(Received for publication, November 17, 1993; and in revised form, November 1, 1994)
From the
Neuraminic acid is the core structure of most known sialic
acids. In natural systems, the amino group at the 5 position of
neuraminic acid residues is usually assumed to be acylated. Previously,
synthetic de-N-acetyl-gangliosides (with free amino groups at
the 5 position of neuraminic acids) have been shown to modulate
cellular proliferation and tyrosine phosphokinase reactions. While
indirect evidence has suggested that traces of these molecules exist
naturally in certain tumor cells, further exploration has been hampered
by the lack of a system showing consistent expression at an easily
detectable level. Using synthetic compounds as antigens, we have
developed highly specific monoclonal antibodies against
de-N-acetyl-G and
de-N-acetyl-G
that require both the free amino
group and the exocyclic side chain of sialic acids for recognition.
Cultured human melanoma cells showed low but variably detectable levels
of reactivity with these antibodies. The ability of various
biologically active molecules to stimulate this reactivity was
explored. Of many compounds tested, only the tyrosine kinase inhibitor
genistein induced reactivity in a dose-dependent manner. Antibody
reactivity with ganglioside extracts from genistein-treated cells was
abolished by chemical re-N-acetylation and/or truncation of
sialic acid side chains by mild periodate oxidation. High performance
thin layer chromatography immuno-overlay analysis confirmed the
presence of the novel compound de-N-acetyl-G
in
these extracts. Several other tyrosine kinase inhibitors tested did not
give the same increase in de-N-acetyl-ganglioside expression.
However, the microtubule inhibitor nocodazole caused a similar
accumulation of these molecules, particularly in non-adherent cells
expected to be arrested at metaphase. Thus, genistein may induce
de-N-acetyl-ganglioside expression by virtue of its known
ability to arrest cells in the G
M phase, rather than as a
general consequence of tyrosine kinase inhibition. These studies also
provide a system in which to analyze the enzymatic basis of
de-N-acetyl-ganglioside expression and their potential roles
as growth regulating molecules.
Gangliosides are structurally diverse amphipathic molecules
enriched in the outer leaflet of animal plasma
membranes(1, 2, 3) . They can mediate or
influence a variety of biological processes including cell-cell
interaction(4, 5, 6, 7, 8) ,
immune modulation(9) , cell growth, and
differentiation(3, 10, 11) , formation of
neurites(12) , and developmental
organization(8, 13, 14) . Since they coexist
with other plasma membrane constituents, gangliosides may modulate the
functions of proteins associated with or spanning the membrane bilayer.
For example, the ganglioside G(
)can inhibit
both epidermal growth factor receptor (EGFR) autophosphorylation on
tyrosine residues (15, 16) and cell growth. In
contrast, insulin receptor-associated cellular proliferation and
tyrosine kinase activity are specifically inhibited by
2-3-sialylparagloboside and not by
G
(17) . Gangliosides can also modulate cellular
interactions mediated by extracellular matrix adhesion receptors (4, 18, 19) and the cell surface expression
of certain proteins(20) .
The defining feature of
gangliosides is the presence of at least 1 residue of a nine carbon,
anionic monosaccharide called sialic acid. ``Sialic acid'' is
a generic term for a family of molecules represented by over 25
members, the commonest being N-acetyl-neuraminic acid
(Neu5Ac). Diversity arising from modification of Neu5Ac can add
considerable structural variability to
gangliosides(21, 22) . We and others have provided
indirect but highly suggestive evidence for a naturally occurring
ganglioside modification in which the C-5 amino group of sialic acid is
unsubstituted, creating ``de-N-acetyl-gangliosides''
(deNAc-gangliosides)(16, 23, 24) . Also, in
contrast to the suppressive effects of G, addition of
synthetic deNAc-G
to various cell lines stimulated
proliferation (16) as well as in vitro tyrosine and/or
serine phosphorylation of the EGFR(24) . Thus far,
deNAc-gangliosides have been detected in extremely low quantities and
are suggested to be preferentially expressed in certain tumors and
tumor cell lines(16, 23, 24) . In human
melanoma cells in culture, we have found that expression of
deNAc-gangliosides is transient and variable, (
)making
further analysis difficult. We therefore developed new monoclonal
antibodies specifically recognizing deNAc-G
and
deNAc-G
and used them to assess the ability of various
agents to stimulate expression of these molecules in cultured melanoma
cells.
