(Received for publication, January 17, 1996)
From the
In an attempt to correlate the cell surface expression of
Le and sialyl-Le
structures in immature and
mature myeloid cells with the genes expressing
1,3-fucosyltransferase(s) we have examined: 1) the properties of
the cellular
1,3-fucosyltransferases and the mRNA transcripts
corresponding to the five cloned genes, Fuc-TIII, Fuc-TIV, Fuc-TV,
Fuc-TVI, and Fuc-TVII, in mature granulocytes and in the myeloid cell
line HL-60, before and after dimethyl sulfoxide-induced differentiation
and 2) the properties of the
1,3-fucosyltransferases expressed in
COS-7 cells transfected with plasmids containing Fuc-TIV and Fuc-TVII
cDNAs. The previously shown increase in cell surface expression of
sialyl-Le
on differentiation of HL-60 cells (Skacel P. O.,
Edwards A. J., Harrison C. T., and Watkins W. M.(1991) Blood 78, 1452-1460) is accompanied by a sharp fall in expression
of Fuc-TIV mRNA and a persistence of expression of Fuc-TVII mRNA. The
properties of the
1,3-fucosyltransferase expressed in COS-7 cells
transfected with Fuc-TIV are consistent with this being the major gene
responsible for the expression of Le
in the immature
myeloid cells. In Northern blot analyses, no transcripts of Fuc-TIII,
Fuc-TV, or Fuc-TVI were detected in total RNA from mature granulocytes
or mRNA from HL-60 cells before or after differentiation. In total RNA
from mature granulocytes, Fuc-TIV transcripts were only faintly
visible, whereas Fuc-TVII transcripts were quite definitely expressed.
The specificity properties of Fuc-TVII expressed in COS-7 cells are
consistent with this gene being the major candidate
1,3-fucosyltransferase controlling the expression of
sialyl-Le
on mature cells. However, Le
continues to be expressed on the surface of mature granulocytes and
cell extracts retain the capacity to transfer fucose to non-sialylated
acceptor substrates. The question therefore remains as to whether these
properties result from the weakly expressed Fuc-TIV gene or whether
another
1,3-fucosyltransferase gene remains to be identified.
In the process of differentiation of hemeopoietic cells the
capacity to express -L-fucosyltransferases catalyzing the
transfer of fucose to different positional linkages in the sugar
acceptor molecules becomes restricted to certain cell lineages.
1,3-Fucosyltransferase (
)activity is present in mature
granulocytes, monocytes, and lymphocytes whereas neither
1,2- nor
1,4-fucosyltransferase activities are detectable in these
cells(1, 2, 3) . Transfer of fucose to the O-3 position of the N-acetylglucosamine residue in a
Type 2 chain ending (Gal
1-4GlcNAc) results in the formation
of Le
when the terminal
-galactosyl residue is
unsubstituted and to the formation of sialyl-Le
when the
-galactosyl unit is substituted with an
2,3-linked sialic
acid residue; once formed the Le
sequence is not a
substrate for the addition of sialic acid (reviewed in (4, 5, 6) ). The Le
determinant
is expressed on mature granulocytes and on all myeloid cells from the
promyelocyte stage
onwards(7, 8, 9, 10) .
Sialyl-Le
is expressed on myeloid progenitor cells but has
been reported to be down-modulated at intermediate stages of
granulocyte maturation and up-regulated to levels similar to those in
progenitor cells after termination of proliferative activity (11) .
High levels of 1,3-fucosyltransferase activity
are expressed in human leukemic myeloblasts (12, 13, 14) and the finding of even higher
levels in normal bone marrow cells (15) suggests that the
activity in the leukemic blasts is a reflection of the stage of
maturation arrest of the cells. Earlier studies on partially purified
preparations of
1,3-fucosyltransferases isolated from granulocytes
from healthy individuals and from patients with chronic myeloid
leukemia, indicated that two different species of this enzyme are
expressed in myeloid cells since the preparation from the leukemic
cells utilized substrates containing terminal
3`-sialyl-N-acetyllactosamine groupings to only a very limited
extent, whereas the preparation from mature granulocytes had a much
greater capacity to make sialyl-Le
determinant
sequences(16) .
