Characterization of a 225 kilodalton rhoptry protein of Plasmodium falciparum

doi:10.1016/0166-6851(88)90033-3

Molecular and Biochemical Parasitology

Volume 27, Issues 2–3, 15 January 1988, Pages 135-141

https://doi.org/10.1016/0166-6851(88)90033-3 Get rights and content

Abstract

A monoclonal antibody (24C6 4F12) raised against Plasmodium falciparum culture supernatant antigens gave a multiple dot picture on schizonts when assayed by immunofluorescence on P. falciparum erythrocytic stages. The corresponding antigen was localized in the peduncle of rhoptries by immunoelectronmicroscopy. On Western blots of P. falciparum schizonts, a major antigen of 225 kDa and a minor one of 240 kDa were recognized by this McAb. Pulse chase analysis of [³⁵S]methionine biosynthetic labeling of P. falciparum culture demonstrated that the 240 kDa molecule was the precursor of the 225 kDa and that its processing occurred between 0 and 4 h after synthesis. Biosynthesis of the 240-225 kDa antigen occurred only during schizogony.

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2020, Toxoplasma Gondii: The Model Apicomplexan - Perspectives and Methods
The Apicomplexa are named for their unique apical secretory organelles: the micronemes, the rhoptries, and the dense granules (DGs). The contents of these organelles are critical for the successful invasion and intracellular survival of Toxoplasma gondii. Microneme proteins are secreted in a calcium-dependent manner and are critical for adhesion and bridging with the host, as well as parasite egress from host cells. Rhoptry neck molecules, with microneme proteins, form the moving junction that enables the parasite to enter the host, while other rhoptry proteins are important for modulation of host cell signaling and formation of the parasitophorous vacuole. DG proteins play important roles in the interaction with the host as well as survival within the host. This chapter provides a detailed overview of the functions of Toxoplasma secretory proteins and their roles in invasion, egress, and host cell parasitism.
Secretory organelle trafficking in Toxoplasma gondii: A long story for a short travel
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Electron microscopy (EM) studies revealed that pre-rhoptries resemble secretory granules and develop into mature organelles by condensation and elongation of the neck region towards the conoid of the developing daughter tachyzoites (Dubremetz, 2007). As studied in Plasmodium falciparum, where PfRON4 was found in the pre-rhoptries (Roger et al., 1988) before being transported to the mature rhoptry neck, the sub-compartmentalization between ROPs and RONs supposedly takes place via lateral protein sorting and not by fusion of distinct compartments. Finally, anchoring of the maturing rhoptries to the apical complex relies on the activity of the Armadillo Repeats Only protein, TgARO (Mueller et al., 2013).
Toxoplasma gondii (T. gondii) possesses a highly polarized secretory system, which efficiently assembles de novo micronemes (MIC) and rhoptries (ROP) during parasite replication. Pioneer works have studied the sorting motifs within MIC and ROP proteins, required for their trafficking towards their final destination. These studies led to the conclusion that protein processing and protein sorting are inter-dependent activities. More recent works have revealed the trafficking routes taken by the MIC and ROP proteins by examining the functions of the endo-exocytic compartments and identified key molecules involved in protein sorting and transport. These recent findings have suggested that T. gondii has repurposed the evolutionarily conserved regulators of the endosomal system to the secretory pathway. This review reports the pioneer as well as the most recent findings on the molecular mechanisms regulating apical organelle and dense granule biogenesis and portrays the parasite as a remarkable secretory machine that has efficiently remodeled its trafficking system to adapt to an intracellular lifestyle.
Compositional and expression analyses of the glideosome during the Plasmodium life cycle reveal an additional myosin light chain required for maximum motility
2017, Journal of Biological Chemistry
Citation Excerpt :
Mouse anti-PfELC antibodies were raised by immunization of female BALB/c mice with recombinant His-tagged protein (production of which is described below). Other primary polyclonal antibodies and mAbs used were anti-PfMTIP (40), anti-PfGAP45 (18), anti-PfGAP50 (26), anti-PfRON4 mAb (57), and anti-PbP28 mAb 13.1 (58), which are described in the referenced works. Rabbit anti-GFP11 and rat anti-HA (monoclonal 3F10; Roche Applied Science) antibodies were used to detect tagged proteins.
Myosin A (MyoA) is a Class XIV myosin implicated in gliding motility and host cell and tissue invasion by malaria parasites. MyoA is part of a membrane-associated protein complex called the glideosome, which is essential for parasite motility and includes the MyoA light chain myosin tail domain–interacting protein (MTIP) and several glideosome-associated proteins (GAPs). However, most studies of MyoA have focused on single stages of the parasite life cycle. We examined MyoA expression throughout the Plasmodium berghei life cycle in both mammalian and insect hosts. In extracellular ookinetes, sporozoites, and merozoites, MyoA was located at the parasite periphery. In the sexual stages, zygote formation and initial ookinete differentiation precede MyoA synthesis and deposition, which occurred only in the developing protuberance. In developing intracellular asexual blood stages, MyoA was synthesized in mature schizonts and was located at the periphery of segmenting merozoites, where it remained throughout maturation, merozoite egress, and host cell invasion. Besides the known GAPs in the malaria parasite, the complex included GAP40, an additional myosin light chain designated essential light chain (ELC), and several other candidate components. This ELC bound the MyoA neck region adjacent to the MTIP-binding site, and both myosin light chains co-located to the glideosome. Co-expression of MyoA with its two light chains revealed that the presence of both light chains enhances MyoA-dependent actin motility. In conclusion, we have established a system to study the interplay and function of the three glideosome components, enabling the assessment of inhibitors that target this motor complex to block host cell invasion.