Figure 1:
mAb reactivity by
HPTLC immuno-overlay and the effect of mild periodate oxidation. Panel A, purified synthetic deNAc-G (1 µg, lanes 1, 3, 5, and 7) and
deNAc-G
reaction mixture (2 µg, lanes 2, 4, 6, and 8) were loaded onto HPTLC plates
and developed using (50:40:10, v/v/v) chloroform/methanol, 0.02%
CaCl
. Lanes 1 and 2 and 5 and 6 were cut out and treated with mild periodate while lanes
3 and 4 and 7 and 8 were incubated with
PBS alone as described under ``Experimental Procedures.'' The
plates were then probed with SMR36 or SGR37 as indicated. Panel
B, the synthetic deNAc-G
reaction mixture (2 µg, lanes 2 and 4) or the G
starting
material (1 µg, lanes 1 and 3) was loaded onto a
HPTLC plate and developed as in panel A. After development,
the plate was split in half and overlaid with either SGR37 (lanes 3 and 4) or an antibody directed against G
,
MB3.6 (lanes 1 and 2).
Figure 2:
DEAE-HPLC profile of SMR36 and SGR37
reactive products. Synthetic deNAc-G (1 µg) and
deNAc-G
(2 µg) were fractionated on a TSK-DEAE-HPLC
column as described under ``Experimental Procedures.''
Aliquots (100 µl) of fractions were dried in 96-well plates and
analyzed by ELISA using hybridoma supernatants from SMR36 (panel
A) or SGR37 (panel B). The elution positions of G
and G
(indicated by bars) were established
using known
C-labeled gangliosides from Melur
cells.
Base hydrolysis of G gave a more
complex array of products (by resorcinol staining, not shown). However,
SGR37 reacts only with two major products migrating slower than
G
(Fig. 1B) that were subsequently
identified as deNAc-G
and lyso-deNAc-G
. On
DEAE-HPLC these SGR37 reactive products elute in the region expected
for disialogangliosides (Fig. 2B, again the free amino
groups do not alter DEAE elution). These were further fractionated by
Iatrobeads HPLC and elution of SGR37-reactive products monitored by
HPTLC immuno-overlay. A single SGR37 reactive fraction was observed and
characterized by FAB-MS in the negative ion mode, yielding a major
cluster of signals shifted from G
by 42 mass units, i.e. de-N-acetylated G
. The most
abundant ions were at m/z 1457, 1471, 1485, 1499, and 1513
corresponding to de-NAc-G
containing fatty acyl chain
lengths C20:0, C21:0, C22:0, C23:0, and C24:0 respectively, with the
last three being most prominent (data not shown). Deuteropermethylation
of the SGR37-reactive fraction afforded major molecular ions at m/z 1827, 1841, and 1855 (Fig. 3), corresponding to fully
deuteromethylated mono-de-NAc-G
species having the fatty
acyl chain lengths of the most abundant components previously defined
by the FAB experiments on the native G
. Fragment ions at m/z 359 (m/z 394 minus deuteromethanol), 369
(de-N-acetylNeuAc
), 394
(NeuAc
), and 745
(de-N-acetylNeuAc-NeuAc
or
NeuAc-de-N-acetylNeuAc
) indicate that
monodeNac-G
is the major component in the reactive
fraction with deacetylation on either the inner or the outer Neu5Ac
residue. Thus, SGR37 reacts with de-N-acetylG
having a single free amino group on the inner and/or the outer
sialic acid residue (since these two isomers do not separate by HPTLC
or Iatrobeads, we cannot resolve this issue). In earlier experiments
the highly retarded lyso form of deNAc-G
also eluted very
late from the column (confirmed by FAB-MS, data not shown) and reacted
with the antibody, showing that reactivity is not dependent on the
presence of the ceramide fatty acyl chain. Re-N-acetylation of
de-NAc-G
reaction mixtures with acetic anhydride abolished
all reactivity with SGR37 confirming the requirement for the free amino
group of neuraminic acid (data not shown). Thus, in contrast to SMR36,
which reacts with both de-N-acetyl G
and
de-N-acetylG
, SGR37 is relatively specific for
the latter, cross-reacting very weakly with
de-N-acetylG
(Fig. 1A). If it is
assumed that SMR36 cross-reacts with the de-NAc-G
species
having the free amino group on the inner residue (i.e. similar
to de-NAc-G
), then substitution by the outer sialic acid
residue at the C-8 position of the inner one does not adversely effect
recognition. Although we examined the synthetic mixture for the
presence of di-de-NAc-GD3, the complexities of the ceramide fatty acyl
chain heterogeneity made it difficult to be certain if this derivative
is present. Therefore, we cannot rule out the possibility that SGR37
also reacts with di-de-NAc-G
(although the latter would be
expected to have a slower migration on HPTLC).
