Molecular & Biochemical Parasitology 144 (2005) 55–67

Structural comparison of apical membrane antigen 1 orthologuesand paralogues in apicomplexan parasites

Marie-Laure Chesne-Secka, Juan Carlos Pizarroa, Brigitte Vulliez-Le Normanda,Christine R. Collinsb, Michael J. Blackmanb, Bart W. Faberc, Edmond J. Remarquec,

Clemens H.M. Kockenc, Alan W. Thomasc, Graham A. Bentleya,∗a Unite d’Immunologie Structurale, CNRS URA 2185, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris, France

b Division of Parasitology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UKc Department of Parasitology, Biomedical Primate Research Centre, 2280 GH Rijswijk, The Netherlands

Received 4 May 2005; received in revised form 25 July 2005; accepted 25 July 2005Available online 16 August 2005

Abstract

anes analysis

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Apical membrane antigen 1 (AMA1) is a membrane protein present inPlasmodium species and is probably common to all apicomplexparasites. The recent crystal structure of the complete ectoplasmic region of AMA1 fromPlasmodium vivax has shown that it comprises threstructural domains and that the first two domains are based on the PAN folding motif. Here, we discuss the consequences of thifor the three-dimensional structure of AMA1 from otherPlasmodium species and other apicomplexan parasites, and for thePlasmodiumparalogue MAEBL. Many polar and apolar interactions observed in the PvAMA1 crystal structure are made by residues that areor highly conserved throughout allPlasmodium orthologues; a subgroup of these residues is also present in other apicomplexan orthoand in MAEBL. These interactions presumably play a key role in defining the protein fold. Previous studies have shown that the ectregion of AMA1 must be cleaved from the parasite surface for host-cell invasion to proceed. The cleavage site in the crystal structreadily accessible to proteases and we discuss possible consequences of this observation. The three-dimensional distribution of psites in PfAMA1 shows that these are all on the surface and that their positions are significantly biased to one side of the ectoplasmOf particular note, a flexible segment in domain II, comprising about 40 residues and devoid of polymorphism, carries an epitope reby an invasion-inhibitory monoclonal antibody and a T-cell epitope implicated in the human immune response to AMA1.© 2005 Elsevier B.V. All rights reserved.

Keywords: Apical membrane antigen 1; MAEBL; Structural homology; Polymorphism; Proteolytic processing

1. Introduction

Apical membrane antigen 1 is a malarial surface proteinfound in all characterizedPlasmodium species[1] and ortho-logues exist in at least two other apicomplexan parasites,Toxoplasma gondii [2,3] and Babesia bovis [4]. AMA1 isstored in the microneme organelles immediately after syn-thesis and is transported to the parasite surface just priorto, or during, host-cell invasion. It comprises an N-terminal

∗ Corresponding author. Tel.: +33 1 45 68 86 10; fax: +33 1 40 61 30 74.E-mail address: bentley@pasteur.fr (G.A. Bentley).

ectoplasmic region, a single transmembrane segment asmall cytoplasmic domain. Sixteen invariant Cys residuesencoded in the ectoplasmic region of all characterized Pmodial AMA1 genes. AMA1 is currently being developed aa malaria vaccine[5,6].

The precise function of AMA1 is not known but many lineof evidence point to a direct role in host cell invasion. Monvalent Fab fragments derived from invasion inhibitory mooclonal anti-AMA1 antibodies can also inhibit erythrocyinvasion, implying direct interference with AMA1 functionrather than cross-linking of the antigen or immune clearanof the parasite as the mechanism of protection[7]. In COS-7

0166-6851/$ – see front matter © 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.molbiopara.2005.07.007

56 M.-L. Chesne-Seck et al. / Molecular & Biochemical Parasitology 144 (2005) 55–67

cells expressing different recombinant ectoplasmic domaincombinations of AMA1 fromP. yoelii, a construct compris-ing domains I and II was able to specifically bind rodenterythrocytes[8]; however, others have failed to demonstrateerythrocyte binding by either cell surface-expressed, solublerecombinant, or naturally processed AMA1 fromP. falci-parum [9,10] or P. vivax (C. Kocken, Unpublished results).In vitro studies with an invasion-inhibitory monoclonal anti-body suggest that AMA1 is probably involved in the invasionphase after the initial non-specific reversible attachment ofthe merozoite to the erythrocyte[11]. These studies implicateAMA1 in some way in the formation of the tight junctionthat forms between the merozoite and erythrocyte surfacesas the parasite penetrates the host cell. Consistent with this,a recent study inT. gondii using an inducible promoter toobtain conditional knock-down of AMA1 expression hasshown that the molecule is not involved in initial attach-ment to the host cell but is required for intimate attach-ment and efficient triggering of rhoptry secretion[12]. Arecent study with different AMA1 domain combinationsexpressed on the surface of CHO cells showed that domainIII interacts with the erythrocyte membrane protein Kx, butonly after treatment with trypsin[13]. While these diverseobservations are consistent with a receptor-binding functionfor AMA1, conclusive confirmation for such a role is stilllacking.

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2. Methods and materials

2.1. Sequence alignment

Sequence alignments were made using the program MUS-CLE [20]. One representative AMA1 sequence was taken foreachPlasmodium species. Minor manual adjustments weremade to the alignment of AMA1 sequences with that ofMAEBL from P. vivax using criteria based on the PvAMA1crystal structure. Sequences used in the alignments are givenin Figs. 2 and 5.

2.2. Structure modelling

The structure of theP. falciparum orthologue, PfAMA1,of the FVO strain[21] was modelled by introducing the 135amino acid changes that exist in the ordered regions of thePvAMA1 crystal structure (an additional 48 changes occurin disordered regions). Atomic coordinates of PvAMA1 weretaken from the Protein Data Bank (PDB) entries 1w81 and1w8k. Dihedral angles of the side chains were adjusted onthe PvAMA1 main-chain scaffold using the mean force fieldalgorithm[22]. No changes were introduced into the main-chain conformation. The limited insertions in the PfAMA1sequence with respect to PvAMA1 are located on disorderedregions and were thus not modelled.

3

3P

n ofP c-t ws:d 249t um-b froma e ofPI hichsa insI ,A nsc ptorb tralfi 3-4 s oft edt c-o ot ites area rder

Although first found in thePlasmodium merozoite, AMA1s also expressed in the sporozoite ofP. falciparum [14] and

onoclonal anti-AMA1 antibodies that inhibit erythrocnvasion by the merozoite can also inhibit hepatocyte iion by the sporozoite. AMA1 could therefore have idenr very similar functions in the pre-erythrocytic and asexlood stages of the parasite life cycle, and might serveffective vaccine against these two critical phases.

