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 ofsurface antigens (SAgs) according to culture con-ditions. These high MT proteins are anchored inthe plasma membrane by a glycolipid, and theycan be isolated in two different forms, an amphi-philic membrane-bound form (mSAg) and a hy-drophilic soluble form (sSAg). Endogenous orexogenous phospholipase C can convert mSAg tosSAg with unmasking of a carbohydrate antigenicdeterminant similar to that found in the solubleform of Trypanosoma variant surface glyco-proteins and called the cross-reacting determi-nant. By immunizing mice with cilia from strain156 of P. primaurelia expressing the G SAg, weobtained six monoclonal antibodies against the156G SAg, which could be classified into twogroups. Y4 and Y8, representative of each group,have been characterized by checking their reac-tivity in situ and in vivo towards a series of allelicG and D SAgs in P. primaurelia and the 5IB SAgin P. tetraurelia.

The monoclonal Y4 recognizes a confor-mational determinant, accessible in vivo andcommon to all the G SAgs. Thus, Y4 defines a G

locus-specific epitope that corresponds to a con-served region inside a polymorphic domain. Themonoclonal Y8 recognizes two homologous deter-minants whose detection depends on the presenceor absence of the SAg membrane-anchor, andwhich are mutually exclusive: one is found in thereduced soluble form of all the SAgs and othersurface proteins, the cross-reacting glycoproteins(CRGs); the other occurs in the unreducedmembrane-bound form of the G SAgs. Thus,Y8 enables us to demonstrate that the mem-brane-anchor of Paramecium SAgs contains anadditional hidden determinant close to the cross-reacting determinant and to discriminate be-tween the membrane-bound and soluble form ofSAgs.

The in situ organization of the 156G SAg mol-ecules is also discussed on the basis of immuno-gold labelling obtained using Y4 and a polyclonalantiserum.

Key words: surface antigen, membrane-anchor, monoclonalantibody, Parameciiim.

Introduction

The surface of Paramecium aurelia, a ciliated proto-zoon, is coated mainly by a single set of surfaceantigens, SAgs (also called immobilization antigens).These molecules belong to a multigene family whoseexpression 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

generally expressed (for reviews, see Beale, 1954;Sonneborn, 1974).

Immunological and biochemical studies of Para-mecium SAgs have revealed that these surface proteinsare highly polymorphic, but the polymorphism appearsto be restricted to the external part of the molecule. In/-". primaurelia two unlinked loci, G and D, expressdifferent allelic forms of SAgs in strains of distinctgeographical origins (Beale, 1954). In comparing these

553

forms we have identified two immunologically distinctregions: one region exposed to antibodies in vivo isallele-specific, while the other is not exposed in vivoand bears antigenic determinants common to all allelieforms of a given SAg locus (Capdeville, 1979). SAgmolecules are made up of one huge polypeptide chain(Mr = 300x103) containing about 10% cysteine resi-dues, 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 DNAcoding sequence of the 156G surface antigen of P.primaurelia (Prat et al. 1986) has revealed a pseudo-periodic primary sequence: this periodicity, mainlydictated by the regular position of the cysteine andtryptophan residues, is made up of 70-75 amino acidson average. The central part of the primary sequenceconsists of five almost identical repeats of 74 aminoacids.

Another feature of Paramecium SAgs is their modeof anchorage in the plasma membrane via a glycosyl-inositol phospholipid, in the same way as Trypanosomavariant surface glycoproteins (VSGs) (Ferguson et al.1985) and other eukaryotic surface proteins (for re-views, see Cross, 1987; Low et al. 1986). This type ofmembrane anchor can be specifically cleaved by aphosphoinositol-specific phospholipase C (for review,sec Cross, 1987). In Paramecium, an endogenousphospholipase C, whose activity can be induced byethanol or Triton X-100, converts the membrane-bound form of SAgs (mSAg) to the soluble form(sSAg) (Capdeville el al. 1986). This conversion alsocan be obtained with heterologous phospholipase Cfrom Trypanosoma bntcei or from Bacillus cereus(Capdeville et al. 1987) but is inhibited by Zn2+, Cd2+

