Plasmodium falciparum TryThrA antigen synthetic peptides block in vitro merozoite invasion to erythrocytes

BBRC Biochemical and Biophysical Research Communications 339 (2006) 888–896 www.elsevier.com/locate/ybbrc

Plasmodium falciparum TryThrA antigen synthetic peptides block in vitro merozoite invasion to erythrocytes q Hernando Curtidor *, Marisol Ocampo, Luis E. Rodrı´guez, Ramses Lo´pez, Javier E. Garcı´a, ´ lvaro Puentes, Jesus Leiton, Lina J. Cortes, John Valbuena, Ricardo Vera, A Yolanda Lo´pez, Manuel A. Patarroyo, Manuel E. Patarroyo Fundacio´n Instituto de Inmunologı´a de Colombia (FIDIC), Universidad Nacional de Colombia, Colombia Received 11 November 2005 Available online 28 November 2005

Abstract Tryptophan–threonine-rich antigen (TryThrA) is a Plasmodium falciparum homologue of Plasmodium yoelii-infected erythrocyte membrane pypAg-1 antigen. pypAg-1 binds to the surface of uninfected mouse erythrocytes and has been used successfully in vaccine studies. The two antigens are characterized by an unusual tryptophan-rich domain, suggesting similar biological properties. Using synthetic peptides spanning the TryThrA sequence and human erythrocyte we have done binding assays to identify possible TryThrA functional regions. We describe four peptides outside the tryptophan-rich domain having high activity binding to normal human erythrocytes. The peptides termed HABPs (high activity binding peptides) are 30884 (61LKEKKKKVLEFFENLVLNKKY80) located at the N-terminal and 30901 (401RKSLEQQFGDNMDKMNKLKKY420), 30902 (421KKILKFFPLFNYKSDLESIM440) and 30913 (641DLESTAEQKAEKKGGKAKAKY660) located at the C-terminal. Studies with polyclonal goat antiserum against synthetic peptides chosen to represent the whole length of the protein showed that TryThrA has fluorescence pattern similar to PypAg-1 of P. yoelii. All HABPs inhibited merozoite in vitro invasion, suggesting that TryThrA protein may be participating in merozoite–erythrocyte interaction during invasion.
2005 Elsevier Inc. All rights reserved. Keywords: Peptides; Tryptophan-threonine-rich antigen; Plasmodium falciparum; Malaria; Invasion

It is estimated that between 300 and 500 million cases and between 700,000 and 2.7 million deaths are caused by malaria around the world each year [1,2]. The resistance of both the parasite to anti-malarial drugs and the mosquito to insecticides, added to the parasiteÕs different immune system evasion mechanisms, have all contributed towards making the problem of malaria become more severe [3,4]. Although several studies have shown

q

Abbreviations: HABPs, high activity binding peptides; TryThrA, tryptophan–threonine-rich antigen; pypAg-1, Plasmodium yoelii particulate antigen-1; MaTrA, merozoite-associated tryptophan-rich antigen; PvTRAg, Plasmodium vivax tryptophan-rich antigen. * Corresponding author. Fax: +57 1 3244672/73x108. E-mail addresses: hernando_curtidor@fidic.org.co, hercur@hotmail. com (H. Curtidor). 0006-291X/$ - see front matter
2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.11.089

that it is feasible to obtain different types of anti-malarial vaccines, no effective vaccine has been found to date; the search thus continues for finding and selecting new antigens for being included in a multi-component, multistage vaccine [5–7]. One of the approaches used for identifying and selecting vaccine candidates is studying immunization-induced protective immune responses in animal models [8–10]. It can thus be established that a mixture of highly enriched Plasmodium yoelii membrane proteins mediates protective immunity in mice, such mixture being associated with the production of antibodies specifically recognizing from six to eight P. yoelii antigens [9]. When sera from these immunized mice were used for screening a P. yoelii cDNA expression library, pypag-1 and pypag-3 genes were identified which encode two of these antigens [11,12]. The

