Allelic antigen and membrane-anchor epitopes of Paramecium primaurelia surface antigens

YVONNE CAPDEVILLE*, FRANCOIS CARON, CLAUDE ANTONY, CIIRISTIANE DEREGNAUCOURT and ANNE-MARIE KELLER Deportement I (In Centre de Genetiqiie Molecnlaire, Centre National tie la Recherche Scientifiqne, 91190 Gif-snr-Yvette, Fiance * Author for correspondence

Summary

Paramecium aurelia can express a repertoire of surface antigens (SAgs) according to culture conditions. These high MT proteins are anchored in the plasma membrane by a glycolipid, and they can be isolated in two different forms, an amphiphilic membrane-bound form (mSAg) and a hydrophilic soluble form (sSAg). Endogenous or exogenous phospholipase C can convert mSAg to sSAg with unmasking of a carbohydrate antigenic determinant similar to that found in the soluble form of Trypanosoma variant surface glycoproteins and called the cross-reacting determinant. By immunizing mice with cilia from strain 156 of P. primaurelia expressing the G SAg, we obtained six monoclonal antibodies against the 156G SAg, which could be classified into two groups. Y4 and Y8, representative of each group, have been characterized by checking their reactivity in situ and in vivo towards a series of allelic G and D SAgs in P. primaurelia and the 5IB SAg in P. tetraurelia. The monoclonal Y4 recognizes a conformational determinant, accessible in vivo and common to all the G SAgs. Thus, Y4 defines a G

locus-specific epitope that corresponds to a conserved region inside a polymorphic domain. The monoclonal Y8 recognizes two homologous determinants whose detection depends on the presence or absence of the SAg membrane-anchor, and which are mutually exclusive: one is found in the reduced soluble form of all the SAgs and other surface proteins, the cross-reacting glycoproteins (CRGs); the other occurs in the unreduced membrane-bound form of the G SAgs. Thus, Y8 enables us to demonstrate that the membrane-anchor of Paramecium SAgs contains an additional hidden determinant close to the crossreacting determinant and to discriminate between the membrane-bound and soluble form of SAgs. The in situ organization of the 156G SAg molecules is also discussed on the basis of immunogold labelling obtained using Y4 and a polyclonal antiserum.

Introduction

generally expressed (for reviews, see Beale, 1954; Sonneborn, 1974). Immunological and biochemical studies of Paramecium SAgs have revealed that these surface proteins are highly polymorphic, but the polymorphism appears to be restricted to the external part of the molecule. In /-". primaurelia two unlinked loci, G and D, express different allelic forms of SAgs in strains of distinct geographical origins (Beale, 1954). In comparing these

The surface of Paramecium aurelia, a ciliated protozoon, is coated mainly by a single set of surface antigens, SAgs (also called immobilization antigens). These molecules belong to a multigene family whose expression displays both mutual intergenic (Beale, 1952) and interallelic (Capdeville, 1971) exclusion: under given conditions of culture, a single SAg is Journal of Cell Science 88, 553-562 (1987) Printed in Great Britain © The Company of Biologists Limited 1987

Key words: surface antigen, membrane-anchor, monoclonal antibody, Parameciiim.