and />-chloromercuriphenyl sulphonie acid (Capdevilleet al. 1987), agents known to also inhibit the trypano-somal phospholipase C (Biilow & Overath, 1985;Cardoso de Almeida & Turner, 1983). Thus, the mSAgand sSAg forms differ biochemically: mSAg is amphi-philic (Capdeville et. al. 1985) and fatty-acylated (Cap-deville et al. 1987) whereas sSAg is not. They alsodiffer in an antigenic determinant, which is hidden inthe mSAg and accessible to antibodies in the sSAg(Capdeville et al. 1986). This determinant containssugars (Capdeville et al. 1986) and cross-reacts with anantigenic determinant common to many TrypanosomaVSGs and called CRD, the cross-reacting determinant(Barbet & McGuire, 1978). In Trypanosoma VSGs,the CRD is also cryptic in the membrane-bound formand becomes exposed in the soluble form after thecleavage of dimyristoylglycerol by an endogenousphospholipase C (Cardoso de Almeida & Turner, 1983;Ferguson et al. 1985).

In paramecia, the phospholipase C conversion pro-cess yields not only sSAg molecules but also two setsof 45-50 (XlO3)yV/r molecules previously called cross-

reacting light chains (Capdeville et al. 1985). Indeed,these molecules share at least two different determi-nants with all the sSAgs: one is of the 'CRD' type; theother is specific to Paramecium and is not present inTrypanosoma VSGs (Capdeville et al. 1986). Bothdeterminants are buried and require the rupture of S-Sbonds to become accessible to antibodies (Capdeville etal. 1985, 1987). More recently, these molecules havebeen shown to be surface proteins also anchored in theplasma 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 SAgmolecules and establish relationships between theirantigenicity and their biochemical structure, we raisedmonoclonal antibodies against cilia of strain 156 of P.primaurelia expressing G SAg. The specificity of twomonoclonal antibodies, Y4 and Y8, towards differentG and D SAgs is described in this paper. Furthermore,the ultrastructural localization of the 156G SAg ana-lysed with these monoclonal antibodies was comparedwith that analysed using a polyclonal antiserum.Altogether the results reported here allow us to de-scribe epitopes that give new information on thestructure of the SAg molecules and on the structuralchange occurring in the conversion process.

Materials and methods

Strains and culturesSeveral homozygous strains of two species of the P. aureliagroup (23) were used: strains of P. primaurelia from differentgeographical origins (Beale, 1954) 33, 60, 90, 156, 168 and513 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 haygrass infusion inoculated with Klebsiella pneumoniae andsupplemented with /3-sistosterol (0-8mgl~'). Cultures wereroutinely checked by immobilization tests performed withhomologous antisera to ascertain the surface antigen ex-pressed and the homogeneity of the population (Capdeville,1971). Cultures were used when 100% of the cells wereimmobilized by their corresponding homologous antiserum.

Cellular fractions, sSAg purification and antiseraPreparation of cilia and ciliary membranes, carried outaccording to Hansma & Kung (1975) with some modifi-cations (Adoutte el al. 1980), and preparation of ethanolicciliary and cellular extracts performed according to Preer(1959), have been described (Capdeville et al. 1985).

Preparation of rabbit antisera against the G SAg expressedby strains 33, 60, 90 and 513 and raised against whole cells hasbeen described (Capdeville, 1971). Rabbit antisera againstthe G or D SAg expressed by strains 156 and 168 were raisedagainst the corresponding purified sSAg. These sSAgs werepurified either in their native state according to the method ofPreer (1959), followed by filtration on Sephadex G-200superfine gel, or in their denatured state by SDS-containing

554 Y. Capdeville et al.

preparative gel eleetrophoresis of unreduced ethanolic cellu-lar extracts (Capdeville el al. 1985). Preparation of antisera2355 (against the 156G sSAg purified in denatured state),2519 and 2543 (against the 168G and the 156D sSAgs purifiedin native state, respectively) has been described (Capdevilleel 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 andvimentin were gifts from Drs C. Klotz and M. Bornens.

