ORCID Profile
0000-0003-1349-4698
Current Organisations
University of Dundee
,
La Trobe University
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In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Veterinary Sciences | Medical Parasitology | Sociobiology And Behavioural Ecology | Nanotechnology | Nanobiotechnology | Parasitology
Infectious diseases | Expanding Knowledge in the Biological Sciences | Prevention—biologicals (e.g. vaccines) | Biological sciences |
Publisher: Elsevier BV
Date: 07-1992
Publisher: American Society for Microbiology
Date: 12-2012
DOI: 10.1128/IAI.00665-12
Abstract: Merozoite surface protein 2 (MSP2) is an abundant glycosylphosphatidylinositol (GPI)-anchored protein of Plasmodium falciparum , which is a potential component of a malaria vaccine. As all forms of MSP2 can be categorized into two allelic families, a vaccine containing two representative forms of MSP2 may overcome the problem of ersity in this highly polymorphic protein. Monomeric recombinant MSP2 is an intrinsically unstructured protein, but its conformational properties on the merozoite surface are unknown. This question is addressed here by analyzing the 3D7 and FC27 forms of recombinant and parasite MSP2 using a panel of monoclonal antibodies raised against recombinant MSP2. The epitopes of all antibodies, mapped using both a peptide array and by nuclear magnetic resonance (NMR) spectroscopy on full-length recombinant MSP2, were shown to be linear. The antibodies revealed antigenic differences, which indicate that the conserved N- and C-terminal regions, but not the central variable region, are less accessible in the parasite antigen. This appears to be an intrinsic property of parasite MSP2 and is not dependent on interactions with other merozoite surface proteins as the loss of some conserved-region epitopes seen using the immunofluorescence assay (IFA) on parasite smears was also seen on Western blot analyses of parasite lysates. Further studies of the structural basis of these antigenic differences are required in order to optimize recombinant MSP2 constructs being evaluated as potential vaccine components.
Publisher: Wiley
Date: 20-02-2004
DOI: 10.1002/PROT.20005
Abstract: The new antigen receptor (IgNAR) is an antibody unique to sharks and consists of a disulphide-bonded dimer of two protein chains, each containing a single variable and five constant domains. The in idual variable (V(NAR)) domains bind antigen independently, and are candidates for the smallest antibody-based immune recognition units. We have previously produced a library of V(NAR) domains with extensive variability in the CDR1 and CDR3 loops displayed on the surface of bacteriophage. Now, to test the efficacy of this library, and further explore the dynamics of V(NAR) antigen binding we have performed selection experiments against an infectious disease target, the malarial Apical Membrane Antigen-1 (AMA1) from Plasmodium falciparum. Two related V(NAR) clones were selected, characterized by long (16- and 18-residue) CDR3 loops. These recombinant V(NAR)s could be harvested at yields approaching 5mg/L of monomeric protein from the E. coli periplasm, and bound AMA1 with nanomolar affinities (K(D)= approximately 2 x 10(-7) M). One clone, designated 12Y-2, was affinity-matured by error prone PCR, resulting in several variants with mutations mapping to the CDR1 and CDR3 loops. The best of these variants showed approximately 10-fold enhanced affinity over 12Y-2 and was Plasmodium falciparum strain-specific. Importantly, we demonstrated that this monovalent V(NAR) co-localized with rabbit anti-AMA1 antisera on the surface of malarial parasites and thus may have utility in diagnostic applications.
Publisher: American Society for Microbiology
Date: 08-2012
DOI: 10.1128/IAI.00206-12
Abstract: Plasmodium vivax invasion of human erythrocytes requires interaction of the P. vivax Duffy binding protein (PvDBP) with its host receptor, the Duffy antigen (Fy) on the erythrocyte surface. Consequently, PvDBP is a leading vaccine candidate. The binding domain of PvDBP lies in a cysteine-rich portion of the molecule called region II (PvDBPII). PvDBPII contains three distinct subdomains based upon intramolecular disulfide bonding patterns. Subdomain 2 (SD2) is highly polymorphic and is thought to contain many key residues for binding to Fy, while SD1 and SD3 are comparatively conserved and their role in Fy binding is not well understood. To examine the relative contributions of the different subdomains to binding to Fy and their abilities to elicit strain-transcending binding-inhibitory antibodies, we evaluated recombinant proteins from SD1+2, SD2, SD3, and SD3+, which includes 24 residues of SD2. All of the recombinant subdomains, except for SD2, bound variably to human erythrocytes, with constructs containing SD3 showing the best binding. Antisera raised in laboratory animals against SD3, SD3+, and SD2+3 inhibited the binding of full-length PvDBPII, which is strain transcending, whereas antisera generated to SD1+2 and SD2 failed to generate blocking antibodies. All of the murine monoclonal antibodies generated to full-length PvDBPII that had significant binding-inhibitory activity recognized only SD3. Thus, SD3 binds Fy and elicits blocking antibodies, indicating that it contains residues critical to Fy binding that could be the basis of a strain-transcending candidate vaccine against P. vivax .
Publisher: Elsevier BV
Date: 10-1994
DOI: 10.1016/0166-6851(94)00136-7
Abstract: Erythrocytes infected with mature-stage malaria parasites accumulate phospholipids from exogenous sources. We show that the transport of N-(7-nitrobenzy-2-oxa-1,3-diazol-4-yl)-1,2- dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (N-NBD-DPPE), from the erythrocyte membrane to the intracellular malaria parasite, is dependent upon metabolic energy. A photoreactive phospholipid analogue, N-[125I]iodo-4-azidosalicylamidyl-1, 2-dilauryl-sn-glycero-3-phosphatidylethanolamine (N-125I-ASA-DLPE), has been synthesised and used in an attempt to identify proteins involved in phospholipid trafficking in malaria-infected erythrocytes. This photoreactive probe was found to preferentially label a protein with an apparent molecular weight of 22 kDa. Photolabelling of the 22-kDa protein was enhanced upon ATP depletion of malaria-infected erythrocytes.
Publisher: Elsevier BV
Date: 25-11-1996
DOI: 10.1016/0166-6851(96)02732-6
Abstract: A photoreactive quinolinemethanol analog, N-[4-[1-hydroxy-2-(dibutylamino)ethyl]quinolin-8yl]-4- azido-2-salicylamide (ASA-MQ) has been synthesized which closely mimics the action of mefloquine. ASA-MQ possesses potent antimalarial activity against a mefloquine-sensitive strain of Plasmodium falciparum and shows decreased activity against a mefloquine-resistant parasite strain. Radioiodinated ASA-MQ has been used in photoaffinity labeling studies to identify mefloquine-interacting proteins in serum, uninfected erythrocytes and Plasmodium falciparum-infected erythrocytes. We have shown that mefloquine interacts specifically with apo-A1, the major protein of serum high density lipoproteins. In addition, mefloquine was shown to interact specifically with the erythrocyte membrane protein, band 7.2b (stomatin). A further two high affinity mefloquine-binding proteins with apparent molecular masses of 22 and 36 kDa were identified in three different strains of Plasmodium falciparum. We suggest that these two mefloquine-binding parasite proteins may be involved in the uptake of mefloquine or may represent macromolecular targets of mefloquine action in malaria parasites.
Publisher: Wiley
Date: 28-09-1987
DOI: 10.1016/0014-5793(87)80189-8
Abstract: A simple method for isolation and fluorescence labelling of Leydig cells (L-cells) from rat testes was developed. Lateral diffusion coefficients of both lipid and protein membrane fluorescent probes were measured by the method of fluorescence recovery after photobleaching (FRAP). Age-dependent changes in diffusibility of membrane lipids and proteins were discovered.