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forms we have identified two immunologically distinct regions: one region exposed to antibodies in vivo is allele-specific, while the other is not exposed in vivo and bears antigenic determinants common to all allelie forms of a given SAg locus (Capdeville, 1979). SAg molecules are made up of one huge polypeptide chain (Mr = 300x10 3 ) containing about 10% cysteine residues, all involved in S-S bonds (Hansma, 1975; Jones, 1965; Rcisner et al. \%9a,b; Steers, 1965; Steers & Davis, 1977). The determination of the complete DNA coding sequence of the 156G surface antigen of P. primaurelia (Prat et al. 1986) has revealed a pseudoperiodic primary sequence: this periodicity, mainly dictated by the regular position of the cysteine and tryptophan residues, is made up of 70-75 amino acids on average. The central part of the primary sequence consists of five almost identical repeats of 74 amino acids. Another feature of Paramecium SAgs is their mode of anchorage in the plasma membrane via a glycosylinositol phospholipid, in the same way as Trypanosoma variant surface glycoproteins (VSGs) (Ferguson et al. 1985) and other eukaryotic surface proteins (for reviews, see Cross, 1987; Low et al. 1986). This type of membrane anchor can be specifically cleaved by a phosphoinositol-specific phospholipase C (for review, sec Cross, 1987). In Paramecium, an endogenous phospholipase C, whose activity can be induced by ethanol or Triton X-100, converts the membranebound form of SAgs (mSAg) to the soluble form (sSAg) (Capdeville el al. 1986). This conversion also can be obtained with heterologous phospholipase C from Trypanosoma bntcei or from Bacillus cereus (Capdeville et al. 1987) but is inhibited by Zn 2 + , Cd 2+ and />-chloromercuriphenyl sulphonie acid (Capdeville et al. 1987), agents known to also inhibit the trypanosomal phospholipase C (Biilow & Overath, 1985; Cardoso de Almeida & Turner, 1983). Thus, the mSAg and sSAg forms differ biochemically: mSAg is amphiphilic (Capdeville et. al. 1985) and fatty-acylated (Capdeville et al. 1987) whereas sSAg is not. They also differ in an antigenic determinant, which is hidden in the mSAg and accessible to antibodies in the sSAg (Capdeville et al. 1986). This determinant contains sugars (Capdeville et al. 1986) and cross-reacts with an antigenic determinant common to many Trypanosoma VSGs and called CRD, the cross-reacting determinant (Barbet & McGuire, 1978). In Trypanosoma VSGs, the CRD is also cryptic in the membrane-bound form and becomes exposed in the soluble form after the cleavage of dimyristoylglycerol by an endogenous phospholipase C (Cardoso de Almeida & Turner, 1983; Ferguson et al. 1985). In paramecia, the phospholipase C conversion process yields not only sSAg molecules but also two sets of 45-50 (XlO3)yV/r molecules previously called cross554

Y. Capdeville et al.

reacting light chains (Capdeville et al. 1985). Indeed, these molecules share at least two different determinants with all the sSAgs: one is of the 'CRD' type; the other is specific to Paramecium and is not present in Trypanosoma VSGs (Capdeville et al. 1986). Both determinants are buried and require the rupture of S-S bonds to become accessible to antibodies (Capdeville et al. 1985, 1987). More recently, these molecules have been shown to be surface proteins also anchored in the plasma membrane by a glycosylinositol phospholipid: they are now called cross-reacting glycoproteins (CRGs) (Deregnaucourt et al. unpublished). In order to analyse the organization of the SAg molecules and establish relationships between their antigenicity and their biochemical structure, we raised monoclonal antibodies against cilia of strain 156 of P. primaurelia expressing G SAg. The specificity of two monoclonal antibodies, Y4 and Y8, towards different G and D SAgs is described in this paper. Furthermore, the ultrastructural localization of the 156G SAg analysed with these monoclonal antibodies was compared with that analysed using a polyclonal antiserum. Altogether the results reported here allow us to describe epitopes that give new information on the structure of the SAg molecules and on the structural change occurring in the conversion process. Materials and methods Strains and cultures Several homozygous strains of two species of the P. aurelia group (23) were used: strains of P. primaurelia from different geographical origins (Beale, 1954) 33, 60, 90, 156, 168 and 513 expressing G or D SAg, at 24°C or 33°C, respectively, and strain 51 of P. let?aurelia expressing B SAg at 27°C. Cells were grown monoxenically at 24°C or at 34°C in hay grass infusion inoculated with Klebsiella pneumoniae and supplemented with /3-sistosterol (0-8mgl~'). Cultures were routinely checked by immobilization tests performed with homologous antisera to ascertain the surface antigen expressed and the homogeneity of the population (Capdeville, 1971). Cultures were used when 100% of the cells were immobilized by their corresponding homologous antiserum.

