Identifying merozoite surface protein 4 and merozoite surface protein 7 Plasmodium falciparum protein family members specifically binding to human erythrocytes suggests a new malarial parasite-redundant survival mechanism.

Plasmodium falciparum merozoite surface proteins (MSP-1 to -11) have been involved in merozoite interaction with the red blood cell (RBC) surface. Peptides covering complete MSP-4 and MSP-7 amino acid sequences were synthesized and tested in RBC binding assays. One MSP-4 high activity binding peptide (HABP) and five MSP-7 HABPs were found having specific binding to RBC surface. MSP-4 and MSP-7 HABP binding was sensitive to enzymatic treatment; they recognized a 52 kDa erythrocyte membrane protein. MSP-4 HABP had low invasion inhibition, suggesting it might bind to RBCs and also be involved in physiological mechanisms, while MSP-7 HABPs displayed different invasion inhibition activity (83-24%) in in vitro tests, suggesting different roles for both proteins during invasion. Structural characteristics found when comparing the MSP-4 HABP with MSP-HABPs displaying epidermal growth factor-like sequences suggested that these redundant MSP-family proteins could be a new parasite strategy for evading host genetic variability and immune pressure.

Monosaccharides modulate HCV E2 protein-derived peptide biological properties.

A hepatitis C virus E(2) protein-derived sequence was selected for studying the effect of N-glycosylation on the peptide chain's conformational structure. The results suggested that the (534)TDVF(537) motif contained in peptide 33402 ((529)WGENDTDVFVLNNTRY(544)) had a type III beta-turn, relevant in antigen recognition of polyclonal antibodies, binding to human cells, and binding to HLA DRB1 *0401 molecules. N-Glycopeptides derived from this sequence contained monosaccharides in Asn(532). N-Glycopeptides presented differences in peptide chain structure compared to non-glycosylated peptides. Peptide 33402 specifically bound to human cells, specificity becoming lost when it was N-glycosylated. N-Glycosylation decreased antigen recognition of mouse polyclonal sera against this sequence. N-Glycopeptide binding to HLA DRB1 *0401 molecules was similar to that presented by non-glycosylated peptide, indicating that N-glycosylation did not affect binding to HLA DRB1 *0401 molecules. N-Glycosylation induced changes at structural and functional level which could be relevant for modulating human cell binding properties and antibody recognition.

Characterisation of Plasmodium falciparum RESA-like protein peptides that bind specifically to erythrocytes and inhibit invasion.

The Plasmodium falciparum ring-erythrocyte surface antigen (RESA)-like putative protein was identified and characterised. PCR and RT-PCR assays revealed that the gene encoding this protein was both present and being transcribed in P. falciparum strain FCB-2 16 h after erythrocyte invasion. Indirect immunofluorescence studies detected this protein in infected erythrocyte (IE) cytosol in dense fluorescent granules similar to Maurer's clefts at 16-20 h (parasites in ring and trophozoite stages) and very strongly on IE membranes at 22 h, suggesting that it is synthesised during early ring stages (16 h) and transported to the infected red blood cell (RBC) membrane surface during the trophozoite stage (22 h). Western blotting showed that antisera produced against polymerised synthetic peptides of this protein recognised a 72-kDa band in P. falciparum schizont lysate. P. falciparum RESA-like peptides used in normal RBC binding assays revealed that peptides 30326 ((101)NAEKI LGFDD KNILE ALDLFY(120)), 30334 ((281)RVTWK KLRTK MIKAL KKSLTY(300)) and 30342 ((431)SSPQR LKFTA GGGFC GKLRNY(450)) bind with high activity and saturability, presenting nM affinity constants. These peptides contain alpha-helical structural elements, as determined by circular dichroism, and inhibit P. falciparum in vitro invasion of normal RBCs by up to 91%, suggesting that some RESA-like protein regions are involved in intra-erythrocyte stage P. falciparum invasion.

Developmental biology of sporozoite-host interactions in Plasmodium falciparum malaria: implications for vaccine design.

