From the Glycobiology Program, Cancer Research
Center, the Burnham Institute, La Jolla, California 92037 and the
§ Howard Hughes Medical Institute, Department of Pathology,
and the Life Sciences Institute, University of Michigan Medical School,
Ann Arbor, Michigan 48104
Received for publication, December 16, 2002, and in revised form, January 9, 2003
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ABSTRACT |
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It has been established that sialyl
Lewis x in core 2 branched O-glycans serves as an E- and
P-selectin ligand. Recently, it was discovered that 6-sulfosialyl Lewis
x in extended core 1 O-glycans,
NeuNAc Mucin-type O-glycans are unique in having clusters of
large numbers of O-glycans. These O-glycans
contain N-acetylgalactosamine residues at reducing ends,
which are linked to serine or threonine residues in a polypeptide (1).
These attached O-glycans can be classified into several
different groups according to the core structure (2). In many cells, a
structure called core 1, Gal When leukocytes are recruited to inflammatory sites, E- and P-selectins
expressed in activated endothelial cells recognize The roles of sialyl Lewis x in core 2 branched O-glycans
have been demonstrated by analyzing mutant mice with deficient
Core2GlcNAcT-I, obtained through homologous recombination (17).
Leukocytes derived from null mutant mice display significantly reduced
adhesion to L-, P-, and E-selectins, demonstrating that ligands for
these selectins are mainly carried by core 2 branched
O-glycans. By contrast, in these mice, lymphocyte adhesion
to HEV in lymph nodes is only marginally impaired, and MECA-79 antibody
staining that decorates HEV is not reduced (17). Recent studies reveal
that L-selectin ligand activity remaining after abrogation of
Core2GlcNAcT-I is due to the activity of 6-sulfosialyl Lewis x in
extended core 1 O-glycans,
NeuNAc Extended core 1 structure is synthesized by core 1 2
3Gal
1
4(Fuc
1
3(sulfo
6))GlcNAc
1
3Gal
1
3GalNAc
1
Ser/Thr, functions as an L-selectin
ligand in high endothelial venules. Extended core 1 O-glycans can be synthesized when a core 1 extension enzyme
is present. In this study, we first show that
1,3-N-acetylglucosaminyltransferase-3 (
3GlcNAcT-3) is
almost exclusively responsible for core 1 extension among seven
different
3GlcNAcTs and thus acts on core 1 O-glycans attached to PSGL-1. We found that transcripts encoding
3GlcNAcT-3 were expressed in human neutrophils and lymphocytes but that their levels were lower than those of transcripts encoding core 2
1,6-N-acetylglucosaminyltransferase I
(Core2GlcNAcT-I). Neutrophils also expressed transcripts encoding fucosyltransferase VII (FucT-VII) and Core2GlcNAcT-I, whereas lymphocytes expressed only small amounts of transcripts encoding FucT-VII. To determine the roles of sialyl Lewis x in extended core 1 O-glycans, Chinese hamster ovary (CHO) cells were stably transfected to express PSGL-1, FucT-VII, and either
3GlcNAcT-3 or
Core2GlcNAcT-I. Glycan structural analyses disclosed that PSGL-1 expressed in these transfected cells carried comparable amounts of
sialyl Lewis x in extended core 1 and core 2 branched
O-glycans. In a rolling assay, CHO cells expressing sialyl
Lewis x in extended core 1 O-glycans supported a
significant degree of shear-dependent tethering and rolling
of neutrophils and lymphocytes, although less than CHO cells expressing
sialyl Lewis x in core 2 branched O-glycans. These results
indicate that sialyl Lewis x in extended core 1 O-glycans
can function as an L-selectin ligand and is potentially involved in
neutrophil adhesion on neutrophils bound to activated endothelial cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
3GalNAc, is the major constituent of
O-glycans. Core 1 oligosaccharides are converted to core 2 oligosaccharides, Gal
1
3(GlcNAc
1
6)GalNAc, when core 2
1,6-N-acetylglucosaminyltransferase
(Core2GlcNAcT)1 is present
(3, 4). Various ligand carbohydrates can be formed in core 2 branched
oligosaccharides. For example, sialyl Lewis x in mucin-type
glycoproteins of blood cells can be found in core 2 branched
oligosaccharides such as
NeuNAc
2
3Gal
1
4(Fuc
1
3)GlcNAc
1
6(NeuNAc
2
3Gal
1
3)GalNAc
1
Ser/Thr (see Fig. 1) (5, 6).
2
3-sialylated,
1
3-fucosylated O-glycans, allowing leukocytes to
eventually extravasate (7-9). It has also been demonstrated that
L-selectin in neutrophils mediates neutrophil rolling on neutrophils
adherent to activated endothelial cells (10, 11). PSGL-1
(P-selectin glycoprotein
ligand-1) contributes to this process, although
the nature of the glycan moieties that decorate this and other
potential L-selectin ligands in neutrophils has not been well
characterized. On the other hand, L-selectin in lymphocytes recognizes
6-sulfosialyl Lewis x in core 2 branched O-glycans,
NeuNAc
2
3Gal
1
4(Fuc
1
3(sulfo
6))GlcNAc
1
6(Gal
1
3)GalNAc
1
Ser/Thr, which are expressed in high
endothelial venules (HEV) in lymph nodes (12-14). Such recognition
leads to lymphocyte adhesion to HEV, allowing lymphocyte movement from
the vascular to the lymphatic system. The formation of 6-sulfosialyl
Lewis x depends on 6-sulfotransferase, designated L-selectin ligand
sulfotransferase (LSST) or high endothelial cell
N-acetylglucosamine 6-O-sulfotransferase
(12, 15, 16).