Figure 3:
Characterization of SGR37 reactive
fraction by fast atom bombardment-mass spectrometry. SGR37 reactive
material was purified and deuteropermethylated and the products
analyzed by positive FAB-MS. The signals separated by increments of 14
mass units from m/z 624 to 666 are derived from the ceramide.
The signal at m/z 377 is probably a b-cleavage ion derived
from a penultimate NeuAc residue. Other signals attributable to
de-N-acetyl G are described in the text. Minor
unassigned signals above m/z 900 are probably derived from
contaminants.
Mild periodate
treatment selectively truncates the unsubstituted exocyclic side chain
of terminal Neu5Ac, forming C-7 and C-8 derivatives without affecting
the underlying oligosaccharide structure (35, 36, 37) and can abolish mAb reactivity
with some gangliosides(33) . Such treatment abrogates
recognition of deNAc forms of G by SMR36 and deNAc forms
of G
by both SMR36 and SGR37 (Fig. 1A).
Identical treatment of a mild periodate-resistant ganglioside,
9-O-acetylatedG
(33) , did not affect
recognition by the specific antibody JONES(38) , indicating
that the insitu periodate treatment did not
nonspecifically affect colorimetric detection on the plate (data not
shown). Thus, both SMR36 and SGR37 require intact exocyclic side chains
on terminal sialic acids of the molecules they recognize. The data
raise the possibility that when SMR36 recognizes deNAc-G
,
it may be recognizing the isomer with the outer sialic acid
de-N-acetylated. Alternatively, oxidation of the side chain of
the outer residue may change the conformation of the molecule such that
an inner residue can no longer be recognized.
In summary, SMR36
recognizes deNAc-G, deNAc-G
, and their lyso
derivatives, while SGR37 reacts only with deNAc-G
and its
lyso derivative. Recognition by each antibody shows an absolute
requirement for a free amino group at the C-5 position of sialic acid
and an intact exocyclic sialic acid side chain on the terminal sialic
acid residue. This dual requirement indicates that these mAbs can be
used as highly specific probes for detection of deNAc-gangliosides in
melanoma cells. Furthermore, mild periodate oxidation and
re-N-acetylation can be used in appropriate situations to
confirm the specificity of antibody reactivity.
Figure 4:
The effect of various compounds on
deNAc-ganglioside expression. Melur cells were grown with added
MeSO solvent (2 µl), PMA (0.5 µM), or
genistein (100 µg/ml) as indicated. After 15 h, the cells were
harvested and stained with SMR36 and SGR37 (1:1) as described under
``Experimental Procedures.'' Dotted lines, isotype
stained non-treated cells; dashed lines, SMR36/SGR37 stained
cells treated with the indicated compound; solid lines,
non-treated control cells stained with
SMR36/SGR37.
Figure 5: Dose dependence of genistein effect on deNAc-ganglioside expression. Melur cells were grown in the presence of genistein at the concentrations indicated, for 15 h, the cells harvested, and stained with SMR36 and SGR37 (1:1 v/v) as described under ``Experimental Procedures.'' Dotted lines, SMR36/SGR37 stained non-treated cells grown with equivalent solvent volumes; dashed lines, genistein-treated cells stained with isotype matched antibodies; solid lines, genistein-treated cells stained with SMR36/SGR37.
Figure 6:
ELISA plate assay for detection of
deNAc-ganglioside expression in Melur melanoma cells treated with
genistein effects of mild periodate and de-N-acetylation.
Synthetic deNAc-G (panel A) and ganglioside
extracts of either control or genistein-treated Melur cells (panel
B) were studied for reactivity with a 1:1 mixture of SMR36 and
SGR37 in ELISA plates. Mild periodate oxidation and
re-N-acetylation destroyed the epitope after one or three
successive rounds of reaction, respectively, as shown in panel
A. Panel B shows the effect of genistein on
deNAc-ganglioside expression in Melur cells. The specificity of the
reaction was confirmed by its abrogation after periodate or
re-N-acetylation treatment. Reactivity in panel B was
adjusted for the amount of G
present in each sample,
determined in parallel by ELISA with an anti-G
antibody.
