Primary structure of a Plasmodium falciparum malaria antigen located at the merozoite surface and within the parasitophorous vacuole

DNA encoding an antigen of 101,000 apparent molecular weight from the human malaria parasite Plasmodium falciparum was cloned and sequenced. Genomic DNA from the Camp strain covering the complete coding region along with cDNA from the FCR3 strain covering 81% of the coding region were obtained. The cloned DNA specified a full-length protein of 743 amino acids which included two tandemly repeated regions, one near the amino terminus containing eight hexapeptide repeats of sequence TVNDEDED, and the second near the carboxyl terminus containing primarily KE and KEE repeats. The latter repeated region is encoded by a 174-base stretch of mRNA containing only a single pyrimidine. Except for a putative leader sequence located at the amino terminus of the protein, the protein is hydrophilic and highly charged with a calculated isoelectric point of 5.6. Sequences from the Camp and FCR3 strains are very close and are also nearly identical to the partial cDNA sequence of the acidic basic repeated antigen (ABRA) protein from the FC27 strain (Stahl, H.D., Bianco, A.E., Crewther, R.F., Anders, R.F., Kyne, A.P., Coppel, R. L., Mitchell, G.F., Kemp, D.J., and Brown, G.V. (1986) Mol. Biol. Med. 3, 351-368). ABRA was previously shown to be located at the merozoite surface and in the parasitophorous vacuole. Because of its location and because it becomes complexed to merozoites when schizonts rupture in the presence of immune serum, ABRA is a candidate component of a malaria vaccine.     

The alanine-rich heptad repeats are intact in the processed form of Plasmodium falciparum MSP3

The potential of Plasmodium falciparum merozoite surface protein 3 as a component of an asexual-stage malaria vaccine is currently being assessed. The precursor form of MSP3 undergoes cleavage during schizogony to generate a mature processed form. It is unknown if this cleavage event is necessary for MSP3 function, but it may be an important consideration for assessing and developing MSP3 as an asexual-stage vaccine candidate. We have therefore determined the cleavage site in MSP3 by sequencing the N-terminus of the processed form of MSP3, which was isolated from parasite material. The position of the cleavage site indicates that the processed form of MSP3 retains the three blocks of alanine-rich heptad repeats, which are predicted to provide the structural framework for an intramolecular coiled-coil. The cleavage-site motif has many features in common with the published cleavage sites of MSP1(30), MSP6(36), and MSP7(22), which are all located on the merozoite surface and are implicated in the erythrocyte invasion process. The common cellular location and similar cleavage-site motifs suggest that these merozoite proteins may be cleaved by the same or related proteases.     

Lipid interactions of the malaria antigen merozoite surface protein 2

With more than half the world's population living at risk of malaria infection, there is a strong demand for the development of an effective malaria vaccine. One promising vaccine candidate is merozoite surface protein 2 (MSP2), which is among the most abundant antigens of the blood stage of the Plasmodium falciparum parasite. In solution, MSP2 is intrinsically unstructured, but little is known about the conformation of native MSP2, which is GPI-anchored to the merozoite surface, or of the implications of that conformation for the immune response induced by MSP2. Initial NMR studies have shown that MSP2 interacts with lipid micelles through a highly conserved N-terminal domain. We have further developed these findings by investigating how different lipid environments affect the protein structure. All of the tested lipid preparations perturbed only the N-terminal part of MSP2. In DPC micelles this region adopts an α-helical structure which we have characterized in detail. Our findings suggest a possible mechanism by which lipid interactions might modulate immune recognition of the conserved N-terminus of MSP2, potentially explaining the apparent immunodominance of the central variable region of this important malaria antigen.     