2
3Gal
1
4[Fuc
1
3(sulfo
6)]GlcNAc
1
3Gal
1
3GalNAc
1
Ser/Thr (18). Moreover, a minimum MECA-79
epitope was found to be a 6-sulfo structure in the extended core 1 O-glycan, and MECA-79 antibody binds efficiently to
6-sulfosialyl Lewis x-containing extended core 1 O-glycans
(18). These findings are consistent with previous findings that MECA-79
antibody inhibits lymphocyte adhesion to HEV without removal of sialic
acid (19) or fucose and that MECA-79 staining remains after expression
of fucose is abrogated by inactivation of fucosyltransferase VII
(FucT-VII) (20).
1,3-N-acetylglucosaminyltransferase (
3GlcNAcT), which
adds
1,3-linked N-acetylglucosamine to
Gal
1
3GalNAc
1
R (Fig. 1). A
cDNA encoding
3GlcNAcT was first cloned by expression cloning,
and the encoded protein was designated both i-antigen forming
1,3-N-acetylglucosaminyltransferase (21) and
3GlcNAcT-1 (22, 23). Expression of
3GlcNAcT-1 does not, however,
result in the synthesis of extended core 1 structure (18). By screening
expressed sequence tag data bases using the cDNA encoding the now
designated
1,3-galactosyltransferase-6 as a probe, cDNA encoding
core 1-
3GlcNAcT was identified (18). This cloning was possible
because it was reported that
1,3-galactosyltransferase and
3GlcNAcT are highly homologous proteins (24). In parallel, at least
five additional
3GlcNAcTs have been molecularly identified based on
similarity to the previously cloned
1,3-galactosyltransferase or to
3GlcNAcT-2 (22, 25-28). These results indicate that core 1-
3GlcNAcT belongs to the
3GlcNAcT gene family, and it is thus designated
3GlcNAcT-3.
View larger version (19K):
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Fig. 1.
Biosynthetic schemes of core 1 and core 2 O-glycans. Core 2 branch can be added to core 1 by Core2GlcNAcT-I, and this branch is galactosylated by
1,4-galactosyltransferase IV (
4GalT-IV) (54).
This is followed by sialylation and fucosylation by
2,3-sialyltransferase and FucT-VII, forming sialyl Lewis x in core 2 branch (left). Core 1 is also modified by core 1
3GlcNAcT, which is also known as
3GlcNAcT-3, forming extended
core 1. Extended core 1 is then galactosylated (most likely by
1,4-galactosyltransferase I), sialylated, and fucosylated, forming
sialyl Lewis x in extended core 1 structure (middle). Core 1 can be sialylated by
-galactoside
2,3-sialyltransferase I
(ST3Gal I) (50) and then by
N-acetylgalactosamine
2,6-sialyltransferase
(ST6GalNAc), forming disialosyl core 1 O-glycan
(right). This biosynthetic pathway precludes either core 1 extension or core 2 branching.
3GlcNAcT-3 transcripts are highly expressed in the small intestine,
colon, and placenta and are moderately expressed in various tissues,
including the liver, kidney, pancreas, and prostate (18). It is not
known whether blood cells express
3GlcNAcT-3.
In this study, we address the function and expression pattern of
3GlcNAcT-3. First, we show that
3GlcNAcT-3 was the only enzyme
that significantly formed extended core 1 among highly related
3GlcNAcTs. We then show that
3GlcNAcT-3 transcripts were present
in both human neutrophils and lymphocytes, but that these cells lacked
LSST. Transfection studies using FucT-VII and
3GlcNAcT-3 or
Core2GlcNAcT-I showed that extended core 1 could be synthesized in
Chinese hamster ovary (CHO) cells and that extended core 1 structure
was fucosylated by FucT-VII more efficiently than core 2 branches.
Finally, we show that sialyl Lewis x synthesized in extended core 1 served as an L-selectin ligand, although it is apparently less potent
than sialyl Lewis x in core 2 branches.
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EXPERIMENTAL PROCEDURES |
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Cloning of cDNA Encoding 3GlcNAcTs--
cDNA cloning
of
3GlcNAcT-3 was described previously (18). cDNAs encoding
human
3GlcNAcT-1 (21),
3GlcNAcT-2 (22),
3GlcNAcT-4 (22),
3GlcNAcT-5 (25, 26),
3GlcNAcT-6 (27), and
3GlcNAcT-7 (28) were
cloned by reverse transcription (RT)-PCR using the Expand High Fidelity
PCR system (Roche Molecular Biochemicals) (29). RT-PCR primers were as
follows:
3GlcNAcT-1, 5'-CGAGAGCCATGCAGATGTCCTAC-3' (5'-primer) and 5'-AAGGGCTCAGCAGCGTCGGGGAG-3' (3'-primer);
3GlcNAcT-2, 5'-GACAAGATATGAGAAATGAGTGTTGG-3' (5'-primer) and
5'-TTTTAGCATTTTAAATGAGCACTCTGC-3' (3'-primer);
3GlcNAcT-4,
5'-AGCACGGAGACAGTCTCCAGCTG-3' (5'-primer) and
5'-AGGCATCAATTTCGCATCACGATAG-3' (3'-primer);
3GlcNAcT-5, 5'-AGACTTGAGTGGATATGAGAATGTTG-3' (5'-primer) and
5'-AAGTACTATTAGATAAACGCAGCCCT-3' (3'-primer);
3GlcNAcT-6,
5'-ACGCTCAAGCACCTGCACTTGCT-3' (5'-primer) and
5'-ACTGGCCTCAGGAGACCCGGTG-3' (3'-primer); and
3GlcNAcT-7, 5'-GCCGCCATGTCGCTGTGGAAGA-3' (5'-primer) and
5'GGGTCAGAGCACCTGGAGCTTG-3' (3'-primer). As templates, we used a human
fetal brain cDNA library in pcDNA1 (30) for
3GlcNAcT-1, human esophagus cDNA in a human digestive system
multiple tissue cDNA panel (BD Biosciences) for
3GlcNAcT-2,
an SK-N-MC cell cDNA library (see below) for
3GlcNAcT-4, human
pituitary gland Marathon-Ready cDNA (BD Biosciences) for
3GlcNAc-5, human stomach cDNA in a human digestive system
multiple tissue cDNA panel (BD Biosciences) for
3GlcNAcT-6, and
human colon Marathon-Ready cDNA (BD Biosciences) for
3GlcNAcT-7.
