Glycosphingolipids in detergent-insoluble substrate attachment matrix (DISAM) prepared from substrate attachment material (SAM)

Experimental

Cell Research 155 (1984) 44W56

Glycosphingolipids in Detergent-insoluble Substrate Attachment Matrix (D&AM) Prepared from Substrate Attachment Material (SAM) Their Possible

Role in Regulating

Cell Adhesion

YOSHIO OKADA,* GABRIELE MUGNAI,** ERIC G. BREMER and SEN-ITIROH HAKOMORI*** Program of Biochemical OncologylMembrane Research, Fred Hutchinson Cancer Research Center, Departments of Pathobiology, Microbiology and Immunology, University of Washington, Seattle, WA 98104, USA

The glycosphingolipids isolated from the detergent-insoluble material (DIM) of whole cells as well as from a similar detergent-insoluble substrate attachment matrix (DISAM) have been investigated in comparison with the glycosphingolipids of whole cells. The proportion of glycolipids in the total lipid extract was enriched in the DISAM as well as DIM fractions as compared to whole cells. The ratio of ganglioside (GMs) to neutral glycolipids was also higher in the DISAM fractions than in whole cells. The radioactivity incorporated into DISAM glycolipids of BHK cells, metabolically labeled with radioactive glucosamine. was greater in confluent cells than in sparsely growing cells; however, label incorporation into glycolipids of the DISAM fraction of BHKpy cells was 2-3-fold higher than that of confluent BHK cells, although the chemical quantity of GM3 in whole cells was much lower in BHKpy cells than in BHK cells. In order to confirm the enhanced label in DISAM glycolipids of BHKpy cells by other procedures, the labeled cells were detached by EGTA, washed, and reattached on plates. The amount of label in DISAM glycolipids of the reattached matrix of BHKpy cells was much higher than that of BHK cells. Cell spreading and cell attachment on plastic plate were inhibited by inclusion of GM3 in the medium. These data suggest that: (i) glycolipids, particularly GM3, at the cell attachment site have different metabolic activity from those of whole cells; the label in glycolipids goes preferentially into cell attachment sites, and may have some functional role in regulating cell attachment of BHK cells; (ii) metabolic activity and turnover of GMs in cell attachment sites of confluent cells are higher than actively growing cells, yet those of transformed cells are much higher than any state of non-transformed cells. @I 1984 Academic Ress, Inc.

Cells are attached to their substrate through loci which may have specific chemical composition and organization in membranes. Detachment of cells from their tissue culture substrate with a Ca2+-specific chelating agent (EGTA) leaves cell substrate adhesion sites which have been called “substrate attachment material” (SAM) by Culp [2, 31. Treatment of cultured cells with aqueous buffer * Present address: Department of Medicine, Okayama University School of Medicine, Okayama, Japan. ** Present address: Institute de Patologia Generale, Firenze 50134, Italy. *** To whom offprint requests should be sent. Copyright 0 1984 by Academic Ress, Inc. All rights of reproduction in any form reserved 0014-4827/84$03.00

Glycolipids

in cell adhesion matrix

449

containing l-2 % non-ionic or zwitterionic detergent leaves numerous filamentous structures with convoluted nuclei which are considered to be pericellular adhesive structures and cytoskeletal meshwork [4-8] and are hereby called the “detergent-insoluble matrix” (DIM). Both DIM and SAM contain the major adhesive proteins such as fibronectin, 170K, and 140K glycoproteins, actin, and myosin [2, 7-91 as common components, although DIM contains, in addition, major nuclear components. Cell recognition involves cell attachment through a specific cellular locus called the adhesion matrix (lot. cit.) or adhesion plaque [lo], and a loss of cell adhesion is associated with oncogenic transformation, Therefore, much attention has been focused on molecular mechanisms associated with the cellular structures involved in cell adhesion and on the changes of the structures associated with oncogenic transformation. Accumulation of the src gene product (~60”“) at the cytoplasmic loci of focal adhesion plaques [ 111, and phosphorylation of the tyrosine group of vinculin at the adhesion site [12] have been observed, although no clear conclusion has been drawn. A set of membrane components, pericellular proteins (140K, 170K, 250K, and fibronectin), and cytoskeletal components (vinculin and actinin) may cooperate in a highly organized fashion [9, 131. Although glycolipids are not generally considered to be functional components of adhesion sites, a few data suggest that they may play an important role in cell adhesion, since polysialogangliosides can inhibit cell adhesion on fibronectincoated substrates [14] as well as on other substrates non-specifically [15]. Transformed mouse cells incubated with ganglioside gain tibronectin deposition at the cell surface [ 161. Liposomes containing sialosyllactoneotetraosylceramide (sialosylparagloboside), and the amphipathic membrane proteins associated with the ganglioside fraction both inhibit cell adhesion (Carter, W G, Rauvala, H & Hakomori, S, unpublished observation) [17]. However, the presence of glycolipids, particularly gangliosides, in the cell adhesion matrix has not been carefully studied, although GM, has been found in detergent-insoluble residue of mouse tibroblasts [18, 191 and a specific association of GM, and a-actinin in lymphocytes has been suggested in a recent study [20]. The present study is therefore focused on the glycolipid composition of the detergent-insoluble fraction prepared from substrate attachment material (designated as “detergent-insoluble substrate attachment matrix”, abbreviated DISAM) and its changes associated with cell confluency and transformation.