Formation of the food vacuole in Plasmodium falciparum: a potential role for the 19 kDa fragment of merozoite surface protein 1 (MSP1(19))

Plasmodium falciparum Merozoite Surface Protein 1 (MSP1) is synthesized during schizogony as a 195-kDa precursor that is processed into four fragments on the parasite surface. Following a second proteolytic cleavage during merozoite invasion of the red blood cell, most of the protein is shed from the surface except for the C-terminal 19-kDa fragment (MSP1(19)), which is still attached to the merozoite via its GPI-anchor. We have examined the fate of MSP1(19) during the parasite's subsequent intracellular development using immunochemical analysis of metabolically labeled MSP1(19), fluorescence imaging, and immuno-electronmicroscopy. Our data show that MSP1(19) remains intact and persists to the end of the intracellular cycle. This protein is the first marker for the biogenesis of the food vacuole; it is rapidly endocytosed into small vacuoles in the ring stage, which coalesce to form the single food vacuole containing hemozoin, and persists into the discarded residual body. The food vacuole is marked by the presence of both MSP1(19) and the chloroquine resistance transporter (CRT) as components of the vacuolar membrane. Newly synthesized MSP1 is excluded from the vacuole. This behavior indicates that MSP1(19) does not simply follow a classical lysosome-like clearance pathway, instead, it may play a significant role in the biogenesis and function of the food vacuole throughout the intra-erythrocytic phase.     

Plasmodium falciparum: an intervening sequence in the GBP 130/96 tR gene

The 96 tR antigen is a heat stable protein produced during the late stages of the intraerythrocytic development of the malaria parasite Plasmodium falciparum and is released into the culture supernatant or the sera of infected patients at the time of schizont rupture. This antigen, identified as a putative protective antigen, was shown to be identical to the glycophorin-binding protein GBP 130 (Perkins 1988, Bonnefoy et al. 1988). We report here that the gene contains a small undescribed intervening sequence located immediately after the sequence coding for the signal sequence. This shows that in P. falciparum, all the genes described so far coding for proteins exported outside the parasitophorous vacuole share a common organization.     

Alphavirus nucleocapsid protein contains a putative coiled coil alpha-helix important for core assembly

The alphavirus nucleocapsid core is formed through the energetic contributions of multiple noncovalent interactions mediated by the capsid protein. This protein consists of a poorly conserved N-terminal region of unknown function and a C-terminal conserved autoprotease domain with a major role in virion formation. In this study, an 18-amino-acid conserved region, predicted to fold into an alpha-helix (helix I) and embedded in a low-complexity sequence enriched with basic and Pro residues, has been identified in the N-terminal region of the alphavirus capsid proteins. In Sindbis virus, helix I spans residues 38 to 55 and contains three conserved leucine residues, L38, L45, and L52, conforming to the heptad amino acid organization evident in leucine zipper proteins. Helix I consists of an N-terminally truncated heptad and two complete heptad repeats with beta-branched residues and conserved leucine residues occupying the a and d positions of the helix, respectively. Complete or partial deletion of helix I, or single-site substitutions at the conserved leucine residues (L45 and L52), caused a significant decrease in virus replication. The mutant viruses were more sensitive to elevated temperature than wild-type virus. These mutant viruses also failed to accumulate cores in the cytoplasm of infected cells, although they did not have defects in protein translation or processing. Analysis of these mutants using an in vitro assembly system indicated that the majority were defective in core particle assembly. Furthermore, mutant proteins showed a trans-dominant negative phenotype in in vitro assembly reactions involving mutant and wild-type proteins. We propose that helix I plays a central role in the assembly of nucleocapsid cores through coiled coil interactions. These interactions may stabilize subviral intermediates formed through the interactions of the C-terminal domain of the capsid protein and the genomic RNA and contribute to the stability of the virion.     

Molecular cloning of nucleobindin, a novel DNA-binding protein that contains both a signal peptide and a leucine zipper structure.