Preparation of monoclonal antibodiesAll the procedures used here are those described by Galfre &Milstein (1981) and Fazekas de Groth & Scheidegger (1980).Balb/c mice were hyperimmunized intraperitoneally with100,1*1 of a suspension in Dryl's (1959) solution of cilia ofparamecia issued from strain 156 expressing the G SAg,emulsified with an equal volume of complete Freund'sadjuvant (Calbiochem-Behring Corp., La Jolla, CA 92073).After a rest period of one month, mice were boosted with asimilar dose. Five days later, their spleen cells were used forfusion with NSO myeloma cells (gift from Dr Milstein,Cambridge, England). After screening by solid-phase en-zyme immunoassay, hybridomas were cloned by limitingdilution using mouse macrophages as feeders. Antibody-richascitic fluids were obtained from pristane-primed Balb/cmice.

Protein eleetrophoresis

One-dimensional SDS-polyacrylamide gradient (5% to15%) slab gel eleetrophoresis was performed according toLaemmli (1970). /3-Mercaptoethanol was omitted from thesample buffer for eleetrophoresis of unreduced proteins. Thewells were loaded as follows: 40-60 f.ig for ethanolic cellularextracts, 15—30 /̂ g for ciliary membranes. Protein concen-trations 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). Inimmunoblotting experiments, ascitic fluids containing anti-bodies 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 againstmouse and rabbit immunoglobulins (Institut Pasteur,France) were used at a 1:200 (v/v) dilution, with 3',3-diaminobenzidin or 4-chloro-l-naphthol, as chromogenicsubstances (Sigma).

Enzyme immunoassays

Enzyme immunoassays (Engvall & Perlmann, 1971) wereperformed in polystyrene microtitrating plates (Nunc). Theimmunoglobulin subtype of the monoclonal antibodies wasdetermined by using specific (anti-IgA, IgCl, IgG2a,IgG2ab and IgM) rabbit anti-mouse immunoglobulins (Nor-dic Immunological Laboratories, The Netherlands) at a1:100 (v/v) dilution. Peroxidase-labelled antibodies raisedagainst mouse and rabbit immunoglobulins (Institut PasteurProduction) were used at a 1:500 (v/v) dilution. The

enzymic reaction was allowed to develop for 3 min with0-05% 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 001 M-phosphate buffer, pi 17-3, for10 min. They were then incubated for lh with the firstantibody in 10 mM-phosphate buffer containing 1 % bovineserum albumin (BSA). The dilutions were the following:rabbit antisera 2355 and 2543, 1:500; ascitic fluids Y4 andY8, 1:100 and 1:5, respectively. Labelling was achieved by asecond incubation for 1 h with goat anti-rabbit lgG coupledwith 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 Nether-lands) was added before the final GAR G5 (diluted 1:5).After several washings, paramecia were fixed in 2% glutaral-dehyde in 0-05M-sodium cacodylate buffer, pi 17-3, for45 min, and post-fixed in 1 % osmium tetroxide. Cells werethen carefully rinsed in distilled water and dehydrated in anethanol series before embedding in Spurr resin (E. FullamInc., NY, USA). Sections were stained with saturatedaqueous uranyl acetate for 30 min. Micrographs were taken at60 kV with a Philips EM 301. Measurements of the antigenlayer were made on micrographs at a magnification ofX230000. Only areas with a well-preserved membranebilayer, cut perpendicularly, were examined.

Other procedures

Immunoelectrophoresis experiments and pcriodate treatmentwere performed as described (Capdeville el al. 1985, 1986).

Results

Preparation and properties of monoclonal antibodiesagainst 156G SAg

Mice were hyperimmunized with whole cilia isolatedfrom strain 156 of P. priinaiirelia that expresses the GSAg (156G cilia). Antibody-secreting hybridomas wereselected by an enzyme immunoassay using an ethanolicextract of 156G cilia that is enriched in sSAg molecules(Capdeville et al. 1985). Six reactive hybridomasobtained in this way were cloned by limiting dilutionand found by immunoblotting experiments to reactwith 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 E1C4these hybrids were stable, and ascitic fluids with a highantibody titre were obtained from pristane-primedmice for Y4, Y5, Y8 and Y10. On the basis of theirimmunoreactivity in enzyme immunoassay against156G ethanolic ciliary extracts, they could be dividedinto two groups: one (E1C4 and Y4) reacted verystrongly, whereas the other (Y2, Y5, Y8 and Y10)reacted weaklv.

