Identifying Plasmodium falciparum EBA175 homologue sequences that specifically bind to human...

www.elsevier.com/locate/ybbrc

Biochemical and Biophysical Research Communications 321 (2004) 835–844

BBRC

Identifying Plasmodium falciparum EBA-175 homologue sequencesthat specifically bind to human erythrocytesq

John Jairo Valbuena *, Ricardo Vera Bravo, Marisol Ocampo, Ramses Lopez,Luis E. Rodriguez, Hernando Curtidor, Alvaro Puentes, Javier E. Garcia,

Diana Tovar, Johana Gomez, Jesus Leiton, Manuel Elkin Patarroyo

Fundacion Instituto de Inmunologia de Colombia, Universidad Nacional de Colombia, Colombia

Received 6 July 2004

Abstract

Erythrocyte binding antigen-160 (EBA-160) protein is a Plasmodium falciparum antigen homologue from the erythrocyte binding

protein family (EBP). It has been shown that the EBP family plays a role in parasite binding to the erythrocyte surface. The EBA-

160 sequence has been chemically synthesised in seventy 20-mer sequential peptides covering the entire 3D7 protein strain, each of

which was tested in erythrocyte binding assays to identify possible EBA-160 functional regions. Five EBA-160 high activity binding

peptides (HABPs) specifically binding to erythrocytes with high affinity were identified. Dissociation constants lay between 200 and

460nM and Hill coefficients between 1.5 and 2.3. Erythrocyte membrane protein binding peptide cross-linking assays using SDS–

PAGE showed that these peptides bound specifically to 12, 28, and 44kDa erythrocyte membrane proteins. The nature of these re-

ceptor sites was studied in peptide binding assays using enzyme-treated erythrocytes. HABPs were able to block merozoite in vitro

invasion of erythrocytes. HABPs� potential as anti-malarial vaccine candidates is also discussed.

� 2004 Elsevier Inc. All rights reserved.

Keywords: Erythrocyte binding antigen-160; High activity binding peptides; Plasmodium falciparum

Erythrocyte invasion by malaria parasites is a com-

plex multi-step process involving parasite and erythro-

cyte receptors [1,2]. Diverse Plasmodium species use

different ligands [3,4]. Identifying erythrocyte binding li-

gands has a direct application in understanding the steps

involved in parasite invasion and thus in designing atailored multi-component, subunit-based synthetic

malarial vaccine.

The erythrocyte binding protein (EBP) family has

been shown to play a role in parasite binding to the

erythrocyte surface. These proteins� extra-cellular part

is characterised by two cys-rich domains and a C-cys

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

doi:10.1016/j.bbrc.2004.07.034

q Abbreviations: RBC, red blood cells; HABP, high activity binding

peptide; TFE, trifluoroethanol.* Corresponding author. Fax: +57-1-4815269.

E-mail address: [email protected] (J.J. Valbuena).

domain adjacent to a transmembrane domain [4,5].

Plasmodium falciparum erythrocyte binding antigen-

175 (EBA-175) is a member of the EBP family that binds

to sialic acid residues on the major erythrocyte glycopro-

tein (glycophorine-A) and has been shown to be an im-

portant mediator in the invasion process [6–8]. EBA-175has two conserved cysteine-rich domains. The amino

terminal cysteine domain contains two copies of a Duffy

binding like domain (DBL) called F1 and F2 [4,7].

The P. falciparum genome sequence identifies at least

five EBA-175 paralogues: EBA-140, EBA-165, EBA-

181, EBL-1, and MAEBL [4,9]. EBA-140 protein is

30% identical to EBA-175 along the whole protein se-

quence and plays a role in invasion, binding to the eryth-rocyte surface via the glycophorine-C receptor [10,11].

The putative weba-165 pseudogene is transcribed but

does not appear to be expressed at protein level [12].

mailto:[email protected]

836 J.J. Valbuena et al. / Biochemical and Biophysical Research Communications 321 (2004) 835–844

The EBA-181 protein binds sialic acid-dependently to a

trypsin-resistant/chymotrypsin-sensitive receptor on the

erythrocyte surface [4,13]. EBL-1 has been identified as

being a second EBP family member in P. falciparum

on the basis of consensus family characteristics, but its

C-cys domain is degenerate, having only four cysteineresidues [14]. The MAEBL malarial ligand is another

EBP family paralogue, having a chimeric structure in

which the DBL domain is functionally replaced by a dis-

tinct cysteine-rich erythrocyte binding domain being

similar to the apical membrane antigen-1 (AMA-1)

ligand domain [15].

Other parasite ligands likely to be homologous to EBP

family members are present in P. falciparum. Other geneshomologous to the known EBPmembers have been iden-

tified in searches of P. falciparum genome project se-

quence data [16,17]. One of these genes (PFD1155w)

was found in chromosome 4, having 4167bps, encoding

a 160kDa, 1388 amino-acid protein which has been

termed ‘‘erythrocyte binding antigen’’ (GenBank Acces-

sion No. CAD49277). This protein�s structure is identicalto that described for EBA-175 and similar to those ofother proteins described for EBP family members.