A cDNA library of SK-N-MC neuroblastoma cells was prepared by
isolation of total RNA with TRIzol (Invitrogen), followed by
first-strand cDNA synthesis using SuperScript II RNase
H
reverse transcriptase (Invitrogen).
PCR products were first inserted into pCR2.1-TOPO (Invitrogen). In the
case of 3GlcNAcT-1, -2, -4, -6, and -7, cDNAs in pCR2.1-TOPO were digested with EcoRI and inserted into the
dephosphorylated EcoRI site of pcDNA3.1(N).
pcDNA3.1(N) is a vector created by the digestion of
pcDNA3.1/Zeo with SphI and BspLU11I (Roche
Molecular Biochemicals), followed by filling in and self-ligation to
remove the Zeocin resistance gene and f1 origin. For
3GlcNAcT-5,
cDNA in pCR2.1-TOPO was digested with EcoRI and
ScaI and subcloned into the
EcoRI-EcoRV sites of pcDNA3.1(N). Similarly,
3GlcNAcT-3 was cloned into the HindIII-XhoI
sites of pcDNA1.1, resulting in pcDNA1.1-
3GlcNAcT-3.
cDNA encoding LSST was cloned as described previously (12, 15).
Expression of i Antigen in HeLa Cells by Different
3GlcNAcTs--
To determine whether all of
3GlcNAcTs direct the
synthesis of poly-N-acetyllactosamine synthesis, HeLa cells
were transiently transfected with one of the
pcDNA3.1(N)-
3GlcNAcTs or pcDNA1.1-
3GlcNAcT-3. Thirty-six
hours after transfection, cells were dissociated into monodispersed
cells using an enzyme-free cell dissociation solution (Hanks'
balanced saline solution-based) purchased from Cell and Molecular
Technologies. Monodispersed cells were incubated with human anti-i
serum (Dench) (31), followed by affinity-purified fluorescein
isothiocyanate (FITC)-conjugated goat anti-human IgM antibodies
(Pierce). The stained cells were subjected to FACS analysis using a
FACScan (BD Biosciences) as described previously (32).
HeLa cells were chosen as recipient cells for transfection because the
molecular mass of lysosome-associated membrane protein-1, a
major carrier of poly-N-acetyllactosamines (33), is the
smallest among Namalwa, HL-60, CHO, HepG2, and HeLa cells. The
results indicated that HeLa cells express minimum amounts of
3GlcNAcTs because poly-N-acetyllactosaminylated
lysosome-associated membrane protein-1 displays a higher molecular mass
than lysosome-associated membrane protein-1 containing minimum amounts
of poly-N-acetyllactosamine (33).
Expression of MECA-79 in Lec2 Cells by LSST and
3GlcNAcT--
To determine which
3GlcNAcT directs expression of
MECA-79 antigen, Lec2 cells were transiently transfected with
pcDNA1-LSST and one of the pcDNA3.1(N)-
3GlcNAcTs or
pcDNA1.1-
3GlcNAcT-3. Thirty-six hours after transfection, cells
were dissociated into monodispersed cells using the cell dissociation
solution as described above. Monodispersed cells were incubated with
MECA-79 antibody (BD Biosciences) (19), followed by affinity-purified
FITC-conjugated goat anti-rat IgM antibody (ICN Biochemicals). The
stained cells were subjected to FACS analysis as described above. CHO
mutant Lec2 cells lack a functional Golgi CMP-sialic acid transporter; and therefore, sialylation is absent in Lec2 cells (34). The absence of
sialylation facilitates core 1 extension because core 1 extension and
sialylation compete with each other for the same acceptor,
Gal
1
3GalNAc
1
R.
Core 1 Extension in PSGL-1 by 3GlcNAcTs--
To determine
which
3GlcNAcT can add
1,3-N-acetylglucosamine
to core 1, Gal
1
3GalNAc
1
R, Lec2 cells were transiently
transfected with pZeoSV-PSGL-1 (kindly provided by Dr. Richard
Cummings) and vectors encoding
3GlcNAcT using LipofectAMINE as
described previously (12). The ratio of pZeoSV-PSGL-1 and
3GlcNAcT
cDNA was 1:5 (w/w) to achieve efficient modification of PSGL-1 by
3GlcNAcT. Forty-eight hours after transfection, cells were harvested
in phosphate-buffered saline with a cell scraper. The cells were subjected to three cycles of freezing and thawing to disrupt the plasma
membrane. The membrane fraction was collected by centrifugation at
12,000 × g for 10 min. The resultant membrane fraction
was first resuspended in 10 mM Tris-HCl and 1 mM EDTA (pH 8.0), and then 10% Triton X-100 was added to a
final concentration of 1%. After gentle rocking at 4 °C for 15 min,
the Triton X-100-soluble membrane fraction, containing PSGL-1, was
obtained by centrifugation at 12,000 × g for 10 min.
The membrane fraction was then lysed in sample buffer and subjected to SDS-PAGE. After blotting onto a polyvinylidene difluoride membrane filter, the blot was reacted with anti-PSGL-1 antibody (KPL-1, BD Biosciences), followed by secondary antibody; and immunoreactive PSGL-1 was visualized using enhanced Luminol reagent (PerkinElmer Life Sciences).
