1,3-Galactosyltransferase
3Gal-T5 Acts on the
GlcNAc
1
3Gal
1
4GlcNAc
1
R Sugar Chains of
Carcinoembryonic Antigen and Other N-Linked
Glycoproteins and Is Down-regulated in Colon Adenocarcinomas*
Roberta
Salvini,
Anna
Bardoni,
Maurizia
Valli, and
Marco
Trinchera
From the Department of Biochemistry, University of Pavia, via
Taramelli 3B, 27100 Pavia, Italy
Received for publication, July 26, 2000, and in revised form, October 6, 2000
 |
ABSTRACT |
We attempted to determine whether
1,3-galactosyltransferase
3Gal-T5 is involved in the biosynthesis
of a specific subset of type 1 chain carbohydrates and expressed in a
cancer-associated manner. We transfected Chinese hamster ovary (CHO)
cells expressing Fuc-TIII with
3Gal-T cDNAs and studied the
relevant glycoconjugates formed.
3Gal-T5 directs synthesis of Lewis
type 1 antigens in CHO cells more efficiently than
3Gal-T1, whereas
3Gal-T2, -T3, and -T4 are almost unable to direct synthesis. In the
clone expressing Fuc-TIII and
3Gal-T5 (CHO-FT-T5), sialyl-Lewis a
synthesis is strongly inhibited by swainsonine but not by
benzyl-
-GalNAc, and sialyl-Lewis x is absent, although it is
detected in the clones expressing Fuc-TIII and
3Gal-T1 (CHO-FT-T1) or Fuc-TIII and
3Gal-T2 (CHO-FT-T2).
Endo-
-galactosidase treatment of N- glycans
prepared from clone CHO-FT-T5 releases
(±NeuAc
2
3)Gal
1
3[Fuc
1
4]GlcNAc
1
3Gal but not
GlcNAc
1
3Gal or type 2 chain oligosaccharides, which are found in
CHO-FT-T1 cells. This result indicates that
3Gal-T5 expression
prevents poly-N-acetyllactosamine and sialyl-Lewis x
synthesis on N-glycans. Kinetic studies confirm that
3Gal-T5 prefers acceptors having the GlcNAc
1
3Gal end,
including lactotriosylceramide. Competitive reverse transcriptase
mediated-polymerase chain reaction shows that the
3Gal-T5 transcript
is expressed in normal colon mucosa but not or poorly in
adenocarcinomas. Moreover, recombinant carcinoembryonic antigen
purified from a CHO clone expressing Fuc-TIII and
3Gal-T5 reacts
with anti-sialyl-Lewis a and carries type 1 chains on oligosaccharides
released by endo-
-galactosidase. We conclude that
3Gal-T5
down-regulation plays a relevant role in determining the
cancer-associated glycosylation pattern of N-glycans.
 |
INTRODUCTION |
Type 1 chain oligosaccharides found in N- and
O-glycans, as well as in glycolipids, contain the
distinctive Gal
1
3GlcNAc disaccharide as their core structure. It
is synthesized by
1,3-galactosyltransferases (
1,3Gal-Ts),1 a family of
enzymes whose genes have been cloned very recently (1). The functional
role of type 1 chains is not known, but several studies have indicated
that some of them are differentially expressed in cancer. In
particular, CEA expressed in colon cancer is characterized by the
absence of such chains (2), which are abundantly present on the
N-glycans of its normal counterparts synthesized by healthy
colon mucosa, also referred to as the nonspecific cross-reacting
antigen-2 (3) and the normal fecal antigen-2 (4). At this regard,
a
1,3Gal-T activity measured using GlcNAc
1
3Gal
1
4Glc as
acceptor was found lower in adenocarcinomas than in normal mucosa (5).
However, type 1 chain Lewis antigens sialyl-Lewis a (sLea)
and Leb are considered tumor markers (6), and serum levels
of sLea (CA19-9 antigen) are used for clinical diagnosis
and follow-up of epithelial cancers of the gastrointestinal tract (7).
sLea from cancer patient serum was found on mucins (8), and
it is reported on both mucins (9) and glycolipids (10) in
adenocarcinoma cell lines. Five
3Gal-T cDNAs are presently
available.
3Gal-T1, cloned first from melanoma cells (11) and then
from colon carcinoma cells (12), as well as
3Gal-T2 (13), were found
to synthesize sLea in CHO cells but to be very poorly
expressed in cancer cell lines expressing sLea.
3Gal-T3
and -T4 (14) were found expressed in various tissues, including colon
and pancreas and in some cancer cell lines, but the expression levels
do not correlate with those of type 1 chain Lewis antigens. Moreover,
there is no evidence yet for their ability to synthesize the type 1 chain. In particular,
3Gal-T3 was very recently reported as a GalNAc
transferase involved in globoside biosynthesis and renamed
3GalNAc-T1 (15).
3Gal-T5, cloned from colon carcinoma cells, was
found to direct synthesis of sLea in different cell lines,
and its expression levels correlate with those of Lewis type 1 antigens
in cancer cell lines from colon and pancreas (16). This enzyme was
suggested to be the one previously characterized as the
1,3Gal-T
active on glycolipids (17) and GlcNAc (18, 19), and involved in the
synthesis of core 3 O-glycans (20). The above data led to
the working hypothesis that the biosynthesis of different type 1 chain
oligosaccharides may depend on the expression of different and
differentially regulated
3Gal-Ts. In particular, an enzyme able to
act on N-glycans would be expected to be less active in
cancer than in normal mucosa, whereas another active on
O-glycans should be more expressed in cancer.
To evaluate whether
3Gal-T5 is actually involved in the biosynthesis
of a specific subset of type 1 chain oligosaccharides and expressed in
a cancer-associated manner, we have transiently transfected CHO
cells expressing Fuc-TIII with
3Gal-T cDNAs and compared the
relative ability of enzymes to direct synthesis of Lewis type 1 antigens. We then constructed CHO clones permanently expressing
Fuc-TIII and
3Gal-T5, Fuc-TIII and
3Gal-T2, or Fuc-TIII and
3Gal-T1, determined the effect of drugs affecting glycosylation on
antigen expression, and characterized the oligosaccharides that became
radioactive in some clones upon metabolic labeling with tritiated Gal.