The leukemia cell line HL-60 consists
predominantly of promyelocytes which can be induced to differentiate
into myelocytes, metamyelocytes, and granulocytes by the addition of
dimethyl sulfoxide (MeSO) to the growth
medium(17) . The surface glycoprotein (18, 19) and glycolipid profiles (20, 21) of the cells specifically change during
differentiation. Therefore, although granulocytic maturation of HL-60
cells has been shown to be defective in some
respects(20, 22, 23, 24, 25) ,
this cell line offers a useful model for studying the enzymatic basis
of changes that take place in expression of carbohydrate cell surface
structures during differentiation of myeloid cells. Our previous
studies demonstrated that Me
SO-induced differentiation of
HL-60 cells resulted in a lowering of
1,3-fucosyltransferase and
2,6-sialyltransferase activities, an increase in cell surface
expression of sialyl-Le
, and a loss of the marked
preference for non-sialylated acceptors shown by the
1,3-fucosyltransferase(s) in undifferentiated cells(14) .
Five human 1,3-fucosyltransferase genes had been cloned:
Fuc-TIII (26) , Fuc-TIV(27, 28, 29) ,
Fuc-TV(30) , Fuc-TVI(31, 32) , and Fuc-TVII (33, 34) . Fuc-TIII, Fuc-TV, and Fuc-TVI are clustered
on chromosome 19 and the catalytic domains share up to 95% nucleotide
and amino acid sequence
homology(26, 30, 31, 32) . Fuc-TIV,
located on chromosome 11(35, 36) , is less closely
homologous but still shares about 60% homology with the three
chromosome 19
1,3-fucosyltransferases(32) . This gene is
expressed in HL-60 cells(27, 28, 29) , and in
other myeloid leukemic cell lines, and by consensus was termed the
``myeloid''
1,3-fucosyltransferase gene. However, when
cDNA corresponding to this gene was transiently expressed in mammalian
cells, there was lack of agreement as to whether the Fuc-TIV gene gave
rise to cell surface expression of sialyl-Le
(27, 28, 29) and when extracts of the
transfected cells were tested for
1,3-fucosyltransferase activity
the expressed enzyme failed to utilize
2,3-sialylated acceptors (27) . The most recently cloned gene, Fuc-TVII, which maps to
chromosome 9, has about 40% homology with the chromosome 19 cluster and
is reported to give rise to cell surface expression of
sialyl-Le
, but not Le
, when transiently
expressed in mammalian cells(33, 34) . Transcripts of
this gene have been detected in mRNA from undifferentiated HL-60
cells(33, 34) and it is therefore a plausible
candidate to be the major gene encoding an enzyme that leads to the
expression of sialyl-Le
in cells of the myeloid lineage.
The aim of the present study was to examine further the changes that
take place in the specificity and general properties of
1,3-fucosyltransferase(s) in the course of maturation of myeloid
cells and to determine whether any direct correlation can be shown
between enzyme activity, mRNA expression of the five cloned
1,3-fucosyltransferase genes, and cell surface expression of
Le
and sialyl-Le
.
The epidermoid carcinoma cell line, A431,
was supplied by Dr. W. Gullick (Imperial Cancer Research Fund Unit,
Hammersmith Hospital, London, U.K.) and COS-7 cells by Dr. P. H.
Johnson (MRC Blood Group Unit, University College London, U.K.) and Dr.
P. J. Mason (Royal Postgraduate Medical School, London, U.K.). These
cells were grown in Dulbecco's modified Eagle's medium
(Life Technologies, Inc. Ltd., U.K.), supplemented with 10% heat
inactivated fetal calf serum, 2 mM glutamine, 100 units/ml
penicillin, and 100 µg/ml streptomycin, in a 5% CO atmosphere. The cells were grown to confluence, the medium
removed, the adherent cells washed with PBS and detached from the flask
by treatment for 5 min with trypsin (0.5%), EDTA (0.2%). The detached
cells were suspended in a small volume of Dulbecco's modified
Eagle's medium, spun at 800
g, washed with PBS,
resuspended in PBS, and counted.
The reaction mixtures containing the glycoprotein acceptors were incubated at 37 °C for 16 h and the radioactive products were separated on columns (0.7 x 16 cm) of Sephadex G-50 by elution with 0.2 M NaCl. The first 2 ml of eluate were discarded and the next 2 ml, containing the product, were mixed with 5 ml of scintillant and counted as above. Control mixtures not containing the exogenous glycoprotein acceptors were included in all these experiments and radioactivity resulting from incorporation into endogenous macromolecular acceptors was subtracted from the product counts.