H. Curtidor et al. / Biochemical and Biophysical Research Communications 339 (2006) 888–896

antigens named pypAg-1 and pypAg-3 were located in the cytoplasm and associated with the membrane of P. yoeliiinfected erythrocytes [11,12]. Both antigens are characterized by containing one unusual tryptophan-rich region and, unlike pypAg-3, pypAg-1 has one domain having 31 copies of a pentapeptide degenerative repeat located in the C-terminal [11,12]. Other studies isolated the Pf208 antigen when polyclonal rabbit sera raised against Plasmodium falciparum-infected erythrocytes, protective monkey sera, and human malaria sera were used for identifying clones expressing potential surface antigens [13]. Furthermore, in vitro phagocytosis inhibition assays showed that a Pf208 recombinant protein inhibited monocyte opsonization of blood stage parasites, suggesting its possible relationship with inducing a protective response [14]. Pf208 was characterized later on and called tryptophan–threonine-rich antigen or TryThrA because it contains a tryptophan-rich region and threonine-rich repeats [15]. This protein is expressed during the entire life cycle of blood stage parasites and has high homology with pypAg-1 (33% identity); it was therefore considered to be a P. falciparum orthologue of pypAg-1 [15]. A P. falciparum tryptophan-rich antigen has been identified, having similar features to the P. yoelii pypAg-3 antigen and, as it has been associated with the merozoite surface, this antigen has been termed merozoite-associated tryptophan-rich antigen (MaTrA) [12,16]. A tryptophan-rich antigen from the human malaria parasite Plasmodium vivax (PvTRAg) has been recently identified and characterized; this antigen shares homology with pypAg-1, pypAg-3, MaTrA, and TryThrA tryptophan-rich proteins. Just like TryThrA, PvTRAg is expressed by all blood stages [17]. It has been suggested that the presence of the unusual tryptophan-rich domain in several proteins from different Plasmodium species may be functionally important [11]. In fact, pypAg-1 and pypAg-3 are part of those particulate antigens inducing a protection-inducing immune response in mice and pypAg-1 contains at least two protective epitopes inducing a four- to sevenfold reduction in P. yoelii blood stage parasitemia [9,11]. Interestingly, pypAg-1 and pypAg-3 secreted from parasitized cells bind to uninfected mouse erythrocytes [12]. Considering their homology with pypAg-1 and pypAg-3 antigens and their expression throughout the asexual life cycle [11,12,15], we aimed at identifying P. falciparum-TryThrA-derived peptides having specific erythrocyte binding capacity and their biological activity in the present study. We found four peptides (HABPs) that bind specifically to erythrocytes and inhibited in vitro merozoite invasion, depending on the peptide concentration. Even though no precise function has yet been found for TryThrA, these findings regarding TryThrA seem to indicate its potential role during merozoite invasion of erythrocytes.

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Materials and methods Peptide synthesis and binding assay. Thirty-four sequential 20-mer peptides, corresponding to the complete P. falciparum TryThrA protein amino acid sequence (GenBank Accession No. NP_704256), were synthesized by the solid-phase multiple peptide system [18,19]; t-Boc amino acids (Bachem) and MBHA resin (0.7 meq/g) were used. Peptides were cleaved by the low–high HF technique [20], purified by RP-HPLC, lyophilized, and analyzed 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-labeling. Synthesized peptides are shown in Fig. 1A in one-letter code. Peptides were 125I-labeled [21,22] and tested in human erythrocyte binding assays. Briefly, erythrocytes (2 · 109 cells/ll) obtained from healthy donors were washed in HBS buffer until the buffy coat was removed. The erythrocytes were then incubated with different radio-labeled-peptide concentrations (10–200 nM) in the absence (total binding) or presence (non-specific binding) of unlabeled peptide (40 lM). The sample reached 200 ll final volume with HBS and was incubated for 90 min at room temperature [21,23]. After incubation, cells were washed five times with HBS and bound cell radio-labeled peptide was quantified in an automatic gamma counter (4/200 plus ICN Biomedicals). The binding assays were performed in triplicate. Jumbled-peptide binding assay. The HABP sequences determined in the binding assay were used in synthesizing the same peptides but now in a jumbled order (i.e., same amino-acid composition as HABPs but having a random sequence selected by the ‘‘out of the bag’’ method) and then tested in binding assays. The assays were carried out in triplicate in identical conditions to those described above. Synthesized peptides are shown in Fig. 1B in one-letter code. Saturation assays. A modified erythrocyte binding assay was used to ascertain saturation with all HABPs: 107–108 cells were used at 170 ll final volume; radio-labeled peptide concentrations were between 20 and 2400 nM. Unlabeled peptide concentration was 40 lM. Cells were washed with HBS and bound cell radio-labeled peptide was quantified [23,24]. Enzymatic treatment. Erythrocytes (5%) suspended in HBS buffer were treated with 0.15 mU/ml neuraminidase (ICN 9001-67-6) at 37 C for 1 h, washed five times with HBS buffer, and centrifuged at 1000g for 5 min. Erythrocytes (5%) were similarly treated with trypsin (Sigma T-1005) or chymotrypsin (Sigma C-4129) in HBS buffer (5 mM Tris–HCl, 140 mM NaCl, pH 7.4) at a final 0.75 mg/ml concentration. After incubation at 37 C for 1 h, the samples were washed five times with HBS buffer to which PMSF (0.1 mM) had been added. After enzyme treatment, these erythrocytes were tested in binding assay with HABPs in previously reported conditions [23,25]. Merozoite invasion inhibition assay. Sorbitol synchronized P. falciparum (FCB-2 strain) cultures were incubated until the late schizont stage at final 0.5% parasitemia and 5% hematocrit in RPMI 1640 + 10% O+ plasma [26,27]. The cultures were seeded in 96-well cell-culture plates (Nunc) in the presence of test peptides at 200, 100, and 50 lM concentrations, in triplicate. After incubation for 18 h at 37 C in a 5% O2/5% CO2/90% N2 atmosphere, the supernatant was recovered and the cells were stained with 15 lg/ml hydroethydine, incubated at 37 C for 30 min, and washed three times with PBS. The suspensions were analyzed using a FACsort in Log FL2 data mode using CellQuest software (Becton Dickinson immunocytometry system) [28]. EGTA (12.5 mM), chloroquine (300 lM), low binding activity peptides 30886 and 30908, and peptide 1513 derived from P. falciparum MSP-1 protein were used as controls. Goat immunization. Two goats (B-26 and B52), previously determined to be non-reactive to P. falciparum lysate by Western blotting, were immunized with five polymeric peptides chosen to represent the whole length of the protein to obtain sera against TryThrA protein regions. A mixture made up of 500 lg from 28946 (CG21LSFLKNENKFLSQGKS LIKF40GC), 28948 (CG201WLEGKNKEYDIWLNHMNSKW220GC), 28950 (CG321KDRKEWLRWKERVTREKLEW340GC), 28952 (CG561N SDKTTNFNTYRTTDLNSDK580GC), and 28954 (CG621SLDHEVRQ

