Identifying Plasmodium falciparum cytoadherence-linked asexual protein 3 (CLAG 3) sequences that specifically bind to C32 cells and erythrocytes

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) 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.     

Specific erythrocyte binding capacity and biological activity of Plasmodium falciparum erythrocyte binding ligand 1 (EBL-1)-derived peptides

Erythrocyte binding ligand 1 (EBL-1) is a member of the ebl multigene family involved in Plasmodium falciparum invasion of erythrocytes. We found that five EBL-1 high-activity binding peptides (HABPs) bound specifically to erythrocytes: 29895 ((41)HKKKSGELNNNKSGILRSTY(60)), 29903 ((201)LYECGK-KIKEMKWICTDNQF(220)), 29923 ((601)CNAILGSYADIGDIVRGLDV(620)), 29924((621)WRDINTNKLSEK-FQKIFMGGY(640)), and 30018 ((2481)LEDIINLSKKKKKSINDTSFY(2500)). We also show that binding was saturable, not sialic acid-dependent, and that all peptides specifically bound to a 36-kDa protein on the erythrocyte membrane. The five HABPs inhibited in vitro merozoite invasion depending on the peptide concentration used, suggesting their possible role in the invasion process.     

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

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 ((61)LKEKKKKVLEFFENLVLNKKY(80)) located at the N-terminal and 30901 ((401)RKSLEQQFGDNMDKMNKLKKY(420)), 30902 ((421)KKILKFFPLFNYKSDLESIM(440)) and 30913 ((641)DLESTAEQKAEKKGGKAKAKY(660)) 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.     

Plasmodium falciparum: red blood cell binding studies using peptides derived from rhoptry-associated protein 2 (RAP2)

Plasmodium falciparum rhoptry-associated proteins 1 (RAP1) and 2 (RAP2) are antigens presenting themselves as candidates for a subunit malaria vaccine. RAP2 protein, non-overlapping, consecutive peptides were synthesised and tested in red blood cell (RBC) binding assays to identify their receptor-ligand interaction in recognising RAP2 regions involved in the in vitro merozoite invasion process. Four high activity binding peptides (HABPs) were identified in the resulting 20 peptides. Peptides 26220 ((61)NHFSSADELIKYLEKTNINT(80)), 26225 ((161)IKKNPFLRVLNKASTTTHAT(180)) and 26229 ((241)RSVNNVISKNKTLGLRKRSS(260)) were located in the amino terminal and central part of the protein and HABP 26235 ((361)FLAEDFVELFDVTMDCYSRQ(380)) was located at the carboxy terminal. All these HABPs showed saturable binding and presented dissociation constants between 500 and 950 nM; the number of binding sites per RBC ranged from 48,000 to 160,000. High binding peptides' critical amino acids involved in RBC binding were determined by competition binding assays; their amino acids appear in bold in the sequences shown above. SDS-PAGE results showed that peptides 26220, 26225 and 26229 had at least two different sets of 62 and 42 kDa HABP receptors on RBCs and that peptide 26235 had at least two different sets of 77 and 62 kDa. HABPs inhibited in vitro merozoite invasion by between 54% and 94% at 200 microM, suggesting that these RAP2 peptides are involved in the in vitro P. falciparum invasion process.     

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.     

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.     

Peptides inducing short-lived antibody responses against Plasmodium falciparum malaria have shorter structures and are read in a different MHC II functional register

The search for a rational method of developing an antimalarial vaccine (malaria caused by Plasmodium falciparum) consists of blocking receptor-ligand interaction. Conserved peptides derived from proteins involved in invasion and having strong red blood cell binding ability have thus been identified; immunization studies using Aotus monkeys revealed that these peptides were neither immunogenic nor protection-inducing. Some of these peptides induced long-lasting and very high antibody titers and protection when their critical red blood cell binding residues were replaced to change their immunological properties. Others induced short-lived antibodies that were not associated with inducing protection. The three-dimensional structure of the short-lived antibody-inducing peptide was determined by (1)H NMR. Their HLA-DRbeta1* molecule binding ability was also determined to ascertain the relationship among three-dimensional structure, their ability to bind to major histocompatibility complex class II molecules (MHC II), and possible short-lived antibody production. These short-lived antibody-inducing peptides were 6.8 +/- 0.5 A shorter between those residues theoretically coming into contact with pocket 1 and pocket 9 of HLA-DRbeta1* molecules to which they bind than immunogenic and protection-inducing peptides. These more compact alpha-helical structures suggest that these short-lived antibody-inducing peptides could have a structure more similar to those of native peptides than immunogenic and protective ones. Such shortening was associated with a shift in HLA-DRbeta1* molecule binding and a consequent shift in functional register reading, mainly by alleles of the same haplotype when compared with immunogenic protection-inducing HABPs, suggesting an imperfect and different conformation of the MHC II peptide-TCR complex.     

