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Protein Science (2005), 14:504-513. Published by Cold Spring Harbor Laboratory Press. Copyright © 2005 The Protein Society
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Identifying Plasmodium falciparum cytoadherence-linked asexual protein 3 (CLAG 3) sequences that specifically bind to C32 cells and erythrocytes

Marisol Ocampo, Luis E. Rodríguez, Hernando Curtidor, Álvaro Puentes, Ricardo Vera, John J. Valbuena, Ramses López, Javier E. García, Luis E. Ramírez, Elizabeth Torres, Jimena Cortes, Diana Tovar, Yolanda López, Manuel A. Patarroyo and Manuel E. Patarroyo

Fundación Instituto de Inmunologia de Colombia and Universidad Nacional de Colombia, Bogotá, Columbia

Reprint requests to: Marisol Ocampo, Avda. Calle 26 No. 50-00, Bogotá, Colombia; e-mail: marisol_ocampo{at}fidic.org.co; fax: +57-1-324-4672.

(RECEIVED May 25, 2004; FINAL REVISION October 12, 2004; ACCEPTED October 18, 2004)


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and methods
 References
 
Adhesion of mature asexual stage Plasmodium falciparum parasite-infected erythrocytes (iRBC) to the vascular endothelium is a critical event in the pathology of Plasmodium falciparum malaria. It has been suggested that the clag gene family is essential in cytoadherence to endothelial receptors. Primers used in PCR and RT-PCR assays allowed us to determine that the gene encoding CLAG 3 (GenBank accession no. NP_473155 [GenBank] ) is transcribed in the Plasmodium falciparum FCB2 strain. Western blot showed that antisera produced against polymerized synthetic peptides from this protein recognized a 142-kDa band in P. falciparum schizont lysate. Seventy-one 20-amino-acid-long nonoverlapping peptides, spanning the CLAG 3 (cytoadherence-linked asexual protein on chromosome 3) sequence were tested in C32 cell and erythrocyte binding assays. Twelve CLAG peptides specifically bound to C32 cells (which mainly express CD36) with high affinity, hereafter referred to as high-affinity binding peptides (HABPs). Five of them also bound to erythrocytes. HABP binding to C32 cells and erythrocytes was independent of peptide charge or peptide structure. Affinity constants were between 100 nM and 800 nM. Cross-linking and SDS-PAGE analysis allowed two erythrocyte binding proteins of around 26 kDa and 59 kDa to be identified, while proteins of around 53 kDa were identified as possible receptor sites for C-32 cells. The HABPs’ role in Plasmodium falciparum invasion inhibition was determined. Such an approach analyzing various CLAG 3 regions may elucidate their functions and may help in the search for new antigens important for developing antimalarial vaccines.

Keywords: Plasmodium falciparum; cytoadherence; C32 cells; peptides

Abbreviations: CLAG, cytoadherence-linked asexual protein • HABPs, high activity binding peptides • PRBCs, parasitized red blood cells • HBS, HEPES buffered saline

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04883905.


   Introduction
TOP
 Abstract
 Introduction
Results
 Discussion
 Materials and methods
 References
 
The Plasmodium falciparum parasite’s complex life cycle with its distinct morphological and antigenic stages has been a major hurdle in developing antimalarial vaccines. It is anticipated that using data from Plasmodium falciparum’s recently completed genomic sequence (Florens et al. 2002) will lead to an increased understanding of parasite biology that will eventually be translated into new drugs and vaccines for malaria (Gardner et al. 2002; Hoffman et al. 2002).

Plasmodium falciparum infection can be distinguished from other forms of malaria due to its ability to cause severe malaria associated with high mortality. The accumulation of parasitized erythrocytes (PRBCs) can cause an obstruction in the flow of blood in the microvasculature, directly by binding to the endothelium or indirectly by binding to other PRBCs (agglutination) or to uninfected erythrocytes (rosetting). Such phenomena are generally referred to as "cytoadherence." Cytoadherence is believed to be fundamental for Plasmodium falciparum’s in vivo survival and a major determinant of this parasite’s virulence. Despite the widely accepted importance of cytoadhesion in the development of severe disease, there are a number of aspects of this highly complex process that remain poorly understood.

Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) was mainly found among those molecules found on the surface of parasitized erythrocytes involved in processes of cytoadherence. This protein has been implicated in antigenic variation and adhesion (Craig and Scherf 2001). Also the rifins (repetitive interspersed family of genes), initially identified as "rosettins," and stevor (subtelomeric variant open reading frame) proteins have been implicated in adhesion and rosetting.

Ligands on the surface of parasitized red cells can bind to a number of endothelial cell receptors, including CD36 (Barnwell et al. 1989), thrombospondin (Roberts et al. 1985), chondroitin-4-sulfate (Rogerson et al. 1995), vascular cell adhesion molecule-1 (Ockenhouse et al. 1992), E selectin (Ockenhouse et al. 1992), and platelet/endothelial cell adhesion molecule-1 (Treutiger et al. 1997).

Holt et al. (1999) have proposed that the clag gene family is essential in cytoadherence to endothelial receptors, with different paralogs involved in binding to different receptors, or that clag gene family paralogs are important in cellular adhesion interactions during different stages of the life cycle. The first gene characterizing the clag (cytoadherence-linked asexual gene) family of Plasmodium falciparum was identified on chromosome 9. The protein product (CLAG 9) was implicated in cytoadhesion, the binding of infected erythrocytes to host endothelial cells, but little information on the biochemical characteristics of this protein is available. RT-PCR has shown that the clag 9 paralogs clag 2 and clag 3.1 are also expressed in blood-stage parasites.

Clags 2, 3.1, and 3.2 (on chromosomes 2 and 3, respectively) have been completely sequenced; they are colinear with clag 9, have identical splicing patterns, and are expressed in asexual blood stages. However, they are considerably divergent in sequence (Gardner et al. 1998; Bowman et al. 1999).

Kaneko et al. (2001) have shown that other members of the clag family, as well as clag 9, encode merozoite rhoptry proteins that may be involved in merozoite–erythrocyte interactions.

Deleting the clag gene from chromosome 9 prevents cytoadherence, indicating that none of the clag genes in chromosome 3 are functionally equivalent to clag 9 (Bowman et al. 1999; Gardiner et al. 2000; Trenholme et al. 2000).

Due to the importance which CLAG 3 (in the currently available malaria genome sequence, in which clag3.2 has the PlasmoDB designation PFC0110w [http://www.PlasmoDB.org]) could have in adhesion and sequestration, there is specific interest in looking for the C32/CD36 cell and erythrocyte-binding sequences in this protein that are possibly used by the parasite as ligands for binding to target cells. This paper assesses CLAG 3.2-encoded gene transcription in the Plasmodium falciparum FCB2 strain and describes those CLAG 3.2 peptides which have been chemically synthesized and tested in C32 cell and erythrocyte binding assays after being radiolabeled with Na125I. Some of their functional activities have been proposed; the molecules to which they bind were determined as well as their possible recognition by polyclonal antibodies.


   Results
TOP
 Abstract
 Introduction
 Results
Discussion
 Materials and methods
 References
 
Molecular assays
CLAG transcription was assessed by RT-PCR amplification using total RNA extracted from schizont-stage parasites as template. Obtained fragment size (474 bp and 419 bp) was the same for both gDNA and cDNA. As PCR product was absent when DNAse-treated RNA was amplified, this confirmed that there was no gDNA contamination in the cDNA sample (Fig. 1Go).



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  		Figure 1. Plasmodium falciparum gDNA cytoadherence-linked asexual protein (CLAG) amplification products using A5/A7 or B1/B3 primer sets (lanes 1 and 4, respectively). Plasmodium falciparum cDNA amplification products using A5/A7 or B1/B3 primer sets (lanes 2 and 5, respectively). Negative controls (lanes 3,6). M, 100-bp molecular weight markers.

 
The 474-bp product (amplified from cDNA using the A5/A7 primer set) was cloned and sequenced to verify whether the amplified product was in fact the NP_473155 [GenBank] gene. Aligning the obtained sequence and its corresponding region within the CLAG protein-coding sequence (GenBank accession NP_473155 [GenBank] ) showed an exact match (Fig. 2Go).



