©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Sialyllactose-mediated Cell Interaction during Granulosa Cell Differentiation
IDENTIFICATION OF ITS BINDING PROTEINS (*)

(Received for publication, December 21, 1994)

Masa-aki Hattori (1)(§) Ryuya Horiuchi (1)(¶) Kohei Hosaka (3) Hiroaki Hayashi (2)(**) Itaru Kojima (1)

From the  (1)Departments of Cell Biology and (2)Molecular Medicine, Institute for Molecular and Cellular Regulation, Gunma University, and the (3)Department of Biochemistry, Gunma University School of Medicine, 3-39-15 Showa-machi, Maebashi, Gunma 371, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The present study was designed to prove the carbohydrate-binding proteins interacting with cell surface sialyllactosylceramide (G, NeuAcalpha23Galbeta14Glcbeta1 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 times 10M) and low (K = 3.1 times 10M) 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.


INTRODUCTION

Gangliosides, sialic acid-containing glycosphingolipids (GSLs), (^1)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^x to Le^x and G to Gg(3) 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(3), LacCer, and Gb(4)(8) ; however, Gg(3) and Gb(4) 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.


EXPERIMENTAL PROCEDURES

Materials

NAcG, other gangliosides, alpha23SL, and Alb-(SL) (17 SL groups per Alb) were purchased from Biocarb Chemicals (Lund, Sweden). NGcG (from horse erythrocytes) was from Seikagaku Co. (Tokyo, Japan), synthetic LSTa-Cer and synthetic SPG were from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), human urinary FSH (Metrodin, 130.4 IU/mg) was from Serono Japan Co. (Tokyo, Japan), [1-^3H]Gal was from DuPont NEN, carrier-free Na-I was from ICN Biomedical Inc. (Costa Mesa, CA), McCoy's 5A and MEM were from Life Technologies, Inc., and bovine insulin, bovine fetuin, bovine prothrombin, and bovine orosomucoid were from Sigma. All the other chemicals used were of reagent grade and obtained from commercial sources.

Cell Culture

Granulosa cells were prepared by puncturing ovaries from immature Wistar rats, which were treated with diethylstilbestrol(20) . The cells (4 times 10^5 in 1 ml of medium) were cultured in McCoy's 5A medium supplemented with 10 mM HEPES, 4 mML-glutamine, 100 nM estradiol-17beta, 4 µg/ml insulin, 1 mg/ml BSA, 50 units/ml penicillin, and 50 µg/ml streptomycin sulfate (pH 7.4) in polystyrene tubes (12 times 75 mm, Becton Dickinson) or Petri dishes (100-mm diameter, Corning, Tokyo, Japan) at 37 °C in a humidified 95% air, 5% CO(2) incubator for the selected times.

G Isolation and Analysis

The granulosa cells were cultured for indicated times with 100 ng/ml FSH and 5 µCi/ml [1-^3H]Gal in polystyrene tubes and washed with ice-cold phosphate-buffered saline. The cells were first sonicated in methanol/water (1:0.1), then the lipids were extracted with successive mixtures of chloroform/methanol/water, 1:1:0.1, 2:1:0, and 1:2:0.1 (v/v)(21) . The lipid fractions were applied on a DEAE-Sephadex A-25 column (10 times 120 mm) to separate neutral and acidic lipids, and then they were treated by mild alkaline hydrolysis (0.1 M NaOH in methanol) at 40 °C for 3 h, followed by neutralization with 2 M HCl(22) . The salts were removed through Sep-Pak C(18) cartridges (Millipore, Milford, MA), and the GSLs eluted from the cartridges were separated on thin-layer plates (Silica Gel 60A, Whatman) using chloroform, methanol, 0.25% aqueous KCl, 60:35:8, as the solvent system and visualized by autoradiofluorography (16) . The location of ^3Hlabeled G corresponded to that of authentic G which was detected with resorcinol-hydrochloric acid. The ^3H-labeled G was extracted from the scraped silica gel with methanol, after removal of the organic solvent, its radioactivity was counted using liquid scintillation spectrometry.