1,4-Galactosyltransferase activity was assayed with a reaction
mixture containing in a total volume of 85 µl: 0.68 µM UDP-[
C]-D-galactose (50,000 cpm),
21 mM MnCl
, 52 mM Tris-HCl buffer, pH
7.2, 5.2 mM ATP, 0.05% Triton X-100, 20 µl of enzyme
source, and 2.6 mMN-acetylglucosamine. The mixtures
were incubated at 37 °C for 30 min and the radioactive N-acetyllactosamine product was separated on Dowex columns and
counted as described for the fucosyltransferase assays.
Five
1,3-fucosyltransferase probes were prepared from plasmids kindly
provided by Dr. J. B. Lowe (Howard Hughes Medical Centre, Ann Arbor,
MI). The probes were: the 1.7-kb XhoI-XbaI fragment
isolated from the insert in plasmid
pCDM7-
-(1,3/1,4)-fucosyltransferase for the detection of Fuc-TIII (26) , the 1.05-kb PvuII fragment from the insert in
pcDNA1-
-(1,3)-fucosyltransferase
for the detection
of Fuc-TIV(27) , the 1.9-kb XbaI-EcoRI
fragment from the insert in pcDNA1-Fuc-TV for the detection of
Fuc-TV(30) , the 1.2-kb HindIII fragment from the
insert in pcDNA1-Fuc-TVI for the detection of Fuc-TVI(32) , and
the 2.0-kb XhoI-XbaI fragment from the insert in
pCDM8-Fuc-TVII for the detection of Fuc-TVII(34) . A
glucose-6-phosphate dehydrogenase (G6PD) cDNA probe, supplied by Dr. C.
M. Corcoran (Royal Postgraduate Medical School, London), was used as a
control for quality and even loading of the mRNA.
P-Labeled cDNA probes were prepared by the random
oligonucleotide priming method according to the manufacturers
instructions (Megaprime, Amersham) to a specific activity equal to, or
greater than, 1
10
cpm/µg DNA. The
P-labeled cDNA probes (50 ng) were added to 5 ml of the
hybridization solution and the membranes were then heated for 16 h at
42 °C. After hybridization the blots were washed once in 0.45 M NaCl, 0.045 M sodium citrate, 1% sodium dodecyl sulfate
(SDS) for 15 min at room temperature, twice with 0.3 M NaCl,
0.03 M sodium citrate, 1% SDS for 30 min at 42 °C and once
with 0.03 M NaCl, 0.003 M sodium citrate, 1% SDS for
30 min at 42 °C. The membranes were air dried and then exposed to
Kodak X-Omat film with an enhancing screen at -70 °C.
Figure 1:
Flow cytometry analyses of
mature granulocytes and HL-60 cells, before and after MeSO (DMSO)-induced differentiation. HL-60 cells, grown with or
without Me
SO (DMSO) in the standard growth medium
for periods up to 4 days and granulocytes isolated from peripheral
blood were washed, stained with monoclonal antibodies, and examined by
indirect immunofluoresence as described under ``Experimental
Procedures.'' The commercial antibodies were used at the following
dilutions: anti-LeuM1 (anti-Le
) 1:50, anti-CDw65
(anti-VIM2) 1:10, anti-CD11b undiluted, and anti-CD34 undiluted. CSLEX1
(anti-sialyl-Le
) was diluted 1:50 for tests on HL-60 cells
and 1:100 for tests on granulocytes. FH6 (anti-sialyl-dimeric
Le
) was used at the concentration supplied. The data are
presented as the percentage of cells giving positive reactions with the
antisera.
Both the differentiated and undifferentiated HL-60
cells reacted with anti-CD15 (anti-Le) and neither the
percentage of positive cells, nor the mean fluorescence intensity (data
not shown), changed significantly on Me
SO-induced
maturation (Fig. 1). As observed earlier(14) ,
differentiation of HL-60 cells was accompanied by an up-regulation in
the expression of sialyl-Le
; the increase in the number of
positive cells was apparent after 24 h and continued to develop up to
48 h (Fig. 1). Treatment with 10 milliunits of Newcastle disease
virus neuraminidase decreased the number of cells reacting with
anti-sialyl-Le
by 89%, thus demonstrating that the antibody
was reacting with newly formed structures terminating with sialic acid
residues. The undifferentiated HL-60 cells reacted quite strongly with
antibody CDw65 which detects the VIM2 epitope(49) , but
differentiation led to a decrease in the number of cells positive with
this antibody (Fig. 1). The antibody FH6, which is reported to
react with sialyl-dimeric-Le
but not sialyl-Le
or the VIM2 epitope(48) , failed to react with untreated
HL-60 cells and no significant change was detectable after 4 days
exposure to Me
SO (Fig. 1).
