Based on the detection of expressed sequence tags
that are similar to known galactosyltransferase sequences, we have
isolated three novel
UDP-galactose:
-N-acetylglucosamine
1,3-galactosyltransferase (
3GalT) genes from a mouse
genomic library. The three genes, named
3GalT-I, -II, and -III,
encode type II transmembrane proteins of 326, 422, and 331 amino acids,
respectively. The three proteins constitute a distinct subfamily as
they do not share any sequence identity with other eucaryotic
galactosyltransferases. Also, the entire protein-coding region of the
three
3GalT genes was contained in a single exon, which contrasts
with the genomic organization of the
1,4- and
1,3-galactosyltransferase genes. The three
3GalT genes were
mainly expressed in brain tissue. The expression of the full-length
murine genes as recombinant baculoviruses in insect cells revealed that
the
3GalT enzymes share the same acceptor specificity for
-linked
GlcNAc, although they differ in their Km for this
acceptor and the donor UDP-Gal. The identification of
3GalT genes
emphasizes the structural diversity present in the
galactosyltransferase gene family.
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INTRODUCTION |
The structural diversity of glycoconjugates results from the
combined action of glycosidases and glycosyltransferases that are
differentially expressed throughout tissues. Until recently, each
carbohydrate linkage was believed to be catalyzed by one single
glycosyltransferase. Deduced from this assumption, it was estimated
that ~250 genes were required for shaping the glycoconjugate repertoire of vertebrates (1). This count is constantly corrected to a
higher mark as many glycosyltransferase activities are found to be
encoded by multiple related genes. This apparent redundancy likely
reflects a selective preference of glycosyltransferase isozymes for
related although distinct acceptor structures, as observed for
1,3-fucosyltransferase (2) and GalNAc
2,6-sialyltransferase (3-5) isozymes.
Various strategies have been applied to identify genes related to known
glycosyltransferase genes. The use of low stringency hybridization was
only successful to detect very closely related genes, as was the case
for
1,3-fucosyltransferases (6, 7). Often, the comparison of related
glycosyltransferase genes unveils conserved regions that correspond to
residues essential for catalytic activity. Such comparisons performed
on sialyltransferase genes led to the detection of a stretch of
conserved sequences called the sialyl motif (8). By designing
degenerate primers according to the sialyl motif, it has been possible
to isolate additional sialyltransferase genes by polymerase chain
reaction (PCR)1 (5, 8, 9).
The recent availability of "single-pass" cDNA sequences, or
expressed sequenced tags (ESTs), represents a tremendous amount of
information that can be analyzed to retrieve sequences sharing limited
but significant sequence similarity. This approach is particularly well
suited to detect similarity at the level of the protein sequence, which
would be difficult to isolate by DNA-based retrieval techniques such as
cross-hybridization and PCR.
We have applied the EST screening procedure to identify genes encoding
1,3-galactosyltransferases, which refers to an activity yet
uncharacterized at the genetic level. To this end, we have probed the
EST division of the GenBankTM/EMBL Data Bank with the sequence of a
-galactosyltransferase protein that was believed to encode a
1,3-galactosyltransferase (
3GalT) (10). This procedure led to the
identification of novel open reading frames (ORFs) that were similar to
the query
-galactosyltransferase, but distinct from the previously
described
1,3-galactosyltransferase (11, 12) and
1,4-galactosyltransferase (13) genes. We have used probes designed
after these ORFs to isolate the corresponding genes from a mouse
genomic library. We have cloned three genes coding for proteins of 326, 422, and 331 amino acids. The three proteins are type II transmembrane
proteins containing a single transmembrane domain of 17-19 amino
acids. The sequence identity between the three proteins was the highest
in the luminal domain, where it ranged from 35 to 51%. Several
conserved motifs were detected in the catalytic domain. None of these
motifs were present in
1,3- and
1,4-galactosyltransferases,
although one motif was identified in some bacterial
galactosyltransferases. The expression of the three murine genes as
recombinant baculoviruses in Sf9 insect cells confirmed them as
encoding UDP-galactose:
-N-acetylglucosamine
1,3-galactosyltransferase enzymes. The genomic organization of the
murine
3GalT-I, -II, and -III genes differed from that of
1,4-galactosyltransferase (14) and
1,3-galactosyltransferase (15)
genes. The entire protein-coding region of the murine
3GalT genes
was contained in a single exon. Northern blot analysis showed that the
three genes were mainly expressed in brain tissue and also at lower
levels in other tissues.