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  		Figure 2. Nucleotide sequence alignment between the 474-bp amplified product (Clone 7A) and the reported CLAG nucleotide sequence (NP_473155 [GenBank] ). Sequences where primers anneal are highlighted in gray. Dots and dashes represent identities and gaps, respectively.

 
CLAG recognition by polyclonal antibodies
Sera obtained in goats by inoculating them with a mixture of polymeric forms of peptides 30376, 30379, 30389, 30392, 30404, 30413, 30431, and 30439 were tested against parasite lysate in Western blot assays. Strong recognition was shown by a 142-kDa protein (Fig. 3Go) in final blood sera (lanes 1, 2) which was not observed in preimmune sera (lanes 3, 4). Bearing in mind the sequence of peptides from the 3D7 strain (GenBank accession NP_473155 [GenBank] ) (Gardner et al. 1998) and parasite lysate corresponding to the FCB2 strain, we can assume that the recognized protein would have been CLAG 3. A negative control used normal uninfected erythrocyte lysate which did not present any reactivity to sera (data not shown).



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  		Figure 3. CLAG recognition by polyclonal antibodies. Western blotting was done with preimmune and final blood sera of goat against a P. falciparum lysate (lanes 1–4,7). (Lanes 1,2) Preimmune goat sera, (lanes 3,4) final blood sera. MW markers are shown on the right-hand side.

 
Identifying specific high activity binding peptides (HABPs)
C-32 cell and erythrocyte binding assays using 71 peptides derived from CLAG 3.2 or PFC0110w-protein (GenBank accession NP_473155 [GenBank] ) allowed 12 C-32 cell HABPs to be identified, of which five also presented high erythrocyte binding activity, as shown in Figure 4AGo. HABPs have been found localized throughout the whole protein even though most of them are localized in the central region having high binding activities (residues 660–1059) in what could be established as being a binding domain.




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  		Figure 4. C-32 cell- and erythrocyte-binding assays using PfCLAG 3.2 peptides. (A) Each of the black bars represents the slope of the specific binding graph, which is named specific binding activity. Peptides with 2% were considered as having high specific erythrocyte binding (HABPs). (B) Specific binding activity for HABP peptide analogs; jumbled peptide sequences are shown to the right, and the bars on the left represent specific binding. The numbers in the first column represent FIDIC’s internal code number for those peptides synthesized.

 
When peptide sequences were jumbled but amino acid composition was maintained (Fig. 4BGo) specific binding to these target cells became less than that of some of the original HABPs while, in other cases, jumbled peptide did not have specific binding. This happened for HABPs to both C-32 and erythrocyte cells.

Analyzing saturation curves obtained from HABPs led to calculating affinity constants (KD), Hill coefficients (nH) and the number of binding sites per cell (NrSC), shown in Table 1Go. The affinity constant range (100–800 nM) indicated high affinity in peptide–cell interaction, as well as positive cooperativity interaction (nH > 1 in all cases). The number of receptor sites for HABPs was greater in C-32 cells than erythrocytes.


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  		Table 1. Binding constants of CLAG-HABPs to C-32 cells and erythrocytes
 
Analyzing HABP structure
Figure 5Go shows some of the trends found for HABP structures. Figure 5AGo shows an {alpha}-helix-like feature according to two 208 and 222 nm minimum values and a 190 maximum ellipticity in the case of peptides 30409, 30421, 30414, 30403, and 30428. Other HABPs such as 30371 and 30387 presented random coil structural elements (Fig. 5BGo). Peptide 30373 (presenting high binding to two cell types) clearly showed a helical conformation (Fig. 5CGo).



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  		Figure 5. PfCLAG 3.2 HABP circular dichroism analysis. (A) Helical structural elements present in most HABPs. (B) Two of the HABPs presented random coil structural elements. (C) Peptide 30373 (presenting high binding to two cell types) clearly showed a helical conformation.

 
Determining receptors for HABPs
Cross-linking HABPs with C-32 cells and erythrocytes is shown in Figure 6Go. It can be seen that HABP is binding mainly to two proteins (26 kDa and 59 kDa) on the erythrocyte surface (Fig. 6BGo) and to C-32 cells with one protein of around 53 kDa (Fig. 6AGo)—the approximate weight corresponds to the calculated weight for CD36 receptor (53 kDa–54.3 kDa). These bands become inhibited in the presence of unlabeled peptide (panel A, lanes 4 and 5; panel B, even lanes).