RT-PCR of 3GlcNAcT-3, Core2GlcNAcT-I, LSST, FucT-VII, and
PSGL-1 on RNA Isolated from Neutrophils and Lymphocytes--
Human
neutrophils and lymphocytes were isolated from the peripheral blood of
a volunteer as described previously (35). Briefly, blood was drawn in a
syringe containing heparin, and erythrocytes were sedimented by
dextran/saline solution, obtaining leukocyte-rich plasma in the upper
layer. Ficoll-Paque Plus solution (Amersham Biosciences) was added
beneath this layer and centrifuged. Mononuclear cells enriched with
lymphocytes were isolated from the saline/Ficoll interface. The pellet
from the above centrifugation was enriched with neutrophils, which were
isolated after hypotonic lysis of erythrocytes. Total RNA was isolated
from neutrophils and lymphocytes using TRIzol. RT-PCR of
3GlcNAcT-3
(18), Core2GlcNAcT-I (4), LSST (12, 15), FucT-VII (20), and PSGL-1 (36,
37) was carried out as follows. Using isolated RNA from respective
cells as a template, first-strand cDNA was synthesized using
RNase H
reverse transcriptase. PCR was carried out
using AmpliTaq DNA polymerase (Applied Biosystems) and the following
PCR primers:
3GlcNAcT-3, 5'-TTCTTCAACCTCACGCTCAAGCAG-3' (5'-primer)
and 5'-AGCATCTCATAAGGTAGGAAGCGG-3' (3'-primer); Core2GlcNAcT-I,
5'-TGAAATGCTTGACAGGCTGCTGAG-3' (5'-primer) and
5'-GGTGTTTCGAGTGGAGGAAGCATT-3' (3'-primer); LSST,
5'-GGGAGATCTCATGATTGACAGTCG-3' (5'-primer) and
5'-TAGTGGATTTGCTCAGGGACAGTC-3' (3'-primer); FucT-VII, 5'-GTCAGCAACTTCCAGGAGCGGCA-3' (5'-primer) and
5'-TCAAGGTCCTCATAGACTTGGCTG-3' (3'-primer); PSGL-1,
5'-CCCTGTCCACAGAACCCAGTGC-3' (5'-primer) and
5'-GAAGCTGTGCAGGGTGAGGTCAT-3' (3'-primer); and
glyceraldehyde-3-phosphate dehydrogenase, 5'-CCTGGCCAAGGTCATCCATGACA-3'
(5'-primer) and 5'-ATGAGGTCCACCACCCTGTTGCT-3' (3'-primer) or
5'-GACCCCTTCATTGACCTCAACTACA-3' (5'-primer) and 5'-ACATGGCCTCCAAGGAGTAAGA-3' (3'-primer). The last primer pair was included in the human digestive system multiple tissue cDNA panel.
PCR products were separated by electrophoresis on 1% agarose gels. To
confirm that the PCR products were derived from respective transcripts,
RT-PCR products were digested with restriction enzymes and subjected to
electrophoresis on 2% agarose.
Stable Expression of 3GlcNAcT-3 and Core2GlcNAcT-I in CHO
Cells--
CHO cells were first transfected with pZeoSV-PSGL-1 and
selected in the presence of 100 µg/ml Zeocin (Invitrogen). CHO
colonies stably expressing PSGL-1 were selected after staining with
anti-PSGL-1 antibody KPL-1, establishing CHO-PSGL-1 cells. CHO-PSGL-1
cells were stably cotransfected with pcDNA1.1-
3GlcNAcT-3 (core 1
3GlcNAcT) and pSV-hygromycin and selected in 100 µg/ml Zeocin and
400 µg/ml hygromycin B (Calbiochem). Expression of
3GlcNAcT-3 was
shown by the expression of larger forms of PSGL-1 that contains
extended core 1 O-glycans (see also "Results"). The
resultant CHO-PSGL-1/C1 cells were then cotransfected with
pCDM8-FucT-VII and pcDNA3 and cultured in the presence of 400 µg/ml Geneticin (Invitrogen). CHO-PSGL-1/C1 cells stably expressing
FucT-VII were selected after staining with anti-sialyl Lewis x antibody
(CSELX-1), resulting in CHO-PSGL-1/F7/C1 cells.
In parallel, CHO-PSGL-1 cells were transfected with pcDNA1.1-Core2GlcNAcT-I together with pcDNA3 and cultured in the presence of 100 µg/ml Zeocin and 400 µg/ml Geneticin. Cells expressing Core2GlcNAcT-I were chosen for expressing larger forms of PSGL-1 that contains core 2 branched O-glycans. Expression of core 2 O-glycans was confirmed after transient transfection of CD43 (also called leukosialin) and staining with T305 antibody, resulting in CHO-PSGL-1/C2 cells. As reported previously, T305 reacts with core 2 branched O-glycans attached to CD43 (4, 38). CHO-PSGL-1/C2 cells were stably transfected with pCDM8-FucT-VII and pSV-hygromycin and cultured in the presence of Zeocin, hygromycin B, and Geneticin. Cells expressing FucT-VII were selected after staining with CSELX-1 antibody, resulting in CHO-PSGL-1/F7/C2 cells.
As a control, CHO-PSGL-1 cells were stably transfected with pCDM8-FucT-VII and pcDNA3 and cultured in the presence of Zeocin and Geneticin. Cells expressing FucT-VII were selected after staining with CSELX-1 antibody, resulting in CHO-PSGL-1/F7 cells.
Isolation of PSGL-1/IgG Chimeric Protein from Transfected CHO Cells-- cDNA encoding PSGL-1/IgG chimeric protein was constructed using pZeoSV-PSGL-1 and pcDNA3.1-IgG essentially as described previously (12), resulting in pcDNA3.1-PSGL-1/IgG. CHO-PSGL-1/F7/C1, CHO-PSGL-1/F7/C2, and CHO-PSGL-1/F7 cells were transfected with pcDNA3.1-PSGL-1/IgG as described previously (12). Twenty-four hours after transfection, cells were cultured in glucose-free Dulbecco's modified Eagle's medium containing 10% dialyzed fetal calf serum, 100 µM sodium pyruvate, 2 mM glutamine, 25 mM HEPES, and 20 µCi/ml [3H]glucosamine (PerkinElmer Life Sciences). PSGL-1/IgG chimeric protein was isolated using ImmunoPure immobilized protein A (Pierce) from the medium obtained after 48 h of additional culture as described previously (12, 18).