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EXPERIMENTAL PROCEDURES |
Materials--
The mouse 129/SvJ genomic library, Duralose-UV
nitrocellulose membranes, and the Bluescript SKII plasmid were
purchased from Stratagene. The Vent polymerase, restriction
enzymes, and DNA-modifying enzymes were obtained from New England
Biolabs Inc. The AmpliTaq FS Dye Terminator Cycle Sequencing kit, the
CentriSep columns, and the ABI 310 Genetic Analyzer were from
Perkin-Elmer/Applied Biosystems. The baculovirus expression system
Bac-to-Bac and Grace's medium were obtained from Life Technologies,
Inc. Carbohydrate acceptors and donors were purchased from Sigma.
Asialo-ovine submaxillary mucin was a gift from Dr. Robert L. Hill
(16). Gal
1,4GlcNAc-
-pNP was generated enzymatically by the action
of recombinant human
1,4-galactosyltransferase (17), and the
Gal
1,3GlcNAc-
-pNP standard was purchased from Sigma. The HPLC
system consisted of a Jour Research X-ACT degasser, two Waters 510 pumps, a Waters 717 autoinjector, a Jasco C0965 Column Heater
(30 °C), and a Waters 490 programmable multi-wavelength detector
(214, 262, and 350 nm). The system was controlled using Waters
Millennium (Version 2.15.2). UDP-[14C]Gal and Hybond-N
nylon membranes were from Amersham Corp. Sep-Pak C18
cartridges were obtained from Waters, and GFA glass-microfiber filters
were from Whatman. [32P]CTP was purchased from Hartmann
Analytics (Braunschweig, Germany).
Genomic Cloning of
3GalT Genes--
The mouse genomic library
was plated onto Luria-Bertani agar, and plaques were lifted onto
Duralose-UV filters. The filters were hybridized to
[32P]CTP-labeled DNA probes designed after the EST
sequences that were similar to a human galactosyltransferase gene (10).
The genomic inserts from the phage clones were subcloned as
NotI-NotI fragments into the NotI site
of the pBluescript SKII vector, and regions that hybridized to the EST
fragments were further subcloned into pBluescript and sequenced.
Sequencing--
Sequencing was done using the AmpliTaq FS Dye
Terminator Cycle Sequencing kit with the vectors T3 and T7 matching
primers flanking the cloned inserts in pBluescript. Sequenced products were purified with CentriSep columns and run on an ABI 310 Genetic Analyzer. Primer walking was used afterward for sequence determination throughout the complete coding region of every
3GalT gene. The sequences of the coding and complementary strands were determined and
edited using the Sequencher program (Gene Codes, Ann Arbor, MI).
Cloning of Recombinant Baculoviruses and Expression in Sf9
Cells--
Recombinant baculoviruses were generated using the
transposon-mediated insertion system developed by Luckow et
al. (18) as purchased from Life Technologies, Inc. The mouse
3GalT-I, -II, and -III genes were amplified by PCR from the genomic
clones using 5
-primers containing an EcoRI site
(
3GalT-I) or a BamHI site (
3GalT-II and -III) just
upstream of the ATG codon. The 3
-primers for the three
3GalT genes
contained a XbaI site following the stop codon. The
conditions for PCR were 20 cycles at 95 °C for 45 s, 55 °C
for 30 s, and 72 °C for 60 s using the Vent
polymerase and 50 ng of plasmid DNA as template. The resulting PCR
fragments were subcloned into the baculovirus donor vector FastBac1
opened at the EcoRI and XbaI sites (
3GalT-I)
or at the BamHI and XbaI sites (
3GalT-II and
-III). Following the instructions of the manufacturer, the
FastBac1-
3GalT plasmids were transformed into DH10Bac cells for
transposition into the bacmid. The integration of the
3GalT genes in
the bacmids was confirmed by PCR using the 5
-primers specific for each
3GalT-I gene and the m13r primer (5
-AACAGCTATGACCATGATTACG-3
),
which binds to a bacmid region outside of the transposition element.
Sf9 cells (106) were transfected with 1 µg of
recombinant bacmid DNA using Cellfectin (Life Technologies, Inc.) and
incubated for 3 days at 27 °C. The culture medium was saved as the
primary baculovirus stock. The primary stocks were amplified three
times to reach titers of ~109 plaque-forming units/ml.