Moreover, we studied the substrate specificity of
3Gal-T5 by
calculating the kinetic constants toward different acceptors, and
measured the expression levels of the transcript in colon
adenocarcinomas and surrounding normal mucosa by competitive RT-PCR. We
further modified the CHO clone expressing
3Gal-T5 to make it able to
stably express human CEA and investigated the presence of type 1 chains
in CEA purified from such clone.
 |
EXPERIMENTAL PROCEDURES |
Materials--
GlcNAc
1
3Gal
1
4Glc was prepared by
digesting lacto-N-tetraose (IsoSep, Lund, Sweden), 5 mg/ml,
in 0.1 M citrate phosphate buffer, pH 4.5, with 40 milliunits/ml bovine testis
-galactosidase (Sigma), for 20 h at
37 °C. The obtained trisaccharide was purified from Gal and
unreacted lacto-N-tetraose by repeated Bio-Gel P-2 columns
monitored by HPTLC, as reported (21). Lactotriosylceramide was
prepared by digesting lacto-N-neotetraosylceramide, 4 mg/ml, in 50 mM cacodylate/HCl buffer, pH 6.5, with
Diplococcus pneumoniae
-galactosidase (Sigma), 0.2 units/ml for 20 h at 37 °C. The obtained compound was purified
by a Silica-Gel column (0.7 × 50 cm) using chloroform/methanol/water, 55:20:3 (v/v), as the eluting solvent system. Lacto-N-neotetraosylceramide was prepared by
digesting sialyl-lacto-N-neotetraosylceramide, 5 mg/ml, in
0.1 M sodium cacodylate buffer, pH 6.0, with
Clostridium perfringens sialidase, 1 unit/ml, for 20 h
at 37 °C. Ganglioside sialyl-lacto-N-neotetraosylceramide was purified from bovine erythrocytes using the procedure reported by
Chien et al. (22). GlcNAc
1
2Man was from Dextra
Laboratories (Reading, UK),
GlcNAc
1
3Gal
1
O-methyl and
GlcNAc
1
6Man
O-methyl were from Sigma.
Anti-sLex monoclonal antibody was precipitated from the
culture media of hybridoma CSLEX1 (ATCC HB-8580) by ammonium sulfate,
resuspended, dialyzed, and kept at a concentration of 2 mg/ml.
Anti-Lea and anti-sLea monoclonal antibodies
were as reported (12, 19). Mouse monoclonal anti-CEA (clone COL-1) was
from NeoMarkers (Lab Vision, Italy).
Cell Cultures, Transfections, and Treatments--
CHO cells
expressing Polyoma virus T antigen and Fuc-TIII (CHO-T-FT), COLO-205,
and WM266-4 cells were cultured as described previously (12, 19). Human
gastric adenocarcinoma MKN-45 cells (a gift of C. Ponzetto, University
of Turin), were cultured in RPMI 1640 medium containing 10% fetal
bovine serum, 100 units/ml penicillin, 1.0 mg/ml streptomycin, and 2 mM L-Glu. For transient transfection, 2.0 × 105 CHO-T-FT cells were plated in 12-well plates 20 h before transfection, washed with serum free
-minimal essential
medium, and incubated with 0.5 ml of transfection solution for 3 h
under usual growing conditions. Transfection solutions, containing 1 µg/ml
3Gal-T cDNA in pcDNAI or pCDM8 vectors plus 0.065 µg/ml pcDNAI-Luc and 18 µl/ml DOTAP (Roche Molecular
Biochemicals) were prepared and used as reported previously (12).
Seventy-two hours after transfection, cells were harvested, washed, and
resuspended with PBS. One-tenth of resuspended cells was processed for
luciferase activity determination, using a commercial kit (Luciferase
assay system, Promega) according to the manufacturer's
recommendations. The remaining material was stained and analyzed by
flow cytometry as previously reported (12, 19). For treating cell lines
and clones with drugs affecting glycosylation, 1 × 105 cells were plated in 12-well plates and incubated
overnight, and the medium was replaced with medium containing 0.1 µg/ml swainsonine (Sigma), 2 mM benzyl-
-GalNAc
(Sigma), or 2 mM sodium butyrate. After 48 h, medium
was replaced with fresh medium containing drugs. After additional
48 h, cells were collected, stained, and analyzed by flow
cytometry (19).
Construction of CHO Clones--
CHO clones expressing either
Fuc-TIII and
3Gal-T5 (CHO-FT-T5), Fuc-TIII and
3Gal-T2
(CHO-FT-T2), or Fuc-TIII and
3Gal-T1 (CHO-FT-T1) were obtained by
the calcium phosphate transfection method (23), using a modification of
the procedure reported (24). Briefly, the DNA mixture (20 µg)
contained 1 µg of EcoRI-linearized pSV2Neo, 10 µg
of ScaI-linearized pcDNAI/ Fuc-TIII, and 10 µg of
ScaI-linearized pCDM8/
3Gal-T5, or 10 µg of
KpnI-linearized pCDM8/
3Gal-T2, or 10 µg of
ScaI-linearized pcDNAI/
3Gal-T1, respectively. Upon
selection with 0.4 mg/ml active G418, 30 (CHO-FT-T5 and CHO-FT-T1) or
60 colonies (CHO-FT-T2) were collected using cloning cylinders, grown
in tissue culture slides, stained with anti-sLea or
anti-Lea antibody followed by secondary FITC-conjugated
anti-mouse IgG, and analyzed by fluorescence microscopy. Two or three
positive colonies were subcloned by limiting dilution in 96-well
plates, and several subclones were analyzed as above. sLea
and Lea expression on positive colonies was quantitated by
flow cytometry. Single colonies expressing a constant level of
sLea and Lea, named CHO-FT-T5, CHO-FT-T2, and
CHO-FT-T1, respectively, were selected and used for further
characterization and experiments. CHO cells expressing CEA
(CHO-FT-T5-CEA) were obtained from clone CHO-FT-T5 by the transfection
procedure described above but using 20 µg of
KpnI-linearized pCDM8-CEA and 1 µg of pHA58 plasmid in the
DNA mixture, and 0.5 mg/ml hygromycin B (Roche Molecular Biochemicals) for selection. pHA58 plasmid, a generous gift of P. Morandini (University of Milan), was derived from pSV72 vector and had the hygromycin B gene under the control of mouse phosphoglycerate kinase-1
promoter. Monoclonal anti-CEA antibody was used, at 1:100 dilution, for
cell staining.
DNA Preparation--
3Gal-T1 cloning in pcDNAI vector has
been previously reported (12). For
3Gal-T2, -T3, -T4, -T5, and CEA
cloning, the corresponding coding sequences were amplified using
single-stranded cDNA as template, obtained by reverse transcription
of total RNA extracted (12) from WM266-4 (
3Gal-T2), MKN-45
(
3Gal-T3 and CEA), or COLO-205 cells (
3Gal-T4 and -T5), in a
reaction mixture containing, in 40-µl volume, 50 mM
Tris-HCl, pH 8.3, 50 mM KCl, 5 mM
MgCl2, 1000 units/ml human placental ribonuclease
inhibitor, 1 mM of each deoxynucleotide triphosphate, 0.4 µM oligo(dT)16 primer, 1000 units/ml avian
myeloblastosis virus reverse transcriptase (Amersham Pharmacia
Biotech), and freshly denatured total RNA (250 µg/ml) as template.
