Stage-specific antigens reacting with monoclonal antibodies against contact site A, a cell-surface glycoprotein of Dictyostelium discoideum

Cell Differentiation, 11 (1982) 1--13 1 Elsevier/North-Holland Scientific Publishers, Ltd,

STAGE-SPECIFIC ANTIGENS REACTING WITH MONOCLONAL ANTIBODIES AGAINST CONTACT SITE A, A CELL-SURFACE GLYCOPROTEIN OF DICTYOSTELIUM DISCOIDEUM

H. OCHIAI 1, H. SCHWARZ 2, R. MERKL 1, G. WAGLE 1 and G. GERISCH 1 *

I Max-Planck-Institut fiir Biochemie, 8033 Martinsried bei Miinchen; and 2 Max-Planck-lnstitut fiir Biologie, 74 Tiibingen, F.R.G.

(Accepted 1 September 1981)

Monoclonal antibodies against a glycoprotein presumably involved in adhesion of aggregating Dictyostelium discoideum cells have been used for labeling of the antigen at the cell surface. The antigen is distr ibuted over the whole surface of the cells, appa ren t ly in form of small clusters. The antigen appears concomitant ly with the acquisition of EDTA-stable adhesiveness typical of aggregation competent celts. In contrast , discoidin I, a lectin whose accumulation during development parallels EDTA~table adhesiveness in another strain (NC-4), is present in nearly the same amounts in growth phase and aggregating cells of AX2-214, the strain used by us. Thus, no correlation exists in this strain between the expremion of discoidin I and the development of cell adhesiveness.

The 80 kflodalton glycoprotein typical of aggregation competent cells has been purified by affinity chromatography on a monoclonal ant ibody column. The purified antigen absorbs adhesion-blocking Fab from rabbits. Another antigen strongly reacting with the same monoclonal antibodies has an apparent molecular weight of 106 000 and is not detectable before slugs are formed.

cell adhesion glycoproteins monoclonal antibodies lec~ins Dictyostelium

1. Introduction

Aggregating cells of Dictyostelium discoi. deum move towards aggregation centers in the form of streams in which they adhere to each other by end-to-end and side-by,side adhesion. The end-toend contact is specific for aggregation-competent cells and is characterized by its EDTA stability. Univalent antibody fragments block cell adhesion. The target sites of Fab that block the EDTA-stable, developmentally regulated type of contact have been called contact sites A (Beug et al., 1973a). Fat) that blocks this contact is neu- tralized by a purified antigen preparation containing one major glycoprotein with a molecular weight of about 80000 (MiUler

* To whom all correspondence should be addressed.

and Gerisch, 1978; Miiller et al., 1979). D. discoideum produces two major lectins

called discoidin I and II (Frazier et al., 1975). These are soluble, cytoplasmic proteins, but a small portion of the total discoidin content of a cell appears to be present on its surface (Chang et al., 1975). Discoidin I has been suggested to function in cell adhesion by interaction with carbohydrate residues on adjacent cells (Bartles and Frazier, 1980; Ray et al., 1979; Breuer and Siu, 1981).

To monitor the developmental regulation of cell surface components probably involved in cell adhesion, monoclonal antibodies against contact sites A and discoidin have been prepared. Here we focus on monoclonal antibodies 12-120-94 and 12-5-58, both directed against contact sites A. These antibodies bind not only to the 80 kilodalton glycoprotein present in aggregation compe-

0045-6039/82/0000--0000/$02.75 © 1982 Elsevier/North-Holland Scientific Publishers, Ltd.

tent cells, but also to an antigen of about 106 kilodalton which is specific for the slug stage. A monoclonal antibody, 7-78-3, against discoidin II was used as a control in cell surface- labeling experiments; it did not detectably bind to living cells.