Sf9 cells were infected at a multiplicity of 10 and further
incubated at 27 °C for 72 h.
Galactosyltransferase Activity Assays--
Baculovirus-infected
Sf9 cells were washed with phosphate-buffered saline and lyzed in
2% Triton X-100 for 15 min on ice. Nuclei were removed from the
lysates by centrifugation at 500 × g. 10 µl of the
lysates were assayed for 30 min at 37 °C in 50-µl reactions of 50 mM cacodylate buffer, pH 6.6, 10 mM
MnCl2, 0.5 mM UDP-Gal, 1% Triton X-100, 10%
Me2SO, and varying acceptors at different concentrations
(see Table II). 105 cpm of UDP-[14C]Gal (164 pmol) were added to standard assays, whereas 2.5 × 105 cpm of UDP-[14C]Gal (410 pmol) were added
when kinetic parameters were determined. The reactions were stopped by
dilution with 500 µl of H2O and applied onto a Sep-Pak
C18 cartridge. The cartridge was washed with 15 ml of
H2O and eluted with 5 ml of methanol. The samples containing asialo-ovine submaxillary mucin as acceptor were stopped by
adding 1 ml of cold 15% trichloroacetic acid and 5% phosphotungstic acid solution. The precipitates were collected on GFA glass-microfiber filters, which were washed with 5 ml of ice-cold ethanol and dried at
80 °C for 30 min. The amount of [14C]Gal in the
methanol eluates and on the GFA filters was measured in a liquid
scintillation counter (Rackbeta, Pharmacia Biotech Inc.).
Analysis of Linkage Specificity of
3GalT Enzymes--
Sep-Pak
C18 eluates of
3GalT-I, -II, and -III assays using
unlabeled UDP-Gal with GlcNAc-
-pNP as acceptor were dried by centrifugal evaporation and redissolved in 100 µl of H2O.
A 20-µl aliquot of this material was diluted with acetonitrile to 100 µl and subjected to normal phase (NP) HPLC. NP-HPLC was carried out
using the 50 mM ammonium formate pH 4.4 buffer system on a GlycoSep-N column (Oxford GlycoSciences, Abingdon, United Kingdom) over
180 min as described previously (19). The pNP-labeled disaccharides were isolated from the sample mixture by comparison to standard UDP,
UDP-Gal, GlcNAc-
-pNP, Gal
1,3GlcNAc-
-pNP, and
Gal
1,4GlcNAc-
-pNP. The fractions containing the disaccharides
were pooled and analyzed by reverse phase HPLC (20) to separate
Gal
1,3GlcNAc-
-pNP and Gal
1,4GlcNAc-
-pNP. Briefly, the first
step of the gradient applied to a GlycoSep-R column (Oxford
GlycoSciences) consisted of isocratic elution with 75% Solvent A (25%
50 mM ammonium formate, pH 4.4, in acetonitrile) and 25%
Solvent B (50 mM ammonium formate, pH 4.4) over 10 min at a
flow rate of 1.0 ml/min. This was followed by a linear gradient of 75 to 25% Solvent A over 20 min, followed by a 5-min isocratic step under
these conditions. The column was re-equilibrated in the initial solvent
composition 20 min prior to the next injection.
Exoglycosidase Digestion Conditions--
Streptococcus
pneumoniae
-galactosidase (Oxford GlycoSciences) digestions
were performed at 37 °C for 16-24 h in 100 mM sodium acetate, pH 6.0, at 27 milliunits/ml. At this concentration, the enzyme
cleaves only the Gal
1,4HexNAc linkage (21). An aliquot of the
aqueous sample was mixed with acetonitrile at a ratio of 20:80 and
applied to the NP-HPLC column.
RNA Preparation and Northern Blotting--
Total RNA from
8-week-old ICR mouse tissues was isolated with guanidinium
isothiocyanate, followed by centrifugation on cesium chloride cushions
(22). 5 µg of total RNA from each tissue were separated on
formaldehyde-agarose gels. RNA was either stained in ethidium bromide
(0.5 µg/ml in 0.1 M ammonium acetate) or transferred to
Hybond-N membranes by capillary elution. Filters were hybridized overnight at 42 °C with [32P]CTP-labeled probes
corresponding to the full-length mouse
3GalT-I, -II, and -III genes.
