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Enzymic Control of the Expression of the X Determinant (CD15) in Human
Myeloid Cells During Maturation: The Regulatory Role of 6’-Sialyltransferase
By Patricia 0. Skacel, Andrew J. Edwards, Coral T. Harrison, and Winifred M. Watkins
To establish the basis for the reduced expression of the X
determinant on leukemic blasts and the changes i n antigenic
expression that occur during myeloid maturation, the presence on myeloid cells of X and related structures was
examined in conjunction with studies on the activities of the
glycosyltransferases involved in their biosynthesis. Expression of X and sialyl-X was weak on blasts in comparison with
neutrophils despite the presence of the requisite precursor
structures. Much higher levels of 3-fucosyltransferase activity were found in blasts than in neutrophils when nonsialylated substrates were used. but, whereas the enzyme in
neutrophils reacted equally well with 3’-sialylated and nonsialylated acceptors, the enzyme in blasts showed a marked
preference for nonsialylated substrates. 6’-Sialyltransferase
activity was strong in blasts but was not detectable in
neutrophils, whereas a much lower level of 3’-sialyltrans-
ferase activity was present in both blasts and neutrophils.
Dimethyl sulfoxide-induced maturation of HL60 cells was
associated with (1) a decrease in both 6’-sialyltransferase
and 3-fucosyltransferase activities, (2) a change in the substrate specificity of 3-fucosyltransferase towards that found
in mature cells, and (3) increased cell surface expression of
sialyl-X. These results suggest that the reduced expression of
X in myeloblasts is related t o the presence of the strong
6’-sialyltransferase, which uses the precursor substrate at
the expense of the 3-fucosyltransferase and prevents the
synthesis of X and sialyl-X. The developmental regulation of
the levels of 3’- and 6’-sialyltransferases, and the level and
specificity of the 3-fucosyltransferases, therefore controls
the expression of X and its degree of sialylation.
0 1991b y The American Society of Hematology.
C
tures and their biosynthetic enzymes during cell differentiation and in different cell type^.'^.'^
On neutrophils, the most abundant surface carbohydrate
structures on glycoproteins and glycolipids are based on
type 2 polylactosamine chains, composed of repeating
N-acetyllactosamine (NAL) (Galpl-4GlcNAc) sub~nits.’~-*~
which is
The X determinant (Gal~l-4[Fucal-3]GlcNAc),
the structure recognised by CD15 antibodies, is a fucosylated derivative of N-acetylla~tosamine.~~~~
The final stage in
its synthesis is catalysed by a 3-fucosyltransferase (GDPfucose: N-acetyl-p-glucosaminide a1,3-fucosyltransferase)
that transfers fucose to the 3-position of subterminal
N-acetlylglucosamine residues at the ends of type 2 chains.%
The determinant is expressed strongly on neutrophils and
on all myeloid cells from the promyelocyte stage onward^.'^
However, expression on leukemic myeloblasts is frequently
negative or weak,26x27
which is surprising in view of the high
levels of activity of 3-fucosyltransferase in leukemic cells
compared with mature neutrophils.28Antigenic masking by
sialic acid occurs to some extent because reactivity of acute
myeloid leukemia (AML) cells with CD15 antibodies can
be enhanced in some, but not all AML cells, by pretreatment with neuraminida~e.~~”
To explore the expression of the X determinant further
and to determine whether it is possible to identify the
activities of glycosyltransferases that influence this expression, we have studied the occurrence of X and its sialylated
derivative, sialyl-Xi, and the activities of fucosyl-, 3‘sialyltransferase (CMP-N-acetylneuraminic acid: j3-Dgalactoside a2,3-~ialyltransferase) and 6’-sialyltransferase
(CMP-N-acetylneuraminic acid: p-D-galactoside a2,6sialyltransferase) in myeloid cells. We present our findings
in normal neutrophils, leukemic myeloblasts, and HL60
cells induced to differentiate with dimethyl sulfoxide
(DMSO).
ELL DIFFERENTIATION and maturation in mammalian cells involves an ordered program of changes
involving cell to cell adhesion and recognition. Many of
these interactions are mediated by cell surface carbohydrates on glycoproteins and glycolipid^,'.^ although their
specific functions are generally not well understood. There
are also striking differences between the cell surface carbohydrate structures found on normal and malignant ceIIs,5-’
which have an important influence on the metastatic
potential and other cell to cell interactions of tumour
cells.&’o
The carbohydrate structures recognized by monoclonal
antibodies (MoAbs) are usually at the nonreducing ends of
carbohydrate chains. Their synthesis is controlled by the
activity of a series of glycosyltransferases, each one catalyzing the transfer of a single sugar in a specific linkage to a
specific acceptor molecule. It is essential to understand the
interactions between these enzymes if the mechanisms
controlling the expression of cell surface carbohydrate
antigens are to be fully understood. Although the most
widely studied examples are the A and B blood grouprelated determinants, whose expression is controlled by the
activity of specific blood group gene encoded glycosyltransferases,11.12
there has been much recent interest in the
relationship between other cell surface carbohydrate struc~~
From the Divisions of Immunochemical Genetics and Transplantation Biology, MRC Clinical Research Centre, Middleseu, UK.