Plasmodium falciparum merozoite surface protein 6 (MSP-6) derived peptides bind erythrocytes and partially inhibit parasite invasion

This work shows that Plasmodium falciparum merozoite surface protein-6 (MSP-6) peptides specifically bind to membrane surface receptor on human erythrocytes. Three high activity binding peptides (HABPs) were found: peptides 31175 (41MYNNDKILSKNEVDTNIESN60) and 31178 (101YDIQATYQFPSTSGGNNVIP120) in the amino terminal region and 31191 (361EIDSTINNLVQEMIHLFSNNY380) at the carboxy terminal. Their binding to erythrocytes was saturable. HABPs 31191 and 31178 recognized 56 and 26 kDa receptors on erythrocyte membrane and inhibited in vitro Plasmodium falciparum merozoite invasion of erythrocytes by between 27% and 46% at 200 microg ml(-1) concentration, suggesting that these MSP-6 protein peptides play a possible role in the invasion process.     

Peptides of the liver stage antigen-1 (LSA-1) of Plasmodium falciparum bind to human hepatocytes

Synthetic peptides from the liver stage antigen-1 (LSA-1) antigen sequence were used in HepG2 cell and erythrocyte binding assays to identify regions that could be involved in parasite invasion. LSA-1 protein peptides 20630 ((21)INGKIIKNSEKDEIIKSNLRY(40)), 20637 ((157)KEKLQGQQSDSEQERRAY(173)), 20638 ((174)KEKLQEQQSDLEQERLAY(190)) and 20639 (191KEKLQEQQSDLEQERRAY(207)) had high binding activity in HepG2 assays. Were located in immunogenic regions; peptide cell binding was saturable. Peptide 20630 bound specifically to 48kDa HepG2 membrane surface protein. LSA-1 peptides 20630 ((21)INGKIIKNSEKDEIIKSNLRY(40)) and 20633 ((81)DKELTMSNVKNVSQTNFKSLY(100)) showed specific erythrocyte binding activity and inhibited merozoite invasion of erythrocytes in vitro. A monkey serum prepared against LSA-1 20630 peptide analog (CGINGKNIKNAEKPMIIKSNLRGC) inhibited merozoite invasion in vitro. The data suggest LSA-1 "High Activity Binding Peptides" could play a possible role in hepatic cell invasion as well as merozoite invasion of erythrocytes.     

P. falciparum: merozoite surface protein-8 peptides bind specifically to human erythrocytes

This work determined Plasmodium falciparum merozoite surface protein-8 (MSP-8) regions specifically binding to membrane surface receptors on human erythrocytes. Five high activity binding peptides (HABPs), whose binding to erythrocytes became saturable and sensitive on being treated with neuraminidase and chymotrypsin were identified from the MSP-8 protein. Those amino acids directly involved in interaction with erythrocytes were also determined for each one of the HABPs. Some of them specifically recognized 28, 46, and 73 kDa erythrocyte membrane proteins. Some HABPs inhibited in vitro P. falciparum merozoite invasion of erythrocytes by up to 98%, suggesting the MSP-8 protein's possible role in the invasion process.     

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.     

Identifying Plasmodium falciparum EBA-175 homologue sequences that specifically bind to human erythrocytes

Erythrocyte binding antigen-160 (EBA-160) protein is a Plasmodium falciparum antigen homologue from the erythrocyte binding protein family (EBP). It has been shown that the EBP family plays a role in parasite binding to the erythrocyte surface. The EBA-160 sequence has been chemically synthesised in seventy 20-mer sequential peptides covering the entire 3D7 protein strain, each of which was tested in erythrocyte binding assays to identify possible EBA-160 functional regions. Five EBA-160 high activity binding peptides (HABPs) specifically binding to erythrocytes with high affinity were identified. Dissociation constants lay between 200 and 460 nM and Hill coefficients between 1.5 and 2.3. Erythrocyte membrane protein binding peptide cross-linking assays using SDS-PAGE showed that these peptides bound specifically to 12, 28, and 44 kDa erythrocyte membrane proteins. The nature of these receptor sites was studied in peptide binding assays using enzyme-treated erythrocytes. HABPs were able to block merozoite in vitro invasion of erythrocytes. HABPs’ potential as anti-malarial vaccine candidates is also discussed.     

