Processing of the Plasmodium chabaudi chabaudi AS merozoite surface protein 1 in vivo and in vitro

MOLECULAR

ELSEVIER

Molecular

and Biochemical

Parasitology

72 (1995) 11 l-l 19

&%WWAL PARASITOLOGY

Processing of the Plasmodium chabaudi chabaudi AS merozoite surface protein 1 in vivo and in vitro Kieran P. O’Dea a, Paul G. McKean a31,Alan Harris b, K. Neil Brown a-* aDivision of Parasitology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 IAA, UK b Sequency and Synthkis Section, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 IAA. UK Received 25 January

1995; accepted

18 May 1995

Abstract Processing of the Plasmodium merozoite surface protein 1 (MSP-1) has been described for parasites maintained under in vitro conditions. We have now demonstrated, using CBA/Ca mice infected with Plasmodium chabaudi chabaudi AS, that MSP-1 processing also occurs in vivo. The major proteolytic cleavage sites and a processing scheme were deduced from N-terminal amino-acid sequences of the MSP-1 breakdown products. Comparison of MSP-1 processing in P. falciparum and P.c. chabaudi indicates a degree of conservation and in two cases the position of protease cleavage appears identical. Significant amounts of MSP-1 polypeptides are found in plasma during schizogony. Various aspects of MSP-1 processing including immunological and physiological reactions in the host during the critical period of schizogony can now be

examined in vivo. Keywords: Plasmodium chabaudi chabaudi; Plasmodium falciparum; Malaria; Merozoite surface protein 1; Processing

1. Introduction One of the most intensively studied molecules of Plasmodium is the merozoite surface protein 1 (MSP-1). Experiments using Plasmodium falcipat-urn parasites adapted to in vitro culture, have shown that the protein is synthesized at the trophozoite stage as a high-molecular-mass precursor, which

Abbreuiations: mAb, monoclonal antibody; MSP-1, merozoite surface protein 1. * Corresponding author. Tel.: (44-181) 959-3666; Fax: (44-181) 906-4477. ’ Present address: Department of Biochemistry, Imperial College of Science, Technology and Medicine, South Kensington, London SW7 2AY, UK 0166-6851/95/$09.50 0 1995 El sevier Science B.V. All rights reserved SSDI 0166-6851(95)00090-9

is then cleaved into a number of smaller polypeptides [l]. The proteolytic cleavage events have been divided into primary and secondary phases. Primary processing, which may occur prior to schizont rupture, breaks the 195kDa precursor into four major fragments of 83, 28-30, 38 and 42 kDa. The secondary phase, presumed to occur sometime after merozoite release, involves the cleavage of the Cterminal 42-kDa polypeptide into 33 and 19-kDa fragments [2,3]. This cleavage is calcium and serine-protease dependent [4]. The 19-kDa fragment alone is carried through into newly invaded erythrocytes, while a complex of non-covalently associated polypeptides, including the 33-kDa species, is shed into the culture supernatant [4]. Evidence for primary processing of the MSP-1

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has also been demonstrated for the simian parasite P. knowlesi and the rodent malaria parasites P. yoelii and P. chabaudi chabaudi [5-71. However, these studies have all been performed on parasites during in vitro culture and the studies on P. knowlesi have indicated the occurrence of artifactual proteolysis during parasite extraction [5]. Indeed some authors [S-lo] have concluded that in P. falciparum the unprocessed MSP-1 molecule is involved in the initiation of the erythrocyte invasion process raising doubts about the significance of primary processing. Recently we presented evidence that a 21-kDa MSP-1 C-terminal polypeptide of the rodent malaria parasite P.c. chabaudi is carried through, during erythrocyte invasion, into the ring-stage parasite in vivo [ll], indicating that at least the secondary phase cleavage event does occur in vivo. In this paper we extend this work by demonstrating that a complex of MSP-1 polypeptides are shed into plasma during schizogony in vivo. Identification of the primary and secondary processing cleavage sites by N-terminal amino-acid sequencing and comparison with those found within the P. falciparum MSP-1 indicates that certain features of MSP-1 processing are conserved. The repeated release at every parasite cell cycle of this large and highly immunogenic protein into the host plasma could have important immunological, and possibly pathophysiological, consequences.