Reactions were kept for 90 min at 42 °C. Amplifications were
performed in the presence of a commercially available "high
fidelity" Taq polymerase (LA Taq, Takara),
according to the manufacturer's protocol, using 2.5 µg/ml of each
oligonucleotide primer, deduced from published sequences, and 4 µl of
reverse transcription reaction for each 100 µl of amplification
mixture. PCR reactions were incubated as follows: a single treatment at 94 °C for 3.5 min followed by a cycle consisting of 1.5 min at 94 °C (melting), 2.0 min at 62 °C (annealing), and 2.5 min at 72 °C (extension), repeated 30 (
3Gal-Ts) or 15 times (CEA); a final extension step was performed at 72 °C for 8 min. The amplified DNA was digested with HindIII and XbaI
(
3Gal-T3) or ligated to BstXI adaptors (all others), and
cloned in pcDNAI (
3Gal-T3) or pCDM8 vectors (all others) using a
procedure reported previously (12). Direct DNA sequencing of the
obtained constructs, performed by the dideoxynucleotide
chain-termination method using an automated procedure, indicated that
the coding sequences are identical to those published. pcDNAI-Luc,
expressing the luciferase gene, was constructed by removing the
luciferase coding sequence from plasmid pGL3 (Promega) using
HindIII and XbaI, and subcloning into the corresponding sites of pcDNAI vector.
Enzyme Assays and Reaction Product
Characterization--
3Gal-T5 and Fuc-TIII were assayed in cell
clones as previously reported (19). For kinetic analysis,
3Gal-T5
was assayed upon transfection of pCDM8-
3Gal-T5 in COS-7 cells. Cells
were transfected, harvested, washed, resuspended, and used as the
enzyme source, as described previously (21). Enzyme activity was
determined in a reaction mixture containing, in a final volume of 10 µl, 0.1 M Tris/HCl buffer, pH 7.0, 10 mM
MnCl2, 0.5 mg/ml Triton X-100, 1 mg/ml
-lactalbumin, 5 mM CDP-choline, 0.8 mM donor
UDP-[3H]Gal, specific radioactivity (10 mCi/mmol), and
0.2-1.0 mg/ml cell protein, in the presence of different acceptors at
various concentrations. In the case of glycolipid acceptor, it was
dissolved in chloroform/methanol, 4:1 (v/v), mixed in the reaction tube with 15 µg of Triton CF-54 dissolved in the same solvent, and dried
before adding the reaction mixture. Incubations were done at 37 °C
for 60 min. At the end of incubation, reaction products were assayed by
Dowex chromatography (oligosaccharide acceptors) or descending paper
chromatography (glycolipid acceptor) according to previously reported
protocols (19, 25). The oligosaccharide reaction products were
identified by pooling several Dowex eluates, which were lyophilized and
purified by Bio-Gel P-2 chromatography (21). The obtained saccharides
were treated with D. pneumoniae or Xanthomonas
manihotis
-galactosidases, specific for
1,4- and
1,3-linkages, respectively, and analyzed using a Bio-Gel P-2 column
(19). Reaction product with glycolipid acceptor, pooled from several
reactions, was purified by Sep-Pack C-18 cartridge, desalted by
partitioning in chloroform/methanol/water, 2:1:1 (v/v), and analyzed by
HPTLC, using chloroform/methanol/water, 60:35:8 (v/v), as eluting
solvent system, and visualized by fluorography (25).
Metabolic Labeling and Carbohydrate Analysis--
CHO clones
(2.0 × 106 cells) were plated in 60-mm dishes
containing 0.1 mCi of [3H]Gal (Amersham Pharmacia
Biotech) in 2.5 ml of culture medium and incubated 40 h under
regular conditions. Labeled cells were harvested as for immunostaining,
resuspended in PBS at a density of 4 × 106 cells/0.1
ml, and kept frozen until used. N-Glycans were released from
cell suspension by N-glycanase (Glyko) digestion of the
material denatured by heating in the presence of SDS, under the
conditions recommended by the manufacturer. Released
N-glycans were isolated by a Sep-Pak C-18 cartridge and
further purified by Sephadex G-50 chromatography, as reported (24).
Oligosaccharides released by endo-
-galactosidase were characterized
following the procedure reported by Seuyoshi et al. (26)
with some modifications. Purified N-glycans were passed
through a Bio-Gel P-4 column (0.7 × 50 cm), and only the
radioactivity eluted with water as a peak close to the exclusion volume
was collected, lyophilized, and used for characterization.
Endo-
-galactosidase digestion was performed on radioactive
N-glycans, 10,000-20,000 cpm/µl, using the enzyme from
Bacteriodes fragilis (Glyko), 0.5 milliunit/µl, in the
buffer supplied by the manufacturer, for 20 h at 37 °C. The
reaction mixture was then applied to the same Bio-Gel P-4 column as
above, and the radioactivity was eluted with the exclusion volume
collected and referred to as the endo-
-galactosidase resistant
N-glycans, whereas the radioactivity eluted as
oligosaccharides was applied to a QAE-Sephadex column to separate
neutral and charged sugars, according to a previous procedure (26).
Material collected in the flow-through was referred to as the neutral
oligosaccharides, whereas that eluted with NaCl was collected, desalted
on a Bio-Gel P-2 column, and treated with Newcastle disease virus
sialidase. Neutral and de-sialylated oligosaccharides were analyzed by
a Bio-Gel P-4 column (0.7 × 100 cm), eluted with water at a flow rate of 0.1 ml/min, 4.5 min/fraction. The obtained peaks were collected, lyophilized, and treated with glycohydrolases for
characterization. X. manihotis
-galactosidase (Glyko),
almond meal
-fucosidase (Oxford Glycosystem), D. pneumoniae
-N-acetylglucosaminidase (Glyko),
D. pneumoniae
-galactosidase (Sigma), and Newcastle disease virus sialidase (Glyko) digestions were performed on
radioactive oligosaccharides, 1000-2000 cpm/µl, according to the
procedures reported (19, 26). The amount of Gal released by
glycohydrolase treatment of endo-
-galactosidase-resistant
N-glycans was determined as reported (19). Glycohydrolase
digestions of the tetrasaccharide obtained from clone CHO-FT-T5 were
analyzed using a Bio-Gel P-2 column (0.7 × 100 cm) eluted with
water at a flow rate of 0.1 ml/min, 4.5 min/fraction, whereas those of
the peaks were collected from CHO-FT-T1 using a Bio-Gel P-4 column
(0.7 × 100 cm) under the same conditions.
CEA Purification and Characterization--
CEA was extracted and
purified following a published procedure (27). Briefly, about 2.5 × 107 metabolically labeled cells, prepared as above
described, were diluted to 1.5 × 107 cells/ml in PBS,
homogenized by vortexing 1 min, and treated with 0.1 unit/ml
phosphatidylinositol phospholipase C (Glyko) at 37 °C for 2 h.
Total reaction was spun at 14,000 rpm for 10 min, and the supernatant
was collected, made 0.6 M with perchloric acid, and
centrifuged as above. The clear supernatant was neutralized with
NaOH, freeze-dried, resuspended, and loaded onto a Sepharose CL6B
column equilibrated and eluted with 50 mM PBS, pH 5.2. Elution was monitored by liquid scintillation counting, and relevant
fractions were analyzed by SDS-PAGE and visualized by fluorography.
