Plasmodium falciparum

EBA-140 kDa protein

peptides that bind to human

red blood cells

L.E. Rodriguez

M. OcampoR. Vera

A. PuentesR. Lopez

J. Garcia

H. CurtidorJ. Valbuena

J. SuarezJ. Rosas

Z. Rivera

M. UrquizaM.E. Patarroyo

Authors' affiliations:

L.E. Rodriguez, M. Ocampo, R. Vera, A. Puentes,

R. Lopez, J. Garcia, H. Curtidor, J. Valbuena,

J. Suarez, J. Rosas, Z. Rivera, M. Urquiza and

M.E. Patarroyo, Fundacion Instituto de

Inmunologıa de Colombia and Universidad

Nacional de Colombia, Cra 50 # 26-00, Bogota,

Colombia.

Correspondence to:

Manuel E. Patarroyo

Fundacion Instituto de Inmunologıa de Colombia

and Universidad Nacional de Colombia

Cra 50 # 26-00

Bogota

Colombia

E-mail: mepatarr@fidic.org.co

Tel.: + 57 1 4815219

Fax: + 57 1 4815269

Key words: BAEBL; EBA140; malaria; peptides; Plasmodium

falciparum

Abstract: The erythrocyte-binding antigen 140 (EBA140) sequence

was chemically synthesized in 61 20-mer sequential peptides

covering the entire 3D7 protein strain, each of which was tested in

erythrocyte-binding assays. Peptides 26135, 26144, 26147, 26160,

26170 and 26177 presented high erythrocyte-binding activity, with

affinity constants ranging from 350 to 750 nM. Critical erythrocyte-

binding residues were determined by competition-binding assays

with glycine analogous peptides. Cross-linking assays with SDS-

PAGE from high erythrocyte membrane protein binding peptides

showed that all these peptides bound specifically to 25, 52 and

75 kDa erythrocyte membrane proteins. The nature of these

receptor sites was studied in peptide-binding assays using enzyme-

treated erythrocytes, showing that these protein receptors are

susceptible to structural changes provoked by enzyme treatment

(neuraminidase, trypsin or chymotrypsin). Inhibition invasion assays

in ‘in vitro’ cultures showed that all specific high binding sequences

were able to inhibit invasion by 11–69% at 200 lM concentration.

Dates:

Received 7 May 2003

Revised 16 June 2003

Accepted 5 July 2003

To cite this article:

Rodriguez, L. E., Ocampo, M., Vera, R., Puentes, A.,

Lopez, R., Garcia, J., Curtidor, H., Valbuena, J., Suarez, J.,

Rosas, J., Rivera, Z., Urquiza, M. & Patarroyo, M.E.

Plasmodium falciparum EBA-140 kDa protein peptides

that bind to human red blood cells.

J. Peptide Res., 2003, 62, 175–184.

Copyright Blackwell Munksgaard, 2003

ISSN 1397–002X

Introduction

The ability of Plasmodium merozoite to invade erythro-

cytes depends on the cascade effect of specific parasite

molecules and host erythrocyte receptor interactions.

P. falciparum is known to use different receptors for inva-

ding erythrocytes; it commonly invades via sialic acid res-

idues present on glycophorin A or B, epitopes associated

with glycophorin C and D, Band 3 and an uncharacterized

receptor ‘X’. These have been defined by analyzing enzyme-

treated and mutant erythrocytes and by their susceptibility

to merozoite invasion (1–8).

175

Merozoite invasion of host cells is a multi-step process

and is thought to require numerous interactions between

apical-complex proteins and erythrocyte receptors. The

compartments making up this complex include the micro-

nemes, rhoptries and dense granules that sequentially dis-

charge proteins facilitating merozoite attachment to, entry

and residence inside the erythrocyte (7–11).

Merozoites are known to use proteins sequestered in

apical-complex organelles to mediate invasion of host

erythrocytes via specific receptor/ligand interactions. One

of them (P. falciparum EBA175 protein) has been identified

in micronemes and shown to be the ligand binding to a

sialic acid-dependent site on glycophorin A (7–9).

A novel P. falciparum ligand has been recently identified,

termed erythrocyte-binding antigen 140 (EBA140), also

known as BAEBL or PfEBP-2, sharing structural features

and homology with EBA175 and EBA-181 (JESEBL) (12,13).

Sub-cellular EBA140 location studies suggest that it is

located in the micronemes (the same location as EBA175

and EBA181); these proteins bind to sialoglycoproteins on

the red blood cell (RBC) surface. Even though the binding of

these parasite ligands to their respective receptors is sialic

acid-dependent, all of them showed different specificities to

erythrocyte receptors, indicating that such binding specif-

icity is defined by the receptor’s nature and composition.