The filters were washed with 0.1 × SSC and 0.1% SDS up to
60 °C and exposed for 3 days at
70 °C using intensifying screens.
 |
RESULTS |
Identification and Isolation of Mouse
3GalT Genes--
The
EST division of the GenBankTM/EMBL Data Bank was searched for
sequences similar to a human
-galactosyltransferase protein (
xGalT) (10) that was regarded as a possible
3GalT. The query sequence was compared with the data base using the tblastn algorithm (Version 1.4.9) (23). ESTs bearing the National Center for
Biotechnology Information sequence identification numbers R13867,
D81474, H14861, R61672, R13064, R15977, T31289, H70585, H70574, Z43147,
and Z45582 were identified as being significantly similar to the query
xGalT sequence (Table I). It is
noteworthy that we have not detected any EST that is identical to the
probed
xGalT, indicating that this gene is not represented yet in
the EST data base. The retrieved EST sequences were aligned with the query sequence, and partially overlapping ESTs were assembled to build
larger ORFs (Table I). DNA fragments corresponding to the
xGalT gene
and to EST probes D81474, H70574, and R15977 were amplified by PCR from
a pool of human T-cell cDNAs. The resulting fragments were used as
DNA probes in screening a mouse genomic DNA library. We could isolate
seven clones, which represented three distinct genomic regions. The
three fragments 6, 10, and 13 encompassed the same genomic region and
hybridized to EST probe D81474. Both genomic fragments 5 and 14 hybridized to EST probes H70574 and R15977, indicating that the
assembled ORF2 and ORF3 were parts of the same gene. Genomic fragments
2 and 15 included the murine
xGalT gene (Table I).
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Table I
Cloning of mouse genomic fragments related to a human
-galactosyltransferase gene
ESTs that were similar to a human -galactosyltransferase gene
( xGalT) were retrieved using a tblastn search of the EST division
of the GenBankTM/EMBL Data Bank. The EST sequences were aligned, and
those that overlapped were assembled to form larger ORFs. ESTs
representative of each ORF as well as the human xGalT gene were used
as DNA probes to screen a mouse genomic DNA library. Seven mouse
genomic fragments were isolated and grouped according to their
specificity for the DNA probes. ORF2 and ORF3 were found to be
different parts of the same gene, as genomic fragments 5 and 14 hybridized to both DNA probes H70574 and R15977.
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The genomic regions hybridizing to the probes were sequenced, and the
deduced genes were tentatively named
3GalT-I, -II, and -III. The
3GalT genes were aligned with the originally identified ESTs (Table
I) and additional related ESTs that were retrieved from the EST data
base with the blastn algorithm (Fig. 1).
This comparison confirmed ORF2 and ORF3 as being parts of the
3GalT-III gene. Also, it appeared that the sequence of the human
3GalT-III gene could be entirely determined by joining contiguous
EST fragments. No EST corresponding to the
3GalT-I gene could be
identified in the
EST data base using the blastn, blastx, and
tblastx programs.

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Fig. 1.
Schematic alignment of EST sequences with the
murine 3GalT genes. The EST fragments similar to the 3GalT
genes were aligned with the corresponding 3GalT genes. The
protein-coding regions of the three murine 3GalT genes are given as
black rectangles. National Center for Biotechnology
Information sequence identification numbers are provided to the left of
the lines representing the EST fragments. The vertical
dotted lines indicate 500-bp increments.
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Primary Structure and Genomic Organization of Murine
3GalT
Genes--
The murine
3GalT-I, -II, and -III genes code for
proteins of 326 amino acids (Fig. 2), 422 amino acids (Fig. 3), and 331 amino acids
(Fig. 4), respectively. The
3GalT-II
and -III genes contain two in-frame ATG codons spaced by 12 and 11 amino acids, respectively. It is unclear whether the first, second, or
both ATG codons are used as the start codon. It appears, however, that in both genes, the proximal ATG codons have stronger contexts for
translation initiation (24). A Kyte-Doolittle hydropathy analysis
detected single membrane-spanning domains of 19, 19, and 17 amino acids
for the
3GalT-I, -II, and -III proteins, respectively (Figs. 2-4).
