 |
INTRODUCTION |
There are three CD1
markers of human leukocytes comprising fucosylated carbohydrate
epitopes. As listed in Fig. 1 below, the distal lactosamine unit (LN;
type 2 chain), Gal
1,4GlcNAc, of the polylactosamine chain is
fucosylated through
1,3-fucosyltransferase (
1,3FUT) activity to
form the CD15 (Lewis x; LeX) epitope (1, 2). The CD15s
(sialylated CD15; sialyl LeX (sLeX)) and CDw65
(VIM-2) epitopes are also fucosylated structures related to CD15,
i.e. CD15s is formed by
2,3-sialylation prior to the
fucosylation of the distal LN unit of polylactosamine by
1,3FUT, and
CDw65 is formed by fucosylation of the inner LN unit of
2,3-sialylated polylactosamine by
1,3FUT (2, 3).
The CD15 epitope is expressed in some tissues, such as epithelial cells
of intestinal tissues (4-6), certain neurons and glial cells in the
central nervous system (7, 8). In human leukocytes, CD15 is expressed
preferentially in monocytes, mature neutrophils, and all myeloid cells
from the promyelocyte stage onwards, making it a useful cell surface
marker (9-11). CD15 is considered to be involved in neutrophil
functions, that is, cell-cell interactions, phagocytosis, stimulation
of degranulation, and respiratory burst, although the function of CD15
is not clear (12-16).
Six human
1,3FUT genes have been cloned to
date, which are FUT3 (Fuc-TIII), FUT4
(Fuc-TIV), FUT5 (Fuc-TV),
FUT6 (Fuc-TVI), FUT7
(Fuc-TVII), and FUT9 (Fuc-TIX) (1,
17-23). FUT9, a new member of the human
1,3FUT family, which we
have recently cloned, is expressed in human leukocytes, glandular
compartments of the stomach, and forebrain (23). The FUT9
gene was mapped on chromosome 6q16 (24). Interestingly, only FUT9 has a
highly conserved amino acid sequence between human and mouse, the level
of conservation being equal to that of
-actin (23). Five human
1,3FUTs (FUT3, 4, 5, 6, and 7) share highly homologous sequences,
whereas FUT9 has a different sequence altogether (23). This indicated
that the substrate specificity of FUT9 is unique among the
1,3FUTs. In fact, we demonstrated in a previous study that FUT9 preferentially transfers a fucose to the GlcNAc residue at the distal LN unit of the
polylactosamine chain, resulting in the LeX (CD15)
structure, whereas the other
1,3FUTs preferentially transfer a
fucose to the GlcNAc residue at the inner LN unit of the
polylactosamine chain (25). This implied that FUT9 exhibits stronger
activity than the other
1,3FUTs for forming the CD15 epitope that is
recognized by anti-LeX (anti-CD15) antibodies.
It has been reported that FUT4 and FUT7 are expressed in human
leukocytes, but FUT3, 5 and 6 are not (23, 26, 27). The carbohydrates
modified by both FUT4 and FUT7 can function as ligands for E-selectin
and P-selectin (28, 29). FUT4, named a myeloid-type
1,3FUT, is
ubiquitously expressed in a variety of tissues and not only in
leukocytes (23). In a previous study (23), we could not find any human
tissues in which FUT4 is absent. However, a number of the tissues do
not necessarily express the CD15 antigen. Overexpression of the
FUT4 gene by its transfection resulted in the expression of
CD15 on the cell surface (1, 22, 23, 30, 31), whereas FUT7 transfection
resulted in the expression of CD15s but not CD15 (20, 21, 31).
Therefore, FUT4 had been believed to be the enzyme solely responsible
for the expression of CD15 in leukocytes before the finding of FUT9 in
those cells. It has been investigated whether the expression of FUT4
and FUT7 correlated with the expression of CD15 and CD15s epitopes
during hematopoiesis. Clarke et al. (26) reported that FUT7
definitely determined the expression of the CD15s epitope, consistent
with other studies (32-34), however, the expression of CD15 does not correlate well with the expression of FUT4, which indicated that an
unknown
1,3FUT is involved in the expression of CD15 in leukocytes. Marer et al. (27) also reported a discrepancy between the
expression of FUT4 and that of CD15, in which the level of FUT4
expression does not necessarily correlate with the level of CD15
expression during myeloid cell maturation.