We have recently reported the crystal structure of the clete ectoplasmic region of AMA1 fromP. vivax (PvAMA1)

rom the strain Sal I[15], showing that it is divided into threomains as previously predicted from the pattern of cysridge formation[16,17]. Here, we discuss the implicatiof the PvAMA1 crystal structure for the three-dimensiotructure of AMA1 from otherPlasmodium species and itrthologues inT. gondii and B. bovis. Merozoite antigerythrocyte-binding ligand (MAEBL), a rhoptry membrarotein identified in severalPlasmodium species[18,19], is aaralogue of AMA1. It contains two closely related tandepeat segments, M1 and M2, in the N-terminal moiethe protein, each homologous to the combined ectoplaomain I/II motif of AMA1, and a Duffy-binding like (DBLomain immediately upstream from the transmembraneytoplasmic regions. We compare the M1 and M2 regionAEBL with PvAMA1 and show that some of the invariand highly conserved residues in domains I and II of AMre also likely to play an important structural role in this plogue. We also examine the distribution of polymorphic s

n PfAMA1 and PvAMA1, and discuss the consequenceaccine development.

. Results and discussion

.1. The structure of the AMA1 ectoplasmic region inlasmodium species

The secondary structure and domain organisatiovAMA1 is illustrated inFig. 1. Based on the crystal stru

ure[15], the domains of PvAMA1 can be defined as folloomain I extends from residues 43 to 248, domain II from

o 385, and domain III from 386 to 487. Residues are nered from the first residue of the signal sequence and,lignment with the PfAMA1 sequence, the prosequencvAMA1 is assumed to terminate at residue 42[9]. Domainhas an N-terminal extension (residues 43–62) in wtrand�1 hydrogen bonds in parallel to�20 of domain IIInd helix �1 lies across domain II. The core of domaand II is based on the PAN folding motif (Plasminogenpple, Nematode[23]), a protein fold describing domaiommonly associated with carbohydrate- or protein-receinding functions. The PAN folding motif consists of a cenve-stranded anti-parallel�-sheet with strand order 1-5--2, an� helix connecting the second and third strand

he sheet, and two anti-parallel�-strands hydrogen bondogether, one connecting the first�-sheet strand to the send and the other connecting the fourth�-sheet strand t

he fifth. The helix and double�-strand lie against opposides of the central�-sheet to one another. PAN domainslso characterized by six Cys residues linked in the o

M.-L. Chesne-Seck et al. / Molecular & Biochemical Parasitology 144 (2005) 55–67 57

Fig. 1. Secondary structure and domain organisation of PvAMA1.� strands are shown as arrows,� helices are shown as rectangles and the 3.010 helix �1 as awavy rectangle. Cystine bridges are drawn in thick line with residue numbers indicated. Domain I secondary structure elements are indicated in white, domainII in light grey and domain III in dark grey.

Cys1-Cys6, Cys2-Cys5 and Cys3-Cys4, although in a num-ber of cases the Cys1-Cys6 bridge is absent[23]. In thePvAMA1 structure, only the Cys2-Cys5 bridge, connectingthe� helix to the fourth�-sheet strand, conforms to the PANpattern (Cys162-Cys192 in domain I and Cys282-Cys354 indomain II); the other cystines are thus not critical for main-taining this protein fold in AMA1. The structural homologybetween domains I and II suggests that they are probablythe product of gene duplication and have since diverged sig-nificantly. Indeed, both domains – domain I in particular –have significant additional secondary structure elements andloop regions appended to the PAN core. Domain III does notbelong to any currently known fold. The structure of the iso-lated domain III from PfAMA1 has been studied in solutionby nuclear magnetic resonance (NMR)[17]. Much of theNMR structure is disordered because domains I and II inter-act with, and presumably stabilize, these regions of domainIII. Indeed, only�22, �23, the first turn of�8 and the threecystine bridges of the NMR structure are similar to the crys-tal structure of PvAMA1. The root-mean-square differencebetween 26 equivalent C� positions of the crystal and NMRstructures after superposition is 3.5A, suggesting that eventhese regions depart significantly from the native structure inthe absence of domains I and II.

Several Plasmodium AMA1 ectoplasmic sequences,which are highly conserved, are compared inFig. 2, witht rystals er-a ind senf re inP ues,u fin-i lar

interactions in the PvAMA1 crystal structure are made byresidues that are invariant or conserved in character betweenPlasmodium orthologues and include both inter- and intra-domain contacts. Polar interactions in PvAMA1 between theside chains of species-invariant residues, listed inTable 1,

Table 1Polar interactions in the PvAMA1 crystal structure between the side chainsof invariant residues inPlasmodium AMA1

Intra-domain contactsDomain I Ser70 Asp75 * *

Arg73 Glu201Glu78 Lys237Glu78 Asn238Lys99 Gln200 * *Tyr147 His165Gln200 Tyr234 * *Lys237 Asn238

Domain II Asn283 Thr373Asp293 Ser337 * *Thr374 Ser377

Domain III Lys392 Asp426Glu453 Arg455Arg455 Tyr474

Inter-domain contactsDomain I–II Ser70 Asp293 * *

Asp79 Lys336

S in thestructure are not shown. Those interactions where residues are also invari-ant with TgAMA1, BbAMA1 are indicated by *. Intra- and inter-domaincontacts are separated according to domain. Using the coordinates from thePDB entry 1w8k, the inter-atomic distance cut-off was set at 3.5A.

he secondary structure components observed in the ctructure of PvAMA1 indicated in the alignment. The ovll sequence identity is 39%, with 40% in domain I, 38%omain II and 33% in domain III for the sequences cho

or this comparison. The most significant conserved featulasmodium AMA1 sequences is the set of 16 Cys residnderlining the importance of the disulphide bridges in de

ng the polypeptide fold. A number of polar and apo

Lys225 Asn283

Domain I–III Trp55 Tyr391

Domain II–III Asp262 Arg420Asn366 Ser442

everal invariant side-chain/main-chain interactions that also occur

58 M.-L. Chesne-Seck et al. / Molecular & Biochemical Parasitology 144 (2005) 55–67

Fig. 2. Sequence alignment ofPlasmodium AMA1 ectoplasmic regions. Strictly conserved residues are indicated with black background and conservativedifferences with grey background. Residue numbering is given forP. vivax and secondary structure assignation from the PvAMA1 crystal structure (calculatedby the program DSSP[51]) is indicated as “e” for� strand, “h” for� helix and “g” for 3.010 helix. Disordered regions that were not modelled are shown with“+” and the domain II loop is indicated. The following sequences were selected for comparison:P. vivax, GenBank entry CAA76546;P. knowlesi, AAA63444;P. cynomolgi, CAA60053;P. fragile, AAA29474;P. reichenowi, CAB66387;P. falciparum, AJ277646;P. chabaudi, AAB36509;P. yoelii yoelli, EAA20929;P. berghei, AAC47192.

