Molecular and Biochemical Parasitology, 46 (1991) 137-148
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© 1991 Elsevier Science Publishers B.V. /0166-6851/91/$03.50 ADONIS 016668519100047M MOLBIO 01518
The ring-infected erythrocyte surface antigen of Plasmodiumfalciparum associates with spectrin in the erythrocyte membrane Michael Foley 1, Leann T i l l e r , William H. S a w y e r 3 and Robin F. Anders 1 1The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria, Australia; 2Biochemistry Department, University of La Trobe, Bundoora, Victoria, Australia; and 3Biochemistry Department, University of Melbourne, Parkville, Victoria, Australia (Received 3 July 1990; accepted 26 November 1990)
The malaria parasite Plasmodiumfalciparum synthesises a protein, RESA, which associates with the membrane of newly invaded erythrocytes. Using spent supernatants from P.falciparum growing in culture as a source of soluble RESA we have developed an assay to examine the characteristics of RESA binding to the erythrocyte membrane in vitro. RESA associated with the Triton X-100 insoluble proteins on the inner face of the host erythrocyte membrane but did not bind to the outer surface of intact erythrocytes. Other proteins present in culture supernatants did not bind to the erythrocyte membrane. RESA was co-sedimented with the ternary complex formed between actin, spectrin and band 4.1 and co-precipitated with spectrin precipitated with anti-spectrin antibodies. The extent of association between RESA and the inner face of the erythrocyte membrane was reduced by the inclusion of excess purified spectrin in the assay. Thus, RESA appears to be associated with spectrin in the erythrocyte membrane skeleton. Key words: Plasmodiumfalciparum; Spectrin; Bindingassay; Ring-infected erythrocyte surface antigen
Introduction
Various structural and biochemical changes in the erythrocyte membrane occur during asexual development of the malarial parasite within erythrocytes [1]. As the parasite matures the shape and deformability of the host membrane are altered, and even in the less mature ring-stage parasites there is a measurable decrease in the deformability of the erythrocyte membrane [2]. The network of cytoskeletal proteins underlying the erythrocyte bilayer is responsible for maintaining the shape and deformability of the erythrocyte membrane [3]. Thus, parasite proteins that interact with this membrane skeleton may be responsible for changes in these Correspondence (present) address: Michael Foley, Institute of Cell, Animal and Population Biology, University of Edinburgh, King's Buildings, West Mains Road, Edinburgh EH9 3JN, U.K. Abbreviations: RESA, ring-infected erythrocyte surface antigen; TX- 100, Triton X- 100; IOVs, inside-outvesicles.
membrane characteristics. Several malarial proteins are known to be localised on the cytoplasmic face of the erythrocyte membrane [4,5] and such interactions may be important in maintaining the peculiar knob structures seen in the membrane of erythrocytes infected with mature stages of P.falciparum. The ring-infected erythrocyte surface antigen (RESA) is a P.falciparum protein that becomes associated with the membrane of newly invaded erythrocytes. After synthesis, RESA is stored in organelles within the mature parasite and is released into the red cell at the time of merozoite invasion. In mature parasites RESA is largely soluble in the anionic detergent Triton X- 100 (TX- 100) but when associated with the membrane of newly invaded erythrocytes it is TX-100-insoluble, presumably due to an interaction with a component of the erythrocyte membrane skeleton [6]. Antibodies to RESA inhibited merozoite invasion in vitro [7] and immunisation of Aotus monkeys with recombinant RESA proteins provided
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partial protection against subsequent challenge with P.falciparum [8] but the function of RESA has not been established. A role for RESA in the process of merozoite invasion was suggested by the timing of the transfer of RESA to the erythrocyte membrane and the observation that RESA is phosphorylated when associated with the membrane [91. However, the finding that merozoites of a parasite line not expressing RESA efficiently invade erythrocytes in vitro argues against a role for RESA at the time of merozoite invasion [10]. As RESA persists in association with the erythrocyte membrane for at least 24 h after invasion its role may be to modify the properties of the membrane during that period when the infected erythrocyte is in the circulation, prior to cytoadherence. A first step in understanding the function of RESA would be to identify the component(s) of the erythrocyte membrane with which RESA associates. To this end we have developed an assay for looking at the interaction of RESA with the host membrane. Several lines of evidence suggest that RESA associates with spectrin in the erythrocyte membrane skeleton. Materials and Methods
P. falciparum isolate FCQ27/PNG (FC27) was obtained through collaboration with the Papua-New Guinea (PNG) Institute of Medical Research. The clonal line D10 was derived from FC27 by limiting dilution culture [11]. Parasites were maintained in asynchronous culture as described [12]. Rabbit antisera against RESA were prepared by immunising rabbits with ~-galactosidase fusion proteins produced in Escherichia coli [ 13], or synthetic peptides corresponding to repeat sequences, coupled to keyhole limpet haemocyanin [ 14]. The monoclonal antibody 18/2 which was used in immunoblotting experiments reacted with both the 5' and 3' repeat regions of RESA [15]. P. falciparum growing in culture was synchronised by treatment with sorbitol [ 16]. Antisera to the S-antigen was obtained as described previously [17].
pared according to Steck and Kant [18]. Centrifugation through a Dextran cushion (Dextran 80; 1.03 g ml m 0.5 mM sodmm phosphate, pH 8.0), separated IOVs from unsealed ghosts and right-side-out vesicles [18]. Contamination of IOVs with rightside-out vesicles was less than 10% and usually, the Dextran step was omitted. 1 -
Preparation of culture supernatants. Spent supernatants from cultures of P. falciparum were used as a source of RESA. A 10-ml dish of infected cells (haematocrit, 2%) was centrifuged at 1 500 rev./min, the pellet containing parasites and red cells discarded and the supernatants centrifuged at 100 000 × g for 20 min to remove cellular debris. The remaining supernatants were either used immediately or frozen at-70°C until required.
Metabolic labelling with [35S]methionine. Parasites from schizont stages to late ring stages were cultured overnight in methionine free medium (RPMI-1640, Selective Kit, Gibco, New York) modified for P. falciparum growth and containing 100 btCi ml -j of [35S]methionine (>1 000 Ci mmol-1; Amersham). The supernatants containing released radiolabelled parasite molecules were collected and processed as described in the previous section.
Parasites, peptides and sera.
Preparation of erythrocyte ghosts and inside-out vesicles. Inside-out vesicles (IOVs) were pre-
Assessment of RESA binding to inside-out vesicles. For most experiments, 100-150 ~tg of IOV protein were added to phosphate-buffered saline (PBS) containing 10-20 ~tl of culture supernatant and incubated for 30 min at 4°C. Binding reactions were performed in 500 ~tl. Incubations were terminated by centrifugation in a microfuge for 20 min and the resultant supernatants were discarded. The vesicle pellets were resuspended in 500 ~tl of PBS and centrifuged again. After a further wash, samples were boiled in 2× concentrated SDS sample buffer and half the pellet volume was then analysed by SDSPAGE. Alternatively, samples were diluted in TNET (0.5 % TX- 100 in 50 mM Tris/150 mM NaC1/5 mM EDTA, pH 8.0), and centrifuged to obtain the TX-100-insoluble pellet which, after 2 further washes in T-NET, was dissolved in sample buffer and analysed by SDS-PAGE [ 19]. Where different conditions of binding were used, these are indicated in Results.
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man 3MM paper and exposed to Agfa X-ray-film. The gels were incubated in Amplify (Amersham) for 10 min immediately before drying. Immunoblotting and SDS-PAGE were performed as described previously [ 19,20].
