further characterization of interactions between gamete surface antigens of plasmodium falciparum

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Molecular and Biochemical Parasitology, 53 (1992) 113-120 113 © 1992 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/92/$05.00 MOLBIO 1750 Further characterization of interactions between gamete surface antigens of Plasmodium falciparum Nirbhay Kumar and Benjamin Wizel Department of Immunology and Infectioas Diseases, School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, MD, USA (Received 9 September 1991; accepted 10 February 1992) Target antigens of malaria transmission blocking immunity include a complex of 3 gamete surface proteins of 230-kDa and 48/45-kDa glycoproteins. Previous studies have shown that epitopes recognized by blocking antibodies are conformational (reduction sensitive) in nature. Studies were conducted to characterize the interactions between the target antigens and role of disulfide groups in the formation of the complex. Treatment of detergent extracts of gametes with chaotropic agents and extremes of pH resulted in dissociation of the complex. The interaction between the 3 proteins was also perturbed when the extract was incubated in the presence of antibodies against the 230-kDa protein but not against the 48/45-kDa doublet. Chemical modifications of disulfide and sulfhydryl groups in the target antigens, otherwise inaccessible either in the total extract or after phase separation in Triton X-114, required prior denaturation of antigens. Key words: Malaria; Transmission blocking immunity; Plasmodiumfalciparum; Gametocyte; Gamete Introduction Antibodies against surface proteins of Plasmodium falciparurn gametes and mosquito midgut stages have been shown to block malaria transmission by reducing infectivity of gametocytes to mosquitoes [1]. Target antigens of transmission-blocking immunity (TBI) have been defined using monoclonal antibodies (mAbs). These include 3 proteins of apparent sizes 230, 48 and 45 kDa, actively synthesized in the vertebrate stage gametocytes and expressed on the surface of male and female gametes and newly fertilized zygotes [1- 4]. A 25-kDa protein synthesized predomi- nantly after induction of gametogenesis and Correspondenceaddress: Nirbhay Kumar, DIID-SHPH-JHU, 615 N. Wolfe St., Baltimore, MD 21205, USA. Abbreviations: TB, transmission blocking; TBI, transmission- blocking immunity; mAb, monoclonal antibody; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; DTT, dithiothreitol; IAA, iodoacetamide during transformation of zygotes into ooki- netes in the mosquito midgut has also been identified as a target of TBI [1, 5-7]. Previous studies based on phase separation in the detergent Triton X-II4, hydrophobic chromatography and reconstitution into lipid vesicles have suggested that the 48/45-kDa proteins are hydrophobic in nature and the 230-kDa protein relatively hydrophilic [8]. Many TB mAbs directed against epitopes in the 48/45-kDa antigens immunoprecipitate not only this doublet but also co-immunoprecip- itate the 230-kDa antigen [2,4,9,10]. Vermeulen et al. [4] and Quakyi et ai. [3] have also reported mAbs which either immunoprecip- itate only the 48/45-kDa or only the 230-kDa antigens respectively. Based on chemical cross- linking using cleavable and non-cleavable cross-linkers and immunodepletion analysis it was shown that a proportion of these antigens are physically associated with each other and exist as a stable membrane-bound complex [10].

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Page 1: Further characterization of interactions between gamete surface antigens of Plasmodium falciparum

Molecular and Biochemical Parasitology, 53 (1992) 113-120 113 © 1992 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/92/$05.00

MOLBIO 1750

Further characterization of interactions between gamete surface antigens of Plasmodium falciparum

Nirbhay Kumar and Benjamin Wizel Department of Immunology and Infectioas Diseases, School of Hygiene and Public Health, The Johns Hopkins University,

Baltimore, MD, USA

(Received 9 September 1991; accepted 10 February 1992)

Target antigens of malaria transmission blocking immunity include a complex of 3 gamete surface proteins of 230-kDa and 48/45-kDa glycoproteins. Previous studies have shown that epitopes recognized by blocking antibodies are conformational (reduction sensitive) in nature. Studies were conducted to characterize the interactions between the target antigens and role of disulfide groups in the formation of the complex. Treatment of detergent extracts of gametes with chaotropic agents and extremes of pH resulted in dissociation of the complex. The interaction between the 3 proteins was also perturbed when the extract was incubated in the presence of antibodies against the 230-kDa protein but not against the 48/45-kDa doublet. Chemical modifications of disulfide and sulfhydryl groups in the target antigens, otherwise inaccessible either in the total extract or after phase separation in Triton X-114, required prior denaturation of antigens.