Publisher: Elsevier BV
Date: 07-1998
DOI: 10.1016/S0163-7258(98)00012-6
Abstract: Quinoline-containing antimalarial drugs, such as chloroquine, quinine and mefloquine, are mainstays of chemotherapy against malaria. The molecular basis of the action of these drugs is not completely understood, but they are thought to interfere with hemoglobin digestion in the blood stages of the malaria parasite's life cycle. The parasite degrades hemoglobin, in an acidic food vacuole, producing free heme and reactive oxygen species as toxic by-products. The heme moieties are neutralized by polymerisation, while the free radical species are detoxified by a vulnerable series of antioxidant mechanisms. Chloroquine, a dibasic drug, is accumulated several thousand-fold in the food vacuole. The high intravacuolar chloroquine concentration is proposed to interfere with the polymerisation of heme and/or the detoxification of the reactive oxygen species, effectively killing the parasite with its own metabolic waste. Chloroquine resistance appears to arise as a result of a decreased level of chloroquine uptake, due to an increased vacuolar pH or to changes in a chloroquine importer or receptor. The more lipophilic quinolinemethanol drugs mefloquine and quinine do not appear to be concentrated so extensively in the food vacuole and may act on alternative targets in the parasite. Resistance to the quinolinemethanols is thought to involve a plasmodial homolog of P-glycoprotein. As the malaria parasites become increasingly resistant to the quinoline antimalarials, there is an urgent need to understand the molecular mechanisms for drug action and resistance so that novel antimalarial drugs can be designed. A number of modified quinolines and bisquinoline compounds show some promise in this regard.
Publisher: Elsevier BV
Date: 03-1995
Publisher: American Society for Microbiology
Date: 05-2006
DOI: 10.1128/IAI.74.5.2628-2636.2006
Abstract: Apical membrane antigen 1 (AMA1) is currently one of the leading malarial vaccine candidates. Anti-AMA1 antibodies can inhibit the invasion of erythrocytes by Plasmodium merozoites and prevent the multiplication of blood-stage parasites. Here we describe an anti-AMA1 monoclonal antibody (MAb 1F9) that inhibits the invasion of Plasmodium falciparum parasites in vitro. We show that both reactivity of MAb 1F9 with AMA1 and MAb 1F9-mediated invasion inhibition were strain specific. Site-directed mutagenesis of a fragment of AMA1 displayed on M13 bacteriophage identified a single polymorphic residue in domain I of AMA1 that is critical for MAb 1F9 binding. The identities of all other polymorphic residues investigated in this domain had little effect on the binding of the antibody. Examination of the P. falciparum AMA1 crystal structure localized this residue to a surface-exposed α-helix at the apex of the polypeptide. This description of a polymorphic inhibitory epitope on AMA1 adds supporting evidence to the hypothesis that immune pressure is responsible for the polymorphisms seen in this molecule.
Publisher: Oxford University Press (OUP)
Date: 13-06-2011
Abstract: Mycobacterium avium subspecies paratuberculosis (Map) is the causative agent of Johne's disease (JD). Current serological diagnostic tests for JD are limited by their sensitivity when used in sub-clinical stages of the disease. Our objective was to identify peptides that mimic diagnostically important Map epitopes that might be incorporated into a new-generation JD diagnostic. Four peptides were isolated from a phage-displayed random peptide library by screening on antibodies derived from Map-infected goats. The peptides were recognised by antibodies from Map-infected goats but not by antibodies from uninfected goats. The peptides elicited immune responses in rabbits, which reacted strongly with bona fide Map antigens proving the peptides were true epitope mimics. To assess the diagnostic value a panel of goat sera was screened for reactivity's with peptides. The peptides were recognised by antibodies from a proportion of goats infected with Map compared with control animals with a diagnostic specificity of 100% and the sensitivity ranged from 50 to 75%. Combinations of any two peptides improved sensitivity 62.5-87.5% and 100% sensitivity was achieved with three of the four peptides in combination. These data suggest peptides representing diagnostically important Map epitopes could be incorporated into a sensitive diagnostic test.
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 12-2002
Abstract: The quinolines have been used in the treatment of malaria, arthritis, and lupus for many years, yet the precise mechanism of their action remains unclear. In this study, we used a functional proteomics approach that exploited the structural similarities between the quinoline compounds and the purine ring of ATP to identify quinoline-binding proteins. Several quinoline drugs were screened by displacement affinity chromatography against the purine binding proteome captured with gamma-phosphate-linked ATP-Sepharose. Screening of the human red blood cell purine binding proteome identified two human proteins, aldehyde dehydrogenase 1 (ALDH1) and quinone reductase 2 (QR2). In contrast, no proteins were detected upon screening of the Plasmodium falciparum purine binding proteome with the quinolines. In a complementary approach, we passed cell lysates from mice, red blood cells, or P. falciparum over hydroxychloroquine- or primaquine-Sepharose. Consistent with the displacement affinity chromatography screen, ALDH and QR2 were the only proteins recovered from mice and human red blood cell lysate and no proteins were recovered from P. falciparum. Furthermore, the activity of QR2 was potently inhibited by several of the quinolines in vitro. Our results show that ALDH1 and QR2 are selective targets of the quinolines and may provide new insights into the mechanism of action of these drugs.
Publisher: Elsevier BV
Date: 04-2009
Publisher: Wiley
Date: 10-1989
DOI: 10.1111/J.1365-2958.1989.TB00114.X
Abstract: Morphological evidence has previously indicated that the periplasmic space of Escherichia coli is compartmentalized at sites corresponding to future sites of cell ision. The borders of these morphological compartments are formed by localized zones of adhesion (periseptal annuli). In the present study, the technique of fluorescence recovery after photobleaching was used to determine whether these structures act as barriers to the free movement of proteins within the periplasm. The recovery of fluorescence in the ftsA filaments was found to be uniformly low over at potential sites of cell ision and at the cell poles, indicating that these regions are biochemically sequestered from the remainder of the periplasmic space. Our results provide direct evidence for local compartments within the periplasm, primarily located at the sites of past or future cell isions. The implications of this finding for cell ision and other periplasmic processes are discussed.
Publisher: Elsevier BV
Date: 06-2016
Publisher: Elsevier BV
Date: 09-2015
DOI: 10.1016/J.CELL.2015.08.031
Abstract: The cytokine TWEAK and its cognate receptor Fn14 are members of the TNF/TNFR superfamily and are upregulated in tumors. We found that Fn14, when expressed in tumors, causes cachexia and that antibodies against Fn14 dramatically extended lifespan by inhibiting tumor-induced weight loss although having only moderate inhibitory effects on tumor growth. Anti-Fn14 antibodies prevented tumor-induced inflammation and loss of fat and muscle mass. Fn14 signaling in the tumor, rather than host, is responsible for inducing this cachexia because tumors in Fn14- and TWEAK-deficient hosts developed cachexia that was comparable to that of wild-type mice. These results extend the role of Fn14 in wound repair and muscle development to involvement in the etiology of cachexia and indicate that Fn14 antibodies may be a promising approach to treat cachexia, thereby extending lifespan and improving quality of life for cancer patients.
