Molecular and Biochemical Parasitology, 49 (1991) 35 44 © 1991 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/91/$03.50 ADONIS 016668519100337W

MOLBIO 01599

35

Processing of the Plasmodiumfalciparum major merozoite surface protein-1" identification of a 33-kilodalton secondary processing product

which is shed prior to erythrocyte invasion

Michael J. B l ackman 1, Hi l ton Whit t le 2 and A n t h o n y A. Ho lde r 1

~National Institute for Medical Research, Mill Hill, London, U.K.; and 2Medical Research Council Laboratories, Fajara, The Gambia

(Received 19 March 1991; accepted 11 May 1991)

We have previously shown that only a single 19-kDa fragment of the Plasmodium falciparum major merozoite surface protein (MSP1) is carried with an invading merozoite into the infected red cell. This fragment (MSP119) is derived from the C- terminal, membrane-bound end of a major product, MSP142, of the primary stage of MSPI proteolytic processing. Using a monoclonal antibody mapped to an epitope within the N-terminal region of MSPI42, we have shown that a soluble 33-kDa polypeptide (MSP133) corresponding to the N-terminal region of MSP142 is shed into culture supernatants during merozoite release and erythrocyte invasion. These observations provide further evidence that the secondary processing of MSPI42 involves a highly site-specific proteolytic activity.

Key words: Malaria; Plasmodium falciparum; Merozoite surface protein-1.

Introduction

A number of reports have demonstrated that the precursor to the major merozoite surface proteins of the malaria parasite Plasmodium falciparum, here referred to as MSP1 (merozoite surface protein 1), is sub- jected to proteolytic processing at or just prior to the release of merozoites from the mature schizont [1-4]. On the surface of free mero- zoites the protein is present in the form of a non-covalently associated complex of at least 4 major polypeptide fragments [5]. The C- terminal, membrane-bound region of the processed MSP1 complex is represented by a

Correspondence address: Michael J. Blackman, Division of Parasitology, National Institute for Medical Research, Mill Hill, London NW7 1AA, U.K.

Abbreviations: mAb, monoclonal antibody; EBV, Epstein Barr virus;/~-ME, beta-mercaptoethanol; IF, indirect immunofluor- escence; B-Gal, beta-galactosidase; IPTG, isopropyl /~-o-thio- galactopyranoside; NCP, nitrocellulose membrane.

major glycosylated 42-45-kDa polypeptide, MSP142, as well as by a minor fragment, MSP119, which is thought to be derived, via a secondary processing event, from the extreme C-terminal part of MSP142 [2,5]. Following erythrocyte invasion only MSP119 is detectable in the newly-invaded (ring-stage) parasite [6], and it has been suggested that this secondary proteolytic event must proceed to completion for a merozoite to invade an erythrocyte [6]. We report here the epitope mapping of an MSPl-reactive human monoclonal antibody (mAb), X509, to the N-terminal region of MSP142, and the use of mAb X509 to investigate the secondary processing of MSP142. As well as reacting with MSP142 on the merozoite surface, mAb X509 was found to react with a soluble 33-kDa polypeptide which appeared in malarial culture supernatants following schizogony. We present evidence that this 33-kDa species, here denoted as MSP133, represents the N-terminal region of MSP142, which is not taken into the erythro-

36

cyte with the invading merozoite, but is shed from the free merozoite surface before or during invasion.

Materials and Methods

Polyclonal and monoclonal antibodies. Human mAb X509 was produced by Epstein Barr virus (EBV) immortalisation of peripheral blood B lymphocytes obtained from an adult Gambian donor clinically immune to P. falciparum malaria. The transformation protocol used was essentially a modification of that descri- bed by Brown et al. [7]. Isolated peripheral blood mononuclear lymphocytes were infected at a multiplicity of infection of approximately 0.1 with EBV, and plated in medium contain- ing 2 #g m l - ~ phytohaemagglutinin (Wellcome Diagnostics), in round-bottomed microtitre wells (Flow) at 1 × 104 cells/well. At 2-4 weeks following infection, individual wells containing proliferating cells were screened for the presence of antimalarial antibodies by indirect immunofluorescence (IF) on acetone-fixed preparations [8] of red cells containing the T9/96 clone of P. falciparum. Cells from antibody positive wells were cloned immedi- ately by limiting dilution. The line X509 was cloned 3 times at 0.3 cells/well, then main- tained in RPMI 1640 growth medium (Gibco) containing 10% foetal calf serum.