Figure 2:
Flow cytometry analyses of COS-7 cells
transfected with plasmids containing Fuc-TIV and Fuc-TVII cDNA. COS-7
cells were transfected with either plasmid pcDNA1-Fuc-TIV (FT4) or pSI-Fuc-TVII (FT7), or with a mixture of the
two plasmids (FT4 + 7), and the transfected cells were
grown for 48 h in standard medium and then harvested, washed, stained
with monoclonal antibodies directed to carbohydrate epitopes and
examined by indirect immunofluoresence as described under
``Experimental Procedures.'' The antibodies anti-LeuM1
(anti-Le), CSLEX1 (anti-sialyl-Le
), and
anti-CDw65 (anti-VIM2) were used at the concentration supplied. The
data are presented as: a, the mean fluorescence intensity with
the antibodies; and b, the percentage of cells giving positive
reactions with the antibodies.
Figure 3:
Acceptor specificity of
1,3-fucosyltransferase(s) expressed in HL-60 cell extracts with
low molecular weight oligosaccharide and glycoprotein acceptors before
and after growth of the cells in the presence of Me
SO.
HL-60 cells were grown in the presence of Me
SO (DMSO) for periods up to 4 days and extracts were prepared and
assayed for
1,3-fucosyltransferase activity with: a, N-acetyllactosamine (NAL) and
3`-sialyl-N-acetyllactosamine (SNAL); and b,
fetuin and asialofetuin as described under ``Experimental
Procedures.'' The percentage incorporation of
[
C]fucose represents the mean values of three
different experiments.
On growth of the
HL-60 cells in the presence of MeSO, the activity of
1,3-fucosyltransferase measured with either N-acetyllactosamine or asialofetuin sharply decreased; the
levels were reduced by 40-50% after exposure to Me
SO
for 48 h (Fig. 3). In contrast, the low level of activity
measured with the sialylated oligosaccharide acceptor
3`-sialyl-N-acetyllactosamine remained largely unchanged on
differentiation of the cells while incorporation into the glycoprotein
acceptor fetuin showed a gradual increase (Fig. 3): after 4 days
exposure the ratio of incorporation of fucose into asialofetuin and
fetuin was changed from approximately 3:1 to 1:1.
The level of
1,4-galactosyltransferase measured with N-acetylglucosamine as acceptor remained virtually unchanged
in HL-60 cells grown for up to 4 days in the presence of
Me
SO (data not shown), indicating that the changes observed
in the
1,3-fucosyltransferase activity were not attributable to a
general down-regulation of glycosyltransferase activity.
Extracts of COS-7 cells transfected with Fuc-TVII
cDNA in the vector pSI, under the same conditions used for transfection
of the Fuc-TIV plasmid, transferred fucose to
3`-sialyl-N-acetyllactosamine and had virtually no activity
with the unsubstituted Type 2 disaccharide or any of the other
oligosaccharides tested (Table 3). Similarly, incorporation into
fetuin and Tamm-Horsfall glycoprotein took place into the intact
sialylated glycoproteins and negligible transfer took place to the
asialoglycoproteins. These results are thus in agreement with the
earlier reports(33, 34) , that, based on specificity
studies with low molecular weight oligosaccharides and cell surface
expression of sialyl-Le, suggested that the Fuc-TVII
gene-encoded enzyme could act only on sialylated acceptor substrates.
No activity with either N-acetyllactosamine or
3`-sialyl-N-acetyllactosamine was detectable in the culture
media of the cells transfected with the Fuc-TVII plasmid.
Co-transfection of the Fuc-TIV and Fuc-TVII plasmids into COS-7
cells led to a decrease in activity of the cellular
1,3-fucosyltransferase(s) measured with the asialo-substrates, N-acetyllactosamine and asialofetuin, and an increase in the
activity with the sialylated substrates,
3`-sialyl-N-acetyllactosamine and fetuin, in comparison with
the activities found in the extracts of the cells transfected with
Fuc-TIV alone. The levels of incorporation of
[
C]fucose into the low molecular weight, or
glycoprotein, sialylated substrates did not change from those observed
when the Fuc-TVII plasmid was transfected on its own (Table 3).