It has been shown that the EBA140 receptor is glycoph-

orin-C by using a combination of enzyme-treated RBCs and

RBC variants lacking different surface proteins. It has also

been reported that a binding domain in glycophorin-C is

restricted from residue 14 to 22 (4).

Sixty-one EBA-140 peptides from the 3D7 strain (2)

deduced sequence were synthesized in 20 non-overlapping

residues to determine Pf-EBA-140 sequences specifically

involved in erythrocyte binding. Six peptides having

selective and specific binding to erythrocytes were iden-

tified in specific RBC binding assays; they were pep-

tides 26135 (361SYTSFMKKSKTQMEVLTNLY380), 26144

(541DLADIIKGSDIIKDYYGKKM560), 26147 (601LKNKETC-

KDYDKFQKIPQFL620), 26160 (861GHSESSLNRTTNAQD-

IKIGRY880), 26170 (1061CNNEYSMEYCTYSDERNSSP1080)

and 26177 (1191VQETNISDYSEYNYNEKNMY1210). All of

them were conserved, according to polymorphism studies

carried out to date.

These high affinity binding peptides (HABPs) showed

affinity constants ranging from 350 to 750 nm. Critical res-

idues for these peptides’ RBC binding were determined by

competition binding assays with glycine scanning analogs.

Cross-linking studies (SDS-PAGE) for all high binding

peptides showed erythrocyte-proteins binding at around 75,

52 and 25 kDa. Enzyme-treated RBCs showed that peptide

binding was susceptible to treatment; such susceptibility

was caused by the great structural modifications suffered by

the cell and consequently these peptides’ receptors. Inhi-

bition invasion assays in ‘in vitro’ cultures showed that all

specific high binding sequences were able to inhibit inva-

sion by 11–69% at 200 lm concentration. All results

reported here suggest that the specific high binding

sequences could be participating in the different recognition

processes and blocking the invasion process.

Materials and Methods

Peptide synthesis

Sixty-one sequential, 20-mer peptides, corresponding to the

EBA140 3D7 strain amino acid sequence (2), were synthes-

ized by solid-phase multiple peptide synthesis for this study

(14,15). MBHA resin (0.7 meq/g), t-Boc amino acids and

low–high HF cleavage techniques were used (16). Peptide

identity and purity were analyzed by MALDI-TOF mass

spectrometry and analytical reverse-phase, high-perform-

ance liquid-chromatography (RP-HPLC). These synthesized

peptide sequences are shown in one-letter code (Fig. 1). An

extra Tyr residue was added to the peptide C-terminus if it did

not contain it for radio-labeling purposes.

Radio-labeling

125I-radio-labeling was done according to previously

described techniques (17–22) in which chloramine T

(2.25 mg/mL) and 3.2 lL Na125I (100 mCi/mL) were added

to 5 lL of peptide solution (1 lg/lL). Fifteen microliters of

sodium bisulphite (2.75 mg/mL) and 50 lL NaI (0.16 m)

were added following 5 min of reaction at 18 �C. The radio-

labeled peptide was then separated from the reaction sub-

products on a Sephadex G-10 column (Pharmacia, Uppsala,

Sweden) (80 · 5.0 mm).

Binding assays

RBCs [1 · 108; previously washed in isotonic phosphate

buffered saline (PBS)] were incubated with four increasing

quantities of each one of the EBA-140 P. falciparum protein125I-radio-labeled peptides (between 0 and 400 nm) at a total

volume of 100 lL for 90 min at 18 �C, in the absence (total

Rodriguez et al . P. falciparum EBA-140 peptides bind to RBC

176 J. Peptide Res. 62, 2003 / 175–184

binding) or presence of 40 lm unlabeled peptide (non-

specific binding), to determine binding specificity. The

unbound radio-labeled peptide was removed by three

washes with PBS and the cell-bound radio-labeled peptide

was measured in a c-counter. Specific binding was calcu-

lated by subtracting the non-specific binding from total

binding. Specific binding slope values taken from the start

of the curve ·100 (specific bound peptide vs. added peptide)

were considered as being the binding activity (18–22).

Assays were carried out in triplicate under identical

conditions; the mean results of the triplicate assays are

reported and shown graphically in Fig. 1.