Previously (23), we cloned a human cDNA encoding FUT9, and examined
the tissue distribution of the six
1,3FUTs. We found that FUT9 is
expressed in peripheral blood leukocytes. This indicated that FUT9
might be the enzyme responsible for determining the expression of CD15
in leukocytes. In this study, we examined which
1,3FUT is
responsible for the CD15 expression, in conjunction with the expression
of CDw65, in human leukocytes and demonstrated that the expression of
CD15 is determined differentially either by FUT9 or FUT4, depending on
the subpopulation of leukocytes.
 |
EXPERIMENTAL PROCEDURES |
Antibodies--
The monoclonal antibodies (mAbs) anti-human CD4
(PRA-T4), CD8 (PRA-T8), CD14 (clone M5E2), CD19 (HIB19), and CD56
(B-159) were obtained from Becton Dickinson Immunocytometry Systems
(San Jose, CA). The mAbs anti-human CD34 (BIRMA-K3), CD15 (80H5), and CDw65 (VIM-2) were obtained from DAKO, Beckman Coulter, and Serotec, respectively.
Cell Preparation--
Peripheral blood mononuclear cells (PBMCs)
and polymorphonuclear cells obtained from normal donors were isolated
from heparinized venous blood by density-gradient sedimentation over
Lymphoprep and Polymorphoprep (Nycomed Pharma, AS, Oslo, Norway),
respectively. Cord blood samples obtained with permission from the
umbilical vein at normal delivery were aspirated in heparinized plastic syringes. Mononuclear cells were also separated by density
centrifugation over Lymphoprep (Nycomed Pharma, AS). The cells
magnetically labeled with CD4, CD8, CD19, or CD34 Dynabeads (Dynal,
Oslo, Norway) were isolated from mononuclear cell preparations
and detached by DETACHaBEAD CD4/CD8, CD19, or CD34 in accordance
with the manufacturer's protocol. The cells magnetically labeled with
CD14, CD15, or CD56 MicroBeads (Miltenyi Biotec, Auburn, CA) were
selectively bound to an MS+ column using the MiniMACS and
eluted from the column in accordance with the manufacturer's instructions.
Cell Culture and Transfection--
Human leukemic cell lines,
HL-60, U937, Jurkat and Namalwa, were cultured in RPMI 1640 (Life
Technologies) supplemented with 10% heat-inactivated fetal calf serum,
2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml
streptomycin. Jurkat, U937, and Namalwa cells were transfected with
pAMo vectors containing FUT9 coding sequences by electroporation using
a Gene-Pulser (Bio-Rad) and selected in the presence of G418 (1 mg/ml)
(Life Technologies) for 2-4 weeks to obtain stable transfectants.
Limited dilution cloning was performed to stabilize and enhance the
prevalence of transfectants with the desired phenotype that was
examined by flow cytometric analysis using anti-CD15 antibodies, as
described previously (22). Namalwa cells were also transfected with
pAMo vectors containing each of the FUT4 and the FUT7 coding sequence and cloned by limited dilution. We selected one clone from each group,
which expressed transcripts at almost an equal level, and used it for
the following experiments (see Fig. 2D).
Purification of FUT Proteins Fused with FLAG Peptide--
The
putative catalytic domain of each of FUT4, FUT7, and FUT9 was expressed
as a secreted protein fused with FLAG peptide in Namalwa cells. pAMoF2
is an expression vector derived from pAMo and contains a fragment
encoding signal peptide of human immunoglobulin
(MHFQVQIFLLISASVIMSRG) and FLAG peptide (DYKDDDDK). Namalwa cells were
transfected with pAMoF2 vector containing each FUT coding sequence by
electroporation and selected with G418 as described above. The
recombinant enzyme secreted in culture medium was purified by anti-FLAG
(M1) antibody resin (Sigma Chemical Co.) in accordance with the
manufacturer's protocol.
Competitive RT-PCR Assay--
Competitive RT-PCR assay was
performed to determine the amount of transcripts of FUT4, FUT7, FUT9,
and
-actin in lymphocytes as previously described (22, 23). In
brief, total RNAs were isolated from the cells using ISOGEN (Nippon
Gene, Tokyo, Japan) as recommended by the manufacturer. cDNAs were
synthesized with oligo(dT) primers from RNAs in a total volume of 20 µl of reaction mixture using SuperScript II reverse transcriptase in
accordance with the superscript preamplification system protocol (Life
Technologies). Competitive RT-PCR was performed using Ampli
Taq Gold with GeneAmp (PerkinElmer Life Sciences) in a total
volume of 50 µl of reaction buffer containing 10 µl of standard
plasmid DNA or sample cDNA, 10 µl of competitor DNA at optimal
concentration, and 0.2 µM of each primer of the
gene-specific primer sets (Table I).
