Molecular and Biochemical Parasitology, 34 (1989) 79-86Elsevier

MBP 01119

79

Polymorphism of a 35-48 kDa Plasmodium falciparum merozoite surfaceantigen

Brian Fenton1, John T, Clark2, Christina F, Wilson2, Jana S. McBride2 andDavid Walliker1

'Departments of Genetics and 2Zoology, University of Edinburgh, Edinburgh, Scotland, u.K.

(Received 6 October 1988; accepted 14 November 1988)

Glycoproteins of the asexual blood stages of Plasmodium falciparum were labelled with radioactive glucosamine and analysedby two-dimensional electrophoresis. Four major glycoproteins were detected in all eight parasite isolates studied. Two of the gly-coproteins, designated GP2 and GP4, were invariant among the isolates, while the other two GP1 and GP3 were found to bepolymorphic in both their biochemical and antigenic properties. By immunoblotting and immunoprecipitation with specific mon-oclonal antibodies, the two polymorphic glycoproteins were identified as surface antigens of merozoites.

Key words: Plasmodium falciparum, Two-dimensional electrophoresis, Monoclonal antibody; Glycoproteins; Antigenic diversity.

Introduction

A large number of antigens expressed by theinfective stages of Plasmodium Jalciparum ma-laria have been identified in an attempt to pro-duce a vaccine against this parasite [1]. Particularattention has been given to antigens associatedwith the surface of the merozoite. The first majorantigen identified was a glycoprotein denotedPMMSA (the precursor to the major merozoitesurface antigens). The antigen exists as a series ofallelic variants distinguishable by monoclonal an-tibodies (McAbs) [2,3].

A second glycoprotein of the merozoite sur-face has been described which is of interest as acandidate vaccine antigen because parasite growth

Correspondence address: Brian Fenton, Genetics Depart-ment, University of Edinburgh, West Mains Road, Edin-burgh EH9 3JN, U.K.

Abbreviations: 2D-PAGE, two-dimensional polyacrylamidegel electrophoresis; SDS, sodium dodecyl sulphate; rEP, is-oelectric point; McAb, monoclonal antibody; GP, glycopro-tein; PBS, phosphate-buffered saline; DMSO, dimethyl sul-phoxide; PPO, 2,5-diphenyloxazole; PMMSA, precursor to themajor merozoite surface antigens.

is inhibited in vitro in the presence of monoclonalantibodies recognising this molecule [4,5].

In the present work, we show that, likePMMSA, different forms of this antigen exist inP. Jalciparum populations, distinguishable by dif-ferential reactivity with McAbs and mobility us-ing two- dimensional polyacrylamide gel electro-phoresis (2D-PAGE). We also describecharacteristics of other major glycoproteins ofblood forms detected by 2D-PAGE.

Materials and Methods

Plasmodium Jalciparum isolates and clones. TheP. Jalciparum clones and isolates used in this studywere the clones T9-94, T9-96, T9-101 (Thailand),HB3 (Honduras), 3D7 (The Netherlands), andthe isolates K29 (Thailand), Palo Alto 17(Uganda) and FCB-1 (Colombia). They differfrom one another in one or more genetically de-termined markers such as alloenzymes, drug sen-sitivity and DNA markers [6-8]. The parasiteswere obtained from the World Health Organisa-tion Registry of Standard Strains of Malaria Par-asites held at the Genetics Department of Edin-burgh University. Asexual stages of the parasite

0166-6851/89/$03.50© 1989 Elsevier Science Publishers B.Y. (Biomedical Division)

80

were grown in vitro [9] and synchronised by sor-bitol treatment [10].

Radiolabelling of parasites. Parasites were la-belled with [3H]_ or [14C]glucosamine or [35S]meth-ionine for 16 h during development from tropho-zoite to schizont stage [11].

Experiments using the immunoblot techniquedescribed below required large amounts of unla-belled parasite material, as well as labelled par-asites. To achieve an identical composition of bothlabelled and unlabelled material, parasites in one25-ml culture were synchronised and grown untilthey consisted of 5-10% trophozoites. 5 ml of thisculture was removed and radiolabelled. Whenschizonts were present in both cultures they werethen harvested.

Parasites pellets were solubilised in bufferssuitable for analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-P AGE)[12] or 2D-PAGE [13].

Monoclonal antibodies and indirect immunoflu-orescence assay. Mouse McAbs were produced ashas been described [14]. Hybridomas 12.3-2-2 and13.4-2-1, producing McAbs 12.3 and 13.4, re-spectively, were derived by fusion of NSI mye-loma cells with spleen cells from BALB/c miceimmunised with schizonts of P. falciparum clonesT9-96 and T9-94, respectively. The characteris-tics of McAbs 12.2 and 12.8 have been reportedelsewhere [2,15].