Cellular fractions, sSAg purification and antisera Preparation of cilia and ciliary membranes, carried out according to Hansma & Kung (1975) with some modifications (Adoutte el al. 1980), and preparation of ethanolic ciliary and cellular extracts performed according to Preer (1959), have been described (Capdeville et al. 1985). Preparation of rabbit antisera against the G SAg expressed by strains 33, 60, 90 and 513 and raised against whole cells has been described (Capdeville, 1971). Rabbit antisera against the G or D SAg expressed by strains 156 and 168 were raised against the corresponding purified sSAg. These sSAgs were purified either in their native state according to the method of Preer (1959), followed by filtration on Sephadex G-200 superfine gel, or in their denatured state by SDS-containing

preparative gel eleetrophoresis of unreduced ethanolic cellular extracts (Capdeville el al. 1985). Preparation of antisera 2355 (against the 156G sSAg purified in denatured state), 2519 and 2543 (against the 168G and the 156D sSAgs purified in native state, respectively) has been described (Capdeville el al. 1985). Purified soluble variant surface glycoproteins BoTat-4 and -28 of Trypanosoma equiperdum were a gift from Dr T. Baltz. IgM monoclonal antibodies directed against myosin and vimentin were gifts from Drs C. Klotz and M. Bornens. Preparation of monoclonal antibodies All the procedures used here are those described by Galfre & Milstein (1981) and Fazekas de Groth & Scheidegger (1980). Balb/c mice were hyperimmunized intraperitoneally with 100,1*1 of a suspension in Dryl's (1959) solution of cilia of paramecia issued from strain 156 expressing the G SAg, emulsified with an equal volume of complete Freund's adjuvant (Calbiochem-Behring Corp., La Jolla, CA 92073). After a rest period of one month, mice were boosted with a similar dose. Five days later, their spleen cells were used for fusion with NSO myeloma cells (gift from Dr Milstein, Cambridge, England). After screening by solid-phase enzyme immunoassay, hybridomas were cloned by limiting dilution using mouse macrophages as feeders. Antibody-rich ascitic fluids were obtained from pristane-primed Balb/c mice. Protein eleetrophoresis One-dimensional SDS-polyacrylamide gradient ( 5 % to 15%) slab gel eleetrophoresis was performed according to Laemmli (1970). /3-Mercaptoethanol was omitted from the sample buffer for eleetrophoresis of unreduced proteins. The wells were loaded as follows: 40-60 f.ig for ethanolic cellular extracts, 15—30 /^g for ciliary membranes. Protein concentrations were determined by the method of Lowry el al. (1951). Western blotting Immunoblotting was carried out according to Tovvbin et al. (1979) with slight modifications (Capdeville et al. 1985). In immunoblotting experiments, ascitic fluids containing antibodies Y4 or Y8 were used diluted 1:25 and 1:10 (v/v), respectively; the rabbit antiserum 2355 was used at a 1:200 (v/v) dilution. Peroxidase-labelled antibodies raised against mouse and rabbit immunoglobulins (Institut Pasteur, France) were used at a 1:200 (v/v) dilution, with 3',3diaminobenzidin or 4-chloro-l-naphthol, as chromogenic substances (Sigma). Enzyme immunoassays Enzyme immunoassays (Engvall & Perlmann, 1971) were performed in polystyrene microtitrating plates (Nunc). The immunoglobulin subtype of the monoclonal antibodies was determined by using specific (anti-IgA, IgCl, IgG2a, IgG2ab and IgM) rabbit anti-mouse immunoglobulins (Nordic Immunological Laboratories, The Netherlands) at a 1:100 (v/v) dilution. Peroxidase-labelled antibodies raised against mouse and rabbit immunoglobulins (Institut Pasteur Production) were used at a 1:500 (v/v) dilution. The

enzymic reaction was allowed to develop for 3 min with 0-05% O-dianisidine (Sigma) as chromogenic substance. The absorbance was read at 490 nm. lmmunoelectron micmscopy Cells were prefixed in 3 % paraformaldchyde with 025 % glutaraldehyde in a 0 0 1 M-phosphate buffer, pi 17-3, for 10 min. They were then incubated for l h with the first antibody in 10 mM-phosphate buffer containing 1 % bovine serum albumin (BSA). The dilutions were the following: rabbit antisera 2355 and 2543, 1:500; ascitic fluids Y4 and Y8, 1:100 and 1:5, respectively. Labelling was achieved by a second incubation for 1 h with goat anti-rabbit lgG coupled with 5 nm gold granules (GAR G5, Janssen Pharmaccutica, Beerse, Belgium). In the case of Y4 monoclonal antibodies, an intermediate rabbit anti-mouse antibody diluted 1: 100 (RAM IgG 7S, Nordic Immunology, Tilburg, The Netherlands) was added before the final GAR G5 (diluted 1:5). After several washings, paramecia were fixed in 2 % glutaraldehyde in 0-05M-sodium cacodylate buffer, pi 17-3, for 45 min, and post-fixed in 1 % osmium tetroxide. Cells were then carefully rinsed in distilled water and dehydrated in an ethanol series before embedding in Spurr resin (E. Fullam Inc., NY, USA). Sections were stained with saturated aqueous uranyl acetate for 30 min. Micrographs were taken at 60 kV with a Philips EM 301. Measurements of the antigen layer were made on micrographs at a magnification of X230000. Only areas with a well-preserved membrane bilayer, cut perpendicularly, were examined. Other procedures Immunoelectrophoresis experiments and pcriodate treatment were performed as described (Capdeville el al. 1985, 1986).