Figure 7:
SMR36/SGR37 immuno-overlay of endogenous
Melur gangliosides treated with and without genistein. Melur cells (2
150-mm plates) were grown in the presence (lanes 2 and 4) or absence (lanes 1 and 3) of 200
µM genistein for 15 h. The cells were harvested, and total
lipids were extracted and fractionated by DEAE-HPLC into mono- and
disialogangliosides. The fractions were loaded onto HPTLC plates and
developed using chloroform/methanol, 0.02% CaCl
(50:40:10,
v/v/v). The plates were overlaid with SMR36 and SGR37 and
deNAc-gangliosides detected using
[
S]streptavidin as described under
``Experimental Procedures.'' Lane 5 is a 2-µg
aliquot of the deNAc-G
reaction
mixture.
Figure 8: Comparative effects of genistein and herbimycin A on de-NAc-ganglioside expression in M21 melanoma cells. M21 cells were grown in the presence of herbimycin A (0.4 µM) or genistein (200 µM), for 8 h, and cells were harvested and analyzed separately for DNA content and expression of deNAc-gangliosides with SMR36 and SGR37, as described under ``Experimental Procedures.''
Figure 9: Induction of de-NAc-ganglioside expression in melanoma cells by nocodazole. Melur melanoma cells were grown in the presence of nocodazole (0.1 µg/ml), for 8 h, adherent and non-adherent cells were harvested and analyzed separately for DNA content and expression of deNAc-gangliosides with SMR36 and SGR37, as described under ``Experimental Procedures.''
Although the existence and potential growth regulatory
properties of deNAc-gangliosides were suggested several years ago,
analysis of these interesting molecules has proven to be difficult
because they are expressed transiently and in very small quantities.
For example, B16 melanoma cells, wherein deNAc-G was first
reported, were estimated to contain
1 pmol of deNAc-G
for every 5
10
cells even though G
is the major ganglioside expressed in this cell
line(16) . Our own previous studies using sensitive
double-label pulse-chase analyses provided indirect evidence for
expression of deNAc-G
and deNAc-G
in human
melanoma cells(23) . Subsequently, we noted that this evidence
for deNAc-gangliosides was variably seen, even between batches of
otherwise apparently identical cells.
This complicated
further analysis but was reminiscent of other transiently expressed
molecules known to regulate cellular proliferation or signal
transduction(50, 51, 52) . We therefore
attempted to stimulate synthesis of deNAc-gangliosides using various
growth altering and biologically active compounds.
To permit rapid
screening, we have developed mAbs highly specific for
deNAc-gangliosides. Targeted generation of mAbs with purified
ganglioside antigens is generally difficult, and most
ganglioside-specific antibodies have been derived fortuitously by
injection of whole cells into
mice(53, 54, 55) . However, one of us (T. T.)
has shown that certain murine strains provide more reliable host
responses to purified
gangliosides(27, 28, 56) . Using this and
other refinements, we have reported success in generating sets of mAbs
specific for ganglio-series gangliosides of the a, b, and
-pathways, and for NeuGc-containing
gangliosides(27, 28, 57, 58) .
Exploiting this knowledge, we produced two mAbs against purified
deNAc-gangliosides (one of them is an IgG). Of several compounds
initially screened, only genistein, a tyrosine kinase inhibitor,
consistently stimulated cell surface expression of deNAc-gangliosides
to a substantial degree. This was confirmed by whole cell lipid ELISA
assays, and by HPTLC immuno-overlays, in which a band corresponding to
deNAc-G
was detected in genistein-treated cells but not in
control cells. Ultimately, it will be necessary to isolate sufficient
quantities of this material to allow complete structural
characterization by FAB-MS, which should also allow for the precise
localization of the free amino group on the inner and/or the outer
sialic acid residue of G
. This will require improvements
in our current methods for derivatization and detection of gangliosides
by FAB-MS. Such efforts are currently under way.
While the transient
and variable nature of endogenous deNAc-ganglioside expression in
human melanoma cells has hindered careful analysis, this may also
suggests the involvement of these molecules in temporally regulated
biological processes. Understanding the mechanism by which genistein
induces deNAc-ganglioside expression may help to elucidate these
matters. Prior studies have provided conflicting results regarding the
direction of modulation of EGFR activity by addition of synthetic
deNAc-G to intact cells and isolated
membranes(16, 24, 59, 60) .