P. falciparum pro-histoaspartic protease (proHAP) protein peptides bind specifically to erythrocytes and inhibit the invasion process in vitro

Plasmodium falciparum histoaspartic protease (HAP) is an active enzyme involved in haemoglobin degradation. HAP is expressed as an inactive 51-kDa zymogen and is cleaved into an active 37-kDa enzyme. It has been proposed that this kind of protease might be implicated in the parasite's invasion of erythrocytes; however, this protein's role during invasion has still to be determined. Synthetic peptides derived from the HAP precursor (proHAP) were tested in erythrocyte binding assays to identify their possible function in the invasion process. Two proHAP high-activity binding peptides (HABPs) specifically bound to erythrocytes; these peptides were numbered 30609 (101LKNYIKESVKLFNKGLTKKS120) and 30610 (121YLGSEFDNVELKDLANVLSF140 ). The binding of these two peptides was saturable, presenting nanomolar affinity constants. These peptides interacted with 26- and 45-kDa proteins on the erythrocyte surface; the nature of these receptor sites was studied in peptide binding assays using enzyme-treated erythrocytes. The HABPs showed greater than 90% merozoite invasion inhibition in in vitro assays. Goat serum containing proHAP polymeric peptide antibodies inhibited parasite invasion in vitro .     

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.     

MAEBL Plasmodium falciparum protein peptides bind specifically to erythrocytes and inhibit in vitro merozoite invasion

MAEBL is an erythrocyte binding protein located in the rhoptries and on the surface of mature merozoites, being expressed at the beginning of schizogony. The structure of MAEBL originally isolated from rodent malaria parasites suggested a molecule likely to be involved in invasion. We thus became interested in identifying possible MAEBL functional regions. Synthetic peptides spanning the MAEBL sequence were tested in erythrocyte binding assays to identify such possible MAEBL functional regions. Nine high activity binding peptides (HABPs) were identified: two were found in the M1 domain, one was found between the M1 and M2 regions, five in the erythrocyte binding domain (M2), and one in the protein's repeat region. The results showed that peptide binding was saturable; some HABPs inhibited in vitro merozoite invasion and specifically bound to a 33kDa protein on red blood cell membrane. HABPs' possible function in merozoite invasion of erythrocytes is also discussed.     

Identifying Plasmodium falciparum merozoite surface antigen 3 (MSP3) protein peptides that bind specifically to erythrocytes and inhibit merozoite invasion

Receptor-ligand interactions between synthetic peptides and normal human erythrocytes were studied to determine Plasmodium falciparum merozoite surface protein-3 (MSP-3) FC27 strain regions that specifically bind to membrane surface receptors on human erythrocytes. Three MSP-3 protein high activity binding peptides (HABPs) were identified; their binding to erythrocytes became saturable, had nanomolar affinity constants, and became sensitive on being treated with neuraminidase and trypsin but were resistant to chymotrypsin treatment. All of them specifically recognized 45-, 55-, and 72-kDa erythrocyte membrane proteins. They all presented alpha-helix structural elements. All HABPs inhibited in vitro P. falciparum merozoite invasion of erythrocytes by ~55%-85%, suggesting that MSP-3 protein's role in the invasion process probably functions by using mechanisms similar to those described for other MSP family antigens.     

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.     

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.     

Peptides from the Plasmodium falciparum STEVOR putative protein bind with high affinity to normal human red blood cells

Synthetic 20-mer long non-overlapped peptides, from STEVOR protein, were tested in RBC binding assays for identifying STEVOR protein regions having high RBC binding activity and evaluating whether these regions inhibit Plasmodium falciparum in vitro invasion. Affinity constants, binding site number per cell and Hill coefficients were determined by saturation assay with high activity binding peptides (HABPs). HABP binding assays using RBCs previously treated with enzymes were carried out to study the nature of the receptor. The molecular weight of RBC surface proteins interacting with HABPs was determined by cross-linking assays and SDS-PAGE analysis. RBC binding assays revealed that peptides 30561 (41MKSRRLAEIQLPKCPHYNND60), 30562 (61PELKKIIDKLNEERIKKYIE80) and 30567 (161ASCCKVHDNYLDNLKKGCFG180) bound saturably and with high binding activity, presenting nanomolar affinity constants. HABP binding activity to RBCs previously treated with neuraminidase and trypsin decreased, suggesting that these peptides bound to RBC surface proteins and that such binding could be sialic acid dependent. Cross-linking and SDS-PAGE assays showed that the three HABPs specifically bound to 30 and 40 kDa molecular weight RBC membrane proteins. Peptides 30561, 30562 and 30567 inhibited P. falciparum in vitro invasion of red blood cells in a concentration-dependent way. Goat sera having STEVOR protein polymeric peptides antibodies inhibit parasite in vitro invasion depending on concentration. Three peptides localized in STEVOR N-terminal and central regions had high, saturable, binding activity to 30 and 40 kDa RBC membrane proteins. These peptides inhibited the parasite's in vitro invasion, suggesting that STEVOR protein regions are involved in P. falciparum invasion processes during intra-erythrocyte stage.