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H. Curtidor et al. / Biochemical and Biophysical Research Communications 339 (2006) 888–896

A

Peptide number

30881 30882 30883 30884 30885 30886 30887 30888 30889 30890 30891 30892 30893 30894 30895 30896 30897 30898 30899 30900 30901 30902 30903 30904 30905 30906 30907 30908 30909 30910 30911 30912 30913 30914

B

1

M N L 21 L S F 41 L I G 61 L K E 81 K K E 101 D T S 121 R N I 141 S K N 161 D E E 181 D E T 201 W L E 221 T N Y 241 KWN 261 Q D F 281 QW I 301 E E D 321 K D R 341 K HW 361 KWK 381 W I R 401 R K S 421 K K I 441 E E D 461 T G D 481 N V G 501 T A D 521 N S D 541 A T D 561 N S D 581 T T N 601 L G K 621 S L D 641 D L E 661 K A K

E L I K N D I E G E G N E K Q E K V K E L L E V K L K L K F T H S A

Q F K N I N K D L K N E N K F L S Q A I F L V V L I F K K K V L E F F E I T A A I A S K E S E D E D H I I N N N P D E K V H N E D K T D E S Y N E V N L E DWK K E E WQ L L K L W K N K E Y D I W L K D I D E E Y D S K Q W E Q WM K T RW L E D S E G Y WK N MK I L E C KW S N L E D T D E W L RWK E R V E MK E N M N I Y N K L A N F N E W K QWN NW I N E E Q Q F G D N M D K F F P L F N Y K N E Y N S F D E N N V G K T E A L N T E D L N VA K T N A E K T T D L N T T D L N P E K T N A N K T A D L N T T N F N T Y R T N T Y K T D L Y A T N H N VA K T T E V R QM I D Q K T A E Q K A E K K K T K V R T V D D

Peptide number

30884 32715 30901 32716 30902 32718 30913 32719

Binding Activity (%)

SEQUENCE A G I N L K V E N L N N E L L Q T N S R K S E V E S T S T E D V G D

2

T N L F S Q Y K S L I K F Y K S S H P A Y L V L N K K Y A D A E T T Y K V K R R K Y K E K N S K Y T S L L S S E W I K WM Y E G E K N K Y HM N S KW V F K D S Y G K E F M L Y K S W L I K M N E WR R Y I R V L N H Y R E K L E WY KWK KW I K N F I E K Y K K Y T S Q M N K L K K Y D L E S I M E E N D E K A K T E G L Y D L N VA K Y E K T A D L Y N F N T Y K D K T T D L Y D L N S D K K T T D V N Q K V V K H Y A Q I M N H Y G K A K A K Y G N E I N V Y

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680

Binding Activity (%)