Saturation binding assays were carried out for each one of

the peptides showing high specific RBC binding activity in

a similar way as mentioned above, but diminishing to

1 · 107 RBCs, using a broad range of radio-labeled peptide

concentrations (100 and 2200 nm) and increasing the total

volume to 205 lL. As before, each assay was performed in

triplicate; bound and free peptides were determined by

measurement in a c-counter. Data from triplicate assays

Peptidenumber 1.0% 2.0% 3.0%

26117 1 M K G Y F N I Y F L I P L I F L Y N V I 20

26118 21 R I N E S I I G R T L Y N R Q D E S S D 40

26119 41 I S R V N S P E L N N N H K T N I Y D S 60

26120 61 D Y E D V N N K L I N S F V E N K S V K 80

26121 81 K K R S L S F I N N K T K S Y D I I P P 100

26122 101 S Y S Y R N D K F N S L S E N E D N S G 120

26123 121 N T N S N N F A N T S E I S I G K D N K Y 140

26124 141 Q Y T F I Q K R T H L F A C G I K R K S 160

26125 161 I K W I C R E N S E K I T V C V P D R K Y 180

26126 181 I Q L C I A N F L N S R L E T M E K F K Y 200

26127 201 E I F L I S V N T E A K L L Y N K N E G 220

26128 221 K D P S I F C N E L R N S F S D F R N S Y 240

26129 241 F I G D D M D F G G N T D R V K G Y I N 260

26130 261 K K F S D Y Y K E K N V E K L N N I K K 280

26131 281 E W W E K N K A N L W N H M I V N H K G Y 300

26132 301 N I S K E C A I I P A E E P Q I N L W I Y 320

26133 321 K E W N E N F L M E K K R L F L N I K D Y 340

26134 341 K C V E N K K Y E A C F G G C R L P C S 360

26135 361 S Y T S F M K K S K T Q M E V L T N L Y 380

26136 381 K K K N S G V D K N N F L N D L F K K N Y 400

26137 401 N K N D L D D F F K N E K E Y D D L C D 420

26138 421 C R Y T A T I I K S F L N G P A K N D V 440

26139 441 D I A S Q I N V N D L R G F G C N Y K S 460

26140 461 N N E K S W N C T G T F T N K F P G T C Y 480

26141 481 E P P R R Q T L C L G R T Y L L H R G H 500

26142 501 E E D Y K E H L L G A S I Y E A Q L L K 520

26143 521 Y K Y K E K D E N A L C S I I Q N S Y A 540

26144 541 D L A D I I K G S D I I K D Y Y G K K M 560

26145 561 E E N L N K V N K D K K R N E E S L K I Y 580

26146 581 F R E K W W D E N K E N V W K V M S A V Y 600

26147 601 L K N K E T C K D Y D K F Q K I P Q F L 620

26148 621 R W F K E W G D D F C E K R K E K I Y S 640

26149 641 F E S F K V E C K K K D C D E N T C K N Y 660

26150 661 K C S E Y K K W I D L K K S E Y E K Q V 680

26151 681 D K Y T K D K N K K M Y D N I D E V K N 700

26152 701 K E A N V Y L K E K S K E C K D V N F D 720

26153 721 D K I F N E S P N E Y E D M C K K C D E 740

26154 741 I K Y L N E I K Y P K T K H D I Y D I D 760

26155 761 T F S D T F G D G T P I S I N A N I N E Y 780

26156 781 Q Q S G K D T S N T G N S E T S D S P V Y 800

26157 801 S H E P E S D A A I N V E K L S G D E S Y 820

26158 821 S S E T R G I L D I N D P S V T N N V N Y 840

26159 841 E V H D A S N T Q G S V S N T S D I T N Y 860

26160 861 G H S E S S L N R T T N A Q D I K I G R Y 880

26161 881 S G N E Q S D N Q E N S S H S S D N S G Y 900

26162 901 S L T I G Q V P S E D N T Q N T Y D S Q 920

26163 921 N P H R D T P N A L A S L P S D D K I N Y 940

26164 941 E I E G F D S S R D S E N G R G D T T S Y 960

26165 961 N T H D V R R T N I V S E R R V N S H D Y 980

26166 981 F I R N G M A N N N A H H Q Y I T Q I E 1000

26167 1001 N N G I I R G Q E E S A G N S V N Y K D 1020

26168 1021 N P K R S N F S S E N D H K K N I Q E Y 1040

26169 1041 N S R D T K R V R E E I I K L S K Q N K Y 1060

26170 1061 C N N E Y S M E Y C T Y S D E R N S S P 1080

26171 1081 G P C S R E E R K K L C C Q I S D Y C L 1100

26172 1101 K Y F N F Y S I E Y Y N C I K S E I K S 1120

26173 1121 P E Y K C F K S E G Q S S I P Y F A A G 1140

26174 1141 G I L V V I V L L L S S A S R M G K S N Y 1160

26175 1161 E E Y D I G E S N I E A T F E E N N Y L 1180

26176 1181 N K L S R I F N Q E V Q E T N I S D Y S 1200

26177 1191 V Q E T N I S D Y S E Y N Y N E K N M Y 1210

Sequence Binding activity

Figure 1. Binding activity for each one of the EBA-140 peptides covering the total length of the 3D7 strain protein. The number given for each

