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(Received for publication, July 10, 1995; and in revised form, August 21, 1995) From the
Glycophorin A is a protein with an abundant glycosylation (60%
carbohydrate by weight), and studies have suggested that resistance of
target cells to natural killing may be correlated with the level of
glycophorin A expression. To assess the role of glycophorin A and of
its carbohydrates in sensitivity to lysis by natural killer (NK) cells,
the glycoprotein was inserted into the membrane of K562 target cells
using electropulsation. Peripheral blood lymphocytes were used as
effector cells. When glycophorin A was inserted into the membrane, the
level of resistance to NK cell attack increased with the number of
glycophorin A molecules electroinserted. The resistance to lysis was
not due to a defect in target cell-effector cell binding.
Electroassociation of glycophorin A did not cause a decrease in the
expression of either ``positive signals'' for NK cells (such
as CD71, CD15, and CD32 antigens) or cellular adhesion molecules (CD18,
CD29, CD54, and CD58). Furthermore, electroinsertion of glycophorin A
did not trigger any ``negative signals,'' such as class I HLA
antigen expression. Finally, it was shown that the sialic acid and O-linked oligosaccharides of glycophorin A did not play any
role in its effect against NK cells. Conversely, the unique N-linked oligosaccharide was shown to be essential for
resistance to occur.
Natural killer (NK) ( Although there has been extensive characterization of many features
of NK cells, the NK cell receptors and their ligands on the target cell
surface have remained elusive for a long time. Thus, the sensitivity of
target cells has been considered alternatively as being due to the
expression of immature structures and viral antigens (2) or to
an increased expression on the cell surface of normal glycoproteins
such as transferrin receptor (CD71) and CD32
antigen(3, 4, 5) , of glycolipids such as
asialo-G In addition to specific NK ligand(s), cell
adhesion molecules (such as CD54, CD58, CD56, lymphocyte
function-associated antigen 1, and CD2) are also involved in cellular
cytotoxic mechanisms(18, 19) . However, these
accessory molecules do not appear to represent NK ligands by
themselves, but rather as strengthening target cell-effector cell
conjugates after the initial cognate interaction between the target
cell and the NK cell(19) . Recently, it has been established
that resistance of K562 cells to NK cells can be correlated with an
increase of glycophorin A on the cell
surface(20, 21) . However, it has never been suggested
that this glycoprotein was capable of protecting tumor cells against
lysis by NK cells. The insertion of this molecule into the membrane of
cells sensitive to NK cell attack gives a direct measure of its
involvement in NK cell-mediated lysis. In addition, the abundant
glycosylation of this molecule allows us to use an experimental
approach to determine the role of the glycoprotein sugars in the
mechanism of resistance to NK cell-mediated lysis. In this report,
glycophorin A was inserted into the K562 cell membrane. This was
achieved by electroinsertion, which has recently been used to insert
proteins with a membrane spanning sequence into mouse red blood cell
membranes (22) and into nuclear
cells(23, 24) . We demonstrate that such modified
cells are resistant to NK cell-mediated lysis. The carbohydrate nature
of the molecular entity that is involved in this modulation has also
been determined.
Peripheral blood mononuclear cells from normal human
volunteers were isolated on Ficoll-Hypaque (MSL, Eurobio). Peripheral
blood lymphocytes (PBL) were obtained after peripheral blood
mononuclear cell depletion of adherent cells by 1 h of incubation at 37
°C in plastic Petri dishes.
After fluorescence intensity calibration of the cytofluorometer
using quantitative fluorescent microbead standards, we used Simply
Cellular
Figure Z1:
Structure 1.
Figure Z2:
Structure 2.
O-Glycanase (TEBU, Le Perray, France)
catalyzes the hydrolysis of the Gal-GalNAc disaccharide core attached
to serine or threonine residues of asialoglycoproteins. In this
experiment, we used asialoglycophorin since sialic acid was observed to
inhibit the enzyme activity(26) . Cells (3 Endoglycosidase F (Sigma, St. Quentin, France) cleaves the link
between the two N-acetylglucosamine residues linking the
glycan moiety to the asparagine of the protein backbone(27) .