M.-L. Chesne-Seck et al. / Molecular & Biochemical Parasitology 144 (2005) 55–67 59

Fig. 3. Conserved polar interactions inPlasmodium AMA1. The ectoplasmic region of PvAMA1 (coordinates from the Protein Data Bank entry 1w81) isshown as a stereo view. Domain I is green, domain II is blue and domain III is mauve. Polar interactions between the side chains of residues that are invariantin Plasmodium are indicated by dotted lines: red for intra-domain interactions and yellow for inter-domain interactions. Disulphide bridges are indicated bysolid yellow lines.

form a network extending over the entire molecule (Fig. 3).A larger number of polar contacts are made between invari-ant side chains and the polypeptide backbone (not shown);these should contribute equally to the structural similarity ofAMA1 from the different species.

The sequence of PfAMA1 from the FVO strain wasmodelled on the PvAMA1 (Sal I) crystal structure (58.9%sequence identity). Sequence differences between these twospecies are distributed over the entire ectoplasmic region.Five occur at solvent-inaccessible sites in the PfAMA1 modeland are conservative changes involving aliphatic side chains(Val, Leu and Ile), whereas the remaining 183 differences arelocated on the surface of the molecule (Fig. 4A). The side-chain conformation of the non-conserved residues could beeasily adapted to the fixed PvAMA1 main-chain conforma-tion, implying that the structure of the two orthologues shouldbe very similar to each other. This probably also appliesto otherPlasmodium species because the limited sequenceinsertions and deletions occur in disordered regions of thePvAMA1 crystal structure, with the exception of one insertedresidue in theP. chabaudi orthologue at a position aligningwith �19 (seeFig. 2).

Domain I and, to a lesser extent, domain II have longpolypeptide segments that are either convoluted with no reg-ular secondary structure or are disordered in the PvAMA1crystal structure. One notable feature in the structure is thep hesem rved

residues and accordingly can be expected in allPlasmod-ium orthologues. Three of these buried water molecules arein contact with each other and contribute importantly toa network of internal hydrogen bonds with several main-chain atoms and the side chains of six invariant buried polarresidues: His68, Ser70, Asp75, Lys99 and Gln200 fromdomain I, and Asp 293 from domain II. This highly polarcavity is separated by the invariant residue, Tyr234, fromanother cavity with two buried solvent molecules, also main-tained in an environment of invariant residues. Other buriedsolvent molecules occur singly, often interacting with main-chain atoms and invariant or highly conserved residues.

3.2. Polymorphism in PfAMA1 and PvAMA1

AMA1 is significantly polymorphic in bothP. falci-parum [24–26]andP. vivax [27]. The predominance of non-synonymous over synonymous mutations in the PfAMA1gene implies that sequence diversity is selected for, andmaintained, to counter the host’s immune response[28–32].Although polymorphic sites are distributed throughout thelength of the ectoplasmic region, domain I is the most vari-able[29–32].

Using the crystal structure of PvAMA1 and our model ofPfAMA1, we examined the three-dimensional distribution ofpolymorphic sites in these two species. Taking 356 PfAMA1s andD hic if

resence of 13 buried water molecules in domain I. Tolecules are located in cavities that are lined with conse

equences that were available in the EMBL, GenBankDJB databases, we considered a site to be polymorp

60 M.-L. Chesne-Seck et al. / Molecular & Biochemical Parasitology 144 (2005) 55–67

Fig. 4. Model of PfAMA1 showing the three-dimensional distribution of (A) PfAMA1(FVO)/PvAMA1(Sal I) sequence differences in magenta and (B) three-dimensional distribution of PfAMA1 polymorphic sites shown in green; unaffected residues are in grey. The two views in both (A) and (B) are related by a180◦ rotation about a vertical axis so that both sides of the molecule are shown.

at least two sequences differed from the consensus amino acidat that position. All polymorphic sites are surface exposed;a total of 32 are located in domain I (15.5% of the residuescomprising this domain), 11 in domain II (8.0%) and 9 indomain III (8.6%), thus showing domain I to be the mostpolymorphic region of PfAMA1, as previously noted by oth-ers[24–26]. Most sites are dimorphic, but 13 sites have threedifferent amino acids (however, at five of these sites the leastfrequent substitution appears in only one sequence), two siteshave four substitutions (the least frequent substitution appearsonly once at one site) and one site shows seven substitutions

(seeTable 2). Unlike the distribution of PvAMA1/PfAMA1sequence differences, the distribution of the PfAMA1 poly-morphic sites is highly biased to one side of the molecule(Fig. 4B). This suggests that this part of the ectoplasmicregion may be more exposed to the exterior on the parasitesurface and/or less susceptible to functional constraints.

Polymorphism has been less studied inP. vivax, andbecause current data give only limited coverage of domains IIand III, our analysis is restricted to domain I. From a compar-ison of 232 partial PvAMA1 sequences taken from the samedata bank sources as PfAMA1, we found 17 polymorphic

M.-L. Chesne-Seck et al. / Molecular & Biochemical Parasitology 144 (2005) 55–67 61

Table 2Comparison of polymorphic sites in PfAMA1 and PvAMA1

PfAMA1 PvAMA1Domain I

121 EK (230/3) 66 R –162 NK (274/81) 107 DA (11/2)167 TK (262/93) 112 KTR (131/93/8)172 EG (194/164) 117 G –173 NKE (305/49/2) 118 D –175 DY (319/37) 120 RKS (219/7/6)185 P – 130 NK (157/75)187 NEK (138/133/85) 132 DN (119/113)188 P – 133 DN (223/9)189 LPH (327/24/5) 134 H –190 MI (240/116) 135 I –195 L – 140 IL (124/108)196 DNY (265/90/1) 141 AE (185/47)197 QGDEHRV (116/85/84/32/26/7/6) 142 N –199 RK (352/4) 144 K –200 DHRL (158/144/27/26) 145 EAG (170/60/2)201 FLSV (281/66/8/1) 146 R –204 DN (206/150) 149 D –206 EK (278/78) 151 V –207 YD (298/58) 152 E –244 MI (351/5) 169 K –225 NI (243/113) 170 V –227 D – 172 AT (229/3)228 NK (345/10) 173 G –230 KEQ (239/97/20) 175 Q –242 YD (183/173) 187 E –243 KNE (223/72/61) 188 K –244 DNY (328/14/14) 189 EKN (113/91/27)245 KN (328/28) 190 KEQ (148/83/1)248 H – 193 HY (229/3)265 K – 210 SP (181/51)267 EQ (216/140) 212 A269 KI (341/15) 214 N –273 M – 218 VL (197/35)282 KI (238/117) 227 EV (165/67)283 SL (249/106) 228 SD (165/67)285 QE (280/75) 230 E –296 DH (282/70) 241 N –300 KE (251/95) 245 K –