peptide is the code assigned for each peptide in our lab. The peptide’s position in the protein appears in the sequence column. Binding activity

represents the slope value on the specific binding curve. Each one of the black bars represents the slope of the specific binding graph. The dotted line

separates peptides having binding activity greater than or equal to 2%. Only peptides 26135, 26144, 26147, 26160, 26170 and 26177 were taken

as being high binding peptides (HABPs).

Rodriguez et al . P. falciparum EBA-140 peptides bind to RBC

J. Peptide Res. 62, 2003 / 175–184 177

were averaged. The saturation curves obtained were

analyzed and the affinity constants were determined by the

Hill equation (17).

Analog peptide competition binding assay

Glycine analogs were synthesized and then scanned to

identify critical residues for RBC binding. Erythrocytes

(1 · 108) were incubated with increasing quantities

(150 nm, 300 nm, 3 lm and 29 lm) of each unlabeled analog

peptide or original unlabeled peptide in the presence of

native 125I-labeled peptide for the competition binding

assays. The unbound radio-labeled peptide was removed

by three washes in PBS and the cell-bound radio-labeled

peptide was measured in a c-counter following 90-min

incubation at 18 �C.

Cross-linking assays

The peptide–receptor complex was identified in SDS-PAGE

electrophoresis using 12% polyacrylamide gel and constant

200 V. The binding test was performed using 1% final

hematocrit; after incubation with the radio-labeled peptide

for 90 min at 18 �C and thorough washing with PBS, the

bound peptide was cross-linked with 10 lm BS3 [bis(sulfo-

succinimidyl)suberate] for 30 min at 4 �C. The cells were

washed again with PBS and treated with lysis buffer (5%

SDS, 10 nm iodoacetamide, 1% Triton X-100, 100 mm

EDTA and 10 mm PMSF). The obtained membrane proteins

were solubilized in Laemmli buffer and separated in

SDS-PAGE. Those proteins cross-linked with radio-labeled

peptides were exposed on Kodak film (X-OMAT) for 24 h at

)70 �C and the apparent molecular weight was determined

by using molecular weight markers ranging from 175 to

6.5 kDa (BIO-RAD Inc., Hercules, CA, USA).

Enzyme-treated RBCs

Human RBCs were washed with PBS; some were then treated

with 0.06 mU/mL neuraminidase (ICN NC-100872, Irvine,

CA, USA) whilst others were treated with 0.37 mg/mL

trypsin (Sigma T-8253, St. Louis, MO, USA) or 0.37 mg/mL

chymotrypsin (Sigma C-9381), at 5% final hematocrit. They

were incubated at 37 �C for 1 h (23,24). Erythrocytes were

washed thrice with PBS and spun at 50 · g for 3 min for each

washing. These RBCs were used in binding assays; the pep-

tide-binding activity was then compared between treated and

non-treated RBCs using the binding assay described above.

Invasion inhibition assay

Sorbitol-synchronized P. falciparum (FCB-2 strain) (25,26)

cultures were incubated until the late schizont stage at

final 0.5% parasitemia and 5.0% hematocrit in RPMI

1640 + 10% O + plasma. Cultures were then seeded in

96-well cell-culture plates (Nunc, Denmark) in the presence

of test peptides at 200, 100 and 50 lm concentrations. Each

peptide was tested in triplicate, after being incubated for

18 h at 37 �C in a 5% O2/5% CO2/90% N2 atmosphere. The

supernatant was skimmed off and the cells were then

stained with 15 lg/mL hydroethidine and incubated at

37 �C for 30 min, after being washed twice with PBS. The

suspensions were analyzed using a FACsort in Log FL2 data

mode using CellQuest software (Becton Dickinson

Immunocytometry System, San Jose, USA). Infected and

uninfected erythrocytes, infected erythrocytes treated with

ethylene glycol-bis-(b-aminoethylether-N, N, N¢, N¢-tetra-

acetic acid) (EGTA), chloroquine and non-binding peptides

were used as controls.