After competitive RT-PCR, a 10-µl aliquot was electrophoresed in 1% agarose gel, and the bands were visualized by ethidium bromide staining. The intensities of the amplified fragments were quantified by
scanning positive pictures. Measurement of the
-actin transcripts in
each sample was performed using the same competitive RT-PCR method. The
values for the transcripts were plotted on the respective standard
curves to obtain the actual amount of each transcript. The actual
amount of transcript of each glycosyltransferase (fg/µl) was divided
by that of
-actin (pg/µl) for normalization.
Western Blot Assay--
We also determined the amount of CD15
antigen in each subpopulation of peripheral blood cells from healthy
individuals by Western blot analysis, as described previously (35).
Briefly, cell pellets were solubilized in a 20 mM HEPES
buffer (pH 7.2) containing 2% Triton X-100 by brief sonication.
Proteins (20 µg) separated by 7.5% SDS-polyacrylamide gel
electrophoresis were transferred to an Immobilon polyvinylidene
difluoride membrane (Millipore, Bedford, MA) using a Transblot SD cell
(Bio-Rad, Richmond, CA). The membrane was blocked with
phosphate-buffered saline containing 5% skim milk at 4 °C overnight
and then incubated with anti-CD15 antibody. The membrane was stained
with the ECL Western blot detection reagents (Amersham Pharmacia
Biotech) as recommended by the manufacturer.
Assay of the Activity of
1,3FUTs toward Neutral or Sialyl
Polylactosamines--
We analyzed the substrate specificity of each
FUT protein fused with FLAG peptide and each cell lysate for the
2-aminobenzamide (2AB)-labeled polylactosamine acceptors,
Gal
1-4GlcNAc
1-3Gal
1-4GlcNAc
1-3Gal
1-4GlcNAc-2AB (3LN-2AB) and 2AB-labeled
2,3-sialylated polylactosamine acceptors, NeuAc
2-3Gal
1-4GlcNAc
1-3Gal
1-4GlcNAc
1-3Gal
1-4GlcNAc-2AB
(S3LN-2AB), as previously described (25). Briefly, S3LN-2AB was
obtained from 3LN-2AB by
2,3-sialylation using recombinant
2,3-sialyltransferase IV. Cell pellets were sonicated in 20 mM HEPES buffer (pH 7.2) containing 2% Triton X-100. Each
cell lysate, containing 60 µg of protein, was incubated in 50 mM cacodylate buffer (pH 6.8), 5 mM ATP, 10 mM L-fucose, 75 mM guanosine
diphosphate fucose, 25 mM MnCl2, and 15 mM acceptor substrate at 37 °C for 2 h, and the
enzyme reactions were subjected to reverse-phase high performance liquid chromatography (HPLC) on a TSKgel ODS-80TS QA column (4.6 × 250 mm; Tosoh, Tokyo, Japan) and eluted with a 20 mM
ammonium acetate buffer (pH 4.0) containing 7% methanol at a flow rate of 1.0 ml/min at 50 °C with monitoring by fluorescence
spectrophotometer (JASCO FP-920; Nihon Bunkoh, Tokyo, Japan). We
measured the amount of each FUT-FLAG by Western blot analysis with
anti-FLAG (M2) and used the same amount of purified FUT-FLAG proteins
for the enzyme activity assays (see Fig. 3C). The peaks were
assigned to
Gal
1-4(Fuc
1-3)GlcNAc
1-3Gal
1-4GlcNAc
1-3Gal
1-4GlcNAc-2AB (P1),
Gal
1-4GlcNAc
1-3Gal
1-4(Fuc
1-3)GlcNAc
1-3Gal
1-4GlcNAc-2AB (P2),
Gal
1-4(Fuc
1-3)GlcNAc
1-3Gal
1-4(Fuc
1-3)GlcNAc
1-3Gal
1-4GlcNAc-2AB (P3),
NeuAc
2-3Gal
1-4(Fuc
1-3)GlcNAc
1-3Gal
1-4GlcNAc
1-3Gal
1-4GlcNAc-2AB (P1s), and
NeuAc
2-3Gal
1-4GlcNAc
1-3Gal
1-4(Fuc
1-3)GlcNAc
1-3Gal
1-4GlcNAc-2AB (P2s). To ascertain the above assignment, a sequential digestion with
neuraminidase,
-galactosidase,
-N-acetylhexosaminidase, and
-fucosidase was
performed, as described previously (25). The area of the P1, P1s, and
P2s peak indicated synthesizing activity for CD15, CD15s, and CDw65
(VIM-2), respectively (Fig. 1).