The antibody secreted by the X509 cells (mAb X509) was affinity purified from culture supernatant on a column of rabbit anti-human IgG (Cappel) coupled to protein A Sepharose CL-4B (Pharmacia) with glutaraldehyde [9]. Purified mAb X509 was coupled to cyanogen bromide-activated Sepharose 4B (Pharmacia) according to the manufacturer's recommenda- tions, using 2-5 mg of protein per ml of swollen gel, for use as an affinity absorbant.

Three MSPl-specific mouse mAbs were used; 89.1 (anti-MSP183 [10]); and 2.2 and 12.8 (anti-MSP142 and MSPlw; [5]), kindly provided by J. McBride (Dept. of Zoology, University of Edinburgh, Edinburgh, U.K.).

Polyclonal rabbit antisera were produced to Escherichia coli beta-galactosidase (//-Gal)

purified on p-aminobenzyl 1-thio-/~-D-galacto- pyranoside agarose (Sigma) by the method of Steers et al. [11], from isopropyl /3-D-thio- galactopyranoside (IPTG)-induced bacteria transformed with the plasmid pXY461 [12], and to affinity-purified MSP1 from the Well- come P. falciparum isolate [13].

Culture and metabolic radio-labelling of P. falciparum. Clones T9/94 and T9/96 [14] were maintained in culture and synchronised when required, essentially as described pre- viously [10]. Metabolic radiolabelling of para- sites was performed by resuspending washed synchronous cultures at late schizont stage in methionine-free complete medium (Selecta- mine, Gibco) containing 50 #Ci ml - I [35S]methionine (Amersham) for 2-4 h at a 10% haematocrit. Cultures were then washed and resuspended in normal complete medium for production of both metabolically labelled merozoites and of medium containing labelled parasite components released during schizo- gony.

Preparation of schizonts and naturally released merozoites. Schizonts were enriched from synchronous parasite cultures by centrifuga- tion over 63% isotonic Percoll and used for merozoite production as described [6]. Wa- shed, pelleted schizont and merozoite prepara- tions were stored at -70°C. For analysis by immunoblotting, samples were solubilised directly into boiling SDS sample cocktail (62 mM Tris-HC1 pH 6.8/ 10% glycerol/ 2.3% SDS/ 0.005% bromophenol blue) with or without the addition of beta-mercaptoethanol (/%ME; 5% v/v) as a reducing agent.

Immunoprecipitations. Metabolically radiola- belled material was thawed directly into lysis buffer (50 mM Tris-HC1 pH 8.2/ 5 mM EDTA/ 5 mM EGTA/ 0.5% (w/v) sodium deoxycholate, supplemented with leupeptin, aprotinin, PMSF and TLCK) and extracted for 1 h on ice. Lysates were clarified by centrifugation then incubated with either 5 #1 mAb 12.8 (bound to formalin-fixed S. aureus cells) or 100 /A Sepharose-bound mAb X509

37

for 4 h at 4°C. Supernatants from cultures containing metabolically-labelled schizonts which had been allowed to undergo lysis and red cell reinvasion were centrifuged at 100 000 x g for 1 h before incubation with Sepharose-

bound mAb X509. Immune complexes were washed 4 times with 50 mM Tris-HC1 pH 8.2/ 0.5% (w/v) sodium deoxycholate, then with 100 mM Tris-HC1 pH 8.0/0.5 M NaC1, before subjecting to SDS-PAGE under reducing or non-reducing conditions. Gels were stained with Coomassie blue, destained, soaked for 30 min in Amplify (Amersham), dried at 60°C, and fluorographed at -70°C using preflashed Fujichrome X-ray film.