This article identifies and characterises erythrocyte

binding antigen-160 protein (EBA-160) in terms of its

erythrocyte binding specificities and investigates its func-

tional relevance to parasite invasion. Seventy EBA-160

peptides from the 3D7 strain-deduced sequence were

synthesised in 20 non-overlapping sequences and tested

in receptor–ligand erythrocyte binding assays. Fiveerythrocyte high activity binding peptides (HABPs) were

identified. It was found that HABP erythrocyte binding

was saturable and sensitive to enzyme treatment. These

sequences inhibited the merozoite in vitro invasion pro-

cess in cultures. It was found that all HABP bound

RBC proteins having around 12, 28, and 44kDa molec-

ular weight in RBC cross-linking and SDS–PAGE bind-

ing assays.Results presented here provide new insight into

knowledge regarding receptor–ligand interactions in-

volved in the invasion process and suggest the potential

of EBA-160 as a candidate for inclusion in an effective

asexual-stage malaria vaccine.

Materials and methods

Peptide synthesis. Seventy 20-mer peptides, covering the complete

P. falciparum erythrocyte binding antigen-160 protein 3D7 strain

(Accession No. CAD49277) [16,17], were synthesised by solid-phase

multiple peptide system [18,19], with 60mg p-methylbenzyhydryl

amine resin (0.74meq/g substitution). Standard N a t-Boc protected

amino-acids were employed (Bachen). Peptides were cleaved by the

low-high HF technique [20], purified by RP-HPLC, and characterised

by MALDI-TOF mass spectrometry. An extra Tyr residue was added

to a peptide�s C-terminus for radio-labelling purposes when such

peptide did not contain Tyr. Fig. 1A shows synthesised sequences in

one-letter code.

Radio-labelling. The synthesised peptides were Na125I labelled for

performing human erythrocyte binding assays [21–26]. Briefly, 2nm

purified peptides were radio-labelled with 5ll Na125I and 0.3lmol

chloramine-T in 25ll final volume for 15min. The reaction was

stopped by adding 15ll sodium metabisulphite (2.75lg/ll). The radio-labelled peptide was separated from reaction products by phosphate

buffered saline (PBS) elution on a Sephadex G10 column (Pharmacia,

Uppsala, Sweden) (80 · 5mm) and then measured by automatic

gamma counter (4/200 plus ICN Biomedicals).

Binding assays. The screening binding assay for each of the peptides

was performed on human erythrocytes obtained from healthy O po-

sitive blood-donors [21–26]. 2 · 108 red blood cells were incubated in

triplicate with increasing quantities of 125I-labelled peptide (between 0

and 285nM) in a 200ll final volume for 90min in the absence (total

binding) or presence of 20lM unlabelled peptide (non-specific binding)

to determine binding specificity. After incubation, unbound peptide

was removed with three 5ml PBS washes. Cell-bound peptide was

measured in an automatic gamma counter.

Specific binding was calculated as being the difference between total

and non-specific binding. The specific binding curve slope (specific

bound-pmol/total added-pmol) was taken as being the indicator for

binding activity and was determined for each peptide [23–26]. The

results are reported and shown graphically in Fig. 1A.

Binding assay with HABP jumbled-peptide. HABP jumbled-peptide

analogue sequences were synthesised (peptides having the same amino-

acid composition as HABPs but random sequences) to determine

whether HABP binding to red blood cells was due to their specific

amino-acid sequences or just their amino-acid composition.

These peptides were tested in binding assays. Briefly, 2 · 108 eryth-

rocytes were incubated with a total of 200ll 125I-labelled jumbled pep-

tide (0–285nM) for 90min at room temperature, in the presence or

absence of 20lM unlabelled jumbled-peptide. After incubation, un-

bound peptide was removed from cells by washing three times with PBS.

The cell-bound peptide was measured in an automatic gamma counter.

The assays were carried out in triplicate under identical conditions.

Saturation assays. Saturation assays were carried out using human

erythrocyte binding assays with each of the HABPs. A total

1.5 · 108cells were incubated with radio-labelled HABPs at concen-

trations between 0 and 1000nM, in the presence or absence of unla-

belled peptide (25lM). PBS was added to achieve a final 255llvolume. Reaction mixtures were incubated for 90min at room tem-

perature [21–26]. Cells were washed with PBS and bound cell radio-

labelled peptide was measured by gamma counter; each assay was

performed in triplicate. Curves were obtained in the same way as those

for the binding assays.

Cross-competition assays. The cross-competition assays were per-

formed between each one of the HABPs. Radio-labelled HABPs

(280nM) were incubated with 2 · 108 erythrocytes for 90min at room

temperature, in the presence or absence of the same or other unlabelled

HABPs (20lM). After incubation, unbound peptide was removed with

three 5ml PBS washes. Cell-bound peptide was measured in an auto-

matic gamma counter. The cross-competition assays were done in

triplicate [27].