Detergent extraction, immunoprecipitation and SDS-polyacrylamide gel electrophoresis. After incubation with supernatants, IOVs were dissolved in T-NET, incubated on ice for 30 min, and then separated into soluble and insoluble fractions by centrifugation at 25 000 x g for 30 min. The TX100-insoluble fraction was then solubilised in 2% SDS in PBS for 30 min at room temperature, followed by addition of 20% TX-100 and T-NET to give final concentrations of 0.2% SDS and 0.7% TX-100. Immunoprecipitation was performed using 5 ~tl of rabbit antiserum and the precipitated antigens were analysed on SDS-PAGE under reducing conditions. For autoradiography the gels were fixed in 5% methanol, 10% acetic-acid, dried onto What-
Purification of skeletal components, sedimentation assay and competition experiments. Rabbit skeletal muscle actin was isolated according to the method of Pardee and Spudich [21] and was purified by two successive cycles of polymerisation and depolymerisation. The depolymerisation buffer was buffer G (10 mM Tris-HC1, pH 7.6/0.2 mM CaC12/ 0.5 mM DTT/0.02% NAN3). Spectrin dimers were prepared from human erythrocytes by the method of Cohen and Foley
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Fig. 1. Association ofRESA with the cytoplasmic face oftheerythrocytemembrane. Spent culture supematants from cultures ofP.
falciparum were incubated with IOVs from uninfected erythrocytes or with intact erythrocytes. Unbound material was washed away and the proteins in the pellets separated by SDS-PAGE, blotted onto nitrocellulose and probed with RESA specific antisera. A sample of culture supernatant was run on lane 1 of each panel for comparison. (A) RESA binding to IOVs (lane 2) and to intact erythrocytes (lane 3). A sample of IOVs and erythrocytes after incubation were treated with TX-100 and the insoluble material blotted and probed with antisera to RESA. Lane 4 shows RESA attached to the insoluble matrix of IOVs and lane 5 RESA attached to the insoluble matrix from erythrocytes. (B) Supernatants alone (lane 1) and IOVs after incubation (lane 2) were separated by SDS-PAGE, blotted onto nitrocellulose and probed with antisera to the S-antigen. (C) Identical to panel B but reprobed with a pool of sera from infected individuals from Papua-New Guinea. Several proteins which do not associate with IOVs are indicated with open arrowheads, and an M r 73 000 protein that interacts with IOVs is indicated by an asterisk.
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[22]. Spectrin dimers were passaged twice through a Biogel A15M column to ensure complete removal of contaminating actin and protein 4.1. A crude preparation of band 4.1 was obtained by high salt extraction of erythrocyte membrane vesicles which had been depleted of band 6, spectrin and actin. This yielded a preparation in which band 4.1 represented about 50% of the Coomassie Blue staining material [23].
Binding of malarial antigens to purified skeletal proteins. Samples were prepared containing 0.12 -I
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mg ml G-actm, or actmplus 0.32 mg ml spectrm and 0.04 mg m1-1 band 4.1. [35S]methionine-labelled culture supernatant was added to 100 ~Ci ml -~. Polymerisation of the actin was initiated by addition of NaC1 to 100 mM and MgC12 to 2 mM. After 1 h at room temperature, the F-actin and associated proteins were pelleted by centrifugation at 290 000 × g for 40 min at 20°C. The pellets were resuspended in 1 ml of buffer G plus 2 m M MgC12 and 100 mM NaC1 and repelleted (290 000 × g, 40 min). The pellets were solubilised in 100 ~tl of Buffer G and stored frozen until analysis by SDS-PAGE. For competition studies erythrocyte ghosts (0.4 mg ml ) were incubated with [ S]methlonlne-labelled culture supernatants (100 ~tCi ml -~) in buffer C (10 mM sodium phosphate, pH 7.5/100 mM NaC1/0.01% NAN3/0.5 mM DTT in the presence of varying concentrations of purified spectrin (1.2 mg 1 • -1 ml ) or protein 4.1 extract (0.05 mg ml ). After a 1-h incubation at room temperature, the membranes were pelleted at 12 000 × g for 15 min. The pellets were resuspended in 1 ml of buffer C and recentrifuged. These pellets were resuspended in 100 ~tl of buffer C and stored frozen until analysis by SDS-PAGE. -1
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used to probe immunoblots of culture supernatants the full length RESA polypeptide of Mr 155 000 was detected together with 2 smaller polypeptides of Mr 130000 and 120000 (Fig. 1A, lane 1). We have assumed that these 2 smaller polypeptides are derived from RESA as both react with a variety of antibodies directed against the 3' or 5' repeats of RESA (data not shown). The immunoblotting resuits indicate that the full length RESA polypeptide bound to IOVs to a greater extent than either of the 2 smaller fragments (Fig. 1, lane 1). RESA bound to IOVs was apparently associated with the membrane skeleton because it was detected in the TX100-insoluble fraction derived from IOVs (Fig. 1A, lane 4). In addition, when a TX- 100-insoluble fraction generated from freshly made IOVs was incubated with culture supernatants for 60 min, RESA bound to the membrane skeleton. Another antigen that is readily detected in spent culture supernatants
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Results
RESA binds to the erythrocyte membrane skeleton. RESA in spent culture supernatants bound to IOVs but did not bind to normal erythrocytes. The associated RESA was detected when IOVs that had been incubated for 60 min in culture supernatants were washed extensively, fractionated by SDS-PAGE and electrophoretically transferred to nitrocellulose filters which were subsequently probed with specific antisera. When anti-RESA antisera were
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Fig. 2. [3~S]Methionine-labelledRESA binds to IOVs. Polypeptides in supematantsfrom 2 P.falciparumstrains grown in culture in the presence of [35S]methioninewere separated by SDS-PAGE and visualised by autoradiography of the dried gels (lanes 1 and 2). Alternativelysupernatantswere incubated with IOVs from uninfected erythrocytes and bound polypeptides visualisedin the same way (lanes 3 and 4). The supematants were from cultures of FC27 (lanes 1 and 3) and FCR3 (lanes 2 and 4).
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Binding t o lOWS
Triton Pellets
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i!~il/ii!)~ii~ii~ii~i/i!!~ i~i ¸ CHRMS
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Fig. 3. RESAbindsto the membraneskeletonoferythrocytesfroma varietyofmammals.Culturesupernatantswereincubatedwith IOVsprepared fromrabbit (R),mouse(M) and sheep(S) as wellas fromhuman(H) and boundRESA,afterextensivewashing,analysedby immunoblotting(as describedin Fig. 1). Culturesupernatants(C) were alsorun alongsidethe IOVs as a comparison.The IOVs, afterwashingawayunboundmaterial,wereextractedwithTX-100 and the insolubleprecipitateexaminedin the sameway.It can be seenthatRESAcan associatewiththe TX-100 insolublefractionin all erythrocytespeciestested. is the S-antigen of P.falciparum. The Mr 220 000 S antigen of isolate FC27 was detected in the supernatants used in these experiments but, in contrast to the finding with RESA, the S-antigen did not bind to IOVs (Fig. 1B). Probing immunoblots of supernatants with a pool of sera from individuals exposed to malaria in Papua-New Guinea revealed several other antigens that did not bind to IOVs, although one antigen of Mr 73 000 did associate with IOVs in a similar manner to RESA (Fig. IC). Additional bands in Fig. 1C, lane 2 (Mr 50 000, 65 000 and approx. 190 000) did not have counterparts in the supernatant and were due to the reaction of the human serum with components of the erythrocyte membrane. These immunoblotting results suggest a specific association between RESA and the erythrocyte membrane skeleton. Further evidence of specificity in this association was obtained from experiments in which IOVs were incubated with heated culture supernatants. Heating culture supernatants at 65°C for 15 min did not affect the anti-
genicity of RESA in the supernatant but dramatically reduced the binding of RESA to IOVs (data not shown) indicating that a heat labile conformation in RESA was important for this interaction. Since RESA in mature parasites is mainly TX100-soluble [6], a sonicate of infected erythrocytes, cleared by high speed centrifugation, was used as an alternative source of RESA for binding experiments in vitro. RESA in these sonicates bound to IOVs in a similar manner to RESA from culture supernatants (data not shown). The association of culture supernatant proteins with IOVs was also investigated using supernatants containing parasite proteins which had been biosynthetically labelled with [35S]methionine. A closely migrating radiolabelled doublet of Mr approx. 155 000 bound to IOVs (Fig. 2, lane 3) but not to normal erythrocytes. This doublet, which has been observed in previous studies [14], was shown to be RESA by immunoprecipitation with defined antisera (data not shown). The nature of the doublet
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is not understood but may be the result of some post-translational modification such as phosphorylation. The FCR3 strain of P. falciparum has been shown not to synthesise the RESA polypeptide [10]. When we used [35S]methionine-labelled supernatants from FCR3 in similar binding experiments, no labelled doublet was found to bind to IOVs (Fig. 2A, lane 4) nor was a doublet immunoprecipitated by antisera to RESA (data not shown). Many other radiolabelled proteins were present in FCR3 culture supernatants (Fig. 2A, lane 2). In these experiments we did not observe binding of the M r 73 000 antigen presumably because it is not labelled with [35S]methionine.
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RESA associates with spectrin.