SDS-PAGE and immunoblotting. Samples were analysed by SDS-PAGE on homoge- neous 7.5% or 12.5% gels, or 10-15% linear gradient gels. Depending on the gel system used, molecular weight markers used were obtained from Pharmacia (low molecular weight; 14400-97000), or Gibco BRL (high molecular weight prestained; 14300-200000). When required, SDS-PAGE-treated proteins were electrophoretically transferred to nitro- cellulose (NCP; Schleicher and Schuell, 0.45 /~m pore size) [15]. Blots were blocked with 5% (w/v) non-fat milk powder in TBS (20 mM Tris-HC1 pH 7.4, 0.5 M NaC1) for 1 h at room temperature, then washed in TBS 0.05% Tween 20 (TBS/T). Blots were probed with first antibody diluted into TBS/T for 2-3 h at 37°C, washed 3 times, then incubated in a 1/ 500 dilution of HRP-conjugated rabbit anti- human, rabbit anti-mouse, or goat anti-rabbit IgG (ICN Immunobiologicals) for 1 h at 37°C. Blots were then washed and developed using 4- chloro-l-napthol (Sigma) as substrate.

Identification of the region of the MAD20 MSP1 gene encoding the epitope recognised by mAb X509. Recombinant pUC8 plasmids MAD/H5.0 and MAD/R0.96 [16], containing overlapping genomic DNA restriction frag- ments of the MSP1 gene of the MAD20 isolate were a kind gift of J. Scaife (Dept. of Molecular Biology, University of Edinburgh, U.K.). Using standard methodology [17],

plasmids were transformed into Escherichia coli strain JM109. The 440-bp BamHI/EcoRI fragment from the MAD/R0.96 insert, and the 846-bp EcoRI/PstI fragment from the MAD/ H5.0 insert were prepared and ligated into BamHI/PstI-digested pUC9 to give a recombi- nant plasmid (denoted TI - IMAD20) with a 1286-bp insert covering the region of the MAD20 MSP1 gene from the BamHI site at nucleotide 3883 to the PstI site at nucleotide 5169 (numbering as in ref. 16). Randomly sized fragments of this insert were expressed as fl-Gal fusion proteins in the vector pXY460, essentially as described previously [2]. Briefly, gel-purified insert from TI - IMAD20 was treated with BAL 31 exonuclease, the reaction being stopped after various times by the addition of EDTA to 50 mM. DNA from the different time points was pooled, made blunt- ended using the Klenow fragment of DNA polymerase, and ligated into SmaI cut, alkaline phosphatase-treated pXY460. JM109 E. coli were transformed with the ligation mix and plated onto selective agar (containing 50 #g ml - l ampicillin) with the addition of 5-bromo- 4-chloro-3-indolyl-fl-D-galactoside (X-Gal) to 40 /~g ml -~. Blue colonies were picked and screened by restriction enzyme analysis of plasmid DNA, and by immunoblotting SDS- PAGE-fractionated, IPTG-induced bacterial lysates, using mAb X509. Inserts of interest were partially sequenced [18] using a Seque- nase kit (United States Biochemical).

Partial purification of mAb X509-reactive MSP133 from culture supernatants. Schizonts enriched from a synchronous culture of T9/96 were recultured in complete medium with fresh red cells at a parasitaemia of 1-5%. After overnight incubation (15 h), culture medium was harvested and centrifuged at 15 000 x g for 10 min at 4°C. The supernatant was made to 50 mM Tris-HC1 pH 7.4, 1 mM EDTA and 0.05% Tween 20, then passed over a pre-eluted 2 ml X509-Sepharose affinity column, via a precolumn of Sepharose 4B, both pre-equili- brated with 50 mM Tris-HCl pH 7.4/ 1 mM EDTA/0.05% Tween 20 (running buffer). The X509-Sepharose was washed with running

38

buffer supplemented with 0.5 M NaC1, then eluted with 0.2 M glycine-HCl pH 2.5/0.15 M NaC1. Fractions were collected directly into 1 M Tris-HCl pH 8.0, and stored at -70°C until analysed by SDS-PAGE and immunoblotting. The eluate contained a 33-kDa polypeptide denoted here as MSP133.

