Synthetic peptides from two Pf sporozoite invasion-associated proteins specifically interact with HeLa and HepG2 cells

Two recently described molecules have been associated with sporozoite traversal ability and hepatocyte entry: sporozoite invasion-associated proteins (SIAP)-1 and -2. The HeLa and HepG2 cell binding ability of synthetic peptides spanning the whole SIAP-1 and -2 sequences has been studied in the search for identifying these proteins’ functionally active specific regions. Twelve HepG-2 and seventeen HeLa cell high-activity binding peptides (HABPs) have been identified in SIAP-1, 8 of them having high specific binding affinity for both cell lines. Four HepG2 HABPs and two HeLa HABPs have been identified in SIAP-2, one of them interacting with both HeLa and HepG2 cells. SIAP-1 and SIAP-2 HABPs bound specifically and saturably to heparin sulfate and chondroitin sulfate-type membrane receptors on host cells. Circular dichroism assays have shown high α-helix content in SIAP-1 and SIAP-2 HABP secondary structure. Immunofluorescence analysis has revealed that specific peptides against SIAP proteins are highly immunogenic in mice and that anti-SIAP-1 and -2 antibodies recognize the native protein in Plasmodium falciparum sporozoites. Polymorphism studies have shown that a most SIAP-1 and -2 HABPs are conserved among P. falciparum strains. Our results have suggested that SIAP-1 and -2 participate in host-pathogen interactions during cell-traversal and hepatocyte invasion and highlighted the relevance of the ongoing identification and study of potentially new molecules when designing a fully protective antimalarial vaccine.     

Identification of peptides with high red blood cell and hepatocyte binding activity in the Plasmodium falciparum multi-stage invasion proteins: PfSPATR and MCP-1

Plasmodium falciparum multi-stage proteins are involved in vital processes for parasite survival, which turns them into attractive targets for studies aimed at developing a fully effective antimalarial vaccine. MCP-1 and PfSPATR are both found in sporozoite and merozoite forms, and have been associated respectively with invasion of hepatocytes and red blood cells (RBCs). Binding assays with synthetic peptides derived from these two important proteins have enabled identifying those sequences binding with high specific activity (named High activity binding peptides-HABPs) to hepatoma-derived HepG2 cells and human RBCs. Twelve RBC HABPs were identified within the MCP-1 amino acid sequence, most of them in the C-terminal region. The MCP-1 HABPs 33387 and 33397 also presented high activity binding to HepG2 cells. PfSPATR presented four RBC HABPs and two HepG2 HABPs, but only one (32686) could bind to both cell types. RBC binding assays evidenced that binding of all HABPs was saturable and differentially affected by the enzymatic treatment of target cells. Moreover, all HABPs inhibited in vitro invasion of merozoites at 200 microM and had particular structural features when analyzed by circular dichroism. The results suggest that these synthetic peptides capable of binding to the two P. falciparum target cells could be potentially included in the design of a multi-stage, subunit-based, chemically synthesized antimalarial vaccine.     

Plasmodium falciparum: binding studies of peptide derived from the sporozoite surface protein 2 to Hep G2 cells.

Plasmodium falciparum sporozoite surface protein 2 (Pf SSP2), also called thrombospondin related anonymous protein (TRAP), is involved in the process of sporozoite invasion of hepatocytes. Pf SSP2/TRAP possesses two different adhesion domains sharing sequences and structural homology with von Willebrand factor A-domains and human repeat I thrombospondin (TSP). Pf SSP2/TRAP has also been implicated in sporozoite mobility and in mosquito salivary gland invasion processes. We tested 15-mer long synthetic peptides having five overlapping residues covering the complete protein Pf SSP2 sequence in binding assays to Hep G2 cells. In these 57 peptides, 21 high-activity binding peptides (HABPs) were identified; five were in the adhesion domains already described and 16 were in two regions toward the protein's carboxy and middle terminal part. Six HABPs showed conserved amino acid sequences: 3243 (21FLVNGRDVQNNIVDE35), 3279 (201FLVGCHPSDGKCNLY215), 3287 (241TASCGVWDEWSPCSV255), 3289 (251SPCSVTCGKGTRSRK265), 3327 (441ERKQSDPQSQDNNGNY455) and 3329 (451DNNGNRHVPNSEDREY465). The HABPs show saturable binding and dissociation constants between 140 and 900 nm with 40 000-855 000 binding sites per cell. The 3279 (201FLVGCHPSDGKCNLY215), 3323 (421NDKSDRYIPYSPLSP435) and 3331 (461SEDRETRPHGRNNENY475) HABPs have B epitopes in their sequences; these have previously been recognized by antibodies partially inhibiting hepatocyte invasion and development of the hepatic state. The 3287 (241TASCGVWDEWSPCSV255) and 3289 (251SPCSVTCGKGTRSRK265) HABPs share common sequences with the Pf SSP2/TRAP region II plus, which is present in a great number of adhesion proteins. Based on this information, six new peptides covering the high binding regions identified previously were synthesized and, using a competition assay, the amino acid involved in the binding were determined.     