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  		Figure 6. Autoradiograph from HABPs cross-linking assays. C-32 cell (A) and erythrocyte membrane (B) proteins were cross-linked with radiolabeled PfCLAG 3.2 peptides. (A) (Lane 1) molecular weight marker, (lane 2) SDS-PAGE for C-32 cells, (lanes 3,5) total binding, (lanes 4,6) binding inhibited in the presence of unlabeled peptide. (B) Odd-numbered lanes show the total binding (i.e., cross-linking in the absence of unlabeled peptide), and even-numbered lanes show inhibited binding (i.e., cross-linking in the presence of unlabeled peptide). Due to space constraints, only some cross-linking with C-32 cells is shown.

 
Determining HABP invasion–inhibition ability
HABPs’ possible role in the process of P. falciparum invasion of erythrocytes was evaluated by invasion inhibition assays. Table 2Go shows that some peptides presenting high erythrocyte binding activity were able to inhibit the invasion process by 60% to 90% at 200 µM concentration and 9% to 48% at 100 µM. Some peptides presenting high specific C-32 cell binding activity did not inhibit the invasion process, such as 30374, 30419, and 30428, indicating that these peptides could be involved in another important process for the parasite’s survival (rosetting or cytoadherence).


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  		Table 2. CLAG peptide inhibition of parasitic invasion of RBC
 

   Discussion
TOP
 Abstract
 Introduction
 Results
 Discussion
Materials and methods
 References
 
Developing antimalarial vaccines has been focused on the merozoite invasion process and the search for antigens intervening in this process presenting receptor–ligand interactions with target cells. Our method is based on the search for sequences presenting high specific target cell binding activity (Urquiza et al. 1996; Rodríguez et al. 2000; Ocampo et al. 2000, 2004; Curtidor et al. 2001) which have shown their role in the invasion process and some of them (after having been modified) have shown protection-inducing ability in experimental challenge in Aotus monkeys(Espejo et al. 2001; Bermudez et al. 2003; Cifuentes et al. 2003; Cubillos et al. 2003; Torres et al. 2003; Alba et al. 2004a, b). The publication of the P. falciparum genome (Gardner et al. 2002) has made it much more possible for many potential antigens to be analyzed now in the search for an antimalarial vaccine.

On the other hand, P. falciparum’s ability to adhere to the lining of capillaries in different tissue is related to severe malaria’s pathogenicity. Even though molecules have been described on the surface of infected erythrocytes involved in adhesion, a family from the clag gene has been recently described (cytoadherence-linked asexual gene) on chromosomes 1, 2, 3, 4, and 9 (Holt et al. 1999).

This work shows that the CLAG protein-encoding gene (GenBank accession NP_473155 [GenBank] ) is present in P. falciparum, and is transcribed and expressed in the FCB-2 strain. Inoculating polymeric peptides from sequences belonging to the CLAG 3 protein has led to obtaining sera strongly recognizing a protein having an apparent 142 kDa molecular weight in P. falciparum late-schizont-stage lysate (FCB-2 strain), which could be identified as being CLAG 3. This 142-kDa protein was not associated with RhopH2 described by Holder et al. (1985) since the inoculated amino acid sequences did not present homology with RhopH2. Further studies are needed for determining the exact localization and expression in different CLAG 3 protein P. falciparum strains.

It was possible to identify 12 peptides having high C-32 cell binding activity in chromosome 3 CLAG protein sequence expressing CD36, the receptor implicated in infected erythrocytes’ cytoadherence to P. falciparum. These peptides were found in the protein’s N-terminal, central, and C-terminal regions, even though an important binding domain could be established between the protein’s residues 660 and 1059. This domain could be involved in adhesion to endothelial cells expressing the CD36 receptor. Multiple CLAG 3 full amino acid sequence alignments were performed using CLUSTAL W (NPS@: Network Protein Sequence Analysis) finding that high specific-binding activity sequences belonged mainly to conserved regions between proteins PFC0110w and PFC0120w or CLAG 3.2 and CLAG 3.1, these being extremely similar (95%), most variability being found in peptide 30371:

PFC0110w 21KVLCSINENENLGENKNENA40

PFC0120w KVICSINENQN—ENDTISQ

In addition, peptide 30387 also presented change S for N in residue 358.