Structural Analysis of Oligosaccharides Attached to PSGL-1/IgG-- PSGL-1/IgG isolated as described above was subjected to alkaline borohydride treatment to release O-glycans as described (39). Released O-glycans were recovered after Sephadex G-50 gel filtration (12, 18). The isolated O-glycans were then applied to a column (1.0 × 150 cm) of Bio-Gel P-4 (200-400 mesh) equilibrated with 0.1 M NH4HCO3. Those oligosaccharide fractions, separated by Bio-Gel P-4 gel filtration, were then desialylated by treatment in 2 M acetic acid at 80 °C for 4 h (40). After neutralization of the sample with ammonium hydroxide and Sephadex G-25 gel filtration in 7% 1-propanol to remove salt, the sample was again subjected to Bio-Gel P-4 gel filtration under the same conditions. Neutral oligosaccharides separated by Bio-Gel P-4 gel filtration were subjected to HPLC using an amino-bonded column (Asahipak NH2 P50-4E, 4.6 × 250 mm) that was equilibrated with solution A (75% acetonitrile, 20% H2O, and 5% 0.25 M KH2PO4/H2O) and that was attached to a Gilson 306 HPLC apparatus. The sample was eluted for 40 min with solution A and then eluted with a linear gradient from solution A to a 60:40 mixture of solution A and solution B (50% acetonitrile, 45% H2O, and 5% 0.25 M KH2PO4/H2O) over the next 50 min. The sample was finally eluted with 100% of solution B over the last 30 min.
Standard O-glycans were obtained from CD34/IgG glycans
synthesized in the presence of 3GlcNAcT-3 and Core2GlcNAcT-I as
described previously (12, 18). Oligosaccharides were digested with
Streptomyces sp.
1,3/4-fucosidase (Panvera/Takara,
Madison, WI) and jack bean
-galactosidase (Sigma) as described
previously (39, 41, 42). The digest was desalted by Sephadex G-25 gel
filtration in 7% 1-propanol before HPLC analysis.
Measurement of L-selectin-mediated Rolling in CHO Cells
Expressing Sialyl Lewis x in Extended Core 1 or Core 2 Branched
O-Glycans--
CHO cells stably expressing PSGL-1, FucT-VII, and
3GlcNAcT-3 or Core2GlcNAcT-I were established as described above.
These cells maintained similar amounts of sialyl Lewis x and PSGL-1 as
assessed by FACS analysis using anti-PSGL-1 antibody KPL-1 and
anti-sialyl Lewis x antibody CSELX-1 (see also
"Structural Analysis of PSGL-1 O-glycans Synthesized in
the Presence of
3GlcNAcT-3 or Core2GlcNAcT-I"). These stably
transfected cells seeded on dishes were used as the bottom plate of a
parallel flow chamber as described previously (18).
Neutrophils or lymphocytes were initially introduced into the flow
chamber at a wall sheer stress of 5 dynes/cm2 for 15 s, followed by the termination of flow to allow the cells to adhere
under static conditions (17). Flow rate was then initiated at different
shear forces. Image analysis was performed and analyzed as described
previously (17).
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RESULTS |
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3GlcNAcT-3 Acts on Core 1 O-Glycans,
Gal
1
3GalNAc
1
Ser/Thr--
Previously, we showed
that, of
3GlcNAcT-1, -2, -3, and -4,
3GlcNAcT-3 can act on
Gal
1
3GalNAc
1
R, forming
GlcNAc
1
3Gal
1
3GalNAc
1
R (18). This extended core 1 structure can be converted to
Gal
1
4(sulfo
6)GlcNAc
1
3Gal
1
3GalNAc
1
R in
the presence of the 6-sulfotransferase LSST and
1,4-galactosyltransferase. Because
Gal
1
4(sulfo
6)GlcNAc
1
3Gal
1
3GalNAc
1
R is a
minimum epitope for MECA-79 antigen, the formation of extended
core 1 can be detected by immunostaining with MECA-79 antibody when
LSST is also expressed.
After the initial report on core 1 3GlcNAcT (or
3GlcNAcT-3) (18),
three additional
3GlcNAcTs highly related to
3GlcNAcT-3 were
molecularly cloned:
3GlcNAcT-5 (25, 26),
3GlcNAcT-6 (27), and
3GlcNAcT-7 (28). We thus determined whether extended core 1 can be
formed by these more recently identified members of the
3GlcNAcT
gene family.
First, we tested whether all of the cloned 3GlcNAcTs are
active in synthesizing i antigen,
Gal
1
4GlcNAc
1
3Gal
1
4GlcNAc
R (21, 31). The
synthesis of i antigen is dependent on
3GlcNAcT, which adds
1,3-linked GlcNAc to N- acetyllactosamine. HeLa
cells were thus transiently transfected with one of the
3GlcNAcTs in mammalian expression vectors, and the transfected cells were stained with anti-i antibody, followed by FITC-conjugated secondary antibody. Fig. 2 shows that the cells transfected
with any of the
3GlcNAcTs tested displayed increased amounts of i
antigen compared with mock-transfected cells. The results also
indicate that the expression efficiency of different
3GlcNAcTs
is essentially invariable because similar amounts of i antigen were
detected in HeLa cells transfected with different
3GlcNAcTs.