Submitted December 7, 1990; accepted May 14,1991.
Suppored by the Medical Research Council and a grant from the
Leukaemia Research Fund, UK.
Address reprint requests to Patricia Skacel, MB, Department of
Haematology, Northwick Park Hospital, WatfordRd, Harrow, Middlesex€€Al3UJ, UK.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with I8 U.S.C. section I734 solely to
indicate this fact.
0 1991 by The American Society of Hematology.
0006-4971/91/7806-0027$3..00/0
1452
MATERIALS AND METHODS
Cell Isolation
Neutrophils from seven normal volunteers were isolated from
EDTA anticoagulated blood by density gradient centrifugation on
Blood, Vol78, No 6 (September 15). 1991: pp 1452-1460
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1453
EXPRESSION OF X DETERMINANT IN MYELOID CELLS
Ficoll-Paque at 40% for 30 minutes.29Erythrocytes contaminating
the residual pellet were removed by hypotonic lysis in water
restored to 0.15 mol/L saline after 30 seconds.
Leukemic blast cells from seven patients with AML were
isolated from marrow taken into heparinised (20 U/mL) RPMI.
Ceils were harvested from the mononuclear layer after centrifugation on Ficoll-Paque at 80% for 20 minutes. Leukemia was
classified into French-American-British (FAB) subtypes on the
basis of morphology and cytochemistry of marrow smears." All
cells were washed twice in 0.15 mol/L NaCl containing 10 mmol/L
Tris-HC1 pH 7.2 before final suspension in 0.15 mol/L NaCl (for
glycosyltransferase assays) or phosphate-buffered saline (PBS)
containing 2% fetal calf serum (FCS) and 0.1% azide (for marker
studies). Cells were counted in a hemocytometer. Purity and
morphology Of Cell SUSpeIISiOIIS was assessed from cytOCentI'ifUge
preparations stained with May-Grunwald-Giemsa.
PBS/FCS/azide) were incubated with MoAb in the dilution
indicated above. h irrelevant antibody was used as a negative
control.
Fluorescein-conjugated F(ab'), rabbit antimouse Ig (Dako Ltd,
High Wycombe, UK) was used as second antibody at a dilution of
1:25. Both incubation stages were performed for 30 minutes at 4°C.
Stained cells were analyzed by cytofluorimetry in a FACStar PLUS
(Becton Dickinson Immunocytometry Systems).
Gb'co~lRansferase
Assays
Materials. GDP-L-[14C]fucose (254 Ci/mol), CMP-[14C]sialic
acid (262 Ciimol), and UDP-['4C]galactose (270 Ci/mol) were
purchased from h e r s h a m (Aylesbury, UK). NAL, lactose, 3'Sialyl-NAL, 6'-sialy]-NAL, 3'-sialyllactose, 6'&lyllactose, and
cold GDP-L-fucose were kind gifts of Dr A.S.R. Donald (Division
of Immunochemical Genetics, CRC). Fetuin and asialofetuin were
obtained from Sigma (Poole, UK) and phenyl P-D-galactoside
Enzyme Treatment
from Koch-Light Ltd.
Neuraminidase was used to remove terminal sialic acid residues
Assays were performed on 2O PL aliquots of cell suspension in
from cells before marker studies. Cells suspended in PBS/FCS
0.15 mol/L NaC1 containing o.8 x lo6 neutroPhi1s or o.4 x lo6
(10 x 106 cells in 300 *L) were incubated with 30 p~ w b ~ o leukemic cells. Immediately before assay, cells were lysed by
cholerae neuraminidase 1.0 U/mL (Koch-Light Ltd, Haverhill,
preincubation in 0.25% Triton-X100 (BDH Ltd, Poole, UK) for 60
UK) for 30 minutes at 37°C. The cells were washed twice before
minutes at 4"c.
final suspension in PBS/FCS/azide.
2-Fucosylrrunsferase. 2-Fucosyltransferase was assayed as described previously with phenyl P-D-galactoside as acceptor moleCell Culture
de.34
3-Fucoyltransferase assay with low molecular weight acceptors.