Antibody affinity select. To demonstrate antigenic cross-reactivity between different SDS-PAGE-separated proteins, a modifica- tion of the 'affinity select' method [3,19] was used. Partially-purified MSP133, or total mero- zoite antigen was subjected to SDS-PAGE and transferred to NCP. Strips with the antigen of interest (MSP133 or MSP119) were cut out, incubated for 1 h at room temperature with a 1/20 dilution in TBS/T of a Gambian human immune serum (TS), washed 6 times in TBS/T, then eluted into a minimal volume of 0.2 M glycine-HC1/ 0.15 M NaC1, pH 2.5. Eluates were neutralised with 2 M Tris base, then used to probe blots as described above.

Chymotryptic peptide mapping. [35S]Methio- nine-radiolabelled MSP142 was immunopreci- pitated from merozoite extracts with mAb 12.8, and radiolabelled MSP133 was immuno- precipitated from culture supernatants with mAb X509, as described above. Washed immune complexes were solubilised in SDS sample cocktail containing 0.2 M dithiothreitol (DTT), then carboxyamidomethylated with iodoacetamide, essentially as described by Hirs [20]. Following SDS-PAGE on a 12.5% gel, bands corresponding to MSP142 and MSP133 were detected by autoradiography, excised from the gel, and subjected to chymo- tryptic digestion as described previously [21]. Chymotryptic peptides were analysed by two- dimensional thin-layer chromatography (TLC) on cellulose plates (Cellulose F254, Merck), using isopropanol/acetic acid/water (4:1:1 v/v) in the first dimension, and butanol/acetic acid/ water/pyridine (15:3:12:10 v/v) in the second dimension. Dried plates were sprayed with EN3HANCE (DuPont), and [35S]methionine- containing peptides were detected by fluoro- graphy.

Results

Antigen specificity and epitope local&ation of mAb X509. In IF on acetone-fixed prepara- tions of T9/96 schizonts, mAb X509 gave an immunofluorescence pattern characteristic of antibodies which react with MSP1, delineating intracellular developing merozoites; the mAb also reacted with the surface of free, unfixed merozoites, but did not react with acetone- fixed ring stages (data not shown). The mAb did not react in IF with the T9/94 clone, and in further tests against 14 culture-adapted P. falciparum isolates, the reactivity was found to be strain specific for those isolates expres- sing the 'MAD20-1ike' allelic version of MSP1

200

9 7 -

J

6 8 -

4 3 -

1 2 3 4 Fig. I. Monoclonal antibody X509 recognises the MSP1 of the T9/96 but not of the T9/94 P. falciparum clone. SDS lysates of T9/96 (lanes 1 and 3) and T9/94 (lanes 2 and 4) schizonts were subjected to SDS-PAGE on a 7.5% gel and transferred to NCP. Blots were probed with mAb 89.1 (lanes 1 and 2) or mAb X509 (lanes 3 and 4). Molecular weight markers indicated are myosin (200 kDa), phosphorylase b (97 kDa), bovine serum

albumin (68 kDa) and ovalbumin (43 kDa).

39

[16] (personal communication, J. McBride). Immunoblot analysis confirmed that the

reactivity of X509 was isolate specific, and that it reacted with MSP1. Fig. 1 shows SDS- PAGE-fractionated T9/96 and T9/94 schizont lysates probed with mAb X509, as well as with the MSPl-reactive murine mAb 89.1. Mono- clonal antibody 89.1, which recognises a conserved epitope situated within the N- terminal region of MSP1 [2] reacted with a 205-kDa protein in the T9/96 extract (lane 1), and a 195-kDa band in the T9/94 extract (lane 2), corresponding to the respective MSP1 molecules [22]. In contrast, mAb X509 reacted only with the T9/96 MSP1 (lanes 3 and 4). The minor band at 83 kDa in lane 2 corresponds to 89.1 reactivity with some MSPls3 in the T9/94 schizont extract.