Figure 4:
The effect of NEM on the activity of
1,3-fucosyltransferases expressed in HL-60 cells before and after
Me
SO-induced differentiation, in COS-7 cells transfected
with Fuc-TIV and FUC-TVII plasmids, in mature granulocytes, and in
plasma. Different concentrations of NEM were added to the cell extracts
and the mixtures were assayed for
1,3-fucosyltransferase activity
with either asialofetuin (
) or fetuin (
) as acceptors as
described under ``Experimental Procedures.'' a,
untreated HL-60 cells. b, HL-60 cells after 4 days exposure to
Me
SO (DMSO). c, COS-7 cells transfected
with a plasmid containing Fuc-TIV cDNA. d, COS-7 cells
transfected with a plasmid containing Fuc-TVII cDNA. e, mature
human granulocytes. f, human plasma from a blood group Bombay
O
donor.
A
similar pattern of activation of the 1,3-fucosyltransferase(s)
transferring fucose to asialofetuin (130% increase), and inhibition of
the transfer to fetuin (60% decrease) was found when extracts of HL-60
cells were treated with 0.1 mMp-mercuribenzoate,
although with this reagent all activity was abolished with both
substrates at 10 mM concentration. In contrast, iodoacetamide,
at concentrations up to 10 mM induced no detectable change in
the activity of the
1,3-fucosyltransferase(s) in HL-60 cells
toward asialofetuin and the activity toward fetuin was reduced by only
30%.
COS-7 cells transfected with
Fuc-TVII did not show any 1,3-fucosyltransferase activity with
asialofetuin but the activity in the cell extracts toward fetuin was
resistant to NEM under the conditions tested, with virtually 100%
activity remaining on exposure to 10 mM NEM (Fig. 4).
Figure 5:
The effect of heat on the activity of
1,3-fucosyltransferases expressed in HL-60 cells before and after
Me
SO-induced differentiation, in COS-7 cells transfected
with Fuc-TIV and FUC-TVII plasmids, in mature granulocytes, and in
plasma. Cell extracts were heated at 50 °C for various lengths of
time and assayed for
1,3-fucosyltransferase activity with either
fetuin (
) or asialofetuin (
) as acceptors as described under
``Experimental Procedures.'' a, untreated HL-60
cells. b, HL-60 cells after 4 days exposure to
Me
SO (DMSO). c, COS-7 cells transfected
with a plasmid containing Fuc-TIV cDNA. d, COS-7 cells
transfected with a plasmid containing Fuc-TVII cDNA. e, mature
human granulocytes. f, human plasma from a blood group Bombay
O
donor.
The difference in heat inactivation of the enzyme measured with the sialylated and non-sialylated glycoprotein acceptors was not found with the low molecular weight substrates. Inactivation proceeded at much the same rate with 50% of the activity toward both N-acetyllactosamine and 3-sialyl-N-acetyllactosamine being destroyed in 50 min.
The
susceptibility to heat of the 1,3-fucosyltransferase acting on
fetuin in COS-7 cells transfected with the plasmid containing Fuc-TVII
cDNA was very similar to that of the enzyme in cells transfected with
the plasmid containing Fuc-TIV (Fig. 5) and only about 20% of
the original activity remained after 30 min at 50 °C.
Figure 6:
Expression of Fuc-TIV transcripts in mRNA
from HL-60 cells, before and after growth in the presence of
MeSO. Poly(A)
RNA (15 µg) prepared
from untreated HL-60 cells and cells grown in 1.3% Me
SO (DMSO) were electrophoresed, transferred to a membrane, and
hybridized with a
P-labeled Fuc-TIV probe as described
under ``Experimental Procedures.'' The Fuc-TIV probe
contained 4.1
10
cpm/µg DNA. A
P-labeled glucose-6-phosphate dehydrogenase (G6PD) cDNA (4.7
10
cpm/µg DNA) was
used to probe 4.5 µg of the same poly(A)
RNA
preparation. The blots were exposed to Kodak X-Omat film with an
intensifying screen for 24 h at -70 °C. The figure shows
transcripts in mRNA isolated from untreated HL-60 cells and cells
cultured for 24 and 48 h in the presence of
Me
SO.