HL60 ce11s were Obtained from the European Co11ection Of
Incubation mixtures contained cell suspension 20 pL, 1%TritonAnimal Cell Cultures (PHLS Centre for Applied Microbiology and
xloo 5 pL, NAL o.75 CLmol,GDP-fucose 7.5 nmol, G ~ ~ - ~ Research, Porton Down, UK) and grown in Falcon tissue culture
[~~Clfucose
o.3 nmol, ATp o.25 pmol, MnCI, 1.o )Lmol, Na
flasks in RPM1 (F1ow Laboratories, High Wycombe, uK) supp1ecacodylate pH 7.0 5.0 *mol. The total volume was 65 IJ.L.After
mented with 10% heat-inactivated (56°C for 30 minutes) FCS and 2
incubation at 37°C for 2 hours the radioactive product, 3-fuc0sylmmol/L glutamine without antibiotics at 37°C in a humidified
Na,
was separated from nucleotide sugar donor and free sugar by
atmosphere Of 5% coy Ce1k were passaged twice weekly to
descending paper chromatography on Whatman DE 81 paper
maintain exponential growth.
(Whatman Ltd, Haidstone, UK) in propan-1-ol/ethyl acetate/
Granulocytic differentiation was induced with DMSO. Cells
pyridine/H,O (5:1:1:3, by vel) for 12 to 16 hours and identified by
were seeded at an initial concentration of 2.5 x 105/mL into
its mobility relative to lactose (R,,, l.o).
duplicate flasks containing fresh medium with or without 1.3%
In SOme experiments, cu-3-fucosyltransferaseactivity with nonDMSo. After periods Of growth between 30 minutes and 9 days,
sialylated and sialylated acceptors was compared. These assays
cells were harvested from the culture medium by centrifugation at
contained o.75 ,,mol NAL or a sialylated derivative, 3 t - or
for 5 minutes and exanlined
for movho1ogic evidence Of
6'-sialyl-N&, and 0.3 nmol GDP-L-['4C]fucose, Cell suspension,
differentiation." Viability was greater than 95% in all experiments,
Triton-X100, ATp, MnCI,, and Na cacodylate were added as
as judged by Trypan B1ue dye exc1usion.In addition, cytochrome c
described above. Assays with NAL were incubated for 30 minutes
reduction was used to assess functional differentiation of induced
only to enSure linear incorporation of [14C]fucoseinto acceptor (up
cells?'
to 15% use of GDP-['4C]fucose) and allow direct comparison with
KG1 ce11swere kind1ysupp1ied bY Dr M.Y. Gordon (Institute Of
the activity measured with sialyl-NAL. A longer incubation period
Cancer Research7 London* uK). They do not differentiate in
of 4 hours was necessary using the sialylated acceptors to ensure
response to DMS033and were cultured in identical conditions to
sufficient incorporation of [14C]fucosefor accurate measurement of
HL60 ce11s as contro1s3 to exc1ude a direct effect Of DMSo On
product. Incorporation was linear during this period. Radioactive
glycosyltransferase activity.
products were separated as described above for assays with NAL
and by high-voltage paper electrophoresis (4,000 V, 90 minutes) on
Indirect Immunofluorescence
Whatman 3 MM paper in 40 mmol/L pyridine acetate buffer pH
MoAbs. LeuMl (CD15) was purchased from Becton Dickinson
5.4 for assays with sialyl-NAL. The product was identified by its
Immunocytometry Systems (Mountain View, CA). CSLEXl was
mobility relative to picric acid (q,c,
0.6). By either separation
kindly provided by Dr P. Terasaki (UCLA Tissue Typing Laboraprocedure the recovery of total counts was quantitative.
tory, Los Angeles, CA) as affinity-purified antibody at a concentraa-3-Fucosyltramferase assay with glycoprotein acceptors. Assays
tion of 2.8 mg/mL. Antibodies 1B2 (hybridoma supernatant) and
were performed with 400 pg of fetuin or asialofetuin in 20 pL 0.25
FH3 (ascitic fluid) were a gift of Prof S. Hakomori (Biomembrane
mol/L cacodylate buffer pH 7.0. In addition, reaction mixtures
Institute, Seattle, WA). Antibodies were used at maximal concencontained the following: cell suspension 20 pL, 1%Triton-XlOO 5
trations to just prevent neutrophil agglutination and thus allow
pL, GDP-L-['4C]fucose 0.3 nmol, ATP 0.25 pmol, and MnCl, 1.0
direct comparison of staining intensity between blasts and neutropmol (total volume 65 pL). Control assays to assess endogenous
phils. Working dilutions were LeuMl 1:100, CSLEXl 1:100, 1B2
acceptor were performed without glycoprotein. After incubation at
1:5, and FH3 1:lOO.
37°C for 8 to 16 hours the reaction mixture was subjected to
Antibody binding was assessed by indirect immunofluorescence
descending paper chromatography on Whatman No, 40 paper in
on fresh and enzyme-pretreated cells. Cells (0.5 x lo6 in 50 pL
propan-I-oliethyl acetate/pyridine/H,O (5:1:1:3, by volume).
8m
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1454
SKACEL ET AL
Radiolabeled glycoprotein remained at the origin and was separated from nucleotide sugar donor and free sugar.