Domain II308 EQK (108/59/1) 253 G325 HDR (162/3/1) 270 Y330 SP (152/15) 275 E332 NI (137/29) 277 E393 HRY (115/51/1) 338 K395 KR (152/13) 340 R404 RT (95/71) 349 S405 KE (85/82) 350 V407 QH (141/26) 352 K435 INT (131/34/2) 380 Q439 HND (98/64/4) 384 L

Domain III448 DN (136/31) 393 D451 KM (98/69) 396 E485 KI (97/70) 427 K493 DA (131/36) 435 E496 IM (94/73) 438 R503 NRH (97/69/1) 445 N505 FY (159/7) 447 Y512 KR (88/79) 454 K544 KN (95/48) 486 L

Sequences of PfAMA1 and PvAMA1 are compared at all residue positions where at least one species is polymorphic. Polymorphic sites in each speciesare highlighted in bold and all observed amino acid substitutions are given. A total of 356 PfAMA1 sequences and 232 PvAMA1 sequences were used. ForPvAMA1, only domain I polymorphisms are shown since the coverage of domains II and III is not significant. The number of sequences contributing to eachpolymorphic substitution at each site is given in parentheses in the same order as the amino acid substitutions themselves.

62 M.-L. Chesne-Seck et al. / Molecular & Biochemical Parasitology 144 (2005) 55–67

sites in domain I (8.3%). As with PfAMA1, all polymorphicresidues are surface exposed. Polymorphic sites on domain Iof PfAMA1 and PvAMA1 have similar distributions, beingmostly situated within the long, solvent-exposed, convolutedpeptide segment between residues 105 and 136 (PvAMA1residue numbering), the helices�3 and�4, and the turnsconnecting�7 to �8 and�9 to �10 (Table 2). Ten of thepolymorphic sites in domain I are in common between thetwo orthologues. In both these species, the presence of a par-ticular amino acid at certain polymorphic sites was found insome cases to be highly correlated with the sequence occur-ring at other polymorphic sites. Since a number of these arewell separated from each other in the tertiary structure, it doesnot seem that their occurrence is directly caused by structuralfactors in the protein itself but rather by genetic factors.

3.3. Other orthologues and paralogues of AMA1 inApicomplexa

Orthologues of AMA1 from the apicomplexan parasitesT. gondii (TgAMA1) [2,3] and B. bovis (BbAMA1) [4]have been identified and sequenced. Many of the invariantresidues and conservative differences in AMA1 acrossPlas-modium species are also present in TgAMA1 and BbAMA1.These two orthologues show the same pattern of Cys residuesi est pectt ee-db I ofP 1aT if-f sr rC ouldf andC -t A1,h eriz-i rei sixC ,a A1,w fc en-t wf sd ol-o thec ectt cea ns int withd

Although domains M1 and M2 of MAEBL are eachhomologous to combined domains I/II of AMA1, theirsequence identity to AMA1 is low (Fig. 5). Ten of the 14Cys residues of M1 and M2 align with the 10 Cys residuesof the domain I/II AMA1 consensus sequence (Cys 2, 6, 9,10, 11, 12 13, 14, 15, 16 of M1 and M2 inFig. 5) and thusimply the same pattern of cystine bridge formation for theseresidues in MAEBL. Cys7 and Cys8 in M1 and M2 (Fig. 5)may also be linked together since this would conform to oneof the consensus PAN cystine bridges[23]. There is not suf-ficient information to predict the pairing of the remainingCys residues (Cys1, Cys3, Cys4, Cys5). Most of the residuesin M1 and M2 that are invariant with respect to AMA1are engaged in conserved hydrophobic interactions in thePvAMA1 structure. Of note here, the invariant hydrophobicpair Phe-Leu (residues 110–111 in PvAMA1) anchors its twoflanking sections that form long solvent-exposed loops. Thesetwo residues are buried in a hydrophobic pocket lined by sev-eral invariant and highly conserved residues. All invariant Glyresidues (Fig. 5B), with the possible exception of those thatare equivalent to Gly125 in PvAMA1 (occurring in a disor-dered region in the crystal structure) are buried and wouldgive rise to steric hindrance if substituted by other aminoacids. The only predicted conserved polar interaction is madeby a Lys residue equivalent to Lys225 in PvAMA1, which isburied and forms charged hydrogen bonds to the main chain.A on-o s ani er-f asw er-t 5r A1.

3

s-i mest eT m thet egionf ageo onet log-i ageo[ ,a leav-a hasa ata geo gt

inals las-m y

n domains I and II ofPlasmodium, and several residuhat are invariant or conservative in character with reso Plasmodium sequences play a critical role in the thrimensional structure of PvAMA1 (Fig. 5A). Interactionsetween invariant polar residues in domains I and Ilasmodium AMA1 that should be preserved in TgAMAnd BbAMA1 are noted inTable 1. Domain III in bothgAMA1 and BbAMA1 shows significant sequence d

erences toPlasmodium AMA1, most notably in the Cyesidues (Fig. 5B). Domain III of BbAMA1 has only fouys residues but their alignment shows that they sh

orm disulphide bridges equivalent to Cys388-Cys444ys432-Cys449 in PvAMA1 (seeFigs. 1 and 5B). The cys

ine bridge corresponding to Cys434-Cys451 in PvAMowever, is absent, and the pair of CXC motifs charact

ng the cystine knot[33] observed in the PvAMA1 structus thus not present in BbAMA1. Although TgAMA1 hasys residues, as in domain III of thePlasmodium orthologueslignment only occurs with Cys432 and Cys444 in PvAMhich are not linked together inPlasmodium; the pattern oystine bridges is thus different in TgAMA1. Sequence idity with Plasmodium AMA1 at other residue positions is loor domain III of TgAMA1 (Fig. 5B). Therefore, whereaomain III of BbAMA1 might bear some structural homgy to the PvAMA1 crystal structure, this is unlikely to bease in TgAMA1. The differences in domain III may reflhe proximity of this part of AMA1 to the parasite surfand its consequent interaction with other surface protei

he different apicomplexan taxa, or perhaps interactionifferent host-cell receptors.

