 has two potential initiation codons; only the second is in agreement with Kozak's rule (16). The coding sequence depicts a type II transmembrane glycoprotein with two potentially different N-terminal cytoplasmic domains of 19 or 7 residues, a transmembrane segment of 14 residues, and a stem region plus catalytic domain of 298 residues (Fig. 2). The 774-bp 3'-UTR obtained from all EST clones did not include a consensus polyadenylation signal. The 3'ESTs (WI-9638) were linked to chromosome 3q25 between D3S1275 and D3S3702 microsatellite markers at 176-179 centimorgan.

The predicted coding region of beta 3Gal-T4 has a single initiation codon, in agreement with Kozak's rule (16). The coding sequence yields a type II transmembrane glycoprotein with an N-terminal cytoplasmic domain of 8 residues, a short transmembrane segment of 11 residues, and a stem region and catalytic domain of 359 residues (Fig. 2). All conserved sequence motifs within the human beta 3-galactosyltransferase genes are found in beta 3Gal-T4. Whereas these align fully within beta 3Gal-T1, -T2, and -T3, there is an inserted sequence element in beta 3Gal-T4 that is illustrated by inserted spacings in the multiple sequence alignment analysis (Fig. 2) and in the hydropathy plot (Fig. 3). The hydropathy plot of beta 3Gal-T4 shows a very hydrophobic putative stem region that differs significantly from the other genes. This has not been observed for other animal glycosyltransferases except for a ceramide galactosyltransferase (17). The human beta 3Gal-T4 exhibits relatively high similarity to a recently reported rat gene (overall amino acid sequence identity of 79%) (18). Although this is lower than the similarity found in comparisons between most rat/human glycosyltransferase genes, it suggests that these two genes are homologues. A 3'-UTR with a polyadenylation signal at position 1387 (250-bp 3'-UTR) was included in the EST clones. The 3'ESTs (stSG4027) were to linked to chromosome 6p21.3 between microsatellite markers D6S276 and D6S439 at 44-48 centimorgan.

Expression of beta 3Gal-T1 and -T2-- Expression of a soluble construct of beta 3Gal-T1 and -T2 in Sf9 cells resulted in a marked increase in galactosyltransferase activity using beta GlcNAc-benzyl as an acceptor substrate, compared with uninfected cells or cells infected with control constructs for polypeptide GalNAc- transferases (12) (Table I). Analysis of the substrate specificity of beta 3Gal-T1 and -T2 activities showed that only saccharides with a terminal beta GlcNAc residue and not alpha GlcNAc or alpha GalNAc were acceptor substrates. It was not possible to determine Km for benzyl-beta GlcNAc or Umb-beta GlcNAc with beta 3Gal-T1 and -T2 due to substrate inhibition at concentrations above 100 mM. The Km for UDP-Gal of beta 3Gal-T1 and -T2 were 90 ± 5 µM and 37 ± 9 µM, respectively, using Benzyl-beta GlcNAc as an acceptor substrate. beta 3Gal-T2 catalyzed glycosylation of hen egg ovalbumin, whereas beta 3Gal-T1 showed poor activity with this substrate (Table II). Analysis of enzyme activities with a panel of glycolipid substrates revealed that both enzymes were capable of catalyzing glycosylation of beta GlcNAc-terminating structures Lc3Cer and nLc5Cer, but beta 3Gal-T1 showed lower activity with nLc5Cer. Both enzymes showed low activities with GlcCer (Table III). beta 3Gal-T2 and to a lesser extent beta 3Gal-T1 incorporated galactose into nLc4Cer. Secreted forms of beta 4-galactosyltransferases were included as controls (2).2 Both beta 3Gal-T1 and -T2 showed strict donor substrate specificity for UDP-Gal and did not utilize UDP-GalNAc or UDP-GlcNAc with the acceptor substrates tested (data not shown). The reaction product formed by beta 3Gal-T2 with Lc3Cer was shown by 1H NMR to be Lc4Cer, thus verifying that T2 formed the Galbeta 1-3GlcNAc linkage. In the downfield region (3.7-5.7 ppm) (Fig. 4), the one-dimensional 1H NMR spectrum indicated the presence of Lc4Cer as well as residual Lc3Cer, and this was in agreement with results of TLC analysis, which indicated approximately 40% conversion of Lc3Cer to Lc4Cer. The spectral features of the two components were virtually identical with those previously observed (19, 20) (Fig. 4) and clearly distinct from those obtained for nLc4Cer (21).