Key words: Malaria; Transmission blocking immunity; Plasmodiumfalciparum; Gametocyte; Gamete

Introduction

Antibodies against surface proteins of Plasmodium falciparurn gametes and mosquito midgut stages have been shown to block malaria transmission by reducing infectivity of gametocytes to mosquitoes [1]. Target antigens of transmission-blocking immunity (TBI) have been defined using monoclonal antibodies (mAbs). These include 3 proteins of apparent sizes 230, 48 and 45 kDa, actively synthesized in the vertebrate stage gametocytes and expressed on the surface of male and female gametes and newly fertilized zygotes [1- 4]. A 25-kDa protein synthesized predomi- nantly after induction of gametogenesis and

Correspondence address: Nirbhay Kumar, DIID-SHPH-JHU, 615 N. Wolfe St., Baltimore, MD 21205, USA.

Abbreviations: TB, transmission blocking; TBI, transmission- blocking immunity; mAb, monoclonal antibody; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; DTT, dithiothreitol; IAA, iodoacetamide

during transformation of zygotes into ooki- netes in the mosquito midgut has also been identified as a target of TBI [1, 5-7].

Previous studies based on phase separation in the detergent Triton X-II4, hydrophobic chromatography and reconstitution into lipid vesicles have suggested that the 48/45-kDa proteins are hydrophobic in nature and the 230-kDa protein relatively hydrophilic [8]. Many TB mAbs directed against epitopes in the 48/45-kDa antigens immunoprecipitate not only this doublet but also co-immunoprecip- itate the 230-kDa antigen [2,4,9,10]. Vermeulen et al. [4] and Quakyi et ai. [3] have also reported mAbs which either immunoprecip- itate only the 48/45-kDa or only the 230-kDa antigens respectively. Based on chemical cross- linking using cleavable and non-cleavable cross-linkers and immunodepletion analysis it was shown that a proportion of these antigens are physically associated with each other and exist as a stable membrane-bound complex [10].

Page 2: Further characterization of interactions between gamete surface antigens of Plasmodium falciparum

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MAbs directed against the epitopes in the 230-kDa and the 48/45-kDa doublet antigens are effective blockers of infectivity of gameto- cytes in the mosquitoes. In almost every case, the epitopes recognized by transmission-block- ing mAbs have been shown to be reduction- sensitive (conformational in nature) as reduc- tion of disulfide bonds in the antigens resulted in loss of mAb reactivity to antigens [4,9]. SDS-PAGE analysis under reducing condi- tions also showed that the 230-kDa and 48/45- kDa doublet proteins migrated with electro- phoretic mobilities of 255 (now described as 260 kDa) and 59/53 kDa respectively [2] suggesting involvement of disulfide bonds in the maintenance of conformation of these proteins. In addition to the disulfide bonds, the oligosaccharide residues in the 48/45-kDa glycoproteins [11] may also participate in the overall conformation and formation of the epitopes of TB mAbs. In view of the fact that the 230-kDa and the 48/45-kDa gamete proteins are target antigens for the develop- ment of a malaria TB vaccine, studies were undertaken to investigate the potential role of the disulfide bonds in the formation of the complex and interactions between the target antigens of P. falciparum TBI.

Materials and Methods

Parasites. Gametocytes of P. falciparum clone 7G8 (a clone of isolate 1MTM22) and 3D7 (a clone of Amsterdam airport isolate NF54) were produced in culture as described [12]. Female gametes and zygotes were purified after induction of gametogenesis and exflagel- lation [2-4]. Briefly, cultures containing ma- ture gametocytes were resuspended in 10 mM Tris/145 mM NaC1/10 mM glucose, pH 7.3 (TSG). Mosquito extract [13] was added to a final concentration of 20% and the pH was adjusted to 8.1-8.3 and incubated for 30 min at room temperature. Extracellular gametes were purified by centrifugation (10000 rev./min, 10 min, 5°C) using 'Nycodenz' discontinuous gradient (6%, 11%, 16%). Gametes which

band at the interphase of 6% and 11% layers were washed in TSG.