Cross-linking assays. High activity binding peptides were cross-

linked to cell membranes for identifying erythrocyte binding sites. The

binding assay used a 1% final cell concentration and, after incubation

with the radio-labelled peptide for 90min at room temperature and

thorough washing with PBS, the bound peptide was cross-linked to

10lM BS3 (bis[sulphosuccinimidyl]suberate) (Pierce) for 30min at

4 �C. The cells were washed again with PBS and treated with lysis

buffer (5% SDS, 10nM iodoacetamide, 1% Triton X-100, 100mM

EDTA, and 10mM PMSF). The obtained membrane proteins were

solubilised in Laemmli buffer and separated in SDS–PAGE. Those

proteins cross-linked with radio-labelled peptides were exposed on

Kodak film (X-OMAT) for 24h at �70�C and the apparent molecular

weight was determined by using 14.4 to 97.4kDa molecular weight

markers (Bio-Rad).

Fig. 1. (A) EBA-160 synthetic peptide erythrocyte binding profile and peptide sequences. In those peptides that did not contain Tyr, this was added

at the carboxy-terminal. Amino-acid sequences are given in the left-hand column, indicating the position of each peptide within the protein. The

column on the right-hand side shows each peptide�s specific binding activity (black bars). This was constructed from specific binding slopes

(specifically bound peptide/added peptide). Peptides with binding activity >2% were considered as having high specific binding to erythrocytes

(corresponding to specific >0.02pmol bound/pmol added binding which, according to previous data, detects >200 binding sites per cell). The assay

was done in triplicate. (B) HABP jumbled-peptide erythrocyte binding profile. Jumbled-peptide binding activity was lower than that for native

HABPs.

J.J. Valbuena et al. / Biochemical and Biophysical Research Communications 321 (2004) 835–844 837

Fig 1. (continued)


Enzyme treatment. HABP binding was compared between enzyme-

treated and untreated human erythrocytes. Five percent red blood cell

suspensionwas incubatedwith 150lU/ml neuraminidase (ICN 9001-67-

6) in sodium acetate buffer, pH 5.1. Enzyme concentration was 0.75mg/

ml in TBS buffer (5mM Tris–HCl, 140mM NaCl, pH 7.4) for trypsin

(SigmaT-1005) and chymotrypsin (SigmaC-4129) treatment.The assays

were incubated for 60min at 37�Cand done in triplicate. They were then

washed five times with PBS buffer to which 0.1mM PMSF had been

added [28,29]. Peptide binding activity was then compared between

treated and non-treated red blood cells using the binding assay as de-

scribed above. The assays were carried out in triplicate under identical

conditions; the mean results of the assays done in triplicate are reported

here.

Invasion inhibition assay. The effect of each peptide on merozoite

invasion was tested by usingP. falciparumFBC-2 strain in vitro cultures

[30,31]. Sorbitol-synchronised cultures were incubated until the late

schizont stage at final 0.5% parasitaemia and 5.0% haematocrite in

RPMI 1640 ± 10% O ± plasma. Culture was then seeded in 96-well cell

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

100, and 50lM concentrations. Each peptide was tested in triplicate,

after being incubated for 18h at 37�C in a 5% O2/5%CO2/90% N2 at-

mosphere. The supernatant was removed. The cells were then stained

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

being washed three times with PBS. The suspensions were analysed by

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

Dickinson immunocytometry system, San Jose, CA, USA) [32]. Ethyl-

ene glycol-bis-(b-aminoethylether-N,N,N 0,N 0-tetraacetic acid) (EGTA)

and chloroquine-treated infected and uninfected erythrocytes were used

as controls.

CD spectroscopy. CD analysis was used for obtaining general in-

formation regarding HABP structure and folding. CD spectra were re-

corded for eachof theHABPs at 20�ConJasco J-810 spectropolarimeter

at wavelengths ranging from 260 to 190nm in 1.00cm cuvettes. The

peptides were dissolved at 0.1mM concentration in pure water or in

aqueous TFE solutions containing 30%TFE by volume. Each spectrum

was obtained from averaging three scans taken at a 20nm/min scan-rate

with 1nm spectra bandwidth, corrected for baseline. The results were

expressed as mean residue ellipticity [H], the units being de-

greescm2dmol�1 according to the [H] = Hk/(100lcn) function, whereHk

was measured ellipticity, l was optical path-length, c peptide concen-

tration, and n the number of amino-acid residues contained in the

sequence.

Results

Erythrocyte binding peptides

Seventy 20-mer peptides, covering the complete P.

falciparum erythrocyte binding antigen 160 protein

3D7 strain (Accession No. CAD49277) [16,17], were

synthesised and tested in human erythrocyte receptor–li-

gand binding assays to determine whether EBA-160 re-

gions were involved in specifically recognising human

erythrocyte surface molecules in the same way as other

P. falciparum EBP family members. Erythrocyte binding

activity was determined for each peptide. Peptide bind-

ing activity was defined as being the amount (pmol) of

peptide that bound specifically to erythrocytes per add-

ed peptide (pmol). High activity binding peptides were

defined as being those peptides showing activity greaterthan or equal to 2%, according to previously established

criteria [23–27]. More than 200 specific binding sites per

cell were detected in these conditions.

Fig. 1A shows the sequence of each one of the tested

synthetic peptides, their position within the EBA-160

protein, and erythrocyte binding activity. The black bars

represent the binding activity (the curve of the specific

binding slope versus added peptide). A peptide havinga slope greater than 0.02 was considered to be a HABP.

The line at 2% in Fig. 1A represents the cut-off value.