Figure 7:
Northern blot analyses of mRNA from HL-60
cells, before and after exposure to MeSO, and from A431
cells, probed with
P-labeled Fuc-TIII, Fuc-TV, and Fuc-TVI
cDNAs. Poly(A
) RNA aliquots (30 µg) prepared from
untreated HL-60 cells and HL-60 cells grown for 96 h in the presence of
1.3% Me
SO were electrophoresed, transferred to a membrane,
and hybridized with the
P-labeled probes as described
under ``Experimental Procedures.'' The Fuc-TIII, Fuc-TV, and
Fuc-TVI probes contained, respectively, 2.4
10
cpm/µg DNA, 1.2
10
cpm/µg DNA, and
1.6
10
cpm/µg DNA. mRNA isolated from A431
cells (10 µg) was hybridized with the same three probes and a
P-labeled glucose-6-phosphate dehydrogenase (G6PD) cDNA (4.8
10
cpm/µg DNA) was
hybridized with 5 µg mRNA isolated from A431 cells and from HL-60
cells before and after growth in the presence of Me
SO. The
membranes were exposed to X-Omat film for 72 h at -70 °C with
an intensifying screen. First lane, mRNA from untreated HL-60
cells, second lane, mRNA from HL-60 cells treated for 96 h
with Me
SO, third lane, mRNA from A431
cells.
Examination of mRNA from undifferentiated HL-60 cells in Northern
blot analyses with the P-labeled Fuc-TVII cDNA probe
revealed three transcripts at
2.0,
2.3, and
3.0 kb (Fig. 8) as reported by Natsuka et al.(34) . In
contrast to the results observed with the Fuc-TIV probe there was no
dramatic change in the level of expression of the Fuc-TVII mRNA
transcripts after Me
SO-induced differentiation and although
there was some reduction in the levels of the
2.3- and
3.0-kb
bands, the major
2.0-kb band was strongly expressed after 4 days
exposure to Me
SO.
Figure 8:
Expression of Fuc-TVII transcripts in
HL-60 cells before and after growth in the presence of
MeSO. Poly(A)
RNA aliquots (15 µg)
prepared from untreated HL-60 cells before and after growth in 1.3%
Me
SO (DMSO) for 24 and 96 h were electrophoresed,
transferred to a membrane, and hybridized to the
P-labeled
Fuc-TVII probe (5.6
10
cpm/µg DNA) as described
under ``Experimental Procedures.'' The blots were exposed to
X-Omat film with an intensifying screen for 72 h at -70 °C.
The same mRNA was subsequently probed with a
P-labeled
glucose-6-phosphate dehydrogenase (G6PD) cDNA (1.9
10
cpm/µg DNA) and exposed to X-Omat film without an
intensifying screen for 24 h at -70
°C.
Figure 9:
Expression of Fuc-TIV and Fuc-TVII
transcripts in total RNA from mature granulocytes and HL-60 cells.
Total RNA (22 µg) from untreated HL-60 cells and mature human
granulocytes isolated from peripheral blood were electrophoresed,
transferred to a membrane, and hybridized to a P-labeled
Fuc-TIV probe (1.2
10
cpm/µg DNA) as described
under ``Experimental Procedures.'' The blot was exposed to
X-Omat film with an intensifying screen at -70 °C for 17
days. The same blot was subsequently reprobed, first with a
P-labeled Fuc-TVII probe (5.6
10
cpm/µg DNA) and exposed to X-Omat film -70 °C for
11 days and second with a
P-labeled glucose-6-phosphate
dehydrogenase (G6PD) cDNA (4.8
10
cpm/µg DNA) and exposed to X-Omat film -70 °C for 3
days. First lane with each probe, total RNA from mature
granulocytes; second lane, total RNA from untreated HL-60
cells.
The carbohydrate determinant, sialyl-Le, is well
expressed on the surface of mature granulocytes and is believed to be a
major ligand recognized by the adhesion molecules, E- and P-selectin,
which are involved in the inflammatory
response(60, 61, 62) . The fact that this
antigen is expressed on myeloid progenitor cells but is down-modulated
during the intermediate stages of granulocyte maturation (11) suggests that it may also have a function in specific
recognition mechanisms in the course of myeloid development in the bone
marrow. The human leukemic cell line, HL-60, contains cells arrested at
the promyelocytic stage(17) , that is, cells which in normal
maturation along the granulocytic pathway would undergo further
differentiation through myelocyte, metamyelocyte, band, and
polymorphonuclear stages before leaving the bone marrow as mature
granulocytes(11) . Sialyl-Le
and Le
are
carried as terminal sequences on oligosaccharide chains in both
glycoproteins and glycolipids on the surface of myeloid cell lines and
on mature
granulocytes(47, 63, 64, 65) .
Le
determinants are strongly expressed on HL-60 cells but
expression of sialyl-Le
is very weak(14) . Both the
glycoprotein and glycolipid profiles on the surface of HL-60 cells
undergo changes when the cells are induced to
differentiate(18, 19, 20, 65) ; the
process of maturation from promyelocyte to granulocyte is accompanied
by a great increase in the quantity and the degree of branching of the
polylactosamine structures of the sialoglycoprotein,
leukosialin(19, 65) , and in the amounts of sialic
acid containing glycosphingolipids(20, 21) .