2. Materials and methods

72 (1995) 111-119

2.3. Polyclonal anti-sera Preparation of anti-sera from P.c. chabaudi AS hyperimmunised mice has been described previously [15]. Anti-sera was raised against purified preparations of P.c. chabaudi AS MSP-1 recombinant fusion proteins: pMCK 118, 113, 110 and 106 [ll], by immunisation of CBA/Ca mice on two occasions with 10 pg protein using saponin adjuvant. 2.4. Merozoite preparation 2-3 h before peak schizogony in heavily infected (30-40% parasitaemia) mice, blood was collected into Krebs glucose saline (KGS) containing 25 U ml-’ of heparin. Cells were washed twice with culture medium (RPM1 1640/Albumax) by centrifugation at 750 X g for 3 min and the buffy coat aspirated with the supernatant. Parasites were then added at 2% haematocrits to flasks containing medium pregassed with 7% 0,/5% CO,/88% N, and incubated at 37°C. Parasite development was followed by examination of Giemsa-stained smears by light microscopy. After two to three hours, when schizont stages began to predominate, cultures supernatants were removed by centrifugation at 750 X g for 5 min. Cultures were then restarted as before. When significant parasite lysis had occured cultures were chilled on ice, and then centrifuged at 750 X g for 10 min. The supernatants were centrifuged at 10000 X g for a further 5 min and the pellets, enriched for merozoites, then frozen at -70°C.

2.1. Parasites 2.5. Metabolic radiolabelling A cloned line of P.c. chabaudi AS obtained from the World Health Organisation Registry of Standard Strains of Malaria Parasites (Edinburgh, UK) was used throughout this investigation. Mice (CBA/Ca strain) and parasites were adapted to and maintained under reversed light conditions as described previously [12]. 2.2. Monoclonal antibodies The production and characterisation of the monoclonal antibodies (mAb) used in this study have been described elsewhere [13,14].

P.c. chabaudi AS parasites were washed and cultured in cysteine + methionine-free RPMI/Albumax and Trans 35S methionine (ICN Biochemicals) was added to cultures at 1 MBq ml- ‘. 2.5.1. In vitro pulse labelling Trophozoite-stage parasites from mice with parasitaemias of 5% or less were pulsed with [ 35S] methionine/cysteine for 30 min at a 10% haematocrit. Cells were collected by centrifugation at 750 X g for 5 min and then subdivided between severa cultures at a 2% haematocrit in RPMI/Albumax

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and Biochemical Parasitology

medium. At l-h intervals, individual cultures were put on ice and then centrifuged at 750 X g for 10 min at 4°C. Pelleted cells were washed twice in KGS by centrifugation at 750 X g for 10 min at 4°C. The culture supematants were clarified by centrifugation at 2.5 000 X g for 20 min at 4°C. Both fractions were then stored at -70°C. 2.5.2. In vitro / in vivo labelling Trophozoite-stage parasites from mice with 2030% parasitaemias were radiolabelled in vitro for 30 min at a 10% haematocrit. After washing, approx. 5 x lo8 parasites were injected intravenously into normal mice. Re-invasion was allowed to occur for the next 4-6 h. Blood from these mice was then collected in KGS/heparin on ice and diluted 4-fold with KGS, before centrifugation at 25 000 X g for 20 min at 4°C. The plasma supernatant was stored at - 70°C.

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2.6. Immunoprecipitation and immunoaffinity purification

All procedures were carried out at 4°C. Intraerythrocytic and merozoite stages were solubilised in lysis buffer: 100 mM Tris . HC1/2 mM phenylmethylsulfonyl fluoride/5 mM EDTA/O.l% Triton X-100 at pH 8.0, and insoluble material removed by centrifugation at 100000 X g for 5 min. Lysates were applied, under gravity, to small columns of the appropriate mAb coupled to CNBr-activated Sepharose (Pharmacia). The Sepharose columns were then washed with 20 volumes of 50 mM Tris . HCl pH 8/5 mM EDTA/O.S% Triton X-100 followed by 20 volumes of 50 mM Tris. HCl pH S/500 mM NaCl. Culture supernatants and plasma were treated in a similar manner, however, lysis and washing steps with Triton X-100 were omitted. For affinity