Five of the 70 peptides were identified as having high

erythrocyte binding activity:HABP30109 (81LPENNKN

FEEKILHEDICKLY100) and 30120 (301MEGLGRSIK

VEFEIDRIFKRY320) were located in the N-terminal re-

gion; 30126 (421QKWDKYKELNFSSIFDQLNA440),

30135(601EGIKANIVSMYPSYADLSLD620), and 30146(821VDDVLSIKENVDLKPFKPKGY840) were located

in the protein�s central region. No HABPs were found in

the C-terminal region.

Binding assay with HABP jumbled-peptide

Analogue peptides containing a jumbled sequence

(the same amino-acid composition as HABPs but hav-ing a random sequence) were synthesised to determine

whether HABP binding was due to their amino-acid

composition or specific sequences. The jumbled pep-

tides� binding activity was low (<1.5% of specific binding

activity), this being lower than the native HABP (data

shown in Fig. 1B) thus showing that binding activity

was due to their specific sequences.

Determining affinity constants

Saturation binding assays were performed to deter-

mine the binding constants for human erythrocyte inter-

action with HABPs. Saturation curves and Hill analysis

allowed affinity constants (Kd) and Hill coefficients (nH)

Fig. 2. Saturation curves for the high activity binding peptides. Increasing quantities of labelled peptide were added in the presence or absence of

unlabelled peptide. The curve represents the specific binding. The Hill plots are shown in the smaller inserted graphs; the axes are as follows: abscissa

is logF and the ordinate is log(B/Bmax �B), where B is the bound peptide, Bmax is the maximum amount of bound peptide, and F represents free

peptide. The assay was done in triplicate.



to be calculated for each HABP and the approximate

number of binding sites per cell (Fig. 2 and Table 1).

The affinity constants lay between 200 and 460nM and

Hill coefficients were between 1.5 and 2.3, suggesting po-

sitive cooperativity. The number of binding sites per cell

was found to be between 2400 and 5700 (Table 1).

Cross-competition assays

Cross-competition assays were carried out for each

one of the HABPs by inhibiting them with unlabelled

native peptide or other unlabelled HABPs to determine

whether they were able to displace the radio-labelled

peptide. The cross-competition assays showed that125I-labelled HABPs were inhibited, in some cases, by

other non-labelled HABPs. For example, it can be ob-

served that radio-labelled peptide 30109 was inhibited

by non-labelled peptide 30135. Radio-labelled peptide

30146 was inhibited by all the other non-radio-labelled

HABPs except for peptide 30126; radio-labelled peptide

30126 however was not inhibited by all the other non-ra-

dio-labelled HABPs. Indeed, non-radio-labelled peptide30146 did not inhibit binding to radio-labelled HABPs.

Most HABPs were not mutually inhibiting. These

results indicate that these peptides do bind to different

receptors (Fig. 3).

Table 1

Binding constants

Peptide Kd (nM) nH Sites per cell

30109 400 2.0 5700

30120 250 1.9 2150

30126 210 1.5 2500

30135 460 2.3 4300

30146 430 1.9 2400

Affinity constants (Kd), Hill coefficients (nH), and number of binding

sites per cell are shown for EBA-160 HABPs. Affinity constants and

number of binding sites were determined from analysing saturation

curves. Hill analysis was performed from the saturation data.

Fig. 3. Cross competition assays. Radio-labelled HABP red blood cell bin

unlabelled HABPs.

Cross-linking assays

HABPs 30109, 30120, 30135, and 30146 were identi-

fied as being able to bind specifically to proteins having

apparent molecular weights of around 12 and 28kDa in

erythrocyte binding and cross-linking assays. HABP30126 also bound to a 12kDa band and another band

having an apparent 44kDa molecular weight. Their

binding to these molecules was inhibited when an excess

of non-radio-labelled peptide was present (Fig. 4).

Enzyme treatment

HABP binding was performed with enzyme-treatedhuman erythrocytes to determine the effect of each en-

zyme on HABP interaction with the human erythrocyte

surface. Non-treated human erythrocytes were used as

positive control (Table 2). Red blood cell neuraminidase

ding inhibited by the presence of unlabelled native peptide or other

Fig. 4. Autoradiograph from HABP cross-linking assays. Erythrocyte

membrane proteins were cross-linked with radio-labelled peptides

30109 (lanes 1 and 2), 30120 (lanes 3 and 4), 30126 (lanes 5 and 6),

30135 (lanes 7 and 8), and 30146 (lanes 9 and 10). Lanes 1, 3, 5, 7, and

9 show the total binding (i.e., cross-linking in the absence of unlabelled

peptide) and lanes 2, 4, 6, 8, and 10 show inhibited binding (i.e., cross-

linking in the presence of unlabelled peptide). The figure shows three

proteins of around 12, 28, and 44kDa.

Table 2

EBA160 HABP binding to enzyme-treated human erythrocytes

Peptide Specific binding (%)a

Controlb Trypsin Chymotrypsin Neuraminidase

30109 100 ± 8 44 ± 3 29 ± 11 34 ± 15

30120 100 ± 10 105 ± 12 223 ± 8 88 ± 4

30126 100 ± 4 181 ± 17 612 ± 4 472 ± 3

30135 100 ± 32 449 ± 8 252 ± 8 0 ± 4

30146 100 ± 3 0 ± 6 104 ± 3 0 ± 5

Peptide binding was compared between enzyme-treated red blood cells

and untreated red blood cells.a Mean ± SD of three experiments.b The binding to normal erythrocytes was considered 100%.