2. Material and methods

2.1. Cell cultures

Cells of D. discoideum were grown axenically as described by Malchow et al. (1972), harvested during exponential growth, washed free of nutrient medium and adjusted to 107 cells/ml in 17 mM SCrensen phosphate buffer, pH 6.0. The times of development are indicated in hours (tn) after harvest from nutrient medium (to). For slug formation, cells were spread at to on non-nutrient agar containing 17 mM SCrensen phosphate buffer, pH 6.0. In shaken suspensions, cells became aggregation competent between t6 and ts, but did not develop into slugs.

2.2. Antigen preparations

Crude membrane preparations were obtained by freezing and thawing of cells and centrifugation at 27 000 X g for 30 rain. Puri- fied plasma membranes were prepared according to Brunette and Till (1971) as described by Mfiller et al. (1979). Butanol extraction of either crude or purified membranes was performed according to Mfiller et al. (1979). Dis- coidins I and II were isolated from the soluble fraction of aggregation~ompetent cells on acid-treated Sepharose 6B and eluted with 0.3 M galactose (Bartles et al., 1979). Discoidin I was purified from the mixture by DEAE-cel- lulose chromatography, discoidin II by HPLC on Spheron-300 (Koch and Light, Coinbrook, U.K.), derivatized with DEAE groups. Start- ing buffer was 20 mM Tris, pH 8.5. The discoidins were separated with a gradient of Tris plus 0.5 M NaC1, pH 6.0. Discoidin II eluted behind discoidin I.

2.3. Monoclonal antibodies

A crude preparation of contact sites A was repeatedly injected into BALB/c mice together with Al-hydroxide as adjuvant. C57B1/6J mice were immunized with a mixture of discoidins I and II similar to a scheme described previously (Bozarro et al., 1981). Spleen cells were fused with Sp2-01 myeloma cells.

Hybridoma cells were cloned under micros- copic observation by deposition of small

droplets into microtiter plates. To droplets containing only one cell, medium with macro- phages was added. IgG was purified after growth of cloned hybridomas in either RPMI 1640 medium with fetal calf serum or in BALB/c × C57B1/6 (F1) mice. IgG was precipitated with 55% saturated ammonium sulfate, dialysed against 0.14 M phosphate buffer, pH 8.0, and isolated on a protein A-Sepharose column (Pharmacia, Uppsala) with a gradient of 0.1 M Na~itrate, pH 3.0. All IgG species used eluted with a peak at pH 6.0, indicating that they belong to the IgG1 class (Ey et al., 1978). Accordingly, all antibodies were precipitated with anti-mouse IgG1 serum (Meloy, Springfield, VA).

Hybridoma antibodies 12-120-94 and 12-5-58 were incubated in 10% mercaptoetha- nol and 8 M urea at pH 8.0 and 37°C for 30 min, and isoelectrofocused in 5% polyacrylamide gel containing 6 M urea (KShler et al., 1976). Only single heavy chain bands were found after staining with Coomassie blue, indicating that only one IgG species was synthesized by the two hybridomas. Two or three light chain bands were located close to each other. The two clones are the products of independent fusion events. The positions of heavy and light chain bands were, however, the same. Thus the spleen cells fused were probably of the same clonal origin.

2.4. Aff ini ty chromatography o f contact sites A

Hybridoma 12-120-94 IgG from mouse ascites was coupled to CNBr-activated Sepha-

rose 4B (Pharmacia, Uppsala). The reaction was stopped with glycine. Butanol/water extract of a crude membrane preparation from aggregation~ompetent cells was concentrated to about 1 mg protein/ml on Amicon XM 50 filters, dialysed against 50 mM Tris-HC1 buffer, pH 7.5, containing 0.2% sodium cholate, and loaded to the column under recycling. The column was washed with the buffer plus cholate, eluted with buffer containing 0.5 M NaC1 plus 0.2% cholate, and finally with buffer plus 4 MgC12 and 0.04% cholate. The MgC12 elution yielded highly purified 80 kflodalton glycoprotein.