Results

High specific binding peptides

A highly specific and sensitive receptor–ligand binding

assay was developed based on previous studies with eryth-

rocytes and other cell lines (18–22). Peptides showed three

types of behavior in the initial screening binding assay.

The first group of peptides did not bind to target cells;

these peptides were considered to be low or non-binding

peptides (data not shown).

The second group consisted of peptides binding non-spe-

cifically to RBCs, as binding was not inhibited by the same

non-labeled peptide.

The third group of peptides interacted strongly and spe-

cifically with the target RBCs (or HABPs), such binding

being inhibited by the same non-radio-labeled peptide. Six

peptides were found to have selective and specific erythro-

cyte binding: 26135, 26144 26147 26160, 26170 and 26177.

Figure 1 shows amino acid sequences from synthesized

peptides and their position within 3D7 strain EBA-140 (2).

The binding activity (specific binding curve slope value) is

denoted for each EBA-140 peptide by black bars. Peptides

Rodriguez et al . P. falciparum EBA-140 peptides bind to RBC

178 J. Peptide Res. 62, 2003 / 175–184

with a slope greater than 0.02 (corresponding to specific

binding greater than 0.02 pmol bound per pmol added) were

denominated HABPs. The line at 2% in Fig. 1 represents

the cut-off value.

Affinity constants

Saturation assays and Hill analyses were carried out for all

HABPs based on initial screening results, using a wider

range of 125I-radio-labeled peptide concentration

(0–2200 nm) (Fig. 2) (17–22). Scatchard and Hill analyses

were then done and affinity constants (Kd) calculated for

HABPs. Hill coefficients (nH) and the number of receptor

sites are shown in Table 1. These HABPs showed affinity

constants between 350 and 750 nm and a number of sites

per cell ranging from 1100 to 6800.

Peptides 26135, 26144, 26147 and 26177 presented Hill

coefficients and saturation curves characteristic of a simple

interaction (i.e. one receptor for each ligand), whilst pep-

tides 26160 and 26170 presented Hill coefficients indicating

positive cooperativity.

Analog peptide competition binding assay

Critical residues were those that, upon replacement with

glycine, rendered an invariable decrease of at least 50% in

their capacity to compete with the original radio-labeled

peptide in a binding assay at four concentrations (150 nm,

300 nm, 3 lm and 29 lm). It shows dramatic change in

peptide binding activity. As shown in Fig. 3, the critical

residues in the binding were (underlined in the sequence):

for peptide 26135 (SYTSFMKKSKTQMEVLTNLY), peptide

26144 (DLADIIKGSDIIKDYYGKKM), peptide 26147

(LKNKETCKDYDKFQKIPQFL), peptide 26160 (GHSES-

SLNRTTNAQDIKIGRY) and peptide 26170 (CNNEYS-

MEYCTYSDERNSSP). Peptide 26177 did not present crit-

ical residues.

Cross-linking assays

All HABPs were identified as being able to bind specifically

to proteins having apparent molecular weights of around

25, 52 and 75 kDa in erythrocyte binding and cross-linking

Free peptide (nM)

Bo

un

dp

epti

de

(pm

ol)

26135

0.7

1.4

–0.5

0.1

2.1 2.9

26144

2.0

4.0

–0.6

0.4

2.1 2.9

26147

3.0

6.0

0 700 1400 2100

–0.6

0.2

2.1 2.9

26160

0.3

0.6

–0.7

0.3

2.1 2.9

26170

–1.4

0.2

2.1 2.9

26177

1.0

2.0

0 700 1400 2100

–1.0

–0.1

2.1 2.9

Figure 2. Saturation curves for the high

binding affinity peptides. Increasing quanti-

ties of radio-labeled peptides were added,

reaching concentrations of 0–2200 nm radio-

labeled peptide in the presence or absence of

non-radio-labeled or cold peptide. The curve

represents the specific binding. The Hill

plots are the smaller inserted graphs; the axes

are: abscissa is log F and the ordinate is

log (B/Bmax ) B), where B is the bound pep-

tide, Bmax the maximum amount of bound

peptide and F the free peptide.

Rodriguez et al . P. falciparum EBA-140 peptides bind to RBC

J. Peptide Res. 62, 2003 / 175–184 179

assays. Their binding to these molecules was inhibited

when an excess of non-radio-labeled peptide was present

(Fig. 4). Only peptide 26135 is shown, as all six peptides

recognized the same proteins.