We have previously reported that KML1-7 cells cloned from a lupus-prone MRL/l mouse produced a soluble factor that preferentially expanded anti-DNA antibody production across the H-2 barrier. We purified this factor, a 55 kD protein that we termed nucleobindin (Nuc), and obtained its cDNA clone. Although the gene for Nuc encodes a signal peptide and, in fact, Nuc was identified as a secreted protein, Nuc had a DNA-binding property. The putative polypeptide predicted from the cDNA sequence featured a signal peptide, a leucine zipper structure and a basic amino acid-rich region. The DNA-binding property of Nuc was destroyed by deletion of either the leucine zipper structure or the basic amino acid-rich region. The amino acid sequences of Nuc are highly conserved between mouse and human. We discuss the possible role of Nuc in autoimmunity.     

Selective inhibition of a two-step egress of malaria parasites from the host erythrocyte

Escape from the host erythrocyte by the invasive stage of the malaria parasite Plasmodium falciparum is a fundamental step in the pathogenesis of malaria of which little is known. Upon merozoite invasion of the host cell, the parasite becomes enclosed within a parasitophorous vacuole, the compartment in which the parasite undergoes growth followed by asexual division to produce 16-32 daughter merozoites. These daughter cells are released upon parasitophorous vacuole and erythrocyte membrane rupture. To examine the process of merozoite release, we used P. falciparum lines expressing green fluorescent protein-chimeric proteins targeted to the compartments from which merozoites must exit: the parasitophorous vacuole and the host erythrocyte cytosol. This allowed visualization of merozoite release in live parasites. Herein we provide the first evidence in live, untreated cells that merozoite release involves a primary rupture of the parasitophorous vacuole membrane followed by a secondary rupture of the erythrocyte plasma membrane. We have confirmed, with the use of immunoelectron microscopy, that parasitophorous vacuole membrane rupture occurs before erythrocyte plasma membrane rupture in untransfected wild-type parasites. We have also demonstrated selective inhibition of each step in this two-step process of exit using different protease inhibitors, implicating the involvement of distinct proteases in each of these steps. This will facilitate the identification of the parasite and host molecules involved in merozoite release.     

Dissection of merozoite surface protein 3, a representative of a family of Plasmodium falciparum surface proteins, reveals an oligomeric and highly elongated molecule

Vaccination with the merozoite surface protein 3 (MSP3) of Plasmodium falciparum protects against infection in primates and is under development as a vaccine against malaria in humans. MSP3 is secreted and associates with the parasite membrane but lacks a predicted transmembrane domain or a glycosylphosphatidylinositol anchor. Its role in the invasion of red blood cells is unclear. To study MSP3, we produced recombinant full-length protein and found by size exclusion chromatography that the apparent size of MSP3 was much larger than predicted from its sequence. To investigate this, we used several biophysical techniques to characterize the full-length molecule and four smaller polypeptides. The MSP3 polypeptides contain a large amount of alpha-helix and random coil secondary structure as measured by circular dichroism spectroscopy. The full-length MSP3 forms highly elongated dimers and tetramers as revealed by chemical cross-linking and analytical ultracentrifugation. The dimer is formed through a leucine zipper-like domain located between residues 306 and 362 at the C terminus. Two dimers interact through their C termini to form a tetramer with an apparent association constant of 3 mum. Sedimentation velocity experiments determined that the MSP3 molecules are highly extended in solution (some with f/f(0) > 2). These data, in light of the recent discoveries of three other Plasmodium proteins containing very similar C-terminal sequences, suggest that the members of this newly identified family may adopt highly extended and oligomeric novel structures capable of interacting with a red blood cell at relatively long distances.     

Plasmodium falciparum: the repetitive MSA-1 surface protein of the RO-71 isolate is recognized by mouse antibody against the nonrepetitive repeat block of RO-33.