Publisher: Elsevier BV
Date: 08-2009
Publisher: Elsevier BV
Date: 07-08-2002
DOI: 10.1016/S0166-6851(02)00117-2
Abstract: The mature human erythrocyte is a simple haemoglobin-containing cell with no internal organelles and no protein synthesis machinery. The malaria parasite invades this cell and develops inside a parasitophorous vacuole (PV). The parasite exports proteins into the erythrocyte to bring about extensive remodelling of its adopted cellular home. Plasmodial homologues of two COPII proteins, PfSar1p and PfSec31p, are exported to the erythrocyte cytosol where they appear to play a role in the trafficking of proteins across the erythrocyte cytoplasm [Eur. J. Cell Biol. 78 (1999) 453 J. Cell Sci. 114 (2001) 3377]. We have now characterised a homologue of the COPI protein, delta-COP. A recombinant protein corresponding to 90% of the Pfdelta-COP sequence was used to raise antibodies. The affinity-purified antiserum recognised a protein with an apparent M(r) of 58 x 10(3) on Western blots of malaria parasite-infected erythrocytes but not on blots of uninfected erythrocytes. Pfdelta-COP was shown to be largely insoluble in non-ionic detergent, possibly suggesting cytoskeletal attachment. Confocal immunofluorescence microscopy of parasitised erythrocytes was used to show that, in contrast to the COPII proteins, Pfdelta-COP is located entirely within the parasite. The location of Pfdelta-COP partly overlaps that of the endoplasmic reticulum (ER)-located protein, PfERC, and partly that of the trans-Golgi-associated protein, PfRab6. Treatment of ring-stage plasmodium-infected erythrocytes with brefeldin A (BFA) inhibited development of the ER structure within the parasite cytosol and prevented the trafficking of the P. falciparum erythrocyte membrane protein-1, PfEMP1, to the erythrocyte cytosol. The Pfdelta-COP and PfSec31p populations each appear to be associated with the restricted ER structure in brefeldin-treated rings. When more mature stage parasites were treated with BFA, erythrocyte cytosol-located populations of parasite proteins were not reorganised, however, the overlap between Pfdelta-COP and PfERC in parasite cytosol was more complete suggesting a possible redistribution of the Golgi compartment into the ER. These data support the suggestion that both COPI and COPII proteins are involved in the trafficking of proteins within the parasite cytoplasm. However, only COPII proteins are exported to the erythrocyte cytosol to establish a vesicle-mediated protein trafficking pathway to the erythrocyte membrane.
Publisher: Public Library of Science (PLoS)
Date: 05-12-2012
Publisher: Wiley
Date: 11-10-2007
DOI: 10.1002/PROT.21663
Abstract: Mimotopes mimic the three-dimensional topology of an antigen epitope, and are frequently recognized by antibodies with affinities comparable to those obtained for the original antibody-antigen interaction. Peptides and anti-idiotypic antibodies are two classes of protein mimotopes that mimic the topology (but not necessarily the sequence) of the parental antigen. In this study, we combine these two classes by selecting mimotopes based on single domain IgNAR antibodies, which display exceptionally long CDR3 loop regions (analogous to a constrained peptide library) presented in the context of an immunoglobulin framework with adjacent and supporting CDR1 loops. By screening an in vitro phage-display library of IgNAR variable domains (V(NAR)s) against the target antigen monoclonal antibody MAb5G8, we obtained four potential mimotopes. MAb5G8 targets a linear tripeptide epitope (AYP) in the flexible signal sequence of the Plasmodium falciparum Apical Membrane Antigen-1 (AMA1), and this or similar motifs were detected in the CDR loops of all four V(NAR)s. The V(NAR)s, 1-A-2, -7, -11, and -14, were demonstrated to bind specifically to this paratope by competition studies with an artificial peptide and all showed enhanced affinities (3-46 nM) compared to the parental antigen (175 nM). Crystallographic studies of recombinant proteins 1-A-7 and 1-A-11 showed that the SYP motifs on these V(NAR)s presented at the tip of the exposed CDR3 loops, ideally positioned within bulge-like structures to make contact with the MAb5G8 antibody. These loops, in particular in 1-A-11, were further stabilized by inter- and intra- loop disulphide bridges, hydrogen bonds, electrostatic interactions, and aromatic residue packing. We rationalize the higher affinity of the V(NAR)s compared to the parental antigen by suggesting that adjacent CDR1 and framework residues contribute to binding affinity, through interactions with other CDR regions on the antibody, though of course definitive support of this hypothesis will rely on co-crystallographic studies. Alternatively, the selection of mimotopes from a large (<4 x 10(8)) constrained library may have allowed selection of variants with even more favorable epitope topologies than present in the original antigenic structure, illustrating the power of in vivo selection of mimotopes from phage-displayed molecular libraries.
Publisher: Elsevier BV
Date: 12-2002
Publisher: Bentham Science Publishers Ltd.
Date: 08-2011
DOI: 10.2174/156802611796575885
Abstract: The invasion of host cells by malaria parasites represents an attractive target for therapeutic intervention. The role played by apical membrane antigen 1 (AMA1) in this process has been elucidated recently with the demonstration that AMA1 forms a complex with parasite rhoptry neck (RON) proteins as part of the moving junction that develops between the host cell and the invading parasite. Structural studies of AMA1 alone and in complexes with antibodies that inhibit host cell invasion have identified a conserved hydrophobic cleft that is essential to the assembly of the AMA1/RON complex. AMA1 is already established as an important candidate for inclusion in a malaria vaccine. Here we review both the structural details and functional significance of interactions at the hydrophobic cleft of AMA1, and argue that this feature of the protein represents an excellent target for the development of drugs that would block host cell invasion by malarial parasites.
Publisher: Elsevier BV
Date: 11-2007
DOI: 10.1016/J.STR.2007.09.011
Abstract: Apical membrane antigen 1 (AMA1) is essential for invasion of erythrocytes and hepatocytes by Plasmodium parasites and is a leading malarial vaccine candidate. Although conventional antibodies to AMA1 can prevent such invasion, extensive polymorphisms within surface-exposed loops may limit the ability of these AMA1-induced antibodies to protect against all parasite genotypes. Using an AMA1-specific IgNAR single-variable-domain antibody, we performed targeted mutagenesis and selection against AMA1 from three P. falciparum strains. We present cocrystal structures of two antibody-AMA1 complexes which reveal extended IgNAR CDR3 loops penetrating deep into a hydrophobic cleft on the antigen surface and contacting residues conserved across parasite species. Comparison of a series of affinity-enhancing mutations allowed dissection of their relative contributions to binding kinetics and correlation with inhibition of erythrocyte invasion. These findings provide insights into mechanisms of single-domain antibody binding, and may enable design of reagents targeting otherwise cryptic epitopes in pathogen antigens.
Publisher: Elsevier BV
Date: 09-1997
DOI: 10.1016/S0166-6851(97)00095-9
Abstract: We have previously shown that a radioiodinated photoreactive analogue of chloroquine, [125I]N-(4-(4-diethylamino-1-methylbutylamino)quinolin-6-yl) -4-azido-2-hydroxybenzamide ([125I]ASA-Q), specifically labels two proteins in Plasmodium falciparum with apparent molecular weights (Mr) of 42 and 33 kDa (Foley M, Deady LW, Ng K, Cowman AF, Tilley L. J Biol Chem 1994:269:6955-6961). We now report the identification of the 33 kDa protein. The 33 kDa protein was purified from Plasmodium falciparum using photoaffinity labeling with [125I]ASA-Q to monitor the enrichment process. N-terminal sequence analysis of the purified protein revealed exact identity of the first 35 amino acids with P. falciparum lactate dehydrogenase (PfLDH). The plasmodial enzyme was cloned and expressed in E. coli and the recombinant protein used to produce a rabbit antiserum. Immunoprecipitation using affinity-purified anti-PfLDH antibodies confirmed the identity of the 33 kDa CQ-binding protein. The enzyme activity of purified PfLDH was not significantly affected by chloroquine indicating that PfLDH is not a direct target of CQ. PfLDH was, however, shown to be exquisitely sensitive to inhibition by free heme and chloroquine protected against this inhibitory effect.
Publisher: Elsevier BV
Date: 07-1996
Abstract: A head-group modified, photoreactive analog of phosphatidylethanolamine, N-([125I]iodo-4-azidosalicy- lamidyl)-1,2-dilauryl-sn-glycero-3-phosphatidylethanolamine ([125I]-N-ASA-DLPE), has been used in photoaffinity labeling studies of proteins of the human erythrocyte membrane. [125I]-N-ASA-DLPE was shown to be preferentially incorporated into a protein with an apparent molecular weight of 31 kDa. Protein sequencing and immunoprecipitation were used to identify this protein as the erythrocyte membrane protein, band 7.2b or stomatin. A sulphydryl-reactive ligand, 4-hydroxy-3-(iodo-[125I])-N-[2-(2-pyridinyldithio)ethyl]- benzenepropanamide ([125I]-PDA), was also shown to preferentially label band 7.2b. We propose that band 7.2b may act as a site of transbilayer reorientation of membrane phospholipids.
