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(Received for publication, July 6, 1995; and in revised form, October 3, 1995) From the
The monoclonal antibody U5, which is a potent inducer of
proliferation in human T-cells, was found to bind to an
alkali-sensitive derivative of ganglioside G
Gangliosides are sialic acid-containing glycosphingolipids
(GSLs) (
CD3 CD16 B-cells were also obtained by the
panning technique. The adherent cell fraction contained 80%
CD20 Monocytes were purified to >90% by adherence to tissue culture
dishes (Greiner, Solingen, FRG) for 60 min at 37 °C. Granulocytes were prepared from the erythrocyte layer obtained by
Ficoll-Hypaque centrifugation of peripheral blood mononuclear cells.
Cells were suspended in 20 ml of PBS, pH 7.2, and 5 ml of dextran 250
solution (5% (w/v) in physiological saline) were added. After 20 min of
incubation at room temperature, the supernatant cells were removed and
washed in 40 ml of PBS (150
Figure 1:
Mitogenic effect of anti G
Figure 2:
Presence of mAb R24, mAb U5, and mAb UM4D4
antigens in the disialoganglioside fraction of human leukocytes.
Disialogangliosides originating from 1.8
Figure 3:
Indirect immunodetection of the U5
antigen. The disialoganglioside fraction of buttermilk (lanes
a) and of unseparated human leukocytes (lanes b) were
separated by thin-layer chromatography as described in the legend to Fig. 2and immunostained before(-) and after (+) mild
alkali treatment as described under ``Experimental
Procedures.'' The arrows indicate the position of the U5
antigen detected by the 9-O-acetyl-G
The changes in the U5 antigen upon
mild and strong alkali treatment in vitro are shown in Fig. 4. Reaction products were separated by TLC and analyzed by
immunostaining with different antibodies and by the nonspecific
detection of all GSL antigens using DIG staining (13). U5 antigen (Fig. 4A, lane 1) purified from bovine
buttermilk was not detectable by the strictly
9-O-acetyl-G
Figure 4:
Immunostaining and DIG staining patterns
of thin layer chromatograms of the U5 antigen (lanes 1), U5
antigen after treatment with 20 mM ammonia (lanes 2),
U5 antigen after treatment with 13.3 M ammonia (lanes
3), G
From these
experiments it was concluded that the U5 antigen was an O-acetylated derivative of ganglioside G
Figure 5:
HPLC analysis of sialic acids released
from purified U5 antigen. A, mixture of standard Neu5Ac and
Neu5,9Ac
Another indication of the 7
position of the O-acetyl group came from periodate oxidation
experiments combined with DIG staining. This latter method is dependent
on periodate oxidation of cis diol groups for the formation of the
digoxigenin hydrazones, which are then manifested immunologically.
Periodate attack of the non-O-acetylated ganglioside G
Figure 6:
Comparative staining of G
Figure 7:
Electrospray mass spectrometry of U5
antigen. A, negative ion electrospray mass spectrometry
showing mainly six doubly charged molecule ions. B, collision
induced decomposition of the doubly charged parent ion at m/z 791.
Fragment
ions incorporating the ceramide portion were detected at m/z 659
(Cer-Hex
Figure 8:
Binding of mAbs U5, R24 and E11 to
7-O-acetyl-G
Figure 9:
Presence of 7-O-acetyl-G
In this study, we have identified the target antigen of the
mAb U5 as 7-O-acetyl-G In human leukocytes, O-acetylated sialic acid residues are
ubiquitous components of disialogangliosides. We found in previous work
that a majority of the disialogangliosides from human leukocytes were O-acetylated and identified the major component as
9-O-acetylated G Theoretically, the O-acetyl group of the U5 antigen could
also be located at the 8 position. However, HPLC separation of
enzymatically released sialic acid showed the characteristic shoulder
of the 7-O-acetylated derivative (the position of the
8-O-acetylated N-acetylneuraminic acid is not known
in this HPLC system because of the extreme lability of this molecule).
In addition, the U5 antigen was susceptible to mild periodate, which
could only be expected for the 7-O-acetyl derivative. The
presence of unsubstituted G Investigations into the
existence and the properties of 7-O-acetyl-G Immunoprecipitates made with mAb U5 from solubilized human
T-cells were shown to contain 7-O-acetyl-G An argument in favor of a surface expression of the
U5 antigen comes through inference, from the evidence that it is
involved in mediating T-cell activation. Our interest in the
characterization of the U5 antigen originated from the observation (Fig. 1) that the T-cell stimulatory capacity of mAb U5 was
severalfold higher than that of mAb R24, although, as noted above, both
bound to ganglioside G Whether or not gangliosides are directly involved in signal
transduction from the cell surface is still largely unknown. An
involvement of gangliosides in signaling through direct binding of
G
Volume 270,
Number 50,
Issue of December 15, 1995 pp. 30173-30180
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
in Human T-lymphocytes Is Detected by a Specific
T-cell-activating Monoclonal Antibody (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
. Using
immunochemical and spectroscopic methods, the structure of the U5
antigen was determined as 7-O-acetyl-G
. The
antibody U5 did not react with 9-O-acetyl-G
and
bound severalfold more stronger to 7-O-acetyl-G
than to G
. U5 is the first antibody known to detect
preferentially 7-O-acetyl-G
. Flow cytometric
analysis showed that each major class of human leukocytes contained a
significant fraction of cells binding the U5 antibody.
