Biochemical and Biophysical Research Communications 315 (2004) 319–329

BBRCwww.elsevier.com/locate/ybbrc

MAEBL Plasmodium falciparum protein peptides bind specificallyto erythrocytes and inhibit in vitro merozoite invasion

Marisol Ocampo,* Hernando Curtidor, Ricardo Vera, John J. Valbuena,Luis E. Rodr�ıguez, �Alvaro Puentes, Ramses L�opez, Javier E. Garc�ıa, Diana Tovar,

Paola Pacheco, Miguel A. Navarro, and Manuel E. Patarroyo

Fundaci�on Instituto de Inmunologia de Colombia, Universidad Nacional de Colombia, Avda. Calle 26 No. 50-00, Bogot�a, Colombia

Received 8 January 2004

Abstract

MAEBL is an erythrocyte binding protein located in the rhoptries and on the surface of mature merozoites, being expressed at

the beginning of schizogony. The structure of MAEBL originally isolated from rodent malaria parasites suggested a molecule likely

to be involved in invasion. We thus became interested in identifying possible MAEBL functional regions. Synthetic peptides

spanning the MAEBL sequence were tested in erythrocyte binding assays to identify such possible MAEBL functional regions. Nine

high activity binding peptides (HABPs) were identified: two were found in the M1 domain, one was found between the M1 and M2

regions, five in the erythrocyte binding domain (M2), and one in the protein’s repeat region. The results showed that peptide binding

was saturable; some HABPs inhibited in vitro merozoite invasion and specifically bound to a 33 kDa protein on red blood cell

membrane. HABPs’ possible function in merozoite invasion of erythrocytes is also discussed.

� 2004 Elsevier Inc. All rights reserved.

Keywords: MAEBL; Peptides; Malaria

Plasmodium falciparum malaria is a major cause of

human morbidity and mortality, thus highlighting theurgent need for an effective vaccine. Identifying those

optimal parasite antigens on which to base a vaccine

represents a challenging task because there are many

antigenic proteins in the parasite; however, the role of

most of these in protective immunity is unknown.

The parasite must engage receptors on RBC for

binding [1] and undergo apical reorientation [2], junc-

tion formation [3], and signalling during the invasionprocess. The parasite then induces a vacuole derived

from the RBC plasma membrane and enters the vacuole

via a moving junction. Three organelles on the invasive

(apical) end of parasites (rhoptries, micronemes, and

dense granules) define the phylum Apicomplex.

Merozoites use proteins sequestered in apical complex

organelles to mediate invasion of host erythrocytes

via specific receptor/ligand interactions [4]. Host cell

* Corresponding author. Fax: +57-1-324-46-72x108.

E-mail address: marisol_ocampo@fidic.org.co (M. Ocampo).

0006-291X/$ - see front matter � 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.bbrc.2004.01.050

invasion by merozoites is a multi-step process thought

to require numerous interactions between apical com-plex proteins and erythrocyte receptors. Receptors for

merozoite invasion of erythrocytes and for sporozoite

invasion of the liver are found in micronemes [5], on the

cell surface, and in the rhoptries. The distribution of

these receptors within an organelle may protect the

parasite from antibody-mediated neutralisation, as their

release after contact with the erythrocyte may limit their

exposure to antibodies.Plasmodium parasite rhoptries are paired club-shaped

organelles located at the apical end of merozoites, the

parasite form that invades RBCs. Following merozoites’

attachment to the RBC surface, rhoptries discharge their

contents onto the RBC membrane [6]. Rhoptry contents

include both protein and lipid components which

assemble to form membrane-like structures. Protein

constituents of the rhoptry contents are still being de-fined. To date, several rhoptry proteins have been

identified and studied. Some have been characterised

at the molecular level whereas others are defined by

320 M. Ocampo et al. / Biochemical and Biophysical Research Communications 315 (2004) 319–329

immunological reagents (reviewed in [7,8]). Most rhoptryproteins are expressed relatively late in the maturation of

the parasite, are either soluble or transmembrane, un-

dergo limited proteolyticmodifications, and are located in

the body of schizont rhoptries. Rhoptry proteins have

been implicated as having a role in the invasion process.

MAEBL is an erythrocyte binding protein located in

the rhoptries and on the surface of mature merozoites

and is expressed at the beginning of schizogony. TheMAEBL family, exhibiting homology to both DBL-

EBP and AMA-1, has been identified in multiple rodent

malaria parasite species [9] and in P. falciparum [10].

The similarity of maebl to the dbl-ebp gene family is seen

in the multi-exon gene structure and in the maebl region

encoding the carboxyl cysteine-rich domain [9]. The

DBL-EBPs represent encoded ebl gene family products

which now contain six members for P. falciparum: baebl,eba-175, ebl-1, jesebl, maebl, and pebl [11]. The common

features of these ebls include: (1) similar multi-exon

structures, (2) conserved exon/intron boundaries, (3)

single copy genes, and (4) amino- and carboxyl cysteine-

rich domains [11,12].

