(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. 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.
Gangliosides are sialic acid-containing glycosphingolipids
(GSLs) ()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.
CD3 T-cells were obtained by depletion
of monocytes and B-cells followed by complement mediated lysis of
CD16
NK cells.
CD16 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.
B-cells were also obtained by the
panning technique. The adherent cell fraction contained 80%
CD20 cells, 4-8% monocytes, 4%
CD16
, and 2-5% CD3
cells. The
above methods have been described in detail elsewhere(7) .
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 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.
Figure 1:
Mitogenic effect of anti G
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.
Figure 2:
Presence of mAb R24, mAb U5, and mAb UM4D4
antigens in the disialoganglioside fraction of human leukocytes.
Disialogangliosides originating from 1.8 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.
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-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.
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-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).
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 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.
From these
experiments it was concluded that the U5 antigen was an O-acetylated derivative of ganglioside G 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.
Figure 5:
HPLC analysis of sialic acids released
from purified U5 antigen. A, mixture of standard Neu5Ac and
Neu5,9Ac. 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.
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 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.
Figure 6:
Comparative staining of G (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.
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-HexNeuAc-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
.
Figure 8:
Binding of mAbs U5, R24 and E11 to
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.''
Figure 9:
Presence of 7-O-acetyl-G 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.
In this study, we have identified the target antigen of the
mAb U5 as 7-O-acetyl-G 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
.
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, 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
.
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 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.
Investigations into the
existence and the properties of 7-O-acetyl-G 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.
Immunoprecipitates made with mAb U5 from solubilized human
T-cells were shown to contain 7-O-acetyl-G (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
.
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 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.
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, 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.