                              
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Table I
Substrate specificity of beta 3GalT-1 and beta 3GalT-2
Enzyme sources were media of infected Sf9 cells. Background values obtained with uninfected cells or cells infected with an irrelevant construct were subtracted. The background rates were not higher than 0.5 nmol/min/ml. Bzl, benzyl; Nph, nitrophenyl; Me, methyl; lumb, 4-methyl-umbellifenyl; OMe, methoxy.

                              
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Table II
Substrate specificity of beta 3 and beta 4-galactosyltransferases with hen egg ovalbumin

                              
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Table III
Substrate specificities with glycolipid acceptors for beta 3Gal-T1 and beta 3Gal-T2


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Fig. 4.   Downfield and NAc regions of 600-MHz 1H NMR spectrum of glycosphingolipid mixture isolated after enzymatic glycosylation of Lc3Cer with beta 3Gal-T2. The substrate (S) and product (P) isolated together from the crude reaction mixture are shown. 2048 free induction decays were accumulated at 308 K. Arabic numerals refer to ring protons of residues designated by Roman numerals or capital letters in the corresponding structures. R refers to protons of the sphingosine backbone, and FA refers to protons of the fatty N-acyl moiety. The downfield region of the spectrum displayed five distinct beta -anomeric resonances (3J1,2 congruent  7-9 Hz). Two of these, at 4.166 ppm (3J1,2 = 7.7 Hz) and 4.263 ppm (3J1,2 congruent  7 Hz), correspond to beta -Glc I-1 and beta -Gal II-1, respectively, of both Lc3Cer and Lc4Cer. A third, at 4.620 ppm (3J1,2 = 8.1 Hz), corresponds to beta -GlcNAc III-1 of Lc3Cer. The two resonances of lower intensity, at 4.781 ppm (3J1,2 = 8.4 Hz) and 4.136 ppm (3J1,2 = 7.0 Hz), correspond to beta -GlcNAc III-1 and beta -Gal IV-1, respectively, of Lc4Cer. By contrast, under these conditions, the resonance for H-1 of the terminal Galbeta 1right-arrow4 of nLc4Cer was previously found at 4.214 ppm (3J1,2 = 6.7 Hz), whereas that of beta -GlcNAc III-1 was found at 4.664 ppm (3J1,2 = 7.9 Hz) (21, 39). Thus, the data clearly show evidence of partial glycosylation of Lc3Cer with Galbeta 1right-arrow3 to make Lc4Cer. Two other pairs of resonances are consistent with the presence in the mixture of both Lc3Cer and Lc4Cer. These are the characteristic beta -Gal II-4 signals, shifted slightly downfield in the latter (3.851 ppm; 3J3,4 = 3.3 Hz) relative to the former (3.837 ppm; 3J3,4 = 2.6 Hz), both found within ±0.002 ppm of previously observed values (19, 20); and the beta -GlcNAc NAc singlets, found at 1.815 ppm in the latter and 1.834 ppm in the former.

Expression of beta 3Gal-T3-- Two different soluble constructs and two full-length coding constructs were expressed in Sf9 cells, but enzyme activity was not detected with any of the substrates listed in Table I, Lc3, GM2, or globoside (not shown).