Surface antigen epitopes of Paramecium 555

Since all the properties of these antibodies werefound to be identical for the members of a given group,results obtained with only one representative of eachgroup, Y4 and Y8, respectively, will be described.Their reactivities were analysed with both whole cellsand cellular extracts from various geographical strains,containing either the soluble form (ethanolic extracts)or the membrane form (ciliary membranes) of a givenSAg.

hmmtnoreactivity with I56G SAg

By enzyme immunoassay, antibody Y4 strongly reactedwith both the soluble form and the membrane form of

Table 1. Imnntnoreactivity of Y4 and Y8 against theG and D SAgs of strains 156 and 168, as expressed

by optical density in EIA

I56G ab

156D ab

168Gab

168D ab

V4

126159

68

140116

88

Y8

1882

,2ftIt

nm1850

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

the 156G SAg, while antibody Y8 mainly reacted withthe membrane form (Table 1).

Considering the importance of S-S bonds in theantigenicity of SAg molecules, we performed immuno-blotting experiments on the reduced and unreducedforms of the 156G SAg. Experiments were carried outwith 156G ethanolic cellular extracts that containedonly the soluble form of SAg and CRG molecules(Capdeville et al. 1985). As previously demonstrated(Capdeville et al. 1985) the reduced 156G sSAgmigrates with an apparent Mr of 300X103, whereas inits unreduced state it displays two major bands withhigher apparent molecular weights (Fig. 1A, I). Y4antibodies were found to decorate only the two majorbands corresponding to the unreduced 156G sSAg(Fig. 1A, III). In contrast, Y8 antibodies reacted onlywith the reduced samples and labelled the 300xl03/l/,.band and the 45-50 X 103/V/r bands corresponding tothe reduced soluble form of the 156G SAg and CRGs,respectively (Fig. 1A, IV). For comparison, a poly-clonal antiserum, AS 2355, raised against the purified156G sSAg was shown to react with the unreducedand reduced 156G sSAg, and the reduced sCRGs(Fig. 1A, II). In contrast to the labelling patterns withethanolic extracts, antibodies Y4 and Y8 gave identicalimmunological patterns when reacted with 156G ciliarymembranes (containing the membrane form of SAgand CRG molecules) (Fig. IB, III and IV): theylabelled only the unreduced 156G mSAg, while thereduced 156G mSAg was also labelled by antiserum2355, but not mCRGs (Fig. IB, II).

From these results we can conclude that Y4 and Y8behave differently towards the 156G SAg. Y4 recog-nizes the unreduced soluble and membrane forms of

I

M 1 2

II

1 2

III

1 2

IV I II III IV

M 1 2 1 2 1 2 1 2

2(200-

9 3 -

4 5 -

3 1 -

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/rX 10~3. I I , III, IV,corresponding immunoblots obtained with AS 2355, raised against the purified 156G sSAg ( I I ) , Y4 (III) and Y8 ( IV) .Star, 45-50 (Xl0 3 ) j l / r bands (cross-reacting glycoproteins, CRGs) .

556 Y. Capdeville et al.

1 2 3 4 5 6 7 8

Fig. 2. Reactivity of Y4 towards various sSAgs. Top:upper part of a Coomassie-Blue-stained SDS-containing gelof unreduced ethanolic cellular extracts of different strainsof 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 sSAgmolecules. Bottom: immunoblotting of the same geldecorated with Y4.

the 156G SAg while Y8 recognizes the reduced solubleform and not the unreduced one; in contrast, itrecognizes the unreduced membrane form of the 156GSAg and not the reduced one. Furthermore, Y8 alsorecognizes the reduced soluble form of CRGs.

