From the 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,
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 .
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ABSTRACT |
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BLAST analysis of expressed sequence tags
(ESTs) using the coding sequence of a human
UDP-galactose:-N-acetyl-glucosamine
-1,3-galactosyltransferase, designated
3Gal-T1, revealed no ESTs
with identical sequences but a large number with similarity. Three
different sets of overlapping ESTs with sequence similarities to
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:
-N-acetyl-glucosamine
-1,3-galactosyltransferase (
3Gal-T2) with similar kinetic
properties as
3Gal-T1. Another gene represented a
UDP-galactose:
-N-acetyl-galactosamine
-1,3-galactosyltransferase (
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.
3Gal-T1 mRNA
was expressed in brain,
3Gal-T2 was expressed in brain and heart, and
3Gal-T3 and -T4 were more widely expressed. The coding regions for each of the four genes were contained in single exons.
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
3-galactosyltransferase genes.
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INTRODUCTION |
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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:-N-acetyl-glucosamine
-1,4-galactosyltransferases (
4Gal-T). At least six novel genes were found of which four have been shown to represent functional
4Gal-Ts (2-5). Perhaps surprisingly, there are no sequence
similarities between the
-4-galactosyltransferases and a putative
UDP-galactose:
-N-acetyl-glucosamine
-1,3-galactosyltransferase gene submitted to GenBank in 1996 and
here designated
3Gal-T1. The disaccharides Gal
1-3GlcNAc
(type
1 chain) and Gal
1-4GlcNAc
(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
-GlcNAc (e.g. the
-3,4-fucosyltransferases (11)), suggesting that some enzymes may recognize common features of the two
acceptor sites.
In the present study, we found that 3Gal-T1 is one member of a
3-galactosyltransferase family. The human EST sequence data base was
used to identify several novel members of a
3-galactosyltransferase gene family. The
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:
GlcNAc
-1,3-galactosyltransferase (
3Gal-T2); and one gene
encoded a UDP-Gal:
GalNAc
-1,3-galactosyltransferase gene
(
3Gal-T4), with different acceptor substrate specificity.
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EXPERIMENTAL PROCEDURES |
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Identification of 3Gal-T1 Homologues Genes
Data base searches were performed with the reported coding
sequence of a human 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
3Gal-T2
Four partly overlapping ESTs with approximately 360 bp open
reading frame with sequence similarity to the C-terminal sequence of
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
3Gal-T1.
Cloning and Sequencing of the Full Coding Sequence of
3Gal-T3
Five partly overlapping ESTs with approximately 900 bp of open
reading frame and sequence similarity to 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
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
3Gal-T1 and 33% to -T2.
Cloning and Sequencing of the Full Coding Sequence of
3Gal-T4
Two overlapping EST clones with sequence similarity to the
N-terminal sequence of 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
3Gal-T1.
Baculo Expression Constructs for 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)
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)
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);
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
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 3Gal-T4 cDNA from EST H2O623, which
contained the full coding sequence). The following primer pairs were
used: (i)
3Gal-T1, EBER306 (5'-AGACAATGGCTTCAAAGGTCTC) and EBER 304;
(ii)
3Gal-T2, EBER400FUL (5'-TACAACATGCTTCAGTGGAGGAG) and EBER415;
(iii)
3Gal-T3, EBER600FUL (5'-TGACCATGGCCTCGGCTCTCTGGACTG) or
EBER611 (5'-GTAGGATGTCACTGAGATCCC) (for second in-frame ATG) in
combination with EBER614; and (iv)
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 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 GlcNAc-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
3Gal-T1 and -T2)
or Triton CF-54 (for
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 3Gal-T2 and
-T4
Terminal glycosylation of Lc3Cer (43) with
3Gal-T2 was performed in a reaction mixture consisting of 1 milliunit of
3Gal-T2 (specific activity determined with
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
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
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
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
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 [-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
3Gal-T1, -T2, and -T3
P1 genomic clones were obtained for 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
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 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
3Gal-T1 was found; thus, the localization of this gene was not
determined.