2. 5. Analytical procedures

SDS-polyacrylamide gel electrophoresis was performed according to Laemmli (1970) in 10% gels. The proteins were transferred to HA millipore filters, pore size 0.45 /am, as described by Towbin et al. (1979) and modified by Vaessen et al. (1981). Filters were incubated in 100--200/ag monoclonal IgG per ml and subsequently in ~2SI-labeled anti- mouse IgG IgG from rabbit (106 c.p.m./ml). Autoradiograms were obtained on Kodak KRXR-5 film. Marker proteins were labeled with [14C]formaldehyde according to Dot- tavio-Martin and Ravel (1978).

Concanavalin A-binding glycoproteins were labeled on the polyacrylamide gel by concanavalin A and horseradish peroxidase (Parish et al., 1977}. Molecular weights were indicated according to the high molecular weight stan- dards of Bio-Rad (bovine serum albumin had changed its apparent molecular weight from 66 000 to 64 000 after labeling).

Two<limensional electrophoresis was performed according to O'Farrell (1976) as modified by Ames and Nikaido (1976). The ampholine mixture consisted of 0.56 ml, pH 3.5--10; 0.19 ml, pH 4--6; 0.19 ml, pH 5--7; 0.31 ml, pH 2.5--4, in a total volume of 25 ml. The pH gradient was measured as described by Ono et al. {1979).

2.6. Fluorescence microscopy and analytical cell sorting

Freshly washed cells were resuspended in barbital buffer, pH 7.3 (Beug et al., 1973a), at a density of 10V/ml. Ceils were labeled on ice for 15 min with monoclonal IgG (50/ag/ ml for fluorescence microscopy, 100 /ag/ml for cell sorting) and FITC-anti-mouse IgG from sheep (Institute Pasteur Production). For cell sorting 5 × 106 ceUs/ml were resuspended in PBS to suppress spontaneous agglutination, and sorted at 4°C with a Becton-- Dickinson FACS IV cell sorter.

2. 7. Immunoelectron microscopy

Amoebae were prefixed with 0.25% glutaraldehyde in 17 mM SCrensen phosphate buffer, pH 6.0, for 20 min in the cold, incubated with 2% lysine, washed and labeled at room temperature with 100 /ag/ml of hybridoma 12-120-94 IgG. After washing ferritin conjugated anti-mouse IgG goat IgG (Otto et al., 1973) was applied. Labeled cells were washed and postfixed with 2.5% glutaraldehyde, followed by 1% osmium tetroxide, stained with 2% uranyl acetate and embedded in Epon.

2.8. Measurement o f cell adhesion and erythrocyte agglutination

EDTA-stable cell adhesion and absorption of adhesion blocking rabbit Fab were assayed according to Beug et al. (1973a) and Miiller and Gerisch (1978).

Discoidin I was determined after freezing and thawing of cells and centrifugation of the homogenate at 12 000 X g for 10 rain. Agglu- tination of formalinized sheep erythrocytes was determined by serial two-fold dilutions of the supernatant according to Barondes et al. (1978).

3. Results

3.1. Developmental regulation of cell surface antigens

Cells of strain AX2-214 were harvested from nutr ient medium and, after washing, shaken in phosphate buffer. At various times cells were labeled with monoclonal IgG and F1TC-conjugated anti-mouse IgG antibody. Since monoclonal antibodies 12-5-58 and 12-120-94 gave similar results, only those obtained with the latter are shown. At to no label was seen in the fluorescence microscope, or only very few, small fluorescent patches (Fig. la , b). The surfaces of to cells Were brilliantly labeled (Fig. lc) . When the cells were kept on ice, numerous fluorescent patches were distributed over their surfaces. At room temperature the label accumulated into caps.

Neither in the earlier nor the later stage of development was significant cell surface labeling observed when 12-120-94 antibody was replaced by another monoclonal IgG1, 7-78.3. This ant ibody reacted preferentially with discoidin II (Table I). IgG 7-78-3 was chosen as a control because, in contrast to other monoclonal anti<iiscoidin IgGs, it did not detectably cross-react with an antigen preparation containing contact sites A.

TABLE I

Specificity of monoclonal ant ibody 7-78-3

Antigen With first ant ibody (c.p_,n.)

Without first ant ibody (c.p.m.)