n epitope mapping study with the invasion-inhibitory mclonal antibody 4G2 has shown that this residue play

mportant role in maintaining the domain I/domain II intace in AMA1 [15] and this probably applies to MAEBLell. The alignment of M1 and M2 shows significant ins

ions immediately preceding�4 and�5 (about 30 and 7esidues, respectively, in M1 and M2) with respect to AM

.4. Natural processing in AMA1

AMA1 in P. falciparum undergoes proteolytic procesng in the parasite after translocation from the microneo the parasite surface[10,34]. The main chain is cut at thhr517-Ser518 peptide bond, 29 residues upstream fro

ransmembrane region, thus cleaving the ectoplasmic rrom the merozoite surface. An additional internal cleavccurs at the Asn464-Asp465 peptide bond in about

hird of the shed ectoplasmic polypeptides, but the biocal significance of this is not known. Proteolytic cleavf the AMA1 ectoplasmic region also occurs inP. knowlesi

35] and is likely to take place in otherPlasmodium specieslthough this has not been experimentally confirmed. Cge of the AMA1 ectoplasmic region from the tachyzoitelso been observed for TgAMA1[2,3], although this occursn intramembrane site[34]. Antibodies that prevent cleavaf AMA1 in P. falciparum inhibit invasion[36], suggestin

hat shedding may be a prerequisite for invasion.We have found two partial cleavage sites by N-term

equencing of the soluble recombinant PvAMA1 ectopic region expressed inPichia pastoris, both presumabl

M.-L. Chesne-Seck et al. / Molecular & Biochemical Parasitology 144 (2005) 55–67 63

Fig. 5. Sequence alignment of (A) domains I and II of PvAMA1, PfAMA1, TgAMA1 and BbAMA1 with domains M1 and M2 of PvMAEBL (GenBank entriesY16950, AJ277646, AF010264, AY486101 and AY042083, respectively), and (B) of domain III of PvAMA1, PfAMA1, TgAMA1 and BbAMA1. Strictlyconserved residues are indicated with black background and conservative differences with grey background. Residue numbering is given forP. vivax and thealigned secondary structure from the PvAMA1 crystal structure (calculated by the program DSSP[51]) is indicated as “e” for� strand and “h” for� helix. Thedomain II loop is indicated by “+”. Cys residues of regions M1 and M2 of PvMAEBL are numbered sequentially from 1 to 16.

64 M.-L. Chesne-Seck et al. / Molecular & Biochemical Parasitology 144 (2005) 55–67

mediated by a protease(s) fromP. pastoris. One site occurs atthe Tyr409-Ser410 peptide bond, which is located within thedisordered loop between residues 403 and 414 in domain III,aligning with the cleavage site, Asn464-Asp465, in PfAMA1that is partially proteolyzed during natural processing. Thesecond partial proteolysis site in recombinant PvAMA1occurs between Lys321 and Ser322 in domain II, and thuslies within the disordered segment 295–334 that we havereferred to as the domain II loop[15]. This was not modelledin the PvAMA1 crystal structure. This cleavage site alignswith a partial cleavage site found in the recombinant ecto-plasmic domain of PfAMA1, also expressed inP. pastoris[37]. For both these sites, the cleaved polypeptide chains arecovalently held together by the disulphide bridges and theproteolyzed PvAMA1 ectoplasmic region thus migrates atessentially the same rate as the intact protein in SDS-PAGEunder non-reducing conditions.

The natural cleavage site Thr517-Ser518 in PfAMA1,which leads to shedding of the ectoplasmic region duringmaturation, aligns with the Lys459-Glu460 peptide bond ofPvAMA1. This peptide bond is intact in the crystal structure,stabilized by extensive hydrogen bonds within in a�-hairpin.In this conformation, we anticipate protection of this pep-tide bond against proteolysis. Thus, if AMA1 inP. vivax(and otherPlasmodium species) is cleaved, like PfAMA1,from the parasite surface during erythrocyte invasion, wes teind eastd t oft therp asitem se ofi cteda osedt itsr

areb thusb n thep thati 1( bel rfacee ain.T las-m indis-t ati oites thee ferredt ecifici sed,s -i va-s tes

3.5. The domain II loop

In the crystal structure of PvAMA1, a 40-residue segmentin domain II, comprising residues 295–334, had no corre-sponding electron density in the Fourier maps and thus couldnot be traced. This region is therefore disordered becauseof conformational heterogeneity or mobility. We have pre-viously shown that the base of the domain II loop includesthe epitope of the anti-PfAMA1 invasion-inhibitory mono-clonal antibody, 4G2[15]. Interestingly, this B-cell epitopeoverlaps with a highly immunogenic T-helper cell epitopein the human response[39]. The location of the 4G2 epi-tope and currently known structure–function correlations ofPAN domains led us to speculate that AMA1 might have areceptor-binding role involving domain II. The loop is strictlyconserved in residue length within the differentPlasmodiumorthologues but has lower sequence identity (21%) comparedto the complete ectoplasmic region (39%). Nonetheless, nopolymorphic sites have been detected to date in the domain IIloop in P. falciparum, suggesting that functional constraintsmay operate to prevent antigenic variation in this surface-exposed segment. Of note, secondary structure predictionsusing PHD[40] consistently assign a high probability to helixformation in the first half of the domain II loop in allPlas-modium orthologues. In BbAMA1, the domain II loop is tworesidues longer compared toPlasmodium AMA1, whereas inT atefi rs inM senti elyd

4

r otif,a withr sim-i omt onall ins Ia re-v s arecg I andI insw ainc ile att n thes tiono ti-g host.O ur in

uggest that the conformation of this region of the proiffers from that observed in the crystal structure, at luring the proteolysis step. The structure of this par

he molecule may be influenced by interactions with oarasite surface molecules, by the proximity of the parembrane or by erythrocyte molecules during the cour

nvasion. It is possible that the cleavage site is thus protegainst premature proteolysis and only becomes exp

o the protease upon interaction between AMA1 andeceptor(s).