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RESULTS |
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Identification and Cloning of Human 3Gal-T2,
3Gal-T3, and
3Gal-T4--
The search and cloning strategy outlined in Fig.
1 produced three novel genes with
significant sequence similarity to
3Gal-T1 (Fig.
2). Additionally, three genes with less
similarity were identified (not shown). Multiple sequence alignment of
the three
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,
3Gal-T2 is most
similar to
3Gal-T1,
3Gal-T3 was the second most similar, and the
least similar is
3Gal-T4. At least three cysteine residues align
within all the human genes; an additional two align within
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
3Gal-T1, five in
3Gal-T2 and -T3, and one in
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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Expression of 3Gal-T1 and -T2--
Expression of a soluble
construct of
3Gal-T1 and -T2 in Sf9 cells resulted in a
marked increase in galactosyltransferase activity using
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
3Gal-T1 and -T2 activities showed that
only saccharides with a terminal
GlcNAc residue and not
GlcNAc or
GalNAc were acceptor substrates. It was not possible to determine
Km for benzyl-
GlcNAc or Umb-
GlcNAc with
3Gal-T1 and -T2 due to substrate inhibition at concentrations above
100 mM. The Km for UDP-Gal of
3Gal-T1
and -T2 were 90 ± 5 µM and 37 ± 9 µM, respectively, using Benzyl-
GlcNAc as an acceptor
substrate.
3Gal-T2 catalyzed glycosylation of hen egg ovalbumin,
whereas
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
GlcNAc-terminating structures Lc3Cer and nLc5Cer, but
3Gal-T1 showed lower
activity with nLc5Cer. Both enzymes showed low activities
with GlcCer (Table III).
3Gal-T2 and
to a lesser extent
3Gal-T1 incorporated galactose into
nLc4Cer. Secreted forms of
4-galactosyltransferases were
included as controls (2).2
Both
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
3Gal-T2 with Lc3Cer was shown by 1H NMR to
be Lc4Cer, thus verifying that T2 formed the
Gal
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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Expression of 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 3Gal-T4--
Expression of the full-length coding
construct for
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
3Gal-T4 with GM2 was analyzed
by 1H NMR to verify that T4 formed the Gal
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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Northern Analysis of 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).
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
3Gal-T1 was not included in this study.
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
3Gal-T2 were
obtained from brain libraries, but two mouse ESTs were from a mammary
gland library.
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
3Gal-T3 were obtained from brain, liver/spleen, and heart
libraries.
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
3Gal-T4 were obtained from brain libraries.
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Genomic Organization and Chromosomal Localization--
Sequence
analysis of P1 clones showed that the coding regions of 3Gal-T2 and
-T3 were located in a single exon. PCR with genomic DNA revealed that
the coding regions of
3Gal-T1 and -T4 were also contained in one
exon. The four
3-galactosyltransferase genes were located on
different chromosomes;
3Gal-T2 at 1q31, T3 at 3q25, and T4 at
6p21.3.
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DISCUSSION |
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In the present study, four members of a human
3-galactosyltransferase gene family were characterized. A human
3-galactosyltransferase gene, here designated
3Gal-T1, indicated
to catalyze the formation of the type 1 core structure,
Gal
1-3GlcNAc, was released in GenBank October 1996 and described in
some detail in a patent application (JP1994181759-A/1).
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
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
4-galactosyltransferases
(2), a novel family of homologous
3-galactosyltransferase genes was identified. Three genes with a high degree of similarity,
3Gal-T2, -T3, and -T4, were studied here, and recombinant forms of two of these
have
3-galactosyltransferase activity.
3Gal-T2 has kinetic
properties similar to
3Gal-T1, but there was a striking difference
in their activities with the glycoprotein ovalbumin.
