(Received for publication, December 21, 1994)
From the
The present study was designed to prove the carbohydrate-binding
proteins interacting with cell surface sialyllactosylceramide
(G, NeuAc
2
3Gal
1
4Glc
1
1`Cer), which is highly expressed during differentiation of rat ovarian
granulosa cells. As a specific ligand for the sialyllactose
(SL)-binding proteins on granulosa cells, we used a radioiodinated
multivalent SL-linked albumin (Alb-(SL)
). The specific
association of the ligand to the putative proteins on the intact cells
was competitively inhibited by G
more effectively than
other gangliosides, sialyllactotetraosylceramide,
sialylneolactotetraosylceramide, and several glycoproteins with N-linked oligosaccharides. However, the proteins had no
specificity for the side chain (N-acetyl or N-glycolyl forms) of sialic acid in G
. Scatchard
analysis of Alb-(SL)
binding showed high (K
= 6.4
10
M) and low (K
= 3.1
10
M)
affinity population of binding sites. By direct binding of
I-Alb-(SL)
to SL-binding proteins on Western
blots, the putative proteins with molecular masses of 35, 18, and 14
kDa were detected. The interaction of the multivalent derivative with
these binding proteins was differently modulated by Ca
and Mn
. The SL-binding proteins occurred in
immature granulosa cells and progressively decreased during
differentiation, whereas their endogenous ligand G
increased. These results indicate that relatively low molecular
weight SL-binding proteins exist on the surface of immature granulosa
cells and that they may serve as receptor sites for newly synthesized
G
during differentiation.
Gangliosides, sialic acid-containing glycosphingolipids (GSLs), ()are located on the plasma membranes of eukaryotic cells
and have evolved for two critical roles in cellular
regulation(1) . They may act as modulators of transmembrane
signal transduction system and mediators for cell-cell interaction,
resulting in regulation of cell proliferation, differentiation, and
oncogenesis. Recently, there is a growing body of evidence that GSLs
may function as ligands in cell-cell interaction through their
oligosaccharide chains exposed to the external
environment(2, 3, 4, 5, 6, 7, 8, 9, 10) .
Hakomori and co-workers (6, 7, 8) have
reported that carbohydrate-carbohydrate interactions such as Le
to Le
and G
to Gg
may play
an important role in controlling cell recognition during cell
aggregation. They have provided a model in the process of cell adhesion
consisting of multiple steps, in which initial cell recognition is
mediated by multiple carbohydrate-carbohydrate
interactions(6) . Moreover, it has been reported using B16
melanoma cells that specific removal of SL from G
by
endoglycoceramidase treatment reduces cell aggregation(11) ,
indicating that the carbohydrates in G
may function as
determinants in the cell recognition system. Although
carbohydrate-carbohydrate interactions have received attention, great
interest has also been focused on cell-surface proteins as receptor
sites for gangliosides. Carbohydrate specificities of proteins such as
endogenous lectins and glycosyltransferases are primarily directed
toward mono- or disaccharide groups. Gangliosides appear to act as
binding sites for cell-surface
fibronectin(12, 13, 14) . There is a few
reports demonstrating that carbohydrate-binding proteins recognize the
specific oligosaccharide sequences in
gangliosides(5, 15) ; however, they are still limited
to the cell surfaces.
We previously described high expression of
G on the surface of ovarian granulosa cells during
differentiation(16) . Differentiation of granulosa cells
involves FSH-stimulated transformation of immature into mature cells.
Treatment with FSH promotes cell aggregation and induces receptors for
hormones and growth factors on their cell surfaces(17) . As
exogenous addition of G
to cultures and binding of
ganglioside-specific ligands such as antibodies to cell-surface
gangliosides stimulate FSH-induced LH receptor
expression(18, 19) , it is speculated that
gangliosides have some function in granulosa cell differentiation.
G
expressed on granulosa cells could be recognized by a
cell surface molecule such as Gg
, LacCer, and
Gb
(8) ; however, Gg
and Gb
are not expressed in granulosa cells, and LacCer content is very
low when compared with G
(16) . The SL recognition
molecules on granulosa cells have not yet been identified. In this
report, we extend our recent studies to prove that the
carbohydrate-binding proteins interacted with the carbohydrate portion
of G
. The strategy that we employed was to use a
multivalent SL derivative that possesses binding ability to the
putative proteins with high affinity. The results presented here
demonstrate relatively low molecular weight SL recognition proteins.