Cells (3 These
conditions were chosen to reach complete deglycosylation of susceptible
asparagine-, serine-, or threonine-linked oligosaccharides. After
enzyme treatment, the cells were washed, checked for staining with
fluorescent lectins, and used in the cytotoxicity assay.
Figure 1:
Electroinsertion of glycophorin A into
K562 cell membrane. A, effect of the concentration of
glycophorin A on the number of molecules inserted. K562 cells
(10
We previously demonstrated using Chinese hamster ovary cells that
electropermeabilization mediates a stable insertion of GPA into the
cell membrane(24) . To determine the stability of the
interaction between GPA and pulsed or non-pulsed K562 cells, the GPA
molecules were stained by mAb at 0, 24, and 48 h after pulsing. The
percentage of fluorescent cells strongly decreased when the cells were
not electropulsed and cultured for 24 and 48 h (27 ± 4 and 6
± 1%, respectively, versus 41 ± 5% at 0 h),
whereas it was stable at 24 and 48 h after electroinsertion (83
± 6 and 79 ± 6%, respectively, versus 89
± 5% directly after the pulse). In addition, the number of bound
GPA molecules/cell decreased from 12,000 to 2500 after 1 day of
non-pulsed cell culture and only decreased by a 2-fold factor every 24
h after electropulsation. Since the growth rate of K562 cells is one
doubling/24 h, the above results indicate that (i) the GPA molecules
bound to the cell membrane (termed electroinserted GPA) were shared
between the daughter cells (Fig. 1B) and (ii) the
GPA-cell membrane interaction is stable after pulsing. Consequently,
the nature of the interaction with the membrane of electropulsed cells
is likely to be different from that of the control cell membrane.
Figure 2:
Effect of glycophorin A electroinsertion
on K562 cell susceptibility to NK cell-mediated lysis. K562 cells
(10
NK cell-mediated cytotoxicity,
calculated from individual effector cell/target cell curves, was
cumulated and expressed as lytic units (LU The spontaneous lysis of target cells induced by natural killer cell
activity is accomplished in two main distinguishable steps: binding
between target and effector cells and post-binding events leading to
target cell destruction. To examine the possibility that the decreased
lysis was due to a defect in the first step of binding, direct
conjugate-forming cell assays were performed after CD3
Figure 3:
Effect of glycophorin sialic acid on
susceptibility of K562 cells to lysis by NK cells. K562 cells
(10
We were
also interested in determining whether changes, other than sialic acid,
in glycosylation of the inserted glycophorin would alter the resistance
of GPA
Figure 4:
Control of enzyme treatments. K562 cells
with electroinserted GPA (GPA+) (6.2
Figure 5:
Effect of glycosidases on K562 cell
sensitivity to NK cell-mediated lysis. K562 cells (10
In this report, we have shown the direct involvement of
glycophorin A in the NK cell-mediated lysis mechanism by
electroinserting this molecule into the K562 cell membrane. In our
system, Our present data show that the resistance to
natural killer cells correlates with the increase in the number of GPA
molecules on the K562 cell membrane. A significant resistance to NK
cell attack was observed with In our system, the protective effect of GPA might be due to masking
(i) of cellular adhesion molecules involved in NK cell mechanisms
and/or (ii) of putative epitopes able to deliver an activating signal
to NK cells. Both explanations are unlikely because we demonstrated
that GPA electroinsertion did not alter the accessibility of cell
adhesion molecules (CD18, CD29, CD54, CD56, and CD58), CD71, and CD15
by mAbs. In addition, no consistent reduction of target cell binding to
NK cells was recorded in the experiments using GPA GPA is a sialoglycoprotein made up of
131 amino acid residues with no disulfide bonds and is composed of 60%
carbohydrate by weight. The sugar chains contain On the contrary, this work indicates a role for the N-linked oligosaccharide of GPA in NK cell activity modulation
because endoglycosidase F, which eliminates N-linked
oligosaccharide structure, completely reverses the resistance to NK
cells. On the other hand, the glycophorin O-linked
oligosaccharide seems to have no effect on NK cell activity. However,
glycosidase treatment of control K562 cells did not clearly alter their
reactivity to lectins or their sensitivity to NK cells. This is
probably due to the biosynthesis of new sugar chains for the membrane
molecules, whereas these mechanisms cannot act on electroinserted GPA.