RESA binding studies using culture supernatants were repeated
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Fig. 4. Interaction of RESA with inside-out-vesicles is reduced by protease treatment of the vesicles. IOVs were incubated for 60 mln at 4 C m the presence of 0 lag ml , lane 1; 50 lag ml , lane 2; and 100 lag ml , lane 3; of ~-chymotrypsln. The reaction was stopped by the addition of a 10-fold excess of chymostatin. After incubation for 60 min at 4°C the vesicles were washed several times in 0.5 mM sodium phosphate plus 10 lag ml -~ chymostatin. Binding of RESA to each preparation was then assessed as described in Materials and Methods. •
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Fig. 5. RESA binds predominantly to the ternary complex in vitro. Actin alone (lane 2) or with actin plus spectrin plus band 4.1 (lane 3) were examined for the ability to bind RESA or other malarial antigens present in culture supernatants (lane 1) as described in Materials and Methods. Bound proteins were visualised by probing nitrocellulose filters with antisera to RESA (panel A) or a pool of sera from malaria infected individuals from Papua-New Guinea (panel B). Quantitative densitometry on an identical gel stained with Coomassie Blue indicated that approx• 11 lag of protein was applied to lane 2 (actin alone) and approx. 19 lag of protein was applied to lane 3 (actin plus spectrin plus band 4.1 ).
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with IOVs from a variety of mammalian species. In each case RESA bound to IOVs and became TX100-insoluble in an identical manner as for human 1OVs (Fig. 3). Thus, the host molecule with which RESA interacts is a highly conserved component of the membrane. The protein nature of this molecule was demonstrated by binding studies using IOVs pretreated with proteases. Fig. 4 shows the reduction in association of RESA to IOVs digested with increasing concentrations of chymotrypsin. Coomassie Blue staining of IOVs after proteolysis revealed extensive degradation of most major protein bands such as spectrin and band 3; however, several polypeptide fragments were still observed, indicating that complete breakdown of cell membrane proteins had not occurred (data not shown). As these results were consistent with RESA associating with a component of the erythrocyte membrane skeleton we purified the 3 major skeletal components: spectrin, actin, and band 4.1, and per-
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Fig. 6. Excess purified spectrinreduces the binding of RESA to inside-outvesicles. IOVs from uninfectederyt,hrocyteswere incubated with culture supernatantswith no additions(lane 1, both panels) or in the presence of 50, 100, 200 I.tgml-' spectrin (panel A, lanes 2,3,4). Addition of excess purified actin (10, 50, 100 ~tgm1-1,panel B, lanes 2,3,4) to the assay did not reduce the binding of RESA to IOVs. Lane S correspondsto supernatantsrun on the same gel for comparison. formed co-sedimentation experiments with RESA as described in Materials and Methods. When actin alone was polymerised in the presence of culture supematant, and sedimented by centrifugation, a small amount of co-sedimented RESA was detected by immunoblotting (Fig. 5A, lane 2). Very much more RESA was co-sedimented with the temary complex of actin, spectrin and band 4.1 (Fig. 5A, lane 3). Probing a replicate immunoblot with a PNG serum pool (Fig. 5B) revealed that this co-sedimentation is specific for RESA and the Mr 73 000 polypeptide consistent with previous resuits in this study. Further evidence of the specificity of this reaction was obtained when this cosedimentation assay was performed using [35S]methionine-labelled culture supernatants. Again, only RESA and a Mr 73 000 polypeptide were sedimented (data not shown). These results indicate that either spectrin or band 4.1 is necessary for efficient co-sedimentation of RESA. Evidence that
spectrin is a major target for RESA attachment was obtained by carrying out a binding assay as already described but including in the assay increasing concentrations of purified spectrin. High concentrations of purified spectrin in the reaction mix substantially inhibited RESA from binding to the IOVs (Fig. 6A). This was not the case when actin was included in the reaction mix in place of spectrin (Fig. 6B). These data are consistent with the hypothesis that RESA binds to spectrin in the erythrocyte membrane. Evidence that RESA interacts with spectrin in the parasitised erythrocyte was obtained by immunoprecipitation with antisera to spectrin (Fig. 7). Parasites labelled with [35S]methionine for 12 h were solubilised in T-NET and immunoprecipitations were carried out from the TX- 100-soluble and insoluble fractions using rabbit antisera to spectrin and control normal rabbit serum. A major radiolabelled polypeptide of approx. Mr 155 000 was co-
144 trin was precipitated from the insoluble fraction. Western blots also revealed that the antiserum recognises c~ spectrin (data not shown). Thus it appears from these results that spectrin is a major target for RESA binding. 200
Discussion
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S N Fig. 7. Antibodiesto spectrin co-precipitate RESA. A culture of P. falciparum infected erythrocytes grown for 12 h in the presence of [35S]methioninewas washed in PBS and fractionated by addition of TX-100 into soluble and insoluble fractions. The soluble fraction was immunoprecipitatedwith antisera to spectrin(S) (lane 1) or normalrabbit serum (N) (lane 3). The insoluble fraction was treated with SDS and an excess of TX-100 and T NET was added (as described in Materials and Methods) followed by immunoprecipitationwith antisera to spectrin (lane 2) or normal rabbit antisera (lane 4). Arrowhead corresponds to Mr 155 000 co-precipitatedby spectrinspecific antisera.
precipitated with anti-spectrin antibodies from the TX- 100-soluble fraction, but not from the TX- 100insoluble fraction which was treated with 1% SDS. Coomassie Blue staining of the gel revealed that both t~ and ~ spectrin were immunoprecipitated from the TX-100-soluble fraction, but only t~ spec-
We report here that RESA can associate with the cytoplasmic face of human erythrocytes but not with the external aspect of intact erythrocytes. This is consistent with previous data which showed that antibodies to RESA failed to bind unfixed ring-infected erythrocytes but did bind to lightly glutaraldehyde-fixed cells [24]. Probing immunoblots of spent culture supernatants with antisera to RESA revealed 2 faster migrating species of Mr 130 000 and Mr 120 000 in addition to the intact RESA polypeptide of Mr 155 000. This pattern of antigenically related of polypeptides has been noted previously and was assumed to be due to proteolysis of the M~ 155 000 polypeptide [25]. Although the Mr 120 000 polypeptide appears to be relatively abundant in supernatants, it binds relatively poorly to erythrocyte membranes and to purified spectrin (Fig. 5A, lane 3). Thus, the Mr 120 000 polypeptide may lack a sequence involved in the binding of RESA to the erythrocyte membrane. The binding of RESA to the erythrocyte membrane appeared specific in that the S antigen and several other antigens present in culture supernatants were not bound under the assay conditions used. However, an Mr 73 000 antigen, present in culture supernatants and recognised by PNG immune sera did bind to the erythrocyte membrane in a similar manner to RESA. This antigen does not appear to be a breakdown product of RESA (unpublished results) arid experiments are underway to characterise it further. The results of the binding experiments analysed by immunoblotting were confirmed by binding experiments using supernatants from parasites labelled with [35S]methionine. Studies using these supernatants revealed many parasite proteins which differed from RESA and the Mr 73 000 antigen in that they did not bind to erythrocyte vesicles under physiological conditions. By continually replenishing the assay with fresh IOVs we were able to remove almost all of the RESA present in a given volume of culture supernatants. Indicating that over 90% of