The transmembrane domains are located close to the N terminus of each
protein, which is typical of the type II transmembrane glycosyltransferase protein family. The protein-coding regions of
3GalT-I, -II, and -III are contained in a single exon. However, more
proximal untranslated exons might exist, as is the case for other
glycosyltransferase genes (25, 26). This assumption is supported by the
fact that no TATA box sequence was detected in front of the
3GalT
genes. Also, we noticed that the cDNAs of the three
3GalT genes
contain ~500 bp of untranslated sequence that differs from the
genomic sequence 5
of the ATG codons (data not shown).

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Fig. 2.
Nucleotide and predicted amino acid sequences
of the murine 3GalT-I gene. The translation initiation codon
and stop codon are boldface and underlined. Amino
acids are in single-letter notation. The amino acids corresponding to
the putative transmembrane domain are boldface and
underlined.
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Fig. 3.
Nucleotide and deduced amino acid sequences
of the murine 3GalT-II gene. The first in-frame ATG codon at 64 bp is given as the translation initiation codon and is
boldface and underlined. The second in-frame ATG
codon at 103 bp is boldface. The stop codon at 1330 bp is
boldface and underlined. Single-letter notation
is used for the amino acids. The amino acids corresponding to the
putative transmembrane domain are boldface and
underlined.
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Fig. 4.
Nucleotide and deduced amino acid sequences
of the murine 3GalT-III gene. The first in-frame ATG codon at
37 bp is boldface and underlined, and the second
in-frame ATG codon at 73 bp is underlined. The stop codon at
position 1030 bp is boldface and underlined.
Amino acids are in single-letter notation. The amino acids
corresponding to the proposed transmembrane domain are
boldface and underlined.
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Homology between
3GalT Proteins--
The three mouse
3GalT
protein sequences were compared using the ClustalW alignment program
(27) (Fig. 5). The greatest identity was
observed in the catalytic domains of the proteins. In this region,
3GalT-I was 51% identical to
3GalT-II and 36% identical to
3GalT-III, whereas
3GalT-II was 35% identical to
3GalT-III.
The
3GalT enzymes shared no sequence similarity in their N-terminal
part corresponding to the cytoplasmic, transmembrane, and stem regions.
We noticed that the larger size of
3GalT-II reflects a longer stem
region, whereas the size of the catalytic domains is conserved among
the three
3GalT enzymes. The positions of six cysteine residues in
the catalytic domains of the three
3GalT proteins were conserved
(Fig. 5). This supports the formation of up to three disulfide bridges
that would confer a similar structure to the three
3GalT enzymes.
Such related conservations of cysteine residues have been previously
observed in the sialyltransferase protein family (28). Several
conserved motifs emerged from the alignment of the
3GalT protein
sequences (Fig. 5). Notably, we did not detect these same motifs in
1,4- and
1,3-galactosyltransferase sequences, indicating that
there are no consensual galactosyl motifs in vertebrate
galactosyltransferases. However, a variation of the
3GalT motif
EDVYVG was recognized in the bacterial galactosyltransferases Lex1 (29), LgtB (30), and LgtE (31) (Fig.
6). This conservation was also detected
by others and described as a putative galactosyltransferase motif
(32).

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Fig. 5.
Alignment of the three murine 3GalT
protein sequences. The 3GalT sequences were aligned using the
ClustalW algorithm. Amino acid identities are marked in
boldface, and conserved regions are shaded.
Cysteines that are conserved in the three 3GalT proteins are
boxed.
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Fig. 6.
Comparison of the (E/D)DV motif in murine
3GalT enzymes and bacterial galactosyltransferases. The (E/D)DV
region of the three murine 3GalT proteins was aligned with the
corresponding region found in the Hemophilus influenzae Lex1
(GenBankTM/EMBL Data Bank accession number U32736) and Neisseria
gonorrhoeae LgtB (GenBankTM/EMBL Data Bank accession number
U14554) and LgtE (GenBankTM/EMBL Data Bank accession number U14554)
galactosyltransferases. The position of the first amino acid of the
(E/D)DV motif in each protein is indicated to the left of the protein
sequences, which are given in single-letter notation. Conserved
residues are boldface.
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Tissue Expression of
3GalT Genes--
The expression pattern of
the three
3GalT genes was investigated in adult mouse tissues. We
have found the
3GalT genes to be predominantly expressed in brain
tissue, whereas lower transcript levels were also detected in other
tissues (Fig. 7). Beside brain tissue,
the 7-kilobase
3GalT-I mRNA was detected at low levels in all
tissues examined, indicating a possible constitutive expression. Two
additional smaller transcripts were also observed in colonic tissue.