Monoclonal antibody X509 was next com- pared to 2 well characterised murine mAbs in immunoblots of merozoite lysates run under reducing or non-reducing conditions. Fig. 2 lanes B1-B5 show that mAb X509 reacted with a polypeptide with an apparent mobility of 43 kDa after reduction (arrowed), but which migrated with an apparent Mr of 39 000 in the absence of reduction. This reduction- sensitive change in mobility is characteristic of MSP142 [2,5]. When used to probe identical preparations, mAbs 2.2 (data not shown) and 12.8 did not react with reduced merozoite antigens, but bound 2 species of 39 kDa and 19 kDa in non-reduced preparations (lanes A1- A5), corresponding to MSP142 and MSPll9. The epitopes recognised by mAbs 12.8 and 2.2 are reduction-sensitive [5], and present in the MSP119 merozoite surface species derived from the C-terminal region of MSP142. The reactiv- ity of mAb X509 with MSP142 in both reduced and non-reduced forms, together with its complete nonreactivity with MSPI~9, indi- cates that the X509 epitope is not disulphide- restrained, and is situated away from the C- terminal region of MSP142.

Monoclonal antibody X509 was used to screen a library of recombinants expressing random fragments of the 3' region of the MAD20 MSP1 gene in the form of /~-Gal fusion proteins. Sixty clones expressing detect-

A B

9 7 -

6 8 -

4 3 -

3 0 -

18-

1 2 3 4 5 1 2 3 4 5

Fig. 2. Reactivity of monoclonal antibody X509 with reduced T9/96 MSP142. SDS lysates of T9/96 merozoites were subjected to SDS-PAGE on a 12.5% gel under reducing (lanes A1, A5, B1 and B5) or non-reducing conditions (A2-A4, B2-B4), and transferred to NCP. Blots were then probed with either mAb 12.8 (panel A) or mAb X509 (panel B). The band at 43 kDa in lanes B1 and B5 (arrowed) represents mAb X509 reactivity with reduced MSP142. The bands at about 50 kDa in panel B are due to contamination of the merozoite extracts with serum-derived human IgG. Molecular weight markers indicated are phos- phorylase b (97 kDa), bovine serum albumin (68 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa) and

soybean trypsin inhibitor (18 kDa).

able/~-Gal activity were picked, induced, and screened by immunoblotting. Twenty-seven were found to react with mAb X509, as well as with a human serum from a malaria- immune Gambian donor, TS (data not shown). The DNA from clone 2-EBBr, which contained the smallest insert, was partially sequenced. Fig. 3 indicates the position of clone 2-EBBr in relation to the complete MAD20 gene map reported by Tanabe et al. [16]. The insert runs from nucleotide 4415- 4734 inclusive (numbering according to the sequence in ref. 16), encoding amino acid residues Tyr1469 to Ile1574. This region is situated downstream of the N-terminal cleav- age site of the MSP142 of the FCB1 P. falciparum isolate [23]. The region Tyr1469 to Ile1574 lies within the dimorphic block 16 of the MAD20 MSP1 primary sequence [16], explain-

40

H P E H B H E P

B I I

c m Fig. 3. Position of 2-EBBr insert in relation to the complete MAD20 MSP1 gene sequence and the predicted primary sequence of MSP142. Random fragments of the 3' region of the MAD20 gene were cloned into plasmid pXY460 and expressed as/%Gal fusion proteins. Of the recombinant clones expressing products reactive with mAb X509 by Western blot, clone 2- EBBr contained the smallest insert, which was end sequenced. The entire MAD20 sequence is represented in (A); the predicted position of MSP142 [23] (B) and the position of the 2-EBBr insert (C) are shown aligned to this complete sequence. The positions of the conserved (white), semi-conserved (coarse cross-hatched) and variable (fine cross-hatched) sequence blocks [16], and sites for the restriction enzymes BarnHI (B),