Imrnunoreactivity with other SAgsVarious allelic G (168G, 60G, 90G and 513G) and D(156D, 168D and 60D) SAgs of P. primaurelia and theSIB SAg of P. tetraiirelia were analysed for theirimmunoreactivities with Y4 and Y8. By enzyme immu-noassay, Y4 reacted strongly with the soluble andmembrane forms of the 168G SAg (Table 1) as well aswith the other allelic G SAgs analysed (data notshown), but not with the D SAgs (Table 1). Incontrast, Y8 reacted weakly with the soluble form of allthe SAgs tested as well as with the membrane form ofthe 156D SAg, but displayed a stronger reaction withthe 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 correspond-ing G mSAgs, but did not react with D mSAgs (datanot shown). Y8 reacted with all the reduced sSAgs(Fig. 3B) and sCRGs analysed, whatever the SAgexpressed, the strain or the species used (Fig. 4). Incontrast, it did not react with all the mSAgs: Y8reacted strongly with the unreduced 168G mSAg, butriot with the 156D mSAg (data not shown). Further-more, Y8 did not react with the membrane form ofCRGs, either unreduced or reduced.

The reaction of Y8 towards reduced sSAgs andsCRGs was shown to be specific (no labelling wasobtained by using two unrelated IgM monoclonal

antibodies raised against myosin and vimentin, data notshown). Since Y8 is found to react in a similar way asantibodies specific to the cross-reacting determinant(CRD) (Capdeville et al. 1986), we wondered if theepitope recognized by Y8 in reduced sSAgs and sCRGscorresponded to the CRD. To check this possibility,we performed Western blot experiments with purifiedsoluble variant surface glycoproteins of Tiypanosomaequiperdum, which present the CRD (see Introduc-tion). Since no labelling of these proteins was observedwith Y8 (data not shown) we conclude that the Y8antibodies are not directed against the CRD structurebut are directed against another determinant, which isrevealed after cleavage of diacylglycerol from mSAgand after reduction. The fact that the immunoreac-tivity of Y8 was not lost after treatment with periodate(data not shown) suggests that this determinant isprobably not glycosidic.

Immunoreactivity with whole cellsTo ascertain the in situ localization of the Y8 epitopes,we performed immunological tests of immobilization invivo and immunolabelling of fixed cells.

Living cells of strains 33, 60, 90, 156, 168 and 513expressing G SAg, and strains 156 and 168 expressingD SAg, were incubated with Y4 or Y8 alone, or firstwith Y4 or Y8, then with anti-mouse immunoglobulinantibodies (Table 2). Y4 antibodies affected all the Gcells, whatever the G allele expressed, but did notimmobilize them: cells were simply sluggish. Immobil-ization was completed only after the addition of themouse Ig antibody. Y8 antibodies did not affect livingcells, whatever the SAg expressed, even after theaddition of mouse Ig antibody. It is worth noting thatY4 did not yield any precipitation line by Ouchterlonydouble immunodiffusion or by immunoelectrophoresiswith native 156G and 168G sSAgs (results not shown).

The reactivity of Y4 and Y8 was also examined byimmunoelectron microscopy with the IgG-gold tech-nique and compared with that of the polyclonal anti-serum, AS 2355, raised against the 156G sSAg. Thepolyclonal antiserum showed a dense and continuousdistribution of antigens both on pellicle and cilia of156G cells, with gold granules aligned at a distance ofabout 23—25 nm from the membrane bilayer (Fig. 5A).In contrast, a 156G cell labelled with an anti-156Dserum (AS 2543) did not show any significant labelling(Fig. 5B). Cells treated by Y8 did not display specificbinding to the G molecules. The presence of few ag-gregates laying apart from the antigen layer (Fig. 5C)was also observed with two unrelated monoclonal IgM(data not shown). A continuous labelling of the antigencoat, similar to that observed with AS 2355, wasobtained 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 asa control (A) and with Y8 (B) from a SDS-polyacrylamide gel of reduced (lanes 1-4) and unreduced (lanes 5-8) ethanoliccellular 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 thereduced sSAgs and sCRGs were labelled. We note in 156D reduced extracts (lane 4) and immuno-decoration of two highmolecular weight bands. This finding, previously discussed (Capdeville et al. 1985), probably corresponds to the co-expression of two SAgs (the D SAg and another one) in strain 156 grown at 34°C. With regard to the high molecularweight bands other than the sSAg band that are labelled in reduced extracts, they probably correspond to proteolyticfragments 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.