The two natural cleavage sites identified in PfAMA1oth in the membrane-proximal domain III and shoulde easily accessible to membrane-bound proteases oarasite. By contrast, the additional partial cleavage

s found in domain II of soluble recombinant PvAMALys321-Ser322) and PfAMA1 (Lys376-Ser377) mayess accessible to such proteases on the parasite suxplaining its absence in the naturally cleaved ectodomhe enzyme responsible for cleaving the PfAMA1 ectopic region is a membrane-bound parasite protease

inguishable, in terms of inhibitor susceptibility, from thnvolved in secondary processing of the major merozurface protein MSP1[10]. It has been suggested thatnzyme involved belongs to a class of proteases often re

o as sheddases, which are not primarily sequence-spn cleavage-site recognition but instead target expotructurally flexible, polypeptide segments[38]. Processng of MSP1 also occurs at the time of erythrocyte inion and, as with PfAMA1[36], is essential for parasiurvival.

,

gAMA1 this region is 17 residues shorter. This may indicunctional differences; whereasBabesia, like Plasmodium,nvades erythrocytes,Toxoplasma is able to invade a widepectrum of cell types. In MAEBL, the equivalent region1 and M2 is short and thus closer in length to that pre

n other PAN domains; the role of this region is most likifferent for this paralogue.

. Conclusion

The crystal structure determination of AMA1[15] hasevealed a hitherto unexpected relationship to the PAN m

polypeptide fold found in proteins often associatedeceptor-binding functions. The generally low sequencelarity between PAN domains, the departure in AMA1 frhe usual PAN Cys signature, and the insertion of additiarge peptide segments onto the PAN scaffold of domand II account for this structural homology not being piously recognized from sequence alone. PAN domainommonly present as tandem repeats in proteins[23], andene duplication may indeed have produced domains

I in AMA1. According to this hypothesis, the two domaould have subsequently co-evolved to form inter-domontacts that are highly conserved between species, whhe same time developing large sequence insertions ourface of the protein that contribute directly to the funcf AMA1, either in receptor binding or in displaying anenic variability to escape immune responses by thef note, however, the only insertions/deletions that occ

M.-L. Chesne-Seck et al. / Molecular & Biochemical Parasitology 144 (2005) 55–67 65

Plasmodial AMA1 sequences are in domain III. The domainI/II motif is also present in the chimericPlasmodium par-alogue MAEBL as the tandem repeats M1 and M2, implyinga second gene duplication; here, insertions into the PAN scaf-fold are even more extensive than in AMA1, although theregion matching the domain II loop is considerably short-ened. The similarity between AMA1 and MAEBL suggeststhat the domain I/II ensemble originated early in apicom-plexan evolution and that the M1/M2 duplication occurredprior to divergence ofPlasmodium species[41]. Like AMA1,MAEBL also plays a role in hepatocyte invasion by the sporo-zoite as well as erythrocyte invasion by the merozoite[18,42].AMA1 is the first protein inPlasmodium shown, via three-dimensional structure analysis, to have PAN folding motifs,and by homology, we can also include MAEBL. The PANmodule and the closely related Apple domain are found inother micronemal proteins inApicomplexa [43], hinting thatthey, AMA1 and MAEBL might be phylogenetically derivedfrom a distant common ancestor.

The use of individual domains or sub-domains of theectoplasmic region could lead to simpler and possibly morestable AMA1-based vaccine products. For example, anti-bodies that were affinity-purified from the plasma from adonor in a malaria-endemic region using the recombinantPfAMA1 domain III showed significant inhibition of erythro-cyte invasion with two different parasite strains[17], thusd thisd rentr enssi ioni m-b m-p , thea g-n of theP in I[ n T-c ep-t forv ainI thep nedw mi-t ingc

ve-n ni-m lenge[ nge[ sitei thath las-m D7[ em ple,

antibodies from rabbits immunised with AMA1 strain 3D7gave 20% inhibition of erythrocyte invasion by FVO parasites[37]. This supports the hypothesis that a non-negligible part ofthe protective immune response induced by AMA1 is againstconserved epitopes. Indeed, the loss of inhibition with het-erologous challenge is approximately correlated linearly withthe number of amino acid differences between the species inquestion[37]. Moreover, most experiments showing lowerinhibition following heterologous challenge have been donewith rabbit or mouse antibodies, species whose immune sys-tems do not always adequately represent the human immunesystem, as is increasingly becoming evident[50]. In supportof this notion, preliminary data obtained from an AMA1 vac-cination study in rhesus macaques show that the differencesin in vitro parasite inhibition levels between homologousand heterologous challenge are much less pronounced whencompared to antibodies derived from rabbits under similarexperimental conditions (Thomas AW and Remarque EJ,Unpublished data).

Irrespective of these considerations, polymorphism inAMA1 will have an impact on vaccine development and thusthe challenge for the vaccine developers is to address thisissue. For instance, in a recent human phase I trial[6], the useof an equal mixture of FVO and 3D7 AMA1 (strains that dif-fer at 24 of the 52 polymorphic sites in the ectoplasmic region)gave equal ELISA titres for both strains, and invasion inhi-b s forh ensa gousi rep-r lemo

A

con-t 7),t nsti-t que,t dicalR

R

pro-ed

isellular

ion

inva-ti-

emonstrating the presence of protective epitopes onomain. However, a recent study in rabbits using diffeecombinant PfAMA1 domain combinations as immunoguggests that the situation may be less straightforward[44];ndividual domains were not effective in inducing invasnhibitory antibodies and only the double domain I/II coination induced growth-inhibitory antibodies levels coarable to the complete ectoplasmic region. Moreoverctivity of the invasion-inhibitory mAb 4G2, which recoizes a reduction-sensitive epitope located at the basefAMA1 domain II loop, requires the presence of doma

15]. But since the domain II loop also contains a humaell epitope[39] and carries no known polymorphisms, pide mimetics of this region would be of particular interestaccine development. Although the 4G2 epitope on domI requires the presence of domain I, we do not excludeossibility that such sub-domain mimetics could be desigith knowledge of the conformation of the ordered extre

ies of the domain II loop in the crystal structure; promisandidates could be selected with mAb 4G2.