EcoRI (E), HindlII (H) and Pstl (P) are shown.

ing the allele specificity of X509 reactivity. Further immunoblot analysis confirmed this specificity (Fig. 4); X509 reacted with the 2-

200

97

68

1 2 3 4 5 6 7 8 9 Fig. 4. Strain specificity of monoclonal antibody X509 reactivity; analysis by Western blot. Lysates of IPTG-induced recombinant E. coli clones expressing/~-Gal fusion proteins 2- EBBr (lanes 1,4 and 7), or pME11 (lanes 2, 5 and 8), or samples of partially-purified/?-Gal only (lanes 3,6 and 9) were subjected to SDS-PAGE under reducing conditions on a 7.5% gel. Loads were adjusted such that equal amounts of fusion protein and/~- Gal were loaded in each lane. After transferral of proteins to NCP, blots were probed with: a rabbit antiserum to /~-Gal (lanes 1 3); a rabbit antiserum to the MSP1 of the Wellcome P. falciparum isolate (lanes 4~6); and mAb X509 (lanes 74). Molecular weight markers indicated are myosin (200 kDa), phosphorylase b (97 kDa) and bovine serum albumin (68 kDa).

EBBr fusion protein, but failed to recognise the /%Gal fusion protein pME11 [2] which encodes Gln1293 to Met1718 of the MSP1 of the Wellcome P. falciparum isolate. These data show that the X509 epitope is located within the N-terminal region of MSP142.

Subprocessing of MSP14e at merozoite inva- sion. shedding of fragment carrying X509 epitope. The lack of any detectable reactivity of mAb X509 with ring stages of the parasite in IF ('and also in immunoblots; see ref. 6)

R NR

-97

- 6 8

- 4 3

- 3 0

-18

-14

Fig. 5. Monoclonal antibody X509 reacts with a soluble 33- kDa parasite-derived polypeptide released into culture super- natants following schizogony. [35S]methionine-radiolabelled T9/96 schizonts were allowed to undergo schizogony, then culture supernatants were collected and incubated with Sepharose-bound mAb X509. Bound proteins were subjected to SDS-PAGE on a 12.5% gel under reducing (R) and non- reducing (NR) conditions, and detected by fluorography. Molecular weight markers indicated are phosphorylase b (97 kDa), bovine serum albumin (68 kDa), ovalbumin (43 kDa), c~- chymotrypsinogen (30 kDa), /~-lactoglobulin (18 kDa) and

lysozyme (14 kDa).

suggested that the polypeptide expressing the X509 epitope might be either degraded or shed from the merozoite surface before erythrocyte invasion. Schizonts metabolically radiolabelled with [35S]methionine were allowed to undergo lysis and erythrocyte reinvasion over a 4 h period, and then culture supernatants were collected and analysed by immunoprecipita- tion using mAb X509. Fig. 5 shows that mAb X509 precipitated a 33-kDa labelled protein, the mobility of which was not reduction sensitive. In further analysis by immunoblot- ting, the 33-kDa species could be detected in samples of culture supernatants subjected to SDS-PAGE, using mAb X509 to probe the blots (data not shown). Furthermore, it was found that the 33-kDa species could be concentrated and partially purified by X509- Sepharose affinity chromatography (data not

1 2 3 4 5 6 7 8 9 10

Fig. 6. The monoclonal antibody X509-reactive 33-kDa polypeptide shares antigenic epitopes with MSP142 but not with MSP1 zg. Antibodies reactive with NCP-immobilised non- reduced 33-kDa protein, or with NCP-immobilised, non- reduced merozoite-derived MSPI~9, were affinity-selected from human immune serum TS. Affinity-purified 33-kDa protein (lanes 1,3,5,7 and 9), or T9/96 merozoite lysates (lanes 2,4,6,8 and 10) were subjected to SDS-PAGE under non- reducing conditions on a 10-15% linear gradient gel, and transferred to NCP. Blots were probed with; mAb 12.8 (lanes I and 2); mAb X509 (lanes 3 and 4); TS serum (lanes 5 and 6); affinity-selected anti-33-kDa TS antibodies (lanes 7 and 8); and affinity-selected anti-MSPl~9 antibodies (lanes 9 and 10). Molecular weight markers indicated are bovine serum albumin (68 kDa), ovalbumin (43 kDa), ~-chymotrypsinogen (30 kDa),

fl-lactoglobulin (18 kDa) and lysozyme (14 kDa).