The GPA molecule contains 15 O-linked tetrasaccharides and a
single complex N-linked oligosaccharide (13 monosaccharide
residues). Several works have emphasized the importance of carbohydrate
molecules in target cell-effector cell interactions(17) .
According to the structures and the location of the extra sugar
residues added to a pentasaccharide common core, all the N-linked sugar chains are classified into three
subgroups(31) : (i) complex-type sugar chains, (ii) high
mannose-type sugar chains, and (iii) hybrid-type sugar chains.
Recently, by using N-glycan processing inhibitors, it has been
demonstrated that the presence of high mannose-type glycans on K562
cells correlates with increased binding of effectors and a greater
susceptibility to lysis. The high mannose-type glycans can influence
the NK cell-target cell interaction at the level of the adhesion
molecules(32) . However, the N-linked sugar chain of
GPA belongs to the complex-type sugar chain, and its presence on the
target cell surface decreases the susceptibility to NK cells without
altering the conjugate formation. Thus, two types of sugar chain with
strong differences in their structure can modulate the NK cell activity
in opposite directions. As mentioned above, NK cell receptors have
been identified that bind MHC class I molecules and inhibit natural
killer cell activation(14, 15) , and it is possible
that carbohydrates of the MHC glycoprotein participate in the
inhibition of NK cell-mediated cytotoxicity(15) . On the other
hand, it has recently been shown in mice that members of the type II
transmembrane lectin family are preferentially expressed on NK cells
and can deliver either positive (NKR-P1 protein) (33) or
negative (Ly49 protein) signals to the effector cell(34) . In
addition, NKR-P1 binds a diversity of oligosaccharides that activate NK
cells and cytotoxicity(16) . In humans, the same type of
membrane proteins (named NKG2 proteins and with carbohydrate-binding
external domains) was also detected on NK cells(34) . Their
ligands and their effect on natural killing are unknown(34) .
It is possible that sugar residues of the N-linked sugar
chain, shared by several glycoproteins, fulfill the role of ligand
molecule for these putative NK cell receptors or for other unknown
molecules characterized by lectin activity. From this point of view,
sugar residues of the N-linked oligosaccharide of GPA could be
a ligand for a putative receptor that delivers a negative signal to NK
cells.
Volume 270,
Number 45,
Issue of November 10, 1995 pp. 26970-26975
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ROLE OF OLIGOSACCHARIDES (*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)cells are generally considered
part of the first host defenses against neoplastic and infectious
diseases. NK cells are CD3 large granular lymphocytes
and can be functionally defined as cells that mediate
non-histocompatibility-restricted killing of some target cells. These
lymphocytes are spontaneously cytotoxic against some tumors and virally
infected cells via nonspecific mechanisms. In addition, NK cells are
able to kill certain normal cells in vitro(1) .
and G
(6, 7) , of
oligosaccharides such as 3-fucosyl-N-acetyllactosamine (CD15
antigen)(8) , and of peptides such as a 42-kDa
polypeptide(9) . These molecules could act as ``positive
signals'' on NK cells. Conversely, it has been shown that a high
level of cell membrane sialylation decreases the sensitivity of target
cells(6, 10) . More recently, it has been demonstrated
that the expression of MHC class I molecules protects tumor cells
(predominantly those of lymphoid origin) against NK cell
attack(11, 12, 13) , acting as a
``negative signal'' for NK cell-mediated lysis. Moreover,
inhibitory receptors for NK cell activation have been identified, and
they bind MHC class I molecules(14, 15) . Finally, in
the past year, a natural killer cell receptor proved to be specific for
ubiquitous oligosaccharides and triggers the NK cell cytolytic
mechanism(16) . In conclusion, the target molecules involved in
recognition of target cells by NK cells may contain peptidic or
carbohydrate residues that can activate or inhibit the NK cell-mediated
killing(17) .
