Characterisation of the binding sites of monoclonal antibodies reacting with the Plasmodium falciparum rhoptry protein RhopH3

Malaria causes a half a million deaths annually. The parasite intraerythrocytic lifecycle in the human bloodstream is the major cause of morbidity and mortality. Apical organelles of merozoite stage parasites are involved in the invasion of erythrocytes. A limited number of apical organellar proteins have been identified and characterized for their roles during erythrocyte invasion or subsequent intraerythrocytic parasite development. To expand the repertoire of identified apical organellar proteins we generated a panel of monoclonal antibodies against Plasmodium falciparum schizont-rich parasites and screened the antibodies using immunofluorescence assays. Out of 164 hybridoma lines, 12 clones produced monoclonal antibodies yielding punctate immunofluorescence staining patterns in individual merozoites in late schizonts, suggesting recognition of merozoite apical organelles. Five of the monoclonal antibodies were used to immuno-affinity purify their target antigens and these antigens were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two known apical organelle protein complexes were identified, the high-molecular mass rhoptry protein complex (PfRhopH1/Clags, PfRhopH2, and PfRhopH3) and the low-molecular mass rhoptry protein complex (rhoptry-associated proteins complex, PfRAP1, and PfRAP2). A novel complex was additionally identified by immunoprecipitation, composed of rhoptry-associated membrane antigen (PfRAMA) and rhoptry neck protein 3 (PfRON3) of P. falciparum. We further identified a region spanning amino acids Q221-E481 within the PfRAMA that may associate with PfRON3 in immature schizonts. Further investigation will be required as to whether PfRAMA and PfRON3 interact directly or indirectly.

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Combination Assays and Molecular Docking can Identify Binding sites of Anti-Microtubule Drugs on Plasmodium falciparum Tubulin

Infectious Disorders - Drug Targets ◽

10.2174/1871526516666160711164905 ◽

2016 ◽

Vol 16 (3) ◽

pp. 204-216

Author(s):

Rimi Chakrabarti ◽

Swati Patankar

Keyword(s):

Plasmodium Falciparum ◽

Molecular Docking ◽

Binding Sites

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Structural analysis of the SRP Alu domain from Plasmodium falciparum reveals a non-canonical open conformation

Communications Biology ◽

10.1038/s42003-021-02132-y ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Komal Soni ◽

Georg Kempf ◽

Karen Manalastas-Cantos ◽

Astrid Hendricks ◽

Dirk Flemming ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Binding Sites ◽

Signal Recognition Particle ◽

Signal Recognition ◽

Closed Conformation ◽

Open Conformation ◽

Structural Database ◽

Alu Rna ◽

Species Specific ◽

Protozoan Species

AbstractThe eukaryotic signal recognition particle (SRP) contains an Alu domain, which docks into the factor binding site of translating ribosomes and confers translation retardation. The canonical Alu domain consists of the SRP9/14 protein heterodimer and a tRNA-like folded Alu RNA that adopts a strictly ‘closed’ conformation involving a loop-loop pseudoknot. Here, we study the structure of the Alu domain from Plasmodium falciparum (PfAlu), a divergent apicomplexan protozoan that causes human malaria. Using NMR, SAXS and cryo-EM analyses, we show that, in contrast to its prokaryotic and eukaryotic counterparts, the PfAlu domain adopts an ‘open’ Y-shaped conformation. We show that cytoplasmic P. falciparum ribosomes are non-discriminative and recognize both the open PfAlu and closed human Alu domains with nanomolar affinity. In contrast, human ribosomes do not provide high affinity binding sites for either of the Alu domains. Our analyses extend the structural database of Alu domains to the protozoan species and reveal species-specific differences in the recognition of SRP Alu domains by ribosomes.

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In vitro and in vivo inhibition of malaria parasite infection by monoclonal antibodies against Plasmodium falciparum circumsporozoite protein (CSP)

Scientific Reports ◽

10.1038/s41598-021-84622-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Merricka C. Livingstone ◽

Alexis A. Bitzer ◽

Alish Giri ◽

Kun Luo ◽

Rajeshwer S. Sankhala ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Monoclonal Antibodies ◽

Disease Burden ◽

Vaccine Candidate ◽

Specific Binding ◽

Circumsporozoite Protein ◽

Target Epitope ◽

Selection Of

AbstractPlasmodium falciparum malaria contributes to a significant global disease burden. Circumsporozoite protein (CSP), the most abundant sporozoite stage antigen, is a prime vaccine candidate. Inhibitory monoclonal antibodies (mAbs) against CSP map to either a short junctional sequence or the central (NPNA)n repeat region. We compared in vitro and in vivo activities of six CSP-specific mAbs derived from human recipients of a recombinant CSP vaccine RTS,S/AS01 (mAbs 317 and 311); an irradiated whole sporozoite vaccine PfSPZ (mAbs CIS43 and MGG4); or individuals exposed to malaria (mAbs 580 and 663). RTS,S mAb 317 that specifically binds the (NPNA)n epitope, had the highest affinity and it elicited the best sterile protection in mice. The most potent inhibitor of sporozoite invasion in vitro was mAb CIS43 which shows dual-specific binding to the junctional sequence and (NPNA)n. In vivo mouse protection was associated with the mAb reactivity to the NANPx6 peptide, the in vitro inhibition of sporozoite invasion activity, and kinetic parameters measured using intact mAbs or their Fab fragments. Buried surface area between mAb and its target epitope was also associated with in vivo protection. Association and disconnects between in vitro and in vivo readouts has important implications for the design and down-selection of the next generation of CSP based interventions.