3D structure and immunogenicity of Plasmodium falciparum sporozoite induced associated protein peptides as components of fully-protective anti-malarial vaccine

SIAP-1 and SIAP-2 are proteins which are implicated in early events involving Plasmodium falciparum infection of the Anopheles mosquito vector and the human host. High affinity HeLa and HepG2 cell binding conserved peptides have been previously identified in these proteins, i.e. SIAP-1 34893 ((421)KVQGLSYLLRRKNGTKHPVY(440)) and SIAP-1 34899 ((541)YVLNSKLLNSRSFDKFKWIQ(560)) and SIAP-2 36879 ((181)LLLYSTNSEDNLDISFGELQ(200)). When amino acid sequences have been properly modified (replacements shown in bold) they have induced high antibody titres against sporozoites in Aotus monkeys (assessed by IFA) and in the corresponding recombinant proteins (determined by ELISA and Western blot). (1)H NMR studies of these conserved native and modified high activity binding peptides (HABPs) revealed that all had α-helical structures in different locations and lengths. Conserved and corresponding modified HABPs displayed different lengths between the residues fitting into MHCII molecule pockets 1-9 and different amino acid orientation based on their different HLA-DRβ1(?) binding motifs and binding registers, suggesting that such modifications were associated with making them immunogenic. The results suggested that these modified HAPBs could be potential targets for inclusion as components of a fully-effective, minimal sub-unit based, multi-epitope, and multistage anti-malarial vaccine.     

Biological and structural characteristics of the binding peptides from the sporozoite proteins essential for cell traversal (SPECT)-1 and -2

The sporozoite microneme proteins essential for cell traversal, SPECT-1 and SPECT-2, are considered attractive pre-erythrocytic immune targets due to the key role they play in crossing of the malaria parasite across the dermis and the liver sinusoidal wall, prior to invasion of hepatocytes. In this study, the sequences of SPECT-1 and SPECT-2 were mapped using 20 mer-long synthetic peptides to identify high-activity binding peptides (HABPs) to HeLa cells. 17 HABPs with enzyme sensitive bindings to HeLa cells were identified: 3 predominantly α-helical in SPECT-1, and 10 α-helical and 4 β-turns/random coils in SPECT-2. Immunofluorescence assays (IFA) with antibodies raised in rabbits against chemically synthesized B-cell epitopes suggests the presence of these two proteins in the micronemes and in sporozoite membrane. ¹H NMR studies showed that HABPs located in the membrane-attack complex/perforin (MACPF) domain of SPECT-2 share high similarity with the 3D structure of C8α. Altogether, the results highlight the potential of including HABPs from SPECT-1 and SPECT-2 as components of a fully effective multistage, multiepitopic, minimal subunit-based synthetic vaccine against Plasmodium falciparum malaria.     

Structural characterisation of sporozoite components for a multistage, multi-epitope, anti-malarial vaccine

A totally effective anti-malarial vaccine must contain epitopes derived from multiple proteins found in different stages of the particular parasite involved in invasion. It must therefore include sporozoite molecules able to induce protective immunity thereby blocking the parasite's access to hepatic cells; thrombospondin-related anonymous protein (TRAP) is one of them. Conserved high activity binding peptides (HABPs) attaching themselves to hepatic cells were used in immunisation studies with the highly malaria-susceptible Aotus monkey. However, they had to be modified to render them immunogenic. The changes induced in lead peptide 3D structure were analysed by correlating such substitutions with the induction of high anti-sporozoite antibody levels in the experimental monkey model. The modification induced structural changes in most modified HABPs, changing them from random-coil or distorted type III beta-turn structures to classical type III or III' beta-turn, thereby allowing a better fit into the MHC-II-peptide-TCR complex since they bound with high affinity to purified HLA-DRbeta1* molecules. These are the first (TRAP) conserved HABPs corresponding to functionally active amino acid sequences in sporozoite invasion and mobility which, when modified, were able to induce very high anti-sporozoite antibody responses, leading to suggesting them as components in the first line of defence of a fully-effective, subunit-based, multi-epitope, multi-stage, synthetic anti-malarial vaccine.     