145 RESA molecules are competent for binding (data not shown). The association of RESA with the erythrocyte membrane is due to an interaction between RESA and a component of the erythrocyte membrane skeleton rather than an association with the lipid bilayer. There are regions of the RESA sequence that are relatively hydrophobic but none of these are of sufficient length and average hydrophobicity to be considered likely membrane spanning domains. Furthermore, there is no evidence that RESA is post-translationally modified with lipid to provide a membrane anchor. The observations reported here that protease pretreatment of IOVs inhibits the binding of RESA, and that RESA binds to the insoluble residue remaining after TX- 100 extraction of RBCs are consistent with RESA binding to a component of the membrane skeleton. This component is conserved among mammalian species as RESA bound to IOVs prepared from erythrocytes of 6 different species. Klotz et al. [26] have reported that when P. falciparum merozoites invade mouse erythrocytes RESA was subsequently found to associate with the erythrocyte membrane, a finding which is consistent with our observations. The erythrocyte membrane skeleton is a meshwork of peripheral proteins consisting primarily of spectrin, actin and band 4.1. The finding that RESA binds to the ternary complex of spectrin, actin and band 4.1 points to one of these proteins being the major target for RESA binding. Two different experimental approaches have provided evidence that spectrin is the component of the skeleton with which RESA associates. In one experimental approach purified spectrin was used to inhibit the binding of RESA to IOVs. The binding of RESA was markedly reduced but not completely inhibited in the presence of excess free spectrin and this suggests the possible involvement of other components in RESA binding. In the second experimental approach RESA was co-precipitated when spectrin was precipitated with anti-spectrin antibodies. Much spectrin is usually lost from the erythrocyte membrane during the preparation of IOVs but analysis by SDS-PAGE and Coomassie Blue staining revealed the presence of significant amounts of spectrin in the IOVs used in this study. Loss of spectrin was minimised by carrying out
vesiculation at 4°C [18]. Our recent observations using radiolabelled purified recombinant RESA, that binding to IOVs was saturable and could be greatly reduced by excess unlabelled RESA supports the conclusion that RESA binds to a specific receptor site (unpublished results). The function of RESA remains unknown. An involvement in the invasion process, as initially proposed [27], appears unlikely since FCR3, a P.falciparum strain grown in vitro over many years, has a subtelomeric deletion onchromosome 1 which prevents expression of the RESA gene [ 10]. As RESA is invariably expressed in field isolates [28], and as it persists at the erythrocyte membrane for some time after invasion the function of RESA may be to modify the properties of the host cell membrane during the period the infected cell persists in the peripheral circulation. Recently, it has been suggested that antigens previously located to micronemes may in fact be present in dense granules, which appear to release their contents into the parasitophorous vacuole after invasion [29]. If RESA is located in dense granules and not in micronemes, as previously reported, this would be consistent with a function after invasion during the early stages of intraerythrocytic development. The evidence presented here for RESA being a spectrin binding protein suggests a mechanism whereby RESA could modify the host cell membrane. The membrane skeleton is an assembly of proteins attached to the erythrocyte lipid bilayer and controls the shape and mechanical properties of the erythrocytes. Spectrin, quantitatively the major component of the skeleton is an elongated flexible molecule consisting of two subunits. Spectrin self associates and interacts with other proteins, principally actin, to form a flexible meshwork. Spectrin also interacts with ankyrin which in turn interacts with the integral membrane protein band 3, thereby linking the membrane skeleton to the lipid bilayer. If RESA binding to spectrin were to disturb any of these interconnections it would have consequences for the arrangement of the membrane skeleton. Our results provide a possible mechanism for the results found by Nash et al. [2] who observed that ring-infected erythrocytes had increased m e m brane rigidity. The authors speculated that this reflect changes in the membrane skeleton most likely
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involving spectrin. However, if RESA binding to spectrin does result in the changes in membrane viscoelasticity observed in the ring-infected cells, another mechanism must be responsible for the more dramatic changes in erythrocytes infected with more mature stages when RESA is apparently lost from the membrane. Spectrin has been implicated in the maintenance of the normal asymmetric distribution of lipids across the erythrocyte plasma membrane [30,31 ], and it is possible that an interaction between RESA and spectrin could alter the normal lipid composition of the erythrocyte membrane in infected cells. Although several studies have been shown that changes of this nature can be detected in trophozoite and schizont infected cells but not in ring-infected erythrocytes [32,33], the persistence of RESA at the membrane for 24 h could conceivably affect the lipid compositional asymmetry. Identification of spectrin as a major target for RESA on the erythrocyte membrane does not rule out the participation of other erythrocyte molecules in the binding of RESA to the host membrane. Further studies will examine the nature of RESA binding and its effects on the structure of the erythrocyte membrane.
Acknowledgements We thank Sakunthala Meera for the preparation of the rabbit skeletal muscle actin and Etty Bonnici for typing this manuscript. This work was supported by the Australian National Health and Medical Research Council, the John D. and Catherine T. MacArthur Foundation and the Australian Malaria Vaccine Joint Venture. Support was also provided under the Generic Technology Component of the Industry Research and Development Act 1986, and the Wellcome Trust Fellowship Fund. References 1 Sherman, I.W. (1985) Membrane structure and function of malaria parasites and the infected erythrocyte. Parasitology 91,609-645. 2 Nash, G.B., O'Brien, E., Gordon-Smith, E.C. and Dormandy, J.A. (1989) Abnormalitiesin the mechanical properties of red blood cells caused by Plasmodiumfalciparum. Blood 24, 855-861. 3 Bennett, V. (1985) The membrane skeleton of human
erythrocytes and its implications for more complex cells. Annu. Rev. Biochem. 54,273-304. 4 Leech, J.H., Bamwell, J.W., Aikawa, M., Miller, L.H. and Howard, R.J. (1984) Plasmodiumfalciparum malaria: Association of knobs on the surface of infected erythrocytes with a histidine-rich protein and the erythrocyte skeleton. J. Cell. Biol. 98, 1256-1264. 5 Coppel, R.L., Culvenor, J.G., Bianco, A.E., Crewther, P.E., Stahl, H.D., Brown, G.V., Anders, R.F. and Kemp, D.J. (1986) Variable antigen associated with the surface of erythrocytes infected with mature stages of Plasmodium falciparum. Mol. Biochem. Parasitol. 20, 265-277. 6 Brown, G.V., Culvenor, J.G., Crewther, P.E., Bianco, A.E., Coppel, R.L., Saint, R.B., Stahl, H.D., Kemp, D.J. and Anders, R.F. (1985) Localization of the ring-infected erythrocyte surface antigen (RESA) of Plasmodiumfalciparum in merozoites and ring-infected erythrocytes. J. Exp. Med. 162,774-779. 7 Whlin, B., Wahlgren, M., Perlmann, H., Berzins, K., Bjtrkman, A., Patarroyo, M.E. and Perlmann, P. (1984) Human antibodies to a Mr 155 000 Plasmodiumfalciparum antigen efficiently inhibit merozoite invasion. Proc. Natl. Acad. Sci. USA 81,7912-7916. 8 Collins, W.E., Anders, R.F., Pappaioanou, M., Campbell, G.H., Brown, G.V., Kemp, D.J., Coppel, R.L., Skinner, J.C., Andrysiak, P.M., Favaloro, J.M., Corcoran, L.M., Broderson, J.R., Mitchell, G.F. and Campbell, C.C. (1986) Protection of Aotus monkeys by immunization with recombinant proteins of the ring-infected erythrocyte surface antigen (RESA) of Plasmodiumfalciparum. Nature 323,259-262. Foley, M., Murray, L.J. and Anders, R.F. (1990) The ringinfected erythrocyte surface antigen protein of Plasmodium falciparum is phosphorylated upon association with the host cell membrane. Mol. Biochem. Parasitol. 38, 6976. 10 Cappai, R., van Schravendijk, M.R., Anders, R.F., Peterson, M.G., Thomas, L.M., Cowman, A.F. and Kemp, D.J. (1989) Expression of the RESA gene in Plasmodium falciparum isolate FCR3 is prevented by a subtelomeric deletion. Mol. Cell. Biol. 9, 3584-3587. 11 Culvenor, J.G., Langford, C.J., Crewther, P.E., Saint, R.B., Coppel, R.L., Kemp, D.J., Anders, R.F. and Brown, G.V. (1987) Plasmodiumfalciparum: identification and localization of a knob protein antigen expressed by a cDNA clone. Exp. Parasitol. 63, 58-67. 12 Trager, W. and Jensen, J.B. (1976) Human malaria parasites in continuous culture. Science 193,673-675. 13 Cowman, A.F., Coppel, R.L., Saint, R.B., Favaloro, J., Crewther, P.E., Stahl, H.D., Bianco, A.E., Brown, G.V., Anders, R.F. and Kemp, D.J. (1984) The Ring-infected Erythrocyte Surface Antigen (RESA) polypeptide of Plasmodium falciparum contains two separate blocks of tandem repeats encoding antigenic epitopes that are naturally immunogenic in man. Mol. Biol. Med. 2, 207-221. 14 Anders, R.F., Shi, P.T., Scanlon, D.B., Leach, S.J., Coppel, R.L., Brown, G.V., Stahl, H.D. and Kemp, D.J. (1986) Ciba Foundation Symposium on Synthetic Peptides as Antigens, Symposium, No. 119. In Ciba Found. Symp.