Figure 1:
The association kinetics of I-Alb-(SL)
using intact granulosa cells.
Granulosa cells were cultured for 48 h with 100 ng/ml FSH. The cells
were washed with binding buffer, then they were incubated with 0.1
nM
I-Alb-(SL)
at indicated
intervals at 4 (
), 15 (
) or 37 °C (
). Data show
the means ± S.E. for triplicate determinations. Specific binding
was determined by subtracting nonspecific binding in the presence of
100 µM NAcG
from total
binding.
Various GSLs were tested for their ability to suppress I-Alb-(SL)
binding to cultured intact cells.
Monosialogangliosides such as G
and G
competitively inhibited the binding (Fig. 2). Two types of
G
, those bearing N-acetyl or N-glycolyl
neuraminic acids, were the most potent of GSLs tested as competitors,
with IC
values in the micromolar concentration, although
they were less potent than Alb-(SL)
(Table 1).
However,
2
3SL and NeuAc had no significant inhibition at
concentration of 1 mM. SPG
(NeuAc
2
3Gal
1
4GlcNAc
1
3Gal
1
4Glc
1
1`Cer)
showed only weak inhibition at 100 µM, and LSTa-Cer
(NeuAc
2
3Gal
1
3GlcNAc
1
3Gal
1
4Glc
1
1`Cer) had no inhibitory potency.
Fetuin, having the NeuAc
2
3Gal
1
4GlcNAc
1 group
in the N-linked oligosaccharides, showed a significant
inhibition, whereas glycoproteins such as prothrombin (having
NeuAc
2
3Gal
1
3GlcNAc
1 in the oligosaccharide
side chain) and orosomucoid showed no inhibition at 100
µM.
Figure 2:
Competitive inhibition of I-Alb-(SL)
binding to intact granulosa cells
by monosialogangliosides. The cells cultured with 100 ng/ml FSH for 48
h were incubated in 0.2 ml binding buffer with 0.1 nM
I-Alb-(SL)
and increasing
concentrations of the competitors such as Alb-(SL)
(
), NAcG
(
), NGcG
(
)
and G
(
) for 3 h at 37 °C. The gangliosides
were suspended in MEM with the aid of a sonicator before appropriate
dilution into binding buffer. Data show the mean ± S.E. of
triplicate determinations.
Figure 3:
Scatchard analysis of I-Alb-(SL)
binding to granulosa cell plasma
membranes. Granulosa cell membranes were incubated with 0.02 nM
I-Alb-(SL)
and increasing
concentrations of cold Alb-(SL)
(10 nM to 2
µM) in 100 µl binding buffer containing 5 mM MnCl
at 37 °C for 3 h. Membrane-bound ligand was
separated from free ligand by rapid filtration through glass microfiber
filters. Nonspecific binding was determined in parallel incubations in
the absence of membranes, and was subtracted from total binding. Data
show the mean ± S.E. of triplicate determinations. Dissociation
constant (K
) and maximum binding capacity (B
) were calculated by the method of
Scatchard(25) .
Figure 4:
Direct binding of I-Alb-(SL)
to granulosa cell membrane
proteins separated by SDS-PAGE and transferred to PVDF filters. 10
µg plasma membrane proteins from cultured granulosa cells were
subjected to SDS-PAGE and the filters were treated as follows. A, proteins stained with Coomassie Brilliant Blue. Lane
1, molecular mass markers; lane 2, membrane proteins. B, the filters were incubated with
I-Alb-(SL)
in the absence (lane 1)
or presence of 10 µM NAcG
(lane 2).
In this case, 0.15 M NaCl, instead of 0.1% Triton X-100, was
contained in binding solution. C, the filters were incubated
with the ligand in the absence (lane 1) or presence (lane
2) of 0.15 M NaCl.