7

8

9

43-

29-

Fig. 1. Autoradiograph of P.c. chabaudi AS [35S]methionine/cysteine-labelled polypeptides immunoprecipitated by the anti-MSP-1 monoclonal antibody NIMP32. Trophozoite stage parasites, metabolically radiolabelled in vitro with [35Slmethionine/cysteine, were washed and returned to individual in vitro cultures without radioisotope or injected intra-venously into normal mice. In vitro cultured parasites (A), culture supernatants (B) and mouse plasma (Cl were immunoprecipitated with mAb NIMP32-Sepharose. Precipitates were solubilised in SDS and resolved on an SDS-PAGE gel (8% polyacrylamide) under reducing conditions. Representitive precipitates of parasite lysates are shown after 0 h (lane 1); 4 h (lane 2); and 5 h (lane 3) in radioisotope-free culture. NIMP3ZSepharose reacted with culture supematant (5 h) was left untreated (lane 4); or washed once with 10 volumes of 50 mM Tris HCl pH 8.2/0.5% sodium deoxycholate (lane 5); sodium deoxycholate wash (lane 6). NIMP3ZSepharose reacted with plasma obtained from mice 5 h after parasite injection were similarly treated; (lanes 7-9, respectively). Molecular masses &Da) of r4C markers are indicated.

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preparations bound material was eluted mM diethylamine/O.l% Triton X-100. 2.7. N-terminal

with

and Biochemical Parasitology

100

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branes (Millipore) by electrophoresis. After staining of the Immobilon membrane with Coomassie blue, the relevant bands were excised. The microsequence analysis was carried out by semi-automated Edman degradation chemistry, followed by conversion to phenylthiohydantoin (PTH) derivatives and performed on Applied Biosystems (Al311 Model 477A pulsed liquid-phase sequencers using ABI reagents and optimised standard programs [17]. On-line HPLC

amino-acid sequencing

Polypeptide extracts were boiled in SDS-PAGE sample buffer, reduced and carboxymethylated [16]. Samples were resolved on 7515% polyacrylamide gradient gels and transferred onto Immobilon mem-

A 12345 s.1 ETIGVYND s.2 SEQVTTSS s.3 SEQVTTSS s.4 TEETKQND s.5 SEDEMFVD s.6 ETIGVYND s.7 STDGEVKD

pMCKII0 581 pMCK113 35/31 kDa

s.l,6 20

52147 kDa

921

1508 pMCK106

66/36 kDa

I

I

s.4 293

s.2,3 712

39137 kDa

I 1%

1766

32143 kDa

I

I

s.7 1366

Fig. 2. Identification and analysis of MSP-1 associated polypeptides released into the plasma of P.c. chabaudi AS infected mice during schizogony. (A) MSP-l-associated polypeptides were affinity purified (NIMP32Sepharose) from plasma of P.c. chubaudi AS infected mice, resolved on a SDS-PAGE gel (8% polyacrylamide) under reducing conditions, electroblotted onto nitrocellulose membranes and probed with: P.c. chobaudi AS byperimmune serum (lane 1); anti-sera raised against the P.c. chabaudi AS MSP-1 recombinant proteins pMCK118 (lane 2); pMCK113 (lane 3); pMCKll0 (lane 4); pMCK106 (lane 5). Apparent molecular masses &Da) and the N-terminal amino-acid sequences (s.l-7) of the major polypeptides (as visualised by Coomassie blue staining) are shown as they relate to P.c. chabaudi hyperimmune serum reactive bands in lane 1. (B) Schematic showing the relationship between the deduced proteolytic cleavage sites (s.l-7) on the native MSP-1 and the predicted reactivity of anti-sera raised against the MSP-1 pMCK recombinant proteins. Amino-acid positions of the N-terminal residue of each cleaved polypeptide and of regions encoded by the pMCK recombinant proteins are shown. The hatched area represents the cleaved N-terminal signal sequence. Apparent/predicted molecular masses are shown.