147 119, 164-175. 15 Anders, R.F., Barzaga, N., Shi, P-T., Scanlon, D.B., Brown, L.E., Thomas, L.M., Brown; G.V., Stahl, H.D., Coppel, R.L. and Kemp, D.J. (1986) Molecular strategies of parasitic invasion, In: Molecular Strategies of Parasitic Invasion, UCLA Symposium on Molecular and Cellular Biology, New Series. Agabian, N. Goodman, H. and Noguiera, N., eds.), Alan R. Liss, New York. 16 Lambros, C. and Vanderberg, J.P. (1979) Synchronization ofPlasmodiumfalciparum erythrocytic stages in culture. J. Parasitol. 65, 418-420. 17 Anders, R.F., Brown G.V. and Edwards, A. (1983) Characterization of an S antigen synthesized by several isolates of Plasmodium falciparum. Proc. Natl. Acad. Sci. USA 80, 6652-6656. 18 Steck, T.L. and Kant, J.A. (1974) Preparation of impermeable ghosts and inside-out vesicles from human erythrocyte membranes. Methods Enzymol. 31, 172-180. 19 Laemmli, U.K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680--685. 20 Crewther, P.E., Bianco, A.E., Brown, G.V., Coppel, R.L., Stahl, H.D., Kemp, D.J. and Anders R.F. (1986) Affinity purification of human antibodies directed against cloned antigens ofPlasmodiumfalciparum. J. Immunol. Methods 86, 257-264. 21 Pardee, J.D. and Spudich, J.A. (1982) Purification of muscle actin. Methods Enzymol. 85, 164-181. 22 Cohen, C.M. and Foley, S.F. (1980) Spectrin-dependent and independent association of F-actin with the erythrocyte membrane. J. Cell. Biol. 86, 694--698. 23 Sawyer, W.H., Hill, J.S., Howlett, G.J. and Wiley, J.S. (1983) Hereditary spherocytosis of man. J. Biochem. 211, 349-356. 24 Coppel, R.L., Cowman, A.F., Anders, R.F., Bianco, A.E., Saint, R.B., Lingelbach, K.R., Kemp, D.J. and Brown, G.V. (1984) Immune sera recognize on erythrocytes a Plasmodium falciparum antigen composed of repeated amino acid sequences. Nature 310, 789-791.
25 Ruangjirachupom, W., Whlin, B., Perlmann, H., Carlsson, J., Berzins, K., Wahlgren, M., Udomsangpetch, R., Wigzell, H. and Perlmann, P. (1988) Monoclonal antibodies to a synthetic peptide corresponding to a repeated sequence in the Plasmodiumfalciparum antigen Pf155. Mol. Biochem. Parasitol. 29, 19-28. 26 Klotz, F.W., Chulay, J.D., Daniel, W. and Miller, L.H. (1987) Invasion of mouse erythrocytes by the human malaria parasite, Plasmodiumfalciparum. J. Exp. Med. 165, 1713-1718. 27 Kemp, D.J., Coppel, R.L. and Anders, R.F. (1987) Repetitive proteins and genes of malaria. Annu. Rev. Microbiol. 41,181-208. 28 Perlmann, H.K., Berzins, K., Whlin, B., Udomsangpetch, R., Ruangjirachupom, W., Wahlgren, M. and Perlmann, P.H. (1987) Absence of antigenic diversity in Pf155, a major parasite antigen in the membrane of erythrocytes infected with Plasmodiumfalciparum. J. Clin. Microbiol. 25, 2347-2354. 29 Torii, M., Adams, J.H., Miller, L.H. and Aikawa, M. (1989) Release of merozoite dense granules during erythrocyte invasion by Plasmodium knowlesi. Infect. Immun. 57, 3230-3233. 30 Haest, C.W.M., Plasa, G., Kamp, D. and Devticke, B. (1978) Spectrin as a stabilizer of the phospholipid asymmetry in the human erythrocyte membrane. Biochim. Biophys. Acta 509, 21-32. 31 Williamson, P., Bateman, J., Kozarsky, K., Mattocks, K., Hermanowicz, N., Choe, H.R. and Schlegel, R.A. (1982) Involvement of spectrin in the maiptenance of membrane phase state asymmetry. Cell 30, 725-733. 32 Joshi, P., Dutta, G.P. and Gupta, C.M. (1987) An intracellular similar malarial parasite (Plasmodium knowlesi) induces stage-dependent alterations in membrane phospholipid organization of it's host membrane. Biochem. J. 246, 103-108. 33 Joshi, P. and Gupta, C.M. (1988) Abnormal membrane phospholipid organisation in Plasmodiumfalciparum-infected human erythrocytes. Br. J. Haematol. 68,255-259.
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© 1991 Elsevier Science Publishers B.V. /0166-6851/91/$03.50 ADONIS 016668519100047M MOLBIO 01518
The ring-infected erythrocyte surface antigen of Plasmodiumfalciparum associates with spectrin in the erythrocyte membrane Michael Foley 1, Leann T i l l e r , William H. S a w y e r 3 and Robin F. Anders 1 1The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria, Australia; 2Biochemistry Department, University of La Trobe, Bundoora, Victoria, Australia; and 3Biochemistry Department, University of Melbourne, Parkville, Victoria, Australia (Received 3 July 1990; accepted 26 November 1990)
The malaria parasite Plasmodiumfalciparum synthesises a protein, RESA, which associates with the membrane of newly invaded erythrocytes. Using spent supernatants from P.falciparum growing in culture as a source of soluble RESA we have developed an assay to examine the characteristics of RESA binding to the erythrocyte membrane in vitro. RESA associated with the Triton X-100 insoluble proteins on the inner face of the host erythrocyte membrane but did not bind to the outer surface of intact erythrocytes. Other proteins present in culture supernatants did not bind to the erythrocyte membrane. RESA was co-sedimented with the ternary complex formed between actin, spectrin and band 4.1 and co-precipitated with spectrin precipitated with anti-spectrin antibodies. The extent of association between RESA and the inner face of the erythrocyte membrane was reduced by the inclusion of excess purified spectrin in the assay. Thus, RESA appears to be associated with spectrin in the erythrocyte membrane skeleton. Key words: Plasmodiumfalciparum; Spectrin; Bindingassay; Ring-infected erythrocyte surface antigen
Introduction
Various structural and biochemical changes in the erythrocyte membrane occur during asexual development of the malarial parasite within erythrocytes [1]. As the parasite matures the shape and deformability of the host membrane are altered, and even in the less mature ring-stage parasites there is a measurable decrease in the deformability of the erythrocyte membrane [2]. The network of cytoskeletal proteins underlying the erythrocyte bilayer is responsible for maintaining the shape and deformability of the erythrocyte membrane [3]. Thus, parasite proteins that interact with this membrane skeleton may be responsible for changes in these Correspondence (present) address: Michael Foley, Institute of Cell, Animal and Population Biology, University of Edinburgh, King's Buildings, West Mains Road, Edinburgh EH9 3JN, U.K. Abbreviations: RESA, ring-infected erythrocyte surface antigen; TX- 100, Triton X- 100; IOVs, inside-outvesicles.
membrane characteristics. Several malarial proteins are known to be localised on the cytoplasmic face of the erythrocyte membrane [4,5] and such interactions may be important in maintaining the peculiar knob structures seen in the membrane of erythrocytes infected with mature stages of P.falciparum. The ring-infected erythrocyte surface antigen (RESA) is a P.falciparum protein that becomes associated with the membrane of newly invaded erythrocytes. After synthesis, RESA is stored in organelles within the mature parasite and is released into the red cell at the time of merozoite invasion. In mature parasites RESA is largely soluble in the anionic detergent Triton X- 100 (TX- 100) but when associated with the membrane of newly invaded erythrocytes it is TX-100-insoluble, presumably due to an interaction with a component of the erythrocyte membrane skeleton [6]. Antibodies to RESA inhibited merozoite invasion in vitro [7] and immunisation of Aotus monkeys with recombinant RESA proteins provided
138
partial protection against subsequent challenge with P.falciparum [8] but the function of RESA has not been established. A role for RESA in the process of merozoite invasion was suggested by the timing of the transfer of RESA to the erythrocyte membrane and the observation that RESA is phosphorylated when associated with the membrane [91. However, the finding that merozoites of a parasite line not expressing RESA efficiently invade erythrocytes in vitro argues against a role for RESA at the time of merozoite invasion [10]. As RESA persists in association with the erythrocyte membrane for at least 24 h after invasion its role may be to modify the properties of the membrane during that period when the infected erythrocyte is in the circulation, prior to cytoadherence. A first step in understanding the function of RESA would be to identify the component(s) of the erythrocyte membrane with which RESA associates. To this end we have developed an assay for looking at the interaction of RESA with the host membrane. Several lines of evidence suggest that RESA associates with spectrin in the erythrocyte membrane skeleton. Materials and Methods
P. falciparum isolate FCQ27/PNG (FC27) was obtained through collaboration with the Papua-New Guinea (PNG) Institute of Medical Research. The clonal line D10 was derived from FC27 by limiting dilution culture [11]. Parasites were maintained in asynchronous culture as described [12]. Rabbit antisera against RESA were prepared by immunising rabbits with ~-galactosidase fusion proteins produced in Escherichia coli [ 13], or synthetic peptides corresponding to repeat sequences, coupled to keyhole limpet haemocyanin [ 14]. The monoclonal antibody 18/2 which was used in immunoblotting experiments reacted with both the 5' and 3' repeat regions of RESA [15]. P. falciparum growing in culture was synchronised by treatment with sorbitol [ 16]. Antisera to the S-antigen was obtained as described previously [17].
pared according to Steck and Kant [18]. Centrifugation through a Dextran cushion (Dextran 80; 1.03 g ml m 0.5 mM sodmm phosphate, pH 8.0), separated IOVs from unsealed ghosts and right-side-out vesicles [18]. Contamination of IOVs with rightside-out vesicles was less than 10% and usually, the Dextran step was omitted. 1 -
Preparation of culture supernatants. Spent supernatants from cultures of P. falciparum were used as a source of RESA. A 10-ml dish of infected cells (haematocrit, 2%) was centrifuged at 1 500 rev./min, the pellet containing parasites and red cells discarded and the supernatants centrifuged at 100 000 × g for 20 min to remove cellular debris. The remaining supernatants were either used immediately or frozen at-70°C until required.