)consisting of an oligosaccharide chain attached to
a lipid core structure. They are plasma membrane constituents of all
mammalian cells. Recently, we showed that normal human leukocytes
contain disialogangliosides with an 9-O-acetyl group on their
terminal sialic acid(1) . Not only was there a very restricted
surface expression of this GSL on human blood cells(2) , but it
was also found to be the first surface marker for helper cells within
the CD8 positive T-cell population(3) . These findings
suggested that slight modifications of cell surface molecules, such as O-acetylation, might suffice to define new functional
subpopulations of leukocytes. This hypothesis is in accordance with
observations that the pattern of glycolipids expressed on human
hematopoietic cells is cell
type-specific(4, 5, 6) . Our studies also
indicated that O-acetylated disialogangliosides other than the
9-O-acetylated forms were present on human cells (1) .
During the Fifth Workshop and Conference on Human Leukocyte
Differentiation Antigens (Boston, 1993), we presented evidence that a
monoclonal antibody (mAb), U5, bound strongly to an alkali labile form
of G, which was different from
9-O-acetyl-G
and, furthermore, that antibodies
specific for 9-O-acetyl-G
failed to bind to this
labile G
derivative. This, taken together with the
observation that binding of mAb U5 to human CD4
and
CD8
cells induced a strong T-cell proliferation, which
was accompanied by up-regulation of antigen expression(7) ,
stimulated our interest in characterizing the structure of the U5
antigen. In this report, we describe the purification of the U5 antigen
and identify it as the ganglioside 7-O-acetyl-G
.
In addition, the distribution of the U5 antigen on human blood cells is
analyzed.
Antibodies
mAb U5, R24, and E11 were prepared as
described previously(7) . mAb UM4D4 (CDw60) (8) was
donated by Dr. D. A. Fox, University of Michigan, Ann Arbor, MI. The
mAb M-T32 (CDw60) (9) was a kind gift of Dr. E.P. Rieber,
University of Dresden, FRG.Purification of the U5 Antigen, of 9-O-Acetyl-G
1460 g of
buttermilk powder, obtained by freeze drying 7.5 liters of buttermilk,
was suspended in 7.5 liters of chloroform/methanol (2:1) (v/v) and
stirred for 1 h at ambient temperature. The suspension was filtered
under reduced pressure through a Buchner funnel. The residue was
reextracted twice as above. The extracts were combined, and the solvent
was evaporated in vacuo at a maximum of 25 °C. The
evaporated lipid extract was dialyzed for 3 days at 4 °C against
several changes of water. The desalted extract was lyophilized and
dissolved in chloroform/methanol/water (30:60:8) (v/v/v). After removal
of insoluble material, the extract was pumped onto a 3.5 and of G
from Bovine Buttermilk
20-cm
column filled with DEAE-Sepharose (acetate form) in three separate
runs. Elution was performed with 1 liter of chloroform/methanol/water
(30:60:8), 1 liter of methanol, 1 liter of 20 mM, 2 liters of
50 mM, and finally with 1 liter of 150 mM ammonium
acetate in methanol, respectively. The 50 mM ammonium acetate
eluates contained all of the U5 antigen, the
9-O-acetyl-G, and the G
, as shown by
immunostaining of thin-layer chromatograms using the mAbs U5, M-T32,
and R24, respectively (see below). The 50 mM ammonium acetate
eluates were pooled, concentrated by evaporation, dialyzed,
lyophilized, dissolved in chloroform/methanol (85:15) (v/v), and pumped
onto a HPLC column (16
500 mm) filled with LiChrosorb Si 60
5-µm particles (Merck). Elution was performed using a linear
gradient from chloroform/methanol/water (v/v/v) (82.6:16.4:1) to
(40:50:10) in 400 min at a flow rate of 2 ml/min. Fractions were
collected every 2 min. Fractions 112-128 contained
9-O-acetyl-G, fractions 129-134 contained
the U5 antigen, fractions 135-140 contained a mixture of the U5
antigen and G
, and fractions 141-159 contained
G
. Fractions 129-134 were pooled and further
purified on an analytical 4
250 mm Partisil 5-µm silica
HPLC column (Whatman Ltd., Maidstone, United Kingdom) using a gradient
from chloroform/methanol/water (v/v/v) (82.6:16.4:1) to (40:52:8) in
200 min at 1 ml/min. Fractions were collected every minute. Fractions
36-40 contained the U5 antigen together with an impurity that
showed a somewhat higher chromatographic mobility than the U5 antigen.