Expression of beta 3Gal-T4-- Expression of the full-length coding construct for beta 3Gal-T4 in Sf9 cells produced no detectable activity with any of the simple sugar derivatives listed in Table I. However, analysis of enzyme activity with a panel of glycolipids revealed that products migrating as Gg4 and GM1 were formed with Gg3 and GM2 glycolipid substrates, respectively (Table IV). The reaction product formed by beta 3Gal-T4 with GM2 was analyzed by 1H NMR to verify that T4 formed the Galbeta 1-3GalNAc linkage to make GM1 (Fig. 5). Aside from the appearance of a number of impurities mainly ascribed to membrane phospholipids not separated from the ganglioside product, the proton NMR spectral data of the reaction product were comparable with those of Koerner et al. (22) for nonexchangeable CH, CH2, and CH3 signals of GM1, allowing for slight differences in temperature (308 versus 303 K), % D2O, and ganglioside concentration.

                              
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Table IV
Substrate specificities with glycolipid acceptors for beta 3Gal-T4


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Fig. 5.   Sections of 600-MHz 1H NMR spectrum of biosynthetic ganglioside produced by enzymatic glycosylation of GM2 with beta 3Gal-T4. 1024 transients were accumulated at 308 K. Designations are as in Fig. 4. Cis-vinyl and -allyl refer to mid-chain unsaturation of fatty acids. Key structure reporter group resonances of the GM1 product include the anomeric protons, beta -Glc I-1 (4.149 ppm), beta -Gal II-1 (4.275 ppm), beta -GalNAc III-1 (4.861 ppm), and beta -Gal IV-1 (4.222 ppm) as well as alpha -NeuAc A-3eq (~2.54 ppm; overlapped with residual protonated Me2SO, not shown) and A-3ax (1.630 ppm), I-2 (3.035 ppm), II-4 (3.940 ppm), III-2 (3.918 ppm), A-NAc, and III-NAc (1.876 and 1.756 ppm, respectively), all within 0.01 ppm of the published values (the chemical shift of NeuAc-A-3eq, incorrectly given as 2.281 ppm by Koerner et al. (22), is compared with the corrected value of 2.53 ppm given by Scarsdale et al. (40)). In most cases, variances from published values are much smaller than 0.01 ppm. Resonances for the ceramide sphingosine backbone (R) and fatty acid chain (FA) are found with chemical shifts and splitting patterns characteristic for sphing-4-enine type bases (i.e. d18:1, d20:1), characteristic of mammalian brain gangliosides, such as the GM2 used as acceptor substrate. For sphingosine, these are R-1b (3.976 ppm), R-3 (3.882 ppm), R-4 (5.347 ppm), R-5 (5.535 ppm), and R-6 (1.931 ppm). The strong triplet for nFA-2 (2.024 ppm) is characteristic for nonhydroxylation at C-2 of the fatty acid component. The major manifestation of residual lipid impurities from the membrane preparation appears to be the disproportionately large cis-vinyl and cis-allyl resonances (5.325 and 1.981 ppm, respectively), which are normally attributed to fatty acyl mid-chain unsaturation (22, 41, 42) but are not characteristic of the type of GM2 preparation used as acceptor substrate in this study. The smaller non-anomeric resonances observed in the region 4.0-5.1 ppm are ascribed primarily to partly unexchanged ganglioside OH signals (43).