3Gal-T4 was
found to catalyze the addition of galactose to the gangliosides
GM2 and Gg3. The function of the human
3Gal-T3 gene reported here was not identified.
During the course of this work, a rat UDP-Gal:GalNAc
-1,3-galactosyltransferase gene was isolated by the
transfection-cloning strategy (18). The rat enzyme has an overall
sequence identity of 79% to human
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
3Gal-T1 and -T2 were reported by Kolbinger et al. (23), and in agreement with our finding that
3Gal-T2 utilized nLc4Cer as acceptor, these authors
reported 16% activity of this enzyme with
Gal
O-(CH2)8-CO2CH3.
Mouse homologues of
3Gal-T1, -T2, and -T3 were reported
simultaneously (24), and in this study, the murine
3Gal-T2 and -T3
were found to have low levels of activity with GlcNAc-
-pNP,
approximately 30-fold lower than
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
3Gal-T1 and
-T2 were 90 and 37 µM, respectively. Furthermore, the
kinetic properties of human
3Gal-T1 and -T2 with simple acceptors were comparable, but we did not detect significant activity with
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
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
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
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
1-6N-acetylglucosaminyltransferase families are encoded
by a single exon (26, 27). In contrast to these gene families, all
members of the
3Gal-T family have different chromosomal
localizations. The significance of this is presently unknown.
The existence of multiple 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),
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,
3Gal-T2 catalyzed transfer of galactose to ovalbumin, whereas
3Gal-T1 did not. Analogously, one member of the
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 GlcNAc
1-2Man and GlcNAc
1-4Man structures (34), and apparently none of these are utilized by
3Gal-T1. However,
3Gal-T1 utilized the disaccharide GlcNAc
1-6Man-1-OMe more
efficiently than
3Gal-T2 (Table I), indicating that perhaps tri- or
tetraantennary N-linked glycoproteins may serve as
substrates for
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
3Gal-Ts
utilized the disaccharide GlcNAc
1-3GalNAc representing mucin-type
core 3 (Table I), whereas four
4Gal-Ts efficiently use this
substrate (2).
3Gal-T1 and -T2 showed similar activities with
-GlcNAc-terminating lactoseries glycosphingolipids. Interestingly,
especially
3Gal-T2 exhibited significant incorporation into
nLc4Cer (Table III). The structure of the product was not
determined, but it is likely to be
Gal
1-3Gal
1-4GlcNAc
1-3Gal
1-4Glc
1-Cer, which was
originally isolated by Stellner and Hakomori (35). Thus, the substrate
specificities of
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 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
3Gal-T2, and EST W26435 is from another
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
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
3Gal-T4 was
aligned. The potential N-glycosylation site conserved in all
human
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 3-galactosyltransferases is described in the present
study. The substrate specificities and expression patterns for the
3-galactosyltransferases characterized to date were different, although some redundancy in function may exist for
3Gal-T1 and -T2.
The finding that
3Gal-T4 transferred galactose to
-GalNAc in
GM2 suggests that other related
3-galactosyltransferases
may belong to this gene family. Candidates include the
Gal
1-3Gb4 glycolipid synthase, the
Gal
1-3GalNAc
1-3(Fuc
1-2)Gal
1-R synthase initiating the
repetitive histo-blood group A-associated glycosphingolipids, as well
as the Gal
1-3GalNAc
1-O-Ser/Thr mucin-type core 1 synthase.
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ACKNOWLEDGEMENT |
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We thank Dr. M. Sobrinho-Simoes for support throughout the project.
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FOOTNOTES |
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* 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.
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; 3Gal-T1,
UDP-galactose:
-N-acetylglucosamine
-1,3-galactosyltransferase, reported in GenBank accession number
E07739;
3Gal-T2,
3Gal-T3, and
3Gal-T4,
UDP-galactose:
-N-acetylglucosamine/
-N-acetylgalactosamine
-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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REFERENCES |
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