Figure 5:
Different dependence of the interaction of
the multivalent SL derivative to the SL-binding proteins on bivalent
cations. After SDS-PAGE of plasma membranes and their electroblotting
to PVDF filters as described in Fig. 4, incubation with I-Alb-(SL)
was carried out in 20 mM Tris buffer containing 2% BSA and 0.1% Triton X-100 in the absence (lane 1) or presence of 5 mM Ca
(lane 2) or 5 mM Mn
(lane
3), thereafter the filters were washed with Tris buffer containing
0.15 M NaCl with or without each bivalent cations,
respectively.
Figure 6:
Time-dependent changes of G synthesis, SL-binding proteins, and LH receptor content during
granulosa cell differentiation. Granulosa cells were cultured for
indicated times with or without 100 ng/ml FSH. A, ganglioside
synthesis was performed by metabolic labeling of cells with
[
H]Gal at 0-48 h and 48-96 h, and
H-labeled G
was isolated by TLC as described
under ``Experimental Procedures.'' The data are means
± S.E. of triplicate determinations. B, the SL-binding
proteins of plasma membranes isolated from granulosa cells cultured for
48 h (lanes 1 and 2) and 96 h (lanes 3 and 4) with (lanes 2 and 4) or without (lanes 1 and 3) FSH were detected by Western blots. C, LH receptor content in the cells cultured was assayed as
described under ``Experimental Procedures.'' The data are
mean ± S.E. of triplicate
determinations.
Immature granulosa cells develop into their mature
counterpart in response to FSH, and concomitantly receptors for
hormones and growth factors are expressed abundantly on the mature
cells. The morphological appearance of the cultured granulosa cells
exhibits extensive aggregation of cells with long cell processes that
form contacts with adjacent cells. The cells also express ganglioside
G during differentiation(16) . Manipulation of
membrane gangliosides leads to modulation of FSH-induced expression of
LH receptor(17, 18) , and inhibition of cell
aggregation is induced by exogenous G
. (
)Based
on these findings, we have focused on the possible role of the
carbohydrate chain,
2
3SL, as a ligand to cell-surface
substances, and have proposed that its interaction may regulate the
cellular behavior during differentiation. As a specific ligand for
identifying the hypothetical SL-binding proteins on granulosa cells, we
used a multivalent SL-linked albumin. In the present study the
multivalency of the derivative is an advantage for proving the
occurrence of the proteins, since its decreased multivalency by trypsin
hydrolysis of protein backbone reduced the binding ability on granulosa
cells (data not shown). Our present study provides clear evidence for
the occurrence and modulation of SL-binding proteins on the surface of
immature and mature granulosa cells.
The binding characteristics of I-Alb-(SL)
using intact cells and plasma
membranes were indicative of specific ligand binding substances. The
interaction was temperature-dependent, and the associated kinetics were
slow even at 37 °C, showing a low affinity. The ligand binding to
intact cells was blocked by micromolar concentrations of G
added in binding solution as micelles, but the SL-binding
proteins did not have structural specificity of the terminal neuraminic
acid, N-acetyl or N-glycolyl forms, bound to the
lactose residue. Other gangliosides such as G
,
G
, and G
had low inhibitory potencies
compared with G
. SPG, LSTa-Cer, prothrombin, and
orosomucoid were at most weakly inhibitory. However, fetuin, having a
structural similarity with SPG in the side chain of oligosaccharides,
showed a significant inhibition. These data indicate that anionic
charge in the neuraminic acid residue is not responsible for the
binding ability to SL-binding proteins. Moreover, although carbohydrate
portions of gangliosides have at least affinity to the binding
proteins, Gal
1
3GalNAc
1
4 grouped on the internal
galactose appears to reduce the inhibitory potency. In addition,
NeuAc
2
3Gal
1
4GlcNAc
1 grouped on the
glycoproteins and glycolipids seems to have affinity to the binding
proteins, although its affinity is very low compared with
G
. These binding studies suggest structural specificify of
the carbohydrate portion in the ligand binding. In this assay system,
however, monovalent
2
3SL failed to prevent binding of the
multivalent derivative even at a high concentration of 1 mM.