Fig. 5. CD spectroscopy. CD data for HABPs were obtained in water

and in the presence of 30% TFE/water (v/v). Only data for 30% TFE/

water are shown.


treatment diminished the binding of peptides 30109,

30120, 30135, and 30146, but it did not affect peptide

30126 binding, suggesting that these peptides bound tosialic acid. Only peptide 30109 binding to these cells be-

came diminished when human erythrocytes were treated

with chymotrypsin. Treatment with trypsin did not af-

fect the binding of peptides 30120, 30126 and 30146,

but it did diminish the binding of peptides 30109 and

30146. Peptide 30109 binding activity was weaker for

all enzyme-treated human erythrocytes; on the contrary,

an increase was observed in peptide 30126 binding to allenzyme-treated cells.

Invasion inhibition assay

The effect of HABPs during merozoite invasion of

erythrocytes was analysed in in vitro P. falciparum cul-

tures. These peptides inhibited merozoite invasion at

200lM concentration (Table 3); there was between59% and 88% invasion inhibition. The highest inhibition

was achieved by peptides 30109 and 30146 and the low-

est inhibition by peptide 30126. By comparison, one

low-binding activity EBA-160 peptide (30149) used as

Table 3

Invasion inhibition assays

Peptide % Inhibition invasiona

200lM 100lM 50lM

30109 88 ± 4 25 ± 3 10 ± 2

30120 62 ± 1 17 ± 4 7 ± 3

30126 59 ± 2 17 ± 4 9 ± 5

30135 69 ± 5 16 ± 2 14 ± 6

30146 87 ± 6 26 ± 4 15 ± 3

30149 23 ± 2 7 ± 2 8 ± 3

Chloroquine control 100 ± 1

EGTA control 100 ± 1

The percentage of merozoite invasion inhibition is shown with its re-

spective standard deviation. Chloroquine and EGTA were used as

controls.a Mean ± standard deviation of three experiments.

control did not have any significant effect on parasite

invasion.

CD spectroscopy

Preliminary indications of the peptide structures in

solution were obtained with circular dichroism (CD)

measurements in water and in 30% TFE/water. All pep-

tides displayed typical unordered structure spectra in

water (data not shown). In the more hydrophobic medi-

um however some peptides� CD profiles indicated a clear

shift towards an ordered structure, possibly typical a-he-lix as characterised by double minima at 208 and220nm. The resulting spectra showed that HABPs

30109, 30120, and 30126 displayed a-helix-like features.

Peptides 30135 and 30146 presented a random coil struc-

ture, showing little evidence of a stable secondary struc-

ture. The CD spectra are summarised in Fig. 5.

Discussion

Malaria merozoites enter erythrocytes by an active

invasion process mediated by parasite ligands interact-

ing with erythrocyte receptors. One of these parasite li-

gand families is called eythrocyte binding protein

(EBP); the first of this family�s antigens to be described

and characterised was EBA-175. EBA-175 binds to sialic

acid and the peptide backbone of glycophorine-A on theerythrocyte surface [4,6–8]. However, ligands similar to

EBA-175 are presumed to mediate parasite invasion

via the alternative pathways to glycophorine-A in the

case of P. falciparum [3,4].

EBA-160 is a protein having an identical structure to

that described for EBA-175 and similar to that of the


other members of the EBP family: a signal sequence in

region I, two cys-rich domains (F1 and F2) in region

II, a transmembrane domain and cytoplasmic tail, and

a C-cys domain [4]. Comparing CLUSTAL W align-

ments for this protein�s predicted amino-acid sequence

with EBA-175 revealed an overall 26.7% similarity. AllEBA 160 cysteine residues in the C-cys showed perfect

conservation in both position and number. EBA 160

can thus be considered to be a possible ligand involved

in the invasion process due to its similarity with EBA-

175 and other EBP family members.

Synthetic peptide receptor–ligand binding assays

have led to specific erythrocyte binding sequences be-

coming identified and allowed it to be demonstrated thatEBA-160 protein is able to interact with RBC. Identify-

ing HABPs from those proteins relevant to the invasion

process (such as EBA-175, MSP1, and NBP-1) has been

used as the first step in the search for possible anti-

malarial vaccine candidates which can stimulate an

appropriate immune response capable of blocking

receptor–ligand interactions [33–38].

Seventy peptides from the EBA-160 protein 3D7strain were tested in receptor–ligand assays to define

those amino-acid sequences involved in EBA-160 inter-

actions with human erythrocytes. The binding assays

showed that five peptides corresponding to EBA-160

protein specifically bound to erythrocytes (Fig. 1A).

HABPs were found in the protein�s central and N-termi-

nal regions. HABP 30109 was found in the N-terminal�sregion I. It is interesting to note that this peptide wasfound in the same position as HABP 1758 reported for

EBA-175 [23], located between residues 80 and 100; even

though the homology of their sequences was low, the

fact that they were located in the same position suggests

that this region is participating in the process of the mer-

ozoite recognising the erythrocyte in this protein family.