SEQUENCE 61

L L 401 R K 421 K I 641 D L

K K K K K L L G

E K S K I K E K

K N L N L F S K

K K E F K F T A

K K Q E F L A E

K F Q K F K E K

V L F K P S Q A

L L G N L D K G

E N D D F Y A E

F E N D N L E Q

F E L V M D Q L Y K F I K K T L

N K K L S E G K

2

L V V K M N MM D L S N G K K A

L E K G E P A E

N E L Q S K K S

K K K S I K A D

K Y F Y K Y R Y M M K Y A Y

80

420

440

660

Fig. 1. Erythrocyte binding assays. (A) Each one of the black bars represents the slope of the specific binding graph (called specific binding activity) when TryThrA peptides are used. To the right of the figure is a schematic representation of the TryThrA protein, showing the putative signal sequence (gray box), the tryptophan-rich region (black box), and the threonine repeat region (white box). The rectangle shown more to the right of the figure represents the Pf208 antigen [13,14]. (B) HABP jumbled-peptide erythrocyte binding profile. MIDQKVAQIMNH640GC) polymeric peptides was inoculated with FreundÕs complete adjuvant on day 0 and incomplete adjuvant on days 20 and 40. The bleedings were done on days 0 (pre-immune), 20 (I-20), and 40 (II-20) for antibody production analysis. Final bleeding was carried out on day 60 (III-20) and sera were collected. Immunizations and bleeding were performed according to Colombian Ministry of Public Health animal-handling procedures. Goat sera adsorption with E. coli and M. smegmatis sonicate and SPf66– Sepharose. Escherichia coli (DH5a strain) proteins were obtained from overnight culture in Luria–Bertani medium, washed, suspended and sonicated for 2 min at 4 C and 10 min at 4500g. M. smegmatis proteins were obtained from 5-day-old Middlebrook 7H9 broth culture, washed, sus-

pended, sonicated for 10 min (as described above), and centrifuged for 10 min at 4500g. Both pellets were suspended in coupling buffer (1.0 M NaHCO3, 0.5 M NaCl, pH 8.3). The suspended lysate and synthetic SPf66 vaccine [29] were collected and used individually for coupling to CNBractivated Sepharose 4B (Pharmacia Biotech) according to manufacturerÕs recommendations. Each goat serum (pre-immune and immune) was preadsorbed with E. coli–Sepharose, M. smegmatis–Sepharose, and SPf66– Sepharose affinity columns to eliminate cross-reactivity. Briefly, each serum (5 ml) was added to Sepharose affinity columns (5 ml) and left in a gently rotating/shaking mode for 30 min at room temperature. This procedure was done twice using a new lysate–Sepharose affinity column each time; sera were stored at
70 C. The sera which were pre-adsorbed with