|
As shown previously, CHO cells and the CHO mutant Lec2 cell line do not
synthesize core 2 O-glycans (43) or extended core 1 oligosaccharides (18). Lec2 cells were used as a reporter cell line to
test 3GlcNAcT-dependent reconstitution of MECA-79 antigen expression because Lec2 cells are deficient in core 1 extension
and thus MECA-79 antigen expression and because the sialylation defect
in Lec2 cells will not inhibit formation of MECA-79 antigen by
sialylation of Gal
1
3GalNAc
1
R. Lec2 cells were thus
transiently transfected with LSST and one of the
3GlcNAcTs. As shown
in Fig. 3, Lec2 cells transfected with
3GlcNAcT-3, also called core 1-
3GlcNAcT, expressed significant
amounts of MECA-79 antigen, but none of the other enzymes formed
MECA-79 antigen. Because all of the
3GlcNAcTs tested were expressed
in similar amounts in HeLa cells, it is reasonable to assume that these
3GlcNAcTs were expressed in similar amounts in Lec2 cells as well
(see also Fig. 4). These results indicate that only
3GlcNAcT-3 can
form extended core 1 structure.
|
Modification of PSGL-1 O-Glycans by 3GlcNAcTs--
To confirm
the above conclusions, Lec2 cells were transiently cotransfected with
vectors encoding PSGL-1 and one of the
3GlcNAcTs. The transfected
Lec2 cells were then subjected to Western blot analysis using
anti-PSGL-1 antibody KPL-1.
Fig. 4 illustrates that PSGL-1 in
mock-transfected Lec2 cells migrated at ~125 kDa. By contrast,
3GlcNAcT-3 converted PSGL-1 into polydisperse higher molecular mass
glycoforms of ~150-200 kDa. Apparently,
3GlcNAcT-2 can form
similar products with very low efficacy, and
3GlcNAcT-6 forms small
amounts of extended core 1, but its product is smaller than that formed
by
3GlcNAcT-3 or
3GlcNAcT-2. These results indicate that
3GlcNAcT-3 is almost exclusively responsible for extending
core 1.
|
Core 1 Extension Enzyme (3GlcNAcT-3) Is Also Expressed in
Neutrophils and Lymphocytes--
Previously, we showed that
3GlcNAcT-3 is expressed in HEV in peripheral lymph nodes and forms
L-selectin ligand critical for lymphocyte homing (18). To determine
whether
3GlcNAcT-3 is also expressed in human neutrophils and
lymphocytes, neutrophils and lymphocytes were isolated from the
peripheral blood, and RT-PCR was used to assay for
3GlcNAcT-3
transcripts. The results shown in Fig. 5
demonstrate that the
3GlcNAcT-3 transcript was expressed in
neutrophils and lymphocytes, although the amount of the transcript was
much less than that of the Core2GlcNAcT-I transcript. In addition, the
level of Core2GlcNAcT-I transcripts was less than that of PSGL-1.
Notably, neutrophils, but not lymphocytes, contained a significant
amount of FucT-VII transcripts. To confirm that these transcripts are
derived from the proper corresponding GlcNAcT locus, the RT-PCR
products were digested with restriction enzymes and analyzed by agarose
gel electrophoresis. The results shown in Fig. 5B
demonstrate that the transcripts from neutrophils and lymphocytes
produced the same restriction digest products as those derived from
plasmids encoding those proteins, supporting our conclusion that the
transcripts derived from these cells represent
3GlcNAcT-3,
Core2GlcNAcT-I, FucT-VII, and PSGL-1, respectively.
|
On the other hand, LSST transcripts were barely detected in neutrophils or lymphocytes, indicating that LSST is expressed, if at all, in very low quantities in these cells (Fig. 5). These results indicate that neutrophils express each of the enzymes necessary to form sialyl Lewis x in extended core 1 structure, whereas lymphocytes express trace amounts of the sialyl Lewis x moiety due to negligible expression of FucT-VII.
Structural Analysis of PSGL-1 O-Glycans Synthesized in the Presence
of 3GlcNAcT-3 or Core2GlcNAcT-I--
Because PSGL-1 is expressed in
neutrophils and lymphocytes as indicated, the above results suggest
that
3GlcNAcT-3 can form extended core 1 structure in PSGL-1, a
counter-receptor for L-, P-, and E-selectins in neutrophils. Such
expression led to the formation of sialyl Lewis x structure in extended
core 1 O-glycans when FucT-VII was also present. Fig.
6 illustrates that either
3GlcNAcT-3
(C1) or Core2GlcNAcT-I (C2) could convert
PSGL-1/IgG chimeric protein into a polydisperse high molecular mass
glycoform, which could be metabolically labeled with
[3H]glucosamine (second and third
lanes). By contrast, PSGL-1 chimeric protein migrated as sharper
bands when isolated from control CHO cells expressing only FucT-VII
(first lane).
|
These samples were treated with alkaline borohydride to release
O-glycans and subjected to Sephadex G-50 gel filtration.
Released O-glycans (Fig. 7,
A and E, horizontal bars) were
subjected then to Bio-Gel P-4 gel filtration. As shown in Fig. 7
(B and F), PSGL-1 O-glycans derived from CHO cells expressing FucT-VII and
3GlcNAcT-3 or Core2GlcNAcT-I mainly produced two or three peaks (I,
I', and II). After desialylation and Bio-Gel P-4 gel filtration,
peak I produced peaks IA and IB (Fig. 7, C and
G). As shown in Fig. 7 (C and G), peak
C2-IA from Core2GlcNAcT-I-expressing CHO cells eluted slightly later
than peak C1-IA from
3GlcNAcT-3-expressing CHO cells.
|
Peaks C1-IA and C2-IA were then analyzed by HPLC (Fig.