Sialyltransferase activity. Assays were performed with NAL or
asialofetuin as acceptor molecules and contained the following:
cell suspension 20 pL, 1% Triton-X100 5 pL, NAL 0.75 pmol or
asialofetuin 400 pg, CMP-[14C]sialicacid 0.4 nmol, MnC1, 0.25
pmol, Na cacodylate, pH 6.4, 5.0 pmol (total volume 60 pL).
Reaction mixtures were incubated for 4 hours at 37°C. With NAL,
the radioactive products were separated from nucleotide donor
and free sugar by high voltage paper electrophoresis (4,000 V, 90
minutes) on 3 MM paper in 40 mmol/L pyridine acetate buffer pH
5.4. The 3'- and 6'-sialyl-NAL reaction products obtained using
NAL as acceptor have identical mobilities in the above system (Rplo
0.7) and, therefore, to distinguish 3'- and 6'4alyltransferase
activities measured with this acceptor further, assays were performed in triplicate. The reaction products were pooled and the
two isomers were further separated as follows. After electrophoresis the total product was identified by radiochromatogram scanning, eluted overnight with 100 mmol/L pyridine acetate buffer pH
5.4, evaporated, resuspended in H,O, and subjected to descending
paper chromatography on Whatman No. 40 paper in ethyl acetate/
pyridineiacetic acid/H,O (5:5:1:3, by volume). 3'- and 6'-sialylNAL were identified by their mobilities relative to 3'-sialyllactose
(1.4 and 1.0, respectively). In sialyltransferase assays with asialofetuin, the separation of products by chromatography was identical
to that used for the 3-fucosyltransferase assay.
P-4-Galactosyltransferaseassays. p-4-Galactosyltransferaseassays contained the following: cell suspension 20 pL, 1.0% TritonXlOO 5 pL, N-acetylglucosamine 0.25 pmol, MnCI, 1.0 pmol,
UDP-['4C]galactose 0.14 nmol, Na cacodylate buffer pH 7.0, 5
pmol. Incubations were performed at 37°C for 10 minutes. The
radioactive product was separated by descending paper chromatography on DE81 paper in ethyl acetate/pyridine/H,O (10:4:3 by
volume) for 24 hours (R,,,,0.7).
Protein was estimated on duplicate samples by a modification of
the Bradford method.35 Glycosyltransferase incubation mixtures
contained between 20 and 50 pg protein. Incorporation of radioactive sugars was linear with respect to protein concentration and
incubation time.
Table 1. Structures Recognized by Antibodies Specific for the X
Determinantand Related CarbohydrateAntigens
MoAb
LeuMl
Antigen
Recognized
Structure
Reference
X determinant (CD15)
Galpl-4GlcNAc
36
3
I
FH3
X determinant
1
Fuca
Galpl-4GlcNAc
37
3
I
CSLEXl Sialyl-X
1
Fuca
NeuAca2-3Galpl-4GlcNAc
38
3
I
182
N-acetyllactosamine
1
Fuca
Galpl-4GlcNAc
39
CSLEXl. In a proportion of cases of AML, the expression of X is enhanced by prior treatment of the cells with
neuraminidase, which suggests that antigenic masking by
sialic acid may occur.26r27
We investigated this possibility
using CSLEX1, an antibody that recognizes sialyl-X, the
sialylated derivative of the X determinant. Neutrophils
were strongly reactive with CSLEXl (Fig lb) and therefore
express sialyl-X as well as X. The majority of cells from
cases 1,2, and 4,by contrast, were only weakly reactive with
the antibody. Again, in case 3 stronger reactivity appeared
to be associated with cell maturity. Therefore, sialylation of
RESULTS
Cell Marker Studies
The FAB subtypes of leukemic samples 1 through 4 were
M4, M1, M2, and M1, respectively. Cytospin preparations
of the mononuclear cell fractions isolated from marrow
contained over 90% monomorphic blast cells in all but case
3, in which approximately 40% of the cells were more
mature (promyelocytes and myelocytes).
The MoAbs used in this study recognize the X determinant and related oligosaccharide structures. Their specificities are shown in Table 1.
LeuM1. LeuMl recognizes the X (CD15) determinant
that is strongly expressed on mature neutrophils. The
results in normal neutrophils and in three of the leukemic
samples are illustrated in Fig l a . The majority of cells from
all four leukemic samples were negative with LeuMl at the
antibody dilutions used in this study. In case 3, the positive
fraction, when separated on the basis of light scattering,"
corresponded to the granulated population of promyelocytes and myelocytes identified on the cytospins. Similar
results were obtained using the antibody FH3.
Log fluorescence
Fig 1. Cytofluorimetric analysis of neutrophils and mononuclear
preparations of leukemic marrows using MoAbs directed to carbohydrate sequences I-).
LeuMl (a). CSLEXl (b), and l B 2 before (c) and
after (d) neuraminidase treatment were used. Negative control trace
using an irrelevantantibody is also shown (-).