Results Preparation and properties of monoclonal antibodies against 156G SAg Mice were hyperimmunized with whole cilia isolated from strain 156 of P. priinaiirelia that expresses the G SAg (156G cilia). Antibody-secreting hybridomas were selected by an enzyme immunoassay using an ethanolic extract of 156G cilia that is enriched in sSAg molecules (Capdeville et al. 1985). Six reactive hybridomas obtained in this way were cloned by limiting dilution and found by immunoblotting experiments to react with 156G SAg (data now shown). Four of them (Y2, Y5, Y8 and Y10) are of the IgM type and the others (E1C4 and Y4) of the IgG type. Apart from E1C4 these hybrids were stable, and ascitic fluids with a high antibody titre were obtained from pristane-primed mice for Y4, Y5, Y8 and Y10. On the basis of their immunoreactivity in enzyme immunoassay against 156G ethanolic ciliary extracts, they could be divided into two groups: one (E1C4 and Y4) reacted very strongly, whereas the other (Y2, Y5, Y8 and Y10) reacted weaklv. Surface antigen epitopes of Paramecium

555

the 156G SAg, while antibody Y8 mainly reacted with the membrane form (Table 1). Considering the importance of S-S bonds in the antigenicity of SAg molecules, we performed immunoblotting experiments on the reduced and unreduced forms of the 156G SAg. Experiments were carried out with 156G ethanolic cellular extracts that contained only the soluble form of SAg and CRG molecules (Capdeville et al. 1985). As previously demonstrated (Capdeville et al. 1985) the reduced 156G sSAg migrates with an apparent Mr of 300X103, whereas in its unreduced state it displays two major bands with higher apparent molecular weights (Fig. 1A, I). Y4 antibodies were found to decorate only the two major bands corresponding to the unreduced 156G sSAg (Fig. 1A, III). In contrast, Y8 antibodies reacted only with the reduced samples and labelled the 300xl0 3 /l/,. band and the 45-50 X 103/V/r bands corresponding to the reduced soluble form of the 156G SAg and CRGs, respectively (Fig. 1A, IV). For comparison, a polyclonal antiserum, AS 2355, raised against the purified 156G sSAg was shown to react with the unreduced and reduced 156G sSAg, and the reduced sCRGs (Fig. 1A, II). In contrast to the labelling patterns with ethanolic extracts, antibodies Y4 and Y8 gave identical immunological patterns when reacted with 156G ciliary membranes (containing the membrane form of SAg and CRG molecules) (Fig. IB, III and IV): they labelled only the unreduced 156G mSAg, while the reduced 156G mSAg was also labelled by antiserum 2355, but not mCRGs (Fig. IB, II). From these results we can conclude that Y4 and Y8 behave differently towards the 156G SAg. Y4 recognizes the unreduced soluble and membrane forms of

Since all the properties of these antibodies were found to be identical for the members of a given group, results obtained with only one representative of each group, Y4 and Y8, respectively, will be described. Their reactivities were analysed with both whole cells and cellular extracts from various geographical strains, containing either the soluble form (ethanolic extracts) or the membrane form (ciliary membranes) of a given SAg. hmmtnoreactivity with I56G SAg By enzyme immunoassay, antibody Y4 strongly reacted with both the soluble form and the membrane form of Table 1. Imnntnoreactivity of Y4 and Y8 against the G and D SAgs of strains 156 and 168, as expressed by optical density in EIA

I56G a b 156D a b

168Ga b

168D a b

V4

Y8

126 159 6 8 140 116 8 8

18 82 ,2ft

It

n

m 18 50

The wells were coated with equal amounts (0-5 fig) of native extracts from strains 156 and 168, expressing either G or D SAg: a, ethanolic ciliary extract; b, ciliary membranes. The culture supcrnatants containing Y4 antibodies (V4) and Y8 antibodies (Y8) were used non-diluted. The values represent the average values of the optical density observed (sec Materials and methods). The background was similar for antibodies Y4 and Y8 with an average value of 7.