After a final HPLC separation using the Partisil column and a gradient
from chloroform/methanol/water (v/v/v) (82.6:16.4:1) to (50:45:5) in
200 min at 1 ml/min (200 fractions), apparently pure U5 antigen (about
87 µg) was found in the fractions 57-65. Fractions
54-56 and 66-75 also contained the U5 antigen as major
component but of less purity. The antigen containing fractions were
dried and stored at -70 °C.Preparation of the Disialoganglioside Fraction from
Unseparated Human Leukocytes
The disialogangliosides were
prepared exactly as has been described previously(1) .Thin Layer Chromatography (TLC)
TLC analysis was
carried out on high performance TLC (HPTLC) silica gel 60 plates
(Merck). The running solvent was chloroform/methanol/water (50:40:10)
(v/v/v) containing 0.05% calcium chloride, and running time was 40 min. Quantitation
For quantitation of TLC-separated
gangliosides, the HPTLC plates were sprayed with resorcinol/HCl, and
then covered with a glass plate and heated at 95 °C for 30 min.
Densitometric measurements were made in transmission mode at 580 nm (10) using a Shimadzu dual wavelength TLC Scanner CS9001 PC
(Shimadzu, Düsseldorf, FRG). 0.5-3.0 µg
of G was used for calibration.
TLC Immunostaining
Ganglioside antigens separated
on HPTLC plates were detected by immunostaining using the method of
Bethke et al.(11) with modifications(12) .Nonspecific Immunochemical Detection of Gangliosides on
HPTLC Plates by Digoxigenin-succinyl-
5-Bromo-4-chloro-indolyl-3-phosphate, p-toluidine salt, was obtained from Biomol, Hamburg, FRG. All
other reagents were purchased from Boehringer Mannheim. DIG labeling
was performed essentially as described previously(13) . In
order to obtain improved sensitivity, a 1:333 dilution of the
DIG--aminocaproic Acid Hydrazide
(DIG) Labeling
-aminocaproic acid hydrazide solution was used, and incubation
with the phosphatase-conjugated antidigoxigenin antibody (1:200
dilution) was prolonged to 48 h at 30 °C. Total gangliosides were
detected after O-deacetylation as described below.
Isomerization of the U5 Antigen to
9-O-Acetyl-G
Dried U5 antigen in a glass test tube
was incubated in 20 mM aqueous ammonia for 30 min at ambient
temperature (14) followed by lyophilization. U5 antigen on
HPTLC plates immobilized with polyisobutylmethacrylate (13) was
isomerized by incubation of the plate in 0.1 M glycine-NaOH
buffer, pH 10.0 for 2 h at 37 °C(1) . The plates were then
washed 3 times for 5 min with phosphate-buffered saline (PBS), pH 7.3.
Isomerized U5 antigen was visualized by immunostaining using mAb UM4D4
as described above.O-Deacetylation of Gangliosides
Alkaline
hydrolysis of gangliosides after separation on HPTLC plates was
performed by incubating the plates for 17 h at ambient temperature in a
chamber with an atmosphere saturated with 13.3 N aqueous
ammonia. The ammonia-treated plates were dried for at least 3 h in
vacuo in the presence of P
O
. In vitro
O-deacetylation was done by treating the dried sample for 1 h at
37 °C with 1 ml of 13.3 N aqueous ammonia followed by
evaporation of the ammonia in vacuo at 30 °C.Release and Characterization of O-Acetylated Sialic
Acids
Sialic acids were released enzymatically from
gangliosides, purified, and analyzed by HPLC as described
previously(15, 14) . To confirm the identity of
7-O-acetyl-5-N-acetylneuraminic acid, which was not
easily separable from 5-N-acetylneuraminic acid, an aliquot of
the released acid was treated with 20 mM aqueous ammonia as
described previously(14) . The expected rearrangement product
9-O-acetyl-5-N-acetylneuraminic acid was identified
using an authentic standard, which was a kind gift from Dr. R. Schauer,
Kiel, FRG.Electrospray Mass Spectrometry
A Finnigan MAT TSQ
700 triple quadrupol mass spectrometer equipped with a Finnigan
electrospray ion source (Finnigan MAT Corp., San Jose, CA) was used.
The O-acetylated G derivative was dissolved in
methanol and injected at a flow rate of 2 µl/min into the
electrospray chamber. A voltage of 4.5 kV was applied to the
electrospray needle. For collision-induced decomposition experiments,
the doubly charged parent ions were selectively transmitted by the
first mass analyzer and directed into the collision cell (argon was
used as collision gas) with a kinetic energy set at +29 eV.
ELISA
The indicated amounts of gangliosides,
dissolved in 50 µl of methanol, were aliquoted into a Polysorb
(Nunc, Wiesbaden-Biebrich, FRG) microtiter plate. The samples (up to 30
ng) were dried at 30 °C for about 3 h. Nonspecific binding sites
were saturated with PBS containing 5% (w/v) bovine serum albumin for 1
h at ambient temperature. The plate was incubated with 50 µl/well
purified antibody (5 µg/ml) overnight at 4 °C. Following four
washes with PBS, the plate was incubated for 2 h at 37 °C with 100
µl/well of an alkaline phosphatase-conjugated goat anti-mouse IgG
antibody (Sigma) diluted 1:2000 in PBS containing 1% bovine serum
albumin. After washing 3 times with PBS and 2 times with 0.1 M
glycine-NaOH buffer, pH 10.0, containing 1 mM MgCl and 1 mM ZnCl, the plate was developed with
0.8 mg/ml p-nitrophenylphosphate (Sigma) in the glycine-NaOH
buffer. Absorbance was read at 405 nm in an SLT Spectra III ELISA
reader (SLT, Crailsheim, FRG) after 1 h.Isolation of Leukocyte Cell
Populations
CD4 and CD8
T-cells were prepared by a combination of ``panning''
technique and complement-mediated lysis. Flow cytometric analysis
showed that CD4
and CD8
T-cells
comprised 90% of the respective preparations, with not more than 5%
CD16
NK cells and less than 2%
CD8
/CD4
T-cells, B-cells, and
monocytes.