Blair et al. [10] described Plasmodium yoelii YM

maebl with characteristics distinguishing it from other

members of the ebl family. Each one of the Plasmodium

ebls encodes the cysteine-rich DBL ligand domain, ex-

cept for maebl which has duplicate amino cysteine-rich

regions (M1 and M2) with similarity to domains 1/2 of

apical membrane antigen-1 (AMA-1) [13]. P. falciparum

MAEBL protein consists of a putative N-terminal signal

peptide, characterised by the presence of a stretch of

hydrophobic amino acids followed by a signal peptide

cleavage site between residues 20 and 21. The signalsequence is followed by a tandem duplication of cys-

teine-rich regions called the M1 and M2 domains. Fol-

lowing this are a repeat region and an EBP region. A 22

amino acid long, trans-membrane domain is present

towards the C-terminus, followed by a cytoplasmatic

domain. The PfM2 domain is predicted to be the pri-

mary ligand domain during blood-stage development

[14]. maebl was identified in all P. falciparum laboratoryclones and strains examined, including 3D7, HB3, Dd2,

Dd2/NM2, and FVO [10]. The M2 domain, which is

considered to be the main MAEBL ligand domain for

merozoites, was highly conserved in 3D7 and FVO,

having perfect amino acid identity in this domain. Dd2

and Dd2/NM2 had perfect amino acid conservation in

the M1 domain and shared only a single amino acid

difference (N682K) in the M2 domain. Its early tran-scription pattern [15] and localization pattern, in rhop-

tries and on the surface of mature merozoites, further

distinguish P. falciparum MAEBL as a unique member

of the ebl family.

This study defined erythrocyte binding regions for

P. falciparum MAEBL protein which could be

functionally significant at the moment of invasion.

The results show that nine peptides bound specifically toerythrocytes; peptides 30181 and 30195 were found in

the N-terminal in the M1 domain and peptide 30198

between the M1 and M2 regions. Peptides 30209, 30212,

30213, 30219, and 30220 were found in the so-called

erythrocyte binding domain (M2) whilst peptide 30253

was found in the protein’s repeat region. It is shown that

peptide binding was saturable and all peptides specifi-

cally bound to a 33 kDa protein on erythrocyte mem-brane. The possible functional role of peptides at the

moment of invasion is also discussed, bearing in mind

that some of them inhibit in vitro merozoite invasion of

erythrocytes.

Materials and methods

Peptide synthesis. One hundred and three sequential 20-mer

peptides, corresponding to the complete P. falciparum MAEBL

protein amino acid sequence [GenBank Accession No. NP_701342],

were synthesised by the solid phase multiple peptide system [16,17];

t-Boc amino acids (Bachem); and MBHA resin (0.7meq/g) were

used. Peptides were cleaved by the low-high HF technique [18],

purified by RP-HPLC, lyophilised, and analysed by MALDI-TOF

mass spectrometry. Tyrosine was added to those peptides which did

not contain this amino acid in their sequences at the C-terminal to

enable 125I-labelling. Synthesised peptides are shown in Fig. 2 in

one-letter code.

Radio-labelling. The peptides were labelled with 125I according to a

previously described methodology [19,20]. Briefly, 3.2 ll Na125I

(100mCi/ml) was oxidised with 12.5ll chloramines-T (2.25 lg/ll) andadded to 5lg peptide for 5min at room temperature. The reaction was

stopped by adding 15 ll sodium bisulphite (2.25lg/ll) and 50lL NaI

(0.16M). The radio-labelled peptide was then separated on a Sephadex

G-10 column (Pharmacia, Uppsala, Sweden).

Binding assays. Based on a previously described methodology,

erythrocytes (2� 108 cells/ll) were incubated with different radio-la-

belled-peptide concentrations (20–100 nM), in the presence or absence

of 20 lM unlabelled peptide for 90min at room temperature [20,21].

After incubation, cells were washed five times with PBS and bound

cell radio-labelled peptide was quantified in an automatic gamma

counter (4/200 plus ICN Biomedicals, Inc.). The binding assays were

performed in triplicate.

Peptide analogues containing the jumbled HABP sequence for

those HABPs thus obtained (Fig. 2A) were then tested in erythrocyte

binding assays to observe whether these peptides’ binding was solely

due to their amino acid composition (Fig. 2B).

Saturation assays. An erythrocyte binding assay was used to as-

certain saturation with all HABPs; the following modifications were

introduced: 1.5� 108 cells were used at 255ll final volume; radio-

labelled peptide concentrations were between 20 and 1000 nM. The

unlabelled peptide concentration was 24 lM. Cells were washed with

PBS and a gamma counter was used to measure bound cell radio-

labelled peptide [19,22,23].

Cross-linking assays. The MAEBL HABPs were cross-linked to

erythrocytes for identifying erythrocyte binding sites. The binding test

was performed by using a final 1.5% cell concentration, following in-

cubation with the radio-labelled peptide in the presence or absence of

20 lM unlabelled peptide for 90min at room temperature. After incu-

bation, cells were washed with PBS and the bound peptide was cross-

linked with 10 lM BS3, bis(sulfosuccinimidyl suberate) (Pierce), for

20min at 4 �C. The reaction was stopped with 20 nMTris–HCl (pH 7.4)

and washed again with PBS. The cells were then treated with lysis buffer

(Tris–HCl 5mM, NaCl 7mM, EDTA 1mM, and PMSF 0.1mM).