The
3GalT-II transcript was 3 kilobases long and was present in
brain and heart tissue and could barely be detected in ovaries, colon,
and lymph nodes. The expression of
3GalT-III yielded a 2.7-kilobase
transcript that was restricted to brain, testes, ovaries, and uterus.
In addition, the three
3GalT genes were expressed in lymphoid cells
as detected by reverse transcriptase-PCR (data not shown).

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Fig. 7.
Differential expression of the three 3GalT
genes in murine tissues. Northern blot analysis was performed
using 5 µg of total RNA from each tissue sample, as is visible on the
ethidium bromide staining (bottom panel). The blots were
probed with full-length 3GalT gene probes as detailed under
"Experimental Procedures."
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Expression of
3GalT Genes in Sf9 Cells--
Sf9
insect cells were infected with recombinant baculoviruses expressing
the three
3GalT genes under the control of the polyhedrin promoter.
The lysates of Sf9 cells harvested at 72 h post-infection were assayed for galactosyltransferase activity using various acceptor
saccharides (Table II). The highest
activity was detected with
-conjugated GlcNAc acceptors like
GlcNAc-
-pNP and GlcNAc-
-benzyl (Bzl). By contrast, the
-conjugated GlcNAc acceptors yielded a 10-20-fold lower activity
compared with the
-conjugates. No significant galactosyltransferase
activity was measurable toward GalNAc acceptors. This finding was
corroborated by assays performed in the presence of the high molecular
weight acceptor asialo-ovine submaxillary mucin, which carries multiple
O-linked GalNAc monosaccharides (16). Also, the three
3GalT enzymes failed to transfer GlcNAc or GalNAc to the acceptors
GlcNAc-
-Bzl, Gal-
-Bzl, and GalNAc-
-Bzl, showing that the
proteins are solely galactosyltransferases (data not shown). The
activity of the three
3GalT proteins was strictly dependent on the
divalent cation Mn2+ (data not shown). Also, the proteins
were more active in the presence of 1% Triton X-100 (data not shown).
The enzymatic properties of the three
3GalT proteins were determined
for the donor UDP-Gal and the acceptor GlcNAc-
-pNP (Table
III). When compared with
3GalT-I, we
found that
3GalT-II and
3GalT-III exhibited 3- and 2-fold higher
Km values for GlcNAc-
-pNP, respectively. The opposite was observed with the Km for the donor
UDP-Gal, for which
3GalT-I showed 4- and 2-fold greater
Km values than
3GalT-II and
3GalT-III,
respectively.
Analysis of Linkage Specificity of
3GalT--
The products of
the galactosyltransferase activity assays of the lysates from
baculovirus-infected Sf9 cells expressing
3GalT-I, -II, and
-III and wild-type baculoviruses were resolved by NP-HPLC. Fig.
8A (panel i) shows
the profile for the products of the
3GalT-I assay. The elution
positions of UDP, UDP-Gal, GlcNAc-
-pNP, and Gal
1,3/4GlcNAc-
-pNP (peak g) were assigned by
comparison with known standards (Fig. 8A, panel
ii). The HPLC fractions of the
3GalT-I, -II, and -III reaction
products containing the disaccharide were pooled, evaporated to
dryness, redissolved in water, and applied to the reverse phase HPLC
column (Fig. 8B, panels ii-iiii), which resolves
the linkage-specific isomers Gal
1,3GlcNAc and Gal
1,4GlcNAc. The
Gal
GlcNAc linkage was shown to be exclusively
1,3. It was
assigned by comparing the elution position of the pNP-labeled
disaccharides with those of standard Gal
1,4GlcNAc-
-pNP (Fig.
8B, panel i) and standard Gal
1,3GlcNAc-
-pNP
(panel v) and by co-injection with the authentic material
(data not shown). The assignment was consistent with the finding that
the Gal
1,3GlcNAc-
-pNP product was not susceptible to
S. pneumoniae galactosidase, in contrast to
Gal
1,4GlcNAc-
-pNP (data not shown).

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Fig. 8.