Cells
K562 cells were grown at 37 °C in a
humidified 5% CO
incubator. The culture medium was
Eagle's minimum essential medium (MEM 0111, Eurobio, Paris)
supplemented with 10% fetal calf serum (Boehringer, Mannheim, Germany),
penicillin (100 IU/ml), streptomycin (100 mg/ml), and L-glutamine (0.58 mg/ml). The cell density was maintained
between 2 
10
and 1.6 10
cells/ml.Chemicals
The pulsing buffer contained 125 mM sucrose, 69 mM KCl, 1 mM MgCl, and
10 mM potassium phosphate buffer, pH 7.4. Its low ionic
content reduced the joule heating associated with the electric field
pulse. The washing phosphate-buffered saline (PBS) contained 42
mM KHPO
, 8.3 mM
NaHPO/HO, and 125 mM NaCl,
pH 7.4. All chemicals were from Sigma. Glycophorin A type MN (GPA) and
asialoglycophorin were obtained from Sigma.Electroinsertion
Electroinsertion was carried out
as described previously for Chinese hamster ovary cells (24) using a CNRS electropulser (Jouan, St. Herblain, France)
able to deliver square wave pulses, the parameters of which (voltage,
pulse duration, and number and frequency of pulses) can all be adjusted
separately. The pulses were monitored using a 15-MHz oscilloscope
(Enertec, St. Etienne, France). Electrodes were parallel and flat with
an anode-cathode distance of 1.5 mm. Since electroinsertion is a
back-effect of electropermeabilization(24) , various values of
field intensity and pulse number and duration were applied to K562
cells to obtain both maximum viability and membrane permeabilization.
Optimal conditions were one square wave pulse of 7-ms duration at 0.6
kV/cm. Consequently, electroinsertion was performed using K562 cells
(10
), which were incubated at 37 °C for 15 min with
glycophorin A or asialoglycophorin in a total volume of 18 µl and
then submitted to a permeabilization pulse. After pulse application,
the cells were washed three times in PBS (400 g for 4
min at room temperature) and analyzed by immunofluorescence. A control
sample was subjected to all these steps, except that no pulse was
applied. Cell viability was checked by using the trypan blue exclusion
test after pulsing (always >95%) and by observing cell growth and
cell viability 24 h (always >98%) after pulsing.Immunofluorescence Assay
GPA, asialoglycophorin,
transferrin receptor (CD71), CD15 antigen, MHC class I antigen, and
cellular adhesion molecules were detected by an indirect staining
method using, respectively, an anti-GPA mouse monoclonal antibody (mAb)
that also stains the asialoglycophorin and that binds to amino acids
27-39 of GPA (Immunotech, France), anti-CD71 mAb and anti-CD15
mAb (Becton Dickinson, Mountain View, CA), anti-MHC class I W6/32
(American Type Culture Collection, Rockville, MD), and anti-adhesion
molecules, anti-CD54 mAb, anti-CD58 mAb, anti-CD29 mAb, anti-CD18 mAb,
and anti-CD56 mAb (Immunotech, Marseille, France). Purified mouse IgG
was used as a control reagent. Fluorescein isothiocyanate-labeled
(F(ab`)) goat antibodies against mouse immunoglobulins were
used as a second-step reagent for indirect staining. The cells were
then washed in PBS and analyzed by flow cytometry (Fac Scan, Becton
Dickinson). Two parameters were used: the percentage of fluorescent
cells after autofluorescence background subtraction and the mean of
fluorescence intensity of positive cells expressed in arbitrary units. microbeads (Becton Dickinson) to quantify the
inserted glycophorin. These microbeads may be considered as
``model lymphocytes'' since they are approximately the size
of lymphocytes. They bind mouse monoclonal antibodies (such as goat
anti-mouse IgG antibodies) that are covalently bound to the microbead
surface. The beads were calibrated in terms of the number of monoclonal
mouse IgG molecules they bind, allowing determination of the effective
fluorescence/protein ratio. We applied the same method of antibody
labeling to these microbeads as the one used for cells.