I

M

III

II 1

2

1

2

1

I

IV

M 1

2

2

II

III

IV

1 2

1 2

1 2

2(

20093-

4531-

Fig. 1. Reactivity of Y4 and Y8 towards 156G SAg. A. Ethanolic cellular extracts containing 1S6G sSAg molecules. B. Ciliary membranes containing 156G mSAg molecules. I. Coomassie-Blue-stained SDS-polyacrylamide gel of reduced (lane 1) and unreduced (lane 2) samples. M, molecular weight markers; numbers indicate A/ r X 10~ 3 . I I , III, IV, corresponding immunoblots obtained with AS 2355, raised against the purified 156G sSAg ( I I ) , Y4 (III) and Y8 ( I V ) . Star, 45-50 ( X l 0 3 ) j l / r bands (cross-reacting glycoproteins, C R G s ) .

556

Y. Capdeville et al.

12

3

4

5 6

7 8

Fig. 2. Reactivity of Y4 towards various sSAgs. Top: upper part of a Coomassie-Blue-stained SDS-containing gel of unreduced ethanolic cellular extracts of different strains of P. primaurelia expressing G or D SAgs. Lanes 1, 513G; 2, 90G; 3, 60D; 4, 60G; 5, 168D; 6, 168G; 7, 156D; 8, 156G. The stained bands correspond to the sSAg molecules. Bottom: immunoblotting of the same gel decorated with Y4.

the 156G SAg while Y8 recognizes the reduced soluble form and not the unreduced one; in contrast, it recognizes the unreduced membrane form of the 156G SAg and not the reduced one. Furthermore, Y8 also recognizes the reduced soluble form of CRGs. Imrnunoreactivity with other SAgs Various allelic G (168G, 60G, 90G and 513G) and D (156D, 168D and 60D) SAgs of P. primaurelia and the SIB SAg of P. tetraiirelia were analysed for their immunoreactivities with Y4 and Y8. By enzyme immunoassay, Y4 reacted strongly with the soluble and membrane forms of the 168G SAg (Table 1) as well as with the other allelic G SAgs analysed (data not shown), but not with the D SAgs (Table 1). In contrast, Y8 reacted weakly with the soluble form of all the SAgs tested as well as with the membrane form of the 156D SAg, but displayed a stronger reaction with the membrane form of the 168G and 168D SAgs (Table 1). By immunoblotting, Y4 reacted strongly with all the .G sSAgs tested, but did not react with D sSAgs (Fig. 2); it also reacted strongly with the corresponding G mSAgs, but did not react with D mSAgs (data not shown). Y8 reacted with all the reduced sSAgs (Fig. 3B) and sCRGs analysed, whatever the SAg expressed, the strain or the species used (Fig. 4). In contrast, it did not react with all the mSAgs: Y8 reacted strongly with the unreduced 168G mSAg, but riot with the 156D mSAg (data not shown). Furthermore, Y8 did not react with the membrane form of CRGs, either unreduced or reduced. The reaction of Y8 towards reduced sSAgs and sCRGs was shown to be specific (no labelling was obtained by using two unrelated IgM monoclonal