Antibodies. MAb IIC5Bi0 directed against epitope in the 48/45-kDa proteins [2] and rabbit anti gamete serum were obtained from the Malaria Section, NIH, Bethesda, MD. For the production of polyclonal monospecific antiserum against the 230-kDa protein, Triton X-100 extract of gametocytes (5X10 9) was immunoprecipitated using the mAb IIC5BI0 and immunoprecipitates separated by SDS- PAGE. The region of the gel containing the 230-kDa protein was excised, washed in PBS and homogenized. The homogenate of the gel slice was emulsified in complete Freund's adjuvant and used for immunization in rabbits (designated as rabbit i10 and 111). Rabbits were boosted at monthly intervals and bleeds tested by immunoprecipitation for specific antibodies.

Surface radio-iodination, immunoprecipitation, SDS-PAGE and two dimensional gel electro- phoresis. Purified gametes were labeled by lactoperoxidase-catalyzed radioiodination [2]. Details of extraction in Triton X-100, immu- noprecipitation and SDS-PAGE are described elsewhere [2,11,14]. For 2-D gels, samples were extracted with NEPHGE sample buffer (9.5 M urea/2% NP-40/2% ampholyte, range 3 10, Pharmacia). First dimension electrophoresis in tube gels was carried out at 2000 V h and the second dimension in 5-15% SDS-PAGE [15].

EJfect of chaotropic agents on the complex of antigens. Triton X-100 extracts of radioiodin- ated gametes were diluted with stock solutions of guanidine hydrochloride, urea, NaCI to desired concentrations, incubated overnight at 4°C, and then dialyzed against 10 mM Tris/150 mM NaC1, pH 7.4/0.1% Triton X-100 for at least 24 h at 4°C with 5-6 changes of the dialysis buffer. Dialyzed extracts were tested by SDS-PAGE after immunoprecipitation.

lmmunodepletion. Typically, Triton X- 100 extract of radioiodinated gametes was incu- bated with 10/A o fmAb IIC5B10 or rabbit 110

Page 3: Further characterization of interactions between gamete surface antigens of Plasmodium falciparum

serum in a final volume of 100-150/~l, for 1-2 h at room temperature. The incubation mix was transferred to another tube containing 25 /~l (packed volume) protein A-Sepharose beads and incubated for another 1 h at room temperature (cycle 1). The supernatant fluid was quantitatively transferred, using a Hamil- ton syringe, to a new tube and incubated with the same antibody to repeat immunodepletion a desired number of times. Beads were washed and extracted in SDS-sample buffer for SDS- PAGE analysis.

Reduction and alkylation of gamete anti- gens. The Triton X-100 extract of radio- iodinated gametes was treated with 10 vols. of 6 M guanidine hydrochloride/8 M urea/5 M NaCI/0.1 M glycine-HCl buffer, pH 2.7 or 0.1 M triethylamine, pH 11.7. Dithiothreitol (DTT) was added from a freshly prepared solution in 0.2 M boric acid/0.16 M NaC1, pH 8.0 (BBS) to a final concentration of 25 mM

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for at least 60 rain at room temperature followed by 50 mM iodoacetamide (IAA) (added from a freshly prepared solution in BBS) for at least 60 min at room temperature in the dark. Final pH of the extract pretreated in 0.1 glycine-HCl buffer was 5.4 during reduction and 6.0 during alkylation and that of extract pretreated with 0.1 M triethylamine was 10.9 during both reduction and alkylation. Equivalent amount of BBS was added to samples when either DTT or IAA treatment of samples was omitted. Treated samples were then dialyzed as above for immunoprecipita- tion and SDS-PAGE analysis.