Fig. 4. Reactivity of Y8 towards various sCRGs.Immunoblot obtained with Y8, corresponding to the lowerpart of an SDS-polyacrylamide gel of reduced ethanoliccellular extracts issued from strain 51 of P. tetraureliaexpressing 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. Theapparent molecular weights of the cross-reactingglycoproteins vary according to the strain used, aspreviously found (Capdeville et al. 1986).

antibodies were inserted between the monoclonal anti-body and the IgG-gold antibody (Fig. 5D). Evalu-ation of distances between the closest gold granules andthe external membrane lipid bilayer gave 23 + 3 nm forthe pellicular (interciliary) membrane, and 2 5 ± 4 n mfor the ciliary membrane. Direct measurements (with-out immunolabelling) of the thickness of the externalprotein layer (156G SAg molecules), which coats the

Discussion

For the epitope defined by Y4 and referred to asepitope Y4, two main features emerge. (1) It isspecifically found in all the native, or unreduced, GSAgs of/3, primaurelia and absent in the reduced form.(2) It is accessible in vivo in all the cells expressing a GSAg. From these characteristics, epitope Y4 is likely tobe a conformational S-S-dependent determinant. Thisis not surprising since SAg molecules contain a greatnumber of S-S bridges (see Introduction). Epitope Y4does not appear to correspond to a repeated determi-nant (although the 156G gene contains several repeatsand segments of homology (Prat et al. 1986)) for tworeasons: (1) antibodies Y4, even when highly concen-trated, cannot immunoprecipitate the solubilized Gmolecules (data not shown); (2) these antibodiescannot immobilize G cells either. Cross-linking of SAgmolecules has been shown to be necessary and suf-ficient to lead to the immobilization of cells, andmonovalent antibody fragments were unable to triggerthis reaction (Barnett & Steers, 1984; Eisenbach et al.

558 V. Capdeville et al.

mp&wt

Fig. 5. Ultrastructural localization of the 156G SAg. A. Surface immunolabelling of a cell expressing the 156G SAg with apolyclonal anti-156G serum (AS 2355). X75 000. B. Surface immunolabelling of a cell expressing the 156G SAg with apolyclonal anti-156D serum (AS 2543). X75 000. C. Surface immunolabelling of a cell expressing the 156G SAg with theY8 monoclonal antibodies. X75 000. D. Surface immunolabelling of a cell expressing the 156G SAg with the Y4monoclonal antibodies. X75 000.

Surface antigen epitopes of Paramecium 559

Table 2. Immunoreactivity of antibodies Y4 and YS in vivo

33G 60G 90G 156G 168G 513G 156D 168D

Y4 s+ s+ + + s+ s+ s+ + + s+ - -Y8 -Mouse Ig Ab - - - -Y4 + mouse Ig Ab i i — —Y8 + mouse Ig Ab — — — —Specific AS i i i i i i 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 dialysedagainst 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 waythat homologous cells were immobilized within 20min. The in vivo immobilization test was performed as described (Capdeville, 1971).s1 , sluggish cells; s+ + + , very sluggish cells; i, immobilized cells; —, unaffected cells.

1983). Finally, the Y4 epitope is a specific marker of allthe allelic G SAgs and is distinct from a highlypolymorphic region, which differs from one allelicantigen to another (Capdeville, 1979). It is also aspecific tool with which to analyse the ultrastrueturallocalization of the G SAgs. The comparison of thelabelling obtained by Y4 and by a polyclonal antiserumwith the IgG-gold technique shows that the closestgranules were at the same distance from the membranebilayer. This similarity of labelling demonstrates thatonly the most external domain of the 156G molecules isaccessible in situ, and leads us to propose that the majorpart of the SAg molecules probably involved in inter-molecular interactions constitutes a compact surfacecoat similar to that found in the African trypanosome(Strickler & Patton, 1982). Furthermore, it is worthpointing out that the thickness of the 156G layer(17-22 nm) is intermediate between those found forother SAgs from micrographs of sections (20-30 nm)(Ramanathan et al. 1981), and from negative stainingof solubilized molecules (10-20 nm) (Mott, 1965;Reisner et al. 19696). This value supports the model ofan oblate ellipsoid proposed by Reisner et al. (19696),and fits well with a model predictable from the 1S6Ggene sequence data (Prat et al. 1986), where the (3-

156G mSAg

pleated sheets should be aligned along an axis perpen-dicular to the plasma membrane.