Metabolic labelling with [35S]methionine. Parasites from schizont stages to late ring stages were cultured overnight in methionine free medium (RPMI-1640, Selective Kit, Gibco, New York) modified for P. falciparum growth and containing 100 btCi ml -j of [35S]methionine (>1 000 Ci mmol-1; Amersham). The supernatants containing released radiolabelled parasite molecules were collected and processed as described in the previous section.
Parasites, peptides and sera.
Preparation of erythrocyte ghosts and inside-out vesicles. Inside-out vesicles (IOVs) were pre-
Assessment of RESA binding to inside-out vesicles. For most experiments, 100-150 ~tg of IOV protein were added to phosphate-buffered saline (PBS) containing 10-20 ~tl of culture supernatant and incubated for 30 min at 4°C. Binding reactions were performed in 500 ~tl. Incubations were terminated by centrifugation in a microfuge for 20 min and the resultant supernatants were discarded. The vesicle pellets were resuspended in 500 ~tl of PBS and centrifuged again. After a further wash, samples were boiled in 2× concentrated SDS sample buffer and half the pellet volume was then analysed by SDSPAGE. Alternatively, samples were diluted in TNET (0.5 % TX- 100 in 50 mM Tris/150 mM NaC1/5 mM EDTA, pH 8.0), and centrifuged to obtain the TX-100-insoluble pellet which, after 2 further washes in T-NET, was dissolved in sample buffer and analysed by SDS-PAGE [ 19]. Where different conditions of binding were used, these are indicated in Results.
139
man 3MM paper and exposed to Agfa X-ray-film. The gels were incubated in Amplify (Amersham) for 10 min immediately before drying. Immunoblotting and SDS-PAGE were performed as described previously [ 19,20].
Detergent extraction, immunoprecipitation and SDS-polyacrylamide gel electrophoresis. After incubation with supernatants, IOVs were dissolved in T-NET, incubated on ice for 30 min, and then separated into soluble and insoluble fractions by centrifugation at 25 000 x g for 30 min. The TX100-insoluble fraction was then solubilised in 2% SDS in PBS for 30 min at room temperature, followed by addition of 20% TX-100 and T-NET to give final concentrations of 0.2% SDS and 0.7% TX-100. Immunoprecipitation was performed using 5 ~tl of rabbit antiserum and the precipitated antigens were analysed on SDS-PAGE under reducing conditions. For autoradiography the gels were fixed in 5% methanol, 10% acetic-acid, dried onto What-
Purification of skeletal components, sedimentation assay and competition experiments. Rabbit skeletal muscle actin was isolated according to the method of Pardee and Spudich [21] and was purified by two successive cycles of polymerisation and depolymerisation. The depolymerisation buffer was buffer G (10 mM Tris-HC1, pH 7.6/0.2 mM CaC12/ 0.5 mM DTT/0.02% NAN3). Spectrin dimers were prepared from human erythrocytes by the method of Cohen and Foley
B
200
C
--~
200 100
I00 69
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67
1
2
3
4
5 1
2
I
2
Fig. 1. Association ofRESA with the cytoplasmic face oftheerythrocytemembrane. Spent culture supematants from cultures ofP.
falciparum were incubated with IOVs from uninfected erythrocytes or with intact erythrocytes. Unbound material was washed away and the proteins in the pellets separated by SDS-PAGE, blotted onto nitrocellulose and probed with RESA specific antisera. A sample of culture supernatant was run on lane 1 of each panel for comparison. (A) RESA binding to IOVs (lane 2) and to intact erythrocytes (lane 3). A sample of IOVs and erythrocytes after incubation were treated with TX-100 and the insoluble material blotted and probed with antisera to RESA. Lane 4 shows RESA attached to the insoluble matrix of IOVs and lane 5 RESA attached to the insoluble matrix from erythrocytes. (B) Supernatants alone (lane 1) and IOVs after incubation (lane 2) were separated by SDS-PAGE, blotted onto nitrocellulose and probed with antisera to the S-antigen. (C) Identical to panel B but reprobed with a pool of sera from infected individuals from Papua-New Guinea. Several proteins which do not associate with IOVs are indicated with open arrowheads, and an M r 73 000 protein that interacts with IOVs is indicated by an asterisk.
140
[22]. Spectrin dimers were passaged twice through a Biogel A15M column to ensure complete removal of contaminating actin and protein 4.1. A crude preparation of band 4.1 was obtained by high salt extraction of erythrocyte membrane vesicles which had been depleted of band 6, spectrin and actin. This yielded a preparation in which band 4.1 represented about 50% of the Coomassie Blue staining material [23].
Binding of malarial antigens to purified skeletal proteins. Samples were prepared containing 0.12 -I
•
•
-1
•
mg ml G-actm, or actmplus 0.32 mg ml spectrm and 0.04 mg m1-1 band 4.1. [35S]methionine-labelled culture supernatant was added to 100 ~Ci ml -~. Polymerisation of the actin was initiated by addition of NaC1 to 100 mM and MgC12 to 2 mM. After 1 h at room temperature, the F-actin and associated proteins were pelleted by centrifugation at 290 000 × g for 40 min at 20°C. The pellets were resuspended in 1 ml of buffer G plus 2 m M MgC12 and 100 mM NaC1 and repelleted (290 000 × g, 40 min). The pellets were solubilised in 100 ~tl of Buffer G and stored frozen until analysis by SDS-PAGE. For competition studies erythrocyte ghosts (0.4 mg ml ) were incubated with [ S]methlonlne-labelled culture supernatants (100 ~tCi ml -~) in buffer C (10 mM sodium phosphate, pH 7.5/100 mM NaC1/0.01% NAN3/0.5 mM DTT in the presence of varying concentrations of purified spectrin (1.2 mg 1 • -1 ml ) or protein 4.1 extract (0.05 mg ml ). After a 1-h incubation at room temperature, the membranes were pelleted at 12 000 × g for 15 min. The pellets were resuspended in 1 ml of buffer C and recentrifuged. These pellets were resuspended in 100 ~tl of buffer C and stored frozen until analysis by SDS-PAGE. -1
.
.
35
•
used to probe immunoblots of culture supernatants the full length RESA polypeptide of Mr 155 000 was detected together with 2 smaller polypeptides of Mr 130000 and 120000 (Fig. 1A, lane 1). We have assumed that these 2 smaller polypeptides are derived from RESA as both react with a variety of antibodies directed against the 3' or 5' repeats of RESA (data not shown). The immunoblotting resuits indicate that the full length RESA polypeptide bound to IOVs to a greater extent than either of the 2 smaller fragments (Fig. 1, lane 1). RESA bound to IOVs was apparently associated with the membrane skeleton because it was detected in the TX100-insoluble fraction derived from IOVs (Fig. 1A, lane 4). In addition, when a TX- 100-insoluble fraction generated from freshly made IOVs was incubated with culture supernatants for 60 min, RESA bound to the membrane skeleton. Another antigen that is readily detected in spent culture supernatants
200
•
100
67
Results
RESA binds to the erythrocyte membrane skeleton. RESA in spent culture supernatants bound to IOVs but did not bind to normal erythrocytes. The associated RESA was detected when IOVs that had been incubated for 60 min in culture supernatants were washed extensively, fractionated by SDS-PAGE and electrophoretically transferred to nitrocellulose filters which were subsequently probed with specific antisera. When anti-RESA antisera were
1
2
3
4
Fig. 2. [3~S]Methionine-labelledRESA binds to IOVs. Polypeptides in supematantsfrom 2 P.falciparumstrains grown in culture in the presence of [35S]methioninewere separated by SDS-PAGE and visualised by autoradiography of the dried gels (lanes 1 and 2). Alternativelysupernatantswere incubated with IOVs from uninfected erythrocytes and bound polypeptides visualisedin the same way (lanes 3 and 4). The supematants were from cultures of FC27 (lanes 1 and 3) and FCR3 (lanes 2 and 4).