Endoglycosidase Assays
Glycophorin A is composed
of 60% carbohydrate by weight, with most of the sugars being 15 O-linked tetrasaccharides that are attached to serine or
threonine and that have the structure shown in Fig. Z1(25) . A single complex N-linked
oligosaccharide is also present. Its structure is as shown in Fig. Z2.
10) were mixed with 25 milliunits/ml O-glycanase.
The mixture was then incubated for 12 h at 37 °C. 10) were mixed with 60 milliunits/ml of
enzyme. Incubation was then conducted at 37 °C for 12 h.Checking Enzyme Treatments
From knowledge of GPA
oligosaccharide structures, fluorescein isothiocyanate-conjugated
lectins (Dolichos biflorus or Lens culinaris (Sigma))
were used to evaluate glycosylation patterns in control and
endoglycosidase-treated cells. To evaluate the efficiency of
endoglycosidase F treatment, K562 cells were incubated for 15 min with L. culinaris lectin (25 µg/ml), which is known to react
specifically with 
-D-mannose. The cells were then washed
in PBS and analyzed by flow cytometry. In the case of O-glycosylation, K562 cells were incubated for 15 min with D. biflorus lectin (50 µg/ml), which is known to react
specifically with -D-GalNAc. The cells were then washed
in PBS and analyzed by flow cytometry. These conditions were chosen
(after testing various lectin concentrations) to demonstrate almost
complete staining (>95% of positive cells) of K562 cells.Cytotoxicity Assay
The NK cell activity of PBL
from healthy donors was tested in a standard 4-h Cr
release assay against target cells labeled with
Cr as
described previously(20) . Briefly, various numbers of PBL
(effector cells) were mixed in triplicate with 10
labeled
target cells in microtiter plates. After 4 h at 37 °C, 100 µl
of the supernatants were counted in a -counter. The percentage of
cell-mediated lysis was calculated as follows: % cell-mediated lysis
= ((cpm
- cpm
)/(cpm
- cpm))
100. cpm was
determined by counting an aliquot of labeled target cells, and
cpm (spontaneous release) by counting the supernatant
from wells without effectors. Cytotoxicity calculated from individual
effector cell/target cell curves is expressed as lytic units
(LU
)/10
effector cells. One LU is
defined as the number of lymphoid cells required to lyse 25% of
10
target cells under the assay conditions used.Target Cell Binding Assay
PBL were depleted of
CD3 cells by using anti-CD3 reactive magnetic
microbeads (Immunotech). The percentage of effectors conjugated to
target cells was determined using the procedure of Grimm and Bonavida (28) . Briefly, 100 µl (10
/ml) of cells from
suspension of the effector and target cell populations in culture
medium + 10% fetal calf serum were mixed in centrifuge tubes. The
tubes were placed in a water bath for 5 min at 30 °C. The cells
were then centrifuged for 5 min at 400 g at room
temperature to promote conjugate formation. Then the pellet was
resuspended 10 times with a micropipette. A small drop of this
suspension was removed, and the percentage of the conjugates was
determined in a hemocytometer.
Electroinsertion of Glycophorin A into the Membrane of
K562 Cells
When K562 control cells were examined with our
anti-GPA mAb, 10% of the cells were weakly positive (
1000
molecules of GPA/cell). Electroinsertion of GPA into the plasma
membrane was mediated by submitting a GPA/K562 cell mixture to an
electric field pulse (one pulse of 7-ms duration at 0.6 kV/cm).