Regarding the CLAG 9 sequence, PFC0110w protein HABPs were strongly similar, except for peptide 30371 which did not present any homology with CLAG 9 (Gen-Bank accession NP_704889 [GenBank] ) (Hall et al. 2002).

HABPs found in C-32 cell binding assays could be involving these sequences in a possible role in CLAG 3 protein adhesion; some of these sequences could be used alternatively by the parasite in binding to normal erythrocytes (high erythrocyte-binding HABPs). These HABPs’ binding is specific and saturable, and has affinity constants in a range allowing high affinity receptor–ligand interactions to be established. It can also be established, by using jumbled sequences, that this binding depends on a particular HABP’s specific sequence and not on unspecific effects such as amino acid composition, net charge, etc. A relationship between approximate structure, determined by circular dichroism, and HABPs’ high specific-binding activity has also not been observed, as these present both structural {alpha}-helical elements (Fig. 2CGo) such as distorted helices (Fig. 2AGo) and random coil structures (Fig. 2BGo).

The possible receptor for HABP-binding erythrocytes (according to cross-linking assays) is a 26-kDa protein which could possibly present a dimer form (59 kDa). Figure 3BGo shows the binding protein for five high erythrocyte binding activity peptides. In the case of HABPs binding to C-32 cells, the possible receptor coincided with CD36’s approximate weight (Fig. 3AGo only shows the protein obtained in the case of two HABPs; however, all of them presented specific binding to the same band), identified as receptor in the cytoadherence phenomenon presented by P. falciparum. Posterior assays will allow determination of the HABPs’ role in CD36 adhesion assays.

A possible role can be established for those HABPs binding to erythrocytes in the erythrocyte invasion process; some of these sequences are able to inhibit up to 94% invasion at 200 µM. This suggests that blocking the binding site of these sequences of high erythrocyte binding activity of CLAG 3 protein considerably affects P. falciparum merozoite invasion and thus these represent antigens worth considering when developing erythrocyte stage antimalarial vaccines. Interestingly, HABPs having more than 64% inhibition presented µ-helix structural elements.

This work has contributed towards the search for antigen candidates for an antimalarial vaccine bearing CLAG 3 protein HABPs in vitro invasion inhibition ability in mind. A C-32 cell cytoadherence function can also be established bearing in mind that HABPs are also presented specifically binding to CD36.


   Materials and methods
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
References
 
Molecular assays
P. falciparum genomic DNA extraction and purification
P. falciparum FCB2 strain schizont-stage parasites were obtained from a sorbitol synchronized culture maintained as described previously (Trager and Jenson 1978; Lambros and Vanderberg 1979). Parasitized erythrocytes (200 µL), having 30% parasitemia, were lysed using 0.2% saponin. Genomic DNA (gDNA) was extracted from isolated parasites and purified using Wizard Genomic DNA Purification Kit (Promega).

P. falciparum RNA extraction and cDNA synthesis
Total RNA was extracted from isolated parasites using TRIzol reagent (Invitrogen). Total RNA (20 ng) were treated with RQ-DNAse (Promega) to avoid gDNA contamination and used as template for RT-PCR. Two primer sets were designed based on sequence data available from P. falciparum cytoadherence-linked asexual protein, CLAG (GeneBank accession NP_473155 [GenBank] ). The first primer pair used was forward A5 (5'-AAAATGCAAATGTA AACACACC-3') and reverse A7 (5'-ATACATTATTTAATTCT TCGAGC-3'). The second pair was forward B1 (5'-GATAAAGC ATATGGATTAAGTG-3') and reverse B3 (5'-GGAGTACATC TTTTGGGATC-3'). These sets were used following the SuperScript III OneStep RT-PCR System protocol (Invitrogen).

Polymerase chain reaction
gDNA (2 µL) were PCR-amplified in a 25 µl reaction consisting of 1U Taq polymerase (Promega), 1x Taq polymerase reaction buffer, 1.5 mM MgCl2, 0.2 µM dNTPs, and 0.4 µM of each primer under the following thermo-cycling conditions: initial 5-min denaturing cycle at 95°C, followed by 40 cycles consisting of 1 min annealing at 55°C, 1 min extension at 58°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 under the same conditions using DNAse-treated RNA as template. Amplified products were visualized by 1% agarose gel electrophoresis and ethidium bromide staining.