Northern Analysis of beta 3Gal-T1, -T2, -T3, and -T4-- Northern analysis with mRNA from eight adult human organs revealed different patterns of expression by the four genes (Fig. 6). beta 3Gal-T1 expression was detected in brain with a transcript size of 6.5 kb, but no expression was detected in the other human organs tested or in 25 human tumor cell lines derived from the pancreas and colon. The human cancer cell line used for the original cloning of beta 3Gal-T1 was not included in this study. beta 3Gal-T2 produced a major transcript of 3.6 kb and a minor transcript of 3.2 kb in heart and brain, but it was not expressed in any of the other organs tested. All human ESTs derived from beta 3Gal-T2 were obtained from brain libraries, but two mouse ESTs were from a mammary gland library. beta 3Gal-T3 produced a major transcript of 3.8 kb and minor transcript of 3.0 kb similarly in heart and brain, and expression was also observed in placenta, kidney, and pancreas. Human ESTs derived from beta 3Gal-T3 were obtained from brain, liver/spleen, and heart libraries. beta 3Gal-T4 yielded multiple transcripts of 5.0, 3.0, and 2.2 kb in all organs with some variations in detected levels of expression, and the expression in brain was relatively low. The two available human ESTs derived from beta 3Gal-T4 were obtained from brain libraries.


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Fig. 6.   Northern blot analysis of human tissues. Multiple human Northern blot from CLONTECH as labeled were probed with 32P-labeled beta 3Gal-T1, -T2, -T3, and -T4 probes with beta -actin as control. Panel A, beta 3Gal-T1; panel B, beta 3Gal-T2; panel C, beta 3Gal-T3; panel D, beta 3Gal-T4; and panel E, glyceraldehyde-3-phosphate dehydrogenase.

Genomic Organization and Chromosomal Localization-- Sequence analysis of P1 clones showed that the coding regions of beta 3Gal-T2 and -T3 were located in a single exon. PCR with genomic DNA revealed that the coding regions of beta 3Gal-T1 and -T4 were also contained in one exon. The four beta 3-galactosyltransferase genes were located on different chromosomes; beta 3Gal-T2 at 1q31, T3 at 3q25, and T4 at 6p21.3.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

In the present study, four members of a human beta 3-galactosyltransferase gene family were characterized. A human beta 3-galactosyltransferase gene, here designated beta 3Gal-T1, indicated to catalyze the formation of the type 1 core structure, Galbeta 1-3GlcNAc, was released in GenBank October 1996 and described in some detail in a patent application (JP1994181759-A/1). beta 3Gal-T1 was isolated from the melanoma cell line WM266-4 by a transfection-cloning strategy that used expression of Lea and sialosyl-Lea in KJM-1 cells for selection. In the present study, the function of beta 3Gal-T1 was verified by expression in insect cells. Through the application of an EST cloning strategy previously used to identify a family of homologous beta 4-galactosyltransferases (2), a novel family of homologous beta 3-galactosyltransferase genes was identified. Three genes with a high degree of similarity, beta 3Gal-T2, -T3, and -T4, were studied here, and recombinant forms of two of these have beta 3-galactosyltransferase activity. beta 3Gal-T2 has kinetic properties similar to beta 3Gal-T1, but there was a striking difference in their activities with the glycoprotein ovalbumin. beta 3Gal-T4 was found to catalyze the addition of galactose to the gangliosides GM2 and Gg3. The function of the human beta 3Gal-T3 gene reported here was not identified.

During the course of this work, a rat UDP-Gal:beta GalNAc beta -1,3-galactosyltransferase gene was isolated by the transfection-cloning strategy (18). The rat enzyme has an overall sequence identity of 79% to human beta 3Gal-T4, and the substrate specificities are similar. Thus, we propose that these represent homologues among these species. However, it remains possible that the two genes are different but closely related variants, since the sequence similarity is low compared with what has been found for other human and mouse homologues of glycosyltransferases. During review of this manuscript, human beta 3Gal-T1 and -T2 were reported by Kolbinger et al. (23), and in agreement with our finding that beta 3Gal-T2 utilized nLc4Cer as acceptor, these authors reported 16% activity of this enzyme with Galbeta O-(CH2)8-CO2CH3. Mouse homologues of beta 3Gal-T1, -T2, and -T3 were reported simultaneously (24), and in this study, the murine beta 3Gal-T2 and -T3 were found to have low levels of activity with GlcNAc-beta -pNP, approximately 30-fold lower than beta 3Gal-T1. Interestingly, the kinetic properties of all three murine enzymes were poor with Km for UDP-Gal between 0.6 and 2.3 mM, which is much higher than the Km of other glycosyltransferases (5-100 µM) (25). In the present study, the Km for UDP-Gal with human beta 3Gal-T1 and -T2 were 90 and 37 µM, respectively. Furthermore, the kinetic properties of human beta 3Gal-T1 and -T2 with simple acceptors were comparable, but we did not detect significant activity with beta 3Gal-T3. Whether these differences are due to experimental problems or to species-related variations is not known.