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EXPRESSION OF X DETERMINANT IN MYELOID CELLS
1455
the X determinant did not account for the reduced expression in myeloblasts compared with neutrophils. As an
alternative means of assessing sialylation of X, the reactivity
of cells with LeuMl was compared before and after
neuraminidase treatment. The results obtained were consistent with those obtained with CSLEXl but this method was
less sensitive.
lB2. To establish whether, despite high levels of the
biosynthetic enzyme 3-fucosyltransferase in blasts, poor
expression of X and sialyl-X might be explained by lack of a
suitable precursor structure, we examined reactivity of the
cells with 1B2, an antibody that recognizes the terminal
disaccharide NAL that is the precursor of the X determinant. Expression of sialyl-NAL, the precursor of sialyl-X,
was assessed by comparing reactivity with 1B2 before and
after neuraminidase treatment of the cells. Untreated
neutrophils were positive as shown in Fig lc, but reactivity
was considerably enhanced by neuraminidase pretreatment
(Fig Id). On mature cells, therefore, that express both X
and sialyl-X, the terminal NAL disaccharide structures
exist in both sialylated and nonsialylated forms. In contrast,
all four blast cell populations were negative initially but
become strongly positive after neuraminidase treatment.
This result suggests that, on leukemic cells, the NAL
structures are present, but only as sialylated derivatives,
sialyl-NAL.
Glycosyltransferase Activities
2-Fucosyltransferase. 2-Fucosyltransferase activity could
not be demonstrated in neutrophils or in the blast cell
preparations, as reported previo~sly.~~
3-Fucosyliransferase. 3-Fucosyltransferase, which catalyzes the final step in the synthesis of the X determinant,
transfers fucose to NAL structures (see Fig 2) at the ends of
type 2 chains on glycoproteins and glycolipids. In confirmation of our previous observations,ZS activity of this enzyme
was readily demonstrable in neutrophils but was present in
much higher levels in leukemic blasts (Table 2).
\
I
A/Galpl -4GlcNAc-R
6
NeuAcaP9Galpl-4GlcNAc-R
I
2
a-3-fucosyif~nsferase
Galpl-4GlcNAc-R
3
I
I
NeuAca2-3Galpl4GlcNAc-R
3
I
1
Fuca
1
Fuca
X
Sialyl-X
Fig 2. Pathways of fucosylation and sialylation of NAL (Galgl4GlcNAc-R) end groups. Transfer of fucose, by 3-fucosyltransferase,
to the subterminal GlcNAc residue results in the synthesis of X,
whereas synthesis of sialyl-X proceeds via initial sialylation of the
terminal Gal residue, by 3’-sialyltransferase, followed by fucosylation.
Transfer of sialic acid in d - 6 linkage to the terminal Gal residue, as
shown by the left-hand pathway, blocks the synthesis of both X and
sialyl-X.
Table 2. GlycosyltransferaseActivities in Neutrophils and
Leukemic Myeloblasts
Cell
Type
2-Fucosyltransferase
3-Fucosyltransferase
Sialyltransferase
Neutrophils
0
(n = 7)
AML blasts
(n = 4)
0
737 2 109
(530-863)
8,466 f 6,014
(4,066-17,250)
347 2 100
(192-447)
2,883 f 223
(2,614-3,133)
Results are expressed as cpm [‘“C] sugar transferred/lO’ cells/h with
NAL as acceptor molecule. Mean value 2 standard deviation is quoted
with ranges given in parentheses.
Sialyltransferase. Sialyltransferase activity was present
in neutrophils but was much higher in the leukemic cells
(Table 2), a finding consistent with strong expression of
sialyl-NAL on blasts. Incorporation of [“C] sialic acid
measured with asialofetuin was virtually identical to that
with NAL (6.7 f 1.3 compared with 6.4 f 0.5 pmol sialic
acid transferred/106cells/h for blasts, mean f SD).
Separation of 3’- and 6’-sialyltransferaseproducts. To
explore further the lack of expression of sialyl-X on
myeloblasts despite apparently strong expression of sialylNAL and high levels of 3-fucosyltransferase and sialyltransferase, we distinguished 3’- and 6’4alyltransferase activities by separation of the reaction products (3’- and 6’4alylNAL) of the sialyltransferase activity with NAL as acceptor.
The results in Table 3 show that the activity in neutrophils
was exclusively due to 3‘-sialyltransferase, whereas in the
leukemic cells over 70% of the total activity was due to
6’4alyltransferase.
DMSO-InducedHL60 Cells
To determine the extent to which the differences between leukemic myeloblasts and normal mature cells might
reflect the changes occurring during normal myeloid maturation, we studied HL60 cells induced to differentiate with
DMSO. Morphologic evidence of maturation was present
in 80% to 90% of treated cells and by 6 or 7 days most had
progressed to myelocyte and metamyelocyte stages. Phorbo1 myristate acetate (PIMA)-stimulatedsuperoxide production (measured by cytochrome c reduction) increased from
0 to 18 nmol/107 cells/h by day 5. The morphologic changes
were accompanied by alterations in both antigen expression
and enzyme activity.