MATERIAL

AND METHODS

Baby hamster kidney (BHK C-21) cells were cultured on glass Petri dishes (diameter 15 cm) and metabolically labeled with [3H]ghrcosamine (3 yCi/ml) or [“‘Clgalactose (0.3 @i/ml) for 24 h. Substrate attachment material (SAM) was prepared by treating with 0.5 mM EGTA in PBS without Ca*+ and Mg’+ [l, 21, and was further treated with 2% Empigen BB in 25 mM NaCl and 1 mM PMSF in 50 mM borate buffer, pH 7.8, as previously described [9]. The insoluble residue left on the glass Petri dishes, called the detergent-insoluble substrate attachment matrix (DISAM), was extracted Exp Cell Res 155 (1984)

450 Okada et al. Cells cultured

on glass

Petri dish

t “SAM”

Detached cells

*Ligen Chloroformmethanol (2:l)

2% Empigen El8 in 25 mM N&I. 1 MM PMS l 50 mM borate

Acetylation procedure 1 Total glycolipid

1

BB in 25 mM

4-7 Insoluble “DIM”

I Total lipid

left on glass

Chloroformmethanol

N&l

t lnsoiuble “DISAM”

Soluble

t Soluble

Chloroformmethanol (2:l)

T&lipid in “DISAM”

1 DIM glycolipid

Acetylation procedure

DISAM

I glycolipid

Fig. 1. Procedure flow sheet for preparing various glycolipid fractions in cell attachment substrate. with chloroform-methanol (2: 1, v/v). The glycolipids were prepared by acetylation procedure according to Saito & Hakomori [21], and separated into neutral glycolipids and gangliosides [22]. The cells that were detached from the glass Petri dishes upon 0.5 mM EGTA treatment were also extracted with 2% Empigen BB under the same conditions as above and labeled as detergentinsoluble matrix (DIM). The detached cells were directly extracted with chloroform-methanol (2 : 1, v/v), and the glycolipid fraction was prepared in the same way. Cells were scraped with rubber and treated directly with 2 % Empigen BB. The glycolipid fraction extracted from the Empigen-insoluble material was prepared. The schemes of extractions are shown in fig. 1, and the procedure for glycolipid analysis is described in the caption to fig. 2. Confluent cell cultures contained 3.3~10’ cells/l5 cm dish and subconfluent cultures contained 1.6~ 10’ cells/l5 cm dish. The activity of glycolipids in the DISAM fraction of reattached cells was determined by the experimental protocol described in the footnote to table 3. Inhibition of BHK cell attachment by GM3 was determined using cells labeled with [3H]thymidine as described in the caption to fig. 3.

RESULTS Glycolipids of the detergent-insoluble substrate attachment matrix (DISAM) of BHK cells The DISAM fraction of BHK cells consistently contained a small but significant amount of labeled glycolipids (fig. 2). Although chemical quantitation of glycolipids in the DISAM fraction was tried, it was technically very difficult (see Discussion). The glycolipid fraction contained the radiolabeled components with the same TLC mobility as ceramide monohexoside (CMH), ceramide dihexoside Exp Cell Res I55 (1984)

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ABCDE 2. Fluorography of glycolipids isolated from BHK cells metabolically labeled with [?]galactose. The cells were grown in medium containing [‘4Cll;llactose (3 @i/ml) for 24 h, and cells were released from the glass Petri dishes with EGTA, leaving substrate attachment material (SAM) and the glycolipids extracted from the SAM or fractions as described in Materials and Methods. Glycolipids were separated on HPTLC in a developing solvent of chloroform-methanol-water (60 : 35 : 8), and fluorography was performed according to the method of Bonner & Stedman [28]. Lane A, glycolipids of whole BHK cells; B, neutral glycolipids of whole BHK cells; C, ganglioside fractions of whole BHK cells; D, neutral glycolipid fraction of DISAM fraction; E, ganglioside fraction of DISAM fraction. Fig.