Preparation of Plasma Membranes from Cultured Granulosa Cells

Granulosa cells cultured for 48-96 h in Petri dishes were harvested, washed with cold phosphate-buffered saline, and homogenized in 20 mM Tris-HCl, pH 8.0, containing 0.5 mM CaCl(2) and 25 mM NaCl using a Dounce-type homogenizer, then centrifuged for 1 min at 200 times g to separate the nuclear fraction. An equivolume of 60% sucrose was added to the supernatant solution, and the solution was placed on the top of a discontinuous sucrose gradient (45/42/39%) which was centrifuged at 90,000 times g for 90 min. The plasma membrane fraction (39/42% interphase) was collected and washed once with 20 mM Tris-HCl, pH 8.0. Protein content was determined with BCA protein assay reagent (Pierce Chemical Co.).

Binding Studies of I-Alb-(SL) Using Intact Cells and Membranes

Granulosa cells cultured in polystyrene tubes were washed once with 2 ml of ice-cold binding buffer (MEM containing 25 mM HEPES and 1 mg/ml BSA, without sodium bicarbonate, pH 7.4) and incubated in 0.2 ml of binding buffer at 37 °C for 3 h with 0.1 nMI-Alb-(SL) (specific activity, 100,000 cpm/ng) which was labeled using chloramine T. The cell-bound and -free radioactive ligands were separated by adding 2 ml of ice-cold binding buffer and centrifuging at 1,000 times g for 15 min. Binding studies using purified membranes were performed at 37 °C for 3 h in a final volume of 100 µl, and each tube contained 2 µg of membrane proteins in 20 mM Tris-HCl, pH 7.4, containing 5 mM MnCl(2), 1 mg/ml BSA, and 0.1 nMI-Alb-(SL). The ligand bound to the membranes was separated from unbound fraction by rapid filtration through glass microfiber filters (Whatman, GF/C). The filters were pretreated with 1% BSA in 20 mM Tris buffer to reduce background binding and were rinsed with 20 mM Tris buffer before use. After filtration of the binding reactions, the filters were washed three times, 1 ml each, with ice-cold 20 mM Tris buffer containing 5 mM MnCl(2) and 1 mg/ml BSA, pH 7.4. When GSLs were added as inhibitors, they were evaporated from methanol stock solutions using a centrifugal evaporator, and the residue was resuspended in MEM or 20 mM Tris buffer (100 nmol/0.5 ml) with the aid of a sonicator before appropriate dilution into binding buffer. After centrifuging the tube or washing the filter, the radioactivity remaining in the tube or the filter (cell- or membrane-bound ligand) was quantified by -spectrometry. Nonspecific binding for studies using intact cells was determined in the presence of 100 µM G, and that for studies using membranes was determined in the absence of membranes.

SDS-PAGE and Western Ligand Blots of SL-binding Proteins

SDS-PAGE was performed according to Laemmli (23) using 15% gels under nonreducing conditions. The size-fractionated proteins were then electroblotted onto PVDF filters, thereafter, the filters were washed three times at room temperature with 20 mM Tris buffer, pH 7.4, and soaked in blocking solution containing 0.15 M NaCl and 1% gelatin at 4 °C overnight. PVDF filters were rinsed with Tris buffer, incubated for 3 h at 37 °C with 0.1 nMI-Alb-(SL) in binding solution containing 5 mM MnCl(2), 2% BSA and 0.1% Triton X-100, then washed once with binding buffer and three times with 20 mM Tris buffer containing 0.15 M NaCl and 5 mM MnCl(2), unless stated otherwise. The filters were dried and exposed to Kodak XAR film at -80 °C for 24 h. Molecular mass markers used were the colored proteins (Amersham International plc. Buckinghamshire, UK).

Measurement of LH Receptor in Granulosa Cells

The LH receptor binding studies were performed using I-hCG, and the LH receptor content of the cells was measured by quantifying the amounts of I-hCG in the intact cells, as described previously(24) .