Publisher: Elsevier BV
Date: 02-1997
DOI: 10.1016/S0020-7519(96)00152-X
Abstract: The quinoline-containing antimalarial drugs, chloroquine, quinine and mefloquine, are a vital part of our chemotherapeutic armoury against malaria. These drugs are thought to act by interfering with the digestion of haemoglobin in the blood stages of the malaria life cycle. Chloroquine is a dibasic drug which diffuses down the pH gradient to accumulate about a 1000-fold in the acidic vacuole of the parasite. The high intravacuolar concentration of chloroquine is proposed to inhibit the polymerisation of haem. As a result, the haem which is released during haemoglobin breakdown builds up to poisonous levels, thereby killing the parasite with its own toxic waste. The more lipophilic quinolinemethanol drugs, mefloquine and quinine, are not concentrated so extensively in the food vacuole and probably have alternative sites of action. The technique of photoaffinity labelling has been used to identify a series of proteins which interact specifically with mefloquine. These studies have led us to speculate that the quinolinemethanols bind to high density lipoproteins in the serum and are delivered to the erythrocytes where they interact with an erythrocyte membrane protein, known as stomatin, and are then transferred to the intracellular parasite via a pathway used for the uptake of exogenous phospholipid. The final target(s) of quinine and mefloquine action are not yet fully characterised, but may include parasite proteins with apparent molecular weights of 22 kDa and 36 kDa. As resistance to the quinoline antimalarials rises inexorably, there is an urgent need to understand the molecular basis for decreased drug sensitivity. A parasite-encoded homologue of P-glycoprotein has been implicated in the development of drug resistance, possibly by controlling the level of accumulation of the quinoline-containing drugs. As our molecular understanding of these processes increases, it should be possible to design novel antimalarial strategies which circumvent the problem of drug resistance.
Publisher: Elsevier BV
Date: 11-1997
DOI: 10.1016/S0166-6851(97)00134-5
Abstract: An endoplasmic reticulum-located, calcium-binding protein, with an apparent molecular weight (Mr) of approximately 40,000 (PfERC), has been identified in the asexual stages of the malaria parasite, Plasmodium falciparum. This protein appears to be equivalent to a previously described gametocyte protein, Pfs40, which was reported to be expressed on the gametocyte surface (Rawlings DJ, Kaslow DC. J Biol Chem 1992 :3976-3982). Sequencing of the 3' end of the gene revealed the omission of a single base in the 3' region of the published sequence. The corrected gene sequence encodes a C-terminal IDEL motif, which indicates residency of the 40 kDa protein within the endoplasmic reticulum. The predicted C-terminal region also appears to contain a sixth EF-hand calcium-binding domain, which suggests that PfERC is related to previously reported ER-localized calcium-binding proteins, namely reticulocalbin and ERC-55 (Ozawa M. J. Biochem. 1995 :1113-1119 Weis K, Griffiths G, Lamond AI. J. Biol. Chem. 1994 :19142-19150). The presence of the 40 kDa calcium-binding protein in malaria parasites was confirmed using 45Ca2+-blotting and partial protein sequencing of the corresponding Coomassie blue-stained polypeptide. Confocal immunofluorescence microscopy of asexual stage parasites was used to show that PfERC co-localizes with the known ER-located protein, Pfgrp. Analysis of immunoblots of tightly synchronized parasites showed that expression of PfERC increases with increasing maturity of the parasite. We propose that PfERC is a member of the reticulocalbin family of calcium-binding proteins and may play a role in protein trafficking in the malaria parasite.
Publisher: Elsevier BV
Date: 1989
DOI: 10.1016/0020-711X(89)90014-1
Abstract: 1. The effect of different doses of six polypeptide hormones on pyrene diffusion in rat testes and liver plasma membranes was tested. Pyrene mobility was reduced in membranes possessing respective receptors. 2. An incubation time of 15 min of testes plasma membranes with 10(-5) M lutropin (LH) reduced pyrene and diphenyl hexatriene mobilities by 10-20%. 3. The addition of 10(-5) M LH to a suspension of intact L-cells from rats at different ages decreased the diffusion of membrane fluorescently labelled lipids and proteins by ca 60%. Diffusion was measured by fluorescence recovery after photobleaching. 4. Observed LH effects were independent of development and ageing.
Publisher: American Society for Microbiology
Date: 03-2006
DOI: 10.1128/JCM.44.3.764-771.2006
Abstract: Epstein-Barr virus (EBV) is a ubiquitous, worldwide infectious agent that causes infectious mononucleosis, affecting % of the world's population. Currently, enzyme-linked immunosorbent assay, mostly with purified preparations of EBV cell extracts to capture immunoglobulin M (IgM) antibodies in patients' serum, is used for primary diagnosis. Our objective was to determine whether a small set of peptides could contain sufficient immunogenic information to replace solid-phase antigens in EBV diagnostics. Using monoclonal antibodies, we selected four peptides that mimic different epitopes of EBV from a phage-displayed random peptide library. To assess their diagnostic value, we screened a panel of 62 in idual EBV IgM sera for their reactivities with the peptides alone. For all peptides, there was a clear distinction between the EBV-positive and the EBV-negative s les, resulting in 100% specificity. The sensitivities were 88%, 85%, 71%, and 54% for peptides F1, A3, gp125, and A2, respectively. Any combination of peptides increased the sensitivity, indicating that in idual peptides react with different subsets of antibodies. Furthermore, when the F1 and the gp125 peptides were coupled to bovine serum albumin and screened against 216 serum s les, there were dramatic improvements in sensitivities (95% and 92%, respectively) and little cross-reactivity with the other peptides encountered during acute viral infections, including rheumatoid factor. This study shows the potential for the use of peptide mimotopes as alternatives to the complex antigens used in current serodiagnostics for EBV infection.
Publisher: Elsevier BV
Date: 06-2001
DOI: 10.1016/S0166-6851(01)00271-7
Abstract: The histidine-rich protein 2 (PfHRP2) of Plasmodium falciparum has been implicated in the detoxification of ferriprotoporphyrin IX (FP) moieties that are produced as by-products of the digestion of haemoglobin. In this work, we have used a spectroscopic analysis to confirm that recombinant PfHRP2 binds FP. A monoclonal antibody that recognises both recombinant and authentic PfHRP2 was used in immunofluorescence microscopy studies. We found that PfHRP2 is mainly located in the erythrocyte cytosol of infected erythrocytes, however, dual labelling studies suggest that the location of a sub-population of the PfHRP2 molecules overlaps with that of the food vacuole-associated protein, P-glycoprotein homologue (Pgh-1). A semi-quantitative analysis of the level of PfHRP2 in infected erythrocytes suggests a concentration of a few micromolar in the food vacuole. Under conditions designed to mimic the parasite food vacuole, we found that 1.2 microM PfHRP2 is sufficient to catalyse the conversion of about 30% of a 100 microM s le of FP to beta-haematin within 24 h. Moreover, PfHRP2 is capable of promoting the H(2)O(2)-induced degradation of FP at pH 5.2. PfHRP2 also efficiently enhances the ability of FP to catalyse the H(2)O(2)-mediated oxidation of the model co-factor, ortho-phenylene diamine (OPD). These data suggest that PfHRP2 may promote the detoxification of FP and reactive oxygen species within the food vacuole. By contrast, PfHRP2 inhibits the destruction of FP by glutathione (GSH) at pH 7.4. This suggests that PfHRP2 is not a catalyst of FP degradation outside the food vacuole.