(CDH), ceramide trihexoside (CTH), and GM3 (see fig. 2). Although the qualitative pattern of glycolipid composition in the DISAM fraction was similar to that of whole cells (see fig. 2), the ratio of radiolabeled ganglioside (GM,) to neutral glycolipids (ganglioside/neutral ratio) in the DISAM fraction was higher than that of whole cells as determined by both GlcNAc and Gal metabolic labeling (table 1). These results suggest that labeling of GMs is enriched in the DISAM fraction of BHK cells. Comparison

of DZSAM glycolipids

of BHK and BHKpy

cells

The radioactivity in the glycolipid fraction of DISAM of confluent BHK cells was three to four times higher than that of subconfluent BHK cells (table 2). The Table 1. Ratio of radiolabeled glycolipid

fraction

gangliosidelneutral glycolipids and in EGi%-released whole cellf

BHK cell fraction

cpm [3H]GlcNAc incorporated

cpm [‘4C]Gal incorporated

EGTA-released whole cells DISAM glycolipid fraction

0.7kO.2 1..5+0.2

0.14+0.05 0.39kO.08

in the DZSAM

a Cells were cultured in glass Petri dishes in Dulbecco’s modified Eagle medium supplemented with 10% FCS and metabolically labeled by [“Hlglucosamine (3 @i/ml) or [i4C]galactose (3 uCi/ml) for 24 h. The glycolipid fraction was prepared from EGTA-released whole cells and from the DISAM fraction, and was separated into gangliosides and neutral glycolipids by chromatography on DEAESephadex. Exp Cell Res 155 (1984)

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Table 2. Incorporation from detergent-insoluble ceW

of [3H]glucosamine into the glycolipid fraction prepared substrate attachment matrix (DISAM) and from whole

DISAM fraction prepared from lOa cells (cpm)

Whole cells from 10s cells (cpmx 1OV)

Chemical quantity of cellular GM3 (pi3 per 10 mg cellular protein)

l.OkO.3

35-40d

BHK cells

ContIuentb Subconfluent’ BHKpy

2 800+700 800+270

0.720.4

S&54’ W8’

cells

Confluent

8600&l 500

0.7kO.4

&Ild

18’

0 Cells with different population densities were grown in glass Petri dishes, labeled with [3H]glucosamine (3 pCi/ml) for 24 h, detached in the presence of 0.5 mM EGTA, followed by preparation of the DISAM fraction, and extraction and preparation of glycolipids. b 3.3~10’ cells/l5 cm glass dish; ’ 1.6~10’ cells/l5 cm glass dish. d Data from Hakomori & Murakami [22]; e Data from Hakomori [24].

radioactivity of DISAM glycolipids prepared from BHKpy cells, however, was three times higher than that of confluent BHK cells (table 2). In striking contrast, the chemical quantity of GM3 in whole BHKpy cells was much less than that in BHK cells (8-11 &IO mg protein in BHKpy cells as compared with 35-40 &IO mg protein in BHK cells) [23]. In a separate experiment, the GM3 content of BHK cells was higher in confluent cells than in sparsely growing cells, but BHKpy cells had much less GM3 [25]. While the chemical quantity of GM3 in BHKpy cells was less than that of BHK cells, the labeled activity of GM3 (determined by [3H]glucosamine incorporation) in the DISAM glycolipid fraction of BHKpy cells was several times higher than that in the DISAM fraction of BHK

Table 3. The activity

of [3H]glucosamine tion prepared from the DISAM fraction

Reattached cell type

cpm/lOs cells

BHK cells BHKpy cells

2 700+540 6900+1400

incorporated

of reattached

into the glycolipid cells”

frac-

0 BHK and BHKpy cells were cultured under normal conditions and metabolically labeled with [3H]glucosamine under the same conditions as described in the footnote of table 2. Cells were detached with 0.5 mM EDTA-PBS without Ca*+ and Mg*+ and seeded in glass Petri dishes at cell population density of 3~10~ cells/l5 cm diameter Petri dish in Dulbecco’s modified Eagle medium containing 10% FCS. Cells were maintained for 2 h to assure cell reattachment. Then cells were detached and the DISAM glycolipids were prepared. Exp Cell Res 15s (1984)