Peptides 30120, 30126, and 30135 were located in the

protein�s region II. Peptides 30120 and 30126 werefound in the F1 domain, whilst peptide 30135 was locat-

ed in the F2 domain. This fact is interesting since EBA-

175 also presented two HABPs in this region, peptides

1779 and 1783 [23], even though as in the case men-

tioned before there was little homology. It has been re-

ported that region II is very important in merozoite

invasion, showing binding activity in EBA-175 and is re-

cognised by antibodies inhibiting merozoite invasion [7].No HABPs were found in EBA-160 C-terminal region,

as opposed to EBA175 (23), suggesting that the parasite

could be using alternative invasion routes with this pro-

tein and possibly using them as an immunological

escape mechanism.

The HABPs showed high affinity and positive

cooperativity, indicating strong HABP interaction with

erythrocytes. Human erythrocytes treated with neur-aminidase before binding assay (cleaving terminal sialic

acids from glycoproteins) did diminish their interaction

with high binding peptides 30109, 30120, 30135, and

30146, indicating that HABP cell receptors are sialic ac-

id dependent. By contrast, peptide 30126 binding was

not affected by being treated with neuraminidase, indi-

cating that the receptor on the erythrocyte was indepen-

dent of sialic acid. This peptide�s binding was also notaffected by treatment with trypsin or chymotrypsin.

On the other hand, peptide 30109 binding was affected

by trypsin and chymotrypsin treatment, indicating that

erythrocyte membrane binding sites are sensitive to

these enzymes. The results presented here suggest that

these peptides� binding is susceptible to structural

changes provoked by enzyme treatment; however, the

presence of other alternative receptors cannot bediscarded.

Cross-linking assays using high activity binding pep-

tides showed that peptides 30109, 30120, 30135, and

30146 did bind two proteins whose molecular weights

were 12 and 28kDa. Even though these bands presented

similar molecular weights, the results of enzyme treat-

ment and cross-inhibition suggest that these receptors

could be different proteins or different segments of thesame protein, since treatment with trypsin and chymo-

trypsin reduced peptide 30109 binding ability whilst

HABP 30120, 30126, and 30146 binding increased and

cross-competition assays showed that inhibition

amongst these peptides was not mutual. Peptide 30126

bound to human erythrocyte proteins of around 12 and

44kDa. This peptide was the only one whose binding

to neuraminidase-treated erythrocytes increased andwas also the only one which was not inhibited by other

HABPs in cross-competition assays. The results suggest

that peptide 30126 binds to a receptor on erythrocytes in-

dependently of sialic acid, such receptor also being one

which is different to that for other HABP binding sites.

The fact that these sequences specifically bind to

erythrocytes suggests that the protein possibly plays a

significant role in early merozoite invasion stages. Infact, when HABP biological relevance was evaluated,

HABPs inhibited in vitro merozoite invasion of erythro-

cytes at 200lM by around 60–90%; this could indicate

that these peptides are possibly competing for receptor

sites on the erythrocyte and this in turn could be block-

ing merozoite invasion, depending on peptide concen-

tration. However, an alternative effect cannot be

discarded. The concentration of those peptides neces-sary for inhibiting merozoite invasion was relatively

high (200lM), due possibly to competition between

the peptides and native protein present in the parasite

which could present greater affinity than that of individ-

ual peptides. The peptides� three-dimensional structure

could be playing an important role in the invasion pro-

cess; circular dichroism analysis has indeed shown that

some HABPs have structural homology, some of thempresenting a-helix structures whilst others present ran-

dom coil structures.


Previous P. falciparum conserved high activity bind-

ing synthetic peptide studies from our laboratory have

shown that they are generally non-immunogenic and

non-protective; however, precise replacement of certain

residues has frequently converted non-immunogenic

peptides into immunogenic ones, eliciting antibodies rec-ognising native proteins by Western blotting and IFA.

Aotus monkeys immunised with these modified immu-

nogenic peptides have become protected against parasite

challenge, thus making them excellent candidates for a

multi-component, subunit-based synthetic malaria vac-

cine. Such assays have been done with HABPs and their

protein analogues, such as EBA-175, AMA-1, and

MSP-1 [33–38]; immunogenicity studies can thus be car-ried out with modified EBA-160 HABP sequences for

determining their suitability for inclusion in new tailored

synthetic anti-malarial vaccines.

Acknowledgments

This study was supported by the President of Colom-bia�s office and the Colombian Ministry of Public

Health. Jason Garry�s collaboration in reviewing and

writing the manuscript is greatly appreciated.

References

[1] J. Barnell, M.R. Galinski, Invasion of Vertebrate Cells: Erythro-

cytes, American Society for Microbiology, Washington, DC,

1998.

[2] C.E. Chitnis, M.J. Blackman, Host cell invasion by malaria

parasites, Parasitol. Today 16 (2000) 411–415.

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

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

85–91.

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

EBL family of Plasmodium falciparum, Trends Parsitol. 17 (2001)

297–299.

[5] J.H. Adams, B.K. SIM, S.A. Dolan, X. Fang, D.C. Kaslow, A

family of erythrocyte binding proteins of malaria parasites, Proc.