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THE MOLECULAR ORGANIZATION OF HUMAN ALPHA 2-MACROGLOBULIN. AN IMMUNO ELECTRON MICROSCOPIC STUDY WITH MONOCLONAL ANTIBODIES

10.1055/s-0038-1643864 ◽

1987 ◽

Author(s):

E Delain ◽

M Barrav ◽

J Tapon-Bretaudière ◽

F Pochon ◽

F Van Leuven

Keyword(s):

Monoclonal Antibodies ◽

Binding Sites ◽

Divalent Cations ◽

The Other ◽

Molecular Organization ◽

Cellular Receptor ◽

Electron Microscopic ◽

Microscopic Method ◽

Bait Region ◽

Electron Microscopic Method

Electron microscopy is a very convenient method to localize the epitopes of monoclonal antibodies (mAbs) at the surface of macromolecules for studying their tree-dimensional organization.We applied this immuno-electron microscopic method to human ct2-macroglobulin (ct2M). 29 anti-α2M mAbs have been tested with the four different forms of a2M : native and chymotrypsin-transformed tetramers, and the corresponding dimers, obtained by dissociation with divalent cations. These mAbs can be classified in three types : those which are specific for 1) the H-like transformed molecules, 2) the native molecules, and 3) those which can react with both forms of α2M.1) Among the H-like α2M specific mAbs, several react with the 20 kD-domain which is recognized by the cellular receptor of transformed a2M. This domain is located at the carboxyterminal end of each monomer. One IgG binds to the end of two adjacent tips of the H-like form.The other mAbs of this type bind to the α2M tips at non-terminal positions. Intermolecular connections built polymers of alternating α2M and IgG molecules.2) Among the native a2M-specific mAbs some are able to inhibit the protease-induced transformation of the native α2M. The binding sites of these mAbs are demonstrated on the native half-molecules. One of these mAbs was also able to react with transformed dimers, in a region corresponding very likely to an inaccessible epitope in the tetrameric transformed α2M molecule.3) Among the mAbs of this type, only two were able to inhibit the protease-induced transformation of α2M. Obviously, their epitopes should be close to the bait region of α2M. The other mAbs reacting with both α2M forms did not inhibit the α2M transformation.All these mAbs can be distinguished by the structure of the immune complexes formed with all forms of α2M. The epitopes are more easily located on the dimers and on the H-like transformed α2M than on the native molecules.From these observations, we propose a new model of the tree-dimensional organization of the human α2M in its native and transformed configurations, and of its protease-induced transformation.

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Antibodies to the Plasmodium falciparum rhoptry protein RAP-2/RSP-2 in relation to anaemia in Cameroonian children

Parasite Immunology ◽

10.1111/j.1365-3024.2010.01259.x ◽

2011 ◽

Vol 33 (2) ◽

pp. 104-115 ◽

Cited By ~ 7

Author(s):

N. AWAH ◽

H. BALOGUN ◽

E. ACHIDI ◽

L. A. MARIUBA ◽

P. A. NOGUEIRA ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Rhoptry Protein

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Inhibition of acanthamoeba actomyosin-II ATPase activity and mechanochemical function by specific monoclonal antibodies.

The Journal of Cell Biology ◽

10.1083/jcb.99.3.1024 ◽

1984 ◽

Vol 99 (3) ◽

pp. 1024-1033 ◽

Cited By ~ 16

Author(s):

D P Kiehart ◽

T D Pollard

Keyword(s):