 |
RESULTS |
FUT9 Exhibits 20-fold Stronger Activity for CD15 Synthesis than
FUT4, whereas FUT4 Exhibits 5-fold Stronger Activity for CDw65
Synthesis than FUT9--
Namalwa cells transfected with each of three
1,3FUTs, FUT4, FUT7, and FUT9, were subjected to flow cytometric
analysis and measurement of CD15 synthesizing activity. The amount of
transcript expressed in each transfectant was found to be almost the
same (Fig. 2D). Namalwa-FUT9
expressed CD15 more strongly than Namalwa-FUT4, and also expressed
CDw65 slightly. In contrast, Namalwa-FUT4 expressed CDw65 more strongly
than CD15 (Fig. 2A). Namalwa-FUT7 did not express CD15 as
described previously (2). As reported (25), we obtained three peaks,
P1, P2, and P3, for
1,3FUT activity toward 3LN-2AB acceptor
substrates (Fig. 2B). The area of P1, P2, and P3 indicates
the synthesizing activity of each product. The carbohydrate structure
of the P1 peak corresponds to CD15, as described under "Experimental
Procedures." As seen in Fig. 2B, the cell homogenate of
Namalwa-FUT9 exhibited about a 20-fold larger P1 peak than that of
Namalwa-FUT4. FUT9 was found to have about 20 times stronger activity
for CD15 synthesis than FUT4. Both enzymes exhibit almost the same
level of activity for the fucosylation of the inner LN unit of 3LN-2AB
(P2). Namalwa-FUT4 showed a very small P3 peak, indicating the
bifucosylation of distal and inner LN units, which is also recognized
by anti-CD15 antibody. Namalwa-FUT7 showed no activity for the
fucosylation of neutral oligosaccharide, 3LN-2AB.

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Fig. 2.
A, flow cytometric analysis of
Namalwa-FUT9, Namalwa-FUT4, and Namalwa-FUT7 cells with anti-CD15 and
anti-CDw65 mAb. B, profiles of 1,3FUT activity toward
3LN-2AB of each of the Namalwa transfectants. C, profiles of
1,3FUT activity toward S3LN-2AB of each of the Namalwa
transfectants. D, relative amounts of transcripts of FUT4,
FUT7, and FUT9. The transcripts in each Namalwa transfectant were
measured by competitive RT-PCR. The actual amount of each
glycosyltransferase (fg/µl) was divided by that of -actin
(pg/µl) for normalization.
|
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FUT4 and FUT9 also exhibited positive activity for synthesizing the
CDw65 (VIM-2) structure by a fucose transfer to the internal LN unit of
a
2,3-sialylated polylactosamine, S3LN-2AB (P2s in Fig.
2C). The cell homogenate of Namalwa-FUT4 exhibited about a
4.5-fold larger area of peak than that of Namalwa-FUT9, demonstrating that FUT4 had about 4.5 times stronger activity for the CDw65 synthesis
than FUT9. The cell homogenate of Namalwa-FUT7 exhibited only a small,
but apparently positive peak (P1s) that is different from the P2s peak
(Fig. 2C). The P1s peak area indicated synthesizing activity
for CD15s, as described under "Experimental Procedures."
Then we analyzed the relative activities of each FUT for 3LN-2AB and
S3LN-2AB using the same amount of each FUT-FLAG protein (Fig.
3C). The 20 times stronger
activity for the CD15 synthesis of FUT9-FLAG than that of FUT4-FLAG was
again confirmed (P1 peaks in Fig. 3A). Both FUT4-FLAG and
FUT9-FLAG again showed positive activity for the CDw65 synthesis.
FUT4-FLAG exhibited about a 2.5-stronger activity for the CDw65
synthesis than FUT9-FLAG (P2s in Fig. 3B). FUT7-FLAG
exhibited positive activity for synthesizing the CD15s structure (P1s
in Fig. 3B) and also the CDw65 structure (P2s in Fig.
3B). FUT7-FLAG exhibited about a 20-fold larger area of P1s
than P2s.

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Fig. 3.