T-cells were obtained by depletion
of monocytes and B-cells followed by complement mediated lysis of
CD16
NK cells.
NK cells were
isolated immunomagnetically using a magnetic cell sorting system
(Miltenyi, Bergisch Gladbach, FRG). The final cell suspension contained
85-90% CD16
with approximatively 6%
CD3
cells.
cells, 4-8% monocytes, 4%
CD16
, and 2-5% CD3
cells. The
above methods have been described in detail elsewhere(7) .
g, 7 min, without brake).
Remaining erythrocytes were lysed using a solution containing 0.82%
NH
Cl, 0.1% KHCO
, 0.1 mM EDTA, pH 7.27.
The preparations consisted of 90% granulocytes as shown by flow
cytometry.Immunoprecipitation
Purified CD3
T-cells (5
10
) were washed 3 times with 50 mM Tris-HCl, pH 8.0, containing 0.15 M NaCl and 5 mM EDTA and solubilized in 1.4 ml of this buffer containing, in
addition, 0.5% Nonidet P-40, 1% phenylmethanesulfonyl fluoride, and
10.5% aprotinin. The suspension was sonicated for 2 min and incubated
15 min on ice. After centrifugation (4000 g, 4 °C,
15 min) the supernatant was precleared by stirring for 30 min at 4
°C with 100 µl of Pansorbin pellet (Pharmacia Biotech Inc.),
followed by incubation with 20 µg of mAb U5/800 µl of
supernatant for 1 h under the same conditions. 500 µl of this cell
lysate/antibody mixture were incubated under rotation with 50 µl of
protein A-Sepharose (Pharmacia) for 3 h or overnight at 4 °C. The
protein A-Sepharose-antibody-antigen complex was washed 3 times with 50
mM Tris-HCl, pH 8.0, containing 0.15 M NaCl and 5
mM EDTA buffer by successive centrifugation (1250 g, 3 min) and resuspension, and then incubated with 30 µl
of lysis buffer (62.5 mM Tris, 2% SDS, 10% glycerol, pH 6.8)
for 30 min in an ultrasonic bath. The suspension was then centrifuged
(4000 g, 4 °C, 15 min), and the resulting
supernatant was shock frozen in liquid nitrogen.Analysis of the Lipid Constituents of the
Immunoprecipitates
The immunoprecipitates were analyzed as
described previously(16) .Flow Cytometric Analysis of Cell Surface
Antigens
For immunofluorescence assays, 2 10 purified cells were incubated with 5 µg of the purified
anti-G antibodies or the control antibody H-141-30
(mouse IgG3 anti-H-2D
, a kind gift from Dr. G.
Hämmerling, Heidelberg, FRG) followed by 50 µl
of goat anti-mouse IgG-fluorescein isothiocyanate (1:100) (Coulter
Electronics, Krefeld, FRG) as secondary antibody. The control antibody
MsIgG-fluorescein isothiocyanate was purchased from Dianova (Hamburg,
FRG). Cells were analyzed with a fluorescence-activated cell sorter
(FACScan, Becton Dickinson, Heidelberg, FRG).Proliferation Assays
Separated T-cells were
incubated in triplicate in microwell culture plates (Nunclon
1-67008, Nunc, Roskilde, Denmark) in the presence or absence of
the anti-G antibodies. The antibodies were used at final
concentrations from 100 to 1.56 µg/ml. Phytohaemagglutinin (HA17,
Wellcome Diagnostics, Dartford, UK) was applied at 0.5 µg/ml. The
cultures were pulsed with 0.5 µCi/well
[
H]thymidine (Dupont NEN) for 18 h on day 5 of
incubation, and incorporated radioactivity was measured in a Betaplate
Counter (Pharmacia).
Different Proliferative Responses of Human T Cells
Induced by Binding of the mAbs R24, U5, and E11
Previous reports
had suggested that mAb R24 could induce T-cell
proliferation(17) . When we compared two different
anti-G antibodies, R24 and E11, with the putative
anti-G
mAb U5 in an effort to corroborate and extend those
findings, we found that there were extreme variations in the capacities
of these mAbs to induce T-cell growth as assayed by thymidine
incorporation (Fig. 1). The mAbs R24 and U5 stimulated
CD4
and CD8
T-cell growth without
addition of exogenous cytokines, whereas E11 did not. Antibody U5
always induced higher levels of proliferation at significantly lower
antibody concentrations than R24. This finding correlated with the
antibody reactivity measured by flow cytometry. mAb U5 stained
30-70% of the T-cell subpopulations; R24 stained 10-30%.