Western blotting was performed as previously reported (24) using
monoclonal anti-CEA (1:200), anti-sLea (1:500), and
anti-sLex (1:100) antibodies, followed by
peroxidase-conjugated goat anti-mouse immunoglobulin (Amersham
Pharmacia Biotech) (1:2000). ECL was developed with Renaissance
chemiluminescence reagent (PerkinElmer Life Sciences).
Homogeneous fractions were collected, lyophilized, and desalted on a
Bio-Gel P-2 column. Purified [3H]CEA was treated with
C. perfringens sialidase and almond meal
-fucosidase, and
then with endo-
-galactosidase, under the conditions reported for
N-glycan digestion. The reaction mixture was then applied to
a Bio-Gel P-2 column (0.7 × 50 cm), eluted with water at a flow
rate of 0.13 ml/min, 2.5 min/fraction. The obtained peaks, as well as
the total purified CEA, were treated with
-galactosidases and
analyzed by Bio-Gel P-2 chromatography as above described.
Competitive RT-PCR Analysis--
For the analysis of human
samples, bioptic specimens were collected at surgery, immediately
frozen in dry ice, and placed in liquid nitrogen until used. For RNA
extraction, 1-2 mm3 of material was homogenized with a
rotary homogenizer in 0.5 ml of the lysis buffer from a Qiagen RNeasy
minikit and processed in the presence of DNase according to the
manufacturer's recommendations. First strand cDNA was synthesized
by Moloney murine leukemia virus reverse transcriptase (Amersham
Pharmacia Biotech) as previously reported (12). Control reactions were
prepared by omitting the reverse transcriptase in the mixture. First
strand cDNA was amplified in a 50-µl reaction and in the presence
of 20 fg of competitor for 35 cycles (glycosyltransferases) or 5 pg of
competitor for 25 cycles (
-actin), under the conditions reported
(12). No amplification was detected when the control reactions were
used as template. Human
-actin, Fuc-TIII, FUT2, and
3Gal-T1
competitors and oligonucleotide primers used were the same as those
described in a previous study (12).
3Gal-T2, -T3, -T4, -T5, and
mouse
-actin oligonucleotide primers, as well as the restriction
enzymes used for competitor preparation, are listed in Table
I. Quantification of human
3Gal-T5,
3Gal-T2,
3Gal-T1, and Fuc-TIII transcripts in CHO clones was
performed by the same procedure but using 60 fg of each
glycosyltransferase competitor, or 20 pg of mouse
-actin competitor,
in 50-µl reactions that were amplified for 30 (glycosyltransferases) or 25 cycles (
-actin).
 |
RESULTS |
Biosynthesis of sLea and Lea in CHO Cells
Transiently Transfected with
3Gal-T cDNAs--
The ability of
different cloned
3Gal-Ts to synthesize the type 1 chain was assessed
to determine the amount of sLea and Lea
antigens expressed upon cDNA transfection on the surface of
CHO-T-FT cells, a clone permanently expressing Fuc-TIII and Polyoma
virus T antigen. For this purpose, cells were transiently cotransfected with
3Gal-T and reporter luciferase cDNAs both placed in
expression vectors having the Polyoma virus origin of replication.
Luciferase activity from different transfections ranged between 0.6 and
1.4 units. Despite such differences in transfection efficiency, both sLea and Lea antigens were easily detected in
cells transfected with
3Gal-T5 (very bright) and
3Gal-T1, poorly
detected with
3Gal-T2, and not at all detected with
3Gal-T3 and
3Gal-T4. In a typical experiment (Fig.
1), a peak representing up to 35% of
cells transfected with
3Gal-T5 appears intensely bright mostly after
anti-sLea antibody staining. Fluorescence intensity on
positive cells was much lower upon
3Gal-T1 transfection, and faintly
or not detectable in the other cases, although transfection efficiency
was similar or sometimes higher, as assessed by luciferase activity
assay. These results suggest that different substrate specificity
exists among the different
3Gal-Ts, and only
3Gal-T5 finds large
amount of suitable acceptors in CHO cells.

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Fig. 1.
Flow cytometry analysis of CHO-T-FT cells
transiently cotransfected with different
3Gal-T and luciferase cDNAs. CHO-T-FT
cells, expressing Polyoma virus T antigen and human Fuc-TIII, were
transfected using liposomes containing a 15:1 mixture of individual
3Gal-T and luciferase cDNAs, each cloned in pCDM8 or pcDNAI
expression vectors. Seventy-two hours later, cells were harvested and
stained with anti-sLea (solid line) or
anti-Lea (stippled line) monoclonal antibodies
followed by FITC-conjugate anti-mouse IgG secondary antibody, or with
secondary antibody alone (dotted line), and analyzed by flow
cytometry. An aliquot of the same cells was homogenized and used for
luciferase activity assay.
|
|
Construction and Characterization of CHO Clones Expressing
3Gal-T5 and Fuc-TIII,
3Gal-T2 and Fuc-TIII, or
3Gal-T1 and
Fuc-TIII--
To evaluate the hypothesis that the amount of
sLea and Lea antigens expressed in CHO cells
transfected with
3Gal-Ts reflects the availability of the proper
precursors, we constructed three CHO clones permanently expressing the
antigens upon stable transfection with either
3Gal-T5 and Fuc-TIII,
3Gal-T2 and Fuc-TIII, or
3Gal-T1 and Fuc-TIII. All clones, named
CHO-FT-T5, CHO-FT-T2, and CHO-FT-T1, respectively, express human
Fuc-TIII transcript in a similar amount, as determined by competitive
RT-PCR, whereas each clone expresses only its corresponding human
3Gal-T transcript (Fig.
2A). Flow cytometry analysis
(Fig. 2B) of the obtained clones shows intense and
homogeneous staining with anti-sLea and
anti-Lea antibodies in CHO-FT-T5 cells, weak and
heterogeneous staining in CHO-FT-T1, and very faint staining in
CHO-FT-T2 despite the high expression level of the transcript, thus
providing fluorescence patterns very similar to those obtained by the
transient transfection experiments (Fig. 1). Moreover, CHO-FT-T2 and
CHO-FT-T1 cells are homogeneously bright with anti-sLex
staining, as expected in CHO cells expressing Fuc-TIII, whereas CHO-FT-T5 cells are totally negative (Fig. 2B). This last
result is surprising and suggests a competition between Gal-Ts in the synthesis of type 1 versus type 2 chain Lewis antigens.

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Fig. 2.