Publisher: American Society for Microbiology
Date: 10-2005
DOI: 10.1128/IAI.73.10.6981-6989.2005
Abstract: Apical membrane antigen 1 (AMA1) is expressed in schizont-stage malaria parasites and sporozoites and is thought to be involved in the invasion of host red blood cells. AMA1 is an important vaccine candidate, as immunization with this antigen induces a protective immune response in rodent and monkey models of human malaria. Additionally, anti-AMA1 polyclonal and monoclonal antibodies inhibit parasite invasion in vitro. We have isolated a 20-residue peptide (R1) from a random peptide library that binds to native AMA1 as expressed by Plasmodium falciparum parasites. Binding of R1 peptide is dependent on AMA1 having the proper conformation, is strain specific, and results in the inhibition of merozoite invasion of host erythrocytes. The solution structure of R1, as determined by nuclear magnetic resonance spectroscopy, contains two structured regions, both involving turns, but the first region, encompassing residues 5 to 10, is hydrophobic and the second, at residues 13 to 17, is more polar. Several lines of evidence reveal that R1 targets a “hot spot” on the AMA1 surface that is also recognized by other peptides and monoclonal antibodies that have previously been shown to inhibit merozoite invasion. The functional consequence of binding to this region by a variety of molecules is the inhibition of merozoite invasion into host erythrocytes. The interaction between these peptides and AMA1 may further our understanding of the molecular mechanisms of invasion by identifying critical functional regions of AMA1 and aid in the development of novel antimalarial strategies.
Publisher: American Chemical Society (ACS)
Date: 16-02-2001
DOI: 10.1021/JM0010724
Abstract: A series of terpene isonitriles, isolated from marine sponges, have previously been shown to exhibit antimalarial activities. Molecular modeling studies employing 3D-QSAR with receptor modeling methodologies performed with these isonitriles showed that the modeled molecules could be used to generate a pharmacophore hypothesis consistent with the experimentally derived biological activities. It was also shown that one of the modeled compounds, diisocyanoadociane (4), as well as axisonitrile-3 (2), both of which have potent antimalarial activity, interacts with heme (FP) by forming a coordination complex with the FP iron. Furthermore, these compounds were shown to inhibit sequestration of FP into beta-hematin and to prevent both the peroxidative and glutathione-mediated destruction of FP under conditions designed to mimic the environment within the malaria parasite. By contrast, two of the modeled diterpene isonitriles, 7-isocyano hilecta-11(20),15-diene (12) and 7-isocyano-15-isothiocyanato hilecta-11(20)-ene (13), that displayed little antimalarial activity also showed little inhibitory activity in these FP detoxification assays. These studies suggest that the active isonitrile compounds, like the quinoline antimalarials, exert their antiplasmodial activity by preventing FP detoxification. Molecular dynamics simulations performed with diisocyanoadociane (4) and axisonitrile-3 (2) allowed their different binding to FP to be distinguished.
Publisher: Oxford University Press (OUP)
Date: 24-11-2008
Publisher: American Society for Clinical Investigation
Date: 22-02-2022
Publisher: Springer Science and Business Media LLC
Date: 17-06-2008
Publisher: Informa UK Limited
Date: 2010
DOI: 10.4161/HV.6.1.10712
Abstract: It is now more than 25 years since asexual blood stage antigens of Plasmodium falciparum were first expressed as recombinant proteins. Although many asexual blood stage vaccine candidates have been identified, none has yet been fully evaluated in clinical trials. The results of studies in animal models, and from in vitro studies with P. falciparum, indicate that antibody responses induced by many of these recombinant proteins can inhibit parasite development, but so far the evidence that protection can be achieved in exposed human populations is limited. Recombinant forms of MSP2 and AMA1 expressed in E. coli have had significant effects in Phase II trials, although for both antigens the effect was against a subset of parasites expressing a form of these polymorphic antigens related to that in the vaccine. More knowledge of the antigenic structure of the native parasite antigens is required so that recombinant protein constructs can be optimized to induce the correct antibody fine specificity. The very different structural characteristics of MSP2 and AMA1 are discussed, as are some approaches being taken to overcoming the problem of ersity in these antigens.
Publisher: American Society for Microbiology
Date: 10-2004
DOI: 10.1128/IAI.72.10.6095-6105.2004
Abstract: Following invasion of human erythrocytes, the malaria parasite, Plasmodium falciparum , exports proteins beyond the confines of its own plasma membrane to modify the properties of the host red cell membrane. These modifications are critical to the pathogenesis of malaria. Analysis of the P. falciparum genome sequence has identified a large number of molecules with putative atypical signal sequences. The signals remain poorly characterized however, a number of molecules with these motifs localize to the host erythrocyte. To examine the role of these atypical signal sequences in the export of parasite proteins, we have generated transfected parasites expressing a chimeric protein comprising the N-terminal region of the P. falciparum ring-infected erythrocyte surface antigen (RESA) appended to green fluorescent protein (GFP). This N-terminal region contains a hydrophobic stretch of amino acids that is presumed to act as a noncanonical secretory signal sequence. Modulation of the timing of transgene expression demonstrates that trafficking of malaria proteins into the host erythrocyte is dependant on both the presence of an appropriate transport signal and the timing of expression. Transgene expression under the control of a trophozoite-specific promoter mistargets the chimeric molecule to the parasitophorous vacuole surrounding the parasite. However, expression of RESA-GFP in schizont stages, under the control of the RESA promoter, enables correct trafficking of a population of the chimeric protein to the host erythrocyte.
Publisher: Elsevier BV
Date: 1987
Publisher: American Society for Microbiology
Date: 11-2014
DOI: 10.1128/IAI.02061-14
Abstract: Apical membrane antigen 1 (AMA1) is a leading malarial vaccine candidate however, its polymorphic nature may limit its success in the field. This study aimed to circumvent AMA1 ersity by d ening the antibody response to the highly polymorphic loop Id, previously identified as a major target of strain-specific, invasion-inhibitory antibodies. To achieve this, five polymorphic residues within this loop were mutated to alanine, glycine, or serine in AMA1 of the 3D7 and FVO Plasmodium falciparum strains. Initially, the corresponding antigens were displayed on the surface of bacteriophage, where the alanine and serine but not glycine mutants folded correctly. The alanine and serine AMA1 mutants were expressed in Escherichia coli , refolded in vitro , and used to immunize rabbits. Serological analyses indicated that immunization with a single mutated form of 3D7 AMA1 was sufficient to increase the cross-reactive antibody response. Targeting the corresponding residues in an FVO backbone did not achieve this outcome. The inclusion of at least one engineered form of AMA1 in a biallelic formulation resulted in an antibody response with broader reactivity against different AMA1 alleles than combining the wild-type forms of 3D7 and FVO AMA1 alleles. For one combination, this extended to an enhanced relative growth inhibition of a heterologous parasite line, although this was at the cost of reduced overall inhibitory activity. These results suggest that targeted mutagenesis of AMA1 is a promising strategy for overcoming antigenic ersity in AMA1 and reducing the number of variants required to induce an antibody response that protects against a broad range of Plasmodium falciparum AMA1 genotypes. However, optimization of the immunization regime and mutation strategy will be required for this potential to be realized.