K.P. O’Dea et al./Molecular

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analysis of PTH amino acids was performed on Al31 Model 120A analysers [18] using Al31 Model 900A data aquisition modules calibrated with 25 pmol PTH standards 1191. Data interpretation was by visual methods.

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19

peared to be identical, although it would appear from the intensity of the polypeptide bands derived from plasma, that the association of the polypeptides with

1

2.8. SDS-PAGE and immunoblotting

2

3

4

5

105-

Standard protocols were followed throughout [20]. Blots were probed with anti-mouse whole IgG alkaline phosphatase conjugate (Sigma) and developed with the substrate bromochloroindolyl phosphate/ nitroblue tretrazolium. Prestained (BRL) and 14C radioisotope (Amersham) molecular mass markers were used.

3. Results 15Immunoprecipitation of in vitro metabolically radiolabelled parasite proteins by the MSP-l-specific mAb NIMP32, resulted in the precipitation of a single major band of 250 kDa, which represents the unprocessed MSP-1 (Fig. 1). The intensity of this band was seen to diminish as the culture time proceeded (lanes l-3) which appeared to correspond to a reduction in the number of schizonts visualised by microscopy. In contrast, a number of radiolabelled polypeptides were co-precipitated from serum-free culture supematants collected towards the end of the culture period (lane 4). rnAb NIMP32, which reacts with schizont but not ring-stage parasites, has previously been shown to recognise an epitope at the C terminus of the MSP-1 [14]. Although the majority of the co-precipitated polypeptides could bc solubilised by limited washing with the ionic detergent sodium deoxycholate (lane 6), a 32-kDa fragment of the MSP-1 remained associated with the Sepharose bound antibody (lane 5). This fragment of the MSP-1 presumably displays the NIMP32 epitope, whilst all the other polypeptides were precipitated merely due to weaker, non-covalent interactions, directly or indirectly, with this 32-kDa fragment. Significantly, this non-covalently associated complex of polypeptides could also be detected in the plasma of mice which had been injected with radiolabelled parasites. The number and relative molecular mass of the in vivo derived polypeptides ap-

B 45143kDa

s.7

21113 kDa

32130kDa ‘F

GIGSNHV 1650

Fig. 3. Secondary processing of the C terminus of the P.c. chabaudi AS MSP-1 during in vitro culture. (A) Trophozoite and schizont-stage parasites cultured at 2% haematocrit in RPMI/Albumax containing: no additions (lane 1); 5 mM EGTA (lane 2); 5 mM EDTA (lane 3); 5 mM EGTA + 5 mM CaClz (lane 4); 5 mM EDTA+S mM MgCl, (lane 5). Merozoites present in culture supernatants after 2 h were concentrated by centrifugation and iysed in solubilisation buffer. Approximately equal amounts of material were resolved on a SDS-PAGE gel (12.5% polyacrylamide) under non-reducing conditions and electroblotted onto a nitrocellulose membrane and probed with the mAb NIMP23. (B). A 21-kDa polypeptide (detected by Coomassie blue staining) was purified from P.c. chabaudi AS culture supernatant derived merozoites and subjected to N-terminal amino-acid analysis. The MSPl-derived sequence and position of the deduced cleavage site are depicted relative to the 32-kDa site (s.7) in a schematic of the MSP-1 C-terminal region. Apparent (under reducing conditions)/predicted molecular masses are shown.

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To identify the co-precipitating polypeptides the complex was affinity purified from plasma using mAb NIMP3ZSepharose and investigated by Nterminal amino-acid sequencing and immunoblot analysis. Seven major polypetides, as visualised by Coomassie blue staining (not shown), were subjected to N-terminal amino-acid sequence analysis and the

the 32-kDa fragment may be less stable in vitro. The 250-kDa precursor molecule was detectable in culture supernatants and, to a lesser degree, in plasma. Increased amounts of 250 kDa and various other radiolabelled species were immunoprecipitated when parasitaemias used for culture were greater than about 10% (not shown).