Table 3. Measurementof 3’- and 6‘-SialyltransferaseActivities in
Normal Neutrophils and AML Blasts
Patient
Sample
Total
Activity
AML 1
2
3
4
Neutrophils
19,437
23,418
12,159
27,282
2,036
3,333
3,834
6’-Sial yltransferase
15,738
19,220
9,156
19,926
(81)
(82)
(75)
(73)
- (0)
- (0)
-(0)
3’-Sialyltransferase
3,699 (19)
4,198 (18)
3,003 (25)
7,356 (27)
2,036 (100)
3,333 (100)
3,834 (100)
Results are expressed as cpm [“C] sugar transferred/assay. Figures
in parentheses indicate percentage of total activity.
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SKACEL ET AL
1456
Marker studies. HL60 cells express the X determinant
and this was unchanged or slightly reduced in most cells
after exposure to DMSO as reported by other groups.41
However, the expression of sialyl-X increased until by day 5
a population (20% to 30%) of the cells was strongly positive
(Fig 3).
Enzyme activities. In uninduced cells elevated levels of
3-fucosyl- and sialyltransferase were present, comparable
with those in leukemic blasts. After 24 hours of exposure to
DMSO, a reduction in the level of both enzymes was
apparent, (as illustrated in Fig 4) by which time the levels
had decreased to 60% and 40%, respectively, of those
measured in uninduced cells. A predominance of 6'sialyltransferase was measured in uninduced cells similar to
that found in leukemic blasts and the decrease in total
sialyltransferase activity that accompanied DMSO-induced
maturation was due to loss activity of this enzyme (Fig 5).
3'-Sialyltransferase remained unchanged at 24 hours and
decreased only slightly by day 6.
The DMSO-related changes in enzyme activities were
not due to an alteration in the rate of cell growth; this
continued at an exponential rate for 48 hours after exposure to DMSO. To exclude a direct effect of DMSO on
glycosyltransferase activities, we measured the activity of
o,
:I s
:i
3%
;3
25E
0
28
LL
.gs
25
$%
gT
E2
2E
$&
Fig 4. DMSO-induced changes in fucosyl- and sialyltransferase
activity in HL60 cells, measuredwith NAL as acceptor molecule. By 24
hours a marked reduction in the activity of both enzymes was
apparent with a continuing decrease during the following 5 days.
P-4-galactosyltransferase in HL60 cells that remained unchanged during the period of exposure (data not shown).
3-Fucosyl- and total sialyltransferase activities in KG1 cells
exposed to DMSO for 48 hours remained at 89% and 100%
of control values, respectively.
3-FucosyltransferaseActivity Measured with Sialylated and
Nonsialylated Acceptors
3-Fucosyltransferase activity in blasts and HL60 cells was
approximately 10-fold greater than in neutrophils when
measured with asialofetuin, as was found also with NAL as
acceptor. However, activity in the mature cells was similar
when measured with fetuin or asialofetuin, with a slight
preference for asialofetuin (see Fig 6), whereas in AML
blasts and HL60 cells there was a marked difference.
Activity measured with asialofetuin was approximately 5
times higher than with fetuin. Activity with nonsialylated
.25
.-275
0
aY
(0%
E!y
23
2g
s x
?E
m Q
5%
Fig 3. Cytofluorimetricanalysis of DMSO-induced changes in the
expression of X (left-hand column) and sialyl-X (right-hand column) in
HL60 cells. The expression of sialyl-X was weak on uninduced cells
but after DMSO was clearly stronger on 20% to 30% of cells.
Fig 5. Separation of ( 0 )6'- and (B)3'-sialyltransferaseactivities in
HL60 cells after exposure to DMSO. The decrease in activity at 24
hours was predominantly due to a reduction in 6'-sialyltransferase
activity. 3'-Sialyltransferase activity decreased only slightly.
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1457
EXPRESSION OF X DETERMINANT IN MYELOID CELLS
.
0
8
0-I
blasts
Days DMSO
PMN
HL60 cells
Fig 6. 3-Fucosyltransferase activity measured with glycoprotein
acceptors. In neutrophils, activity was low but similar with (0)
fetuin
and ( 0 )asialofetuin, whereas in leukemic cells activity was much
higher with asialofetuin but decreased in HL60 cells after DMSO
induction.
and sialylated low molecular weight acceptors (NAL and
3'-sialyl-NAL) showed a similar pattern (see Table 4), with
the blasts again showing 5 times greater preference than the
mature cells for NAL, as compared with 3'4alyl-NAL.