Synthetic peptides from Plasmodium falciparum apical membrane antigen 1 (AMA-1) specifically interacting with human hepatocytes

Plasmodium falciparum apical membrane antigen 1 (AMA-1) is expressed during both the sporozoite and merozoite stage of the parasite's life cycle. The role placed by AMA-1 during sporozoite invasion of hepatocytes has not been made sufficiently clear to date. Identifying the sequences involved in binding to hepatocytes is an important step towards understanding the structural basis for sporozoite-hepatocyte interaction. Binding assays between P. falciparum AMA-1 peptides and HepG2 cell were performed in this study to identify possible AMA-1 functional regions. Four AMA-1 high activity binding peptides (HABPs) bound specifically to hepatocytes: 4310 ((74)QHAYPIDHEGAEPAPQEQNL(93)), 4316 ((194)TLDEMRHFYKDNKYVKNLDE(213)), 4321 ((294)VVDNWEKVCPRKNLQNAKFGY(313)) and 4332 ((514)AEVTSNNEVVVKEEYKDEYA(533)). Their binding to these cells became saturable and resistant to treatment with neuraminidase. Most of these peptides were located in AMA-1 domains I and III, these being target regions for protective antibody responses. These peptides interacted with 36 and 58 kDa proteins on the erythrocyte surface. Some of the peptides were found in exposed regions of the AMA-1 protein, thereby facilitating their interaction with host cells. It is thus probable that AMA-1 regions defined by the four peptides mentioned above are involved in sporozoite-hepatocyte interaction.     

CelTOS, a novel malarial protein that mediates transmission to mosquito and vertebrate hosts

The malarial parasite has two hosts in its life cycle, a vertebrate and a mosquito. We report here that malarial invasion into these hosts is mediated by a protein, designated cell-traversal protein for ookinetes and sporozoites (CelTOS), which is localized to micronemes that are organelles for parasite invasive motility. Targeted disruption of the CelTOS gene in Plasmodium berghei reduced parasite infectivity in the mosquito host approximately 200-fold. The disruption also reduced the sporozoite infectivity in the liver and almost abolished its cell-passage ability. Liver infectivity was restored in Kupffer cell-depleted rats, indicating that CelTOS is necessary for sporozoite passage from the circulatory system to hepatocytes through the liver sinusoidal cell layer. Electron microscopic analysis revealed that celtos-disrupted ookinetes invade the midgut epithelial cell by rupturing the cell membrane, but then fail to cross the cell, indicating that CelTOS is necessary for migration through the cytoplasm. These results suggest that conserved cell-passage mechanisms are used by both sporozoites and ookinetes to breach host cellular barriers. Elucidation of these mechanisms might lead to novel antimalarial strategies to block parasite's transmission.     

Plasmodium yoelii S4/CelTOS is important for sporozoite gliding motility and cell traversal

Gliding motility and cell traversal by the Plasmodium ookinete and sporozoite invasive stages allow penetration of cellular barriers to establish infection of the mosquito vector and mammalian host, respectively. Motility and traversal are not observed in red cell infectious merozoites, and we have previously classified genes that are expressed in sporozoites but not merozoites (S genes) in order to identify proteins involved in these processes. The S4 gene has been described as criticaly involved in Cell Traversal for Ookinetes and Sporozoites (CelTOS), yet knockout parasites (s4/celtos¯) do not generate robust salivary gland sporozoite numbers, precluding a thorough analysis of S4/CelTOS function during host infection. We show here that a failure of oocysts to develop or survive in the midgut contributes to the poor mosquito infection by Plasmodium yoelii (Py) s4/celtos¯ rodent malaria parasites. We rescued this phenotype by expressing S4/CelTOS under the ookinete‐specific circumsporozoite protein and thrombospondin‐related anonymous protein‐related protein (CTRP) promoter (S4/CelTOS^CTRP), generating robust numbers of salivary gland sporozoites lacking S4/CelTOS that were suitable for phenotypic analysis. Py S4/CelTOS^CTRP sporozoites showed reduced infectivity in BALB/c mice when compared to wild‐type sporozoites, although they appeared more infectious than sporozoites deficient in the related traversal protein PLP1/SPECT2 (Py plp1/spect2¯). Using in vitro assays, we substantiate the role of S4/CelTOS in sporozoite cell traversal, but also uncover a previously unappreciated role for this protein for sporozoite gliding motility.     