We have expressed in Escherichia coli the nonrepetitive repeat zone of the MSA-1 surface protein of the RO-33 isolate of Plasmodium falciparum. The recombinant protein was used to immunize mice and the resulting RO-33 monospecific serum was used to screen our P. falciparum strain collection in order to recover additional alleles lacking tripeptide repeats in block 2 of MSA-1. Only 1 (RO-71) out of 30 isolates tested reacted strongly with the serum by indirect immunofluorescence assay. Surprisingly, block 2 of the RO-71 MSA-1 allele contains tripeptide repeats resembling those of the K1 isolate of P. falciparum. Additional sequence analysis of the entire DNA coding for the 80-kDa MSA-1-derived surface component did not reveal any amino acid stretches similar to block 2 of RO-33 which could rationalize the immunological cross-reactivity. We eliminated the possibility that the RO-71 culture was contaminated with RO-33 type alleles of MSA-1 by Southern blotting and PCR analysis. The RO-33-specific mouse serum used for the initial selection of RO-71 did not react with the antigen in the denatured state (Western blot). This and the sequence analysis suggest that the cross-reactive epitope in the MSA-1 protein of RO-71 is conformational. The possibility that a truncated frame-shift protein encoded by mutated MSA-1 mRNA is recognized by the serum is discussed.     

Plasmodial ortholog of Toxoplasma gondii rhoptry neck protein 3 is localized to the rhoptry body

The proteins in apical organelles of Plasmodium falciparum merozoite play an important role in invasion into erythrocytes. Several rhoptry neck (RON) proteins have been identified in rhoptry proteome of the closely-related apicomplexan parasite, Toxoplasma gondii. Recently, three of P. falciparum proteins orthologous to TgRON proteins, PfRON2, 4 and 5, were found to be located in the rhoptry neck and interact with the micronemal protein apical membrane antigen 1 (PfAMA1) to form a moving junction complex that helps the invasion of merozoite into erythrocyte. However, the other P. falciparum RON proteins have yet to be characterized. Here, we determined that "PFL2505c" (hereafter referred to as pfron3) is the ortholog of the tgron3 in P. falciparum and characterized its protein expression profile, subcellular localization, and complex formation. Protein expression analysis revealed that PfRON3 was expressed primarily in late schizont stage parasites. Immunofluorescence microscopy (IFA) showed that PfRON3 localizes in the apical region of P. falciparum merozoites. Results from immunoelectron microscopy, along with IFA, clarified that PfRON3 localizes in the rhoptry body and not in the rhoptry neck. Even after erythrocyte invasion, PfRON3 was still detectable at the parasite ring stage in the parasitophorous vacuole. Moreover, co-immunoprecipitation studies indicated that PfRON3 interacts with PfRON2 and PfRON4, but not with PfAMA1. These results suggest that PfRON3 partakes in the novel PfRON complex formation (PfRON2, 3, and 4), but not in the moving junction complex (PfRON2, 4, 5, and PfAMA1). The novel PfRON complex, as well as the moving junction complex, might play a fundamental role in erythrocyte invasion by merozoite stage parasites.     

Genetic analysis of the leucine heptad repeats of Lac repressor: evidence for a 4-helical bundle.

Gel-filtration experiments indicate that a peptide (P2) composed of the basic region of GCN4 fused to the leucine heptad repeats of Lac repressor forms tetrameric aggregates. Gel-shift experiments were performed to determine the orientation of the helices in the tetrameric P2 aggregate. Sandwich-complex formation of peptide P2 with two DNA fragments containing two symmetrical CRE binding sites (5'-ATGACGTCAT-3') at a distance of 21 bp suggests antiparallel aggregation of the Lac leucine heptad repeats. Thus, we conclude that the leucine heptad repeats of Lac repressor have the ability to form homomeric 4-helical bundles with an antiparallel arrangement of the helices. This topology enables the two DNA fragments in the sandwich complexes to be held together by two tetramers of peptide P2. Replacement of the uncharged amino acids of the helical g and e positions of peptide P2 by the corresponding charged residues of GCN4 (peptide P4) results in a dimeric and parallel aggregation of the leucine heptad repeats, and consequently abolishes the potential to form sandwich structures. Similarly, a hybrid Lac repressor in which the GCN4 leucine zipper replaces the natural Lac leucine heptad repeats forms dimers only. It regains the ability to form tetramers when the charged amino acids in helical positions g and e are replaced by uncharged alanines.     