RESULTS

Binding of I-Alb-(SL) to Cultured Granulosa Cells

After differentiation by treatment of granulosa cells for 48 h with 100 ng/ml FSH, the properties of SL-binding molecules were characterized using I-Alb-(SL) as a ligand. The association of the ligand to the intact cells was temperature-dependent. In the studies on the association kinetics, I-Alb-(SL) binding increased at 37 °C in a time-dependent manner, reaching the equilibrium state after 8 h (Fig. 1). At 4 and 15 °C, the ligand was very slow in the rate of association, and the maximum binding was less than that at 37 °C after incubation for 20 h.


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 nMI-Alb-(SL) at indicated intervals at 4 (circle), 15 (box) or 37 °C (bullet). 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, alpha23SL and NeuAc had no significant inhibition at concentration of 1 mM. SPG (NeuAcalpha23Galbeta14GlcNAcbeta13Galbeta14Glcbeta11`Cer) showed only weak inhibition at 100 µM, and LSTa-Cer (NeuAcalpha23Galbeta13GlcNAcbeta1 3Galbeta14Glcbeta11`Cer) had no inhibitory potency. Fetuin, having the NeuAcalpha23Galbeta14GlcNAcbeta1 group in the N-linked oligosaccharides, showed a significant inhibition, whereas glycoproteins such as prothrombin (having NeuAcalpha23Galbeta13GlcNAcbeta1 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 nMI-Alb-(SL) and increasing concentrations of the competitors such as Alb-(SL) (circle), NAcG (bullet), 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.





Scatchard Analysis of I-Alb-(SL) Binding

Scatchard analysis of I-Alb-(SL) binding was performed using differentiated granulosa cell membranes. From studies on the binding isotherms performed by incubation of membranes with 0.1 nMI-Alb-(SL), increasing concentrations of cold Alb-(SL) and 5 mM MnCl(2), Scatchard transformation of Alb-(SL) binding isotherm data was curvilinear (Fig. 3), indicating the presence of high and low affinity population of binding sites when fit to a two-site model (high affinity class K(d) = 6.4 times 10M, B(max) = 98.8 pmol/mg of membrane proteins; low affinity class K(d) = 3.1 times 10M, B(max) = 16.1 nmol/mg of membrane proteins).


Figure 3: Scatchard analysis of I-Alb-(SL) binding to granulosa cell plasma membranes. Granulosa cell membranes were incubated with 0.02 nMI-Alb-(SL) and increasing concentrations of cold Alb-(SL) (10 nM to 2 µM) in 100 µl binding buffer containing 5 mM MnCl(2) 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(max)) were calculated by the method of Scatchard(25) .



Direct Binding of I-Alb-(SL) to SL-binding Proteins on Western Blots

To directly detect SL-binding proteins, granulosa cell membrane proteins were separated by SDS-PAGE, transferred to PVDF filters, and probed with I-Alb-(SL). Treatment of plasma membranes at 4 °C overnight with sample buffer under reducing or nonreducing conditions gave similar blotting patterns, and subsequent experiments were performed under nonreducing conditions. I-Alb-(SL) bound to several membrane proteins with molecular masses of 35, 18, and 14 kDa (Fig. 4, B and C), referred to as SLBP-35, SLBP-18, and SLBP-14, respectively. The intensity of I-Alb-(SL)-bound bands with these molecular species was greatly diminished when the protein-blotted PVDF was incubated in the presence of 10 µM G (Fig. 4B). G replacement was carried out in the binding solution containing 0.15 M NaCl instead of 0.1% Triton X-100, because the I-Alb-(SL) binding was not replaced by G in the presence of the detergent (data not shown). However, I-Alb-(SL) binding to SL-binding proteins reduced in the presence of 0.15 M NaCl (Fig. 4C). These binding proteins were visualized by Coomassie Brilliant Blue staining (Fig. 4A).