Polymorphism is a critical factor governing the effectiess of a vaccine. Although vaccination with AMA1 in aal model systems protects against homologous chal

45–47], it is less effective against heterologous challe29,37,48]. Nonetheless, significant levels of in vitro paranhibition are still observed when challenged with strainsave over 20 amino acid differences in the AMA1 ectopic region[37,49] (24 amino acids between FVO and 3

37], and 23 between 3D7 and HB3[49]), which is close to thaximum found in naturally occurring strains. For exam

ition of heterologous challenge was at the same level aomologous challenge. Thus, multiple-allele immunogppear to be additive in their protection against heterolo

nfection[6] and vaccine constructs based on two or moreesentative strains may offer effective solution to the probf polymorphism.

cknowledgements

This work was funded by the European Commission (racts QLK2-CT-1999-01293 and QLK2-CT-2002-0119he European Malaria Vaccine Initiative, the Pasteur Iute, the Centre National de la Recherche Scientifihe Biomedical Primate Research Centre and the Meesearch Council, UK.

eferences

[1] Waters AP, Thomas AW, Deans JA, et al. A merozoite receptortein from Plasmodium knowlesi is highly conserved and distributthroughoutPlasmodium. J Biol Chem 1990;265:17974–9.

[2] Donahue CG, Carruthers VB, Gilk SD, Ward GE. TheToxoplasmahomolog of Plasmodium apical membrane antigen-1 (AMA-1)a microneme protein secreted in response to elevated intraccalcium levels. Mol Biochem Parasitol 2000;111:15–30.

[3] Hehl AB, Lekutis C, Grigg ME, et al.Toxoplasma gondii homologueof Plasmodium apical membrane antigen 1 is involved in invasof hosT-cells. Infect Immunol 2000;68:7078–86.

[4] Gaffar FR, Yatsuda AP, Franssen FF, de Vries E. Erythrocytesion by Babesia bovis merozoites is inhibited by polyclonal an

66 M.-L. Chesne-Seck et al. / Molecular & Biochemical Parasitology 144 (2005) 55–67

sera directed against peptides derived from a homologue ofPlas-modium falciparum apical membrane antigen 1. Infect Immunol2004;72:2947–55.

[5] Saul A, Lawrence G, Allworth A, et al. A human phase 1 vaccineclinical trial of thePlasmodium falciparum malaria vaccine candidateapical membrane antigen 1 in Montanide ISA720 adjuvant. Vaccine2005;23:3076–83.

[6] Malkin EM, Diemert DJ, McArthur JH, et al. Phase 1 clinical trialof apical membrane antigen 1: an asexual blood-stage vaccine forPlasmodium falciparum malaria. Infect Immunol 2005;73:3677–85.

[7] Thomas AW, Deans JA, Mitchell GH, Alderson T, Cohen S. TheFab fragments of monoclonal IgG to a merozoite surface antigeninhibit Plasmodium knowlesi invasion of erythrocytes. Mol BiochemParasitol 1984;13:187–99.

[8] Fraser TS, Kappe SH, Narum DL, VanBuskirk KM, Adams JH.Erythrocyte-binding activity ofPlasmodium yoelii apical membraneantigen-1 expressed on the surface of transfected COS-7 cells. MolBiochem Parasitol 2001;117(1):49–59.

[9] Howell SA, Withers-Martinez C, Kocken CH, Thomas AW, Black-man MJ. Proteolytic processing and primary structure ofPlas-modium falciparum apical membrane antigen-1. J Biol Chem2001;276:31311–20.

[10] Howell SA, Well I, Fleck SL, Kettleborough C, Collins CR, Black-man MJ. A single malaria merozoite serine protease mediates shed-ding of multiple surface proteins by juxtamembrane cleavage. J BiolChem 2003;278:23890–8.

[11] Mitchell GH, Thomas AW, Margos G, Dluzewski AR, BannisterLH. Apical membrane antigen 1, a major malaria vaccine candidate,mediates the close attachment of invasive merozoites to host redblood cells. Infect Immunol 2004;72:154–8.

[12] Mital J, Meissner M, Soldati D, Ward GE, Conditional expression ofn-ol

[ IItoSA

[ rane

[ stalntigen

[ ondem

[ of

53.[ of

Natl

[ ion,oro-otein5.

[ with113.

[ vel

nti-;70:

[ fieldtheir

[23] Tordai H, Banyai L, Patthy L. The PAN module: the N-terminaldomains of plasminogen and hepatocyte growth factor are homolo-gous with the apple domains of the prekallikrein family and witha novel domain found in numerous nematode proteins. FEBS Lett1999;461:63–7.

[24] Oliveira DA, Udhayakumar V, Bloland P, et al. Genetic conservationof thePlasmodium falciparum apical membrane antigen-1 (AMA-1).Mol Biochem Parasitol 1996;76:333–6.

[25] Marshall VM, Zhang L, Anders RF, Coppel RL. Diversity of thevaccine candidate AMA-1 ofPlasmodium falciparum. Mol BiochemParasitol 1996;77:109–13.

[26] Kocken CH, Narum1 DL, Massougbodji A, et al. Molecular char-acterisation ofPlasmodium reichenowi apical membrane antigen-1(AMA-1), comparison withP. falciparum AMA-1, and antibody-mediated inhibition of red cell invasion. Mol Biochem Parasitol2000;109:147–56.

[27] Figtree M, Pasay CJ, Slade R, et al.Plasmodium vivax synonymoussubstitution frequencies, evolution and population structure deducedfrom diversity in AMA 1 and MSP 1 genes. Mol Biochem Parasitol2000;108:53–66.

[28] Hughes MK, Hughes AL. Natural selection onPlasmodium surfaceproteins. Mol Biochem Parasitol 1995;71:99–113.

[29] Crewther PE, Matthew ML, Flegg RH, Anders RF. Protectiveimmune responses to apical membrane antigen 1 ofPlasmod-ium chabaudi involve recognition of strain-specific epitopes. InfectImmunol 1996;64:3310–7.

[30] Hughes MK, Hughes AL. Natural selection onPlasmodium surfaceproteins. Mol Biochem Parasitol 1995;71:99–113.

[31] Escalante AA, Grebert HM, Chaiyaroj SC, et al. Polymorphism inthe gene encoding the apical membrane antigen-1 (AMA-1) ofPlas-modium falciparum X. Asembo Bay Cohort Project. Mol Biochem

[ ainsne.

[ .[ gov-

xan

[ of al

[ ion-1 of

[ om-):f a

[ lfide-rtingistry

[ ll

tion

[ ofilehodsNew

[ rela-es of

[ andfect

Toxoplasma gondii apical membrane antigen-1 (TgAMA1) demostrates that TgAMA1 plays a critical role in hosT-cell invasion. MBiol Cell, in press.

13] Kato K, Mayer DC, Singh S, Reid M, Miller LH. Domain Iof Plasmodium falciparum apical membrane antigen 1 bindsthe erythrocyte membrane protein Kx. Proc Natl Acad Sci U2005;102:5552–7.