S,train reactivities of the McAbs were deter-mined by indirect immunofluorescence assay(IF A) titrations of ascitic fluids on acetone-fixedschizonts of a panel of P. falciparum isolates[2,14].

lmmunoprecipitation. Parasite antigens were ex-tracted in 1% (v/v) NP40 and immunoprecipi-tated with McAbs as described [2]. Human anti-bodies were purified from pooled Gambianimmune sera by adsorption to nitrocellulose-bound antigen immunopurified using McAb 13.4from T9-94 parasites [16].

SDS-polyacrylamide gel electrophoresis. SDS-PAGE of uniform or gradient acrylamide wasused to fractionate proteins according to Mr.

Standard proteins (myosin, 205 kDa; galactosid-ase, 116 kDa; phosphorylase b, 97.4 kDa, bovineserum albumin, 66 kDa; ovalbumin, 45 kDa; andcarbonic anhydrase, 29 kDa (Sigma ChemicalCo.) were also included.

Two-dimensional polyacrylamide gel electropho-resis andfluorography. 2D-PAGE separates pro-teins firstly by charge, and secondly by Mr [13].Using a BioRad multigel cell, up to six gels wererun at once using the method as described [11].After electrophoresis, gels containing labelledproteins were fluorographed [17].

In some experiments the position of certain ra-diolabelled immunoprecipitated antigens wascompared to co-electrophoresed unlabelled totalparasite proteins on the same 2D-PAGE gel bythe following method. The position of the radio 1-abe lied antigen under study was marked on thegel by a pin-prick. The gel was then rehydratedby sequential 2-h incubations in 100%, 75%, 50%and 10% glycerol (v.v in H20). 2,5-Diphenylox-azole (PPO) was removed from the gel by washesin dimethyl sulphoxide (DMSO), and the gel wasthen stained with silver [18]. The position of themarked antigen among the other silver-stainedproteins of P. falciparum was then identified.

lmmunoblotting. Approximately 0.5 mg of unla-belled parasite proteins, together with 20000 cpmof [14C]glucosamine-labelled or 200000 cpm of[35S]methionine-labelled components added asmarkers, separated by either SDS-PAGE or 2D-PAGE, were transferred onto nitrocellulose [19].Blots were incubated with ascitic fluids (11100 di-lution) and bound McAbs detected using perox-idase-conjugated anti-mouse IgG (11300 dilu-tion), followed by development with 4-chloro-l-naphthol [20]. Blots containing 14C-labelled anti-gens were dried and exposed to Hyper-MP film(Amersham) in an X-ray cassette (Kodak) at-70°C.

Results

Glycoproteins of P. falciparum. Glycoproteins oftrophozoite and schizont stages were labelledmetabolically with [3H]glucosamine in six cul-tures of P. falciparum, and compared by 2D-

\

94

81

*GP2_GP3

GP4

307kO.

·200

a

HB3

GPl

-GP2

GPl

b

GP2

~ GP3

GP4 _ 40

• 50

96 • 200-GPl

~GP2 • 50

GP4 ..• GP3

d6.0 6.8 4.7

Isoelectric poi n t

PAGE. In all parasites, four major glycosylatedcomponents were identified which are denotedhere as GPl, GP2, GP3 and GP4. Fig. 1 illus-trates these glycoproteins in four cloned para-sites, T9-94, T9-96, 3D7 and HB3.

Characterisation of CP3. GP3 was the glycopro-tein which exhibited most variation in differentparasites. Five different variants could be identi-fied by 2D-PAGE, ranging in size from 35.2 kDato 48.4 kDa and in isoelectric point (IEP) from4.6 to 5.2 (Table I). The characteristics of GP3 inclones and isolates of P. falciparum were studiedfurther by immunoblotting, immunoprecipitationand immunofluorescence experiments. The re-sults are summarised in Table 1.

GP3 from three clones was reacted with McAbs12.3 and/or 13.4 on immunoblots of componentsseparated by 2D-PAGE (Fig. 2). In clone 3D7,the antigen reacted positively with both anti bod-

• 40

6.0 6.8

Fig. 1. Two-dimensional PAGE of glycoproteins. The four parasite clones T9-94, 3D7, HB3 and T9-96 were labelled with glu-cosamine. 2D-PAGE was carried out, resolving four predominant glycoproteins GP1, GP2, GP3 and GP4, as indicated.

c

4.7

ies (Fig. 2b and c) and had an Mr of 46100 andIEP of 4.7.