M. Ocampo et al. / Biochemical and Biophysical Research Communications 315 (2004) 319–329 321

The obtained membrane proteins were solubilised in Laemmli

buffer and SDS–PAGE analysis was performed with 12% (w/v) poly-

acrylamide gels; these were stained with Coomassie. Those proteins

cross-linked with radio-labelled peptides were exposed on Bio-Rad

Imaging Screen K (Bio-Rad Molecular Imager FX; Bio-Rad Quantity

One, Quantitation Software) for two days and the apparent molecular

weight was determined by using molecular weight markers (NEB).

Enzyme treatment. HABP binding was compared between enzyme-

treated and untreated RBCs [21]. A 30% RBC suspension was incu-

bated with 250lU/ml neuraminidase (ICN 9001-67-6) in sodium

acetate buffer (140mM NaCl, 10mM CaCl2, pH 5.1). The enzyme

concentration was 1mg/ml in TBS buffer (5mM Tris–HCl, 140mM

NaCl, pH 7.4) for trypsin (Sigma T-1005) and chymotrypsin (Sigma C-

4129) treatment. Incubation with each one of the enzymes was carried

out for 60min at 37 �C. The binding assay was performed after ex-

tensive RBC washing with PBS buffer. Untreated RBCs were used as a

control.

Cross-competition assays. The cross-competition assay was done

with radio-labelled MAEBL HABPs versus non-radio-labelled

HABPs. The 125I-labelled HABPs (100 nM) were incubated with

2� 108 cells for 90min at room temperature in the presence of the same

or other unlabelled HABP peptides (40lM). Cells were washed three

times with 1ml PBS and cell bound peptide was quantified, as de-

scribed above for the binding assay.

CD measurement. CD assays were performed at room temperature

on nitrogen-flushed cells using a Jasco J-810 spectro-polarimeter.

Spectra were recorded in 190–260nm wavelength intervals using a 1-

cm path-length rectangular cell. Each spectrum was obtained from

averaging three scans taken at a 20 nm/min scan-rate with 1 nm spec-

tral bandwidth, corrected for baseline, using Jasco software. TFE ti-

tration was carried out by dissolving the lyophilised purified peptides

in the appropriate solvent: (i) 0.1mM sodium phosphate buffer, pH

6.07, and varying concentrations of TFE or (ii) 0–30% aqueous TFE.

Typical peptide concentration was 0.1mM. The results were expressed

as mean residue ellipticity [Q], the units being degrees cm2 dmol�1 ac-

cording to the ½Q� ¼ Qlkð100lcnÞ function where Ql is the measured

ellipticity, l is the optical path-length, c is the peptide concentration,

and n is the number of amino acid residues in the sequence.

Merozoite invasion inhibition assay. Sorbitol synchronised P. falci-

parum (FCB-2 strain) cultures were incubated until the late schizont

stage at final 0.5% parasitemia and 5% haematocrit in RPMI

1640+ 10% O+plasma [24]. The culture was seeded in 96-well cell

culture plates (Nunc, Denmark) in the presence of test peptides at 200,

100, and 10 lM concentrations. Each peptide was tested in triplicate.

After incubation for 18 h at 37 �C in a 5% O2/5% CO2/90% N2 at-

mosphere, the supernatant was recovered and the cells were stained

with 15lg/ml hydroethidine, incubated at 37 �C for 30min, and

washed three times with PBS. The suspensions were analysed on a

FACsort in Log FL2 data mode using CellQuest software (Becton–

Dickinson Immunocytometry System, San Jose, CA) [25]. Infected and

uninfected erythrocytes treated with EGTA and chloroquine were used

as controls.

Results

MAEBL peptides bind specifically to human erythrocytes

Binding assays were used to determine specific

erythrocyte binding activity for 103 synthetic peptidescovering the total length of the MAEBL protein [Gen-

Bank Accession No. NP_701342]. Peptide binding ac-

tivity was defined as being the amount (pmol) of peptide

that bound specifically to erythrocyte per added peptide

(pmol). High activity binding peptides (HABPs) were

defined as those peptides showing activity greater thanor equal to 2%, according to previously established

criteria [20,21,26].

Three types of RBCs’ binding behaviours were found

for the MAEBL peptides (Fig. 1): high specific binding

peptides (i.e., HABP-30212), peptides which did not

bind to RBCs (i.e., 30193) and high no-specific binding

peptides (i.e., 30190) where peptides bound to erythro-

cytes but where there was no inhibition with the samenon-radio-labelled peptide.

Nine erythrocyte HABPs were found in MAEBL-

peptides: 30181 (121KYKLPIEIPLNKSGLSMYQG140),

30195 (401TGSCYFLKKKPTCVLKKENH420), 30198

(461QTNKRVLYENNKKSKRNVRT480), 30209 (681LN

FLNEIRTGYLNKYFKKDV700), 30212 (741KSKIFSN

RFTMKEYDPKTRL760), 30213 (761FMYYGLYGLG

GRLGANIKRD780), 30219 (881YVSSFIRPDYETKCPPRYPL900), 30220 (901KSKVFGTFDQKTGKCKSLM

DY920), and 30253 (1561RAEILKQIEKKRIEEVMK

LY1580). The 30181, 30195, and 30198 HABPs were lo-

cated in the MAEBL amino terminal region (M1), the

30209, 30212, 30213, 30219, and 30220 HABPs were

located within the M2 erythrocyte binding domain,

reported byGhai et al. [27].Only peptide 30253was found

within the protein’s repeat region and no HABPs werefound in the C-terminal region (Fig. 2A).