141
Binding t o lOWS
Triton Pellets
~!~ ~ii/~ii,ii!ii!i~ii i~!!iiii!i~!~ 200
--~
I00
--~
67
--~
43
--~
i!~il/ii!)~ii~ii~ii~i/i!!~ i~i ¸ CHRMS
CHRMS
Fig. 3. RESAbindsto the membraneskeletonoferythrocytesfroma varietyofmammals.Culturesupernatantswereincubatedwith IOVsprepared fromrabbit (R),mouse(M) and sheep(S) as wellas fromhuman(H) and boundRESA,afterextensivewashing,analysedby immunoblotting(as describedin Fig. 1). Culturesupernatants(C) were alsorun alongsidethe IOVs as a comparison.The IOVs, afterwashingawayunboundmaterial,wereextractedwithTX-100 and the insolubleprecipitateexaminedin the sameway.It can be seenthatRESAcan associatewiththe TX-100 insolublefractionin all erythrocytespeciestested. is the S-antigen of P.falciparum. The Mr 220 000 S antigen of isolate FC27 was detected in the supernatants used in these experiments but, in contrast to the finding with RESA, the S-antigen did not bind to IOVs (Fig. 1B). Probing immunoblots of supernatants with a pool of sera from individuals exposed to malaria in Papua-New Guinea revealed several other antigens that did not bind to IOVs, although one antigen of Mr 73 000 did associate with IOVs in a similar manner to RESA (Fig. IC). Additional bands in Fig. 1C, lane 2 (Mr 50 000, 65 000 and approx. 190 000) did not have counterparts in the supernatant and were due to the reaction of the human serum with components of the erythrocyte membrane. These immunoblotting results suggest a specific association between RESA and the erythrocyte membrane skeleton. Further evidence of specificity in this association was obtained from experiments in which IOVs were incubated with heated culture supernatants. Heating culture supernatants at 65°C for 15 min did not affect the anti-
genicity of RESA in the supernatant but dramatically reduced the binding of RESA to IOVs (data not shown) indicating that a heat labile conformation in RESA was important for this interaction. Since RESA in mature parasites is mainly TX100-soluble [6], a sonicate of infected erythrocytes, cleared by high speed centrifugation, was used as an alternative source of RESA for binding experiments in vitro. RESA in these sonicates bound to IOVs in a similar manner to RESA from culture supernatants (data not shown). The association of culture supernatant proteins with IOVs was also investigated using supernatants containing parasite proteins which had been biosynthetically labelled with [35S]methionine. A closely migrating radiolabelled doublet of Mr approx. 155 000 bound to IOVs (Fig. 2, lane 3) but not to normal erythrocytes. This doublet, which has been observed in previous studies [14], was shown to be RESA by immunoprecipitation with defined antisera (data not shown). The nature of the doublet
142
is not understood but may be the result of some post-translational modification such as phosphorylation. The FCR3 strain of P. falciparum has been shown not to synthesise the RESA polypeptide [10]. When we used [35S]methionine-labelled supernatants from FCR3 in similar binding experiments, no labelled doublet was found to bind to IOVs (Fig. 2A, lane 4) nor was a doublet immunoprecipitated by antisera to RESA (data not shown). Many other radiolabelled proteins were present in FCR3 culture supernatants (Fig. 2A, lane 2). In these experiments we did not observe binding of the M r 73 000 antigen presumably because it is not labelled with [35S]methionine.
A
RF~A
B
PNG
200
LI iii iiiii ! i i
I00 67
RESA associates with spectrin.
RESA binding studies using culture supernatants were repeated
200
--~
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67
--~
2
3
Fig. 4. Interaction of RESA with inside-out-vesicles is reduced by protease treatment of the vesicles. IOVs were incubated for 60 mln at 4 C m the presence of 0 lag ml , lane 1; 50 lag ml , lane 2; and 100 lag ml , lane 3; of ~-chymotrypsln. The reaction was stopped by the addition of a 10-fold excess of chymostatin. After incubation for 60 min at 4°C the vesicles were washed several times in 0.5 mM sodium phosphate plus 10 lag ml -~ chymostatin. Binding of RESA to each preparation was then assessed as described in Materials and Methods. •
o
2
3
1
2
3
Fig. 5. RESA binds predominantly to the ternary complex in vitro. Actin alone (lane 2) or with actin plus spectrin plus band 4.1 (lane 3) were examined for the ability to bind RESA or other malarial antigens present in culture supernatants (lane 1) as described in Materials and Methods. Bound proteins were visualised by probing nitrocellulose filters with antisera to RESA (panel A) or a pool of sera from malaria infected individuals from Papua-New Guinea (panel B). Quantitative densitometry on an identical gel stained with Coomassie Blue indicated that approx• 11 lag of protein was applied to lane 2 (actin alone) and approx. 19 lag of protein was applied to lane 3 (actin plus spectrin plus band 4.1 ).
1
-1
1
•
1
-I
-
with IOVs from a variety of mammalian species. In each case RESA bound to IOVs and became TX100-insoluble in an identical manner as for human 1OVs (Fig. 3). Thus, the host molecule with which RESA interacts is a highly conserved component of the membrane. The protein nature of this molecule was demonstrated by binding studies using IOVs pretreated with proteases. Fig. 4 shows the reduction in association of RESA to IOVs digested with increasing concentrations of chymotrypsin. Coomassie Blue staining of IOVs after proteolysis revealed extensive degradation of most major protein bands such as spectrin and band 3; however, several polypeptide fragments were still observed, indicating that complete breakdown of cell membrane proteins had not occurred (data not shown). As these results were consistent with RESA associating with a component of the erythrocyte membrane skeleton we purified the 3 major skeletal components: spectrin, actin, and band 4.1, and per-
143 A
Speetrin
1
2
B
Actin
200 M
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S
Fig. 6. Excess purified spectrinreduces the binding of RESA to inside-outvesicles. IOVs from uninfectederyt,hrocyteswere incubated with culture supernatantswith no additions(lane 1, both panels) or in the presence of 50, 100, 200 I.tgml-' spectrin (panel A, lanes 2,3,4). Addition of excess purified actin (10, 50, 100 ~tgm1-1,panel B, lanes 2,3,4) to the assay did not reduce the binding of RESA to IOVs. Lane S correspondsto supernatantsrun on the same gel for comparison. formed co-sedimentation experiments with RESA as described in Materials and Methods. When actin alone was polymerised in the presence of culture supematant, and sedimented by centrifugation, a small amount of co-sedimented RESA was detected by immunoblotting (Fig. 5A, lane 2). Very much more RESA was co-sedimented with the temary complex of actin, spectrin and band 4.1 (Fig. 5A, lane 3). Probing a replicate immunoblot with a PNG serum pool (Fig. 5B) revealed that this co-sedimentation is specific for RESA and the Mr 73 000 polypeptide consistent with previous resuits in this study. Further evidence of the specificity of this reaction was obtained when this cosedimentation assay was performed using [35S]methionine-labelled culture supernatants. Again, only RESA and a Mr 73 000 polypeptide were sedimented (data not shown). These results indicate that either spectrin or band 4.1 is necessary for efficient co-sedimentation of RESA. Evidence that
spectrin is a major target for RESA attachment was obtained by carrying out a binding assay as already described but including in the assay increasing concentrations of purified spectrin. High concentrations of purified spectrin in the reaction mix substantially inhibited RESA from binding to the IOVs (Fig. 6A). This was not the case when actin was included in the reaction mix in place of spectrin (Fig. 6B). These data are consistent with the hypothesis that RESA binds to spectrin in the erythrocyte membrane. Evidence that RESA interacts with spectrin in the parasitised erythrocyte was obtained by immunoprecipitation with antisera to spectrin (Fig. 7). Parasites labelled with [35S]methionine for 12 h were solubilised in T-NET and immunoprecipitations were carried out from the TX- 100-soluble and insoluble fractions using rabbit antisera to spectrin and control normal rabbit serum. A major radiolabelled polypeptide of approx. Mr 155 000 was co-
144 trin was precipitated from the insoluble fraction. Western blots also revealed that the antiserum recognises c~ spectrin (data not shown). Thus it appears from these results that spectrin is a major target for RESA binding. 200
Discussion
100
67
--~
43
--~
1
2
3
4
S N Fig. 7. Antibodiesto spectrin co-precipitate RESA. A culture of P. falciparum infected erythrocytes grown for 12 h in the presence of [35S]methioninewas washed in PBS and fractionated by addition of TX-100 into soluble and insoluble fractions. The soluble fraction was immunoprecipitatedwith antisera to spectrin(S) (lane 1) or normalrabbit serum (N) (lane 3). The insoluble fraction was treated with SDS and an excess of TX-100 and T NET was added (as described in Materials and Methods) followed by immunoprecipitationwith antisera to spectrin (lane 2) or normal rabbit antisera (lane 4). Arrowhead corresponds to Mr 155 000 co-precipitatedby spectrinspecific antisera.