Characterization of CHO-FT-T5, CHO-FT-T2, and
CHO-FT-T1 clones. CHO clones permanently expressing human Fuc-TIII
and 3Gal-T5 (CHO-FT-T5), Fuc-TIII and 3Gal-T2
(CHO-FT-T2), or Fuc-TIII and 3Gal-T1
(CHO-FT-T1) were constructed as described under
"Experimental Procedures." A, total RNA was extracted
from the clones and reverse-transcribed, and the obtained first strand
cDNA was amplified by PCR using primers specific for human
glycosyltransferases or mouse -actin, in the presence of the proper
competitor DNA. One-fifth aliquot of each PCR reaction was analyzed by
1% agarose gel electrophoresis, visualized by staining with ethidium
bromide, and photographed, and the spots were quantitated by
densitometric scanning of the negative film. The amounts of amplified
target cDNAs were calculated from their respective standard curves
and normalized by those for -actin. B, cell clones were
stained with monoclonal anti-sLea (solid line),
anti-Lea (dotted line) (both IgG), or
anti-sLex (stippled line) (IgM) monoclonal
antibody, followed by FITC-conjugate anti-mouse IgG or IgM,
respectively, and analyzed by flow cytometry. Negative controls
(shaded areas) represent the cell clones stained with
FITC-conjugate anti-mouse IgG alone.
|
|
Effect of Drugs Affecting Glycosylation on the Expression of
sLea and sLea in CHO Clones--
CHO cells are
known to express mostly complex type N-glycans and simple
core 1 type O-glycans (28). Because the latter are not
precursors of type 1 chains, our findings suggest that
3Gal-T5 has a
distinctive ability to act on complex type N-glycans. To address this issue and to study the contribute of N- and
O-linked glycosylation on the expression of sLea
and sLex, cells were treated with swainsonine and
benzyl-
-GalNAc, selectively affecting complex type
N-glycan and O-glycan biosynthesis, respectively, before staining with anti-sLea or anti-sLex
antibodies. sLea staining is 80% inhibited by swainsonine
treatment in CHO-FT-T5, whereas benzyl-
-GalNAc treatment is
ineffective and sLex staining is absent (Fig.
3). Conversely, in clone CHO-FT-T2
sLea staining is almost absent, whereas sLex
staining is reduced by swainsonine treatment (about 65%) and slightly
stimulated by sodium butyrate. In clone CHO-FT-T1 sLea
staining is almost 70% inhibited by benzyl-
-GalNAc, whereas swainsonine treatment is ineffective; moreover, sodium butyrate treatment stimulates sLea synthesis 5-fold.
sLex staining in this clone is reduced by swainsonine
treatment and slightly stimulated by sodium butyrate, as in CHO-FT-T2.
In control COLO-205 cells, sLea and sLex
reactivity is totally abolished by benzyl-
-GalNAc, and only about
30% inhibited by swainsonine treatment (Fig. 3). These results indicate that sLea expressed in CHO-FT-T5 cells is carried
by complex type N-glycans, presumably the same carrying the
sLex epitope in CHO-FT-T1 and CHO-FT-T2 cells.

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Fig. 3.
Effect of drugs affecting glycosylation on
the expression of sLea and sLex antigens in
different cells. Cells were grown under regular conditions
(controls), in the presence of 0.1 µg/ml swainsonine, 2 mM benzyl- -GalNAc, or 2 mM sodium butyrate.
At the end of incubation, cells were harvested, stained, and analyzed
by flow cytometry as in Fig. 2.
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Characterization of Complex N-Glycans Formed in Clone
CHO-FT-T5--
To investigate the actual nature of the saccharides
used as substrates by
3Gal-T5 in CHO cells, we studied the
radioactive structures formed upon metabolic labeling with
[3H]Gal in CHO-FT-T5 as well as in CHO-FT-T1 and CHO-T-FT
cells, a clone not expressing
1,3Gal-T but Fuc-TIII only. The
N-glycans were prepared from the total incorporated
radioactivity and treated with endo-
-galactosidase, and the released
oligosaccharides were separated in the neutral and charged fractions.
As shown in Table II, the radioactivity
recovered after each step is rather similar in the three clones. On the
other hand, the composition of the neutral saccharides released by
endo-
-galactosidase treatment of CHO-FT-T5 N-glycans
is dramatically different from that of CHO-FT-T1 N-glycans.
In fact, almost all radioactivity derived from clone CHO-FT-T5 is
eluted as a single peak in the area of tetrasaccharides by Bio-Gel P-4
fractionation, whereas that from clone CHO-FT-T1 is distributed in
several peaks, including some of higher molecular weight as well as one
in the disaccharide area (Fig. 4,
upper panel). The radioactive peak obtained from CHO-FT-T5
N-glycans is converted to a trisaccharide by almond meal
-fucosidase, to an equal mixture of disaccharide and monosaccharide by the action of both almond meal
-fucosidase and X. manihotis
-galactosidase, and to a monosaccharide by the
combination of the above enzymes and D. pneumoniae
-N-acetylglucosaminidase (Fig. 4, lower
panel). Digestion of the tetrasaccharide peak with almond meal
-fucosidase and D. pneumoniae
-galactosidase
determines the formation of a single peak in the area of trisaccharides
but not detectable monosaccharides (not shown). The elution profile obtained after de-sialylation of the charged oligosaccharides is
identical, indicating that Gal
1
3[Fuc
1
4]GlcNAc
1
3Gal
and NeuAc
2
3Gal
1
3[Fuc
1
4]GlcNAc
1
3Gal account
for the vast majority of oligosaccharides released by
endo-
-galactosidase digestion of the N-glycans formed in
clone CHO-FT-T5. However, the elution profile of the oligosaccharides
released by endo-
-galactosidase treatment of CHO-FT-T1 and CHO-T-FT
cells are very similar. Among such peaks, the one eluted as a
disaccharide was identified as GlcNAc
1
3Gal, because it is
converted to a monosaccharide by D. pneumoniae
-N-acetylglucosaminidase. Such a disaccharide is formed
by the action of endo-
-galactosidase on multiple lactosamine repeats
(29). The peak eluted at the size of tetrasaccharides was identified as
Gal
1
4[Fuc
1
3]GlcNAc
1
3Gal, because it is converted to
a monosaccharide only by the sequential action of almond meal
-fucosidase, D. pneumoniae
-galactosidase, and
D. pneumoniae
-N-acetylglucosaminidase. The
larger peaks (fractions 51-55 and 46-50 in Fig. 4, upper
panel) contain one and two other major oligosaccharides,
respectively. They were identified by sequential exoglycosidase
digestions and Bio-Gel P-4 filtration as the pentasaccharide,
hexasaccharide, and heptasaccharide already reported (26). Such
oligosaccharides contain double lactosamine repeats fucosylated on the
inner GlcNAc, a substitution that makes the Gal
1
4GlcNAc linkage
resistant to endo-
-galactosidase under usual reaction conditions
(30). These results strongly suggest that the
(±NeuAc
2
3)Gal
1
3[Fuc
1
4]GlcNAc
1
3Gal
oligosaccharide synthesized in CHO-FT-T5 replaces the
poly-N-acetyllactosamine chains and sLex
structure present in CHO-FT-T1 as well as in CHO cells expressing Fuc-TIII only. To assess the presence of type 1 chain carbohydrates in
endo-
-galactosidase-resistant N-glycans, we have
determined the amount of radioactive Gal released by X. manihotis
-galactosidase. As shown in Table
III, X. manihotis
-galactosidase is active only on the glycans derived from CHO-FT-T5,
but not on those from CHO-FT-T1. The amount of Gal released by the
enzyme is strongly dependent on the concurrent action of sialidase, but
not on that of
-fucosidase, and is much lower than that released by
D. pneumoniae
-galactosidase under the same reaction
conditions. These results indicate that type 1 chains synthesized on
endo-
-galactosidase-resistant N-glycans by
3Gal-T5 are
present in limited amount and are not significantly substituted by
fucose residues.