RBC enzyme treatment

Peptide binding was compared between enzyme-treated

RBCs and untreated RBCs. RBC neuraminidase treatment

did not affect the binding of peptides 26135, 26144 and

26177, but it did diminish the binding of peptides 26147,

26160 and 26170, suggesting that these peptides are

bound to sialic acid. When RBCs were treated with

chymotrypsin, peptide binding to erythrocytes was

diminished; however, this could mainly be seen with

peptides 26135, 26160, 26170 and 26177. Treatment with

trypsin did not affect the binding of peptides 26144,

26160 and 26170; the binding of only peptide 26147 was

diminished by 20%. On the contrary, an increase was

observed in the binding of peptides 26135 and 26177

(Table 2).

Table 1. Affinity constants (Kd), Hill coefficients (nH) and numberof binding sites per cell are shown for EBA-140 HABPs

Peptide Kd (nM) Hill coefficient (nH) Sites per cell

26135 350 1 1800

26144 350 1.2 3800

26147 500 1.1 6832

26160 590 1.5 1100

26170 600 2 3100

26177 750 1 3052

Affinity constants and number of binding sites were determinedfrom analyzing saturation curves. Hill analysis was performed fromthe saturation data.

Residues replace by glycine

Sp

ecif

ic b

ind

ing

(%

)

26135 29 µµµµM

0

50

100

* S Y T S F M K K S K T Q M E V L T N L Y

26135 3 µµµµM

* S Y T S F M K K S K T Q M E V L T N L Y

ND ND

26144 29

0

50

100

* D L A D I I K S D I I K D Y Y K K M

26144 3 µµµµM

* D L A D I I K S D I I K D Y Y K K M

26160 29

0

50

100

* H S E S S L N R T T N A Q D I K I R

26160 3

* H S E S S L N R T T N A Q D I K I R

26170 29

0

50

100

* C N N E Y S M E Y C T Y S D E R N S S P

26170 3

* C N N E Y S M E Y C T Y S D E R N S S P

26147 29

0

50

100

* L K N K E T C K D Y D K F Q K I P Q F L

26147 3

* L K N K E T C K D Y D K F Q K I P Q F L

NDND

µµµµM

µµµµM µµµµM

µµµµM µµµµM

µµµµM µµµµM

Figure 3. Competition binding assay with

peptide analogs. The specific binding of

original radio-labeled peptide inhibited by

the analogous peptide at four concentrations

is only shown at 29 and 3 lm. The inhibi-

tion assay was performed with original non-

radio-labeled peptide and its analogous pep-

tides. *Represents original peptide’s specific

binding.

Rodriguez et al . P. falciparum EBA-140 peptides bind to RBC

180 J. Peptide Res. 62, 2003 / 175–184

Invasion inhibition assay

The effect of each peptide from the P. falciparum EBA-140

3D7 strain on merozoite invasion was tested in ‘in vitro’

cultures. Table 3 displays the effects of high binding affin-

ity peptides on parasites in RBC invasion. Peptides 26160

and 26170 inhibited parasite invasion (69 ± 2% and

39 ± 2%, respectively). Peptide 26160 showed a higher

inhibitory effect on parasite invasion at 200 lm and it

affected parasite development too. A significant effect was

found in invasion assays for all peptides at 200 and 100 lm.

By comparison, the two low-binding affinity peptides tested

and controls did not have any effect on parasite invasion or

development (data not shown).

Discussion

Plasmodium parasite invasion of erythrocytes is a complex

process consisting of a series of receptor–ligand molecular

interactions, involving a large number of molecules that

have been identified on the merozoite surface, especially on

apical organelles. Ligands necessary for junction or primary

binding have been found; the first antigen to be described

and characterized amongst them was the EBA175 protein,

which has been shown to be an important mediator in the

invasion process.

Plasmodium proteins EBA 175, EBA181 (JESEBL) and

EBA140 (BAEBL or EBP2) share common structures: (i) extra-

cellular domains with peptide signal; (ii) trans-membrane

domains; (iii) putative cytoplasmatic domains and (iv) each

83

62

47.5

32.5

25

52 kDa

1 2

75 kDa

25 kDa

MWMkDa

Figure 4. Cross linking assay. Membrane proteins were obtained from

erythrocytes after binding and cross-linking assays. Lanes 1 and 2 rep-

resent total and inhibited peptide 26135 binding to RBC. The figure

shows three proteins of around 75, 52 and 25 kDa.