antibodies raised against myosin and vimentin, data not shown). Since Y8 is found to react in a similar way as antibodies specific to the cross-reacting determinant (CRD) (Capdeville et al. 1986), we wondered if the epitope recognized by Y8 in reduced sSAgs and sCRGs corresponded to the CRD. To check this possibility, we performed Western blot experiments with purified soluble variant surface glycoproteins of Tiypanosoma equiperdum, which present the CRD (see Introduction). Since no labelling of these proteins was observed with Y8 (data not shown) we conclude that the Y8 antibodies are not directed against the CRD structure but are directed against another determinant, which is revealed after cleavage of diacylglycerol from mSAg and after reduction. The fact that the immunoreactivity of Y8 was not lost after treatment with periodate (data not shown) suggests that this determinant is probably not glycosidic. Immunoreactivity with whole cells To ascertain the in situ localization of the Y8 epitopes, we performed immunological tests of immobilization in vivo and immunolabelling of fixed cells. Living cells of strains 33, 60, 90, 156, 168 and 513 expressing G SAg, and strains 156 and 168 expressing D SAg, were incubated with Y4 or Y8 alone, or first with Y4 or Y8, then with anti-mouse immunoglobulin antibodies (Table 2). Y4 antibodies affected all the G cells, whatever the G allele expressed, but did not immobilize them: cells were simply sluggish. Immobilization was completed only after the addition of the mouse Ig antibody. Y8 antibodies did not affect living cells, whatever the SAg expressed, even after the addition of mouse Ig antibody. It is worth noting that Y4 did not yield any precipitation line by Ouchterlony double immunodiffusion or by immunoelectrophoresis with native 156G and 168G sSAgs (results not shown). The reactivity of Y4 and Y8 was also examined by immunoelectron microscopy with the IgG-gold technique and compared with that of the polyclonal antiserum, AS 2355, raised against the 156G sSAg. The polyclonal antiserum showed a dense and continuous distribution of antigens both on pellicle and cilia of 156G cells, with gold granules aligned at a distance of about 23—25 nm from the membrane bilayer (Fig. 5A). In contrast, a 156G cell labelled with an anti-156D serum (AS 2543) did not show any significant labelling (Fig. 5B). Cells treated by Y8 did not display specific binding to the G molecules. The presence of few aggregates laying apart from the antigen layer (Fig. 5C) was also observed with two unrelated monoclonal IgM (data not shown). A continuous labelling of the antigen coat, similar to that observed with AS 2355, was obtained with Y4 when intermediate rabbit anti-mouse Surface antigen epitopes of Paramecium

557

1 2

3

4 5 6 7 8

1 2

3

5

6 7

8

Mil

:.

,^

B

Fig. 3. Reactivity of Y8 towards various sSAgs and sCRGs. A,B- Two sister immunoblots obtained with AS 2355 used as a control (A) and with Y8 (B) from a SDS-polyacrylamide gel of reduced (lanes 1-4) and unreduced (lanes 5-8) ethanolic cellular extracts of the 156 and 168 strains of P. piimaiirelia expressing either G or D SAg. Lanes 1,5, 168D; 2,8, 156G; 3,7, 168G; 4,6, 156D. In A the reduced sSAgs and sCRGs, and the unreduced sSAgs were labelled, while in B only the reduced sSAgs and sCRGs were labelled. We note in 156D reduced extracts (lane 4) and immuno-decoration of two high molecular weight bands. This finding, previously discussed (Capdeville et al. 1985), probably corresponds to the coexpression of two SAgs (the D SAg and another one) in strain 156 grown at 34°C. With regard to the high molecular weight bands other than the sSAg band that are labelled in reduced extracts, they probably correspond to proteolytic fragments of the sSAg molecules.

1

4

5

6

cell surface, were found to be 17 ± 2 nm and 22 ± 3 nm, respectively, for the pellicular and the ciliary regions. Discussion

Fig. 4. Reactivity of Y8 towards various sCRGs. Immunoblot obtained with Y8, corresponding to the lower part of an SDS-polyacrylamide gel of reduced ethanolic cellular extracts issued from strain 51 of P. tetraurelia expressing B SAg, and from different strains of P. priniaurelia expressing G or D SAgs. Lanes 1, 51B; 2, 513G; 3, 90G; 4, 168G; 5, 156D; 6, 156G. The apparent molecular weights of the cross-reacting glycoproteins vary according to the strain used, as previously found (Capdeville et al. 1986).

antibodies were inserted between the monoclonal antibody and the IgG-gold antibody (Fig. 5D). Evaluation of distances between the closest gold granules and the external membrane lipid bilayer gave 23 + 3 nm for the pellicular (interciliary) membrane, and 2 5 ± 4 n m for the ciliary membrane. Direct measurements (without immunolabelling) of the thickness of the external protein layer (156G SAg molecules), which coats the 558