Analogues containing ‘jumbled’ sequences were syn-

thesised to investigate whether MAEBL HABP binding

was due solely to their amino acid composition or their

specific sequence. These ‘jumbled’ peptides had the same

amino acid composition as the high binding ones but in

a random sequence (32239, 32240, 32241, 32242, 32243,

32244, 32245, 32246, and 32247). These peptides’ spe-cific binding was less than that presented by the original

HABPs (Fig. 2B).

Binding constants for high activity binding peptides

Affinity constants, number of binding sites per cell,

and Hill coefficients were determined for HABPs by

saturation assays and Hill analysis [19] (Fig. 3). The

affinity constants ðKdÞ ranged from 185 to 380 nM and

Hill coefficients were between 1.0 and 1.7, suggesting

positive cooperativity. The number of binding sites per

cell was found to be between 5000 and 32,000 (Table 1).

Recognising MAEBL HABP binding protein (cross-

linking assays)

All HABPs recognised one erythrocyte membrane

protein having an apparent 33 kDa molecular weightwhen erythrocyte membranes and HABPs were cross-

linked with BS3 followed by separation in SDS–PAGE.

Radio-labelled peptide interaction with this protein was

inhibited when the binding was performed in the pres-

ence of unlabelled peptide, indicating that it was a

Fig. 1. Erythrocyte binding assays. The erythrocyte binding for three peptides from the PfMAEBL protein is shown. The X-axis represents peptide

radio-labelled cpm added to the cells and the Y-axis represents the radio-labelled cell bound peptide. The graphs on the left present total radio-

labelled peptide binding (j, cells incubated with radio-labelled peptide) and inhibited binding or non-specific binding (d, cells incubated with radio-

labelled peptide in the presence of non-radio-labelled peptide). The three graphs on the right-hand side show specific binding for each one of the

MAEBL peptides (m). Three types of behaviours are presented: specific high binding peptide (peptide 30212), non-binding peptide (peptide 30190),

and non-specific high binding peptide (peptide 30193).

322 M. Ocampo et al. / Biochemical and Biophysical Research Communications 315 (2004) 319–329

specific interaction. Fig. 4 shows the MAEBL HABP

cross-linking assays.

MAEBL HABP binding to enzymatically treated ery-

throcytes

RBCs were enzymatically treated and tested in

binding assays with HABPS to determine the probable

nature of receptors on erythrocytes. Fig. 5 shows thechanges in HABPs’ specific binding when assayed for

binding to enzyme-treated erythrocytes’ compared to

untreated erythrocytes specific binding. Only peptide

30220 binding to enzymatically treated erythrocytes

became reduced in all cases (44%, 5%, and 15%, re-

spectively, with neuraminidase, chymotrypsin or tryp-

sin). Binding to erythrocytes treated with chymotrypsin

became diminished for peptides 30195, 30209, 30219,and 30253. In the case of peptides such as 30181 there

was an increase in binding when erythrocytes had been

enzymatically treated.

MAEBL protein HABP cross-competition assays

Due to the cross-linking assays revealing an approx-

imately 33 kDa protein as being the possible receptor for

all the MAEBL HABPs, cross-competition assays were

Fig. 2. Erythrocyte binding assays using PfMAEBL peptides. (A) Each

one of the black bars represents the slope of the specific binding graph,

which is named specific binding activity. Peptides with 2% were con-

sidered as having high specific erythrocyte binding (HABPs). A sche-

matic representation of the MAEBL protein regions M1 and M2 is

shown to the right of the figure. (B) Specific binding activity for HABP

peptide analogues; original and jumbled peptide sequences are shown

to the right and the bars on the left represent specific binding. The

numbers in the first column represent FIDIC’s internal code number

for those peptides synthesised.

M. Ocampo et al. / Biochemical and Biophysical Research Communications 315 (2004) 319–329 323

done between the different HABPs, finding that peptide

30253 binding was inhibited by the other HABPs from

the same protein (except for peptide 30198), whilst

peptide 30213 inhibited all HABPs’ binding (data notshown). The foregoing could indicate that the peptides

presented specific binding to different sites on the 33 kDa

protein.

Circular dichroism analysis

HABPs’ crossed inhibition showed that the binding

of some of these peptides to erythrocytes could be in-

hibited by other different non-radio-labelled HABPs

(belonging to the same protein). However, these pep-

tides’ amino acid sequences did not present homology;

structural analysis approximated by circular dichroism

(CD) showed that there are some probable structuralelements (Fig. 6).

Even though most MAEBL HABPs presented a

random coil structure (Fig. 6A), peptides 30209 and

30253 displayed a-helix-like feature according to two

208 and 222 nm minimum values and a 190 nm maxi-

mum ellipticity (Fig. 6B).