H. Curtidor et al. / Biochemical and Biophysical Research Communications 339 (2006) 888–896 E. coli–Sepharose, M. smegmatis–Sepharose, and SPf66–Sepharose affinity columns were called pB26 (preimmune), iB26 (immune), pB52 (preimmune), and iB52 (immune). SDS–PAGE and immunoblotting. Proteins from P. falciparum intraerythrocyte schizont lysate and erythrocyte membrane proteins were separated in a discontinuous SDS–PAGE system, using an acrylamide gradient (7.5–15% wt/vol). A total of 1 mg/ml lysate was loaded per gel and then transferred to nitrocellulose membrane (Hybond 203c, Pharmacia) using the semidry blotting technique [30]. The nitrocellulose membranes (0.4 mm) were incubated for 1 h with 1:100 dilution of the sera in TBS-T blocking solution (1% Tween 20 and 5% skimmed milk in TBS). One hour incubation with 1:5000 alkaline phosphatase-conjugated antigoat IgG antibody (ICN) was carried out after five TBS-T washes. The reaction was then developed with NBT/BCIP (Promega); commercial molecular mass markers (NEB) were used for calibration. Immunofluorescence assay (IFA). Immunofluorescence microscopy of schizont-stage parasites was conducted as described elsewhere [31]. Briefly, the cells were washed in PBS and then fixed with 4% EM (electron microscopy) grade paraformaldehyde and 0.0075% EM grade glutaraldehyde in PBS for 30 min. Fixed cells were washed once in PBS and then permeabilized with 0.1% Triton X-100/PBS for 10 min. Cells were washed again in PBS and then treated with 0.1 mg/mL sodium borohydride (NaBH4)/PBS for 10 min. Following another PBS wash, cells were blocked in 3% BSA/PBS for one hour and then incubated with goat polyclonal pB26, iB26, pB52, and iB52 sera for 30 min. After three washes, cells were incubated with FITC-conjugated rabbit anti-goat IgG, as secondary antibody for 30 min, washed in PBS, and then mounted in 50% glycerol. Immunoreactivity was observed by using an Axioskop fluorescence microscope (Carl Zeiss). An AxioCam MRm CCD camera (CarlZeiss) was used for recording images. CD spectroscopy. CD analysis was used for obtaining general information regarding HABP structure and folding. Spectra were acquired on Jasco J-810 equipment using a 1 cm path length quartz cell at 20 C in pure water or in aqueous TFE (30% v/v) solution. Peptide concentration was 0.1 mM in all conditions. Each spectrum was obtained from averaging three scans taken at a 20 nm/min scan-rate with 1 nm spectral bandwidth and corrected for baseline. Molar ellipticity was estimated using SELCON3, CDSSTR or CONTINlLL software [32,33]. DNA extraction from P. falciparum and PCR. Plasmodium falciparum strain FCB2 schizonts were obtained from a sorbitol synchronized culture, maintained as described previously [26,27]. Two hundred microliters of parasitized erythrocytes, reaching 30% parasitemia, was lysed using 0.2% saponin. Genomic DNA (gDNA) from isolated parasites was extracted and purified using Wizard Genomic DNA Purification Kit (Promega). Two microliters gDNA were PCR amplified in a 25 ll reaction consisting of 1 U Taq polymerase (Promega, Madison, WI), 1· Taq polymerase reaction buffer, 1.5 mM MgCl2, 0.2 lM dNTPs, and 0.4 lM of each primer (D9 forward 5 0 -AGAATAAGTTTTTGAGCC AGG-3 0 , D11 reverse 5 0 -ATCTTCTGAATCGCTTGTATC-3 0 , E1 forward 5 0 -AAAATGGAAGAAAAATAAATTAGC-3 0 , E3 reverse 5 0 -TT CCTACATTAACATCTCCTG-3 0 , and E5 forward 5 0 -ACAGATCAAA AGGTAGTAAAAC-3 0 , and E7 reverse 5 0 -TATTTATTATCCATACA TTCATAC-3 0 ) in the following thermocycling conditions: initial 5 min denaturing cycle at 95 C, followed by 40 cycles consisting of 1 min annealing at 58 C, 1 min extension at 72 C, and 1 min denaturing at 95 C. A final 5-min extension cycle was run at 72 C. A control reaction was carried out in the same conditions using DNAse-treated RNA as template. Amplified products were visualized by 1% agarose gel electrophoresis and ethidium bromide staining. RNA extraction and RT-PCR. Total RNA from isolated parasites was extracted from ring (3 h) and schizont stages (48 h) using TRIzol reagent (Invitrogen). Two microliters of total RNA was treated with RQ-DNAse (Promega) to avoid gDNA contamination and used as template for SuperScript III OneStep RT-PCR (Invitrogen). Three regions encoding all the HABPs were amplified using D9/D11, E1/E3, and E5/E7 primer sets.

891

Results TryThrA peptides specifically bound to human erythrocytes Binding assays were used for determining specific erythrocyte binding activity for 34 synthetic peptides covering the total length of the TryThrA protein (GenBank NP_704256). Peptide binding activity was defined as being the amount (pmol) of peptide that bound specifically to erythrocyte per added peptide (pmol). High activity binding peptides were defined as being those peptides showing activity greater than or equal to 2%, since these peptides recognize more than 200 specific binding sites per cell at low concentrations of radio-labeled peptide (200 nM) [21,34]. Four erythrocyte HABPs were found in TryThrA-peptides: 30884 (61LKEKKKKVLEFFENLVLNKKY80), 30901 (401RKSLEQQFGDNMDKMNKLKK420), 30902 (421KKILKFFPLFNYKSDLESIM440), and 30913 (641DLESTAEQKAEKKGGKAKAK660). HABP 30884 was located in the TryThrA N-terminal region, just after the putative signal sequence (Fig. 1A). HABPs 30901 and 30902 were located towards the TryThrA protein C-terminal region following the tryptophan-rich region. HABP 30913 (displaying the highest specific binding) was located at the C-terminal at the end of the threonine-repeat region within the region called Pf208 antigen (Fig. 1A) [14]. No HABPs were found in the tryptophan-rich region, even though two specific low-binding regions can be seen (121R-K280 and 301E-Q400) (Fig. 1A). Fig. 1B shows that the jumbled peptides presented nominal specific binding activity. Binding constants for HABPs Saturation assays and Hill analysis were used for determining affinity constants, the number of binding sites per cell, and Hill coefficients for the HABPs (Fig. 2) [35,36]. The saturation curves were constructed using GraphPad Prism version 4.03 (Trial) for Windows, GraphPad Software, San Diego, California, USA (www.graphpad.com). The affinity constants (Kd) were: 30884 = 403 nM, 30901 = 310 nM, 30902 = 190 nM, and 30913 = 357 nM. The Hill coefficients were 1.6, 1.4, 1.5, and 1.4, respectively. The number of binding sites per cell ranged from 7000 to 27,000. Enzyme treatment The effect of enzymatic treatment on HABP-erythrocyte interaction was determined in binding assays with enzymetreated human erythrocytes. HABP binding to non-treated human erythrocytes was considered as positive control (100%). Table 1 shows that enzyme treatment of erythrocytes similarly affected the binding of peptides 30902 and 30913; this became lessened when they were treated with neuraminidase and increased when this was done with