E11 bound to only 4% of the T-cells, and thus did not differ from the
mouse IgG
subclass control antibody. The similar affinity
constants of the antibodies R24 (2 10 liters/mol)
and U5 (1.6 10 liters/mol) for the ganglioside
G(18) could not explain these differences. The
data thus suggested that the mAb U5 might recognize an additional
ganglioside on human T-cells.
antibodies on human CD4
and CD8
T-cells. The proliferative response of CD4
and
CD8
T-cells, separated as described previously (7) , was measured after stimulation with mAb U5 or R24 at
final concentrations between 100 and 1.56 µg/ml. The S.D. ranged
within 15%. The values of phytohaemagglutinin stimulation (0.5
µg/ml), mAb E11 (100 µg/ml), and medium control for
CD4
T-cells were 152,930, 189, and 286 cpm,
respectively; for CD8
T-cells 90,411, 49, and 65 cpm,
respectively.
Differences in the Ganglioside Immunostaining Patterns of
the mAbs E11, R24, and U5
We next compared the binding of the
antibodies E11, R24, and U5 with glycosphingolipids in the
disialoganglioside fraction obtained from unseparated human leukocytes (Fig. 2). mAb R24 (Fig. 2, lane A) and mAb E11
(not shown) both detected a double band with the chromatographic
mobility of standard G (Fig. 2, S1, the reference
contained only the upper band of the doublet). mAb U5 not only
recognized the same double band but recognized in addition an unknown
glycolipid migrating somewhat faster (Fig. 2, lane B, arrow). The unknown GSL was not identical to any of the
antigens recognized by mAb UM4D4 (Fig. 2C), which binds
strictly to 9-O-acetylated G
(Fig. 2, S2, only the upper band was present in the reference material)
and to similar gangliosides possessing a terminal
9-O-acetylated disialosyl group(1) . Our interest
focused on the differences in the antigen specificities of the two
mAbs, which induced T-cell proliferation, R24 and U5. As a working
hypothesis, we proposed that the functional difference between the two
mAbs could be ascribed to the recognition by mAb U5 of the additional
GSL band indicated by an arrow in Fig. 2, lane
B. We therefore undertook the isolation and characterization of
this ganglioside.
10
unseparated human leukocytes were separated on silica HPTLC plates for
40 min in chloroform/methanol/water (50:40:10) (v/v/v) containing 0.05%
(w/v) CaCl and immunostained as described with mAb R24 (A); mAb U5 (B); mAb UM4D4 (C). Reference
lanes were as follows: S1, G from bovine
buttermilk immunostained with mAb R24; S2,
9-O-acetyl-G
from bovine buttermilk immunostained
with mAb UM4D4. The arrow in lane B indicates the
position of the additional antigen detected only by mAb U5.
Abbreviations are as follows: 9-O-Ac-GD3,
9-O-acetyl-G
; 9-O-Ac-DSPG,
9-O-acetyldisialosylparagloboside; 9-O-Ac-DSnHC,
9-O-acetyldisialosyllacto-N-norhexaosylceramide.
Purification and Immunological Properties of the U5
Antigen
The overall concentration of disialogangliosides in
human leukocytes is very low (about 122 µg of lipid-bound sialic
acid in 10 unseparated leukocytes(1) ), making
this a poor source for the purification of the U5 antigen. We therefore
searched for an alternative source of the antigen and detected it in
buttermilk, which has been reported to contain several
disialogangliosides of the G
type(19) . The
purification of the U5 antigen from bovine buttermilk was achieved by
ion-exchange chromatography and three consecutive fractionations on
HPLC silica columns as described under ``Experimental
Procedures.'' Two different methods were used to identify the U5
antigen in the course of the purification. The first was direct TLC
immunostaining using the mAb U5 as already shown in Fig. 2.
However, because of the cross-reactivity of this antibody with the
non-O-acetylated ganglioside G
(Fig. 2, lane B) and because the upper G
band and the U5
antigen migrated very close together, it was, especially in the
presence of the large amounts of G
found in bovine
buttermilk, often difficult to distinguish between these two GSLs using
mAb U5. For this reason, we developed a second method to identify the
U5 antigen, which took advantage of the alkali-induced (pH 10)
rearrangement of the U5 antigen to 9-O-acetyl-G
,
an antigen that could be detected with mAb UM4D4 (Fig. 3). In
the left half of Fig. 3the characteristic binding
patterns of the 9-O-acetylated disialogangliosides from bovine
buttermilk (lane a) and from unseparated human leukocytes (lane b) are shown. After treatment of the plate at pH 10, one
new major band appeared in both lanes (Fig. 3, + panel, large arrows). As shown below, this major band
originated from the U5 antigen. The band could clearly be distinguished
from 9-O-acetyl-G
because of its different
chromatographic mobility. Using both methods, the U5 antigen could be
distinguished with certainty from both 9-O-acetyl-G
(by U5 staining) and G
(by staining with
9-O-acetyl-G
-specific antibodies after the
alkali-induced rearrangement).
-specific mAb
UM4D4 after mild alkali treatment. The less pronounced new peaks (thin arrows) in the more polar region of the chromatogram
most likely originate from long chain analogs of the major U5
antigen.