Results and Discussion

Earlier studies on phase separation of Triton X-114-extracted gamete proteins resulting in partitioning of the 230-kDa proteins in the aqueous phase and that of the 48/45-kDa in

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Fig. 1: Dissociation of complex of gamete surface antigens. Triton X-100 extract of surface radioiodinated gametes was incubated for approx. 16 h at 4°C in the presence of indicated concentrations in (M) of various chaotropic salts or pH. After dialysis (overnight at 4°C), the extracts were tested by immunoprecipitation either with the mAb IICSB10 (A) or rabbit anti- gamete serum (B) and immunoprecipitates analyzed by 5--15% SDS-PAGE under non-reducing condition. Lanes marked None were samples either stored at - 70°C or subjected to all the above procedures but without any denaturation treatment. Positions of the 230-kDa and 48/45-kDa antigens are indicated by arrows. BioRad high-molecular weight and low-molecular

weight standards were used to calibrate the gels.

Page 4: Further characterization of interactions between gamete surface antigens of Plasmodium falciparum

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the detergent phase [8] had suggested a role for hydrophobic interactions in the complex of these 3 gamete surface antigens. These antigens remain complexed for prolonged periods of times after extraction in non-ionic detergents.

Dissociation by chaotropic agents. To investi- gate further the nature of the interaction(s) between these antigens, detergent extracts of surface radio-iodinated gametes of P. falciparum were subjected to treatment under a variety of conditions which cause disruption of hydrophobic interactions, ionic and hydro- gen bonds between macromolecules. As shown in Fig. 1 (A,B), treatment of extracts with increasing concentrations of the chaotropic agents guanidine hydrochloride, urea and NaC1 resulted in dissociation of the complex of the 230-kDa and the 48/45-kDa antigens,

the order of effectiveness being guanidine hydrochloride > urea > 5 M NaCI. A brief incubation either at acidic pH 2.7 or basic pH 10.7, likewise resulted in dissociation of the complex between the 230-kDa and 48/45-kDa doublet. To differentiate the complex form of these antigens from the dissociated forms we took advantage of mAbs like IIC5B10 which recognize epitopes in the 48/45-kDa doublet proteins but co-immunoprecipitate the com- plexed form of the 230-kDa protein. Extracts after treatment were also tested in immuno- precipitation assay with a rabbit anti-gamete polyclonal antiserum to confirm that all the antigens were actually intact and still retained antigenicity for other antibodies after various treatment steps (Fig. I B). The absence of or a reduction in the amount of the 230-kDa protein band in the immunoprecipitates with

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Fig. 2. Dissociation of complex of gamete surface antigens by rabbit anti-230-kDa serum. Triton X-100 extract of surface radioiodinated gametes was subjected to successive immunoprecipitation with the mAb IIC5BI0 (A) or rabbit anti-230-kDa serum (B). (A) After 3 cycles of immunodepletion (lanes numbered 1 3) with IIC5BI0, the final supernatant was analyzed with rabbit anti-230-kDa serum (lane marked 3 in parentheses). Immunoprecipitation of extract (before depletion with the mAb IIC5BI0) with rabbit-anti 230-kDa serum after overnight incubation is shown in lane 4. (B) Immunoprecipitation patterns at each cycle after 4 (lanes I-4), 8 (lanes 1-8), 12 (lanes 1-12) and 16 (lanes 1-16) cycles of successive immunodepletion of extract with rabbit anti-230-kDa serum. Final supernatant fluids obtained after 4, 8, 12, 16 cycles of immunodepletion were finally incubated overnight with the mAb IIC5BI0 to detect the complex forms of the 230-kDa and the 48/45-kDa antigens (lanes marked with respective cycle numbers in parentheses, a lane identified by a zero in parentheses represent immunoprecipitation with I IC5BI0 without any prior immunodepletion). In lanes 8 and lane 13, the extract and

anti-230 kDa antibodies were incubated overnight. Arrows indicate the positions of 230-kDa and 48/45-kDa antigens.

Page 5: Further characterization of interactions between gamete surface antigens of Plasmodium falciparum

the mAb IIC5BI0 is indicative of dissociation of the complex (Fig. 1A). Once perturbed either by chaotropic agents or by phase separation, dissociated forms of antigens do not spontaneously reassociate.