Unseparated discoidins I and II 1 302 301

Discoidin I 432 213 Discoidin H 1 642 195 80 000 mol.wt.

glycoprotein 266 262 None 208 222

Protein concentration for coating of PVC micro- ti ter plates with antigen was 50/~g/rnl for discoidins and 40/~g/ml for the glycoprotein. As first ant ibody solution, 100 p.I supernatant of a hybr idoma culture was used. The second ant ibody was iodinated anti- mouse IgG.

Bacteria~rown cells of D. discoideum strain V12/M2 reacted with IgG 12-120-94 similar to AX2-214. Cells harvested during exponential growth showed little, if any, fluorescence, aggregation-competent cells were strongly labeled.

The change of cell surface labeling during the development of AX2-214 cells was more quantitatively examined by monitoring fluorescence in a cell sorter (Fig. 2). The fluorescence signal increased from non<ietectabil-

Fig. 1. Stage-specific cell surface labeling by antibodies, a) to cells, bright field, b) The same cells labeled with IgG 12-120-94 and FITC-anti-mouse IgG ant ibody, c) t6 cells labeled in the same way.

t o t 2 t 4 t 6 "t 8

Fig. 2. Analysis of cell populat ions at various developmental stages with a FACS IV cell sorter. Abscissas: relative light scattering (a) or fluorescence intensity (b--d). Ordinates: relative cell numbers, a, b) Cells labeled by anti- contact site A IgG 12-120-94. c) Control with anti-discoidin II IgG 7-78-3. d) Control wi thout monoclonal IgG. All samples were labeled with FITC-anti-mouse IgG ant ibody. In each case 10 s cells were analysed. Light scattering indicates changes in size distr ibution of the cells during development. These changes are small compared to the drastic increase in fluorescence intensity observed during development in b.

ity to a significant height within 6--8 h of harvest (Fig. 2b). As in the fluorescence microscope, no significant cell surface labeling was detected in the cell sorter when the anti- discoidin II antibody 7-78-3 was used instead of anti-contact site A antibody (Fig. 2c).

3.2. Cell surface labeling coincides with EDTA-stable cell adhesiveness and with the appearance o f an 80 000 mol.wt, antigen

Acquisition of aggregation competence can be monitored by measurement of EDTA- stable cell adhesion (Beug et al., 1973a). Growth phase and t2 cells were almost completely dissociated by 10 mM EDTA (Fig. 3). Weak EDTA stability of cell adhesion was observed at the t4 stage, and a maximum was reached between t~ and ts.

Despite the stringent developmental control of EDTA-stable cell adhesiveness, the discoidin I activity did not detectably increase within the limits of the two-fold dilution assay (Fig. 3, bottom). These results show that it is no t the absence or presence of discoidin I which is responsible for the developmental control of EDTA.stable contact formation. Another factor must be involved.

In contrast to discoidin I, the 80 kilodalton glycoprotein to which ant i~ontact site A antibody binds was stringently regulated in the strain used by us, and the appearance of this antigen closely paralleled the acquisition of EDTA~table cell adhesiveness (Figs. 4a, 6a, c).

3.3. A slug-specific antigen

The two monoclonal ant i~ontact site A antibodies tested showed similar specificities. In suspension cultures the major membrane antigen labeled was the 80 kilodalton glycoprotein. In addition, a weak band was seen in the 140 kilodalton region (Figs. 4a, 6a, c). The 140 kilodalton antigen was regulated during development similar to the 80 kilodalton glycoprotein.

Whereas in suspension cultures the pattern

E/E o 1.0

0.~ ( .