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H. Curtidor et al. / Biochemical and Biophysical Research Communications 339 (2006) 888–896

2.6 2.6

0.2

0.2 1.3

1.3

-0.5

Bound peptide (pmol)

-0.7

-1.2

-1.6 1.4

2.2

1.2

3.0

0.0

1.9

2.6

0.0 0

300

600

0

250

500

30913

30902 3.6

1.4

0.2

0.6 1.8

0.7

-0.6

-0.2

-1.0

-1.4 1.4

2.2

3

0.0

1.2

2.0

2.8

0.0 0

300

600

0

250

500

Added peptide (nM) Fig. 2. Saturation curves for HABP 30884, 30901, 30902, and 30913. Increasing quantities of labeled peptide were added in the presence or absence of unlabeled peptide. The curve represents the specific binding. In the Hill plot (inset), the abscissa is log F and the ordinate is log (B/Bmax-B), where F is free peptide, B is bound peptide, and Bmax is the maximum amount of bound peptide. Table 1 Binding of synthetic TryThrA peptides to enzyme-treated erythrocytes Peptide

Control (%)a

Neuraminidase

Chymotrypsin

Trypsin

30884 30901 30902 30913

100 ± 6b 100 ± 4 100 ± 5 100 ± 4

91 ± 7 15 ± 7 65 ± 1 28 ± 2

123 ± 7 17 ± 1 215 ± 3 220 ± 1

102 ± 5 27 ± 5 217 ± 5 180 ± 7

Peptide binding was compared between enzyme-treated erythrocytes and untreated erythrocytes. a The data are presented as specific binding percentages (%) related to untreated erythrocyte. b Mean ± SD of three experiments.

low binding activity peptides, were added to cultures at the schizont stage before merozoite release from infected erythrocytes. The results show that all HABPs inhibited merozoite invasion by over 60% (Table 2). Even though HABPs 30902 and 30913 did not inhibit at 50 lM, it can be observed that invasion inhibition depended on peptide concentration and specific sequences, but was independent of affinity constants and/or Hill coefficients. Both jumbled and low activity binding peptides displayed minimal inhibitory activity in merozoite invasion (Table 2). CD spectroscopy

chymotrypsin or trypsin. Peptide 30901 binding lessened when erythrocytes were treated with chymotrypsin, trypsin or neuraminidase, whereas peptide 30884 binding was minimally affected by any treatment. Merozoite invasion inhibition assays The role of TryThrA HABPs in in vitro merozoite invasion was investigated. The HABPs, as well as jumbled and

Circular dichroism analysis was used for obtaining general information regarding HABP structure and folding. All HABPs displayed a typical unordered structure spectra in water (data not shown). In the more hydrophobic medium (30% TFE/water) however, HABP 30884 CD profile indicated a clear shift towards an ordered structure, possibly typical a-helix as characterized by double minima at 208 and 220 and 190 nm maximum ellipticity. HABPs

H. Curtidor et al. / Biochemical and Biophysical Research Communications 339 (2006) 888–896 Table 2 Inhibition of parasite invasion to erythrocytes by synthetic TryThrA peptides Peptide

% Inhibition invasion (lM) 200

30884 30901 30902 30913 32715 32716 32718 32719 30886 30908 1513 Cloroquina EGTA a b

a

91 ± l 79 ± 1 60 ± 9 85 ± 2 3±1 2±1 0±2 1±3 0±4 0±l 65 ± 8

100

50

20 ± 1 15 ± 1 20 ± 1 11 ± 1 ndb nd nd nd nd nd nd 100 ± 1 100 ± 1

8±1 5±1 0±1 0±4 nd nd nd nd nd nd nd

Mean ± SD of three experiments. Not determined.

30902 and 30913 presented little evidence of stable a-helix whilst HABP 30901 presented a random coil structure. CD spectra are summarized in Fig. 3.

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(Fig. 4B). No fluorescence of P. falciparum-parasitized erythrocytes incubated with preimmune rabbit serum B52 (pB52) was observed. Pre-immune pB26 and immune iB26 sera did not present reactivity (data not shown). TryThrA gene transcription TryThrA-rich antigen encoding gene transcription was assessed by PCR amplification using total RNA extracted from ring and schizont erythrocyte stage FCB2 strain P. falciparum parasites. The cDNA-amplified fragment size (242 bp) was smaller than that obtained by gDNA amplification (408 bp) when D9/D11 primer set was used, indicating that splicing of the intron located within this gene region was occurring (Fig. 4C, lanes 1–3). Fragment sizes observed when amplifying with E1/E3 and E5/E7 primers sets were the same when either cDNA or gDNA was used as template (323 bp for E1/E3 and 255 bp for E5/E7), confirming that there were no introns within these regions (Fig. 4C, lanes 5–7 and 9–11). The absence of a PCR product when DNAse treated RNA was amplified confirmed that no gDNA contamination was present in the cDNA sample (Fig. 4C, lanes 4, 8, and 12).