When HL60 cells were induced with DMSO, a marked
decrease in total 3-fucosyltransferase activity measured
with asialofetuin occurred (as with NAL as acceptor), but,
in addition, a change in enzyme specificity was apparent in
the direction of a less marked preference for asialofetuin as
cell maturation progressed (Fig 6). In blasts, in contrast to
the findings with sialyltransferase, a direct comparison
between incorporation of ["C] fucose measured with NAL
and asialofetuin showed that it was more than 100-fold
greater with NAL (549 & 391 compared with 1.1 f 0.6
pmol fucose transferred/106 cells/h), which suggests that
asialofetuin is a rather poor acceptor for this enzyme.
DlSCUSSlON
These results show that (1) expression of X and sialyl-X
on leukemic myeloblasts is markedly reduced compared
with neutrophils and (2) the activity of the biosynthetic
3-fucosyltransferase and sialyltransferases is different in
these two cell types. We suggest that the differences
between surface carbohydrate expression in immature and
mature cells can be accounted for by their different enzyme
profiles.
The virtual absence of reactivity of blasts with LeuMl
described here is not strictly comparable with results of
Table 4. Comparison of 3-FucosyltransferaseActivity in Neutrophils
and AML Blasts Using Nonsialylatedand Sialylated Low Molecular
Weight Acceptor Molecules
Patient Sample
AML 5
6
7
Neutrophils
NAL
J'-Sialyl-NAL
8,146
7,905
5,942
1,065
936
85 1
33 1
408
302
245
206
181
~
Results are expressed as cpm [14C1sugar transferred/106 cells/h.
other
because we deliberately used nonsaturating concentrations of antibody and, in all but case 2, weak
reactivity could be induced by increasing the antibody
concentration to a level at which marked agglutination of
neutrophils occurred. Lack of reactivity was not due to
antigenic masking of X by sialic acid, to any major extent,
because the expression of sialyl-X was also weak. The
enzymic studies suggest that lack of X expression is most
probably related to high levels of 6'-sialyltransferase, which
uses the precursor substrate, thereby preventing the synthesis of either X or sialyl-X as shown in Fig 2. In blasts,
3-fucosyltransferase and 6'-sialyltransferase would be expected to be the major competitors for NAL substrate. How
much X or 6'-sialyl-NAL is formed is therefore likely to be
determined by the relative activities of the two enzymes. A
strongly active 6'-sialyltransferase would use NAL end
groups to form 6'-sialyl-NAL structures, at the expense of
3-fucosyltransferase. In neutrophils, 6'-sialyltransferase activity is absent, leaving 3-fucosyltransferase and 3'sialyltransferase as the major competitors for NAL. The
synthesis of X and sialyl-X will therefore depend on the
relative activities of these two enzymes. Initial fucosylation
of NAL by 3-fucosyltransferase would result in X but not
sialyl-X, whose synthesis proceeds via sialylation, followed
by fucosylation as in Fig 2.'' Initial sialylation of NAL by
3'-sialyltransferase would result in 3'-sialyl-NAL, which
can then be converted to sialyl-X. The marker studies in
neutrophils are in agreement with this pattern of activity
and show surface NAL, sialyl-NAL, X and sialyl-X structures. The appearance of unsubstituted NAL end groups is
likely to be a result of lower overall activities of both
enzymes.
The apparent predominance of 6'-sialyltransferase activity in blasts and lack of effective competition for NAL by
3-fucosyltransferase is surprising in view of the markedly
elevated level of 3-fucosyltransferase activity compared
with sialyltransferase activity. There was no evidence for
the presence of fucosidases on prolonged incubations of
reaction mixtures, or for inhibitors. Limited availability of
GDP-fucose or divalent cations might be important controlling influences; 3-fucosyltransferase in neutrophils has an
absolute requirement for Mn", whereas 3'- and 6'sialyltransferase do not (unpublished observations). However, the higher comparative levels of 3-fucosyltransferase
were evident only when the low molecular weight compound NAL was used as acceptor; with asialofetuin as
acceptor molecule, 3-fucosyltransferase activity was actually considerably less than that of 6'-sialyltransferase
(1.1 & 0.6 compared with 6.7 2 1.3pmol sugar transferred/
106 cells/h). It is probable, therefore, that 3-fucosyltransferase demonstrates fine substrate specificity such that the
complexity of the oligosaccharide structures internal to
NAL or the surface conformation of glycoprotein and
glycolipid molecules influence its activity in a major way.
The length and complexity of oligosaccharide moieties
isolated from blasts and neutrophils are very different,42and
analysis of neutrophil glycoproteins suggests that glycosyltransferases acting on NAL structures at the ends of
tetra-antennary N-linked chains do exhibit branch specific-
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1458
SKACEL ET AL
ity.” Gangliosides with terminal 2-6 sialyl type 2 chains and
internal fucose residues have been shown to be much
higher in malignant than in normal t i ~ s u e s ~and
~ ~ ”it is
possible that 2-6 sialyl type 2 chains on leukemic myeloblasts could also be internally fucosylated. However, such
fucose would not give rise to X or sialyl-X structures and
would not have been detected in this study. Although the X
determinant can be demonstrated on both glycoprotein and
glycolipid isolated from HL60
it is not known
whether on blasts it occurs preferentially on one or the
other. Glycolipid acceptors were not used in this study but
clearly the use of different and more complex acceptors,
together with detailed examination of the exact distribution
of X during early stages of cell maturation, will be necessary
to fully appreciate these complex interdependent mechanisms.