Monoclonal Antibodies ◽

Atpase Activity ◽

Conformational Changes ◽

Binding Sites ◽

Polyclonal Antibodies ◽

Myosin Ii ◽

Antigenic Site ◽

Actin Binding ◽

Filamentous Form ◽

Antibody Effects

Monoclonal and polyclonal antibodies that bind to myosin-II were tested for their ability to inhibit myosin ATPase activity, actomyosin ATPase activity, and contraction of cytoplasmic extracts. Numerous antibodies specifically inhibit the actin activated Mg++-ATPase activity of myosin-II in a dose-dependent fashion, but none blocked the ATPase activity of myosin alone. Control antibodies that do not bind to myosin-II and several specific antibodies that do bind have no effect on the actomyosin-II ATPase activity. In most cases, the saturation of a single antigenic site on the myosin-II heavy chain is sufficient for maximal inhibition of function. Numerous monoclonal antibodies also block the contraction of gelled extracts of Acanthamoeba cytoplasm. No polyclonal antibodies tested inhibited ATPase activity or gel contraction. As expected, most antibodies that block actin-activated ATPase activity also block gel contraction. Exceptions were three antibodies M2.2, -15, and -17, that appear to uncouple the ATPase activity from gel contraction: they block gel contraction without influencing ATPase activity. The mechanisms of inhibition of myosin function depends on the location of the antibody-binding sites. Those inhibitory antibodies that bind to the myosin-II heads presumably block actin binding or essential conformational changes in the myosin heads. A subset of the antibodies that bind to the proximal end of the myosin-II tail inhibit actomyosin-II ATPase activity and gel contraction. Although this part of the molecule is presumably some distance from the ATP and actin-binding sites, these antibody effects suggest that structural domains in this region are directly involved with or coupled to catalysis and energy transduction. A subset of the antibodies that bind to the tip of the myosin-II tail appear to inhibit ATPase activity and contraction through their inhibition of filament formation. They provide strong evidence for a substantial enhancement of the ATPase activity of myosin molecules in filamentous form and suggest that the myosin filaments may be required for cell motility.

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Euonymus europaeus lectin as an endothelial and epithelial marker in canine tissues

Laboratory Animals ◽

10.1258/002367792780745850 ◽

1992 ◽

Vol 26 (2) ◽

pp. 114-121 ◽

Cited By ~ 2

Author(s):

F. Roussell ◽

J. Dalion ◽

J. C. Wissocq

Keyword(s):

Endothelial Cells ◽

Monoclonal Antibodies ◽

Binding Site ◽

Binding Sites ◽

Human Tissues ◽

Probable Structure ◽

Euonymus Europaeus ◽

Epithelial Marker ◽

Endothelial Marker

The Euonymus europaeus agglutinin (EEA) is an endothelial marker in mammalia. In canine tissues, 4 types of endothelial cells (general, nervous, arterial, hepatic) were identified by the presence of the EEA receptor and by its sensitivity to neuraminidase enhancement. In adult dogs, EEA binding saccharides had endothelial or epithelial distributions and reactivities similar to those described for human tissues. Different EEA reactivities were observed between fetal, neonatal and adult canine tissues mainly at the arterial level. These findings suggest that the development of the binding sites is not identical in dog and man. Related lectins and monoclonal antibodies were used to characterize the EEA binding site, and the probable structure of the EEA binding saccharide in endothelial cells appeared to be αGal (1,3) βGal (1,4) GIcNAc.

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Cell adhesion and phagocytosis promoted by monoclonal antibodies not directed against fibronectin receptors

Journal of Cell Science ◽

10.1242/jcs.90.2.201 ◽

1988 ◽

Vol 90 (2) ◽

pp. 201-214 ◽

Cited By ~ 1

Author(s):

F. Grinnell ◽

C.H. Ho ◽

T.L. Tuan

Keyword(s):

Monoclonal Antibodies ◽

Cell Adhesion ◽

Binding Sites ◽

Cell Spreading ◽

The Other ◽

Immunofluorescence Staining ◽

Baby Hamster Kidney ◽

Bhk Cells ◽

Reducing Conditions ◽

Latex Beads

In this report we describe cell adhesion and phagocytosis promoted by two monoclonal antibodies that were selected for immunofluorescence staining of non-permeabilized baby hamster kidney (BHK) cells. Anti-BHK1 staining was heaviest along cell margins, whereas anti-BHK2 staining was continuous along cell margins. Neither antibody stained elongated plaque structures such as were observed when cells were reacted with antibodies to fibronectin (FN) receptors. The monoclonal antibodies functioned as adhesion ligands in four different assays: attachment to culture dishes, spreading, binding of latex beads and phagocytosis. Anti-BHK1 and anti-BHK2 promoted attachment to culture dishes similarly, but anti-BHK2 was more effective at promoting cell spreading. Antibody-promoted cell spreading was inhibited by the peptides Ser-Asp-Gly-Arg and Gly-Arg-Gly-Asp-Ser-Pro but not by other, related, peptides tested. The monoclonal antibodies also promoted binding of latex beads, and the bead binding sites were motile, on the basis of their ‘capping’ response. Nevertheless, anti-BHK2 beads were phagocytosed by cells 5- to 20-fold more efficiently than anti-BHK1 beads. The binding sites for anti-BHK1 and anti-BHK2 were characterized by immunoprecipitation experiments. Anti-BHK1 binding sites contained 50K (K = 10(3) Mr) and 88K components under non-reducing conditions that migrated as a 51/53K doublet and a 93K component under reducing conditions. On the other hand, anti-BHK2 binding sites contained 88K and 110K components under non-reducing conditions that shifted to apparent 107K and 128K values when measured under reducing conditions.

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