Such a marked difference between G
and its monovalent
oligosaccharide has been reported for other glycolipid systems such as
G
and
sulfoglucuronylneolacto-glycolipid(5, 10) . A
monovalent neoganglioprotein, BSA linked with one G
molecule to brain membranes, shows less binding ability than its
tetravalent counterpart(5) . The multivalency of the SL
derivative appears to increase its apparent affinity for cell surface
proteins. Since all glycosphingolipids at the outer surface of cell
membrane lipid bilayer are believed to exist as large
clusters(26, 27, 28) , the multivalency of
carbohydrates may be suitable for the interaction with the proteins of
high affinity.
In the Scatchard analysis of I-Alb-(SL)
binding, the binding isotherm was
curvilinear, showing multiple classes of binding sites. The binding
affinities were K
of 6.4
10
M and 3.1
10
M when
calculated as two sites. A large population of the binding proteins was
expected from maximum binding values (B
=
98.8 pmol/mg, 16.1 nmol/mg). In fact, it was revealed that
I-Alb-(SL)
was able to associate with
several proteins on protein-blotted PVDF filters under certain ionic
conditions and that these proteins occurred abundantly. These proteins
were estimated as molecular masses of 35, 18, and 14 kDa, and these
were tentatively referred to SLBP-35, SLBP-18, and SLBP-14,
respectively. Binding of
I-Alb-(SL)
to these
proteins on protein-blotted PVDF filters was clearly replaced by
G
The association of I-Alb-(SL)
to these binding proteins required bivalent cations such as
Mn
and Ca
. It is important to note
that the SL-binding proteins differed in their requirements of these
cations. The multivalent SL derivative bound to SLBP-18 in a manganese-
or calcium-dependent manner, whereas its binding to SLBP-14 was
predominantly manganese-dependent. However, SLBP-35 was independent on
these bivalent cations. Carbohydrate interactions with lectins and
glycosyltransferases usually require Ca
and
Mg
(29, 30, 31, 32, 33, 34, 35, 36) .
Binding of
I-sulfoglucuronylneolacto-coupled Alb to rat
sciatic nerve myelin is increased by Ca
more
effectively than other bivalent cations(10) .
Carbohydrate-carbohydrate interaction is also sensitive to bivalent
cations(6) . However, we do not know different properties of
bivalent cations in SL interactions with proteins or carbohydrates. It
has been reported that sulfoglucuronylneolacto-glycolipid interaction
with L- and P-selectins is calcium-independent and their interaction of
sialyl Le
glycolipid is calcium-dependent(37) .
Based on their molecular mass and their dependence of bivalent cations,
the SL-binding proteins presented here do not seem to correspond to
certain defined proteins, such as
fibronectin(12, 13, 14) , myelin basic
protein(38) , and sialoadhesin(9) , which bind to
gangliosides. Particularly, sialoadhesin, a glycoprotein of 185 kDa, is
able to bind to G
more than to other
monosialogangliosides. Similar to sialoadhesin, a sialic acid binding
protein derived from human placenta(39) , the lymphocyte homing
receptor (LECAM-1) (40, 41) and the endothelial
leukocyte adhesion molecule (ELAM-1) (42, 43) have
been shown to bind specific oligosaccharide structure containing
terminal sialic acid on both glycoproteins and glycolipids. These
carbohydrate-binding proteins are much larger in molecular mass than
our SL-binding proteins.
The SL-binding proteins described in the
present study occurred abundantly in the immature cells and reduced
progressively during differentiation, although, at present, we do not
know exactly what physiological functions these SL-binding proteins
have in granulosa cell differentiation. These proteins may interact
with their endogenous ligand G newly synthesized in
response to differentiation-inducing factors, including FSH. Initial
cell recognition has been proposed to be dependent on multivalent
carbohydrate-carbohydrate interactions followed by nonspecific adhesion
molecules such as adhesion proteins or carbohydrate-binding proteins (6, 7, 8) . However, even after extensive
aggregation of granulosa cells and full expression of LH receptors,
G
synthesis was further increased. Thus, although newly
synthesized G
may interact with SL-binding proteins in the
earlier stage of cell differentiation, successively synthesized
G
appears to be involved in cellular regulation rather
than cell recognization. At any rate, cell recognition and successive
cell aggregation during granulosa cell differentiation may be
controlled primarily by G
expression, and this finding of
novel SL-binding proteins will lead to clarification of
G
-dependent regulation mechanism in the cell
differentiation.