Natl. Acad. Sci. USA 89 (1992) 7085–7089.

[6] B.K. Sim, C.E. Chitnis, K. Wasniowska, T.J. Hadley, L.H.

Miller, Receptor and ligand domains for invasion of erythrocytes

by Plasmodium falciparum, Science 264 (1994) 1941–1944.

[7] B.K. Sim, P.A. Orlandi, J.D. Haydes, F.W. Klotz, J.M. Carter,

D. Camus, M.E. Zegans, J.D. Chulay, Primary structure of the

175kDa Plasmodium falciparum erythrocyte binding antigen and

identification of a peptide which elicits antibodies that inhibit

malaria merozoite invasion, J. Cell Biol. 111 (1990) 1877–1884.

[8] P.A. Orlandi, B.K. Sim, J.D. Chulay, J.D. Haynes, Characteriza-

tion of the 175-kilodalton erythrocyte binding antigen of Plasmo-

dium falciparum, Mol. Biochem. Parasitol. 40 (1990) 285–294.

[9] P. Michon, J.R. Stevens, O. Kaneko, J.H. Adams, Evolutionary

relationships of conserved cysteine-rich motifs in adhesive mole-

cules of malaria parasites, Mol. Biol. Evol. 19 (2002) 1128–1142.

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

Millar, Characterization of a Plasmodium falciparum erythrocyte-

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

USA 98 (2001) 5222–5227.

[11] C.A. Lobo, M. Rodriguez, M. Reid, S. Lustigman, Glycophorin

C is the receptor for the Plasmodium falciparum erythrocyte

binding ligand PfEBP-2 (baebl), Blood 101 (2003) 4628–4631.

[12] T. Triglia, J.K. Thompson,A.F.Cowman,AnEBA175 homologue

which is transcribed but not translated in erythrocytic stages of

Plasmodium falciparum,Mol. Biochem. Parasitol. 116 (2001) 55–63.

[13] T.W. Gilberger, J.K. Thompson, T. Triglia, R.T. Good, M.T.

Duraisingh, A.F. Cowman, A novel erythrocyte binding antigen-

175 paralogue from Plasmodium falciparum defines a new trypsin-

resistant receptor on human erythrocytes, J. Biol. Chem. 278

(2003) 14480–14486.

[14] D.S. Peterson, T. Wellems, EBL-1, a putative erythrocyte binding

protein of Plasmodium falciparum, maps within a favored linkage

group in two genetic crosses, Mol. Biochem. Parasitol. 105 (2000)

105–113.

[15] 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.

[16] M.J. Gardner, N. Hall, E. Fung, O. White, M. Berriman, R.W.

Hyman, J.M. Carlton, A. Pain, K.E. Nelson, S. Bowman, I.T.

Paulsen, K. James, J.A. Eisen, K. Rutherford, S.L. Salzberg, A.

Craig, S. Kyes, M.S. Chan, V. Nene, S.J. Shallom, B. Suh, J.

Peterson, S. Angiuoli, M. Pertea, J. Allen, J. Selengut, D. Haft,

M.W. Mather, A.B. Vaidya, D.M. Martin, A.H. Fairlamb, M.J.

Fraunholz, D.S. Roos, S.A. Ralph, G.I. McFadden, L.M.

Cummings, G.M. Subramanian, C. Mungall, J.C. Venter, D.J.

Carucci, S.L. Hoffman, C. Newbold, R.W. Davis, C.M. Fraser,

B. Barrell, Genome sequence of the human malaria parasite

Plasmodium falciparum, Nature 419 (2002) 498–511.

[17] N. Hall, A. Pain, M. Berriman, C. Churcher, B. Harris, D. Harris,

K.Mungall, S. Bowman,R. Atkin, S. Baker, A. Barron,K. Brooks,

C.O. Buckee, C. Burrows, I. Cherevach, C. Chillingworth, T.

Chillingworth, Z. Christodoulou, L. Clark, R. Clark, C. Corton,

A. Cronin, R. Davies, P. Davis, P. Dear, F. Dearden, J. Doggett,

T. Feltwell, A. Goble, I. Goodhead, R. Gwilliam, N. Hamlin, Z.

Hance, D. Harper, H. Hauser, T. Hornsby, S. Holroyd, P.

Horrocks, S. Humphray, K. Jagels, K.D. James, D. Johnson, A.

Kerhornou, A. Knights, B. Konfortov, S. Kyes, N. Larke, D.

Lawson, N. Lennard, A. Line, M. Maddison, J. McLean, P.

Mooney, S. Moule, L. Murphy, K. Oliver, D. Ormond, C. Price,

M.A. Quail, E. Rabbinowitsch, M.A. Rajandream, S. Rutter,

K.M. Rutherford, M. Sanders, M. Simmonds, K. Seeger, S. Sharp,

R. Smith, R. Squares, S. Squares, K. Stevens, K. Taylor, A. Tivey,

L. Unwin, S. Whitehead, J. Woodward, J.E. Sulston, A. Craig, C.

Newbold, B.G. Barrell, Sequence of Plasmodium falciparum chro-

mosomes 1, 3-9 and 13, Nature 419 (2002) 527–531.

[18] R.B. Merrifield, Solid phase peptide synthesis I. The synthesis of a

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

[19] 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 amino acid, Proc.