Table 2. Binding of EBA140 peptides to enzyme-treated erythro-cytes. Peptide binding was compared between enzyme-treatedRBCs and untreated RBCs

Peptide Neuraminidase Trypsin ChymotrypsinControl(%)a

26135 413 ± 7 900 ± 7 65 ± 3 100 ± 7

26144 91 ± 3 102 ± 1 79 ± 4 100 ± 4

26147 48 ± 10 74 ± 6 73 ± 9 100 ± 11

26160 21 ± 4 104 ± 5 15 ± 4 100 ± 4

26170 24 ± 2 108 ± 11 64 ± 8 100 ± 11

26177 229 ± 8 179 ± 6 19 ± 3 100 ± 4

aAll data shown in this table are presented as specific binding per-centages (%) related to untreated erythrocytes.

Table 3. Invasion and development inhibition assays. The assayswere performed as described in Materials and Methods at threeconcentrations (50, 100 and 200 lM); high binding affinity pep-tides are shown. The percentage of merozoite invasion inhibitionor intra-erythrocyte development inhibition is shown with itsrespective standard deviation. Chloroquine was used as a controlfor the inhibition assays

PeptideConcentration(lM)

Invasioninhibition(% ± SD)

Developmentinhibition(% ± SD)

26135 200 30 ± 2 0 ± 2

100 15 ± 4 2 ± 1

50 3 ± 4 0 ± 2

26144 200 11 ± 9 0 ± 1

100 4 ± 9 2 ± 2

50 1 ± 1 2 ± 0

26147 200 23 ± 7 1 ± 1

100 9 ± 1 0 ± 1

50 2 ± 3 1 ± 1

26160 200 69 ± 2 44 ± 2

100 18 ± 2 0 ± 1

50 8 ± 7 1 ± 0

26170 200 39 ± 2 0 ± 1

100 8 ± 4 0 ± 1

50 3 ± 1 0 ± 1

26177 200 26 ± 0 0 ± 2

100 2 ± 1 0 ± 1

50 0 ± 0 0 ± 1

Chloroquine 200 100 ± 1 100 ± 1

Rodriguez et al . P. falciparum EBA-140 peptides bind to RBC

J. Peptide Res. 62, 2003 / 175–184 181

gene has a concise structure with two cystein-rich domains,

eight conserved cysteines and a high level of amino acid

conservation in these genes (9–13). All these characteristics

lead to these antigens being proposed as having the ability to

generate alternate or mediating mechanisms in the invasion

process. All such alternate mechanisms require binding

sequences having high affinity since this is a short-term

process, thus consolidating the first interaction between the

merozoite and the erythrocyte (junction).

Sixty-one peptides from the EBA140 protein 3D7 strain

were tested in receptor–ligand assays to define more clearly

those amino-acid sequences involved in EBA140 interac-

tions with RBCs; this led to six specific HABPs being

identified. HABPs 26135, 26144 and 26147 were located in

region II; 26135 was located in F1 and 26144 and 26147 in

F2. Region II is very important in merozoite invasion of

RBCs, as this region shows erythrocyte binding activity in

EBA-175 and is recognized by merozoite-invasion-inhibit-

ing antibodies. All these HABPs showed saturable binding

with a finite number of binding sites per cell. The affinity

constants suggested that these are important sequences due

to their high affinity (nm).

HABP amino acid composition consisted of 14% positively

charged residues, 15% negatively charged residues, 35%

apolar residues and 36% non-charged polar residues. Critical

residue amino acid composition consisted of 27% positively

charged residues, 4% negatively charged residues, 34% apo-

lar residues and 35% non-charged residues. This clearly

suggests the importance of positively charged residues in

RBC binding. However, the results also showed that the

residues’ charge was not only important for RBC binding, but

also the position in the sequence. For example, 559K was

critical for RBC binding in HABP 26144 but 558K was not.

Critical residues could not be found in HABP 26177. One

possible explanation is that this peptide has a binding motif

that is present several times in its sequence, considering that

residues Y, E and N are present several times in the same

sequence. This means that when one of these residues was

replaced by glycine there was no decrease in binding activity

because the peptide could have been binding to the RBC

because of another motif present in the HABP sequence:

V Q E T N I S D Y S E Y N Y N E K N M Y

The critical residues could be directly involved in binding

to target cells or be an important part of the peptide’s

structure leading to specific binding. Identifying critical

residues in HABP binding to erythrocytes has been recog-

nized as a useful tool in designing peptides having immu-

nological and structural properties different to those of the

original peptides. It has been reported that the precise

replacement of HABP critical residues frequently converted

non-immunogenic peptides into immunogenic ones elicit-

ing antibodies recognizing native protein by blot and IFI.