In vitro merozoite invasion inhibition

The possible role of MAEBL HBAPs in in vitro

merozoite invasion was investigated. The peptides were

added to cultures at the schizont stage before the mer-

ozoites were liberated from infected erythrocytes. Theresults show that all the HABPs (except for peptides

30209 and 30213) inhibited merozoite invasion by over

60%; peptides 30195, 30220, and 30253 inhibited it by

over 97%. It can also be observed that invasion inhibi-

tion depended on peptide concentration and did not

Fig. 3. Saturation curve for HABPs. Increasing quantities of labelled peptide were added in the presence or absence of unlabelled peptide. The curve

represents the specific binding. In the Hill plot (inset), the abscissa is log F and the ordinate is logðB=Bmax � BÞ, where F is free peptide, B is bound

peptide, and Bmax is the maximum amount of bound peptide.

324 M. Ocampo et al. / Biochemical and Biophysical Research Communications 315 (2004) 319–329

Fig. 4. Autoradiograph from HABPs’ cross-linking assays. Erythrocyte m

Odd-numbered lanes show the total binding (i.e., cross-linking in the absence

(i.e., cross-linking in the presence of unlabelled peptide).

Fig. 5. Specific binding to enzymatically treated erythrocytes. The bars repr

labelled HABPs were incubated with erythrocytes treated with neuraminid

erythrocytes (j).

Table 1

Binding constants of MAEBL HABPs to erythrocytes

Peptide Kd

(nM)

Hill

coefficient

Binding sites

per cell

30181 295 1.6 16,061

30195 220 1.4 16,463

30198 260 1.6 14,054

30209 250 1.0 31,721

30212 300 1.1 18,069

30213 300 1.7 19,274

30219 270 1.2 5220

30220 380 1.7 8031

30253 185 1.5 5220

M. Ocampo et al. / Biochemical and Biophysical Research Communications 315 (2004) 319–329 325

correlate with peptide charge, binding affinity or Hillcoefficient (Table 2).

Discussion

A better understanding of the complex process of

P. falciparum merozoite invasion requires that the

numerous potential parasite ligands become identifiedand characterised. Our studies have required an in-

novative methodology to be employed in the search

for sequences which can be precisely modified for use

as candidates for a vaccine against malaria [28–31].

embrane proteins were cross-linked with radio-labelled PfMAEBL.

of unlabelled peptide) and even-numbered lanes show inhibited binding

esent the percentage of specific binding activity obtained when radio-

ase (�), chymotrypsin ( ), and trypsin ( ) with respect to untreated

Table 2

MAEBL peptide inhibition of parasitic invasion of RBCa

Peptide Invasion inhibition (%)

10 lM 100lM 200lM

30181 15� 1 17� 4 62� 3

30195 5� 3 25� 4 98� 1

30198 12� 2 25� 2 79� 1

30209 1� 4 19� 1 57� 1

30212 5� 3 13� 1 61� 8

30213 0� 4 5� 1 51� 5

30219 4� 1 8� 1 71� 1

30220 5� 2 13� 1 97� 3

30253 12� 2 47� 2 102� 1

30201 1� 1 4� 1 54� 2

Chloroquine 100� 2

aMeans� SD of three experiments.

Fig. 6. Circular dichroism analysis of PfMAEBL HABPs. (A) Random coil structural elements present in most HABPs. (B) Two of the HABPs

presented 208 and 222 nm minimum values and helical structural elements.

326 M. Ocampo et al. / Biochemical and Biophysical Research Communications 315 (2004) 319–329

Some of the important ligand determinants for inva-

sion belong to the members of the ebl family [11].

Differences in merozoite location are understood to

relate to differences in function during merozoite in-

vasion of erythrocytes. Microneme proteins are crucial

for the later steps in the invasion process. For ex-

ample, P. knowlesi DBP/Duffy blood group interaction

is necessary for junction formation during invasion ofhuman erythrocytes [32].

It has been suggested that early parasite interaction

with host cell receptors signals microneme release. The

presence of MAEBL in a different apical organelle and

on the merozoite surface suggests a functional role dif-

ferent from that of microneme proteins such as EBA-

175 or BAEBL. A role for MAEBL in an early phase of

the invasion process is appropriate, since the protein is

in the right place at the right time for involvement in the

early stages of merozoite invasion. These proteins may

function in selecting a particular cell to be invaded

during the initial recognition and attachment to a hosterythrocyte [33,34].

Adhesion molecules are likely to have flexible func-

tions, multiple or variable, in either their receptor

specificity and/or their role during invasion of a host

cell.

Bearing the MAEBL protein’s possible role in mind

regarding the P. falciparum invasion process according

to this protein’s previously presented characteristics, thisstudy has identified high specific erythrocyte activity

binding peptides (HABPs) by using receptor-ligand

binding assays. Most of these HABPs were found in the

M2 region suggested as being the protein’s erythrocyte

binding domain, due to its homology with other pro-

teins from the same family or with other MAEBL from

different Plasmodium species. However, there have been

no reports to date giving results indicating that thewhole P. falciparum MAEBL protein binds to human

erythrocytes; it has solely been reported that r-PfM2

protein has bound erythrocytes in in vitro binding ex-

periments [27]. It has been reported that Plasmodium

yoelii yoelii MAEBL M1 and M2 domains expressed on

COS-7 cells do not present binding to human RBCs [35]

but do present in vitro rat erythrocyte binding activity.