HPLC analysis of cell lysates from control
and 3GalT-expressing systems. A, NP-HPLC profiles of the
lysate from 3GalT-I-expressing cells (panel i) and the
sugar standards GlcNAc- -pNP (peak a), Gal 1,3/4GlcNAc- -pNP (peak b), UDP (peak c),
and UDP-Gal (peak d) (panel ii). In panel
i, peak p indicates that the peak contains protein.
B, reverse phase HPLC profiles of the
Gal 1,4GlcNAc- -pNP standard (panel i); the disaccharide
peak (peak g) from A (panel i) for
3GalT-I (panel ii), 3GalT-II (panel iii),
and 3GalT-III (panel iiii); and the
Gal 1,3GlcNAc- -pNP standard (panel v).
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DISCUSSION |
Based on the retrieval of ESTs sharing similarity with a
-galactosyltransferase gene sequence, we have identified three novel murine
3GalT genes. The identification of multiple genes encoding a
3GalT activity is consistent with recent similar findings made for
other glycosyltransferase families (33). The presence of several
isozymes can be interpreted as a way to maintain a greater potential to
glycosylate different related acceptor structures, thereby increasing
the versatility of glycosyltransferases in shaping the
glycoconjugate repertoire.
The three
3GalT proteins are heterologous in size, with
3GalT-I
and
3GalT-III representing to date the smallest mammalian glycosyltransferases after the GalNAc
2,6-sialyltransferase-III (5).
As observed for many glycosyltransferase genes, two in-frame ATG codons
are present in the
3GalT-II and
3GalT-III genes. The two ATG
codons are located 5
of the region encoding the transmembrane domain,
and an alternate initiation of translation would unlikely affect the
localization and activity of the two proteins. The three
3GalT
proteins represent a distinct family from the
1,4- and
1,3-galactosyltransferase proteins, as no sequence similarity can be
detected between
3GalT enzymes and the other galactosyltransferases. This difference is emphasized by comparing the genomic organization of
3GalT genes with that of
1,4- and
1,3-galactosyltransferase genes, which contain multiple exons coding for protein sequence (14,
15). These distinctions suggest that the family of
3GalT genes
separated early in evolution from the other galactosyltransferases and
possibly expanded by gene duplication. It remains open as to whether
the
3GalT gene family comprises additional related members. The
search for ESTs similar to the three murine
3GalT sequences supports
the notion of a gene family consisting of 7-10 members.
The alignment of the three
3GalT protein sequences highlighted
various conserved regions that were all located in the postulated catalytic domain. These
3GalT putative motifs were absent in other
vertebrate galactosyltransferase protein sequences. However, a short
3GalT motif was identified in bacterial galactosyltransferases, suggesting that these amino acids are essential for
galactosyltransferase activity. Some of the
3GalT motifs were also
present in the Drosophila proteins Fringe and Brainiac as
outlined by Yuan et al. (32). Fringe (34) and Brainiac (35)
have been described as secreted signaling molecules, although their
mechanism of action has not been elucidated yet. The similarity between
Fringe, Brainiac, and
3GalT enzymes suggests that the two
Drosophila proteins may represent additional members of the
3GalT family.
The acceptor specificity of the three
3GalT enzymes was restricted
to
-conjugated GlcNAc. However, the high Km values measured for the acceptor GlcNAc-
-pNP indicate that the physiological acceptors are likely more specific oligomeric structures. The
3GalT enzymes enable the formation of type 1 carbohydrate chains, which are widely distributed on glycolipids and glycoproteins throughout tissues. Being the acceptor of the Lewis enzyme, the type 1 disaccharide Gal
1,3GlcNAc is related to the occurrence of
Lea and Leb antigens (36, 37). Although mainly
expressed in brain tissue, the three
3GalT genes are differentially
expressed in mouse tissues, which suggests that these three
3GalT
enzymes may participate in the formation of Lea and
Leb antigens in mouse tissues. The identification of three
3GalT genes highlights the structural diversity encountered in the
galactosyltransferase gene family. The difference in sequence and
genomic organization between
3GalT and other vertebrate
galactosyltransferase genes suggests a distinct evolution. The
structural information gained from the cloning of the three
3GalT
genes provides a way to identify additional members of this branch of
the galactosyltransferase family.
We thank Dr. Martine Malissard for advice and
criticism throughout this work and Marianne Farah for technical
assistance.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF029790-AF029792.