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Fig. 3. Inhibition of cell-to-substrate attachment and cell spreading of BHK cells by GM+ Cells were detached with 0.5 mM EGTA in phosphate-saline and dispersed at 0°C. Cells were washed and diluted in phosphate-saline containing 1 mM Ca2+ and 1 mM Mg’+ to a concentration of 1x lO?ml. 50 pl of the cell suspension (5x 104cells) was added to 50 ul of phosphate-saline plus 1 mM Ca’” and 1 mM Mg2+ containing 10 ug GMJwell in a 96-well plate. The cell suspensions were then incubated at 37°C for 60 min in 5% CO2 atmosphere. (A) No ganglioside added; (B) 10 &well GMs. Picture shows unwashed plate.

cells. These results indicate that the rate of [3H]GlcNAc incorporation into the DISAM of BHKpy cells is very different from that of BHK cells. Glycolipids

in the DZSAM fraction

of reattached

cells

In order to study possible differences in the labeling of the glycolipid of the established cell attachment site and that of the early attachment site of a highly labeled cell, cells were preliminarily labeled, released by EGTA treatment, washed, and reattached on a glass surface. The label of the DISAM glycolipid in the reattached matrix was compared with that in the established matrix. The same experiment was performed with both BHK and BHKpy cells. The amount of label in the DISAM glycolipid in the reattached matrix site was as high as that in the established confluent matrix, and the amount of label in the matrix of reattached BHKpy cells was only slightly lower than that in the established matrix of BHKpy cells (table 3). Chemical quantitation of glycolipids in the DISAM fraction was, however, technically very diffkult. Effect of GM3 on BHK cell attachment

The metabolic turnover of glycolipids, particularly GM3, in the DISAM fraction could be different from that in whole cells, suggesting a specific role of GM3 in cell attachment. Therefore, the effect of GM3 on cell attachment and spreading was studied. Cell attachment, followed by spreading, was significantly inhibited in the presence of exogenously added GM3, as shown in figs 3 and 4. BHK cell attachment was inhibited 25 % by 2 ug/well GM3 and maximum inhibition of 50 % could be obtained with 10 ug/well of GM3. Exp Cell Res 155 (1984)

454

Okada

et al.

4. Inhibition kinetics of cell-to-substrate attachment of BHK cells with various concentrations of GMs. BHK cells were labeled with 2 @i/ml [3H]thymidine for 2 h prior to detachment with EGTA. Specific activity of radiolabeled cells was about lo5 cpm/5x lo4 cells. Substrate attachment assay was performed similar to that described in the caption to fig. 3. After incubation at 37°C for 1 h, the cells were washed three times with phosphate-saline (I mM Ca’+ and 1 mM Mg2+) to remove nonattached cells. Attached cells were solubilized with 0.1% SDS in 0.1 N NaOH. After neutralization, the radioactivity was counted in a liquid scintillation counter. Data is expressed as the percent of cell attachment (radioactivity remaining on plate/total radioactivity added to each well) vs the concentration of GM3 added per well. Each data point is the mean of the three determinations k SD.

Fig.

DISCUSSION Cell attachment is mediated through specific loci which can be separated from, and left on, the substrate surface when adherent cells are treated with a Ca*+chelating reagent (EGTA) and are called “substrate attachment material” (SAM) [2, 31. SAM consists of specific components of the plasma membrane, pericellular matrix, and some cytoskeletal proteins. When cells are treated with physiological buffer solution containing l-2 % neutral or zwitterionic detergents, essentially the same components of the pericellular matrix and cytoskeletal proteins remain insoluble, together with nuclear components, while essentially all membrane and cytoplasmic components are solubilized [7-91. GM1 has been described as a detergent-insoluble component when cells are treated with non-ionic detergent, and this finding indicates a possible association between a cytoskeletal component and GM, [I& 191. No study has been focused on a possible presence of glycolipids at the cell-adhesion site. In this study, detergent solubilization was applied to SAM, and the detergent-insoluble matrix was separated from SAM. Thus, the “detergent-insoluble substrate attachment matrix” (DISAM), which is believed to be functionally essential in cell adhesion, has been isolated and its glycolipid composition has been studied. The results clearly indicate that glycolipids are present in the DISAM fraction, although the specific role of these glycolipids remains to be studied. The differences in radioactivity of the DISAM glycolipids associated with cell confluency and transformation strongly indicate that metabolic activity of DISAM glycolipids is different from glycolipids present in other parts of cells. In all these studies, however, only the metabolically labeled activity of the DISAM glycoliExp Ceil Res 155 (1984)