0.8

07

0.6

~ 0.5

~ O.4

0.3

0.2

0.1 0 I j

0 1 2 3 4 5 6 7 8 hours of starvation

T t 512 discoidin I activity 512 [titer of sheep erythrocyte agglutination]

Fig. 8. Development of EDTA-etab]e ceLl adhesiveness and discoidin I content . Top) Agglutination of AX2- 214 cells in the presence of 10 mM EDTA was quanti- ta ted by measuring light scattering in an agglutino- meter according to Beug and Gerisch (1972). E was the optical density in the sample, Eo was that of a control with completely d iuoc ia ted cells. Thus E/E o values close to 1 indicate that ceLl adhesion was EDTA-asmdtive. Bot tom) ~ i d i n I content of to and ts cells was determined in the same culture as EDTA-stable ceLl adhesion. The soluble fraction from 2 × 10 s eelle/ml was serially two-fold diluted for erythrocyte agglutination.

of labeled antigens did not change between ts and t~, new cross-reacting antigens appeared when the cells were allowed to develop on agar. Only under these conditions slugs and finally fruiting bodies were formed. Slug formation was associated with the appearance of a strongly labeled antigen of 106 kilodalton. Simultaneously, the degree of labeling of the 80 kilodalton glycoprotein declined, and a diffuse zone of labeled material appeared in the lower molecular weight region (Figs. 4b, 6b, d).

The 80 kilodalton giycoprotein and the

kd

- 1 1 6 - 9 3

- 6 4

- - 4 5

- 31

kd

- 1 1 6 - 9 3

- 6 4

- 4 5

- - 31

0 4 8 12 16 0 4 8 12 16 2.0

0 4 8 1 2 1 6 2 0

Fig. 4. Plasma membrane proteins labeled with monoclonal an t ibody 12-120-94 (a, b) or stained with Coomassie blue (c, d). a, c) Cells starved in a shaken suspension where development ended with acquisit ion of aggregation competence, b, d) Cells starved on agar where development proceeded to the slug stage and fruiting body formation. Total membrane protein applied per slot was 50 ~tg in a--c, and 40 ~tg in d.

106 kilodalton antigen are distinguished by their isoelectric points (Fig. 5). The antibody- labeled lower molecular weight material formed in slugs was detected at the same pH as the 80 kilodalton antigen. Probably the glycoprotein is degraded during slug development, giving rise to heterogeneous fragments.

Later in development, during culmination, an antigen with a molecular weight of about 90000 became detectable. This antigen is probably identical with a protein that cross- reacts with rabbit antibodies against the 80 kilodalton glycoprotein, and has been detected at very late stages of development and in spores (Murray, Yee and Loomis, personal communication). Additional cross-reacting antigens giving rise to faint labeled bands are seen in Fig. 4. If one compares, however, the number of membrane proteins distin- guishable in one<limensional gels (Fig. 4c, d) with the number and relative intensity of labeled bands (Fig. 4a, b), one gets an impres-

sion of the selectivity of the monoclonal antibodies used. None of the prominent bands seen in Coomassie blue,stained gels corre- sponds to one of the heavily labeled antigens.

The major antigens reacting with monoclonal antibodies 12-120-94 and 12-5-58 were recovered in the water phase after butanol extraction. Very clear labeling patterns have been obtained with butanol/water extracts of plasma membranes from various stages of development (Fig. 6). In such extracts the developmentally regulated 80 kilodalton glycoprotein is enriched, so that it can be detected even by Coomassie blue staining (Fig. 7a). Since plasma membranes of D. discoideum contain many concanavalin A-binding glycoproteins, and the 80 kilodalton glycoprotein is one of them, we have compared the pattern of butanol/water extracted concanavalin A-reactive glycoproteins (Fig. 7c, d) with labeling by monoclonal antibodies. The result is that labeling by the anti-

i ¸ i ~ ~ / i ! ~i i i~ii ~ ~ i i ~ ~

Fig. 5. T w o - d i m e n s i o n a l e l ec t rophores i s o f m e m b r a n e antigens reacting with IgG 12-5-58. a) A f t e r development for 8 h o n agar. b ) A f t e r 20 h o n agar.

0 4 81- 12 16 0 4 8 12 16 20

kd

_ 1 ~ - 9 3

- 6 4

- 4 5

- 3 1

1 6 0 4 ~ 8 1 2 1 6

star: on Fig. 6. Monoclonal antibody labeling of membrane antigens extracted with butanol/water. Starved cells were shaken in suspension (a, c) or allowed to develop on agar (b, d). a, b) Labeled with IgG 12-5-58. c, d) Labeled with IgG 12-120-94. To each slot 13/~g of butanol/water extracted proteins were applied.