A, profiles of 1,3FUT activity toward
3LN-2AB of each FUT-FLAG. B, profiles of 1,3FUT activity
toward S3LN-2AB of each FUT-FLAG. C, the protein amounts of
FUT-FLAG were determined by Western blot analysis against FLAG
tag.
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Differential Expression of FUT9 in CD15+ Cells in
Peripheral and Cord Blood Cells--
We evaluated the transcript
levels of three
1,3FUTs, FUT4, FUT7, and FUT9, in CD15+
cells in peripheral and cord blood cells. CD14+ monocytes
and mature granulocytes in peripheral blood cells showed dull staining
and bright staining with anti-CD15 mAb, respectively (Fig.
4A). Interestingly, monocytes
did not express FUT9 at all, whereas granulocytes expressed substantial
amounts of FUT9 (Fig. 4B). The difference of the CD15
staining intensity between monocytes and granulocytes could be
explained by the strong activity of FUT9 for the CD15 synthesis. Both
cells were brightly stained with CDw65, probably directed by FUT4
activity (Fig. 4A). Two populations, CD15+ and
CD34+, were sorted from cord blood mononuclear cells.
Neither expressed detectable amounts of FUT9 (Fig.
5A). Thus, the expression of CD15 in immature promyelocytes in cord blood is not directed by FUT9,
and probably directed by FUT4.

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Fig. 4.
Flow cytometric analysis of CD15 expression
versus relative amounts of
1,3FUT transcripts and profiles of
1,3FUT activity in a series of CD15+
cells. A, the cells were analyzed by flow cytometry
with anti-CD15 and anti-CDw65 mAb. B, relative amounts of
transcripts of FUT4, FUT7, and FUT9 were measured by competitive
RT-PCR. The actual amount of each glycosyltransferase (fg/µl)
was divided by that of -actin (pg/µl) for normalization.
C, profiles of 1,3FUT activity toward 3LN-2AB of HL-60,
Jurkat, and Jurkat-FUT9 cells.
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Fig. 5.
A, relative amounts of transcripts of
FUT4, FUT7, and FUT9. The transcripts in each subpopulation of
peripheral blood and cord blood mononuclear cells, obtained from normal
subjects (n = 20), were measured by competitive RT-PCR.
The actual amount of each glycosyltransferase (fg/µl) was divided by
that of -actin (pg/µl) for normalization. Values are the mean ± S.D. B, the expression levels of the CD15 antigen in
respective subpopulations of peripheral blood cells were determined by
Western blot analysis.
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|
As seen in Fig. 4A, HL-60 was brightly stained with
anti-CD15 mAb. U937 and Jurkat cells were stained to a lesser degree
with anti-CD15 mAb. FUT9 could not be detected in HL-60, U937, or
Jurkat cells, at all, whereas FUT4 and FUT7 were clearly detected (Fig. 4B). HL-60 cells expressed enormous amounts of FUT4
transcripts (18.6). The CD15 intensity in these cell lines
correlated with the amount of FUT4 transcripts. All these cell lines
expressed substantial amounts of the CDw65 epitope. This indicated that the expression of CD15 and CDw65 in these cell lines is directed by FUT4.
The Expression of CD15 Epitope on Cell Surfaces Induced by FUT9 in
Myeloid and Lymphoid Cell Lines--
Stable FUT9 transfectants were
established in Jurkat and U937 cells, and named Jurkat-FUT9 and
U937-FUT9, respectively. Both transfectants showed increases in the
expression of CD15 on flow cytometric analysis, but different results
in the expression of CDw65 (Fig. 4A). U937-FUT9 exhibited an
increase in CDw65 expression in correlation with the increase of CD15,
but Jurkat-FUT9 showed a prominent decrease in the expression of CDw65.
The transfection of the FUT9 gene to U937 and Jurkat cells
demonstrated that FUT9 could potentially express CD15 epitope on their
surfaces in myeloid and lymphoid cells.