Electrical field application allowed us to insert the GPA, detected by
anti-GPA monoclonal antibody, on >90% of the K562 cells. However, a
relatively large percentage (44 ± 5% for 89 µM GPA; n = 3) of fluorescent cells was detected even in the
absence of electric field and for weak field intensities. In this cell
population, the number of stained GPA molecules was always
10
/cell. With an electrical pulse, the number of
detectable glycophorin molecules/fluorescent cell rose with the
increase in glycophorin concentration in medium to reach 6
10 molecules in the cell membrane when there was 89
µM GPA in the pulsing medium (Fig. 1A).
) incubated with glycophorin A (various concentrations)
were subjected (
) or not (
) to electropulsation (one pulse
of 7-ms duration at 0.6 kV/cm), washed, and then stained by an
anti-glycophorin monoclonal antibody and subsequently by
fluorescein-conjugated goat anti-mouse antibodies. The mean number of
stained molecules/cell was determined by the quantification of the
fluorescence intensity using Simply Cellular microbeads. B, stability of the glycophorin insertion. K562 cells
(10
) were treated (89 µM glycophorin) as
described above, and immunostaining was run for 0, 24, and 48 h after
pulsing (one pulse of 7-ms duration at 0.6 kV/cm) (black bars)
or after incubation in the absence of electrical field (white
bars). The results shown in A and B are the
means ± S.D. of three separate
experiments.
Electroinserted Glycophorin A Decreases K562 Cell
Susceptibility to Attack by NK Cells
Fig. 2(A1 and A2) shows a representative experiment of the effect
of glycophorin insertion on susceptibility to NK cell-mediated lysis.
The sensitivity of K562 cells to NK cell attack increased when the
cells were pulsed without GPA (Fig. 2A1), whereas the
presence of 10
GPA molecules at the surface of 40% of
the non-electropulsed cells did not alter susceptibility to lysis.
Considering that the electropulsed cells were the effective target
control, glycophorin had an inhibiting effect at all concentrations
tested, and the resistance to NK cell lysis increased with the number
of glycophorin molecules on the cell surface (Fig. 2A2).
) were subjected to electropulsation (one pulse of 7-ms
duration at 0.6 kV/cm) with or without GPA. After washing, K562 cell
samples were tested in triplicate in the Cr cytotoxicity
assay. A1 shows the effect of electrical treatment in the
absence of GPA (
). Controls were K562 cells incubated in the
absence of the pulse in pulsing buffer with (+) or without ()
89 µM GPA. Results are expressed as a function of various
effector cell/target cell ratios. A2 shows the decrease of
K562 cell sensitivity to NK cell-mediated lysis as a function of the
number of electroinserted glycophorin molecules/cell: 0 (), 28
10
(
), 33 10 (),
40
10 (
), and 60 10
(). A1 and A2 are results from the same
experiment. S.D. values were always <10% of the mean of triplicates.
Two other separate experiments, using PBL from two different normal
donors, gave identical results. B shows the cytotoxicity
potential expressed as lytic units (LU
) and calculated
from the three experiments described above (mean ± S.D.). K562
cells were electropulsed in the absence (P-K562) or presence
(GPA
) of GPA (89 µM) or were incubated
without pulsing in pulsing buffer in the absence (K562) or
presence (+GPA) of GPA.
) (Fig. 2B). The sensitivity of the K562 cells with
electroassociated GPA (termed GPA
cells) was reduced
by a factor of
2 as compared with the electropulsed K562 cells in
the absence of GPA (P-K562; p < 0.001) and with the
non-pulsed K562 cells with or without GPA in the pulsing medium (p < 0.001 and p < 0.001, respectively) (Fig. 2B). In addition, no effect was observed when GPA
was directly added at different concentrations (from 8.9 to 89
µM) to the NK cell cytotoxicity assay (data not shown).
cell depletion from PBL suspensions. Table 1illustrates
that the GPA
K562 cells were as efficient in binding
NK cells as the control samples.
Effect of the Glycophorin Insertion on the Expression of
Surface Antigens
To determine whether electroinsertion of GPA
into K562 cell membranes triggers a modulation of the expression of
negative or positive signals for NK cells, CD71, CD15, and MHC class I
antigens and certain adhesion molecules were stained by specific mAbs.