V. Capdeville et al.

For the epitope defined by Y4 and referred to as epitope Y4, two main features emerge. (1) It is specifically found in all the native, or unreduced, G SAgs of/3, primaurelia and absent in the reduced form. (2) It is accessible in vivo in all the cells expressing a G SAg. From these characteristics, epitope Y4 is likely to be a conformational S-S-dependent determinant. This is not surprising since SAg molecules contain a great number of S-S bridges (see Introduction). Epitope Y4 does not appear to correspond to a repeated determinant (although the 156G gene contains several repeats and segments of homology (Prat et al. 1986)) for two reasons: (1) antibodies Y4, even when highly concentrated, cannot immunoprecipitate the solubilized G molecules (data not shown); (2) these antibodies cannot immobilize G cells either. Cross-linking of SAg molecules has been shown to be necessary and sufficient to lead to the immobilization of cells, and monovalent antibody fragments were unable to trigger this reaction (Barnett & Steers, 1984; Eisenbach et al.

mp&wt

Fig. 5. Ultrastructural localization of the 156G SAg. A. Surface immunolabelling of a cell expressing the 156G SAg with a polyclonal anti-156G serum (AS 2355). X75 000. B. Surface immunolabelling of a cell expressing the 156G SAg with a polyclonal anti-156D serum (AS 2543). X75 000. C. Surface immunolabelling of a cell expressing the 156G SAg with the Y8 monoclonal antibodies. X75 000. D. Surface immunolabelling of a cell expressing the 156G SAg with the Y4 monoclonal antibodies. X75 000.

Surface antigen epitopes of Paramecium

559

Table 2. Immunoreactivity of antibodies Y4 and YS in vivo 33G Y4 Y8 Mouse Ig Ab Y4 + mouse Ig Ab Y8 + mouse Ig Ab Specific AS

s+

i

60G s+ + +

i

90G

156G

s+

s+ i — i

i

168G s+ + + i — i

513G s+

i

156D -

168D -

— — i

— — i

Strains 33, 60, 90, 513 expressing G SAg, and strains 156 and 168 expressing either G or D SAg, were used. Ascitic fluid dialysed against Dryl's (1959) solution was used at the dilution 1:20 (v/v) for Y4 and not diluted for Y8. Anti-mouse inimunoglobulin antibodies (mouse Ig Ab) dialysed against Dryl's solution were used not diluted. Specific rabbit antisera (specific AS) used were diluted in such a way that homologous cells were immobilized within 20min. The in vivo immobilization test was performed as described (Capdeville, 1971). s 1 , sluggish cells; s + + + , very sluggish cells; i, immobilized cells; —, unaffected cells.

1983). Finally, the Y4 epitope is a specific marker of all the allelic G SAgs and is distinct from a highly polymorphic region, which differs from one allelic antigen to another (Capdeville, 1979). It is also a specific tool with which to analyse the ultrastruetural localization of the G SAgs. The comparison of the labelling obtained by Y4 and by a polyclonal antiserum with the IgG-gold technique shows that the closest granules were at the same distance from the membrane bilayer. This similarity of labelling demonstrates that only the most external domain of the 156G molecules is accessible in situ, and leads us to propose that the major part of the SAg molecules probably involved in intermolecular interactions constitutes a compact surface coat similar to that found in the African trypanosome (Strickler & Patton, 1982). Furthermore, it is worth pointing out that the thickness of the 156G layer (17-22 nm) is intermediate between those found for other SAgs from micrographs of sections (20-30 nm) (Ramanathan et al. 1981), and from negative staining of solubilized molecules (10-20 nm) (Mott, 1965; Reisner et al. 19696). This value supports the model of an oblate ellipsoid proposed by Reisner et al. (19696), and fits well with a model predictable from the 1S6G gene sequence data (Prat et al. 1986), where the (3156G mSAg

pleated sheets should be aligned along an axis perpendicular to the plasma membrane. The behaviour of Y8 is rather puzzling, since it reacts differently with the membrane and the soluble forms of SAgs and CRGs. A single epitope cannot explain all the properties of Y8, even if we put forward the hypothesis that, in the process of converting mSAg to sSAg by cleavage of the lipid moiety, the structure recognized by Y8 in mSAg is modified in such a way that its recognition requires the rupture of S-S bonds in sSAg. Such an hypothesis implies the recognition of the membrane form of all the SAgs and CRGs by Y8. Since this is not the case, this hypothesis is not valid. Thus, we are led to propose that the SAg molecules display intramolecular cross-reactivity and that Y8 recognizes two different related epitopes. The existence of intramolecular cross-reactivity is not surprising for a protein containing several repeats (Prat et al. 1986); and intramolecular cross-reactivity has been described in the case of human serum albumin, for which two monoclonal antibodies have both been found to react with two different related epitopes (Doyen et al. 1985). We will call the epitopes assumed to be recognized by the Y8 antibody Y8a and Y8b: Y8a corresponds to the epitope revealed at the level of the 156G sSAg