The expression of X during normal myeloid maturation
does appear to be developmentally regulated. It first
appears at or just before the promyelocyte stage”.46but is
absent in earlier precursors. Lack of expression on leukemic blasts due to high levels of 6’4alyltransferase may,
therefore, simply be a feature of immaturity rather than
leukemic transformation with the occurrence of X and/or
sialyl-X influenced in a major way by the interaction
between 6’-sialyltransferase, 3‘-sialyltransferase, and 3-fucosyltransferase which, in turn, will be determined by the
precise stage of maturation arrest of the leukemic cells.
The strong expression of X on HL60 cells, which also
have high levels of 3-fucosyltransferase and 6’-sialyltransferase, suggests that, in these cells, both enzymes compete
effectively for substrate. The changes in enzyme activity
after exposure to DMSO also suggest a developmentally
regulated reduction in 3-fucosyl- and 6‘-sialyltransferase
activities. These changes were accompanied by a population of cells with increased expression of sialyl-X,
presumably because the decrease in both enzyme levels
encouraged more effective competition for NAL by 3‘sialyltransferase.
The reduced activity with sialylated substrates demonstrated by the 3-fucosyltransferase in blasts is also likely to
have an important regulatory influence on the expression of
sialyl-X. The marker studies did not distinguish between
surface 3’- and 6’4alyl-NAL structures, but even if a
significant proportion of the sialyl linkages were a-2,3 it is
probable that these structures could not have been converted to sialyl-X by the 3-fucosyltransferase present at this
stage of maturation. Whether the observed differences in
substrate specificity of the 3-fucosyltransferase during cell
maturation are due to changes in a single enzyme or
whether two distinct enzymes are expressed independently
is not clear. However, Johnson and Watkin~~’.~’
reported
similar differences in purified 3-fucosyltransferases from
CGL cells and normal neutrophils, and both enzymes
appeared to be distinct from the 3-fucosyltransferase isolated from plasma. Two 3-fucosyltransferases are also
present in mutant CHO cells lines and show different
specificities for sialylated
Interestingly, the
introduction of a 3-fucosyltransferase cDNA from HL60
cells into wild type CHO cells by transfection resulted in
demonstrable 3-fucosyltransferase activity that was distinct
from the two described in mutant cells but resembled the
HL60 enzyme described here?’
The biologic role of sialyl-X is unknown. However, it is
tempting to speculate that it may be important in cellular
adhesion mechanisms, particularly those that determine
the release of mature neutrophils from the bone marrow
into the circulation. X and sialyl-X structures are abundant
on most, if not all, N-linked glycoproteins isolated from
mature neutrophil^,'^^'^ and the integrin glycoproteins, LFAl
Macl, and p150,95, which are important mediators of cell
adherence, have predominantly N-linked oligosaccharides.
They are rich in X structures and it is highly probable that
they also express sialyl-X. It has been suggested that
increased sialylation of cell surface oligosaccharides in
CGL is responsible for the premature release of cells into
the circulation and other in vitro abnormalities of adherence: The precise nature of this aberrant sialylation is not
known, but whether it involves increased expression of
sialyl-X and how this relates to altered glycosyltransferase
activity is worthy of further study. Elevated activity of an
a2,3-sialyltransferase that acts on Galpl3GalNAc residues
in 0-linked oligosaccharides has recently been demonstrated in CGL cells:’ but to what extent this is responsible
for increased sialylation of functional importance is not
clear because in neutrophils only one glycoprotein, leukosialin, has been found to contain predominantly 0-linked
structures.4’ The development of molecular techniques
such as transfection of cloned glycosyltransferases to alter
terminal glycosylation sequence^^^^^' will greatly improve
our understanding of the biologic importance of cell surface
carbohydrate structures and the factors regulating their
synthetic control in malignant transformation and during
normal differentiation.
ACKNOWLEDGMENT
We are grateful to Dr A.S.R. Donald for the unlabeled GDP-Lfucose and oligosaccharidesand to Prof S-I. Hakomori and Prof P.
Terasaki for the gifts of MoAbs. We thank Prof A.W. Segal for help
with cytochrome c measurement and Dr M.Y. Gordon for the KG1
cells.
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1991 78: 1452-1460
Enzymic control of the expression of the X determinant (CD15) in
human myeloid cells during maturation: the regulatory role of 6sialytransferase
PO Skacel, AJ Edwards, CT Harrison and WM Watkins
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