No significant difference was observed between GPA
cells and the control samples (Table 2). Similar results were
obtained when the cell positivity to mAbs was expressed as mean
fluorescence intensity (data not shown). These results indicate that
the resistance to NK cell attack induced by glycophorin insertion into
the membrane depends neither on the triggering of MHC class I antigen
expression nor on the modulation of the expression of other molecules.
In addition, they demonstrate that insertion of GPA in the cell
membrane does not interfere with accessibility of mAbs to natural
epitopes of the cell surface.
Effect of the Glycosylated Structures on NK Cell-mediated
Lysis
It has been reported that cell-surface sialic acid may
contribute to the development of NK cell resistance directly or by
masking the target structure(s) to NK cells(6, 10) .
As GPA is a highly sialylated protein, we tested the hypothesis that
the resistance induced by GPA insertion could be correlated to a simple
contribution from the sialic acid. Asialoglycophorin was
electroinserted into K562 cells. Fig. 3shows that the same
number of asialoglycophorin and GPA molecules induced the same
resistance of target cells to NK cell-mediated cytotoxicity.
) were subjected to electropulsation (one pulse of 7-ms
duration at 0.6 kV/cm) in the absence of proteins () and in the
presence of GPA (89 µM) () or asialoglycophorin (89
µM) (
) corresponding to 5.8 and 6.1 10 electroassociated molecules/cell, respectively. Controls were
K562 cells incubated without pulsing in pulsing buffer (). After
washing, samples were tested in the Cr cytotoxicity assay.
The results are the means of triplicates (S.D. < 10% of the mean).
Similar results were obtained in two separate experiments using PBL
from two other normal donors.
cells to NK cell-mediated lysis. After GPA
electroinsertion into the cell membrane, N- and O-glycosylation were eliminated by enzymatic treatment as
indicated under ``Materials and Methods.'' A comparative
study of lectin binding to enzyme-treated and -untreated K562 cells
(GPA
and K562) revealed a significant reduction of
binding after treatment of GPA
cells, suggesting a
decrease of N-linked (Fig. 4A) or O-linked (Fig. 4B) oligosaccharides, whereas
the enzyme treatment did not affect the glycosylation of control cells.
The effect of enzyme treatments on the susceptibility of K562 cells to
NK cell-mediated lysis was then determined while fluorescein-labeled
lectins were used in parallel to control deglycosylation on the target
cells. K562 cells treated with endoglycosidase F or O-glycanase were as sensitive to NK cell lysis as the control
samples. GPA
cells treated with the O-glycanase enzyme were lysed at the same level as the
untreated GPA
cells (Fig. 5A), whereas
the elimination of N-linked oligosaccharides led to the
restoration of the K562 cell sensitivity to NK cell attack (Fig. 5B).
10 ± 0.4 10 molecules/cell) and those
without (K562) were treated (+ Enzyme) or not (- Enzyme) with endoglycosidase (37 °C, 12 h). After
endoglycosidase F treatment, the cells were washed and then stained
with fluorescein-conjugated L. culinaris lectin (50 µg/ml,
15 min) (A). After O-glycanase treatment, cells were
stained with fluorescein-conjugated D. biflorus lectin (25
µg/ml, 15 min) (B). Cells were analyzed by flow cytometry.
Results are means ± S.D. of three separate experiments. AU, arbitrary units.
) were
subjected to electropulsation (one pulse of 7-ms duration at 0.6 kV/cm)
in the presence (89 µM) ( and ) or absence
(
) of GPA or were incubated without pulsing in pulsing buffer
( and ). K562 cells () and K562 cells with
electroinserted GPA () were treated with O-glycanase
(5.4 10 GPA molecules/cell) (A) or
endoglycosidase F (5.9 10 GPA molecules/cell) (B). Samples were then tested in the Cr
cytotoxicity assay. A and B are results (S.D. <
10% of the mean of triplicates) from two separate experiments performed
with effector cells from two different donors. Each is representative
of three separate experiments using PBL from different
donors.