= hidden) = hidden)

560

V. Capdeville et al.

Fig. 6. A hypothetical model of the location of epitopes Y8a and Y8b. The structure of the membrane-anchor and the location of the CRD are based on information derived from the VSG of T. bnicei (Ferguson et al. 1985), since Paranieciunt SAgs have similar features (Capdeville et al. 1986, 1987). The locations of epitopes Y8a and Y8b are deduced from the properties of the monoclonal antibody Y8 by assuming that it recognizes two related different epitopes (see Discussion).

unreduced membrane form of the 156G and 168G SAgs, and Y8b to the epitope revealed at the level of the reduced soluble form of all SAgs and CRGs. The epitope Y8a, which is a surface epitope requiring the integrity of S-S bonds and the presence of the lipidic membrane anchor to be detected, would result from interactions between a peptidie loop containing at least one S-S bridge and the membrane anchor itself (see Fig. 6). Therefore, the epitope Y8a, which constitutes an antigenic marker of the membrane form of the 156G and 168G SAgs, should be very close to or partly buried in the plasma membrane. The epitope Y8b, which is a masked epitope requiring the rupture of S-S bonds and the cleavage of the lipidic membrane-anchor to be detected (see Fig. 6), might correspond to a peptidie determinant, since it is not destroyed by periodate treatment. The same properties, i.e. immunologieal detection requiring the rupture of S-S bonds and the cleavage of the lipid membrane anchor, have been previously found in the case of another determinant in P. primaurelia, the CRD (Capdeville et al. 1986, 1987). Such findings suggest that these two different determinants, Y8b and CRD, would be located in the same region, in proximity to the lipidic membrane-anchor, which would mask them (see Fig. 6). Finally, we conclude that Y8a and Y8b are two related epitopes whose immunologieal detection is mutually exclusive, depending strictly on the presence or absence of the lipidic membrane-anchor, i.e. on the action of a phosphatidylinositol-specific phospholipase C.

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KELLER, A. M. (1986). Immunologieal evidence of a common structure between Paramecium surface antigens and Tiypanosoma variant surface glycoproteins. Expl Cell Res. 167, 75-86. CAPDEVILLE, Y., CARDOSO DE ALMEIDA, M. L. &

DEREGNAUCOURT, C. (1987). The membrane-anchor of Paramecium temperature-specific surface antigens is a glycosylinositol phospholipid. Biochem. biophys. Res. Commun. 147, 1219-1225. CAPDEVILLE, Y., DEREGNAUCOURT, C. & KELLER, A. M.

(1985). Surface antigens of Paramecium primaurelia. Membrane-bound and soluble forms. Expl Cell Res. 161, 495-508. CARDOSO DE ALMEIDA, M. L. & TURNER, M. J. (1983).

The membrane form of variant surface glycoproteins of Tiypanosoma bnicei. Nature, IJOIUI. 302, 349-352. CROSS, G. A. M. (1987). Eukaryotic protein modification and membrane attachment via phosphatidylinositol. Cell 48, 179-181. DOYEN, N., LAPRESLE, C , LAFAYE, P. & MAZIE, J. C.

We thank Drs T. Batlz, M. Bornens, C. Klotz and C. Milstein for their gifts, and Dr T. Rosenberry for a critical reading of the manuscript and for many helpful comments. We are also grateful to Dr Benedetti, who advised us in the immunocytochemical field. This work was supported in part by the Ministere de l'lndustrie et de la Recherche (grant 82L 1089) and the Institut National de la Sante et de la Recherche Medicate (grant 12600S). C D . was supported by a fellowship from the Ministere de l'lndustrie et de la Recherche.

(1985). Study of the antigenic structure of human serum albumin with monoclonal antibodies. Molec. Iminun. 22, 1-10. DRYL, S. (1959). Antigenic transformation in Paramecium aurelia after homologous antiserum treatment during autogamy and conjugation..7- Pmlozool. 6 (Suppl), 25.

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