A multifunctional serine protease primes the malaria parasite for red blood cell invasion

The malaria parasite Plasmodium falciparum replicates within an intraerythrocytic parasitophorous vacuole (PV). Rupture of the host cell allows release (egress) of daughter merozoites, which invade fresh erythrocytes. We previously showed that a subtilisin-like protease called PfSUB1 regulates egress by being discharged into the PV in the final stages of merozoite development to proteolytically modify the SERA family of papain-like proteins. Here, we report that PfSUB1 has a further role in ‘priming' the merozoite prior to invasion. The major protein complex on the merozoite surface comprises three proteins called merozoite surface protein 1 (MSP1), MSP6 and MSP7. We show that just before egress, all undergo proteolytic maturation by PfSUB1. Inhibition of PfSUB1 activity results in the accumulation of unprocessed MSPs on the merozoite surface, and erythrocyte invasion is significantly reduced. We propose that PfSUB1 is a multifunctional processing protease with an essential role in both egress of the malaria merozoite and remodelling of its surface in preparation for erythrocyte invasion.     

The signal sequence of exported protein-1 directs the green fluorescent protein to the parasitophorous vacuole of transfected malaria parasites

The malaria parasite, Plasmodium falciparum, spends part of its life cycle inside the erythrocytes of its human host. In the mature stages of intraerythrocytic growth, the parasite undertakes extensive remodeling of its adopted cellular home by exporting proteins beyond the confines of its own plasma membrane. To examine the signals involved in export of parasite proteins, we have prepared transfected parasites expressing a chimeric protein comprising the N-terminal region of the Plasmodium falciparum exported protein-1 appended to green fluorescent protein. The majority of the population of the chimeric protein appears to be correctly processed and trafficked to the parasitophorous vacuole, indicating that this is the default destination for protein secretion. Some of the protein is redirected to the parasite food vacuole and further degraded. Photobleaching studies reveal that the parasitophorous vacuole contains subcompartments that are only partially interconnected. Dual labeling with the lipid probe, BODIPY-TR-ceramide, reveals the presence of membrane-bound extensions that can bleb from the parasitophorous vacuole to produce double membrane-bound compartments. We also observed regions and extensions of the parasitophorous vacuole, where there is segregation of the lumenal chimera from the lipid components. These regions may represent sites for the sorting of proteins destined for the trafficking to sites beyond the parasitophorous vacuole membrane.     

The DNA-binding domain of the Cys-3 regulatory protein of Neurospora crassa is bipartite.

Cys-3, the major sulfur regulatory gene of Neurospora crassa, encodes a regulatory protein that is capable of sequence-specific interaction with DNA. The interaction is mediated by a region within the CYS3 protein (the bzip region) which contains a potential dimer-forming surface, the leucine zipper, and an adjacent basic DNA contact region, NH2-terminal to the leucine zipper. To investigate the bipartite nature of the bzip region, a series of cys-3 mutants obtained by oligonucleotide-directed mutagenesis were expressed and tested for dimer formation as well as DNA binding and in vivo function. The results demonstrate that CYS3 protein exists as a dimer in the presence and absence of the target DNA and that dimerization of CYS3 is mediated strictly by the leucine zipper, which is required for both cys-3 function in vivo and DNA-binding activity in vitro. Furthermore, a truncated CYS3 protein corresponding to just the bzip region was found to mediate dimer formation and to possess DNA-binding activity. A CYS3 mutant protein with a pure methionine zipper showed significant, although reduced, function in vivo and in vitro.     