The behaviour of Y8 is rather puzzling, since itreacts differently with the membrane and the solubleforms of SAgs and CRGs. A single epitope cannotexplain all the properties of Y8, even if we put forwardthe hypothesis that, in the process of converting mSAgto sSAg by cleavage of the lipid moiety, the structurerecognized by Y8 in mSAg is modified in such a waythat its recognition requires the rupture of S-S bondsin sSAg. Such an hypothesis implies the recognition ofthe 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 moleculesdisplay intramolecular cross-reactivity and that Y8recognizes two different related epitopes. The exist-ence of intramolecular cross-reactivity is not surprisingfor a protein containing several repeats (Prat et al.1986); and intramolecular cross-reactivity has beendescribed in the case of human serum albumin, forwhich two monoclonal antibodies have both beenfound to react with two different related epitopes(Doyen et al. 1985). We will call the epitopes assumedto be recognized by the Y8 antibody Y8a and Y8b: Y8acorresponds to the epitope revealed at the level of the

156G sSAg

= hidden)

= hidden)

Fig. 6. A hypothetical modelof the location of epitopes Y8aand Y8b. The structure of themembrane-anchor and thelocation of the CRD are basedon information derived fromthe VSG of T. bnicei(Ferguson et al. 1985), sinceParanieciunt SAgs havesimilar features (Capdeville etal. 1986, 1987). The locationsof epitopes Y8a and Y8b arededuced from the properties ofthe monoclonal antibody Y8by assuming that it recognizestwo related different epitopes(see Discussion).

560 V. Capdeville et al.

unreduced membrane form of the 156G and 168GSAgs, and Y8b to the epitope revealed at the level ofthe reduced soluble form of all SAgs and CRGs. Theepitope Y8a, which is a surface epitope requiring theintegrity of S-S bonds and the presence of the lipidicmembrane anchor to be detected, would result frominteractions between a peptidie loop containing at leastone S-S bridge and the membrane anchor itself (seeFig. 6). Therefore, the epitope Y8a, which constitutesan antigenic marker of the membrane form of the 156Gand 168G SAgs, should be very close to or partlyburied in the plasma membrane. The epitope Y8b,which is a masked epitope requiring the rupture of S-Sbonds and the cleavage of the lipidic membrane-anchorto be detected (see Fig. 6), might correspond to apeptidie determinant, since it is not destroyed byperiodate treatment. The same properties, i.e. immu-nologieal detection requiring the rupture of S-S bondsand the cleavage of the lipid membrane anchor, havebeen previously found in the case of another determi-nant in P. primaurelia, the CRD (Capdeville et al.1986, 1987). Such findings suggest that these twodifferent determinants, Y8b and CRD, would belocated in the same region, in proximity to the lipidicmembrane-anchor, which would mask them (seeFig. 6). Finally, we conclude that Y8a and Y8b aretwo related epitopes whose immunologieal detection ismutually exclusive, depending strictly on the presenceor absence of the lipidic membrane-anchor, i.e. on theaction of a phosphatidylinositol-specific phospholipaseC.

We thank Drs T. Batlz, M. Bornens, C. Klotz and C.Milstein for their gifts, and Dr T. Rosenberry for a criticalreading of the manuscript and for many helpful comments.We are also grateful to Dr Benedetti, who advised us in theimmunocytochemical field. This work was supported in partby the Ministere de l'lndustrie et de la Recherche (grant 82L1089) and the Institut National de la Sante et de la RechercheMedicate (grant 12600S). CD. was supported by a fellowshipfrom the Ministere de l'lndustrie et de la Recherche.

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(Received 15 July 1987 - Accepted 10 September 1987)