At the
stage where the strongly active 1,3-fucosyltransferase, with a
marked preference for the non-sialylated Type 2 acceptors (Table 1), is expressed in the undifferentiated HL-60 cells,
transcripts of the Fuc-TIV gene are also readily detectable in mRNA
from these cells (Fig. 6). Transfection of COS-7 cells with a
plasmid containing Fuc-TIV cDNA gives rise to an
1,3-fucosyltransferase with specificity properties (Table 3)
and responses to heat and sulfhydryl reagents that are very similar to
those found for HL-60 cells ( Fig. 4and Fig. 5). The
differences in the heat and NEM inactivation profiles of the
1,3-fucosyltransferase(s) in HL-60 cells when measured with the
intact glycoproteins or the asialoglycoproteins were also found for the
enzyme in COS-7 cells transfected with the plasmid containing Fuc-TIV
cDNA; thus these differences do not, as we at first thought, arise from
the presence of two distinct enzymes. Structural changes accompanying
the cleavage of an -S-S- bond has been suggested as a cause of
activation of an enzyme by sulfhydryl reagents (66, 67) and it could be postulated that a
conformational change in the enzyme molecule induced by heat or by the
cleavage of -S-S- bonds might enable the
1,3-fucosyltransferase to
combine more readily with the unsubstituted Type 2 chain ending in a
glycoprotein but be insufficient to enable increased binding to the
bulkier sialylated-Type 2 structure.
Flow cytometric analysis of the
HL-60 cells (Fig. 1) showed major reactivity with antibodies
directed toward Le and VIM2 determinants and a similar
pattern was exhibited by the COS-7 cells transfected with the plasmid
containing Fuc-TIV cDNA (Fig. 2). The expression of antigens on
the cell surface, together with evidence from enzyme specificity
studies and susceptibility to inactivation by heat or sulfhydryl
compounds is therefore consistent with the interpretation that Fuc-TIV
is the major gene encoding the
1,3-fucosyltransferase expressed in
the myeloid cells at the promyelocyte stage.
In an attempt to see
whether the increased expression of sialyl-Le on
differentiation of HL-60 cells, and the switch in specificity of the
1,3-fucosyltransferase with regard to sialylated acceptors, could
be correlated with loss of expression of one fucosyltransferase gene
and onset of transcription of one or more of the other cloned genes, we
compared mRNA from differentiated and undifferentiated cells in
Northern blot analysis with
P-labeled probes for the five
cloned
1,3-fucosyltransferase genes. On Me
SO-induced
differentiation of the HL-60 cells a dramatic change occurs in the
level of expression of mRNA corresponding to Fuc-TIV (Fig. 6)
and this drop is concomitant with a fall in the level of
1,3-fucosyltransferase activity, and a loss in the preference of
the enzyme(s) for non-sialylated acceptors ( Table 1and Fig. 3). At the same time there is a rise in the cell surface
expression of sialyl-Le
and a fall in the expression of the
VIM-2 epitope. No significant expression of Fuc-TIII, Fuc-TV, or
Fuc-TVI mRNA transcripts could be detected by Northern blot analyses of
mRNA from undifferentiated HL-60 cells and none of the signals were
intensified in mRNA from differentiated cells (Fig. 7).
Transcripts corresponding to the Fuc-TVII gene were, however, well
expressed in mRNA from the untreated HL-60 cells (Fig. 8) and
the major transcript (
2.0 kb) continued to be strongly expressed
on Me
SO-induced differentiation of the cells (Fig. 8). As had been reported by others (34) transfection of COS-7 cells with a plasmid containing
Fuc-TVII cDNA led to the expression of sialyl-Le
and to a
cellular
1,3-fucosyltransferase that reacted only with sialylated
acceptors. The increase in expression of sialyl-Le
did not
therefore appear to correspond to a newly switched on gene but to a
change in the balance of enzymes that allowed expression of the
1,3-fucosyltransferase encoded by the Fuc-TVII gene. This change
could be associated with the sharp fall in the level of expression of
Fuc-TIV which would decrease competition for the Type 2 acceptor
substrate and therefore allow more
2,3-sialylated precursor
structures to be synthesized as substrate for the product of the
Fuc-TVII gene. A decrease in the expression of the strongly expressed
2,6-sialyltransferase which had been shown to occur on
differentiation of HL-60 cells(14) , would also remove a
competitor for the Type 2 acceptor substrate and thus act as a
regulator for the appearance of sialyl-Le
. At the same time
the increase in polylactosamine branching structures that occurs as
differentiation proceeds (64, 65) would provide
further substrates for all the competing enzymes. The reactivity of the
differentiated cells with antibody to the CD11b marker, but failure to
express the CD16 antigen, indicated that the majority of the cells had
matured to the myelocyte stage but not further along the granulocytic
pathway; hence the decrease in reactivity with the antibody detecting
the VIM2 epitope (Fig. 1) may indicate a specific
down-regulation of cell surface expression of this antigen at the
myelocyte stage concomitant with the drop in expression of the Fuc-TIV
gene-encoded
1,3-fucosyltransferase.