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Table II
Radioactivity distribution in CHO clones metabolically radiolabeled
with [3H]Gal
Values are expressed as cpm × 106/mg cell protein.
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Fig. 4.
Characterization of radioactive
oligosaccharides released by
endo- -galactosidase. Upper
panel, the neutral radioactive oligosaccharides released by
endo- -galactosidase from the N-glycans isolated from
CHO-FT-T5 (empty circles) or CHO-FT-T1 (solid
circles), were submitted to Bio-Gel P-4 filtration. Lower
panel, the peak from CHO-FT-T5 cells eluted at the tetrasaccharide
size (fractions 55-59 in upper panel) was
submitted to Bio-Gel P-2 chromatography upon no treatment (empty
circles), almond meal -fucosidase digestion (full
circles), almond meal -fucosidase plus X. manihotis
-galactosidase digestion (empty squares), or to almond
meal -fucosidase plus X. manihotis -galactosidase and
D. pneumoniae -N-acetylglucosaminidase
digestion (full squares). Calibration of each column is
indicated at the top of each panel.
Tetrasaccharide, Gal 1 4GlcNAc 1 3
Gal 1 4Glc; trisaccharide, Fuc 1 2Gal 1 3GlcNAc;
disaccharide, Gal 1 3GlcNAc; monosaccharide,
Gal.
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Table III
[3H]Gal released by D. pneumoniae or X. manihotis
-galactosidase treatment of endo- -galactosidase resistant
N-glycans prepared from CHO clones
The whole reaction mixtures (about 5000 cpm) were applied to Bio-Gel
P-2 column, and radioactivity in the eluted fractions was determined by
liquid scintillation counting. Results are the means for two
independent experiments and are presented as percentage of recovered
radioactivity in the galactose peak.
|
|
In Vitro Properties of
3Gal-T5--
Using a cell homogenate
prepared from COS cells transiently transfected with
3Gal-T5
cDNA as the enzyme source, we determined the optimal reaction
conditions using GlcNAc as acceptor and found that the activity
requires Mn2+, is maximal at neutral pH, is saturated by
donor UDP-Gal concentrations above 0.5 mM, and is not
affected by
-lactalbumin. Under the same reaction conditions,
various oligosaccharides are also used. The obtained reaction products
are over 95% affected by X. manihotis
-galactosidase and
almost unaffected by D. pneumoniae
-galactosidase. In the
presence of the proper detergent concentration, the same homogenate
very efficiently transfers Gal to the glycolipid
lactotriosylceramide, and the reaction product was identified
as lactotetraosylceramide (Gal
1
3GlcNAc
1
3Gal
1
4
Glc
1
1'Cer) by HPTLC mobility and differential sensitivity to
-galactosidases. The calculated Km and Vmax values (Table
IV) indicate that the enzyme prefers the
GlcNAc
1
3Gal end but also distinguish among the different carrier
molecules, with a preference for the glycolipid acceptor. GlcNAc linked
to
-Man through
1,2- or
1,6-linkages is also a suitable
acceptor, but with much lower affinity. These in vitro
results are in good agreement with those obtained by the structural
analysis of CHO-FT-T5 clone, where we found that
endo-
-galactosidase-sensitive saccharides, having the
GlcNAc
1
3Gal acceptor sequence, are preferentially operated upon
by
3Gal-T5, whereas endo-
-galactosidase-resistant N-glycans, mostly having GlcNAc
1
2/6Man as acceptor
sequences, are poorly utilized.
Expression of
3Gal-T5 Transcript in Normal Colon Mucosa
and in Adenocarcinomas--
To evaluate whether the
3Gal-T5
transcript is differentially expressed during carcinogenesis, we
analyzed its amount by competitive RT-PCR performed on total RNA
extracted from human colon specimens collected at surgery, representing
both adenocarcinomas and surrounding normal mucosa. Clinical features
and tumor staging are outlined in Table
V. The
3Gal-T5 transcript is detected
in all normal mucosa samples, although it is faintly detectable or
undetectable in adenocarcinomas whose cDNA is well amplified using
control primers such as those for
-actin or other glycosyltransferases (Fig.
5). In quantitative terms (Fig.
6), the
3Gal-T5 transcript is on the
average 30-fold less expressed in adenocarcinomas than in normal
mucosa. In individual cases, the levels in adenocarcinomas range from
4-fold to over 100-fold less than in normal mucosa. For comparison we
looked at the expression levels of the other
3Gal-T transcripts, as
well as of Fuc-TIII and FUT2, two fucosyltransferases involved in type
1 chain fucosylation.
3Gal-T4 transcript is expressed at high and
heterogeneous levels in normal mucosa and remains detectable in all
adenocarcinomas.
3Gal-T1 transcript is detectable in eight normal
mucosa and four adenocarcinoma cases, whereas
3Gal-T2 transcript is
detectable in four normal mucosa and seven adenocarcinoma cases.
3Gal-T3, Fuc-TIII, and FUT2 transcripts are heterogeneously
expressed in both normal mucosa and adenocarcinomas. Altogether, these
data suggest that the
3Gal-T5 transcript, almost undetectable in
colon adenocarcinomas, is strongly down-regulated during
carcinogenesis.
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Table V
Major clinical features of the patients with colon adenocarcinoma whose
biopsy were analyzed by competitive RT-PCR
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Fig. 5.
Competitive RT-PCR analysis of
3Gal-T5 transcript in normal mucosa and in
adenocarcinomas. RNA extracted from adenocarcinomas and
surrounding normal mucosa, collected at surgery, was
reverse-transcribed, and the obtained first strand cDNA was mixed
with competitor (truncated) cDNAs (5 pg and 20 fg for -actin and
glycosyltransferases, respectively), and subjected to PCR (25 and 35 cycles for -actin and glycosyltransferases, respectively). Primers
and PCR product length are indicated in Table I. One-fifth aliquot of
each amplification reaction was electrophoresed in 1% agarose gel and
visualized by staining with ethidium bromide. N, normal
mucosa; A, adenocarcinoma. Numbers denote
patients according to Table V. Samples 1-6 and
7-10 were run on different gels.
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Fig. 6.
Competitive RT-PCR quantification of
glycosyltransferase transcripts expressed in normal colon mucosa and in
adenocarcinomas. Quantification was performed by densitometric
scanning of the negative films of gels in Fig. 5. The amounts of
amplified target cDNAs were calculated from their respective
standard curves and normalized by those for -actin.