Publisher: American Chemical Society (ACS)
Date: 04-2008
DOI: 10.1021/BC7003523
Abstract: Fusion proteins based on the crystalline bacterial cell surface layer (S-layer) proteins SbpA from Bacillus sphaericus CCM 2177 and SbsB from Geobacillus stearothermophilus PV72 2 and a peptide mimotope F1 that mimics an immunodominant epitope of Epstein-Barr virus (EBV) were designed and overexpressed in Escherichia coli. Constructs were designed such that the peptide mimotope was presented either at the C-terminus (SbpA/F1) or at the N-terminus (SbsB/F1) of the respective S-layer proteins. The resulting S-layer fusion proteins, SbpA/F1 and SbsB/F1, fully retained the intrinsic self-assembly capability of the S-layer moiety into monomolecular lattices. As determined by immunodot assays and ELISAs using monoclonal antibodies, the F1 mimotope was well-presented on the outer surface of the S-layer lattices and accessible for antibody binding. Further comparison of the two S-layer fusion proteins showed that the S-layer fusion protein SbpA/F1 had a higher antibody binding capacity than SbsB/F1 in aqueous solution and in immune sera, illustrating the importance of epitope orientation on the performance of solid-phase immunoassays. To assess the diagnostic values of S-layer mimotope fusion protein SbpA/F1, we screened a panel of 83 in idual EBV IgM-positive, EBV negative, and potential cross-reactive sera for their reactivities. This resulted in 98.2% specificity and 89.3% sensitivity, and furthermore no cross-reactivity with related viral disease states including rheumatoid factor was observed. This study shows the potential of S-layer fusion proteins as a matrix for site-directed immobilization of small ligands in solid-phase immunoassays using EBV diagnostics as a model system.
Publisher: Springer Science and Business Media LLC
Date: 13-07-2011
DOI: 10.1038/NATURE10167
Publisher: Proceedings of the National Academy of Sciences
Date: 10-06-2003
Abstract: Multicomponent therapies, originating through deliberate mixing of drugs in a clinical setting, through happenstance, and through rational design, have a successful history in a number of areas of medicine, including cancer, infectious diseases, and CNS disorders. We have developed a high-throughput screening method for identifying effective combinations of therapeutic compounds. We report here that systematic screening of combinations of small molecules reveals unexpected interactions between compounds, presumably due to interactions between the pathways on which they act. Through systematic screening of ≈120,000 different two-component combinations of reference-listed drugs, we identified potential multicomponent therapeutics, including ( i ) fungistatic and analgesic agents that together generate fungicidal activity in drug-resistant Candida albicans , yet do not significantly affect human cells, ( ii ) glucocorticoid and antiplatelet agents that together suppress the production of tumor necrosis factor-α in human primary peripheral blood mononu-clear cells, and ( iii ) antipsychotic and antiprotozoal agents that do not exhibit significant antitumor activity alone, yet together prevent the growth of tumors in mice. Systematic combination screening may ultimately be useful for exploring the connectivity of biological pathways and, when performed with reference-listed drugs, may result in the discovery of new combination drug regimens.
Publisher: Elsevier BV
Date: 07-1994
DOI: 10.1016/0166-6851(94)90036-1
Abstract: The ring-infected erythrocyte surface antigen (RESA) associates with spectrin in the erythrocyte membrane (Foley, M., Tilley, L., Sawyer, W. H. and Anders, R. F. (1991) Mol. Biochem. Parasitol., 46, 137-148). A fragment of the RESA protein, which was expressed in Escherichia coli, was found to bind to inside-out vesicles of erythrocyte membranes in an apparently saturable manner. Upon extraction of inside-out vesicles with Triton X-100, the RESA fragment remained associated with the erythrocyte cytoskeleton. Using the technique of steady-state fluorescence polarisation, we have studied the thermal denaturation of fluorescein-labelled spectrin in the presence of recombinant RESA. We found that the RESA fragment partially protected spectrin against heat-induced conformational changes. Furthermore, erythrocytes infected with a RESA (-) laboratory strain (FCR3) were shown to be more susceptible to heat-induced fragmentation than erythrocytes infected with a RESA (+) strain of the parasite. RESA does not, however, appear to play an essential role in the invasion process per se as erythrocytes resealed to contain anti-RESA antibodies were efficiently invaded.
Publisher: Rockefeller University Press
Date: 09-1986
Abstract: The technique of fluorescence recovery after photobleaching was used to measure the lateral diffusion of fluorescent lipid analogues in the surface membrane of Schistosoma mansoni. Our data reveal that although some lipids could diffuse freely others exhibited restricted lateral diffusion. Quenching of lipid fluorescence by a non-permeant quencher, trypan blue, showed that there was an asymmetric distribution of lipids across the double bilayer of mature parasites. Those lipids that diffused freely were found to reside mainly in the external monolayer of the outer membrane whereas lipids with restricted lateral diffusion were located mainly in one or more of the monolayers beneath the external monolayer. Formation of surface membrane blebs allowed us to measure the lateral diffusion of lipids in the membrane without the influence of underlying cytoskeletal structures. The restricted diffusion found on the normal surface membrane of mature parasites was found to be released in membrane blebs. Quenching of fluorescent lipids on blebs indicated that all probes were present almost entirely in the external monolayer. Juvenile worms exhibited lower lateral diffusion coefficients than mature parasites: in addition, the lipids partitioned into the external monolayer. The results are discussed in terms of membrane organization, cytoskeletal contacts, and biological significance.
Publisher: Public Library of Science (PLoS)
Date: 28-09-2007
Publisher: Elsevier BV
Date: 05-1991
DOI: 10.1016/0166-6851(91)90207-M
Abstract: The malaria parasite Plasmodium falciparum synthesises a protein, RESA, which associates with the membrane of newly invaded erythrocytes. Using spent supernatants from P. falciparum growing in culture as a source of soluble RESA we have developed an assay to examine the characteristics of RESA binding to the erythrocyte membrane in vitro. RESA associated with the Triton X-100 insoluble proteins on the inner face of the host erythrocyte membrane but did not bind to the outer surface of intact erythrocytes. Other proteins present in culture supernatants did not bind to the erythrocyte membrane. RESA was co-sedimented with the ternary complex formed between actin, spectrin and band 4.1 and co-precipitated with spectrin precipitated with anti-spectrin antibodies. The extent of association between RESA and the inner face of the erythrocyte membrane was reduced by the inclusion of excess purified spectrin in the assay. Thus, RESA appears to be associated with spectrin in the erythrocyte membrane skeleton.
Publisher: Elsevier BV
Date: 11-1998
DOI: 10.1016/S0020-7519(98)00132-5
Abstract: The malaria parasite invades the human erythrocyte and converts this simple "sack of haemoglobin" back into a functional eukaryotic cell. Parasite-encoded proteins are trafficked to the red blood cell membrane where they modify its properties to meet the needs of the intracellular parasite. Trafficking of proteins within the parasite probably occurs via a "classical" vesicle-mediated secretory pathway however, the transit of proteins from the parasite plasma membrane to the erythrocyte membrane appears to involve both a novel vesicle-mediated pathway and a direct protein-translocation system. The polypeptide signals that direct parasite proteins into these novel export pathways may include an unusual "internal" hydrophobic sequence, as well as a series of basic motifs.
Publisher: Cambridge University Press (CUP)
Date: 02-1988
DOI: 10.1017/S0031182000081671
Abstract: Merocyanin 540 (Mc540) is a fluorescent compound which is thought to bind to membranes in which there are substantial amounts of lipid in the lipid-crystalline phase. It is shown here to be of value in detecting the transformation by both mechanical and skin-penetration methods of the cercaria to the schistosomulum. The cercaria does not appear to bind Mc540, but the schistosomulum, binds Mc540 initially, in its anterior region, and at later times over the entire surface. The suggestion that transformation involves changes in the surface membrane lipid phase from gel to liquid-crystalline phase is supported by fluorescence recovery after photobleaching results with 5- N -(octadecanoyl)-amino fluorescein, a lipophilic dye which appears to be immobile in the cercaria, but fully mobile in the 40 min schistosomulum.