Conserved regions from Plasmodium falciparum MSP11 specifically interact with host cells and have a potential role during merozoite invasion of red blood cells

Despite significant global efforts, a completely effective vaccine against Plasmodium falciparum, the species responsible for the most serious form of malaria, has not been yet obtained. One of the most promising approaches consists in combining chemically synthesized minimal subunits of parasite proteins involved in host cell invasion, which has led to the identification of peptides with high binding activity (named HABPs) to hepatocyte and red blood cell (RBC) surface receptors in a large number of sporozoite and merozoite proteins, respectively. Among these proteins is the merozoite surface protein 11 (MSP11), which shares important structural and immunological features with the antimalarial vaccine candidates MSP1, MSP3, and MSP6. In this study, 20‐mer‐long synthetic peptides spanning the complete sequence of MSP11 were assessed for their ability to bind specifically to RBCs. Two HABPs with high ability to inhibit invasion of RBCs in vitro were identified (namely HABPs 33595 and 33606). HABP‐RBC bindings were characterized by means of saturation assays and Hill analysis, finding cooperative interactions of high affinity for both HABPs (n_H of 1.5 and 1.2, K_d of 800 and 600 nM for HABPs 33595 and 33606, respectively). The nature of the possible RBC receptors for MSP11 HABPs was studied in binding assays to enzyme‐treated RBCs and cross‐linking assays, finding that both HABPs use mainly a sialic acid‐dependent receptor. An analysis of the immunological, structural and polymorphic characteristics of MSP11 HABPs supports including these peptides in further studies with the aim of designing a fully effective protection‐inducing vaccine against malaria. J. Cell. Biochem. 110: 882–892, 2010. © 2010 Wiley‐Liss, Inc.     

High affinity interactions between red blood cell receptors and synthetic Plasmodium thrombospondin-related apical merozoite protein (PTRAMP) peptides

Plasmodium falciparum thrombospondin-related apical merozoite protein (PTRAMP) has a thrombospondin related (TSR) domain which in many proteins has been reported as a fragment involved in pathogen-host and cell-interactions. Receptor-ligand studies using eighteen non-overlapping 20-aminoacid-long synthetic peptides from this protein were carried out to determine regions involved in parasite invasion of red blood cells (RBC). Two high activity binding peptides (HABPs) were determined, 33405 (21YISSNDLTSTNLKVRNNWEH40) and 33413 (180LEGPIQFSLGKSSGAFRINY199), presenting high dissociation constants and positive cooperativity. One of the HABPs displayed a modified Plasmodium export element (PEXEL), suggesting that this protein could be involved in the merozoite cytoplasmic reticulum, parasitophorous vacuole, red blood cell (RBC) cytosol, and probably infected RBC (iRBC) membrane transport of some other molecules and nutrients. Enzymatic treatment of RBCs increased HABP 33405 binding to them whilst it decreased HABP 33413 binding. Merozoite invasion assays revealed that HABPs have around 57% ability to inhibit new RBC invasion. Circular dichroism revealed the presence of possible alpha-helical elements in both HABPs structures. RBC binding interaction specificity and the presence of a PEXEL motif make these 2 HABPs good candidates for being included in further studies to develop a new multi-antigenic, multi-stage, subunit-based, chemically-synthesised, anti-malarial vaccine.     

Implications of conformational flexibility,lipid binding,and regulatory domains in cell-traversal protein CelTOS for apicomplexan migration

Malaria and other apicomplexan-caused diseases affect millions of humans, agricultural animals, and pets. Cell traversal is a common feature used by multiple apicomplexan parasites to migrate through host cells and can be exploited to develop therapeutics against these deadly parasites. Here, we provide insights into the mechanism of the Cell-traversal protein for ookinetes and sporozoites (CelTOS), a conserved cell-traversal protein in apicomplexan parasites and malaria vaccine candidate. CelTOS has previously been shown to form pores in cell membranes to enable traversal of parasites through cells. We establish roles for the distinct protein regions of Plasmodium vivax CelTOS and examine the mechanism of pore formation. We further demonstrate that CelTOS dimer dissociation is required for pore formation, as disulfide bridging between monomers inhibits pore formation, and this inhibition is rescued by disulfide-bridge reduction. We also show that a helix-destabilizing amino acid, Pro127, allows CelTOS to undergo significant conformational changes to assemble into pores. The flexible C terminus of CelTOS is a negative regulator that limits pore formation. Finally, we highlight that lipid binding is a prerequisite for pore assembly as mutation of a phospholipids-binding site in CelTOS resulted in loss of lipid binding and abrogated pore formation. These findings identify critical regions in CelTOS and will aid in understanding the egress mechanism of malaria and other apicomplexan parasites as well as have implications for studying the function of other essential pore-forming proteins.     