Dissociation by antibodies. Earlier studies [2] have shown that mAb IIC5B10 directed against epitopes in the 48/45-kDa proteins also co-immunoprecipitates the 230-kDa anti- gen which is physically associated with the former antigens (Fig. 2A lane 1). On the other hand, polyclonal monospecific antisera pro- duced against purified 230-kDa antigen when tested in immunoprecipitation showed only the 230-kDa band (Fig. 2A lane 4) rather than the complex as seen with mAbs. A simple explana- tion for this result could be that antibodies in the rabbit sera recognize only the uncomplexed form of the 230-kDa protein. A careful analysis of these gels however, suggested that the total amount of 230-kDa immunoprecipi- tared by the rabbit 110 antibody was much more than that anticipated for the uncom- plexed form (total minus the 230-kDa in the immunoprecipitates with mAb IIC5B10). This led to the experiments aimed at evaluating the possibility that in the presence of anti-230-kDa antibodies, the complex of the 230-kDa and the 48/45-kDa gamete surface antigens might be partially dissociated and the uncomplexed form of the 230-kDa is then immunoprecipi- tated by such antibodies.

Triton X-100 extract of radio-iodinated gametes was subjected to sequential immuno- depletion for 4, 8, 12 and 16 cycles with rabbit anti-230-kDa serum. The resulting superna- tants at the end were tested with the mAb IIC5BI0 to quantitate the ratio of 230-kDa and the 48/45-kDa doublet before and after immunodepletion. As shown in Fig. 2B, rabbit antiserum against the 230-kDa continues to bring down the 230-kDa protein even at the 16th cycle of successive immunoprecipitation of the same extract (Fig. 2B lanes 1-16). On the other hand, the 48/45-kDa (uncomplexed and the 230 kDa-associated) was completely im- munoprecipitated by the mAb IIC5B10 in just 2 or 3 rounds (Fig. 2A lanes 1-3) leaving

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behind uncomplexed form of the 230-kDa which could then be immunoprecipitated by rabbit anti-230-kDa serum (Fig. 2A lane 3, in parentheses).

These results clearly suggest that rabbit anti- 230-kDa antibodies react with the uncom- plexed form of the 230-kDa antigen and also that in the presence of such antibodies, the complex of the 230-kDa and the 48/45-kDa antigens undergoes slow dissociation. Such disruption of the complex would imply a progressive reduction in the ratio of 230 kDa:48/45 kDa in the complex form of these antigens immunoprecipitated by mAb IIC5B10. To quantitate the ratio of 230 kDa:48/45 kDa in the complex, final super- natant fluids after 4, 8, 12 and 16 immunode- pletion cycles with rabbit anti-230-kDa serum were tested by immunoprecipitation with the mAb IIC5BI0 (Fig. 2B, lanes marked as 0, 4, 8, 12, 16 in parentheses). Radioactivity in the regions of the gels corresponding to the 230 and 48/45-kDa doublet was measured using a gamma counter. As predicted, the ratio of 230 kDa:48/45 kDa decreased from 0.74 (before depletion) to 0.13 after at least 8 cycles of immunodepletion

The results in Fig. 2 suggest that rabbit antibodies against the 230-kDa protein (rab- bits 110 and 111) can bind the uncomplexed form as well as the 48/45 kDa-associated form of the 230-kDa antigen. Reactivity of rabbit anti-230-kDa antibodies to complexed form of the 230-kDa antigen was earlier shown by immunoprecipitation after chemical cross-link- ing of 230-kDa with the 48/45-kDa antigens in the complex [10]. We have also found that rabbit 110 antibodies can immunoprecipitate the 230-kDa and 48/45-kDa complex eluted from the mAb IIC5Bl0-Sepharose immunoaf- finity beads (not shown). For reasons not yet clear, most antibodies against the 48/45-kDa doublet do not cause dissociation as they co- immunoprecipitate the 230-kDa antigen also. In contrast, antibodies against the 230-kDa protein, like rabbit 110 and 111 sera (present studies), mAbs reported elsewhere [3] and others (> 10 independent mAbs) produced in our laboratory (Wizel and Kumar, unpub-

Page 6: Further characterization of interactions between gamete surface antigens of Plasmodium falciparum

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lished), all appear to perturb the interaction between the 230 and 48/45-kDa antigens. We have not found any possible association between ability of these antibodies to disrupt the complex and their effect on the infectivity ofgametocytes in the mosquitoes since some of these antibodies block infection and others do not.