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.



Ionic Requirements of I-Alb-(SL) Binding to SL-binding Proteins

The ligand binding was examined in the presence of bivalent cations using differentiated granulosa cell membranes. Some of the bivalent cations increased I-Alb-(SL) binding to membranes, and Mn was the most potent in specific binding, with maximal binding at 10 mM (5,238 ± 80 cpm/µg of protein, mean ± S.E., n = 3). However, in the presence of more than 30 mM, Mn increased the nonspecific binding (without membranes) of I-Alb-(SL). Ca also increased the specific binding, with about 50% of maximal binding induced by 10 mM Mn (data not shown). To explore the effects of Ca and Mn on the SL-binding proteins, they were probed on Western blots with I-Alb-(SL) in the presence of these cations. When I-Alb-(SL) binding was carried out in binding and washing solutions containing 5 mM Ca or 5 mM Mn, the ligand binding to SLBP-18 was enhanced (Fig. 5). Both cations had similar potencies in enhancement of the ligand binding to SLBP-18. However, the ligand binding to SLBP-14 was predominantly enhanced by Mn rather than Ca. Particularly, in the absence of Mn, I-Alb-(SL) was almost unable to bind to SLBP-14. The ligand binding to SLBP-35 remained unchanged even in the presence of Ca or Mn.


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.



Alterations of G Synthesis and SL-binding Proteins during Granulosa Cell Differentiation

To further explore the relationship of SL-binding proteins with ganglioside G, their changes were determined during cell differentiation and compared with LH receptor content, which is a differentiation marker of granulosa cells. When granulosa cells were labeled with [1-^3H]Gal at 0-48 h and 48-96 h of culture in the presence of FSH, the level of ^3H-labeled G was 5 times higher in the cells labeled at 48-96 h than in the cells labeled at 0-48 h (Fig. 6A). The intensity of I-Alb-(SL)-bound SLBP-35, SLBP-18, and SLBP-14 decreased evenly in FSH-treated groups in a time-dependent manner, whereas they remained unchanged in control groups (Fig. 6B). Thus, the level of G correlated inversely with levels of the SL-binding proteins. During cell differentiation, LH receptor did not appear on the cells at 24 h, reached a maximum level at 48 h, and reduced to about 50% of a maximum at 96 h (Fig. 6C).


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 [^3H]Gal at 0-48 h and 48-96 h, and ^3H-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.




DISCUSSION

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. (^2)Based on these findings, we have focused on the possible role of the carbohydrate chain, alpha23SL, 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, Galbeta13GalNAcbeta14 grouped on the internal galactose appears to reduce the inhibitory potency. In addition, NeuAcalpha23Galbeta14GlcNAcbeta1 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 alpha23SL 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(d) of 6.4 times 10M and 3.1 times 10M when calculated as two sites. A large population of the binding proteins was expected from maximum binding values (B(max) = 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^x 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.


FOOTNOTES

*
This work was supported in part by a Grant-in-Aid for Developmental Scientific Research No. 02660269 from the Japanese Ministry of Education, Science and Culture (to M. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Animal Science, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812, Japan. Tel.: 81-92-641-1101; Fax: 81-92-641-2928.

Present address: Dept. of Pharmacy, Gunma University School of Medicine, 3-39-15 Showa-machi, Maebashi, Gunma 371, Japan.

**
Present address: Gunma Prefectural College of Health Sciences, 232-1 Kamioki-machi, Maebashi, Gunma 371, Japan.

(^1)
The abbreviations used are: GSL, glycosphingolipid; G, sialyllactosylceramide, NeuAcalpha23Galbeta14Glcbeta11`Cer; Gg(3), gangliotriaosylceramide; SL, sialyllactose; FSH, follicle-stimulating hormone; LH, luteinizing hormone; Alb-(SL), alpha23SL human serum albumin conjugate; NAcG, G containing N-acetylneuraminic acid; NGcG, G containing N-glycolylneuraminic acid; MEM, minimum essential medium; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis; PVDF, poly(vinylidene fluoride); hCG, human chorionic gonadotropin; LacCer, lactosylceramide; Gb(4), globoside; G, II^3NeuAc-GgOse(4)Cer; G, IV^3NeuAc, II^3NeuAc-GgOse(4)Cer; G, IV^3NeuAc, II^3(NeuAc)(2)-GgOse(4)Cer; SPG, IV^3NeuAcalpha-nLc(4)Cer; LSTa-Cer, IV^3NeuAcalpha-Lc(4)Cer.