Glycolipids

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pids was compared in confluent and sparsely growing BHK cells and transformed BHKpy cells. It is highly desirable to determine the chemical quantity of glycolipids in the DISAM fraction, although reliable values are difficult to obtain by the existing technique, unless the number of plates used for analysis were increased 10000 times. If comparisons of two or three conditions were analyzed, the number of plates would increase still further, and would be difficult to perform. The 3H-labeling technique at the sphingosine double bond [26] of SAM glycolipids is available. However, the method labels unknown components, and a significant quantity of glycolipid can be adsorbed on platinum oxide catalyst. The method, therefore, lacks quantitative evaluation. Although the chemical quantity of GM3 in whole BHKpy cells was significantly less than that of GM3 in whole BHK cells [15], the radioactivity incorporated into the DISAM glycolipids was much greater in BHKpy cells. It is assumed that metabolic turnover of a small population of glycolipids organized in the DISAM structure must be greatly enhanced, although the chemical quantity of GM3 in the DISAM fraction may not be greatly altered. Similarly, enhanced activity in the DISAM fraction of confluent BHK cells may reflect enhanced turnover of glycolipids at confluency. It has previously been reported that the chemical quantity and metabolic turnover of GM3 in whole BHK cells were enhanced when cells were at confluency [24]. The role of enhanced metabolic turnover in non-growing cells as compared with growing cells has also been well documented [25]. Compartmentalization of the same molecular species in structurally and metabolically distinct moieties of cellular fractions is not unique to glycolipids in the cell attachment site. Most of the fibronectin in SAM is contained within a highly resistant adhesive structure rich in heparan sulfate; however, newly synthesized fibronectin is present in a much more labile structure, along with hyaluronate and chondroitin [27]. Thus, a metabolically distinct moeity of fibronectin in SAM reflects such an organizational difference. A similar situation is assumed for GM3 in the DISAM fraction; it has a distinctively different metabolic activity than that in whole cells and it is different in the DISAM of confluent vs sparsely growing cells as well as in transformed vs non-transformed cells. A higher amount of label in DISAM glycolipids of confluent cells than in those of sparsely growing cells may indicate that confluent cells have more adhesive sites. In contrast, the only explanation for a much higher amount of label in the DISAM glycolipids of transformed BHKpy cells is that the metabolic turnover of DISAM glycolipids of BHKpy cells is much higher than that of non-transformed cells, since total chemical quantity of GM3 in BHKpy cells was much less than that of BHK cells. It is possible that a labeled glycolipid, particularly GM3, represents a newly synthesized GM3 which would preferentially be incorporated into DISAM. All these data indicate that glycolipids, particularly gangliosides, may regulate the function of cell adhesion, probably through regulating the adhesive protein. A possible role for GM3 in regulating cell adhesion is further suggested by the clear inhibition of cell attachment and cell spreading of BHK cells. Glycolipids in the Exp Cell Res 155 (1984)

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DISAM fraction may be important in regulation of the function and organization of adhesive proteins and their association with cytoskeletal components in the adhesion loci. This investigation has been supported by research grants from NCI, NlH, CA20026 and CA23907. A part of this paper was presented at the Annual Meeting of the American Society of Biological Chemists 1982 [l]. G. M. was supported by a fellowship from Associazione Italiana per la Ricerca sul Cancro and a Fulbright travel grant and E. G. B. has been supported by an Otsuka Research Foundation Fellowship.

Note added in proof Recently, a study with two monoclonal antibodies directed to GD2 and GDJ gangliosides detected these gangliosides at the microprocesses as well as in adhesion plaques by immunofluorescence (Cheresh, D A, Harper, J R, Schultz, G & Reisfeld, R A, Proc natl acad sci US (1984). In press). This finding supports the results as described in this paper.

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Exp Cell Res 155 (1984)

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