Accessibility of disulfide and sulJhydryl bonds. SDS-PAGE analysis had earlier shown [2] that the -S-S bonds are not directly involved in the formation of complex between the 230-kDa and 48/45-kDa antigens. They, however ap- pear to be crucial for the formation of TB epitopes of most blocking antibodies reported so far [4,9]. We have recently reported identification of a reduction-insensitive epi- tope, shared among several gametocyte anti- gens, which is a potential target of P. falciparum TBI [16]. Thus both, reduction- sensitive and -insensitive epitopes are targets of TBI.

A series of experiments was conducted to investigate the role of disulfide bonds by analyzing accessibility of disulfide bonds in various components of the complex of 3 target antigens. To assess reduction of these antigens, an assay based on shift in the electrophoretic mobilities from 230, 48/45 kDa to 260, 59/53 kDa, and in some cases loss of mAb IIC5B10 reactivity to reduced antigens, was employed. Initially intact gametes (surface radio-iodina- ted) were treated with DTT (5-10 mM, 30 min at room temperature), followed by alkylation with IAA (10-20 mM, 30 min at room temperature). SDS-PAGE patterns were very similar between untreated and treated parasites and therefore the disulfide and sulfhydryl groups were not accessible for modification in intact parasites (not shown).

Since the disulfide and sulfhydryl groups were not reduced in the intact parasite associated antigens, next we tested Triton X- 100 solubilized parasite antigens for reduction and/or alkylation. After solubilization of gametes in non-ionic detergents, these groups were not accessible for reduction and alkyla- tion (Fig. 3A lanes 1-4). On the other hand, if

A B

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Fig. 3. Reduction and alkylation of gamete surface antigens. In panel A, Triton X-100 extracts of surface radio-iodinated gametes were treated with 0 (lane I), 25 mM DTT (lane 2), 50 mM IAA (lane 3) and DTT/IAA (lane 4). In panel B, Triton X-100 extracts were first treated with 0 (lane I), 0.1 M glycine-HCl pH 2.7 (lane 2), 0.1 M triethylamine pH 11.7 (lane 3), 8 M urea (lane 4), 6 M GnHCI (lane 5) and 5 M NaCI (lane 6) for at least 60 min at room temperature prior to reduction and alkylation with 25 mM DTT (60 min) and 50 mM IAA (60 min). Samples were immediately analyzed by 5-15% SDS-PAGE. Pre-stained high molecular weight standards from Bethesda research laboratories were used to calibrate the gels. Positions of nonreduced and reduced forms of 230-kDa and 48/45-kDa proteins are indicated by diamonds on the left of panel A

and the right of panel B.

the extracts were pretreated with chaotropic agents or at acidic and basic pH, the disulfide and sulfhydryl groups were now easily mod- ified by treatment with DTT and IAA respectively (Fig. 3B lanes 1-6). In these studies we also observed a direct effect of IAA treatment alone, especially on the 48-kDa member of the 48/45-kDa doublet. After IAA treatment the 48-kDa migrated with the 45- kDa molecule. Since the sequence of any of these proteins is not known it is difficult to explain this phenonmenon. It is, however, possible that free -SH groups in the 48-kDa molecule are alkylated, resulting in the ob- served shift in electrophoretic mobility. Mod-

Page 7: Further characterization of interactions between gamete surface antigens of Plasmodium falciparum

ifications of disulfide and sulfhydryl groups in the 230- and 48/45-kDa proteins do produce significant changes in their electrophoretic behavior. However, when analyzed in NEPHGE 2-gel electrophoresis, the pl values were not different; 6.3 -t- 0.2 and 6.5 for the 230-kDa before and after reduction respec- tively and 5.8 _+ 0.1 and 5.9 for the 48/45-kDa before and after reduction (data not shown).