Immunofluorescence and immunoblotting assays Discussion Goats were inoculated with polymeric peptides to obtain polyclonal sera against different TryThrA protein regions. The sera were absorbed with E. coli, M. smegmatis sonicate, and SPf66–Sepharose to eliminate non-specific reactivity. Fig. 4A shows that immune B52 sera (iB52 III-20) strongly recognized 80 and 76 kDa molecular weight proteins and, to a lesser extent, a protein having a lower molecular weight. The 80 kDa protein was very close to the predicted molecular mass (80.4 kDa). The iB52 serum (III-20) also recognized proteins in P. falciparum schizont infected erythrocytes in immunofluorescence assays

Fig. 3. Circular dichroism profiles of TryThrA peptides. The results were expressed as mean residue ellipticity [H], the units being degrees · cm2 · dmol
1 according to the [H] = Hk/(100lcn) function, where the Hk is measured ellipticity, l is the optical path-length, c is peptide concentration, and n is the number of amino acid residues contained in the sequence.

Antigens on the surface both of the P. falciparum-infected erythrocytes and asexual merozoite stage are exposed to the immune system during the intraerythrocytic development or during the invasion process; therefore, they have been considered potential vaccine candidates against malaria [6,37]. Different approaches have been used for investigating the ability of each of these P. falciparum antigens to elicit a protective immune response [8–10,14]. We have used binding assays as a screening approach for identifying peptides from different P. falciparum proteins, some of which have been used for designing immunogenic and protection-inducing peptides from non-immunogenic ones [21,23,34,38,39]. By virtue of its homology with pypAg-1, pypAg-3, and MaTrA antigens, and its expression throughout the asexual life cycle [15,16], the TryThrA protein represents a good candidate for studying the merozoite–erythrocyte interactions. The sequence of pypAg-1, pypAg-3, and MaTrA antigens is different from that of the other adherence domain tryptophan-rich antigens. However, taking their localization and erythrocyte binding activity into account, it has been suggested that similar to the EBA-175 antigen, these proteins may be involved in erythrocyte invasion by merozoites or in the formation of rosettes between infected and infected erythrocytes facilitating re-invasion [12]. This is why this work has focused on analyzing TryThrA erythrocyte binding sequences and their in vitro effect on merozoite invasion of erythrocytes. We found four TryThrA-derived HABPs (30884, 30901, 30902, and 30913) which specifically bound to erythrocytes

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H. Curtidor et al. / Biochemical and Biophysical Research Communications 339 (2006) 888–896 M.W.M

kDa..

175

83

80 76

62

C

400 bp 300 200 Fig. 4. (A) Western blot assay. Immunoblot of P. falciparum/FCB-2 schizont lysate tested with preimmune goat serum (lane 1) or immune goat antiserum (iB52) raised against TryThrA protein polymeric peptides (lane 2). Lane 3, shows immune sera (iB52) tested with erythrocyte membrane proteins. Molecular mass markers are indicated. (B) Immunofluorescence assays with fixed P. falciparum-parasitized erythrocytes incubated with immune goat serum B52 (iB52). (C) TryThrA gDNA and cDNA amplification. Amplified product from Plasmodium falciparum/FCB-2 cDNA (lanes 1, 2, 5, 6, 9, and 10) and gDNA (lanes 3, 7, and 11). Ring stage parasites (3 h) (lanes 1, 5, and 9), schizont stage parasites (lanes 2, 6, and 10), and negative control (lanes 4, 8, and 12). M, 100 bp molecular weight markers.