A 95 kDa 35 kDa

I I

I

52 kDa

66/63 kDa

39 kDa

I

a

b

45 kDa

1 Plasmodium chabaudi chabaudi AS MSP-1 (250 kDa) \ I I

I Id I I

c

I I

I

: 32 kDa

63 kDa

26/30 kDa

39 kDa

II

21 kDi\

42 kDa

I b

c

td I , I I

1 33 kDa

1 , I I

,lp kDa\

e

B a

VTEGQITTEGN

F?c.cAS F?c.cAS

b

a

*TEETKQNDAAQ

l?f T9/96

II KI KL

Fig. 4. Comparison of MSP-1 processing in P.c. chabaudi AS and P. falciparum. (A) Schematic comparison of P.c. chabaudi and P. falciparum MSP-l~processing. The primary (a-d) and secondary (e) cleavage sites are indicated and apparent molecular masses of the cleaved polypeptides under reducing conditions. (B) Alignment of the equivalent MSP-1 cleavage sites ( *) of P.c. chabaudi AS and P. falciparum T9/96 [24], FCB-1 [25] and ‘l9/94 [26]. Identical amino acids are boxed. Gaps are introduced to align homologous regions. The conserved LN motif in (e) is shaded.

K.P. O’Dea et al. /Molecular

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identity of the first 8 amino acids assigned (Fig. 2A). These sequences were compared to the deduced amino-acid sequence derived from nucleotide sequence analysis of the P.c. chabaudi AS MSP-1 [14] and all seven sequences were found to be MSP-1 derived. An additional sequence was obtained from a 68-kDa polypeptide (DQESSKKA) which we have not identified to date. The identities of the constituent polypeptides were also confirmed using sera raised against overlapping P.c. chabaudi AS MSP-1 recombinant polypeptides. As can be seen from Fig. 2B, anti-sera raised against discrete parts of the MSP-1 molecule were found to recognise different components of the complex. Partial cleavage of the 95kDa polypeptide is apparent from its shared N-terminal amino-acid sequence with the 35kDa polypeptide and the pattern of reactivity of pMCKl18 and pMCKl13 anti-sera with the 95, 35 and 52-kDa polypeptides. The difference in apparent molecular mass between the 66 and 63-kDa polypeptides, which share the same N-terminal amino-acid sequence, could arise from two distinct cleavage events at their C-termini rather than secondary modification, which in the P. yoelii MSP-1 appears to be limited [21,22]. The discrepancy between apparent and predicted molecular mass, i.e., 63/66 and 36 kDa, is perhaps not suprising since the native precursor molecule has apparent and predicted molecular masses of 250 and 197 kDa, respectively, aberrant migration on SDS-PAGE is thought to be a common feature of malaria polypetides [21]. The comparitively low yield of the 32-kDa fragment (not shown) may have contributed to the weak signal observed (lanes 1 and 5). Having established that the basic characteristics of P.c. chabaudi AS MSP-1 primary processing were essentially the same in vivo and in vitro we proceeded to investigate the secondary processing of the MSP-1 from culture derived merozoites. mAb NIMP23, which recognises an epitope on the 21-kDa MSP-1 fragment carried into newly invaded erythrocytes [ll], was used in immunoblotting experiments to monitor the processing event. As is evident from Fig. 3 (lane 1) 21 and 43-kDa polpypetides are detectable in merozoite preparations. Addition of 5 mM EGTA and 5 mM EDTA (lanes 2 and 3, respectively) to cultures inhibits the production of the 21-kDa fragment with a reciprocal increase in the