8). Both peaks C1-IA and C2-IA produced
two peaks. The first peaks C1-IA1 and C2-IA1 eluted corresponding
to Gal1
4GlcNAc
1
3Gal
1
3GalNAcOH and
Gal
1
4GlcNAc
1
6(Gal
1
3)GalNAcOH, respectively. After
1,3-specific fucosidase digestion, the second peaks C1-IA2 and
C2-IA2 were converted to peaks C1-IA1 and C2-IA1, respectively (Fig. 8,
B and D), indicating that peaks C1-IA2 and C2-IA2
were derived from NeuNAc
2
3Gal
1
4(Fuc
1
3)GlcNAc
1
3Gal
1
3GalNAcOH and
NeuNAc
2
3Gal
1
4(Fuc
1
3)GlcNAc
1
6(NeuNAc
2
3Gal
1
3)GalNAcOH, respectively.
|
After desialylation, peaks C1-I and C2-I (Fig. 7, B and
F) also produced peaks C1-IB and C2-IB, which eluted at the
same positions as Gal1
3GalNAcOH and sialic acid monomer (Fig. 7,
C and G). These results indicate that peaks C1-I
and C2-I (Fig. 7, B and F) also contained
NeuNAc
2
6(NeuNAc
2
3Gal
1
3)GalNAcOH, which was recovered
in peaks C1-IB and C2-IB after desialylation. After desialylation, peak
C1-II (monosialylated fraction in Fig. 7B) produced peak
C1-IIB and a small amount of peak C1-IIA (Fig. 7D). Peak
C1-IIA did not change its elution position after desialylation and
corresponds to
Gal
1
4(±Fuc
1
3)GlcNAc
1
3Gal
1
3GalNAcOH. Peak
C1-IIB was, on the other hand, found to be a mixture of
Gal
1
3GalNAcOH and sialic acid. Similarly, peak C2-II (Fig.
7F) produced sialic acid and Gal
1
3GalNAcOH
(data not shown). The amount of Gal
1
3GalNAcOH in
peaks IB and IIB was determined after sialic acid was
removed by QAE-Sephadex A-25 column chromatography.
On the other hand, peak C2-I' produced two peaks
corresponding to Gal1
4GlcNAc
1
6(Gal
1
3)GalNAcOH (peak
C2-I'A) and GlcNAc
1
6(Gal
1
3)GalNAcOH (peak C2-I'B), in
addition to sialic acid (peak C2-I'C) (Fig. 7H and Table
I). The latter oligosaccharide (peak
C2-I'B) was also obtained previously in CHO cells transfected with
Core2GlcNAcT-I (38).
|
These combined results indicate that extended core 1 O-glycans can be fucosylated more efficiently than core 2 branched O-glycans (Fig. 8, compare A and C; and Table I). However, the conversion of core 1 structure to extended core 1 is less efficient than the conversion of core 1 structure to core 2 branched structure (Fig. 7, B versus F, compare peaks I and II; and Table I). These results as a whole indicate that sialyl Lewis x in core 2 branched O-glycans is expressed at levels equivalent to sialyl Lewis x in extended core 1 O-glycans (Table I).
Sialyl Lewis x in Extended Core 1 Functions as an L-selectin
Ligand--
To determine the role of sialyl Lewis x in extended core 1 structure, tethering and rolling of neutrophils and lymphocytes were
assayed in CHO cells expressing PSGL-1 and sialyl Lewis x in extended
core 1 O-glycans or core 2 branched O-glycans.
Because CHO cells lack endogenous 3GlcNAcT-3 (18) or Core2GlcNAcT
(4, 39), CHO cells expressing only PSGL-1 and FucT-VII lack sialyl Lewis x in mucin-type O-glycans. As shown in Fig.
9A, neutrophil tethering and
rolling were not detected in this CHO cell line lacking both
3GlcNAcT-3 and Core2GlcNAcT-I (open circles). By contrast, CHO cells expressing sialyl Lewis x in extended core 1 (closed circles) or core 2 (open triangles)
oligosaccharides supported neutrophil tethering and rolling under
shear. Essentially identical results were obtained when lymphocyte
tethering and rolling were assayed in the same transfected CHO cells
(Fig. 9B). These combined results indicate that non-sulfated
sialyl Lewis x in extended core 1 or core 2 oligosaccharides functions
as an L-selectin ligand, although sialyl Lewis x in core 2 branched O-glycans functions as a more efficient L-selectin ligand
than does sialyl Lewis x in extended core 1 O-glycans (Fig.
9, compare open triangles and closed
circles).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study, we have demonstrated that 3GlcNAcT-3, formerly
called core 1
3GlcNAcT, is almost exclusively responsible for adding
1,3-linked GlcNAc to Gal
1
3GalNAc
1
R, forming extended core 1 oligosaccharide. Among other
3GlcNAcTs,
3GlcNAcT-2 may add
1,3-linked GlcNAc to core 1 O-glycans with low
efficiency, whereas
3GlcNAcT-6 apparently forms small amounts
of extended core 1 structure.
3GlcNAcT-2 is the most efficient
enzyme to add N-acetyllactosamine repeats
(22),2 whereas
3GlcNAcT-6
acts on GalNAc
1
Ser/Thr (27). These results suggest that
3GlcNAcT-2 and
3GlcNAcT-6 may act as core 1 extension enzymes
with very low efficiency under certain conditions. Indeed, the amino
acid sequence of
3GlcNAcT-3 is highly related to that of
3GlcNAcT-6 (51.6 8% identify), which acts on GalNAc
1
R.
This study unexpectedly demonstrated that extended core 1 is most
likely present in neutrophils and lymphocytes, although the amount of
extended core 1 structure is less than that of core 2 branched
structure. Interestingly, LSST, which is required to form
6-sulfo-GlcNAc structure, is apparently not expressed in neutrophils
and lymphocytes. This finding is consistent with the fact that
neutrophils and lymphocytes are negative for MECA-79 antigen.