-specific mAb (15) UM4D4 (Fig. 4B, lane 1) but could be detected by DIG
staining (Fig. 4C, lane 1). Treatment of the
U5 antigen with mild alkali (20 mM aqueous ammonia, 30 min, 22
°C) (Fig. 4, lanes 2) abolished binding of mAb U5 (Fig. 4A, lane 2), but the rearranged product
was now recognized by mAb UM4D4 and showed the mobility of
9-O-acetyl-G
(Fig. 4B, lane
2). This change could also be seen in the DIG stain (Fig. 4C, lane 2, band I). Also
visible in the same lane was another band (II) of minor
intensity that had the same mobility as reference ganglioside G
(Fig. 4C, lane 4). Treatment of the U5
antigen with strong alkali (13.3 N ammonium hydroxide, 1 h, 37
°C) resulted in a single product with the mobility of G
(Fig. 4C, lane 3).
standard (lanes 4), and the
disialogangliosides from unseparated human leukocytes (lanes
5). Panel A, immunostain with mAb U5; panel B,
immunostain with mAb UM4D4 (CDw60); panel C, DIG stain for the
nonspecific detection of all gangliosides. Solvent and running time
were as in Fig. 2.
different from 9-O-acetyl-G
. Because the
conditions of the in vitro mild alkali treatment were the same
as those used by Diaz et al.(14) to achieve an
intramolecular migration of O-acetyl groups from the 7- to the
9-position of the O-acetylated sialic acid, we predicted that
the U5 antigen should be identical with or closely related to
7-O-acetyl-G
. HPLC analysis of sialic acids
released by Arthrobacter ureafaciens sialidase treatment of
the purified U5 antigen showed the presence of a small peak as a
shoulder eluting before the 5-N-acetylneuraminic acid main
peak (Fig. 5B). This shoulder (R
= 0.95) has been reported to be
7-O-acetyl-5-N-acetylneuraminic acid(20) .
Although the 7-O-acetyl-5-N-acetylneuraminic acid
(Neu5,7Ac
) derivative could only be partially separated
from the 5-N-acetylneuraminic acid originating from the
penultimate sialic acid residue (elution times in this system were 57 versus 60 min for 7-O-acetylated and unsubstituted
5-N-acetylneuraminic acid, respectively, Fig. 5B, arrow), its disappearance concomitant
with the appearance of 9-O-acetylneuraminic acid (standard in Fig. 5A) after treatment with 20 mM aqueous
ammonia was a further indication of the identity of the shoulder at 57
min as the 7-O-acetylated product (Fig. 5C).
The fact that the peak areas of Neu5Ac (from the penultimate sialic
acid residue) and of Neu5,9Ac were not equal is most likely
the result of some overall de-O-acetylation occurring during
the induction of acetyl group migration.
. B, sialic acids released from the U5
antigen upon treatment with A. ureafaciens neuraminidase. The arrow indicates the position of Neu5,7Ac. C, sialic acids enzymatically released from the U5 antigen and
treated with 20 mM ammonia. The sialic acids were separated on
a Bio-Rad Aminex HPX-72S (300 7.8 mm; inner diameter, 11
µm; sulfate form) anion-exchange column at 0.2 ml/min and detected
in the UV at 210 nm.
can only take place in the exocyclic glycerol-like side chain of
the terminal sialic acid residue(21) . The presence of an O-acetyl substitution in this side chain in either the 9 or 8
position would prevent an attack by mild periodate and subsequent DIG
staining. For 7-O-acetyl-G
, a cleavage between
the terminal (C-9) and the subterminal (C-8) carbon atoms in the sialic
acid side chain could be expected with attendant detectability by DIG
staining. An in situ periodate oxidation followed by DIG
labeling with unsubstituted G
(lane 1), with the
U5 antigen (lane 2), and with 9-O-acetylated G
(lane 3) is shown in Fig. 6. In panel A,
G
and the U5 antigen but not the
9-O-acetyl-G
were oxidized by periodate as shown
by DIG staining. In panels B, C, and D,
control stains with the mAbs U5, UM4D4 after alkali-induced
rearrangement, and UM4D4 without alkali-induced rearrangement,
respectively, are shown. Thus, the detectability of the U5 antigen by
DIG staining also suggested that the O-acetyl group was
located in the 7 position.
(lane 1), 7-O-acetyl-G
(lane 2)
and 9-O-acetyl-G
(lane 3). A,
DIG staining; B, immunostaining with mAb U5; C,
immunostaining with mAb UM4D4 after pretreatment of the plate with
glycine-NaOH buffer pH10; D, immunostaining with mAb UM4D4
without pretreatment of the plate. The lanes contained approximately
100 ng of the three antigens. Solvent and running time were the same as
in Fig. 2.
Mass Spectrometric Analysis of the U5
Antigen
Structural analysis of the U5 reactive ganglioside from
bovine buttermilk was also performed using negative ion electrospray
mass spectrometry (Fig. 7). A cluster of six intense doubly
charged molecular ions was detected at m/z 770.6,
777,4, 783.9, 791.1, 798.0, and 805.1, whereas the corresponding singly
charged species were detectable but of rather low abundance (Fig. 7A). These molecular ions suggest the presence of
a series of gangliosides incorporating a homogeneous carbohydrate
moiety, i.e. a monoacetylated tetrasaccharide of the
composition AcNeuAc-NeuAc-Hex linked to a heterogeneous
ceramide portion with C19-C24 fatty acids bound to a C18 sphingosine.