The M2 domain seems to be the main binding domainsince it shows more activity than the M1 domain. In the

case of those peptides conforming to PfMAEBL pro-

tein, two HABPs have been identified in the M1 region

M. Ocampo et al. / Biochemical and Biophysical Research Communications 315 (2004) 319–329 327

and five in the M2 region; these regions could beimplicated in PfMAEBL protein being recognised by

human erythrocytes. When these HABPs’ sequence is

modified regarding the order of those amino acids

comprising it (but keeping their net charge and final

amino acid composition) then specific binding to ery-

throcytes becomes diminished in such a way that they

can no longer be considered as having high binding

activity (i.e., as they are no longer relevant to the pro-tein’s binding). These results also show that these

HABPs’ binding depends on the amino acid sequence

and that variations in such sequence may alter eryth-

rocyte binding activity.

Binding between HABPs and erythrocytes has

high affinity, presenting a positive cooperativity re-

ceptor–ligand interaction as can be established from

determining physical–chemical constants; this is ex-pected for binding sequences which are involved in

merozoite invasion. The role of these peptides in

invasion becomes evident in in vitro assays where

most HABPs inhibit invasion depending on

concentration.

Regarding a possible receptor for these HABPs on

erythrocyte surface, SDS–PAGE and auto-radiography

have identified a 33 kDa band which becomes specifi-cally inhibited in the presence of the same non-radio-

labelled peptide. However, according to specific

binding results, once erythrocytes have been enzymat-

ically treated, the receptor site on the same 33 kDa

protein can vary for some HABPs. First, neuramini-

dase treatment removing sialic acid from erythrocyte

surface only affected peptide 30220 binding; when

erythrocytes were treated with trypsin there was adrastic lessening in this peptide’s binding (as expected,

following the above). This peptide’s binding also be-

came lessened when erythrocytes were treated with

chymotrypsin (i.e., the receptor site for peptide 30220

was sensitive to all three modifications made to

erythrocyte surface following enzymatic treatment).

Second, receptor sites for most HABPs were sensitive

to chymotrypsin treatment. Also, cryptic receptor siteswere presented for peptide 30181, becoming evident

when the surface of erythrocytes was modified with

whichever enzymatic treatment. The diversity in dif-

ferent HABPs’ binding sites was correlated to the fact

that P. falciparum clones and isolates have alternative

invasion pathways and are therefore not dependent on

a single erythrocyte receptor [36–38]. Erythrocyte re-

ceptors for P. falciparum invasion thus include gly-cophorin A, glycophorin B, epitopes associated with

glycophorin C and D, and as yet non-characterised

receptors ‘X, Y, Z, E’ [36,39,40].

On the other hand, this study wished to determine

whether there was cross-competition between the dif-

ferent HABPs, finding that peptide 30253 binding was

inhibited by the other HABPs and that peptide 30213

was able to inhibit the binding of all the HABPs. Cir-cular dichroism results for these HABPs showed that

peptide 30253 presented a helical structure seeming to be

specifically located on the surface of the erythrocytes

and whichever of the other HABPs having a random

coil structure prevented peptide 30253 from being able

to “fit” into the specific binding site on erythrocyte

surface. Peptide 30213 also presenting random coil

structural elements however seemed to confer theproperty of competing for binding site on the other

HABPs. Bearing in mind that most HABPs have ran-

dom coil structures, many configurations could inhibit

the other HABPs’ binding by adopting the configuration

necessary for inhibition.

Nine high specific erythrocyte binding capacity pep-

tides were determined found distributed in the M1 and

M2 regions as well as the PfMAEBL protein’s repeatregion. This fact confirms the importance of the M2

region which has been described as being important for

P. yoelii MAEBL; most HABPs were found in this re-

gion and were also those presenting the greatest specific

binding ability. These HABPs could be directly involved

in the protein’s binding to erythrocytes and, bearing in

mind that this protein could be involved in invasion,

these HABPs are thus relevant in the invasion process.In fact, these peptides are able to significantly inhibit in

vitro P. falciparum merozoite invasion, indicating that

these peptides can block the merozoite-erythrocyte in-

teraction to some degree; however, it is not clear whe-

ther the 33 kDa protein (specifically recognised by

HABPs on erythrocyte membrane) is directly involved

in such invasion inhibition.

Further in vitro and in vivo studies are necessary toclarify whether MAEBL plays a specific role in the

parasite’s life cycle, specifically at the moment of mer-

ozoite invasion of erythrocytes. Additional in vitro

studies are necessary for identifying the interaction

mechanism between HABPs and erythrocytes as well as

the nature of the protein to which HABPs specifically

bind on erythrocyte membrane.

They are also necessary for identifying the possibilityof using P. falciparum MAEBL HABP protein se-

quences for designing tools for the specific inhibition of

P. falciparum merozoite interaction with erythrocytes.

The above results suggest that MAEBL HABPs

might be involved in one of the several interactions be-

tween merozoites and erythrocytes and support the idea

that they might be bound to erythrocyte surface, in-

hibiting merozoite binding to erythrocytes.

Acknowledgments

This research project was supported by the President of the Re-

public of Colombia’s office and the Colombian Ministry of Public

Health. We thank Jason Garry for reading the manuscript.