Earlier studies have suggested that in general the disulfide groups are buried in the interior of the globular molecules [17,18]. In the above experiments on chemical modification of these groups, we argued that antigens could become susceptible to treatment with DTT and IAA either because they are denatured or due to dissociation of the complex exposing various groups for modification. To differentiate between these 2 possibilities, we separated the 230-kDa protein from the 48/45-kDa by phase separation in Triton X-114 [8]. Aqueous and the detergent phases were then tested for reduction and alkylation with or without denaturation with guanidine hydrochloride. In these experiments too, the disulfide and sulfhydryl groups were not accessible unless subjected to denaturation treatments (not shown). Inaccessibility even after separation of the 230-kDa from the 48/45-kDa suggest that these groups are not buried in the areas of polypeptide chains interactions in the complex.

The studies presented here have focused on the complex of P. falciparum gamete surface antigens (230 kDa and 48/45 kDa) which have been independently shown to be targets of malaria TBI. Treatment of parasite extracts with chaotropic agents, at extremes of pH and even antibodies against the 230-kDa antigen, always caused dissociation of the complex. Studies on the accessibility of disulfide and sulfhydryl groups for chemical modifications could provide further information on their role in the target antigens of P.falciparum TBI. The conditions described here for reduction of gamete surface antigens also facilitated identi- fication of a reduction-insensitive (non-con- formational) epitope target of P. falciparum TBI. In addition to being continuous in nature, this epitope is shared among proteins (230-

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kDa, 48/45-kDa, and 27-kDa) produced in the gametocytes and conserved in isolates of diverse geographic locations [16].

Acknowledgements

These studies were supported by NIH grant AI24704 and funds from the John D. and Catherine T. MacArthur foundation. B.W. received a fellowship from the Fulbright program during the initial phase of these studies.

References

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2 Rener, J., Graves, P. M., Carter, R., Williams, J. L. and Burkot, T. R. (1983) Target antigens of transmission- blocking immunity on gametes of Plasmodium falciparum. J. Exp. Med. 158, 976--981.

3 Quakyi, I. A., Cartcr, R., Rener, J., Kumar, N., Good, M. F. and Miller, L. H. (1987) The 230-kDa gamete surface protein of Plasmodiumfalciparum is also a target for transmission-blocking antibodies. J. Immunol. 139, 4213-4217.

4 Vermeulen, A. N., Ponnudurai, T., Beckers, P. J. A., Verhave, J., Smith, M. A. and Meuwissen, J. H. E. (1985) Sequential expression of antigens on sexual stages of Plasmodiumfalciparum accessible to transmis- sion blocking antibodies in the mosquito. J. Exp. Med. 162, 1460 1476.

5 Grotendorst, C. A., Kumar, N., Carter, R. and Kaushal, D. C. (1984) A surface protein expressed during the transformation of zygotes of Plasmodium gallinaceurn is a target of transmission blocking antibodies. Infect. lmmun. 45, 775-777.

6 Kaslow, D. C., Quakyi, I. A., Syin, C., Raum, M. G., Keister, D. B., Coligan, J. E., McCutchan, T. F. and Miller, L. H. (1988) A vaccine candidate from the sexual stage of human malaria that contains EGF-like domains. Nature 333, 74-76.

7 Fries, H.C.W., Lamers, M.B.A., Deursen, J.B., Ponnu- dural, T. and Meuwissen, J.H.E. (1990) Biosynthesis of the 25 kDa protein in the macrogametes/zygotes of Plasmodium falciparum. Exp. Parasitol. 71,229-235.

8 Kumar, N. (1985) Phase separation in Triton X-114 of antigens of transmission blocking immunity in Plasrno- dium gallinaceum. Mol. Biochem. Parasitol. 17, 343- 358.

9 Carter, R., Graves, P. M., Keister, D. B. and Quakyi, I. A. (1990) Properties of epitopes of Pfs 48/45, a target of transmission blocking monoclonal antibodies on ga-

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12 Ifediba, T. and Vanderberg, J. P. (1981) Complete in vitro maturation of Plasmodium falciparum gameto- cytes. Nature 294, 364-366.

13 Nijhout, M. M. (1979) Plasmodium gallinaceum: exflagellation stimulated by a mosquito factor. Exp. Parasitol. 48, 75 80.

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16 Wizel, B. and Kumar, N. (1991) Identification of a continuous and cross-reacting epitope for Plasmodium falciparurn transmission blocking immunity. Proc. Natl. Acad. Sci. USA. 88, 9533 9537.

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