(^2)
M. Hattori, and R. Horiuchi, unpublished observation.


REFERENCES

  1. Hakomori, S. (1990) J. Biol. Chem. 265, 18713-18716 [Abstract/Free Full Text]
  2. Fenderson, B. A., Zehavi, U., and Hakomori, S. (1984) J. Exp. Med. 160, 1591-1596 [Abstract]
  3. Bird, J. M., and Kimber, S. J. (1984) Dev. Biol. 104, 449-460 [Medline] [Order article via Infotrieve]
  4. Cheresh, D. A., Pytela, R., Pierschbacher, M. D. Klier, F. G., Ruoslahti, E., and Reisfeld, R. A. (1987) J. Cell Biol. 105, 1163-1173 [Abstract]
  5. Tiemeyer, M., Yasuda, Y., and Schnaar, R. L. (1989) J. Biol. Chem. 264, 1671-1681 [Abstract/Free Full Text]
  6. Eggens, I., Fenderson, B., Toyokuni, T., Dean, B., Stroud, M., and Hakomori, S. (1989) J. Biol. Chem. 264, 9476-9484 [Abstract/Free Full Text]
  7. Kojima, N., and Hakomori, S. (1989) J. Biol. Chem. 264, 20159-20162 [Abstract/Free Full Text]
  8. Kojima, N., and Hakomori, S. (1991) J. Biol. Chem. 266, 17552-17558 [Abstract/Free Full Text]
  9. Crocker, P. R., Kelm, S., Dubois, C., Martin, B., McWilliam, A. S., Shotton, D. M., Paulson, J. C., and Gordon, S. (1991) EMBO J. 10, 1661-1669 [Abstract]
  10. Needham, L. K., and Schnaar, R. L. (1993) J. Cell Biol. 121, 397-408 [Abstract]
  11. Ito, M. (1994) Seikagaku 66, 99-125 [Medline] [Order article via Infotrieve]
  12. Kleinman, H. K., Martin, G. R., and Fishman, P. H. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 3367-3371 [Abstract]
  13. Yamada, K. M., Kennedy, D. W., Grotendorst, G. R., and Momoi, T. (1981) J. Cell. Physiol. 109, 343-351 [Medline] [Order article via Infotrieve]
  14. Yamada, K. M., Critchley, D. R., Fishman, P. H., and Moss, J. (1983) Exp. Cell Res. 143, 295-302 [Medline] [Order article via Infotrieve]
  15. Tiemeyer, M., Swank-Hill, P., and Schnaar, R. L. (1990) J. Biol. Chem. 265, 11990-11999 [Abstract/Free Full Text]
  16. Hattori, M., and Horiuchi, R. (1992) Biochim. Biophys. Acta 1137, 101-106 [Medline] [Order article via Infotrieve]
  17. Hsueh, A. J. W., Bicsak, T. A., Jia, X.-C., Dahl, K. D., Fauser, B. C. J. M., Galway, A. B., Czekala, N., Pavlou, S. N., Papkoff, H., Keene, J., and Boime, I. (1989) Recent Prog. Horm. Res. 45, 209-273 [Medline] [Order article via Infotrieve]
  18. Hattori, M., and Horiuchi, R. (1992) Mol. Cell. Endocrinol. 88, 47-54 [Medline] [Order article via Infotrieve]
  19. Hattori, M., Kanzaki, M., Kojima, I., and Horiuchi, R. (1994) Biochim. Biophys. Acta 1221, 47-53 [Medline] [Order article via Infotrieve]
  20. Hattori, M., Takahashi, M., and Horiuchi, R. (1991) Mol. Cell. Endocrinol. 81, 69-76 [Medline] [Order article via Infotrieve]
  21. Nakaishi, H., Sanai, Y., Shibuya, M., and Nagai, Y. (1988) Biochem. Biophys. Res. Commun. 150, 766-774 [Medline] [Order article via Infotrieve]