Thrombospondin related anonymous protein (TRAP) of Plasmodium falciparum binds specifically to sulfated glycoconjugates and to HepG2 hepatoma cells suggesting a role for this molecule in sporozoite invasion of hepatocytes.

Thrombospondin related anonymous protein (TRAP) of Plasmodium falciparum contains an amino acid motif based around the sequence WSPCSVTCG which is also found in region II of the circumsporozoite (CS) proteins of different species of Plasmodium. This amino acid motif confers on the CS protein the ability to bind specifically to sulfated glycoconjugates and to hepatocytes. This suggests that the interaction of CS protein with sulfated glycoconjugates on the surface of the hepatocytes may represent the first molecular event of sporozoite invasion of liver cells. Experimental evidence indicates that TRAP is localized both on the micronemes and on the surface of P. falciparum sporozoites implying that TRAP with its putative sulfated glycoconjugate binding motif may also be involved in recognition and/or entry of hepatocytes by the sporozoite. We show here that different TRAP constructs expressed in Escherichia coli bind to sulfogalactosyl-cerebrosides (sulfatides) and to the surface of HepG2 cells. These interactions are dependent on the presence of the conserved amino acid motif WSPCSVTCG within the sequences of the constructs and are completely inhibited by several sulfated glycoconjugates as well as by suramin, a polysulfonated drug with anti-protozoan activity. Moreover, sporozoite invasion of HepG2 cells is inhibited by antisera raised against these different TRAP constructs and by the presence of low concentrations of suramin. We concluded that TRAP may be one of the parasite encoded molecules in the host-parasite interaction that results in sporozoite invasion of hepatocytes.     

Cellular trajectories of peptide-modified gold particle complexes: comparison of nuclear localization signals and peptide transduction domains

Gold nanoparticles modified with nuclear localization peptides were synthesized and evaluated for their subcellular distribution in HeLa human cervical epithelium cells, 3T3/NIH murine fibroblastoma cells, and HepG2 human hepatocarcinoma cells. Video-enhanced color differential interference contrast microscopy and transmission electron microscopy indicated that transport of nanoparticles into the cytoplasm and nucleus depends on peptide sequence and cell line. Recently, the ability of certain peptides, called protein transduction domains (PTDs), to transclocate cell and nuclear membranes in a receptor- and temperature-independent manner has been questioned (see for example, Lundberg, M.; Wikstrom, S.; Johansson, M. (2003) Mol. Ther. 8, 143-150). We have evaluated the cellular trajectory of gold nanoparticles carrying the PTD from HIV Tat protein. Our observations were that (1) the conjugates did not enter the nucleus of 3T3/NIH or HepG2 cells, and (2) cellular uptake of Tat PTD peptide-gold nanoparticle conjugates was temperature dependent, suggesting an endosomal pathway of uptake. Gold nanoparticles modified with the adenovirus nuclear localization signal and the integrin binding domain also entered cells via an energy-dependent mechanism, but in contrast to the Tat PTD, these signals triggered nuclear uptake of nanoparticles in HeLa and HepG2 cell lines.     

Identification of Plasmodium falciparum RhopH3 protein peptides that specifically bind to erythrocytes and inhibit merozoite invasion

The identification of sequences involved in binding to erythrocytes is an important step for understanding the molecular basis of merozoite-erythrocyte interactions that take place during invasion of the Plasmodium falciparum malaria parasite into host cells. Several molecules located in the apical organelles (micronemes, rhoptry, dense granules) of the invasive-stage parasite are essential for erythrocyte recognition, invasion, and establishment of the nascent parasitophorous vacuole. Particularly, it has been demonstrated that rhoptry proteins play an important role in binding to erythrocyte surface receptors, among which is the PfRhopH3 protein, which triggers important immune responses in patients from endemic regions. It has also been reported that anti-RhopH3 antibodies inhibit in vitro invasion of erythrocytes, further supporting its direct involvement in erythrocyte invasion processes. In this study, PfRhopH3 consecutive peptides were synthesized and tested in erythrocyte binding assays for identifying those regions mediating binding to erythrocytes. Fourteen PfRhopH3 peptides presenting high specific binding activity were found, whose bindings were saturable and presented nanomolar dissociation constants. These high-activity binding peptides (HABPs) were characterized by having alpha-helical structural elements, as determined by circular dichroism, and having receptors of a possible sialic acid-dependent and/or glycoprotein-dependent nature, as evidenced in enzyme-treated erythrocyte binding assays and further corroborated by cross-linking assay results. Furthermore, these HABPs inhibited merozoite in vitro invasion of normal erythrocytes at 200 microM by up to 60% and 90%, suggesting that some RhopH3 protein regions are involved in the P. falciparum erythrocyte invasion.     