Aotus monkeys immunized with these now immunogenic

peptides became protected against parasite challenge,

making them excellent candidates for a multi-component

subunit synthetic malaria vaccine (27,28).

EBA140 and EBA 175 protein sequence alignment showed

24% identity. Bearing in mind that these are structurally

similar proteins having high homology, it was expected that

they would present shared RBC binding motifs, after com-

paring them with other binding studies employing the

EBA-175 protein (20). No common binding sequences were

found, in spite of such HABPs presenting between 15

and 45% identity amongst them. This suggests that

HABP binding to RBC depends on a specific sequence and

therefore to the conformation adopted by each one of them.

They could thus be using different routes and/or with dif-

ferent receptors.

Polymorphism studies reported so far have been focused

on regions I and II, finding that region I presents poly-

morphism in position 112. No high binding peptides have

been found in this region. Region II has F1 and F2 cystein-rich

domains. HABP 26135 (361SYTSFMKKSKTQMEVL-

TNLY380), found in the F1 domain, had a conserved sequence

in the strains studied, peptides 26144 (541DLADIIKGSDI-

IKDYYGKKM560) and 26147 (601LKNKETCKDYDK-

FQKIPQFL620) were found in the cystein-rich F2 domain;

their sequences were also conserved in the strains studied to

date, bearing in mind that polymorphism has been found in

positions 185, 239, 261 and 285 (29). Region II is very

important as this is an erythrocyte-binding region in the

proteins in which it has been found. Furthermore, it has been

reported as being one of the regions recognized by antibodies

able to inhibit merozoite invasion of erythrocytes.

Cross-linking studies for all high binding peptides

showed that these peptides bind three proteins whose

molecular weights were 75, 52 and 25 kDa. Their binding to

these molecules was inhibited when an excess of non-radio-

labeled peptide was present; this provides evidence of a

specific interaction (Fig. 4). It has been reported that

EBA-140 binds to glycophorin C having a molecular weight

of 28 kDa and 50 000 molecules present on the erythrocyte

surface. Accordingly, we suggest that HABP-receptors could

be glycophorin C forming part of a homo- or hetero-dimeric

and trimeric complex. This could partly explain the three

bands revealed in cross-linking assays, the low number of

Rodriguez et al . P. falciparum EBA-140 peptides bind to RBC

182 J. Peptide Res. 62, 2003 / 175–184

binding sites determined and that these bands did not

become stained with Coomasie blue. Furthermore, we

suggest that there are at least three different binding regions

on HABPs receptors: a cryptic region exposed after neura-

minidase and trypsin treatment (26135 and 26177 HABP

receptors); a sialic acid dependent region (26147, 26160 and

26170 HABP receptors); and a neuraminidase and trypsin

resistant region (26144 HABP receptor).

The results presented here imply that these high binding

peptides’ binding sites are susceptible to structural changes

provoked by enzyme treatment. However, the presence of

alternative receptors cannot be discarded as characterizing

the receptors for these high binding peptides requires more

study.

When high specific binding peptides were tested in

‘in vitro’ P. falciparum culture, it was observed that all

peptides were capable of 11–69% invasion inhibition at

200 lm concentration. At a concentration of 50 lm HABP

did not present any significant effect on merozoite invasion

inhibition. Only peptide 26160, inhibiting invasion by

69 ± 2%, was able to affect intra-erythrocyte development

by 44 ± 2%, suggesting that inhibition could have been

mediated by toxic effects.

These results show that these high binding sequences are

involved in one or more interactions between merozoites

and erythrocytes; however, other effects on invasion cannot

be ruled out.

Six P. falciparum EBA140 protein high RBC binding pep-

tides were identified; all of them were conserved. Some of

them presented the same RBC binding behavior as the whole

protein. It is thus worth restating that all HABPs made some

contribution towards the invasion process. Furthermore,

three of them were located in region II, suggesting that

EBA140 high binding peptides could be directly involved in P.

falciparum merozoite invasion of human RBCs. Identifying

these peptides and their critical amino acids represents a

useful tool in designing peptides having immunological and

structural properties different to those of original peptides.

As the precise replacement of critical residues converted

non-immunogenic peptides into immunogenic, protective

ones, making them excellent candidates for a multi-compo-

nent subunit synthetic malaria vaccine.

Acknowledgments: This research project was supported by the

Colombian Ministry of Public Health. Jason Garry’s collaboration in

writing this manuscript is greatly appreciated.

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