72 (199.5) 1 I1 -I I9

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intensity of the 43-kDa band. The proteolytic cleavage giving rise to the 21-kDa fragment, at the expense of the 43-kDa species, is restored when equimolar calcium is added with EGTA (lane 4) but not magnesium with EDTA (lane 5). These results are in accordance with the previously reported calcium-dependent cleavage at the C terminus of P. fatciparum MSP-1 [4]. The 21-kDa polypeptide was purified from merozoite preparations and its N-terminal amino-acid sequence determined (Fig. 3B). The cleavage site was identified by comparison of this sequence with the nucleotide sequence deduced P.c. chabaudi AS MSP-1 amino-acid sequence. Probing of Western blotted merozoite preparations with NIMP32 indicate that its epitope is present on the 43 kDa (45 kDa when reduced) but not the 21-kDa fragment (not shown). It is therefore most probable that cleavage at the s.7 site gives rise to the 43-kDa precursor polypeptide which is then further broken down during secondary processing into the 32 and 21 kDa, complex and merozoite-associated polypeptides, respectively. On the basis of the above data a processing scheme has been devised for the P.c. chabaudi AS MSP-1. Comparison of MSP-1 processing in P. falciparum and P.c. chabaudi AS (Fig. 4A) shows they follow a similar pattern in terms of fragment size and number. Cleavage sites, that occur in equivalent regions of the MSP-1, were aligned to compare flanking amino-acid sequence (Fig. 4B). The alignment of homologous amino acids either side of cleavage sites d and e suggests that despite some changes in primary structure, the position of protease cleavage is conserved. A common feature of the primary processing sites (a-d) is the presence of a glycine or alanine residue in the P2 position. The frequency of other conservatively substituted residues, Val/Ile and Thr/Ser N-terminal, and Ser/Thr C-terminal to the cleaved bond, is also apparent in a-d.

4. Discussion It is evident from the data presented in this paper that processing of the MSP-1, which has hitherto been described in parasites maintained under in vitro

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conditions, does in fact occur in vivo. The P. c.chabaudi AS MSP-1 is broken down into distinct polypeptides, which apart from the 21-kDa fragment, are released in quantity into the plasma. The protease cleavage sites of the P.c. chabaudi AS MSP-1 have been defined by N-terminal aminoacid sequencing of the major breakdown products. Comparison with P. falciparum demonstrates that the overall pattern of processing is similar and that the relative position, in terms of primary structure, of two cleavage sites is identical. To identify a generalized motif involved in primary processing, MSP-1 cleavage sites of other Plasmodium species need to be defined; however, it is already apparent from comparison of P. falciparum and P.c. chabaudi AS MSP-1 sites that some elements of primary structure are important. The relevance of primary processing during erythrocyte invasion is uncertain but it now seems unlikely that it is a random or inconsequential event during infection. In contrast to the ambiguity surrounding primary processing, it is generally considered that secondary processing is a pre-requisite for successful erythrocyte invasion. The proteolytic activity responsible for secondary processing at the MSP-1 C terminus of P. falciparum merozoites has been partially characterised, and shown to be conserved between two MSP-1 dimorphic strains [23]. Secondary cleavage in merozoites of the P. falciparum T9/94 clone takes place between the conserved leucine asparagine motif [26], which is close to, and yet distinct from, the P.c. chabaudi AS MSP-1 cleavage site. The positional conservation of this cleavage site and the requirement for calcium during proteolysis, in vitro by both species indicates a degree of conservation in protease mechanism and specificity. The redundancy of the potential Leu-Asn cleavage site in P.c. chabaudi AS does, however, suggest that higher order structures take precedence in determining protease specificity. Identification of the cleavage sites in newly invaded ring-stage parasites, as opposed to free merozoites, will be necessary to confirm the validity of this interpretation. It is clear from these studies that investigations into MSP-1 processing can be carried out in vivo in a well-established rodent malaria model. Thus, there is now an opportunity to elucidate facets of MSP-1 structure and processing significant for the physiol-

72 (1995) 111-119

ogy and immunobiology of malaria and malaria parasites. Structural changes resulting from single amino-acid differences at the 21-kDa C-terminal polypeptide of P.c. chabaudi have been shown to completely ablate the binding of a mAb with in vivo inhibitory activity and also, to determine the reactivity of highly strain-specific hyper-immune serum [ill; the implications to human infection were considered. An additional question is whether antisera raised against intact MSP-1 are truly representitive of epitopes present on the processed products released in vivo. Anti-MSP-1 monoclonal antibodies have been raised by immunising mice with P. falciparum schizont stage antigen, i.e., not by an infection. A number of these appear to show preferential reactivity towards the intact rather than processed MSP-1 [4,26], indicative of changes in the tertiary structure as a result of processing. The mere presence of intact or processed MSP-1 on the surface of free merozoites does not establish whether or not their putative role in erythrocyte invasion is either direct or indirect. The conserved nature of at least some MSP-1 cleavage sites makes it desirable, nevertheless, that conformational changes resulting from processing are investigated. Immune recognition of such alternative tertiary structures provides an opportunity not only to determine their location but also to examine their possible significance in the modulation of the host anti-parasite immune response.