Analysis of sequence similarities between the four beta 3-galactosyltransferase genes using the ClustalW algorithm (Fig. 2) revealed comparatively low overall sequence identities of 29-42%; however, several conserved short sequence motifs were found. Interestingly, a single potential N-glycosylation site is conserved in all genes, a feature not generally found among homologous glycosyltransferases, although a single conserved site is also found in most of the beta 4-galactosyltransferases (2, 3). Support for an evolutionary relationship among the three genes was provided by analysis of their genomic organizations, which showed that the coding regions of all four genes were located in a single exon. The same organization was found for the three mouse genes (24). At least four beta 4-galactosyltransferase genes have the same genomic organization, including the conservation of five intron positions (2). It appears that genes for several glycosyltransferase families have similar organizations. Several members of the fucosyltransferases and the beta 1-6N-acetylglucosaminyltransferase families are encoded by a single exon (26, 27). In contrast to these gene families, all members of the beta 3Gal-T family have different chromosomal localizations. The significance of this is presently unknown.

The existence of multiple beta 3-galactosyltransferases suggests a surprising redundancy in genes with seemingly similar functions, which could represent a comprehensive genetic back-up. However, it is equally likely that this high number of enzymes have evolved as a result of specific requirements for enzymes with different functions. Apparent redundancies in substrate specificities of glycosyltransferases have been found in sialyltransferases (28, 29), fucosyltransferases (30-32), beta 4-galactosyltransferases (2-4), and polypeptide GalNAc-transferases (14, 33). There are differences in the kinetic parameters of some members of each of these families that relate to type and complexity of acceptor glycoconjugate or acceptor peptide sequence for the polypeptide GalNAc-transferases. In the present study, beta 3Gal-T2 catalyzed transfer of galactose to ovalbumin, whereas beta 3Gal-T1 did not. Analogously, one member of the beta 4-galactosyltransferase family failed to utilize this substrate (Table II). It is not clear whether this indicates that the enzymes have different preferences for glycoprotein and glycolipid substrates or have selective specificities for particular antennae of branched mannose and poly-N-acetyllactosamine structures. Ovalbumin contains unsubstituted GlcNAcbeta 1-2Man and GlcNAcbeta 1-4Man structures (34), and apparently none of these are utilized by beta 3Gal-T1. However, beta 3Gal-T1 utilized the disaccharide GlcNAcbeta 1-6Man-1-OMe more efficiently than beta 3Gal-T2 (Table I), indicating that perhaps tri- or tetraantennary N-linked glycoproteins may serve as substrates for beta 3Gal-T1. Unfortunately, a complete panel of disaccharide acceptors for different antennae of N-linked structures were not available for this study. None of the beta 3Gal-Ts utilized the disaccharide GlcNAcbeta 1-3GalNAc representing mucin-type core 3 (Table I), whereas four beta 4Gal-Ts efficiently use this substrate (2). beta 3Gal-T1 and -T2 showed similar activities with beta -GlcNAc-terminating lactoseries glycosphingolipids. Interestingly, especially beta 3Gal-T2 exhibited significant incorporation into nLc4Cer (Table III). The structure of the product was not determined, but it is likely to be Galbeta 1-3Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4Glcbeta 1-Cer, which was originally isolated by Stellner and Hakomori (35). Thus, the substrate specificities of beta 3Gal-T1 and -T2 are different, but further studies are required to define in detail the full range of functions of each enzyme.