Publisher: Elsevier BV
Date: 08-1996
DOI: 10.1016/0006-2952(96)00306-1
Abstract: We report the synthesis of two series of novel bisquinoline compounds that inhibit the growth of both chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum. To study the molecular basis of the action of these novel antimalarial drugs, we examined their ability to inhibit haem polymerisation in the presence and absence of parasite extracts. The level of antimalarial potency was correlated with the level of inhibition of haem polymerisation, suggesting that these bisquinolines exert their antimalarial activity by antagonising the sequestration of toxic haem moieties.
Publisher: Public Library of Science (PLoS)
Date: 26-12-2013
Publisher: American Chemical Society (ACS)
Date: 30-07-2003
DOI: 10.1021/BI034376B
Abstract: Apical membrane antigen 1 (AMA1) of the human malaria parasite Plasmodium falciparum is synthesized by schizont stage parasites and has been implicated in merozoite invasion of host erythrocytes. Phage-display techniques have recently been used to identify two 15-residue peptides, F1 and F2, which bind specifically to P. falciparum AMA1 and inhibit parasite invasion of erythrocytes [Li, F., et al. (2002) J. Biol. Chem. 277, 50303-50310]. We have synthesized F1, F2, and three peptides with high levels of sequence identity, determined their relative binding affinities for P. falciparum AMA1 with a competition ELISA, and investigated their solution structures by NMR spectroscopy. The strongest binding peptide, F1, contains a beta-turn that includes residues identified via an alanine scan as being critical for binding to AMA1 and inhibition of merozoite invasion of erythrocytes. The three F1 analogues include a 10-residue analogue of F1 truncated at the C-terminus (tF1), a partially scrambled 15-mer (sF1), and a disulfide-constrained 14-mer (F1tbp) which is related to F1 but has a sequence identical to that of a disulfide-constrained loop in the first epidermal growth factor module of the latent transforming growth factor-beta binding protein. tF1 and F1tbp bound competitively with F1 to AMA1, and all three contain a type I beta-turn encompassing key residues involved in F1 binding. In contrast, sF1 lacked this structural motif, and did not compete for binding to AMA1 with F1 rather, sF1 contained a type III beta-turn involving a different part of the sequence. Although F2 was able to bind to AMA1, it was unstructured in solution, consistent with its weak invasion inhibitory effects. Thus, the secondary structure elements observed for these peptides in solution correlate well with their potency in binding to AMA1 and inhibiting merozoite invasion. The structures provide a valuable starting point for the development of peptidomimetics as antimalarial antagonists directed at AMA1.
Publisher: American Society for Microbiology
Date: 02-2004
DOI: 10.1128/IAI.72.2.1126-1134.2004
Abstract: Apical membrane antigen 1 (AMA1) is expressed on the surfaces of Plasmodium falciparum merozoites and is thought to play an important role in the invasion of erythrocytes by malaria parasites. To select for peptides that mimic conformational B-cell epitopes on AMA1, we screened a phage display library of 8 in idual peptides for peptides bound by a monoclonal anti-AMA1 antibody, 4G2dc1, known to inhibit P. falciparum invasion of erythrocytes. The most reactive peptides, J1, J3, and J7, elicited antibody responses in rabbits that recognized the peptide immunogen and both recombinant and parasite AMA1. Human antibodies in plasma s les from in iduals exposed to chronic malaria reacted with J1 and J7 peptides and were isolated using immobilized peptide immunoadsorbents. Both rabbit and human antibodies specific for J1 and J7 peptides were able to inhibit the invasion of erythrocytes by P. falciparum merozoites. This is the first ex le of phage-derived peptides that mimic an important epitope of a blood-stage malaria vaccine candidate, inducing and isolating functional protective antibodies. Our data support the use of J1 and J7 peptide mimics as in vitro correlates of protective immunity in future AMA1 vaccine trials.
Publisher: Elsevier BV
Date: 06-2002
DOI: 10.1016/S0264-410X(02)00197-4
Abstract: Antibodies have the potential to be therapeutic reagents for malaria. Here we describe the production of a novel phage antibody display library against the C-terminal 19kDa region of the Plasmodium yoelii YM merozoite surface protein-1 (MSP1(19)). In vivo studies against homologous lethal malaria challenge show an anti-parasite effect in a dose dependent manner, and analysis by plasmon resonance indicates binding to the antigen is comparable to the binding of a protective monoclonal antibody. The data support the lack of a need for any antibody Fc-related function and hold great significance for the development of a therapeutic reagent for malaria.
Publisher: Wiley
Date: 06-01-2011
DOI: 10.1002/BIP.21582
Publisher: Elsevier BV
Date: 02-2003
Publisher: American Society for Microbiology
Date: 2007
DOI: 10.1128/IAI.01041-06
Abstract: Apical membrane antigen 1 (AMA1) of the malaria parasite Plasmodium falciparum is an integral membrane protein that plays a key role in merozoite invasion of host erythrocytes. A monoclonal antibody, 4G2dc1, recognizes correctly folded AMA1 and blocks merozoite invasion. Phage display was used to identify peptides that bind to 4G2dc1 and mimic an important epitope of AMA1. Three of the highest-affinity binders—J1, J3, and J7—were chosen for antigenicity and immunogenicity studies. J1 and J7 were found to be true antigen mimics since both peptides generated inhibitory antibodies in rabbits (J. L. Casey et al., Infect. Immun. 72:1126-1134, 2004). In the present study, the solution structures of all three mimotopes were investigated by nuclear magnetic resonance spectroscopy. J1 adopted a well-defined region of structure, which can be attributed in part to the interactions of Trp11 with surrounding residues. In contrast, J3 and J7 did not adopt an ordered conformation over the majority of residues, although they share a region of local structure across their consensus sequence. Since J1 was the most structured of the peptides, it provided a template for the design of a constrained analogue, J1cc, which shares a structure similar to that of J1 and has a disulfide-stabilized conformation around the Trp11 region. J1cc binds with greater affinity to 4G2dc1 than does J1. These peptide structures provide the foundation for a better understanding of the complex conformational nature of inhibitory epitopes on AMA1. With its greater conformational stability and higher affinity for AMA1, J1cc may be a better in vitro correlate of immunity than the peptides identified by phage display.
Publisher: Elsevier BV
Date: 06-1990
DOI: 10.1016/0005-2736(90)90090-B
Abstract: Time-resolved phosphorescence anisotropy was used to study the molecular organisation of band 3 in the erythrocyte membrane. Three different rotational relaxation regimes of mobile band 3 were resolved. These populations may represent different aggregation states of band 3 within the membrane, or they may result from association of band 3 with other proteins at the cytoplasmic surface. The polycation spermine decreases the apparent mobility of band 3 by a mechanism that does not involve the underlying cytoskeleton. A monoclonal antibody directed against the cytoplasmic portion of band 3 can also cause an increase in the immobile fraction of band 3 molecules. This monoclonal antibody will inhibit invasion of erythrocytes by malaria parasites. Membranes prepared from erythrocytes infected with mature stages of the malaria parasite, Plasmodium falciparum, show altered dynamic properties corresponding to a marked restriction of band 3 mobility.
Publisher: Elsevier BV
Date: 07-1999
Publisher: Elsevier BV
Date: 11-1995
DOI: 10.1016/0169-4758(95)80032-8
Abstract: In real-estate agent's terms, the red blood cell is a renovator's dream. The mature human erythrocyte has no internal organelles, no protein synthesis machinery and no infrastructure for protein trafficking. The malaria parasite invades this empty shell and effectively converts the erythrocyte back into a fully functional eukaryotic cell. In this article, Michael Foley and Leann Tilley examine the Plasmodium falciparum proteins that interact with the membrane skeleton at different stages of the infection and speculate on the roles of these proteins in the remodelling process.