10

a b c d

6 6 - -

4 5 - -

1 .--

kd 0 4 ~8 12 16 0 4

hours o f s t a r v a t i o n

Fig. 7. Plasma membrane proteins (a), concanavalin A-binding glycoproteins (c, d) extracted with butanol/water, and contact sites A purified by affinity chromatography (b). Cells were starved and cultivated in suspension (a, c) or on agar (d). Cultures were the same as in Fig. 6. SDS-polyacrylamide g e l were stained with Coomassie blue (a) or with silver according to Oakley et al. (1980), or labeled with concanavalin A and peroxidue (c, d).

bodies is h ighly specific. This means that binding of the monoclonal antibodies used is not due to a carbohydrate moiety common to most of these glycoproteins.

3.4. Affinity chromatography of contact sites A

The particle fraction of aggregation~ompetent cells was extracted with butanol--water and the water phase fractionated by affinity chromatography o n IgG 12-120-94. The column was eluted with 0.5 M NaCI and subsequently with 4 M MgC12. The second elution step yielded highly purified glycoprotein (Fig. 7b). In addition to the 80 kilodalton band a faint band in the 140 kilodalton region was seen in silver-stained SDS-polyacrylamide gels of some of our preparations. This 140 kilodalton antigen seems to be the same as in autoradiograms of antibody-labeled mere-

brahe antigens (Figs. 4a and 6a). The relation- ship of this material to the 80 kilodalton glycoprotein remains to be clarified.

As shown in previous studies, rabbit Fab against membrane antigens of aggregation competent cells blocks EDTA-stable cell adhesion (Beug et al., 1973a). A fraction containing the 80 kilodalton glycoprotein as the only major protein completely eliminated the blockage of EDTA-stable contacts by Fab (Miiller and Gerisch, 1978). To confirm that the glycoprotein is a target antigen of adhesion blocking Fab, this experiment has been repeated with the affinity purified material. By 5/~g protein the adhesioh blocking activity of 0.3 mg Fab was reduced by 50%, which is slightly less than previously found for the conventionatly purified antigen (Miiller and Gerisch, 1978).

During the NaC1 step preceding dissocia- tion of the antibody--antigen complex by

11

MgCI2, only little of the 80 kilodalton glycoprotein was eluted. Nevertheless, absorption of adhesion blocking Fab was observed in the NaCl~luted fractions. The nature of the Fab- absorbing compound in these fractions remains to be clarified.

3.5. Distribution of antigens on the cell surface

Aggregating cells are characterized by their end-to-end adhesion. The possibility that the 80 kilodalton glycoprotein plays a role in

cohesiveness of aggregating cells raises the question whether this antigen is specifically located at the ends of elongated cells. By labeling with rabbit antibodies specific for aggregation-competent cells, no accumulation of antigens at the ends of the cells was dis- covered (Gerisch et al., 1974). Also labeling of glutaraldehyde-fixed aggregation~ompetent cells with monoclonal 12-120-94 IgG and ferritin~onjugated anti-mouse IgG did not reveal heterogeneous distribution of antigens.

Although the ferritin-labeled antigens were distributed over the whole surface of aggrega-

i }iil ii ~/ • a

Fig. 8. Labeling of cell surfaces with hybridoma antibody 12-120-94 and ferrltin~onjugated anti-mouse IgG antibody. a) to cells, b, c) t6 cells after development in suspension. Bars represent 0.5/Jm.

12

t ion-competent cells, they did not form a homogeneous layer. The ferritin molecules were often grouped together, with unlabeled surface areas between the ferritin clusters (Fig. 8b, c). This pattern suggests that the antigen molecules exist in the membrane as oligomers.

As expected from fluorescent antibody- labeling of living cells, there was almost no label detected on growth phase cells, confirm- ing that the antigens were stringently regulated during development (Fig. 8a).