In clone T9-96, GP3 was recognised only byMcAb 12.3, and on 2D-PAGE gels possessed twolobes, with an IEP of 5.0 and 5.2 and an Mr of36300 and 35200, respectively (compare Fig. Idand Fig. 2d). Clone T9-94 reacted only withMcAb 13.4 and possessed an IEP of 4.6 and anMr of 48400 (Figs. la and 2a). McAb 13.4 alsospecifically immunoprecipitated the antigen fromdetergent extracts of this parasite, metabolicallyradiolabelled with a mixture of amino acids, his-tidine or· glucosamine (results not shown).

Fig. 3 shows further examples of differentialrecognition of GP3 variants in all the parasite linesstudied either by monoclonal antibodies in im-munoprecipitation (panel a) or immunoblottingtests (panel b). Thus, McAb 13.4 immunoprecip-itated a single [3H]glucosamine-labelled antigenfrom parasites 3D7, T9-94 and FCB-l (Fig. 3a,

82

TABLE r

Characteristics of GP3 in different isolates of P. falciparum

Parasite Electrophoresis McAb 12.3 reactivity McAb 13.4 reactivity Variant formMr IEP Biota rPTNa rFAb Biota rPTNa rFAb

HB3 45.0 4.8 - <10 nt <10 V3D7 46.1 4.7 ++ nt 104 ++ + 103 vrT9-94 48.4 4.6 104 ++ ++ 105 VIIFCB-1 48.4 4.6 nt nt 104 ++c ++c 105 VIIT9-96 35.2/36.3 5.2/5.0 ++ ++ 104 - <10 rK29 45.0 4.7 nt nt <10 nt nt <10 IIIT9-101 36.8 ntd ++ nt 104 nt nt <10 IIPalo Alto 17 37.3 ntd ++ nt 104 nt nt <10 rvaResuits of immunoblots and of immunoprecipitations were scored as + and + + where positive and as - where negative; nt, nottested.brFA results are given as titres of ascitic fluids, i.e., the greatest dilutions positively reactive with approx. 104 acetone-fixed schi-zonts.cFor results on isolate FCB-1 see also refs. 4 and 21.dResults were obtained only by SDS-PAGE.

94 kO• 200307

• 50_13.4

• 40a b

307

• 40

96 ·200

• 50

c J6~8 •6.8

d.4.7 •6.0

Isoe/ectric poi n t

Fig. 2. Two-dimensional PAGE and immunoblotting. Proteins from the three parasite clones T9-94, 3D7 and 96 were separatedon 2D-PAGE gels and blotted on to nitrocellulose. These blots were then reacted with either monoclonal antibody 12.3 or 13.4,

identifying antigens in the 2D-PAGE gels.

kD

200 If

13.4McAb Hum.cx.GP3

kD

b _ 200i•

McAb 12.3

83

• 5050 -l ..111111 ...·.··~~

• 30 .'30 -I

col 0)

CD<.)LL

co0)

r-o,....(")coI

Fig. 3. Antigenic and electrophoretic polymorphism of GP3 in different isolates of P. falciparum. Parasite proteins were reactedwith antibodies either by immunoprecipitation (a) or by immunoblotting (b). The parasites used were T9-96, 3D7, T9-94,

FCB-1, T9-96, Palo Alto 17, T9-101 and HB3.

J

lanes 2-4) but failed to react with antigen of par-asite T9-96 (lane 1). However, GP3 from T9-96was recognised both by human polyclonal anti-bodies against GP3 of T9-94 (Fig. 3a, lane 4), andby McAb 12.3 (Fig. 3b, lane 1). McAb 12.3 alsoreacted positively with GP3 variants of parasitePalo Alto 17 and T9-101 (37.3 and 36.8 kDa, re-spectively; Fig. 2b, lanes 1, 2 and 4), but failed torecognise the antigen of T9-94 and HB3 (Fig. 2b,lanes 2 and 5).

The results were confirmed by IFA tests withthe McAbs on schizonts from the eight parasitelines (Table I). In addition, in repeated IFA test-ing of parasites maintained in long-term culturesfor up to 5 years, no change in phenotype was de-tected in any of the strains. Thus, the serotypicvariants of GP3 detected by McAbs in IFA arephenotypically stable characteristics of P. fa lci-parum grown in vitro.

Characterisation of GPI. GPI is the precursor ofthe major merozoite surface antigens denotedPMMSA, PSA or P195 [2,22,23]. The identity ofGPI has been confirmed by 2D-PAGE of im-munoprecipitates from labelled proteins of para-site T9-94 using antibodies to this molecule. On2D-PAGE, four forms of this glycoprotein can bedetected, ranging in Mr from 185000 to 205000and in IEP from 6.47 to 6.6 [11].