The Plasmodium falciparum sporozoite infects different types of cells in a mosquito's salivary glands and human epithelial and Kuppfer cells and hepatocytes. These become differentiated later on, transforming themselves into the invasive red blood cell form, the merozoite. The ability of sporozoites to interact with different types of cells requires a wide variety of mechanisms allowing them to survive in both hosts: mobility, receptor-ligand interactions with different cellular receptors, and transformation and development into other invasive parasite forms, which are vitally important for parasite survival. Sporozoite complexity is reflected in the large quantity of proteins that can be expressed. Some of them have been extensively studied, such as CSP, TRAP, STARP, LSA-1, LSA-3, SALSA, SPECT1, SPECT2, MAEBL, and SPATR, due to their importance in infection and their potential use as vaccines. Our work has been focused on the search for the molecular mechanisms of parasite-host cellular receptor-ligand interactions by identifying amino acid sequences and the critical binding residues from these proteins relevant to parasite invasion. Once such sequences have been identified, it will be possible to modify them to induce a strong immune response against P. falciparum in the experimental Aotus monkey model. This all leads towards developing multistage, multicomponent, subunit-based vaccines that will be effective in eradicating or controlling malaria caused by P. falciparum.

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.

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.

Identifying putative Mycobacterium tuberculosis Rv2004c protein sequences that bind specifically to U937 macrophages and A549 epithelial cells.

Virulence and immunity are still poorly understood in Mycobacterium tuberculosis. The H37Rv M. tuberculosis laboratory strain genome has been completely sequenced, and this along with proteomic technology represent powerful tools contributing toward studying the biology of target cell interaction with a facultative bacillus and designing new strategies for controlling tuberculosis. Rv2004c is a putative M. tuberculosis protein that could have specific mycobacterial functions. This study has revealed that the encoding gene is present in all mycobacterium species belonging to the M. tuberculosis complex. Rv2004c gene transcription was observed in all of this complex's strains except Mycobacterium bovis and Mycobacterium microti. Rv2004c protein expression was confirmed by using antibodies able to recognize a 54-kDa molecule by immunoblotting, and its location was detected on the M. tuberculosis surface by transmission electron microscopy, suggesting that it is a mycobacterial surface protein. Binding assays led to recognizing high activity binding peptides (HABP); five HABPs specifically bound to U937 cells, and six specifically bound to A549 cells. HABP circular dichroism suggested that they had an alpha-helical structure. HABP-target cell interaction was determined to be specific and saturable; some of them also displayed greater affinity for A549 cells than U937 cells. The critical amino acids directly involved in their interaction with U937 cells were also determined. Two probable receptor molecules were found on U937 cells and five on A549 for the two HABPs analyzed. These observations have important biological significance for studying bacillus-target cell interactions and implications for developing strategies for controlling this disease.

Mycobacterium tuberculosis Rv2536 protein implicated in specific binding to human cell lines.

The gene encoding the Mycobacterium tuberculosis Rv2536 protein is present in the Mycobacterium tuberculosis complex (as assayed by PCR) and transcribed (as determined by RT-PCR) in M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. bovis BCG, and M. africanum strains. Rabbits immunized with synthetic polymer peptides from this protein produced antibodies specifically recognizing a 25-kDa band in mycobacterial sonicate. U937 and A549 cells were used in binding assays involving 20-amino-acid-long synthetic peptides covering the whole Rv2536 protein sequence. Peptide 11207 (161DVFSAVRADDSPTGEMQVAQY180) presented high specific binding to both types of cells; the binding was saturable and presented nanomolar affinity constants. Cross-linking assays revealed that this peptide specifically binds to 50 kDa U937 cell membrane and 45 kDa A549 cell membrane proteins.

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.

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.

Characterising Mycobacterium tuberculosis Rv1510c protein and determining its sequences that specifically bind to two target cell lines.

The process of Mycobacterium tuberculosis infection of the macrophage implies a very little-known initial recognition and adherence step, important for mycobacterial survival; many proteins even remain like hypothetical. The Rv1510c gene, encoding a putatively conserved membrane protein, was investigated by analysing the M. tuberculosis genome sequence data reported by Cole et al. and a previous report that used PCR assays to show that the Rv1510 gene was only present in M. tuberculosis. This article confirmed all the above and identified the transcribed gene in M. tuberculosis, Mycobacterium africanum, and in M. tuberculosis clinical isolates. Antibodies raised against peptides from this protein recognised a 44 kDa band, corresponding to Rv1510c theoretical mass (44,294 Da). Assays involving synthetic peptides covering the whole protein binding to U937 and A549 cell lines led to recognising five high activity binding peptides in the Rv1510 protein: 11094, 11095, 11105, 11108, and 11111. Their affinity constants and Hill coefficients were determined by using U937 cells. Cross-linking assays performed with some of these HABPs showed that they specifically bound to a U937 cell line 51 kDa protein, but not to Hep G2 or red blood cell proteins, showing this interaction's specificity.

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 .