Immunogenicity of C-terminus of Plasmodium falciparum merozoite surface protein 1 expressed as a non-glycosylated polypeptide in yeast

The C-terminal region of the merozoite surface protein 1 (MSP1_(19)) is one of the mostpromising vaccine candidates against the erythrocytic forms of malaria.In the present study,a gene encodingPlasmodium falciparum MSP1_(19) was expressed in yeast Pichia pastoris.A non-glycosylated form of therecombinant protein MSP1_(19) was purified from culture medium.This recombinant protein maintains itsantigenicity.Significant immune responses were seen in C57BL/6 mice after the second immunization.Moreover,the specific antibodies recognized the native antigens of P.falciparum,The prevailing isotypesof immunoglobulin (Ig)G associated with immunization were IgG1,IgG2a and IgG2b.The antibodiesisolated from mouse sera immunized with MSP1_(19) can inhibit parasite growth in vitro.Based on theseimmunological studies,we concluded that MSP1_(19) deserves further evaluation in pre-clinical immunizationsagainst P.falciparum.     

An undecided coiled coil: the leucine zipper of Nek2 kinase exhibits atypical conformational exchange dynamics

Leucine zippers are oligomerization domains used in a wide range of proteins. Their structure is based on a highly conserved heptad repeat sequence in which two key positions are occupied by leucines. The leucine zipper of the cell cycle-regulated Nek2 kinase is important for its dimerization and activation. However, the sequence of this leucine zipper is most unusual in that leucines occupy only one of the two hydrophobic positions. The other position, depending on the register of the heptad repeat, is occupied by either acidic or basic residues. Using NMR spectroscopy, we show that this leucine zipper exists in two conformations of almost equal population that exchange with a rate of 17 s(-1). We propose that the two conformations correspond to the two possible registers of the heptad repeat. This hypothesis is supported by a cysteine mutant that locks the protein in one of the two conformations. NMR spectra of this mutant showed the predicted 2-fold reduction of peaks in the (15)N HSQC spectrum and the complete removal of cross peaks in exchange spectra. It is possible that interconversion of these two conformations may be triggered by external signals in a manner similar to that proposed recently for the microtubule binding domain of dynein and the HAMP domain. As a result, the leucine zipper of Nek2 kinase is the first example where the frameshift of coiled-coil heptad repeats has been directly observed experimentally.     

Merozoite surface protein 2 of Plasmodium falciparum: expression, structure, dynamics, and fibril formation of the conserved N-terminal domain

Merozoite surface protein 2 (MSP2) is a GPI-anchored protein on the surface of the merozoite stage of the malaria parasite Plasmodium falciparum. It is largely disordered in solution, but has a propensity to form amyloid-like fibrils under physiological conditions. The N-terminal conserved region (MSP2(1-25)) is part of the protease-resistant core of these fibrils. To investigate the structure and dynamics of this region, its ability to form fibrils, and the role of individual residues in these properties, we have developed a bacterial expression system that yields > or =10 mg of unlabeled or (15)N-labeled peptide per litre of culture. Two recombinant versions of MSP2(1-25), wild-type and a Y7A/Y16A mutant, have been produced. Detailed conformational analysis of the wild-type peptide and backbone (15)N relaxation data indicated that it contains beta-turn and nascent helical structures in the central and C-terminal regions. Residues 6-21 represent the most ordered region of the structure, although there is some flexibility around residues 8 and 9. The 10-residue sequence (MSP2(7-16)) (with two Tyr residues) was predicted to have a higher propensity for beta-aggregation than the 8-mer sequence (MSP2(8-15)), but there was no significant difference in conformation between MSP2(1-25) and [Y7A,Y16A]MSP2(1-25) and the rate of fibril formation was only slightly slower in the mutant. The peptide expression system described here will facilitate further mutational analyses to define the roles of individual residues in transient structural elements and fibril formation, and thus contribute to the further development of MSP2 as a malaria vaccine candidate.