6
10 GPA molecules/cell were correctly
oriented so as to be detected by an anti-GPA monoclonal antibody that
reacts with an extracellular epitope of GPA. As previously demonstrated
with Chinese hamster ovary cells(23, 24) , the
inserted protein was stable in the membrane, was transferred from
mother to daughter cells, and was able to diffuse freely across the
surface of the cell membrane (data not shown). On the non-electropulsed
cell membrane, it is likely that the GPA molecule was only adsorbed, as
previously reported(24) . On these control cells, the presence
of 10
GPA molecules did not induce resistance to
lysis, suggesting that the putative inhibitive structure is not
efficiently presented to NK cells when GPA was only adsorbed on the
target cell surface.3
10 electroinserted molecules/cell (i.e. 1 GPA molecule/2
10 nm of cell surface or 1 GPA
molecule/2 10 phospholipids). Consequently, the
resistance to NK cell-mediated lysis can be effective when the GPA
number is higher than a threshold value of between 10 and 3
10 molecules. On the other hand, the target
resistance induced by electroinserted GPA may be attributed, at least
partly, to the stabilization of the membrane. Indeed, a GPA molecule
incorporated into experimental bilayers interacts with
500-1000 phospholipids(29, 30) , which
could alter the membrane stability. However, this possibility seems
somewhat unlikely since (i) the interaction between GPA and
phospholipids would involve only 5-10% of the membrane
phospholipids; (ii) GPA electroinserted into Chinese hamster ovary cell
membrane showed a free lateral diffusion with a diffusion coefficient
in agreement with what would be expected for an intrinsic protein
embedded in a viable cell membrane(23) ; and (iii) removal of N-linked oligosaccharide moieties suppresses the resistance.
This relationship between the resistance to NK cell-mediated lysis and
the inserted glycophorin A level is in accordance with previous results
that showed a reduced sensitivity to NK cell lysis of (i) K562 cells
differentiated in vitro by drugs that increased the levels of
GPA on the cell surface (10, 21) and (ii) a K562 cell
clone expressing a very high number of GPA molecules (21) . It
is important to stress that the presence of
6
10 glycophorin molecules on the cell surface does not confer total
resistance to NK cell lysis. This can be explained by the intervention
of several NK cell subsets using different mechanisms to lyse K562
cells in chromium assay or by the choice of each effector cell between
different recognition strategies. Moreover, the resistance to NK cells
might only be partial because GPA cells maintain
malignant features, i.e. positive signals for NK cells such as
the absence of MHC class I antigens and/or the presence of ligands able
to activate NK cells such as CD71 and CD15 antigens or some
carbohydrate determinants (3, 4, 5, 6, 7, 8, 9, 16) .
cells. However, it is well known that conjugate formation
measures integrin binding and represents an early stage of binding that
is followed by a cellular reorientation and firm adhesion that probably
uses other molecules. Consequently, GPA might modulate a limiting step
at the level of the membrane molecular interactions involved in
post-conjugating mechanisms, such as in the stabilization of binding
and/or in the lethal hit (e.g. it is quite possible that GPA
blocks perforin adherence).
45% sialic acid
by weight(25) . As mentioned above, several works have shown
that the level of sialic acid on target cells may modulate their
sensitivity to NK cell-mediated lysis, e.g. neuraminidase
mediates an increase of the susceptibility to NK cell
lysis(10) . However, in agreement with previous
data(20) , we have demonstrated that sialic acid is not an
essential residue in the inhibition of NK cell-mediated lysis due to
inserted GPA.
), ganglioside; MHC, major histocompatibility complex;
PBL, peripheral blood lymphocyte(s); PBS, phosphate-buffered saline;
GPA, glycophorin A type MN; mAb, monoclonal antibody.
We thank Dr. J. J. Fournié, Dr.
M. Thomsen, and Dr. G. J. Fournié for helpful
discussions and suggestions. Thanks are due to Dr. J. Robb and Dr. P.
Winterton for checking the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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