(Fig. 1A). Even though HABPs presented between 30% and 50% charged amino acids and are mostly hydrophilic, binding assays with analogue peptides containing a jumbled sequence from each HABP indicated that HABP binding activity depended on each specific sequence in particular and not on amino acid composition. Jumbled peptidesÕ binding activities were much lower than those for native HABPs (Fig. 1B). HABP–erythrocyte interaction had high affinity and positive cooperativity, since affinity constants were between 190 and 403 nM, and Hill coefficients 1.4 and 1.6 (Fig. 2). These values indicate strong HABP–erythrocyte interaction as expected for receptor–ligand interactions involved in merozoite–erythrocyte interaction. No HABPs were found in the P. falciparum TryThrA tryptophan-rich domain (Fig. 1A). A similar result has been previously reported by [12], showing that residues lying outside the P. yoelii pypAg-1 tryptophan-rich domain might be involved in pypAg-1 binding to normal erythrocytes [12]. It has been suggested that the presence of the unusual tryptophan-rich domain in these different proteins could be related to similar biological properties [11,12]. Our results along with those of others indicate that the tryptophan-rich domain present in different malarial proteins is not related to binding; perhaps, it is associated with generating an immune response. A fourfold reduction in P. yoelii parasitemia was induced when mice were immunized with recombinant pAg-1N alone, which included the tryptophan-rich domain [11]. Additionally, MaTrA and TryThrA recombinant tryptophan-rich domains were specifically recognized by IgG serum antibodies from P. falciparum

exposed individuals [16]. Interestingly, HABP 30913 (displaying the highest specific binding) was located in the TryThrA N-terminus within the region corresponding to Pf208 antigen. Pf208 inhibits blood-stage parasite opsonization by monocytes [14]. Three types of behavior were seen when the binding assays were performed with enzyme-treated human erythrocytes (Table 1). Neuraminidase treatment reduced the binding of peptides 30901, 30902, and 30913, indicating that binding is sialic-acid dependent and sialic-acid removal avoids the interaction between erythrocyte and peptides. Chymotrypsin or trypsin treatment of erythrocytes similarly increased the binding of peptides 30902 and 30913, suggesting that glycophorin or band 3 protein removal allows a better interaction between erythrocytes and peptides. Peptide 30901 binding lessened when erythrocytes were treated with chymotrypsin, trypsin or neuraminidase, whereas peptide 30884 binding was minimally affected by any treatment. The results suggest the presence of at least three different receptor sites; one of them was sialic-acid dependent (HABPs 30901, 30902, and 30913). It has been reported that P. falciparum merozoites interact with different erythrocyte receptors and that they use sialic-acid-dependent and acid-independent pathways for invading erythrocytes [40,41]. TryThrA protein interaction with erythrocytes (or at least that of HABPs derived from them) became even more evident when HABPs were tested in P. falciparum in vitro cultures. It was observed that all HABPs inhibited merozoite invasion, suggesting that the HABPs were blocking the merozoite–erythrocyte interaction, thereby

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inhibiting invasion (Table 2). It was also observed that HABP invasion inhibition and binding activity were not related to amino-acid charge or HABP hydrophobicity, affinity constants or structure (Figs. 1A and B, 2, 3, and Table 2). The results also indicated that, as seen in the binding experiments, HABPs inhibited merozoite invasion of erythrocytes due to their specific sequences. HABP jumbled peptide has no effect on in vitro invasion (data not shown). Preliminary circular dichroism analysis showed different structural elements for each HABP (Fig. 3). We expect that additional studies will determine whether there is a structure–binding activity relationship, similar to that found for the structure–protection relationship for some peptides in previous work [38,39]. Western blot analysis showed that goat sera immunized with TryThrA peptides recognized a protein in P. falciparum/FCB-2 late schizont lysate which migrated at around 80 kDa, close to its predicted molecular weight (Fig. 4A). These results agree with the molecular weight, lower than 94 kDa and nearer to 80 kDa, reported by Uhlemann et al. [15] for the TryThrA protein in strains different from the Kenyan Binh1 strain. Strong recognition of a protein migrating to 76 kDa and weaker recognition of another protein having less molecular weight were possibly due to crossed-reaction with proteins which might have been expressed during this stage. However, it cannot be ruled out that these could be TryThrA protein cleavage products, since a mixture of inoculated peptides blocked polyclonal sera recognition of all proteins (data not shown). Immunofluorescence studies with fixed cells have shown that TryThrA has a fluorescence pattern similar to P. yoelii PypAg-1 [11], indicating that TryThrA was associated with infected erythrocyte membranes (Fig. 4B). We have studied the transcription of the encoding gene in the P. falciparum FCB2 strain (due to the varying molecular weight observed for TryThrA protein) to further confirm this geneÕs identity. Gene transcription in P. falciparum FCB2 strain ring and schizont stages was verified by PCR amplification of three different regions from both gDNA and cDNA; these contained all 4 HABPs from this protein. In summary, we have identified four TryThrA proteinderived HABPs binding to the erythrocyte surface and inhibiting merozoite–erythrocyte interaction. Based on previous binding studies [38,39,42,43], we propose these HABPs should be considered in designing tools for the specific inhibition of P. falciparum merozoite interaction with erythrocytes. Acknowledgments This study was supported by the President of ColombiaÕs office and the Colombian Ministry of Public Health. We thank Jason Garry for reading the manuscript.

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