HL-60 Exhibited a Pattern of
1,3FUT Activity Directed by FUT4,
whereas Jurkat-FUT9 Cells Exhibited a FUT9-specific Pattern--
Upon
measurement of
1,3FUT activity in the cell homogenates using 3LN-AB
as an acceptor, HL-60 exhibited a typical pattern of
1,3FUT activity
directed by FUT4. As seen in Fig. 4, the enormous amounts of FUT4
transcript (18.6) in HL-60 cells gave a very large P2 peak, leading to
the bright expression of CDw65, and a relatively small, but substantial
P1 peak that is enough for the bright expression of CD15. Jurkat cells
also exhibited a FUT4-specific pattern of
1,3FUT activity, in which
the P1 and P2 peak areas were well correlated with the amount of FUT4
transcripts (Fig. 4). The relatively low amount of FUT4 in Jurkat
cells, in comparison to that in HL-60 cells, would explain the very
weak expression of CD15 and CDw65 in Jurkat cells. The profile of
1,3FUT activity in Jurkat-FUT9 cells was almost converted to the
FUT9-specific pattern. The large P1 peak in Jurkat-FUT9 cells gave rise
to the bright expression of CD15, and interestingly, decreased the
expression of CDw65 (Fig. 4).
Differential Expression of FUT9 in Each Subpopulation of
PBMC--
Each subpopulation of peripheral blood mononuclear cells
(PBMC) was isolated from peripheral blood by immunomagnetic labeling. Flow cytometric analysis revealed that monocytes (CD14+)
showed dull staining with anti-CD15 mAb, and CD56+ cells
slightly expressed CD15, but CD4+ T cells, CD8+
T cells, and B cells (CD19+) did not express it (data not
shown). We again found the ubiquitous expression of FUT4 and FUT7 in
all subpopulations of PBMC, whereas FUT9 was expressed in
subpopulations of PBMC except monocytes (Fig. 5A). The
average levels of relative transcripts of FUT4 and FUT7 were not so
different from each other. FUT4 transcript was in the range of 0.5 to
1.7, and FUT7 transcript was in the range of 0.8 to 2.5. CD4+ and CD8+ T cells showed the maximal and
minimal amounts of FUT7 transcripts, respectively, among the
subpopulations examined. B cells (CD19+), monocytes
(CD14+), and natural killer cells (CD56+)
expressed FUT7 at moderate levels. In contrast to the FUT4 and FUT7
expression, the average level of transcript of FUT9 in each subpopulation was different and ranged 0.02 to 0.3 (Fig.
5A). Monocytes did not express FUT9, although they expressed
CD15 on their surface. On the other hand, CD8+ T cells, B
cells, and CD56+ cells, which showed negative or faint
staining with CD15 by flow cytometry, expressed FUT9 in an amount
similar to that of FUT4 and FUT7 in each subpopulation (Fig.
5A).
CD15 Antigen Is Present Intracellularly in All Subpopulations of
Peripheral Blood Cells--
We also determined the expression of the
CD15 antigen in subpopulations of PBMC by Western blot analysis (Fig.
5B). An apparent molecular mass of about 200 kDa was
detected by anti-CD15 mAb in all subpopulations, including
CD4+ T cells, CD8+ T cells, B cells
(CD19+), monocytes (CD14+), NK cells
(CD56+), and mature granulocytes. Although CD4+
T cells and B cells (CD19+) showed two additional bands,
they were not CD15+ glycoprotein, but mouse IgM derived
from magnetic beads. Thus, the subpopulations, which do not express
CD15 antigen on the cell surface, apparently possess CD15 antigen intracellularly.
 |
DISCUSSION |
The CD15 antigen is expressed on mature granulocytes and on
myeloid cells from the promyelocyte stage onwards. Anti-CD15 antibody reacts with promyelocytes, and less strongly with myelocytes and metamyelocytes, but not bone marrow myeloblasts, as detected by flow
cytometric analysis and an immunohistochemical method (10, 11, 36).
CD15 epitopes are carried as terminal sequences on the oligosaccharide
chains in both glycoproteins and glycolipids on mature granulocytes
(37). Some anti-CD15 antibodies also have a high immunoreactivity for
normal peripheral blood monocytes (10), but the majority of T and B
lymphocytes do not express CD15 antigens (36).
The preferential activity of FUT9 to transfer fucose to the distal
GlcNAc residue suggested that FUT9 synthesizes the CD15 epitope more
efficiently in vivo than FUT4. The relative initial rate of
transfer to the distal LN unit of neutral polylactosamine, 3LN-2AB, was
~20 times higher for FUT9 than FUT4. On the other hand, FUT4
preferentially fucosylates the inner LN unit of polylactosamine on both
the neutral and
2,3-sialylated polylactosamine chains, so that FUT4
can efficiently synthesize CDw65 epitope (2, 25). In the present study,
we demonstrated that FUT9 can also transfer a fucose to the inner
LN unit of the sialylated polylactosamine chain with a relative
activity one fifth that of FUT4, resulting in the synthesis of CDw65
epitope (Fig. 2). In fact, the FUT9-transfected cells, Namalwa-FUT9 and
U937-FUT9 cells, showed increased CDw65 expression. This confirmed the
FUT9 activity to transfer fucose to the inner LN unit of the sialylated
polylactosamine chain.