41

shown). Supernatant from a synchronous, schizont stage culture which had been allowed to undergo reinvasion overnight was run over the affinity column, and the eluted protein subjected to SDS-PAGE under non-reducing conditions and transferred to NCP for use in antibody affinity-select experiments.

Antibody affinity-select experiments were carried out to determine the antigenic relation- ship between the supernatant-derived 33-kDa species, and the merozoite-derived MSP142 and MSP119 proteins. Fig. 6 summarises the results of these experiments. Antibodies reactive with the 33-kDa protein were adsorbed from a human immune serum (TS), eluted and used to probe immunoblots of total merozoite ex- tracts. The antibodies reacted with the MSP142 band recognised by mAb X509 (see lanes 4 and 8), but not with MSP119. The presence of MSPlt9 in the merozoite extracts was confirmed by its reactivity with mAb 12.8 (lane 2). Monoclonal antibody 12.8, however, did not react with the 33-kDa species (lane 1). Antibodies affinity-adsorbed on MSP119 ( f r o m

extracts of total merozoites) reacted with both MSP142 and MSP1]9 (lane 10), but not with the X509-reactive 33-kDa species (lane 9). The simplest interpretation of these data is that the 33-kDa protein shares antigenic determinants with MSP142, but not with MSP119.

To further analyse the relationship between MSPla2 and the 33-kDa species, peptides derived by chymotryptic digestion of immuno- precipitated [35S]methionine-labelled MSP142 and 33-kDa proteins were subjected to two- dimensional TLC followed by fluorography to detect methionine-containing peptides. As shown in Fig. 7, the resultant peptide maps were indistinguishable from each other. In view of the fact that the T9/96 MSPll9 fragment does not incorporate [35S]methionine (unpublished observations, M.Blackman and A. Holder), these results are consistent with the antibody affinity select data in that they strongly suggest that the 33- kDa species is derived from the N-terminal, non-membrane-bound region of MSP142. Pre- sumably, at or just before invasion it is shed from the merozoite surface, leaving the intact

42

MSP142 MSP133

e~

! Fig. 7. Two-dimensional chymotryptic peptide mapping of [35S]methionine-labelled MSP142 and 33-kDa proteins gives identical profiles. Radiolabelled MSP142 immunoprecipitated from merozoite extracts with mAb 12.8, as well as radiolabelled 33-kDa protein immunoprecipitated from culture supernatants with mAb X509, were reduced and carboxyamidomethylated, then digested with chymotrypsin. The resultant peptides were applied to cellulose TLC plates (the point of application is indicated by an arrow in the lower right hand corner of each plate), and subjected to two-dimensional TLC in the first dimension with isopropanol/acetic acid/ water (4:1:1, v/v), and in the second dimension with butanol/acetic acid/water/pyridine (15:3:12:10). Radiolabelled peptides were

detected by fluorography.

MSP119 fragment to be taken into the invaded erythrocyte. It was therefore decided to refer to the 33-kDa species as MSP133.

Discussion

We have presented evidence that the final processing event of the MSPl-derived MSP142 merozoite surface species results in the forma- tion of 2 differentially targeted polypeptide products. One of these, MSPll9 , is known to be carried with the invading merozoite into the erythrocyte, and probably remains membrane- bound throughout the process [6]. The other fragment, MSP133, we have shown to be found in culture supernatants following schizogony, in the form of a soluble protein of apparent Mr 33 000.