Regardless, these studies all provide evidence linking
deNAc-gangliosides to tyrosine kinase related signal transduction. In
this regard, the fact that genistein is a known inhibitor of tyrosine
kinases in intact cells (41, 46) is of obvious
interest. However, while some other tyrosine kinase inhibitors slightly
induce de-N-acetyl-ganglioside expression, none stimulate
expression to the extent achieved by genistein. Since genistein has
recently been shown to impede cell cycle progression beyond
G
M(43) , it is possible that it exerts its effect
on deNAc-ganglioside expression at least partly by blocking cell cycle
progression. If the expression of deNAc-gangliosides is normally
increased during this particular phase of the cell cycle, blocking
further progression would lead to increased expression of
deNAc-gangliosides. The cdc2 kinase is a component of the maturation
promoting factor that initiates mitosis. Specific tyrosine residues on
cdc2 are phosphorylated from the beginning of DNA synthesis to the
G
phase of the cell
cycle(61, 62, 63) . The activity of
maturation promoting factor is then induced by dephosphorylation of
tyrosine residues of cdc2 kinase to initiate mitosis(62) . A
previous study suggested that genistein may alter the tyrosine
phosphorylation/dephosphorylation process of cdc2 kinase, thereby
blocking the cell cycle at G
M. Our data indicate that when
melanoma cells are treated with genistein over a 15-h time period, the
number of cells in G
M is also increased relative to
non-treated control cells. As alternative evidence that this may be the
operative mechanism, we have shown that the mitotic spindle inhibitor
nocodazole also causes a buildup of deNAc-ganglioside expression.
Indeed, the selective buildup in expression in the non-adherent cells
in this case indicates a close association with metaphase. This
hypothesis is also attractive because it would help to explain the
highly variable expression of deNAc-gangliosides found in human
melanoma cells in different experiments under slightly varying culture
conditions.
While the association of deNAc-ganglioside expression
with the GM phase of the cell cycle is interesting, no
cause-and-effect conclusions can be reached because pharmacological
agents were used. Alternatively, it is well documented that
pharmacologic intervention in cellular processes can be counteracted by
the cell to maintain homeostasis, e.g. adrenergic receptor
blockade over long periods of time result in up-regulation of
adrenergic receptors(64) . This could also explain why the
maximal inductive effect of genistein on deNAc-ganglioside expression
does not occur until 6-15 h after addition of the compound.
Finally, it is possible that genistein is working by a mechanism
independent of its effects upon tyrosine
kinases(65, 66) . Studies are currently underway to
explore these possibilities further.
The N-acetyl group of
Neu5Ac originates from conversion of GlcNH-6-P to
GlcNAc-6-P(67, 68, 69) . GlcNAc-6-P is
converted via several steps to CMP-Neu5Ac, which is the donor for
sialyltransferases that synthesize
gangliosides(21, 22, 67, 68) . Since
the de-N-acetylated form of sialic acid, neuraminic acid, is
unstable in its free unbound form, it is reasonable to assume that the N-acetyl group remains covalently attached throughout these
steps. However, glycosidically bound neuraminic acid is at least as
stable as its N-acetylated counterpart(70) . Thus, the
most plausible explanation for deNAc-gangliosides is a specific
de-N-acetylase working on the intact ganglioside. These N-acetyl groups could also be rapidly replaced by an N-acetyltransferase. In fact, we have previously presented
pulse-chase data suggesting such a
de-N-acetylation/re-N-acetylation process in Melur
cells(23) . Thus, the delayed onset of the genistein effect on
deNAc-ganglioside expression could imply a feedback loop which may
cause up-regulation of a de-N-acetylase, or
inhibition/down-regulation of N-acetyltransferase. We are
currently searching for such enzyme activities. Further understanding
of these systems may also allow manipulations that permit accumulation
of sufficient quantities of deNAc-gangliosides for structural analysis
by methods such as FAB-MS or NMR.
In summary, this work provides a
new system in which to study deNAc-ganglioside biosynthesis and
function and lends further support for the involvement of
deNAc-gangliosides in growth regulation. Additionally, this report
provides the first evidence for the existence of deNAc-G in any cell type and raises the possibility that
deNAc-gangliosides may play a role in the regulation of the cell cycle.
This report also raises many new questions. For instance, what is the
mechanism of deNAc-ganglioside biosynthesis? Is the genistein effect
due to new ganglioside synthesis or to de-N-acetylation of
existing gangliosides? While the subcellular site of
re-N-acetylation appears to be in the Golgi apparatus or
Golgi-like elements(23) , where is the site of
de-N-acetylation? Is deNAc-G
involved in tyrosine
kinase signal transduction mechanisms as has been suggested for
deNAc-G
? Is the genistein effect specific for
de-N-acetylation of G
, or is deNAc-G
also induced at lower levels? Which isomer of mono-deNAc-G
predominates, and does di-deNAc-G
exist at all?
Finally, is deNAc-ganglioside expression indeed cell cycle dependent,
and if so, does it play an active role in the regulation of this vital
biological process? The present work has set the stage for exploration
of many of these questions.