  22. Ledeen, R. W., and Yu, R. K. (1983) Methods Enzymol. 83, 139-191
  23. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  24. Hattori, M., Hachisu, T., Shimohigashi, Y., and Wakabayashi, K. (1988) Mol. Cell. Endocrinol. 57, 17-23 [Medline] [Order article via Infotrieve]
  25. Scatchard, G. (1949) Ann. N. Y. Acad. Sci. 51, 660-672
  26. Tillack, T. W., Allietta, M., Moran, R. E., and Young, W. W., Jr. (1983) Biochim. Biophys. Acta 733, 15-24 [Medline] [Order article via Infotrieve]
  27. Rock, P., Allietta, M., Young, W. W., Jr., Thompson, T. E., and Tillack, T. W. (1990) Biochemistry 29, 8484-8490 [Medline] [Order article via Infotrieve]
  28. Rock, P., Allieta, M., Young, W. W., Jr., Thompson, T. E., and Tillack, T. W. (1991) Biochemistry 30, 19-25 [Medline] [Order article via Infotrieve]
  29. Frazier, W., and Glaser, L. (1979) Annu. Rev. Biochem. 48, 491-523 [Medline] [Order article via Infotrieve]
  30. Barondes, S. H. (1981) Annu. Rev. Biochem. 50, 207-231 [CrossRef][Medline] [Order article via Infotrieve]
  31. Ashwell, G., and Hanford, J. (1982) Annu. Rev. Biochem. 51, 531-534 [CrossRef][Medline] [Order article via Infotrieve]
  32. Shur, B. D. (1982) J. Biol. Chem. 257, 6871-6878 [Abstract/Free Full Text]
  33. Sharon, N. (1984) Biol. Cell 51, 239-246 [Medline] [Order article via Infotrieve]
  34. Gabius, H.-J., Engelhardt, R., and Cramer, F. (1986) Anticancer Res. 6, 573-578 [Medline] [Order article via Infotrieve]
  35. Drickamer, K. (1988) J. Biol. Chem. 263, 9557-9560 [Free Full Text]
  36. Malhotra, R., Laursen, S. B., Willis, A. C., and Sim, R. B. (1993) Biochem. J. 293, 15-19 [Medline] [Order article via Infotrieve]
  37. Needham, L. K., and Schnaar, R. L. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1359-1363 [Abstract]
  38. Yohe, H. C., Jacobson, R. I., and Yu, R. K. (1983) J. Neurosci. Res. 9, 401-412 [Medline] [Order article via Infotrieve]
  39. Ahmed, H., and Gabius, H. J. (1989) J. Biol. Chem. 264, 18673-18678 [Abstract/Free Full Text]
  40. Rosen, S. D., Chi, S.-I., True, D. D., Singer, M. S., and Yodnock, T. A. (1989) J. Immunol. 142, 1895-1902 [Abstract/Free Full Text]
  41. Imai, Y., True, D. D., Singer, M. S., and Rosen, S. D. (1990) J. Cell Biol. 111, 1225-1232 [Abstract]
  42. Phillips, M. L., Nudelman, E., Gaeta, F. C. A., Perez, M., Singhal, A. K., Hakomori, S., and Paulson, J. C. (1990) Science 250, 1130-1132 [Medline] [Order article via Infotrieve]
  43. Walz, G., Aruffo, A., Kolanus, W., Bevilacqua, M., and Seed, B. (1990) Science 250, 1132-1135 [Medline] [Order article via Infotrieve]

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