328 M. Ocampo et al. / Biochemical and Biophysical Research Communications 315 (2004) 319–329

References

[1] C.E. Chitnis, Molecular insights into receptors used by malaria

parasites for erythrocyte invasion, Curr. Opin. Hematol. 8 (2001)

85–91.

[2] J.A. Dvorak, L.H. Miller, W.C. Whitehouse, T. Shiroishi,

Invasion of erythrocytes by malaria merozoites, Science 187

(1975) 748–750.

[3] M. Aikawa, L.H. Miller, J. Johnson, J. Rabbege, Erythrocyte

entry by malarial parasites. A moving junction between erythro-

cyte and parasite, J. Cell Biol. 77 (1978) 72–82.

[4] G.E. Ward, C.E. Chitnis, L.H. Miller, The invasion of erythro-

cytes by malarial merozoites, in: D.G. Russell (Ed.), Clinical

Infectious Diseases, vol. 1, Bailliere Tindall, London, 1994, pp.

155–187.

[5] J.H. Adams, D.E. Hudson, M. Torii, G.E. Ward, T.E. Wellems,

M. Aikawa, L.H. Miller, The Duffy receptor family of Plasmo-

dium knowlesi is located within the micronemes of invasive malaria

merozoites, Cell 63 (1990) 141–153.

[6] L.H. Bannister, G.H. Mitchell, G.A. Butcher, E.D. Dennis,

Lamellar membranes associated with rhoptries in erythrocytic

merozoites of Plasmodium knowlesi: a clue to the mechanism of

invasion, Parasitology 92 (1986) 291–303.

[7] P. Preiser, M. Kaviratne, S. Khan, L. Bannister, W. Jarra, The

apical organelles of malaria merozoites: host cell selection,

invasion, host immunity and immune evasion, Microbes Infect.

2 (2000) 1461–1477.

[8] M.J. Blackman, L.H. Bannister, Apical organelles of Apicom-

plexa: biology and isolation by subcellular fractionation, Mol.

Biochem. Parasitol. 117 (2001) 11–25.

[9] S.H. Kappe, G.P. Curley, A.R. Noe, J.P. Dalton, J.H. Adams,

Erythrocyte binding protein homologues of rodent malaria

parasites, Mol. Biochem. Parasitol. 89 (1997) 137–148.

[10] P.L. Blair, S.H. Kappe, J.E. Maciel, B. Balu, J.H. Adams,

Plasmodium falciparum MAEBL is a unique member of the ebl

family, Mol. Biochem. Parasitol. 122 (2002) 35–44.

[11] J.H. Adams, P.L. Blair, O. Kaneko, D.S. Peterson, An expanding

ebl family of Plasmodium falciparum, Trends Parasitol. 17 (2001)

297–299.

[12] J.H. Adams, B.K.L. Sim, S.A. Dolan, X. Fang, D.C. Kaslow,

L.H. Miller, A family of erythrocyte binding proteins of

malaria parasites, Proc. Natl. Acad. Sci. USA 89 (1992) 7085–

7089.

[13] S.H. Kappe, A.R. Noe, T.S. Fraser, P.L. Blair, J.H. Adams, A

family of chimeric erythrocyte binding proteins of malaria

parasites, Proc. Natl. Acad. Sci. USA 95 (1998) 1230–1235.

[14] A.R. Noe, D.J. Fishkind, J.H. Adams, Spatial and temporal

dynamics of the secretory pathway during differentiation of the

Plasmodium yoelii schizont, Mol. Biochem. Parasitol. 108 (2000)

169–185.

[15] P.L. Blair, A. Witney, J.D. Haynes, J.K. Moch, D.J. Carucci, J.H.

Adams, Transcripts of developmentally regulated Plasmodium

falciparum genes quantified by real-time RT-PCR, Nucleic Acids

Res. 30 (2002) 2224–2231.

[16] R.B. Merrifield, Solid phase peptide synthesis l. The synthesis of a

tetrapeptide, J. Am. Chem. Soc. 85 (1963) 2149–2154.

[17] R.A. Houghten, General method for the rapid solid phase

synthesis of large numbers of peptides: specificity of antigen

antibody interaction at the level of individual aminoacids, Proc.

Natl. Acad. Sci. USA 82 (1985) 5131–5135.

[18] J.P. Tam, W.F. Heath, R.B. Merrifield, SN2 deprotection of

synthetic peptides with a low concentration of HF in dimethyl

sulfide: evidence and application in peptide synthesis, J. Am.

Chem. Soc. 105 (1983) 6442–6455.

[19] H.I. Yamamura, S.J. Enna, M.J. Kuhar, Neurotransmitter

Receptor Binding, Raven Press, New York, 1978.

[20] M.Urquiza, L.E. Rodr�ıguez, J.E. Suarez, F.Guzman,M.Ocampo,

H. Curtidor, C. Segura, E. Trujillo, M.E. Patarroyo, Identification

of Plasmodium falciparum MSP-1 peptides able to bind to human

red blood cells, Parasite Immunol. 18 (1996) 515–526.

[21] M. Ocampo, M. Urquiza, F. Guzman, L.E. Rodriguez, J. Suarez,

H. Curtidor, J. Rosas, M. Diaz, M.E. Patarroyo, Two MSA 2

peptides that bind to human red blood cells are relevant to

Plasmodium falciparummerozoite invasion, J. Pept. Res. 55 (2000)

216–223.