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

[20] T.P. Tam, W.F. Heath, R.B. Merrifield, SN2 deprotection of

synthetic peptides with a low concentration of HF in RB dimethyl

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

Chem. Soc. 105 (1983) 6442–6445.

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

Receptor Binding, Raven Press, New York, 1978.

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

proach, Oxford University Press, New York, 1993.

[23] L.E. Rodriguez, M. Urquiza, M. Ocampo, J. Suarez, H. Curtidor,

F. Guzman, L.E. Vargas, M. Trivinos, M. Rosas, M.E. Patar-

royo, Plasmodium falciparum EBA-175kDa protein peptides

which bind to human red blood cells, Parasitology 120 (2000)

225–235.


[24] L.E. Rodriguez, M. Ocampo, R. Vera, A. Puentes, R. Lopez, J.

Garcia, H. Curtidor, J. Valbuena, J. Suarez, J. Rosas, M.

Urquiza, M.E. Patarroyo, Z. Rivera, Plasmodium falciparum

EBA-140kDa protein peptides that bind to human red blood cells,

J. Pept. Res. 62 (2003) 175–184.

[25] H. Curtidor, M. Urquiza, J.E. Suarez, L.E. Rodriguez, M.

Ocampo, A. Puentes, J.E. Garcia, R. Vera, R. Lopez, L.E.

Ramirez, M. Pinzon, M.E. Patarroyo, Plasmodium falciparum

acid basic repeat antigen (ABRA) peptides: erythrocyte binding

and biological activity, Vaccine 19 (2001) 4496–4504.

[26] A. Puentes, J. Garcia, M. Ocampo, L. Rodriguez, R. Vera, H.

Curtidor, R. Lopez, J. Suarez, J. Valbuena, M. Vanegas, F.

Guzman, D. Tovar, M.E. Patarroyo, P. falciparum: merozoite

surface protein-8 peptides bind specifically to human erythrocytes,

Peptides 24 (2003) 1015–1023.

[27] J.E. Garcia, A. Puentes, J. Suarez, R. Lopez, R. Vera, L.E.

Rodriguez, M. Ocampo, H. Curtidor, F. Guzman, M. Urquiza,

M.E. Patarroyo, Hepatitis C virus (HCV) E1 and E2 protein

regions that specifically bind to HepG2 cells, J. Hepatol. 36 (2002)

254–262.

[28] D. Camus, T.J. Hadley, A Plasmodium falciparum antigen that

binds to host erythrocytes and merozoites, Science 230 (1985) 553–

556.

[29] J.J. Valbuena, R. Vera, J. Garcia, A. Puentes, H. Curtidor, M.

Ocampo, M. Urquiza, Z. Rivera, F. Guzman, E. Torres,

Plasmodium falciparum normocyte binding protein (PfNBP-1)

peptides bind specifically to human erythrocytes, Peptides 24

(2003) 1007–1014.

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

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

418–420.

[31] W. Trager, J. Jensen, Human malaria parasites in continuous

culture, Science 193 (1976) 673–675.

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

assessing viability of intraerythocytic hemoparasites, J. Immunol.

Methods 140 (1991) 23–30.

[33] G. Cifuentes, F. Guzman, M.P. Alba, L.M. Salazar, M.E.

Patarroyo, Analysis of a Plasmodium falciparum EBA 175 peptide

with high binding capacity to erythrocytes and their analogues

using 1HNMR, J. Struct. Biol. 141 (2003) 115–121.

[34] F. Guzman, K. Jaramillo, L.M. Salazar, A. Rivera, A. Torres,

M.E. Patarroyo, 1HNMR structures of the Plasmodium falcipa-

rum 1758 erythrocyte binding peptide analogues and protection

against malaria, Life Sci. 70 (2002) 2773–2785.

[35] J. Purmova, L.M. Salazar, F. Espejo, M.H. Torres, M. Cubillos,

E. Torres, Y. Lopez, R. Rodriguez, M.E. Patarroyo, NMR

structure of Plasmodium falciparum malaria peptide correlates

with protective immunity, Biochim. Biophys. Acta 96 (2002) 217–

229.

[36] M. Cubillos, F. Espejo, J. Purmova, J.C. Martinez, M.E.

Patarroyo, Alpha helix shortening in 1522 MSP-1 conserved

peptide analogs is associated with immunogenicity and protection

against P. falciparum malaria, Proteins 50 (2003) 400–409.

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

M. Ocampo, Y. Silva, R. Rodriguez, E. Lioy, M.E. Patarroyo,

Structure, immunogenicity, and protective relationship for the

1585 malarial peptide and its substitution analogues, Angew.

Chem. Int. Ed. 40 (2001) 4654–4657.

[38] L.M. Salazar, M.P. Alba, M.H. Torres, M. Pinto, X. Cortes, L.

Torres, M.E. Patarroyo, Protection against experimental malaria

associated with AMA1 peptide analogue structures, FEBS Lett.

527 (2002) 27–33.

Identifying Plasmodium falciparum EBA175 homologue sequences that specifically bind to human...

Documents

Transcript of Identifying Plasmodium falciparum EBA175 homologue sequences that specifically bind to human...