Co-transfection of COS-7
cells with both Fuc-TIV and Fuc-TVII plasmids led to changes in the
levels of cell surface expression of Le and sialyl-Le
in comparison with the levels expressed when the cells were
transfected with either of the plasmids singly (Fig. 2) and the
activity of the cellular
1,3-fucosyltransferases with sialylated
or non-sialylated acceptors was modified. In these experiments the
enzyme encoded by the Fuc-TVII gene appeared to use up acceptor at the
expense of the enzyme encoded by the Fuc-TIV gene since the levels of
activity with the non-sialylated acceptors were the ones that decreased
when both enzymes were present (Table 3). These experiments
cannot be strictly compared with what is happening on differentiation
of HL-60 cells because it is unknown how much of each cDNA is
transfected, and also the nature and amount of the precursor acceptor
molecules in the COS-7 cells probably differ from those in the myeloid
cells and the number and activity of other competing enzymes, such as
the sialyltransferases, may also be different. Moreover, the COS-7
cells are not undergoing the continuum of change in precursor
structures and enzyme levels that are taking place in the maturing
HL-60 cells. The results do, however, demonstrate the changes in cell
surface structures that can occur when there is competition between two
fucosyltransferases for the available substrate.
Examination of
total RNA from mature granulocytes in Northern blot analyses with
probes for Fuc-TIV and Fuc-TVII indicated very weak expression of
Fuc-TIV transcripts and much more definite expression of Fuc-TVII
transcripts (Fig. 9). The cell surface expression of
sialyl-Le on the granulocytes was considerably stronger
than on the differentiated HL-60 cells, which is in agreement the
interpretation that the Fuc-TVII-encoded
1,3-fucosyltransferase is
responsible for the appearance of sialyl-Le
on the mature
cells. Little is yet known about the rate of turnover of the cell
surface carbohydrate antigens, the half-life of the
1,3-fucosyltransferase enzyme proteins, or their mRNA transcripts.
However, the persistence of Le
expression at about the same
level as on the HL-60 cells and the continued activity of the cellular
enzyme(s) with both sialylated and non-sialylated acceptors (Table 2) indicates that Fuc-TVII is not the only
1,3-fucosyltransferase gene that is being expressed in the
granulocytes. The ratios of enyzme activity with sialylated and
non-sialylated acceptors found for the
1,3-fucosyltransferases in
granulocytes, in HL-60 cells, before and after differentiation, and in
COS-7 cells transfected with plasmids containing Fuc-TIV and Fuc-TVII
cDNAs, are summarized in Table 4. The ratios found with both the
low molecular weight and glycoprotein acceptors in the granulocyte
extracts resemble most closely the ratios found in the differentiated
HL-60 cells. In these cells the ratios could be assumed to arise from a
mixture of the enzymes encoded by the Fuc-TIV and Fuc-TVII genes, with
a preponderance still at that stage of the Fuc-TIV-encoded enzyme.
However, the
1,3-fucosyltransferase in the granulocytes acting on
asialofetuin appears to be more resistant to heat than the enzyme in
the COS-7 cells transfected with the Fuc-TIV plasmid and the enzyme
acting on fetuin to be more resistant than the
1,3-fucosyltransferase utilizing this substrate in the extracts of
the cells transfected with the Fuc-TVII plasmid (Fig. 5). The
possibility that these differences might result from the protective
effect of other proteins in the crude extracts cannot be excluded but
the apparent anomalies raise the question as to whether the residual
expression of the Fuc-TIV gene, together with the Fuc-TVII gene, can
account for the specificity and properties of the
1,3-fucosyltransferases expressed in mature granulocytes or
whether yet another myeloid
1,3-fucosyltransferase gene remains to
be identified.