precipitated with anti-spectrin antibodies from the TX- 100-soluble fraction, but not from the TX- 100insoluble fraction which was treated with 1% SDS. Coomassie Blue staining of the gel revealed that both t~ and ~ spectrin were immunoprecipitated from the TX-100-soluble fraction, but only t~ spec-
We report here that RESA can associate with the cytoplasmic face of human erythrocytes but not with the external aspect of intact erythrocytes. This is consistent with previous data which showed that antibodies to RESA failed to bind unfixed ring-infected erythrocytes but did bind to lightly glutaraldehyde-fixed cells [24]. Probing immunoblots of spent culture supernatants with antisera to RESA revealed 2 faster migrating species of Mr 130 000 and Mr 120 000 in addition to the intact RESA polypeptide of Mr 155 000. This pattern of antigenically related of polypeptides has been noted previously and was assumed to be due to proteolysis of the M~ 155 000 polypeptide [25]. Although the Mr 120 000 polypeptide appears to be relatively abundant in supernatants, it binds relatively poorly to erythrocyte membranes and to purified spectrin (Fig. 5A, lane 3). Thus, the Mr 120 000 polypeptide may lack a sequence involved in the binding of RESA to the erythrocyte membrane. The binding of RESA to the erythrocyte membrane appeared specific in that the S antigen and several other antigens present in culture supernatants were not bound under the assay conditions used. However, an Mr 73 000 antigen, present in culture supernatants and recognised by PNG immune sera did bind to the erythrocyte membrane in a similar manner to RESA. This antigen does not appear to be a breakdown product of RESA (unpublished results) arid experiments are underway to characterise it further. The results of the binding experiments analysed by immunoblotting were confirmed by binding experiments using supernatants from parasites labelled with [35S]methionine. Studies using these supernatants revealed many parasite proteins which differed from RESA and the Mr 73 000 antigen in that they did not bind to erythrocyte vesicles under physiological conditions. By continually replenishing the assay with fresh IOVs we were able to remove almost all of the RESA present in a given volume of culture supernatants. Indicating that over 90% of
145 RESA molecules are competent for binding (data not shown). The association of RESA with the erythrocyte membrane is due to an interaction between RESA and a component of the erythrocyte membrane skeleton rather than an association with the lipid bilayer. There are regions of the RESA sequence that are relatively hydrophobic but none of these are of sufficient length and average hydrophobicity to be considered likely membrane spanning domains. Furthermore, there is no evidence that RESA is post-translationally modified with lipid to provide a membrane anchor. The observations reported here that protease pretreatment of IOVs inhibits the binding of RESA, and that RESA binds to the insoluble residue remaining after TX- 100 extraction of RBCs are consistent with RESA binding to a component of the membrane skeleton. This component is conserved among mammalian species as RESA bound to IOVs prepared from erythrocytes of 6 different species. Klotz et al. [26] have reported that when P. falciparum merozoites invade mouse erythrocytes RESA was subsequently found to associate with the erythrocyte membrane, a finding which is consistent with our observations. The erythrocyte membrane skeleton is a meshwork of peripheral proteins consisting primarily of spectrin, actin and band 4.1. The finding that RESA binds to the ternary complex of spectrin, actin and band 4.1 points to one of these proteins being the major target for RESA binding. Two different experimental approaches have provided evidence that spectrin is the component of the skeleton with which RESA associates. In one experimental approach purified spectrin was used to inhibit the binding of RESA to IOVs. The binding of RESA was markedly reduced but not completely inhibited in the presence of excess free spectrin and this suggests the possible involvement of other components in RESA binding. In the second experimental approach RESA was co-precipitated when spectrin was precipitated with anti-spectrin antibodies. Much spectrin is usually lost from the erythrocyte membrane during the preparation of IOVs but analysis by SDS-PAGE and Coomassie Blue staining revealed the presence of significant amounts of spectrin in the IOVs used in this study. Loss of spectrin was minimised by carrying out
vesiculation at 4°C [18]. Our recent observations using radiolabelled purified recombinant RESA, that binding to IOVs was saturable and could be greatly reduced by excess unlabelled RESA supports the conclusion that RESA binds to a specific receptor site (unpublished results). The function of RESA remains unknown. An involvement in the invasion process, as initially proposed [27], appears unlikely since FCR3, a P.falciparum strain grown in vitro over many years, has a subtelomeric deletion onchromosome 1 which prevents expression of the RESA gene [ 10]. As RESA is invariably expressed in field isolates [28], and as it persists at the erythrocyte membrane for some time after invasion the function of RESA may be to modify the properties of the host cell membrane during the period the infected cell persists in the peripheral circulation. Recently, it has been suggested that antigens previously located to micronemes may in fact be present in dense granules, which appear to release their contents into the parasitophorous vacuole after invasion [29]. If RESA is located in dense granules and not in micronemes, as previously reported, this would be consistent with a function after invasion during the early stages of intraerythrocytic development. The evidence presented here for RESA being a spectrin binding protein suggests a mechanism whereby RESA could modify the host cell membrane. The membrane skeleton is an assembly of proteins attached to the erythrocyte lipid bilayer and controls the shape and mechanical properties of the erythrocytes. Spectrin, quantitatively the major component of the skeleton is an elongated flexible molecule consisting of two subunits. Spectrin self associates and interacts with other proteins, principally actin, to form a flexible meshwork. Spectrin also interacts with ankyrin which in turn interacts with the integral membrane protein band 3, thereby linking the membrane skeleton to the lipid bilayer. If RESA binding to spectrin were to disturb any of these interconnections it would have consequences for the arrangement of the membrane skeleton. Our results provide a possible mechanism for the results found by Nash et al. [2] who observed that ring-infected erythrocytes had increased m e m brane rigidity. The authors speculated that this reflect changes in the membrane skeleton most likely
146
involving spectrin. However, if RESA binding to spectrin does result in the changes in membrane viscoelasticity observed in the ring-infected cells, another mechanism must be responsible for the more dramatic changes in erythrocytes infected with more mature stages when RESA is apparently lost from the membrane. Spectrin has been implicated in the maintenance of the normal asymmetric distribution of lipids across the erythrocyte plasma membrane [30,31 ], and it is possible that an interaction between RESA and spectrin could alter the normal lipid composition of the erythrocyte membrane in infected cells. Although several studies have been shown that changes of this nature can be detected in trophozoite and schizont infected cells but not in ring-infected erythrocytes [32,33], the persistence of RESA at the membrane for 24 h could conceivably affect the lipid compositional asymmetry. Identification of spectrin as a major target for RESA on the erythrocyte membrane does not rule out the participation of other erythrocyte molecules in the binding of RESA to the host membrane. Further studies will examine the nature of RESA binding and its effects on the structure of the erythrocyte membrane.
Acknowledgements We thank Sakunthala Meera for the preparation of the rabbit skeletal muscle actin and Etty Bonnici for typing this manuscript. This work was supported by the Australian National Health and Medical Research Council, the John D. and Catherine T. MacArthur Foundation and the Australian Malaria Vaccine Joint Venture. Support was also provided under the Generic Technology Component of the Industry Research and Development Act 1986, and the Wellcome Trust Fellowship Fund. References 1 Sherman, I.W. (1985) Membrane structure and function of malaria parasites and the infected erythrocyte. Parasitology 91,609-645. 2 Nash, G.B., O'Brien, E., Gordon-Smith, E.C. and Dormandy, J.A. (1989) Abnormalitiesin the mechanical properties of red blood cells caused by Plasmodiumfalciparum. Blood 24, 855-861. 3 Bennett, V. (1985) The membrane skeleton of human
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