The human beta 3-galactosyltransferase genes appear to be distantly related to the Drosophila gene Brainiac, which is involved in contact and adhesion between germ-line and follicle cells (36, 37) (Fig. 2). Previously, Yuan et al. (15) compared sequences of Brainiac and a related gene, Fringe, to a number of known bacterial glycosyltransferases and suggested that the two Drosophila genes may represent glycosyltransferases. These investigators also analyzed the human EST data base for potential related human genes, and in fact two ESTs (GenBank accession numbers R13867 and W26435) were suggested to represent human genes homologous to Brainiac. EST R13867 was shown in the present study to be derived from beta 3Gal-T2, and EST W26435 is from another beta 3-galactosyltransferase homologous gene that is presently under study. Yuan et al. (15) identified five conserved sequence motifs between Lex1, Fringe, and Brainiac subfamilies, and all of these motifs fall within the highly conserved sequence regions between the four human beta 3-galactosyltransferase genes (Fig. 2). Although several sequence motifs are shared between these genes, none of the three conserved cysteine residues in these are found in Brainiac. One cysteine in the C-terminal region of Brainiac and beta 3Gal-T4 was aligned. The potential N-glycosylation site conserved in all human beta 3-galactosyltransferases, is also found in Brainiac. Preliminary attempts to express Brainiac and identify glycosyltransferase activity were not successful with the substrates described here.3

A family of beta 3-galactosyltransferases is described in the present study. The substrate specificities and expression patterns for the beta 3-galactosyltransferases characterized to date were different, although some redundancy in function may exist for beta 3Gal-T1 and -T2. The finding that beta 3Gal-T4 transferred galactose to beta -GalNAc in GM2 suggests that other related beta 3-galactosyltransferases may belong to this gene family. Candidates include the Galbeta 1-3Gb4 glycolipid synthase, the Galbeta 1-3GalNAcalpha 1-3(Fucalpha 1-2)Galbeta 1-R synthase initiating the repetitive histo-blood group A-associated glycosphingolipids, as well as the Galbeta 1-3GalNAcalpha 1-O-Ser/Thr mucin-type core 1 synthase.

    ACKNOWLEDGEMENT

We thank Dr. M. Sobrinho-Simoes for support throughout the project.

    FOOTNOTES

* This work was supported by The Danish Cancer Society, the Velux Foundation, the Danish Medical Research Council, the Lundbeck Foundation, PECS/P/SAU/253/95, NIH 1 RO1 CA66234, RO1 CA41521, RO1 CA70740, and National Institute of Health Resource Center for Biomedical Complex Carbohydrates Grant NIH 5 P41 RR05351.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 GenBankTM/EMBL Data Bank with accession number(s) Y15060, Y15061, and Y15062.

Dagger Dagger To whom the correspondence should be addressed: Tel.: 45 35326835; Fax: 45 35326505; E-mail: henrik.clausen{at}odont.ku.dk.

1 The abbreviations used are: EST, expressed sequence tags; beta 3Gal-T1, UDP-galactose:beta -N-acetylglucosamine beta -1,3-galactosyltransferase, reported in GenBank accession number E07739; beta 3Gal-T2, beta 3Gal-T3, and beta 3Gal-T4, UDP-galactose:beta -N-acetylglucosamine/beta -N-acetylgalactosamine beta -1,3-galactosyltransferases cloned and expressed in this paper; UTR, untranslated region; bp, base pair(s); RT-PCR, reverse transcription-polymerase chain reaction; Glycosphingolipids are designated according to the recommendations of the IUPAC-IUB Commission on Nomenclature (Structures shown in Tables III & IV).; kb, kilobases.

2 T. Schwientek, R. Almeida, and H. Clausen, manuscript in preparation.

3 M. Amado and H. Clausen, unpublished observation.

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Abstract
Introduction
Procedures
Results
Discussion
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