PCR product cloning and sequencing
Two independent products, amplified using A5/A7 primer set from P. falciparum cDNA, were purified using Rapid PCR Purification System (Concert) and then cloned into the pGEM vector (Promega) according to the manufacturer’s instructions. Single colorless colonies were isolated, grown overnight and plasmid DNA was further extracted using Wizard Plus Minipreps DNA Purification System (Promega). Two independent recombinant clones were analyzed per amplified product in both directions using an automatic sequencer (ABI PRISM 310 Genetic Analyzer, PE Applied Biosystems).

CLAG 3 protein recognition
Obtaining polyclonal sera
To obtain a greater quantity of serum for later assays, two goats (previously determined to be nonreactive to P. falciparum lysate as assessed by Western blot) were subcutaneously inoculated with a mixture of polymeric peptides corresponding to peptides 30376 (121YIIPTVQSSFHDIVKYEHL139), 30379 (180KQNNDLK SALEELNNVFTNKY199), 30392 (440NRQISAFNLYLNYYY FMKRY459), 30404 (680TLEKMKKSLTFLVHVNSFLQY699), 30413 (860SVNFYKYGIIYGFKVNKEIL879), and 30439 (1380TS TYIDTEKMNEADSADSDD1399), mixed with Freund’s Complete Adjuvant on day 0 and Freund’s Incomplete Adjuvant on days 20 and 40. Final bleeding was carried out on day 60 and sera collected. The immunization and bleeding were done according to the handling procedures required by the Colombian Ministry of Public Health.

The sera thus obtained were preadsorbed by using E. coli-Sepharose, M. smegmatis-Sepharose, and SPf66-Sepharose affinity columns to eliminate cross-reactivity. Briefly, 5 mL of each serum was added to 4 mL lysate (or SPf66)-Sepharose affinity columns and left in a gently rotating/shaking mode for 20 min at room temperature. This procedure was done twice using a new lysate–Sepharose affinity column each time and sera were stored at –70°C.

SDS-PAGE and immunoblotting
Proteins from P. falciparum (FCB-2 strain) lysate were separated in a discontinuous SDS-PAGE system, using 10% to 20% (w/v) acrylamide gradient and then transferred to nitrocellulose paper using the semidry blotting technique. Commercial molecular mass markers (NEB) were used for calibration. The paper strips were incubated with a 1:100 dilution of the sera in TBST (0.02 M TRIS-HCl [pH 7.5] 0.05 M NaCl, 1% Tween 20) and 5% skimmed milk. They were incubated with 1:3,000 alkaline phosphatase-conjugated anti-goat IgG antibody (ICN) for 1 h after five TBST washes. The reaction was developed with NBT/BCIP (KPL).

Peptide synthesis and radio-labeling
Seventy-one sequential 20-mer-amino-acid-long peptides from Plasmodium falciparum CLAG 3.2 -PFC0110w-(GeneBank accession no. NP_473155 [GenBank] ) (Gardner et al. 1998) were chemically synthesized using the Solid Phase Multiple Peptide Synthesis technique (Merrifield 1963; Houghten 1985). Synthesized peptide sequences are shown in Figure 1Go in one-letter code. Tyrosine was added to the sequence of some peptides which did not contain it to allow them to be radiolabeled.

Peptides in 1 mg/mL concentration were radiolabeled with Na125I using chloramine-T (Yamamura et al. 1978), according to previous reports (Urquiza et al. 1996; Ocampo et al. 2000; Rodríguez et al. 2000). Radiolabeled peptide was purified on a Sephadex G-10 column (Pharmacia).

Cell binding assay
C-32 cell binding
C32 cells maintained in culture were used. Cells (1.5 x 106) were placed in 96-well cell culture plates to final 100 µl volume incubated with increasing quantities of radiolabeled peptide (0–300 nM) in the presence or absence of excess unlabeled peptide (20 µM) for 2 h at 4°C with constant shaking. An aliquot of this reaction mixture was passed through a 60:40 dioctylphthalate-dibutylphthalate cushion (density 1.015 g/mL), spinning at 15,000g for 1.5 min. Cell-associated radioactivity was quantified in an Automatic Gamma Counter (4/200 plus, ICN).