Characterisation of GP2 and GP4. GP2 and GP4,were invariant in all the parasites studied. GP2,(Mr 51200, IEP 4.9) is also labelled with methi-onine. GP4 could not be identified with any pre-viously studied methionine-labelled proteins by2D-PAGE analysis; in all the isolates studied,GP4 was found in the same position, with an Mrof 40300 and an IEP of 4.7. It is not known ifeither of these glycoproteins is a breakdownproduct of PMMSA.

84

Discussion

The principal novel finding in this study is thestructural diversity of a glycoprotein, denotedGP3, among different P. falciparum isolates. Wehave also identified variant forms of a second gly-coprotein, denoted GP1, and two other glycopro-teins GP2 and GP4 which appear to exhibit novariation.

The antigen GP3 is a surface component ofmerozoites. It is recognised by the McAb 13.4which can inhibit parasite growth in vitro [4,21].However, McAb 13.4 reacts with only a minorityof parasite isolates [4].

In the present study a molecule was found toreact with both McAb 13.4 and McAb 12.3 in theparasite clone 3D7, suggesting that theepitopesrecognised by these monoclonals are on the samemolecule. It was also found that human poly-clonal antibodies to the antigen recognised byMcAb 13.4, reacted with the antigen in T9-96parasites, a clone which did not react with McAb13.4. The molecular weight of the antigen recog-nised by these human antibodies was the same asthat of the antigen recognised by 12.3. These ob-servations, and the information obtained by 2D-PAGE, suggested that McAb 12.3 and 13.4 reactwith different epitopes of the same antigen.

A survey of parasite strains tested with bothMcAbs 12.3 and 13.4 found that a large propor-tion bound to one or both of these monoclonalantibodies [4 and McBride unpublished]. How-ever, a small number of P. falciparum parasitesreact with neither antibody (e.g., HB3).

Using genetic analysis, it was demonstrated thatdifferent forms of GP1 (PMMSA) were inheritedas alternatives and behaved as allelic forms of thesame gene [3]. A second antigen, GP3 ((recog-nised by McAb 12.3), underwent recombinationwith GPl. GP3 and GP1 were therefore productsof different genes. However, in the study, onlyGP3 of the parental cone 3D7 was detected byMcAb 12.3; the other parental clone, HB3, wasnegative. The lack of reactivity could have beendue to the deletion of this gene, as has been notedfor other parasite genes [24-27], or to lack ofreactivity with the antigen. We have now dem-onstrated that the glycoprotein GP3 exists in HB3,and that it does not react with the McAb. It seems

likely that the different forms of the GP3 antigenwere inherited as alternatives and were thereforeby allelic forms of the same gene. Conclusiveproof may be obtained by using a McAb whichreacts positively with the GP3 of HB3, but notwith 3D7.

Glycoproteins in the range of 45-56 kDa havepreviously been identified in a number of P. fal-ciparum isolates, and some or all of these maycorrespond to GP3 described in this work. How-ard and Reese [28] used 2D-P AGE to study theglycoprotein composition of the FVO isolate. Intheir study, an acidic glycoprotein of 46 kDa wasidentified in two-dimensional gels of glucosa-mine-labelled parasites. Stanley et al. [29] de-scribed a surface glycoprotein of 50 or 56 kDa indifferent strains. Ramasamy [30] described amyristilated glycoprotein of 45 kDa in the FCQ27strain. Epping et al. [5] identified a molecule of51 kDa on the merozoite surface. The two differ-ent MrS calculated [5,30] appear to be artefac-tual, as the same molecule has now been studiedusing molecular biological techniques and thecomplete DNA sequence of one variant, denotedthe 45000 Da MSA, obtained [31].

Studies on this antigen have been complicatedby the fact that different laboratories have ob-tained different Mr estimations for the same var-iant of the antigen. Because of this, it has beendifficult to determine the extent of real variationin this antigen. In this study we have used McAbs,radiolabelling and electrophoresis to demonstratethat the same merozoite surface antigen variesextensively in antigenic structure, apparent Mrand IEP in genetically distinct isolates and clones.The different forms of the antigen appear to be-have as alternatives, at the population level andfollowing genetic exchange, suggesting that thedifferent forms are encoded by allelic variants ofthe same gene. Given the surface location of theantigen, the polymorphism of this molecule maybe of significance in enabling the parasite to avoidhost immunity.

Acknowledgements

We would like to thank Mr. Richard Fawcettand Mrs. Jane Robinson for expert technical as-sistance. We also thank Prof. Geoffrey Beale and

Dr. Ann Fenton for critical reading of the man-uscript. The study was financed by grants from theMedical Research Council of Great Britain, TheWellcome Trust and the UNDPlWorldBankIWHO Special Programme for Research and

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,