14] Silvie O, Franetich JF, Charrin S, et al. A role for apical membantigen 1 during invasion of hepatocytes byPlasmodium falciparumsporozoites. J Biol Chem 2004;279:9490–6.

15] Pizarro JC, Vulliez-Le Normand BV, Chesne-Seck ML, et al. Crystructure of the malaria vaccine candidate apical membrane a1. Science 2005;308:408–11.

16] Hodder AN, Crewther PE, Matthew ML, et al. The disulfide bstructure ofPlasmodium apical membrane antigen-1. J Biol Ch1996;271:29446–52.

17] Nair M, Hinds MG, Coley AM, et al. Structure of domain IIIthe blood-stage malaria vaccine candidate.Plasmodium falciparumapical membrane antigen 1 (AMA1). J Mol Biol 2002;322:741–

18] Kappe SH, Noe AR, Fraser TS, Blair PL, Adams JH. A familychimeric erythrocyte binding proteins of malaria parasites. ProcAcad Sci USA 1998;95:1230–5.

19] Ghai M, Dutta S, Hall T, Freilich D, Ockenhouse CF. Identificatexpression, and functional characterization of MAEBL, a spzoite and asexual blood stage chimeric erythrocyte-binding prof Plasmodium falciparum. Mol Biochem Parasitol 2002;123:35–4

20] Edgar RC. Muscle: a multiple sequence alignment methodreduced time and space complexity. BMC Bioinformat 2004;5:

21] Kocken CH, Withers-Martinez C, Dubbeld MA, et al. High-leexpression of the malaria blood-stage vaccine candidatePlasmod-ium falciparum apical membrane antigen 1 and induction of abodies that inhibit erythrocyte invasion. Infect Immunol 20024471–6.

22] Koehl P, Delarue M. Application of a self-consistent meantheory to predict protein side-chains conformation and estimateconformational entropy. J Mol Biol 1994;239:249–75.

Parasitol 2001;113:279–87.32] Polley SD, Conway DJ. Strong diversifying selection on dom

of the Plasmodium falciparum apical membrane antigen 1 geGenetics 2001;158:1505–12.

33] Isaacs NW. Cystine knots. Curr Opin Struct Biol 1995;5:391–534] Howell S, Hackett F, Jongco AM, et al. Distinct mechanisms

ern proteolytic shedding of a key invasion protein in apicomplepathogens. Mol Microbiol 2005;57:1342–56.

35] Deans JA, Thomas AW, Alderson T, Cohen S. Biosynthesisputative protectivePlasmodium knowlesi merozoite antigen. MoBiochem Parasitol 1984;11:189–204.

36] Dutta S, Haynes JD, Barbosa A, et al. Mode of action of invasinhibitory antibodies directed against apical membrane antigenPlasmodium falciparum. Infect Immunol 2005;73:2116–22.

37] Kennedy MC, Wang J, Zhang Y, et al. In vitro studies with recbinantPlasmodium falciparum apical membrane antigen 1 (AMA1production and activity of an AMA1 vaccine and generation omultiallelic response. Infect Immunol 2002;70:6948–60.

38] Schwager SL, Chubb AJ, Woodman ZL, et al. Cleavage of disubridged stalk domains during shedding of angiotensin-conveenzyme occurs at multiple juxtamembrane sites. Biochem2001;40:15624–30.

39] Lal AA, Hughes MA, Oliveira DA, et al. Identification of T-cedeterminants in natural immune responses to thePlasmodium fal-ciparum apical membrane antigen (AMA-1) in an adult populaexposed to malaria. Infect Immunol 1996;64:1054–9.

40] Bost B. PHD: predicting one-dimensional protein structure by prbased neural networks. In: Carter CW, Sweet RM, editors. Metin enzymology: macromolecular crystallography Part A, 266.York: Academic Press; 1997. p. 525–39.

41] Michon P, Stevens JR, Kaneko O, Adams JH. Evolutionarytionships of conserved cysteine-rich motifs in adhesive moleculmalaria parasites. Mol Biol Evol 2002;19:1128–42.

42] Preiser P, Renia L, Singh N, et al. Antibodies against MAEBL ligdomains M1 and M2 inhibit sporozoite development in vitro. InImmunol 2004;72:3604–8.

M.-L. Chesne-Seck et al. / Molecular & Biochemical Parasitology 144 (2005) 55–67 67

[43] Brown PJ, Gill AC, Nugent PG, McVey JH, Tomley FM. Domains ofinvasion organelle proteins from apicomplexan parasites are homol-ogous with the Apple domains of blood coagulation factor XI andplasma pre-kallikrein and are members of the PAN module super-family. FEBS Lett 2001;497:31–8.

[44] Lalitha PV, Ware LA, Barbosa A, et al. Production of the subdo-mains of thePlasmodium falciparum apical membrane antigen 1ectodomain and analysis of the immune response. Infect Immunol2004;72:4464–70.

[45] Deans JA, Knight AM, Jean WC, Waters AP, Cohen S, MitchellGH. Vaccination trials in rhesus monkeys with a minor, invari-ant.Plasmodium knowlesi 66 kD merozoite antigen. Parasit Immunol1988;10:535–52.

[46] Collins WE, Pye D, Crewther PE, et al. Protective immunityinduced in squirrel monkeys with recombinant apical membrane anti-gen-1 of Plasmodium fragile. Am J Trop Med Hyg 1994;51:711–9.

[47] Narum DL, Ogun SA, Thomas AW, Holder AA. Immunization withparasite-derived apical membrane antigen 1 or passive immuniza-tion with a specific monoclonal antibody protects BALB/c miceagainst lethalPlasmodium yoelii yoelii YM blood-stage infection.Infect Immunol 2000;68:2899–906.

[48] Healer J, Murphy V, Hodder AN, et al. Allelic polymorphisms inapical membrane antigen-1 are responsible for evasion of antibody-mediated inhibition in Plasmodium falciparum. Mol Microbiol2004;52:159–68.

[49] Hodder AN, Crewther PE, Anders RF. Specificity of the protectiveantibody response to apical membrane antigen 1. Infect Immunol2001;69:3286–94.

[50] Mestas J, Hughes CCW. Of mice and men: differences betweenmouse and human immunology. J Immunol 2004:2731–8.

[51] Kabsch W, Sander C. Dictionary of protein secondary structure:pattern recognition of hydrogen-bonded and geometrical features.Biopolymers 1983;22:2577–637.