Human mature granulocytes strongly express CD15 and CDw65 on their
surface (10, 11, 38). Lactosaminoglycan on glycoprotein is a major
carrier for the CD15 epitope in human granulocytes, because the CD15
epitope is three times more abundant in glycoproteins than in
glycolipids (37, 39). A typical feature of granulocyte lactosaminoglycan was a multiple fucosylated structure on LN units. Also, some of the side chains contain two or more fucose residues. However, the majority of fucose-containing neutral oligosaccharides possess a Gal
1-4(Fuc
1-3)GlcNAc terminal structure (37). This is
consistent with the finding in the present study that mature granulocytes express substantial amounts of FUT9, which preferentially fucosylates the GlcNAc residue at the distal LN unit (25). The repertoire of
1,3FUTs expressed in human mature granulocytes is
distinct from that in promyelocytes, that is, a substantial amount of
FUT9 transcript was detected in mature granulocytes, but not in
promyelocytes. FUT4 can also be detected in mature granulocytes and
promyelocytes, although its activity for the synthesis of CD15 is known
to be minor in contrast to that for the synthesis of CDw65.
The absence of FUT9 in the CD15+ and CD34+
cells indicated that FUT4 is mainly responsible for the CD15 expression
in the immature promyelocytes in cord blood cells. The human
promyelocytic leukemic cell line, HL-60, which is composed of cells
arrested at the promyelocytic stage, showed strong staining with both
anti-CD15 and CDw65 antibodies, as described by others (26). The
enormous amount of FUT4 expressed in HL-60 cells was able to give rise
to bright CD15 and CDw65 staining of HL-60 even in the absence of FUT9.
HL-60 cells are able to differentiate into myeloid mature cells and
monocytes when cultured in the presence of dimethyl sulfoxide
(Me2SO) and all-trans-retinoic acid (RA),
respectively (40, 41). However, we could not detect FUT9
transcripts in Me2SO-differentiated and RA-differentiated
HL-60 cells (data not shown). FUT9 was not involved in the CD15
expression even in the differentiated HL-60 cells. It is difficult to
exclude the presence of unidentified
1,3FUT in HL-60 cells and
monocytes. However, each profile of the peaks (P1, P2, and P3) of HPLC
in Fig. 2 is very characteristic of each
1,3FUT, enabling one to
demonstrate that the
1,3FUT activity detected in the HL-60 cell
homogenates corresponded to a typical pattern of FUT4 specificity. The
peak areas were large enough for the CD15 and CDw65 expression,
respectively, on the cell surface. These findings are consistent with
the interpretation that FUT4 is responsible for the CD15 expression in
the myeloid cells at the promyelocyte stage (26).
U937 and Jurkat cells expressed less FUT4 (9.5 and 7.0, respectively)
than HL-60 cells and no detectable FUT9 leading to the weaker
expression of CD15 and CDw65 on the cell surface. The transfection of
the FUT9 gene to U937 cells demonstrated that FUT9 can
potentially synthesize the CD15 epitope in myeloid cells. In U937-FUT9
cells, the intensity of CDw65 staining increased in conjunction with the increase of CD15 reactivity. The transcript levels for
FUT4 and FUT9 genes expressed in Jurkat-FUT9
cells were found to be almost the same as those in mature granulocytes
isolated from peripheral blood cells. The profile of
1,3FUT activity
in Jurkat cells was typical of that of FUT4, and the profile in
Jurkat-FUT9 cells converted to that of FUT4 plus FUT9. The P1 peak
responsible for the CD15 synthesis was demonstrated to be directed by
FUT9 in the Jurkat-FUT9 cells, not by FUT4. This fact strongly
suggested that the CD15 expression in mature granulocytes is directed
by FUT9, not by FUT4.
The CDw65 expression in the Jurkat-FUT9 cells contrasted with that in
the U937-FUT9 cells. The CDw65 intensity in the Jurkat-FUT9 cells
decreased in comparison to that in the wild-type Jurkat cells, even
though the FUT9 activity was increased. One explanation for this
discrepancy may be that U937 cells possess enough of the precursor
structure, i.e. the sialylated polylactosamine chains, to be
fucosylated at the inner LN unit resulting in the CDw65 expression.