4. Discussion

Monoclonal antibodies have been used in this study for labeling the surfaces of aggregation-competent cells. The major membrane antigen to which the antibodies bind in these cells is a developmentally regulated glycoprotein of 80 kilodaiton which has been im- plicated in cell-to-cell adhesion (Miiller and Gerisch, 1978; Miiller et ah, 1979). As it is often observed with monoclonal antibodies, the hybridoma antibodies used by us proved to be not absolutely specific for the glycoprotein. Nevertheless, they showed a high degree of selectivity. Although the 80 kilodalton antigen is not one of the major glycoproteins of the plasma membrane, its particu- lar band is strongly and preferentially labeled in SDS-polyacryiamide gels of t o ~ membrane proteins.

The finding that the 80 kilodaiton antigen is not accumulated a t the ends of the cells shows that the sites of stable intercellular adhesion are not determined by the location of the glycoprotein. Probably the contact sites are specifically activated at the ends of a cell, e.g. by interaction with the cytoskeleton at the inner side of the membrane.

The strong developmental regulation of the 80 kilodalton antigen in strains AX2 and V12/M2 confirms previous findings in which adhesion-blocking Fab was u~mcl for the assay of the antigen (Beug et al., 1973a, b), or in which the glycoprotein was labeled with [14C]acetate (Parish and Schmidlin, 1979).

In contrast to the 80 kilodalton glycoprotein, discoidin I did not show significant developmental control in axenically grown AX2-214 cells. The presence of discoidin I in growth phase ceils has been previously observed in another axenically grown strain, A3 (Rosen et ai., 1973). However, this strain showed also early formation of EDTA-stable contacts.

From antibody-labeling experiments, Parish et ai. (1978) concluded that the 80 kilodaiton glycoprotein disappears in the slugs, whereas Murray, Yee and Loomis (personal communication) found that the glycoprotein persisted during the later stages of development and was detectable even in spores. Also in our preparations, the 80 kilodalton glycoprotein was observed by antibody labeling in the slug stage, although the amount was substantially lower than during aggregation. The appearance of a diffuse zone of labeled material of lower molecular weight in SDS-polyacrylamide gels suggests partial degradation of the glycoprotein in the slug.

A strongly labeled band of a specific antigen of 106 kilodaiton indicates the post- aggregative formation of a protein that cross- reacts with the 80 kilodaiton glycoprotein synthesized prior to aggregation. In starved cells shaken in suspension, the 106 kilodaiton antigen did not appear, indicating that this membrane component is expressed in close association with the developmental processes involved in slug formation rather than at a certain time after starving the cells.

There is no evidence that the 106 kilodalton antigen takes over the function of the 80 kilodaiton glycoprotein in the slug. But Fab from antisera against aggregation-competent cells has been found to dissociate also slug cells (Beug et al., 1971). This finding suggests that a molecule which cross-reacts with antibodies against contact sites X acts as an adhesion site in the slugs.

Acknowledgements

Our work was supported by the Deutsche Forschungsgemeinschaft. The Sp2-01 myelo-

m a line was k ind ly suppl ied to Dr . G. K~hler , Basel, w h o also he lped us to es tabl ish m o n o - c lonal a n t i b o d y t echn iques . We have to t h a n k m a n y col leagues fo r c o o p e r a t i o n : E. Ei t le fo r p rov id ing ant i<l iscoidin h y b r i d o m a s , Dr . M. J a n k u and G. M o n o k for pu r i f i ca t ion of discoidins , K. O p a t z and M. Westpha l fo r se t t ing u p a f f in i ty c h r o m a t o g r a p h y and b lo t t i ng tech- n iques , Dr . P. P r e h m for the c o n t a c t site A p r e p a r a t i o n used for i m m u n i z a t i o n , C. Sund fo r i m m u n i z i n g mice , K. Gri in and B. Schon- b a u e r fo r cul t ivat ing h y b r i d o m a s .

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Stage-specific antigens reacting with monoclonal antibodies against contact site A, a cell-surface glycoprotein of Dictyostelium discoideum