These assumptions were confirmed by tandem mass
spectrometric-experiments. After collision-induced decomposition of the
doubly charged parent ion at m/z 791, the spectrum
depicted in Fig. 7B was obtained. The location of the O-acetyl group in the terminal sialic acid moiety was
unequivocally demonstrated by the detection of a weak daughter ion at m/z 350 (NeuNAcOAc) accompanied by more intense
fragments at m/z 332 (NeuNAc-O-Ac-HO), m/z 290 (NeuNAcOAc-CHCOOH), and m/z 272
(NeuNAcOAc-HO-CHCOOH). A signal at m/z 641 characteristic of the
mono-O-acetylated disialosyl moiety was not observed, but an
intense fragment at m/z 623
(NeuNAcOAcNeuNAc-HO) and weaker signals at m/z 581, 563, and 535, which can be explained by loss of
CHCOOH, HO+CHCOOH, and
HCOOH+CHCOOH, respectively, were detected.
NeuAc-CO-COO), at m/z 958 (Cer-Hex
),
together with weak signals at m/z 796
(Cer-Hex)
, accompanied by peaks at m/z 778 and 760 generated by the loss of one or two molecules of
H
O and at m/z 634
(Cer). Only the latter signals shifted by the
expected mass increment when a different parent ion was decomposed,
confirming the assignments. These results confirmed the structure of
the U5 antigen as a terminally O-acetylated derivative of
ganglioside G
.
Quantitative Binding of mAbs U5, R24, and E11 to G
As shown above, mAb U5 did
not bind to 9-O-acetyl-G and 7-O-acetyl-G
(Fig. 2, lane
B). However, the antibody bound to some extent to G
.
We therefore compared quantitatively binding of the mAbs U5, R24, and
E11 to 7-O-acetyl-G
and G
in an
ELISA assay (Fig. 8). The affinity of mAb U5 for
7-O-acetyl-G
was severalfold higher than that of
the two other mAbs for this antigen, whereas all three mAbs had
relatively low affinities for G
. The high binding affinity
and specificity of mAb U5 for 7-O-acetylated- versus nonacetylated G
classifies this mAb as the first with
a preferential specificity for 7-O-acetyl-G
.
and G
tested by ELISA.
The indicated amounts of each antigen were assayed with the three mAbs
as described under ``Experimental
Procedures.''
Detection of 7-O-Acetyl-G
Direct evidence for the presence of
7-O-acetyl-G in Human
T-Cells
in human T-cells was obtained by
analysis of the lipids extracted from mAb U5 immunoprecipitates of
purified human T-cells (Fig. 9). Lipid extracts from total
leukocytes (A) or T-cell immunoprecipitates (B) were
separated on TLC plates and detected by immunostaining with mAb UM4D4
before and after alkali treatment. The band indicated by the large
arrow originated from 7-O-acetyl-G
as
inferred from the facts that it could only be detected after pH 10
treatment of the lipid extract and that it migrated between the
positions of 9-O-acetyl-G
and
9-O-acetyl-DSPG.
in the U5 immunoprecipitate from human T-cells. The
immunoprecipitate was prepared as described under ``Experimental
Procedures.'' Gangliosides were extracted from the precipitate as
described previously(16) . Immunostaining was performed using
mAb UM4D4 before(-) and after (+) pH 10 treatment. Lane
A, disialoganglioside fraction from unseparated human leukocytes; lane B, lipid extract from the U5 immunoprecipitate of human
T-cells. Abbreviations were as follows: 9-O-acetyl-DSPG,
9-O-acetyldisialosylparagloboside; 9-O-acetyl-DSnHC,
9-O-acetyldisialosyl
lacto-N-norhexaosylceramide.
Expression of U5 Positive Gangliosides by Different Human
Leukocyte Populations
The distribution of the U5 antigen in
different leukocyte populations as determined by flow cytometry is
shown in Table 1. Although our findings suggest that this antigen
serves as a receptor for the functional activation of T-cells, it is
not a specific marker for them, as surface expression of U5 antigen was
also found in a significant fraction of the cells in all other classes
of leukocytes analyzed.
and have shown that this
GSL is present in the disialoganglioside fraction of human leukocytes,
where it was heretofore unknown. 7-O-Acetyl-G
has
recently been identified in bovine buttermilk and in melanoma cells of
hamsters and humans(19, 22, 23) . Its
occurrence in normal human leukocytes may have been overlooked for two
reasons. First, this antigen shows a migration on TLC very similar to
that of unsubstituted G
; second, the classical
method for the detection of alkali labile GSL, a characteristic
decrease in their TLC mobility upon ammonia treatment(24) ,
failed in this case since there is essentially no difference in the
mobilities of G and 7-O-acetyl-G
.