Erythrocyte binding
Blood (obtained by venipuncture and collected in heparinized tubes) was washed with isotonic HBS; white blood cells were removed. It was washed again several times with HBS and centrifuged at 500g for 5 min (at room temperature). 2 x 108 cells were taken to a final 200 µL volume. Increasing amounts of radiolabeled peptide (0–300 nM) were added to each tube in the presence or absence of excess unlabeled peptide (20 µM), according to the methodology previously described for this purpose (Urquiza et al. 1996; Ocampo et al. 2000; Rodríguez et al. 2000). After incubation for 90 min at room temperature, cells were washed five times with HBS and bound cell radiolabeled peptide was quantified in an automatic {gamma} counter. These assays were carried out in triplicate for each point on the curve.

Binding assays were also done using two types of cell (C-32 and erythrocytes) and peptides derived from obtained HABPs (Fig. 1AGo) where the amino acid sequences had been jumbled. This was done to observe whether these HABPs’ binding was determined by amino acid sequences or just their amino acid composition (Fig. 1BGo).

Determining physical–chemical constants
Binding assays were done with erythrocytes and C32 cells using a wide range of radiolabeled peptides (0–1200 nM) using the binding assay mentioned in 2.2 to reach saturation and determine physical–chemical constants in the HABP-cell interaction (Weiland and Molinoff 1981; Hulme 1993; Urquiza et al. 1996; Ocampo et al. 2000, 2004; Rodríguez et al. 2000; Curtidor et al. 2001).

Determining HABP structure by circular dichroism
CD assays were performed according to a previously described method (Ocampo et al. 2004) using a Jasco J-810 spectro-polarimeter. Spectra were recorded in 190–260 nm wavelength intervals. The results were expressed as mean residue ellipticity [Q], the units being degrees x cm2 x dmol–1 according to the [Q] = Ql/(100lcn) function where Ql is the measured ellipticity, l is the optical path length, c is the peptide concentration, and n is the number of amino acid residues in the sequence (Sreerama et al. 1999).

Cross-linking assays
HABPs were cross-linked to cell membranes to identify possible receptor sites for CLAG 3.2 protein HABPs on erythrocyte and C-32 cell surfaces. Cells were incubated with each one of the radiolabeled HABPs. Following thorough washing with HBS, the bound peptide was cross-linked to 10 µM BS3 (Bis[sulfosuccinimidyl]suberate), as previously described (López et al. 2004; Ocampo et al. 2004). The proteins which had been cross-linked with radiolabeled HABPs were exposed on BioRad Imaging Screen K (BioRad Molecular Imager FX; BioRad Quantity One, Quantitation Software) and the apparent molecular weight was determined by using molecular weight markers (NEB).

Merozoite invasion inhibition assay
As previously reported (Urquiza et al. 1996; Ocampo et al. 2000, 2004; Rodríguez et al. 2000; Curtidor et al. 2001), late-schizont stage P. falciparum (FCB-2 strain) cultures were seeded in 96-well cell culture plates (Nunc) in the presence of test peptides at 200, 100, and 10 µM concentrations. Each peptide was tested 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 stained with 15 µg/mL hydroethydine, incubated at 37°C for 30 min and washed three times with PBS. The suspensions were analyzed on a FACsort in Log FL2 data mode using CellQuest software (Becton Dickinson immunocytometry system) (Wyatt et al. 1991). Infected and uninfected EGTA- and chloroquine-treated erythrocytes were used as controls.


   Acknowledgments
 
This research project was supported by the President of the Republic of Colombia’s office and the Colombian Ministry of Public Health. We would like to thank Jason Garry for carefully reading the manuscript.


   References
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Alba, M.P., Salazar, L.M., Purmova, J., Vanegas, M., Rodriguez, R., and Patarroyo, M.E. 2004a. Induction and displacement of an helix in the 6725 SERA peptide analogue confers protection against P. falciparum malaria. Vaccine 22:1281–1289.[CrossRef][Medline]

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