Thus, FUT4 and FUT9 in the U937-FUT9 cells additively fucosylated the
sialylated polylactosamine chains for the CDw65 expression. On the
other hand, Jurkat cells may not possess enough of the precursor
structure, because of weak activity of sialyltransferase(s), which is
supplied for both FUT4 and FUT9 activity. In such a case, FUT9 may
preferentially fucosylate the distal LN unit by overwhelming the
sialyltransferase activity, resulting in the CD15 increase and CDw65 decrease.
FUT9 was detected in each subpopulation of human PBMCs, although the
expression level differed among the populations. Unexpectedly, monocytes do not express FUT9 despite expressing CD15 on their surface.
The flow cytometric analysis indicated that the CD15 intensity in
monocytes was dull and lower than that of CDw65, consistent with the
substrate specificity of FUT4. FUT4 was found to synthesize CDw65 more
than CD15, because P2s is 3.5 times larger than P1 (Fig. 2,
B and C). Monocytes expressed FUT4 to almost the
same extent as granulocytes, however, granulocytes showed bright CD15
staining with a much higher level of CD15 expression than monocytes due
to the additional expression of FUT9. CD4+ T cells,
CD8+ T cells, B cells, and CD56+ cells
expressed FUT9, although flow cytometric analysis did not usually show
CD15-positive staining in those cells. However, the transfection of the
FUT9 gene to Jurkat cells demonstrated that FUT9 can
potentially express the CD15 epitope on the cell surfaces of lymphoid
cells (Fig. 5). Western blot analysis clearly demonstrated that all
subpopulations of PBMCs can produce CD15-carrying glycoproteins intracellularly, which are not transported to be expressed on the cell
surface. Considering the stronger activity of FUT9 for the synthesis of
the CD15 epitope, the intracellular CD15 epitopes in PBMCs must be
mainly synthesized by FUT9. The reasons why there were very few CD15
epitopes on the cell surface of PBMCs is unclear, but it is possible
that either the intracellular CD15-positive glycoproteins are not
translocated to the surface of resting PBMCs for some reason, or the
expression level of FUT9 in each subpopulation of PBMCs is not
sufficient to synthesize enough CD15-positive glycoproteins to be
expressed on the cell surface. In fact, the level of FUT9
expression in PBMCs is lower than that in granulocytes and Jurkat-FUT9
cells. Among many Jurkat clones transfected with the FUT9
gene, we have selected some, which expressed FUT9 at a range of 0.5 to
1.0. These clones with a relatively low expression level of FUT9 did
not express CD15 on their cell surface (data not shown). These findings
indicated that peripheral lymphoid cells do not express a high enough
level of FUT9 to induce the cell surface expression of CD15.
Although the function of CD15 in leukocytes is still not clear, CD15
may be involved in cell-cell interaction. The carbohydrate structure
associated with CD15 on myeloid cells may be another ligand for human
CD2 (16). As reported by others (14, 42, 43), the CD15-positive
glycoproteins in mature granulocytes showed characteristically broad
bands spanning 140-180 kDa and 95-110 kDa on Western blot analysis.
Surface-labeling studies revealed that only the 165-kDa and 105-kDa
CD15-reactive glycoproteins are localized on the cell surface (43, 44).
They were identified to be a member of the LFA1/CR3/p150,95 (CD11/CD18)
family (12, 14) and NCA160 (44). We found that bands corresponding to the molecular sizes of CD11/CD18 members and NCA160 were present not
only in mature granulocytes but also in all subpopulations of PBMCs.
Monocytes showed the highest density of these bands, compatible with
the strong cell surface expression of CD15.
Recently, it has been reported that not only FUT7 but also FUT4
generate selectin ligands that support in vivo rolling of some leukocytes, whereas FUT7-dependent carbohydrates
determine the rolling fraction for most leukocytes (28, 29). FUT9 can synthesize the CD15 epitope with very strong activity by transferring a
fucose to the distal GlcNAc residue, although FUT7 can only fucosylate
this precursor after sialylation by
2,3-sialyltransferases. This
distinct substrate preference may lead to competition between the three
1,3FUTs if the availability of acceptor substrates is limited. This
would result not only in the synthesis of CD15 but also in the
interference of the generation of some carbohydrate epitope(s), such as
CD15s and CDw65. The availability of FUT9 provides a tool for
investigating the biological functions of the polylactosamine chain
with or without fucosylation in human leukocytes under certain
inflammatory conditions.