Acknowledgements We thank M. Blackman and W. Jarra for helpful discussions. This work was funded by the Medical Research Council (UK) and the UNDP/World Bank/ WHO special Programme and Training in Tropical Disease (TDR).

References 111Holder, A.A., Blackman,

M.J., Burghaus, P.A., Chappel, J.A., Ling, LT., McCallum-Deighton and Shai, S. (1992) A malaria merozoite surface protein (MSPlbstucture, processing and function. Mem. Inst. Oswald0 CNZ, Rio de Janeiro 87, 37-42 (Suppl. III). 121Blackman, M.J., Heidrich, H.G., Donachie, S., McBride, J.S. and Holder, A.A. (1990) A single fragment of a malaria

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merozoite surface protein remains on the parasite during red cell invasion and is the target of invasion-inhibiting antibodies. J. Exp. Med. 172, 379-382. Blackman, M.J., Whittle, H. and Holder, A.A. (1991) Processing of the Plasmodium falciparum merozoite surface protein-l: identification of a 33-kilodalton secondary processing product which is shed prior to erythrocyte invasion. Mol. Biochem. Parasitol. 49, 35-44. Blackman, M.J. and Holder, A.A. (19921 Secondary processing of the Plasmodium falciparum merozoite surface protein1 (MSPl) by a calcium-dependent membrane-bound serine protease: shedding of MSPl,, as a noncovalently associated complex with other fragments of the MSPl. Mol. Biochem. Parasitol. 50, 307-316. David, P.H., Hadley, T.J., Aikawa, M. and Miller, L.H. (1984) Processing of a major parasite surface glycoprotein during the ultimate stages of differentiation in Plasmodium knowlesi Mol. Biochem. Parasitol. 11, 276-282. Holder, A.A. and Freeman R.R. (1981) Immunisation against blood-stage rodent malaria using purified parasite antigens. Nature 294, 361-364. Wood, J.C., Sales de Aguiar, C. Jarra, W., Ogun, S.A., Snounou, Cl. and Brown, K.N. (1989) In vivo selection of populations of Plasmodium chabaudi chabaudi AS resistant to a monoclonal antibody that reacts with the precursor to the major merozoite surface antigen. Infect. Immun. 57, 21282135. Pirson, P.J. and Perkins, M.E. (1985) Characterisation with monoclonal antibodies of a surface antigen of Plasmodium falciparum merozoites. J. Immunol. 134, 1946-1951. Perkins, M.E. and Rocco, L.J. (19881 Sialic acid-dependent binding of Plasmodium falciparum merozoite surface antigen, Pf200, to human erythrocytes. J. Immunol. 141, 31903196. Barnwell, J.W. and Galinski, M.R. (1991) The adhesion of malaria merozoite proteins proteins to erythrocytes: a reflection of function? Res. Immunol. 142, 617-735. Mckean, P.G., O’Dea, K.P. and Brown, K.N. (1993) A single amino acid determines the specificity of a monoclonal antibody which inhibits Plasmodium chabaudi AS in vivo. Mol. Biochem. Parasitol. 62, 211-222. Newbold, C.I., Boyle, D.B., Smith, C.C. and Brown, K.N. (19821 Stage specific protein and nucleic acid synthesis during the asexual cycle of the rodent malaria Plasmodium chabaudi. Mol. Biochem. Parasitol. 5, 33-44. Boyle, D.B., Newbold, CL, Smith, CC. and Brown, K.N. (1982) Monoclonal antibodies that protect in vivo against Plasmodium chabaudi recognise a 250 000 Dalton parasite polypeptide. Infect. Immun. 38, 94-102. Mckean, P.G., O’Dea, K.P. and Brown, K.N. (1993) Nucleotide sequence analysis and epitope mapping of the mero-

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