Numbers denote patients according to Table V. Note the
different scales used.
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Characterization of CEA Purified from a CHO Clone Expressing
3Gal-T5--
To study the ability of
3Gal-T5 to act on CEA, a
CHO clone permanently expressing this antigen, as well as
3Gal-T5
and Fuc-TIII, was obtained by transfecting CHO-FT-T5 cells with CEA
(CHO-FT-T5-CEA). As assessed by flow cytometry, the expression
levels of sLea and Lea antigens in this clone
are identical to those of the starting one, whereas those of CEA were
comparable to that of human cancer cell lines. In particular, we
measured a mean fluorescence intensity lower than in MKN-45 cells, but
higher than in COLO-205 cells (not shown). The tritiated material
purified from clone CHO-FT-T5-CEA metabolically radiolabeled with
[3H]Gal runs on SDS-PAGE as a single radioactive
band of an apparent molecular mass of 200 kDa (Fig.
7). Immunoblot analysis shows that this
band strongly reacts with anti-CEA antibody. Sequential stripping and
reprobing of the filter with anti-Lewis antigen antibodies reveals that
the purified CEA reacts with anti-sLea but not with
anti-sLex antibodies, indicating that type 1 chains are
specifically present on CEA expressed in the clone. To directly assess
the presence of type 1 chain carbohydrates on CEA and to determine
whether they are preferentially bound to
GlcNAc
1
3Gal
1
4GlcNAc
1
R outer sequences, the purified
[3H]CEA, once de-fucosylated and de-sialylated, was
treated with different
-galactosidases before and after
endo-
-galactosidase treatment. To this end, the
endo-
-galactosidase reaction mixture was submitted to Bio-Gel P-2
filtration and the endo-
-galactosidase-resistant and -sensitive
radioactivity was recovered with the exclusion volume and at the size
of trisaccharides, respectively (Fig. 8, upper panel). A small but detectable amount of Gal was
removed by X. manihotis
-galactosidase from total
de-sialylated and de-fucosylated CEA, and the amount released from the
endo-
-galactosidase-resistant material (fractions 18-22
in Fig. 8) was minimal, whereas that released by D. pneumoniae
-galactosidase was more consistent in both cases.
Conversely, almost 40% of the radioactivity from the
endo-
-galactosidase-sensitive trisaccharide (fractions
32-39 in Fig. 8) was released by X. manihotis
-galactosidase, but poorly affected by the diplococcal
enzyme (Fig. 8, lower panel). These data indicate
that
3Gal-T5 preferentially acts on the
GlcNAc
1
3Gal
1
4GlcNAc
1
R side chains present in
CEA.

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Fig. 7.
SDS-PAGE of CEA purified from clone
CHO-FT-T5-CEA. CHO-FT-T5 cells were transfected with human CEA
cDNA and a clone expressing CEA, named CHO-FT-T5-CEA, was isolated
and metabolically radiolabeled with [3H]Gal. Radioactive
material recovered upon phosphatidylinositol-specific phospholipase C
extraction, perchloric acid precipitation, and Sepharose CL6B
chromatography was desalted on a Bio-Gel P-2 column, and 10,000-cpm
aliquots were loaded on two lanes of a 6% SDS-PAGE. After the run, one
lane was processed for fluorography using liquid En3Hance
(PerkinElmer Life Sciences) (10-day exposure). The other one was
blotted onto a nitrocellulose filter that was probed with monoclonal
anti-CEA antibody using peroxidase-conjugated goat anti-mouse Ig as
secondary antibody, and visualized by ECL. The same filter was stripped
and reprobed sequentially with monoclonal anti-sLea and
anti-sLex antibodies.
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Fig. 8.
Sensitivity to
endo- -galactosidase and different
-galactosidases of tritiated CEA purified from clone
CHO-FT-T5-CEA labeled with [3H]Gal. Upper
panel, radioactive CEA prepared as described in Fig. 7 was
de-sialylated, de-fucosylated, and then subjected to Bio-Gel P-2
chromatography before (full circles) and after (empty
circles) endo- -galactosidase treatment. Column calibration is
indicated at the top of the panel, standards are
as in Fig. 4. Lower panel, the total de-fucosylated and
de-sialylated material, as well as endo- -galactosidase-resistant
(fractions 19-22 in upper panel) and -sensitive
(fractions 32-38 in upper panel) radioactivity
were treated with D. pneumoniae or X. manihotis
-galactosidases, and the reaction mixtures were analyzed by Bio-Gel
P-2 chromatography. Released Gal was quantitated by liquid
scintillation counting.
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 |
DISCUSSION |
This report shows that
3Gal-T5, in transfected CHO cells,
directs synthesis of type 1 chain oligosaccharides on CEA and
other N-glycans having the
GlcNAc
1
3Gal
1
4GlcNAc
1
R outer sequence, prevents
poly-N-acetyllactosamine and sLex synthesis on
N-glycans, and is down-regulated in colon adenocarcinomas.
We have found that
3Gal-T5 synthesizes sLea and
Lea antigens very efficiently in CHO cells, much more than
3Gal-T1, which is still more efficient than
3Gal-T2, whereas
3Gal-T3 and -T4 are unable to synthesize these antigens at all. In
the stable clone CHO-FT-T5, sLea expression is selectively
affected by swainsonine, an inhibitor of complex type
N-glycan processing (31), but not by benzyl-
-GalNAc, an
inhibitor of O-glycan biosynthesis (32), although the
opposite occurs in clone CHO-FT-T1. These data support the hypothesis
that
3Gal-T5 acts on complex type N-glycans in CHO cells
and suggest that
3Gal-T1 presumably affects unknown
O-glycans available in these cells in a low amount. In this
regard, it is interesting that sLea expression in CHO-FT-T1
is strongly stimulated by sodium butyrate, a commonly used activator
reported to enhance core 2 GnT activity in CHO cells (33). However,
results obtained with CHO-FT-T2 suggest that
3Gal-T2 requires
dedicated substrates not available in these cells. Moreover, CHO-FT-T1
cells, as well as CHO-FT-T2, were found to express a large amount of
sLex that is undetectable in CHO-FT-T5. We found that the
oligosaccharides released from N-glycans by
endo-
-galactosidase are completely different in the clones. In fact,
(±NeuAc
2
3)Gal
1
3[Fuc
1
4]GlcNac
1
3Gal is the
only oligosaccharide identified in CHO-FT-T5, whereas in clone
CHO-FT-T1 several oligosaccharides are released, which correspond to
those reported in CHO expressing Fuc-TIII only (26). They include the
sLex tetrasaccharide as well as
poly-N-acetyllactosamine side chains, but not type 1 chain
oligosaccharides. Among endo-
-galactosidase-resistant N-glycans, type 1 chain oligosaccharides are present in
small amounts and not fucosylated in clone CHO-FT-T5 but not detectable in CHO-FT-T1 cells. On this basis we conclude that the expression of
3Gal-T5 in CHO cells inhibits the synthesis of
poly-N-acetyllactosamines and sLex in
N-glycans, replacing them with a short type 1 chain, whereas that of
3Gal-T1 does not affect N-glycans. Expression of
N-linked poly-N-acetyllactosamines, which are
often modified to express functional oligosaccharides such as
sLex, is considered to be associated with tumor progression
and malignancy (34). Recent work on poly-N-acetyllactosamine
extension in N-glycans (35) demonstrated that it is achieved
mainly by
4Gal-T1 and
1,3-N-acetylglucosaminyltransferase iGnT (36) and is
favored by
1,6GlcNAc branching, because the branched
structure serves as a much better acceptor for such enzymes (37).