, and two minor components as
9-O-acetyl-G
analogs containing in addition one
and two lactosamine disaccharide units(1) . We also showed that
treatment of the disialogangliosides from unseparated leukocytes with
mild alkali caused a considerable increase in the amount of
9-O-acetylated gangliosides(1) . This suggested the
presence of unknown O-acetylated forms of the gangliosides
that had rearranged to the 9-O-acetates during mild alkali
treatment, a supposition that we have now confirmed with the
identification of the U5 antigen as 7-O-acetyl-G
.
in our purified antigen could
be excluded by mass spectrometry. It was not possible to quantitate the
proportion of 7-O-acetylated, 9-O-acetylated, and
nonacetylated forms of disialogangliosides originally present in human
leukocytes or in purified T-cells since it could not be excluded that
the O-acetylated gangliosides were partially deacetylated
during purification. Indeed, it is conceivable that the
non-O-acetylated disialogangliosides originate entirely
through deacetylation during purification.
have
been conducted primarily in two
laboratories(19, 22, 23) . However, their
results concerning the general properties of this molecule differed in
several points. The first matter of controversy is the stability of the
antigen. Manzi et al.(23) found that
7-O-acetyl-G
was an extremely labile compound
with a strong tendency to rearrange to the 9-O-isomer, which
is in agreement with our present results. In contrast, Ren et al.(22) reported that the 7-O-isomer could be
purified from hamster melanoma cells without extensive degradation.
Second, Ren et al.(19) concluded that 7- and
9-O-acetyl-G
were practically indistinguishable
because of their very similar physicochemical properties, whereas we
found differences in the chromatographic mobilities of the two isomers
that were sufficient to permit their separation. Third, Ren et al.(22) reported no difference in the binding of the
9-O-acetyl-G
-specific mAb ``JONES'' (25) to either 7- or 9-O-acetylated forms of
G
, whereas Manzi et al.(23) found that
9-O-acetyl-G
-specific mAbs failed to bind to the
7-O-isomer. Our data (Fig. 4) support the specificities
determined by Manzi et al.(23) . Furthermore, in our
hands mAb JONES also failed to bind to the 7-O-isomer. (
)The reasons for these discrepancies, which could only be
resolved by an exchange of materials and antibodies, are at present
unknown. (Fig. 9), which offered unequivocal proof of its presence
in T-cells but did not distinguish between an intracellular and a cell
surface distribution. The presence of 7-O-acetyl-G
on the cell surface should be a rather unexpected finding in view
of its lability at physiological pH. The existence of
7-O-acetyl-G
in acidic compartments of human
melanoma cells has been well documented(23) , but it was
presumed that the O-acetyl group underwent a rapid migration
from the 7 to the 9 position following its translocation to the cell
surface. The binding of mAb U5 to intact T-cells again does not prove
the existence of 7-O-acetyl-G
on the cell surface
since this binding could equally well have been caused by
cross-reaction as a result of the expression of high concentrations of
G
.
with comparable affinities. This
suggested that the primary antigen recognized by U5 and responsible for
T-cell activation was different from G
. The U5 antigen as
well as non-O-acetylated G
have also been
implicated in earlier studies as activation molecules on
T-cells(7) . In contrast, eight different monoclonal antibodies
specific for 9-O-acetylated derivatives of G
,
tested under the auspices of the Fifth Workshop and Conference on Human
Leukocyte Differentiation Antigens (26) , were found to not
induce T-cell proliferation (data not shown). Further detailed
functional studies with a panel of related antibodies will be necessary
to clarify and confirm the roles that these different
disialogangliosides may or may not play in T-cell activation.
, G
, G
, G
, and
G
to calmodulin and the calmodulin-dependent enzyme cyclic
nucleotide phosphodiesterase has been
demonstrated(27, 28) . Moreover, Hannun (29) and Yuan et al.(30) have suggested roles
for the GSL metabolites ceramide and sphingosine 1-phosphate in the
regulation of cell growth, differentiation and
apoptosis(29, 30) . Recently, a tight and specific
association of the signal transducing GPI-linked surface molecule CD59
with the ganglioside GM3 was described(16) . Thus, as a working
hypothesis for future investigations, it might be speculated that
7-O-acetyl-G
operates in a similar manner by
forming a close association in a membrane microdomain with a
T-cell-activating molecule such as CD2 or CD3.
),
Neu5Ac
28Neu5Ac
23Gal
14Glc
11`-ceramide;
7-O-acetyl-G
,
Neu5,7Ac
28Neu5Ac
23Gal
14Glc
11`-ceramide;
9-O-acetyl-G
,
Neu5,9Ac
28Neu5Ac
23Gal
14Glc
11`-ceramide;
HPTLC, high performance thin layer chromatogram; DIG,
digoxigenin-succinyl-
-aminocaproic acid hydrazide; PBS,
phosphate-buffered saline; Neu5,7Ac
,
5-N-acetyl,7-O-acetylneuraminic acid;
Neu5,9Ac, 5-N-acetyl,9-O-acetylneuraminic
acid; 9-O-acetyl-DSPG,
Neu5,9Ac28Neu5Ac
23Gal
14GlcNAc3
1Gal
14Glc
11`-ceramide;
G
,
IV
NeuAc,IINeuAc-GgOseCer;
G,
II
(NeuAc)-GgOseCer; G,
II
NeuAc-GgOseCer; G,
II
NeuAc-GgOseCer.)
We thank Maria Dittmeyer for performing the
immunoprecipitations of the U5 antigen and Reiner Munder for helping in
the preparation of glycolipids. We also thank Dr. E. Kniep for valuable
comments reading the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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