Amino terminal peptides from the Plasmodium falciparum EBA-181/JESEBL protein bind specifically to erythrocytes and inhibit in vitro merozoite invasion.

Several EBA-175 paralogues (EBA-140, EBA-165, EBA-175, EBA-181, and EBL-1) have been described among the Plasmodium falciparum malaria parasite proteins, which are important in the red blood cell (RBC) invasion process. EBA-181/JESEBL is a 181 kDa protein expressed in the late schizont stage and located in the micronemes; it belongs to the Plasmodium Duffy binding-like family and is able to interact with the erythrocyte surface. Here, we describe the synthesis of 78, 20-mer synthetic peptides derived from the reported EBA-181/JESEBL sequence and their ability to bind RBCs in receptor-ligand assays. Five peptides (numbered 30030, 30031, 30045, 30051, and 30060) displayed high specific binding to erythrocytes; their equilibrium binding parameters were then determined. These peptides interacted with 53 and 33 kDa receptor proteins on the erythrocyte surface, this binding being altered when RBCs were pretreated with enzymes. They were able to inhibit P. falciparum merozoite invasion of RBCs when tested in in vitro assays. According to these results, these five EBA-181/JESEBL high specific erythrocyte binding peptides, as well as the entire protein, were seen to be involved in the molecular machinery used by the parasite for invading RBCs. They are thus suggested as potential candidates in designing a multi-sub-unit vaccine able to combat the P. falciparum malaria parasite.

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.

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.

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.

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.

Liver stage antigen 3 Plasmodium falciparum peptides specifically interacting with HepG2 cells.

Binding assays were carried out with 20 amino acid long peptides covering the complete 200-kDa Liver stage antigen (LSA) 3 protein sequence to identify its HepG2 cell binding regions. Seventeen HepG2 cell high-activity binding peptides (HABPs) were identified in the LSA-3 protein. Seven HABPs were found in the nonrepeat (NRA) region A; five of these formed a 100 amino acid long HepG2 cell binding region located between residues 21Ile and 120Thr. Six HABPs were found in the R2 region and another four in the NRB2 region. LSA-3 protein HABPS bound saturably to HepG2 cells having nanomolar affinity constants and bound specifically to 31, 44, and 70 kDa HepG2 cell membrane proteins. Some of them were located in antigenic and immunogenic LSA-3 protein regions. Immunofluorescence and immunoblotting assays using goat sera immunized with LSA-3 protein peptides recognized P. falciparum (FCB-2 strain) erythrocyte stage proteins (58, 68, 72, 81, 86, 160, and 175 kDa). This reactivity was due mainly to the VEESVAEN motif present in some erythrocyte stage proteins. However, our results suggest that antibodies against LSA-3 regions had a crossed reaction with another 86-kDa protein, and that this crossed reaction was due to a motif present in the NRA region.

Specific erythrocyte binding capacity and biological activity of Plasmodium falciparum-derived rhoptry-associated protein 1 peptides.

Rhoptry-associated protein 1 (RAP1) is a merozoite antigen within Plasmodium falciparum rhoptries as yet having no specific function described for it. Synthetic peptides spanning the RAP1 sequence were tested in erythrocyte binding assays to identify possible RAP1 functional regions. Five high activity binding peptides (HABPs) were identified; 26201, 26202, 26203 and 26204 spanned residues 461C-K540 within RAP1 Cys region, whilst 26188 (201T-Y220) was located in p67 amino terminal. The results showed that peptide binding was saturable, some HABPs inhibited in vitro merozoite invasion and specifically bound to a 72 kDa protein in red blood cell membrane. HABP possible function in merozoite invasion of erythrocytes is also discussed.

Sporozoite and liver stage antigen Plasmodium falciparum peptides bind specifically to human hepatocytes.

Sporozoite and Liver Stage Antigen (SALSA) sequence synthetic peptides were used in HepG2 cell binding assays to identify regions involved in parasite invasion. SALSA 20608 ( 21IWASEKKDEKEASEQGEESHY40) and 20611 ( 64KKDDGTDKVQEKVLEKSPKY83) peptides were determined as having high binding activity in HepG2 cell assays, some of them were located in immunogenic regions. Immune-fluorescence antibody test with 24276 (20608 peptide analogue, CGIWSSMKMDEKMAAMQGEESHCG) showed sporozoite and merozoite reactivity. This data suggests SALSA high activity binding peptides' (HABPs) possible role in hepatic cell invasion and merozoite invasion of erythrocytes.