3GlcNAcT-3 belongs to the
3GlcNAcT gene family, which consists of
at least eight different
3GlcNAcTs. The members of this gene family
include
3GlcNAcTs encoded by fringe and
brainiac (44-47). Fringe was identified as a protein that
regulates Notch signaling by adding
1,3-linked GlcNAc to
-fucose
attached to the extracellular domain of Notch (44, 45). By contrast,
Brainiac apparently acts on glycolipids and thereby modulates Notch
activity by another mechanism (46, 47). Although
3GlcNAcT-1 does not have discernible homology to the
3GlcNAcT gene family, recent studies show that one protein predicted by DNA sequence within the
human or mouse Large locus has some homology to
3GlcNAcT-1 (48). Premature translation termination of this putative
glycosyltransferase within the Large locus in mice results
in myodystrophy (48), whereas the LARGE locus is deleted in
human patients with meningioma, a tumor of the meninges of the central
nervous system (49). These result suggest that
3GlcNAcT-1 may play
an important role in development and cancer because
3GlcNAcT-1 is
ubiquitously expressed (21).
This study demonstrated that, in transfected CHO cells, fucosylation of
N-acetyllactosamine in extended core 1 structure takes place
more efficiently than does fucosylation in core 2 branched O-glycans. This finding is consistent with the previous
finding that a significant portion of extended core 1 structure is
fucosylated in HEV (18). Our results show that extended core 1 structure is efficiently fucosylated once core 1 structure is formed.
On the other hand, the amount of extended core 1 O-glycans
is less than that of core 2 branched O-glycans (Table I). As
an aggregate, extended core 1 and core 2 branched O-glycans
contain similar amounts of sialyl Lewis x. Synthesis of both extended
core 1 and core 2 branches competes with 2,3-sialylation of core 1, which is catalyzed by
-galactoside
2,3-sialyltransferase I (50). It is thus possible that both the levels of expressed
3GlcNAcT-3 and
its catalytic activity are not as great as those of expressed Core2GlcNAcT-I in transfected CHO cells.
It is also possible that localization of 3GlcNAcT-3, Core2GlcNAcT-I,
and
-galactoside
2,3-sialyltransferase I in different Golgi
compartments is a key factor in determining the amount of oligosaccharides synthesized by
3GlcNAcT-3 or Core2GlcNAcT-I (51,
52). Previously, we have shown that Core2GlcNAcT-I resides in the
cis- to medial-Golgi, whereas the majority of
N- and O-glycan sialyltransferases are thought to
reside in the medial- to trans-Golgi (51). This
difference allows Core2GlcNAcT-I to add core 2 branch before core 1 oligosaccharide is sialylated. If core 1 oligosaccharide is sialylated
first, it becomes unavailable for Core2GlcNAcT-I action (see Fig. 1 in
Ref. 2). It is tempting to speculate that
3GlcNAcT-3 resides in
later compartments of the Golgi than does Core2GlcNAcT-I, thus directly
competing with
-galactoside
2,3-sialyltransferase I for the same
acceptor, Gal
1
3GalNAc
Ser/Thr. Such direct competition,
if it occurs, should lead to moderate synthesis of extended core 1 structure.
Previously, we showed that 6-sulfosialyl Lewis x in extended core 1 O-glycans serves as an L-selectin ligand as efficiently as
6-sulfosialyl Lewis x in core 2 branched O-glycans (18). In
this study, we extended this finding by showing that sialyl Lewis x in
extended core 1 O-glycans also functions as an L-selectin ligand, although it is not as efficient as sialyl Lewis x in core 2 branched O-glycans. Our results are also consistent with
previous reports showing that sialyl Lewis x functions as an L-selectin ligand, although extended core 1 structure was not evaluated in that
study (53). In our previous study, we found that the sialyl Lewis x
structure is present in extended core 1 O-glycans of
HEV-derived GlyCAM-1, which were converted to neutral oligosaccharides
after desialylation (18). These results combined indicate that sialyl Lewis x in extended core 1 serves as an L-selectin ligand in HEV. L-selectin-mediated neutrophil rolling was shown to take place in
adherent neutrophils bound to activated endothelial cells (10, 11).
Very recently, we obtained mice heterozygous for 3GlcNAcT-3 deficiency and knock-in of green fluorescent protein under the control
of the
3GlcNAcT-3 promoter. Analysis of these mice indicated that
3GlcNAcT-3 is expressed in neutrophils and T lymphocytes because
neutrophils and T lymphocytes stained with markers CD11b (Mac-1) and
CD3, respectively, were also positive for green fluorescent protein as
determined by FACS analysis.2 These studies did not,
however, inform at to how much sialyl Lewis x in extended core 1 structure is present in neutrophils. Further studies on the knockout
mice are important to determine the degree to which sialyl Lewis x
moieties in extended core 1 structure contribute to L-selectin-mediated
adhesion in HEV and neutrophil-neutrophil interaction.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Richard Cummings for pZeoSV-PSGL-1, Dr. Shigeru Tsuboi and Misa Suzuki for helpful discussion, Dr. Elise Lammer for critical reading of the manuscript, and Tracy Keeton and Thu Gruenberg for organizing the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by NCI Grant R01CA428737 and in part by NCI Grant P01CA71932 from the National Institutes of Health.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.
¶ Present address: Pathology Div., National Cancer Center Research Inst., 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.
Present address: La Jolla Bioengineering Inst., 505 Coast
Blvd. South, La Jolla, CA 92037.
** Investigator of the Howard Hughes Medical Institute.
To whom correspondence should be addressed: The Burnham Inst.,
10901 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-646-3144; Fax:
858-646-3193; E-mail: minoru@burnham.org.
Published, JBC Papers in Press, January 15, 2003, DOI 10.1074/jbc.M212756200
2 J. Mitoma and M. Fukuda, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
Core2GlcNAcT-I, core
2 1,6-N-acetylglucosaminyltransferase I;
HEV, high
endothelial venule(s);
LSST, L-selectin ligand sulfotransferase;
FucT-VII, fucosyltransferase VII;
3GlcNAcT,
1,3-N-acetylglucosaminyltransferase;
CHO, Chinese hamster
ovary;
RT, reverse transcription;
FITC, fluorescein isothiocyanate;
FACS, fluorescence-activated cell sorting;
HPLC, high
performance liquid chromatography.
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