Protective cellular immunity against P. falciparum malaria merozoites is associated with a different P7 and P8 residue orientation in the MHC-peptide-TCR complex

Developing a logical and rational methodology for obtaining vaccines, especially against the main parasite causing human malaria (P. falciparum), consists of blocking receptor-ligand interactions. Conserved peptides derived from proteins involved in invasion and having high red blood cell binding ability have thus been identified. Immunization studies using Aotus monkeys have revealed that these peptides were neither immunogenic nor protection inducing. When modified in their critical binding residues, previously identified by Glycine scanning, some of these peptides were immunogenic and non-protection inducers; others induced short-lived antibodies whilst a few were both immunogenic and protection inducing. However, very few of these modified high activity binding peptides (HABPs) reproducibly induced protection without inducing antibody production, but with high cytokine liberation, suggesting that cellular mechanisms had been activated in the protection process. The three-dimensional structure of these peptides inducing protection without producing antibodies was determined by 1H-NMR. Their HLA-DRbeta1* molecule binding ability was also determined to ascertain association between their 3D structure and ability to bind to Major Histocompatibility Complex Class-II molecules (MHC-II). 1H Nuclear Magnetic Resonance analysis and structure calculations clearly showed that these modified HABPs inducing protective cellular immune responses (but not producing antibodies against malaria) adopted special structural configuration to fit into the MHC II-peptide-TCR complex. A different orientation for P7 and P8 TCR contacting residues was clearly recognized when comparing their structure with modified peptides, which induced high antibody titers and protection, suggesting that these residues are involved in activating the immune system associated with antibody production and protection.     

Identifying Plasmodium falciparum merozoite surface protein-10 human erythrocyte specific binding regions

Receptor-ligand interactions between synthetic peptides and normal human erythrocytes were studied to determine P. falciparum merozoite surface protein-10 (MSP-10) regions specifically binding to membrane surface receptors on human erythrocytes. Three MSP-10 protein High Activity Binding Peptides (HABPs) were identified, whose binding to erythrocytes became saturable and sensitive on being treated with neuraminidase, trypsin and chymotrypsin. Some of them specifically recognised a 50 kDa erythrocyte membrane protein. Some HABPs inhibited in vitro P. falciparum merozoite invasion of erythrocytes by 70%, suggesting that MSP-10 protein's possible role in the invasion process probably functions by using similar mechanisms to those described for other MSP family antigens. In addition to above results, the high homology in amino-acid sequence and superimposition of both MSP-10, MSP-8 and MSP-1 EGF-like domains and HABPs 31132, 26373 and 5501 suggest that tridimensional structure could be playing an important role in the invasion process and in designing synthetic multi-stage anti-malarial vaccines.     

Identification of Plasmodium falciparum reticulocyte binding protein RBP-2 homologue a and b (PfRBP-2-Ha and -Hb) sequences that specifically bind to erythrocytes

Plasmodium falciparum reticulocyte binding protein RBP-2 homologues a and b (PfRBP-2-Ha and -Hb) have been described as being high molecular weight proteins, expressed at the P. falciparum merozoite apical extreme, belonging to a family of proteins found in other Plasmodium involved in the search for erythrocyte populations before being invaded by merozoites. 185, 20-mer-long non-overlapping peptides, spanning the entire PfRBP-2-Ha and -Hb sequences, were synthesised, radiolabelled and tested in erythrocyte binding assays. Fifteen PfRBP-2-Ha and -Hb high binding activity peptides (HBAPs) specifically binding to erythrocytes with high affinity were identified. Dissociation constants were between 70 and 300 nM and Hill coefficients were 1 approximately. HBAPs residues critical for binding to erythrocytes were determined. Cross-linking was performed allowing possible receptors for PfRBP-2-Ha and -Hb to be identified on the surface of the erythrocytes. Some of the HABPs showed merozoite invasion inhibition greater than 90% in in vitro assays.