Expression of GlcNAc-TV, responsible for branching, also correlates
with metastatic potential of cell lines (38) as well as with metastasis and poor prognosis in colon cancer (39). Because
3Gal-T5 has a very
high affinity for acceptors having the GlcNAc
1
3Gal outer sequence, but much lower for those with the GlcNAc
1
2Man or
GlcNAc
1
6Man sequence, the following scenario can be envisaged in
CHO cells. In the presence of
3Gal-T5,
4Gal-T1 still acts on
GlcNAc linked to Man, and complex type N-glycans are
elongated by a single lactosamine unit. This is acted upon by iGnT,
presumably on a
1,6-branched chain, which forms the
GlcNAc
1
3Gal
1
4GlcNAc
1
R outer group. At this stage,
3Gal-T5 competes with
4Gal-T1 adding a
1,3-galactosyl residue
that prevents further chain elongation by iGnT. Consequently, poly-N-acetyllactosamine chains could not be efficiently
extended nor sLex synthesized.
It is worth noting that the Km values of
3Gal-T5
for GlcNAc
1
3Gal
1
4Glc and GlcNAc
1
2Man are virtually
identical to those reported for a
1,3Gal-T expressed in normal colon
mucosa whose activity was found low in adenocarcinomas (5). These data
prompted us to evaluate the expression level of
3Gal-T5 transcript
in normal mucosa versus colon adenocarcinomas. Competitive RT-PCR analysis performed on 10 colon adenocarcinoma cases indicate that
3Gal-T5 transcript is constantly down-regulated in cancer, confirming that it is the enzyme previously detected by Seko et al. (5) and corroborating the hypothesis that in vivo
it would prevent the expression of poly-N-acetyllactosamines
and sLex on N-glycans. This behavior is unique
among
3Gal-T and other glycosyltransferase transcripts studied to
date (40-43).
Because CEA synthesized in normal mucosa is reported to express
N-glycans having type 1 chains as
Gal
1
3GlcNAc
1
3Gal
1
4GlcNAc
1
R outer sequences
(3, 4), whereas CEA produced by colon adenocarcinomas lacks type 1 chains but have poly-N-acetyllactosamine and
1,6 branched
type 2 chains on N-glycans (2, 44), our results suggest that
3Gal-T5 may act on CEA in vivo. To assess this
hypothesis, we permanently expressed CEA in clone CHO-FT-T5
(CHO-FT-T5-CEA). We found that CEA purified from the clone reacts with
anti-sLea antibody by Western blot. Moreover, the type 1 chain carbohydrates present in the molecule are more abundant in
endo-
-galactosidase-sensitive oligosaccharides than in those
resistant. These results confirm that
3Gal-T5 acts on CEA and
suggest that it is responsible for the differential glycosylation
pattern of this protein in colon cancer.
The kinetic data calculated in this paper for lactotriosylceramide, as
well as those reported (20) for GlcNAc
1
3GalNAc as acceptors,
suggest that
3Gal-T5 is able to act on glycolipids as well as on
core 3 O-linked glycoproteins. The possibility of a broad
range of specificity of the enzyme is confirmed by the recent report
that it also acts on the GalNAc end of globotetraosylceramide (45). On
this basis, the down-regulation of
3Gal-T5 in cancer is expected to
affect other glycoconjugates synthesized by the enzyme in normal
mucosa. However,
3Gal-T5 expression is maintained in some colon
adenocarcinoma cell lines (16), and we suggest that the results
obtained with these cells must be cautiously interpreted, or
reinterpreted (12, 19). In particular, further experimental data are
needed to establish whether or not the CA19.9 antigen present in
patient serum is synthesized by
3Gal-T5 and secreted from the tumor mass.
 |
ACKNOWLEDGEMENTS |
We thank Marco Bellaviti for computer
artwork, Dr. Nozomu Hiraiwa (Aichi Cancer Center, Nagoya, Japan) for
helpful suggestions, and Dr. Fabio Dall'Olio (Department of
Experimental Pathology, University of Bologna, Italy) for critical
reading of the manuscript.
 |
FOOTNOTES |
*
This work was supported in part by a grant from the
Associazione Italiana Ricerca sul Cancro (to M. T.) and by a grant
from the University of Insubria (FAR 1999).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.
A researcher at the University of Insubria Medical School
(formerly University of Pavia Medical School II). To whom
correspondence should be addressed. Tel.: 39-0382-507-233; Fax:
39-0382-423-108; E-mail: dbioc@unipv.it.
Published, JBC Papers in Press, October 31, 2000, DOI 10.1074/jbc.M006662200
 |
ABBREVIATIONS |
The abbreviations used are:
Gal-T, galactosyltransferase;
GnT, N-acetylglucosaminyltransferase;
FUT2, secretor type
1,2-fucosyltransferase;
Fuc-TIII,
1,(3/4)-fucosyltransferase;
sLea, sialyl-Lewis
a (NeuAc
2
3Gal
1
3[Fuc
1
4]GlcNAc);
Lea, Lewis a (Gal
1
3 [Fuc
1
4]GlcNAc);
Leb, Lewis
b (Fuc
1
2Gal
1
3[Fuc
1
4] GlcNAc);
sLex, sialyl-Lewis x (NeuAc
2
3Gal
1
4[Fuc
1
3]GlcNAc);
CEA, carcinoembryonic antigen;
RT-PCR, reverse transcriptase
mediated-polymerase chain reaction;
lactotriosylceramide, GlcNAc
1
3Gal
1
4Glc
1
1'Cer;
lacto-N-tetraosylceramide, Gal
1
4GlcNAc
1
3Gal
1
4Glc
1
1'Cer;
PBS, phosphate-buffered saline;
CHO, Chinese hamster ovary;
HPTLC, high
performance thin layer chromatography;
PAGE, polyacrylamide gel
electrophoresis;
FITC, fluorescein isothiocyanate;
CHO-T-FT, CHO cells
expressing Polyoma virus T antigen and Fuc-TIII;
CHO-FT-T1, CHO clone
permanently expressing Fuc-TIII and
3Gal-T1;
CHO-FT-T2, CHO clone
permanently expressing Fuc-TIII and
3Gal-T2;
CHO-FT-T5, CHO clone
permanently expressing human Fuc-TIII and
3Gal-T5.
 |
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