On the basis of the criteria used here, the MSP133 and MSP119 polypeptides appear to be

non-overlapping derivatives of MSP142. If this is the case, one or both proteins clearly exhibits anomalous mobility in SDS-PAGE. This is perhaps most likely for MSP119; this species is highly disulphide-bonded and probably pos- sesses a glycosyl phosphatidylinositol anchor [24], both characteristics which could lead to abberant migration. In support of this predic- tion, N-terminal amino acid sequencing of affinity-purified MSPlI9 isolated from T9/94 merozoites has located the N-terminus of MSPll9 to amino acid residue Asn1631 (numbering as in ref. 16) [25]. The N-terminal amino acid residue of the MSP142 of the FCB1 P. falciparum isolate and the T9/94 clone has been identified as an alanine residue analogous to Ala1348 of the MAD20 sequence [23,25]; the sequence of' MSP133 might therefore be expected to run from Ala1348 to Leu1630 (the residue before Asn1631). The total molecular weight of the primary amino acid sequence

from residue Ala1348 to LeUl638 in the MAD20 sequence is 32456 Da, very close to the estimated size of MSP133.

It is significant that under no circumstances was it possible to detect MSP133 associated with washed merozoites, strongly suggesting that the cleavage of MSP142 to MSP133 and MSPlw occurs simultaneously with shedding of MSP133 from the merozoite surface, possibly along with the other non membrane- bound fragments of the MSPl-derived com- plex. This is in contrast to the primary stage of the MSP1 processing, following which all the components of the complex can still be found associated with the merozoite surface [2,5]. Furthermore, and again in contrast to the products of the first stage of MSP1 processing, MSP133 is never found in extracts of very late (segmented) schizonts (A. Holder and M. Blackman, unpublished observations), suggest- ing that the cleavage and shedding of this moitey is a truly extracellular event and does not occur to any significant extent prior to merozoite release. The biological significance of the shedding of MSP133 is presently unknown. It is, however, of interest that at concentrations of up to 100/lg m l - l, purified mAb X509 was found to have no detectable inhibitory effect on erythrocyte invasion in in vitro cultures of T9/96 (M. Kamber, H. Whittle and M. Blackman, unpublished ob- servations). Similarly, rabbit polyclonal anti- bodies to another shed fragment of the merozoite surface complex, MSPls3, have been reported to have little effect on erythro- cyte invasion in vitro' [26].

The fact that supernatant-derived MSP133 reproducibly appeared as a single discrete band in SDS-PAGE, rather than as a series of X509- reactive polypeptides of different molecular weights, suggests that the proteolytic activity responsible for the cleavage of MSP142 is highly site-specific. An investigation of the effects upon erythrocyte invasion of specific, non-toxic inhibitors of this proteolytic activity might provide some clues as to its importance in the invasion process. Given the extracellular location of the secondary processing, and the fact that the relevant cleavage site is within a

43

highly conserved region of the molecule, such a promising approach is currently under investi- gation.

Acknowledgements

This work received financial support from the Medical Research Council (U.K.) and from the United Nations Development Program/ World Bank/World Health Organisation Spe- cial Programme for Research and Training in Tropical Diseases (TDR).

References

1 Holder A.A. and Freeman R.R. (1984) The three major antigens on the surface of Plasmodium falciparum merozoites are derived from a single high molecular weight precursor. J. Exp. Med. 160, 624-629.

2 Holder, A.A., Sandhu, J.S., Hillman, Y., Davey, L.S., Nicholls, S.C., Cooper, H. and Lockyer, M.J. (1987) Processing of the precursor to the major merozoite antigens of Plasmodium Jalciparum. Parasitology 94, 199 208.

3 Lyon, J.A., Geller, R.H., Haynes, J.D., Chulay, J.D. and Weber, J.L. (1986) Epitope map and processing scheme for the 195,000-dalton surface glycoprotein of Plasmodiumfalciparum merozoites deduced from cloned overlapping segments of the gene. Proc. Natl. Acad. Sci. USA 83, 2989 2993.

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