[22] E.C. Hulme, Receptor–Ligand Interactions. A Practical Ap-

proach, IRL Press, Oxford, 1993.

[23] G.A. Weiland, P.B. Molinoff, Quantitative analysis of drug-

receptor interactions. 1. Determination of kinetic and equilibrium

properties, Life Sci. 29 (1981) 313–330.

[24] C. Lambros, J.P. Vanderberg, Synchronisation of Plasmodium

falciparum erythrocyte stages in culture, J. Parasitol. 65 (1979)

418–420.

[25] C.R. Wyatt, W. Goff, W.C. Davis, A flow cytometric method for

assessing viability of intraerythrocytic hemoparasites, J. Immunol.

Methods 140 (1991) 23–30.

[26] L.E. Rodr�ıguez, M. Urquiza, M. Ocampo, J. Suarez, H.

Curtidor, F. Guzman, L.E. Vargas, M. Trivi~nos, M. Rosas,

M.E. Patarroyo, Plasmodium falciparum EBA-175 kDa protein

peptides which bind to human red blood cells, Parasitology

120 (2000) 225–235.

[27] M. Ghai, S. Dutta, T. Hall, D. Freilich, C.F. Ockenhouse,

Identification, expression, and functional characterization of

MAEBL, a sporozoite and asexual blood stage chimeric erythro-

cyte binding protein of Plasmodium falciparum, Mol. Biochem.

Parasitol. 123 (2002) 35–45.

[28] F. Espejo, M. Cubillos, L.M. Salazar, F. Guzman, M. Urquiza,

M. Ocampo, Y. Silva, R. Rodr�ıguez, E. Lioy, M.E. Patarroyo,

Structure, immunogenicity, and protectivity relationship for the

1585 malarial peptide and its substitution analogues, Angew.

Chem. Int. Ed. Engl. 40 (2001) 4654–4657.

[29] M. Cubillos, L.M. Salazar, L. Torres, M.E. Patarroyo, Protection

against experimental P. falciparum malaria is associated with

short AMA-1 peptide analogue a-helical structures, Biochimie. 84

(2003) 1181–1188.

[30] A. Bermudez, G. Cifuentes, F. Guzman, L.M. Salazar, M.E.

Patarroyo, Immunogenicity and protectivity of Plasmodium falci-

parum EBA-175 peptide and its analog is associated with alpha-

helical region shortening and displacement, Biol. Chem. 384

(2003) 1443–1450.

[31] M.H. Torres, L.M. Salazar, M. Vanegas, F. Guzman, R.

Rodriguez, Y. Silva, J. Rosas, M.E. Patarroyo, Modified mero-

zoite surface protein-1 peptides with short alpha helical regions

are associated with inducing protection against malaria, Eur. J.

Biochem. 270 (2003) 3946–3952.

[32] L.H. Miller, M. Aikawa, J.G. Johnson, T. Shiroishi, Interaction

between cytochalasin B-treated malarial parasites and erythro-

cytes. Attachment and junction formation, J. Exp. Med. 149

(1979) 172–184.

[33] A.A. Holder, R.R. Freeman, Immunization against blood-stage

rodent malaria using purified parasite antigens, Nature 294 (1981)

361–364.

[34] R.R. Freeman, A.J. Trejdosiewicz, G.A. Cross, Protective mono-

clonal antibodies recognising stage-specific merozoite antigens of

a rodent malaria parasite, Nature 284 (1980) 366–368.

[35] S.H. Kappe, A.R. Noe, T.S. Fraser, P. Blair, J.H. Adams, A

family of chimeric erythrocyte binding proteins of malaria

parasites, Proc. Natl. Acad. Sci. USA 95 (1998) 1230–1235.

[36] S.A. Dolan, J.L. Proctor, D.W. Alling, Y. Okubo, T.E. Wellems,

L.H. Miller, Glycophorin B as an EBA-175 independent Plasmo-

dium falciparum receptor on human erythrocytes, Mol. Biochem.

Parasitol. 64 (1994) 55–63.

M. Ocampo et al. / Biochemical and Biophysical Research Communications 315 (2004) 319–329 329

[37] G.H. Mitchell, T.J. Hadley, M.H. McGinniss, F.W.

Klotz, L.H. Miller, Invasion of erythrocytes by

Plasmodium falciparum malaria parasites: evidence for

receptor heterogeneity and two receptors, Blood 67 (1986)

1519–1521.

[38] M.E. Perkins, E.H. Holt, Erythrocyte receptor recognition varies

in Plasmodium falciparum isolates, Mol. Biochem. Parasitol. 27

(1988) 23–34.

[39] D.C. Mayer, O. Kaneko, D.E. Hudson-Taylor, M.E. Reid, L.H.

Miller, Characterization of a Plasmodium falciparum erythrocyte-

binding protein paralogous to EBA-175, Proc. Natl. Acad. Sci.

USA 98 (2001) 5222–5227.

[40] J.K. Thompson, T. Triglia, M.B. Reed, A.F. Cowman, A novel

ligand from Plasmodium falciparum that binds to a sialic acid-

containing receptor on the surface of human erythrocytes, Mol.

Microbiol. 41 (2001) 47–58.