Publisher: Elsevier BV
Date: 02-2009
Publisher: Elsevier BV
Date: 11-1994
DOI: 10.1016/0166-6851(94)00149-9
Abstract: A cDNA clone encoding part of a novel polymorphic merozoite antigen from Plasmodium falciparum was isolated by screening a cDNA library with human immune serum from Papua New Guinea. Immunofluorescence microscopy and immunoblotting with affinity-purified antibodies recognized a highly polymorphic antigen, Ag956, present in schizonts and merozoites. Biosynthetic labeling and immunoprecipitation experiments demonstrated that Ag956 is proteolytically cleaved during merozoite maturation. The complete genomic sequence of Ag956 from the D10 clone of P. falciparum isolate FC27 encodes a secreted protein of calculated molecular mass 43,243 that is very hydrophilic and contains a region of unusual heptad repeats of the general structure AXXAXXX. This antigen has been named the secreted polymorphic antigen associated with merozoites (SPAM). The sequence of a second SPAM allele from the 3D7 clone of isolate NF54 reveals that the alanine heptad repeats and the hydrophilic C-terminal half of the protein are conserved. Variation among SPAM alleles is the result of deletions and amino acid substitutions in non-repetitive sequences within and flanking the alanine heptad-repeat domain. Heptad repeats in which the a and d position contain hydrophobic residues generate hipathic alpha-helices which give rise to helical bundles or coiled-coil structures in proteins. Thus, SPAM is the first ex le of a P. falciparum antigen in which a repetitive sequence has features characteristic of a well-defined structural element.
Publisher: Elsevier BV
Date: 11-1996
DOI: 10.1016/S0006-2952(96)00556-4
Abstract: MDR1 P-glycoprotein in membranes of human tumor cells of the CEM/VBL100 line was selectively labelled using photoreactive analogs of verapamil, N-(p-azido-3-[125I]salicyl)amino-verapamil ([125I]ASA-V) and prazosin, 2-[4-(4-azido-3-[125I]iodobenzoyl)piperazin-1-yl]4 -amino-6,7-dimethoxyyquinazoline ([125I]ASA-P). Mefloquine, a quinolinemethanol antimalarial drug, was shown to inhibit the labelling of P-glycoprotein with an efficiency similar to that for verapamil, a known chemosensitizer. By contrast, chloroquine competed poorly for the binding site on P-glycoprotein. Mefloquine also inhibited the functional activity of P-glycoprotein. It decreased the rates of extrusion of [3H]vinblastine and the fluorescent dyes, fluo-3 acetomethoxy ester and rhodamine 123, from drug-resistant cells and decreased the level of resistance of these cells to vinblastine. The ability of mefloquine to inhibit P-glycoprotein function may be involved in the neurotoxic side-effects occasionally associated with the use of mefloquine as an antimalarial drug.
Publisher: Elsevier BV
Date: 11-1986
Publisher: Elsevier BV
Date: 11-1994
Abstract: The ring-infected erythrocyte surface antigen (RESA) associates with spectrin in the erythrocyte membrane (Foley, M., Tilley, L., Sawyer, W. H., and Anders, R. F., 1991, Mol. Biochem. Parasitol. 46, 137-148). In this study, we have used deletion mutagenesis combined with an in vitro binding assay to identify a region of the RESA polypeptide that is involved in the attachment of this parasite protein to the host membrane skeleton. It was found that the tandem repeat sequences of RESA do not appear to be involved in the association of this protein with the erythrocyte membrane and that large deletions of the N-terminal region of RESA did not affect binding. The membrane-binding domain has been mapped to a 48-amino-acid region of RESA located between the two blocks of repetitive amino acid sequence. This binding domain does not overlap the region of RESA that shares homology with the Escherichia coli molecular chaperone, DnaJ. Identification of the amino acid sequence which is critical for the binding of RESA to erythrocyte spectrin may provide important clues to the functional consequences of RESA attachment to the host membrane.
Publisher: Bentham Science Publishers Ltd.
Date: 02-2002
Abstract: Phage display has become a widely used tool for the identification of proteins or peptides with affinity for a variety of biomolecules. The versatility, simplicity and cost effectiveness of this application has pervaded a wide variety of research areas. Although not without its limitations, phage display has provided a convenient methodology for obtaining ligands to study the function, structure and diagnostic or therapeutic potential of various macromolecules. This review highlights some recent research employing this technology that serves to illustrate its utility in various research and clinical applications.
Publisher: MDPI AG
Date: 25-01-2013
DOI: 10.3390/ANTIB2010066
Publisher: Elsevier BV
Date: 09-2002
DOI: 10.1016/S0022-2836(02)00806-9
Abstract: Apical membrane antigen 1 of the malarial parasite Plasmodium falciparum (Pf AMA1) is a merozoite antigen that is considered a strong candidate for inclusion in a malaria vaccine. Antibodies reacting with disulphide bond-dependent epitopes in AMA1 block invasion of host erythrocytes by P.falciparum merozoites, and we show here that epitopes involving sites of mutations in domain III are targets of inhibitory human antibodies. The solution structure of AMA1 domain III, a 14kDa protein, has been determined using NMR spectroscopy on uniformly 15N and 13C/15N-labelled s les. The structure has a well-defined disulphide-stabilised core region separated by a disordered loop, and both the N and C-terminal regions of the molecule are unstructured. Within the disulphide-stabilised core, residues 443-447 form a turn of helix and residues 495-498 and 503-506 an anti-parallel beta-sheet with a distorted type I beta-turn centred on residues 500-501, producing a beta-hairpin-type structure. The structured region of the molecule includes all three disulphide bonds. The previously unassigned connectivities for two of these bonds could not be established with certainty from the NMR data and structure calculations, but were determined to be C490-C507 and C492-C509 from an antigenic analysis of mutated forms of this domain expressed using phage display. Naturally occurring mutations in domain III that are located far apart in the primary sequence tend to cluster in the region of the disulphide core in the three-dimensional structure of the molecule. The structure shows that nearly all the polymorphic sites have a high level of solvent accessibility, consistent with their location in epitopes recognised by protective antibodies. Even though domain III in solution contains significant regions of disorder in the structure, the disulphide-stabilised core that is structured is clearly an important element of the antigenic surface of AMA1 recognised by protective antibodies.
Publisher: Elsevier BV
Date: 1990
DOI: 10.1016/0166-6851(90)90206-2
Abstract: The ring-infected erythrocyte surface antigen (RESA) is a 155-kDa malarial polypeptide which is released from merozoites and becomes associated with the erythrocyte membrane at the time of invasion. Inside-out vesicles (IOVs) prepared from Plasmodium falciparum-infected erythrocytes contain RESA, presumably bound to the membrane skeleton, as it is largely insoluble in Triton X-100. When these IOVs were incubated with [gamma-32P]ATP, a 155-kDa polypeptide was labeled in IOVs from infected, but not from uninfected erythrocytes. Immunoprecipitation using specific rabbit antisera confirmed that RESA is indeed a phosphoprotein. Phosphoamino acid analysis revealed phosphoserine and a small amount of phosphothreonine, but no phosphotyrosine. Labeling of intact parasitized erythrocytes with inorganic [32P]phosphate for several hours in culture resulted in RESA in Triton-insoluble extracts being phosphorylated. Labeling of synchronized parasites showed that RESA was phosphorylated only when it became associated with the erythrocyte membrane, and although RESA was abundant in mature parasites, it was not phosphorylated. RESA, released into the culture supernatants during the growth of P. falciparum, bound to IOVs prepared from normal uninfected erythrocytes, and subsequent labeling with [gamma-32P]ATP resulted in the phosphorylation of RESA. The evidence suggests that RESA is phosphorylated by an erythrocyte membrane kinase and probably not by a parasite-encoded enzyme.
Location: United Kingdom of Great Britain and Northern Ireland
Start Date: 2020
End Date: 09-2023
Amount: $506,000.00
Funder: Australian Research Council
View Funded ActivityStart Date: 09-2004
End Date: 08-2009
Amount: $1,500,000.00
Funder: Australian Research Council
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