Toxoplasma Secretory Proteins and Their Roles in Cell Invasion and Intracellular Survival
2013, Toxoplasma Gondii: The Model Apicomplexan - Perspectives and Methods: Second Edition
The Apicomplexa are named for their unique apical secretory organelles: the micronemes, the rhoptries and the dense granules. The contents of these organelles are critical for the successful invasion and intracellular survival of T. gondii. Microneme proteins are secreted in a calcium-dependent manner and are critical for adhesion and bridging with the host, as well as parasite egress from host cells. Rhoptry neck molecules, with microneme proteins, form the moving junction that enables the parasite to enter the host, while other rhoptry proteins are important for modulation of host cell signalling and formation of the parasitophorous vacuole. Dense granule proteins play important roles in the interaction with the host as well as survival within the host. This chapter provides a detailed overview of the functions of Toxoplasma secretory proteins and their roles in invasion, egress, and host cell parasitism.
Wherever i may roam: Protein and membrane trafficking in P. falciparum-infected red blood cells
2012, Molecular and Biochemical Parasitology
Citation Excerpt :
In transmission electron micrographs, the neck appears electron-lucent while the bulb is electron-dense and contains internal membranes reminiscent of multivesicular endosomes in higher eukaryotes [142–144]. Individual proteins are not distributed throughout the rhoptry but are sub-compartmentalized within either the bulb or the neck [136,145,146]. Proteins that have so far been identified residing in the neck of the rhoptries are involved in irreversible attachment and tight junction formation [133,135] whereas bulb proteins are thought to be involved in the generation and establishment of the PV and RBC remodeling [147].
Quite aside from its immense importance as a human pathogen, studies in recent years have brought to light the fact that the malaria parasite Plasmodium falciparum is an interesting eukaryotic model system to study protein trafficking. Studying parasite cell biology often reveals an overrepresentation of atypical cell biological features, possibly driven by the parasites’ need to survive in an unusual biological niche. Malaria parasites possess uncommon cellular compartments to which protein traffic must be directed, including secretory organelles such as rhoptries and micronemes, a lysosome-like compartment referred to as the digestive vacuole and a complex (four membrane-bound) plastid, the apicoplast. In addition, the parasite must provide proteins to extracellular compartments and structures including the parasitophorous vacuole, the parasitophorous vacuolar membrane, the Maurer's clefts and both cytosol and plasma membrane of the host cell, the mature human red blood cell. Although some of these unusual destinations are possessed by other cell types, only Plasmodium parasites contain them all within one cell.
Here we review what is known about protein and membrane transport in the P. falciparum-infected cell, highlighting novel features of these processes. A growing body of evidence suggests that this parasite is a real “box of tricks” with regards to protein traffic. Possibly, these tricks may be turned against the parasite by exploiting them as novel therapeutic targets.
Dissecting the apicomplexan rhoptry neck proteins
2010, Trends in Parasitology
Citation Excerpt :
A summary of the characterized apicomplexan rhoptry bulb proteins is provided in Box 2, and more extensive discussions can be found elsewhere [31,32]. A 1980 study was the first to identify a protein that localized to the rhoptry neck in Plasmodium[33]; however, more recent investigations have shown that the rhoptry neck contains a distinct set of proteins, some of which are required for host cell invasion (Box 1), either in binding to the host cell plasma membrane or in the formation of the tight junction [6–9,11,12,34–41]. The proteins that bind to the host cell plasma membrane, such as the reticulocyte binding-like (RBL) protein family, are not a primary focus of this review, and are mentioned only in Box 3.
Apicomplexan parasites possess specialized secretory organelles (rhoptries and micronemes) that release their contents during host cell invasion. Although the rhoptries were once thought to be merely a bulbous ‘protein reservoir’ connected to an anterior neck region, the localization of a protein specifically to the neck suggested that this region was more than just a duct. Recent studies have shown that the rhoptry neck sub-compartment possesses a distinct protein repertoire. Some of these proteins share common features, including conservation across the phylum and involvement in tight-junction formation. A sub-group of rhoptry neck proteins, the RONs, their association with the microneme protein apical membrane antigen AMA1, and their involvement in invasion are discussed.

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Molecular and Biochemical Parasitology

Abstract

References (23)

Isolation of a Plasmodium falciparum rhoptry antigen

Mol. Biochem. Parasitol.

A rhoptry antigen of Plasmodium falciparum contains conserved and variable epitopes recognized by inhibitory monoclonal antibodies

Mol. Biochem. Parasitol.

In vivo time course of synthesis and processing of major schizont membrane polypeptides in Plasmodium falciparum

Mol. Biochem. Parasitol.

A 50 kilodalton exoantigen specific to the merozoite release-reinvasion stage of Plasmodium falciparum

Mol. Biochem. Parasitol.

Early events in the biosynthesis of the lysosomal enzyme cathepsin D

J. Biol. Chem.

Localization, biosynthesis, processing and isolation of a major 126 kDa antigen of the parasitophorous vacuole of Plasmodium falciparum

Mol. Biochem. Parasitol.

Protective monoclonal antibodies recognizing stage specific merozoite antigens of a rodent malarial parasite

Nature

Immunization against blood stage rodent malaria using purified parasite antigens

Nature

Ultrastructural localization of protective antigens of Plasmodium yoelii merozoites by the use of monoclonal antibodies and ultrathin cryomicrotomy

Am. J. Trop. Med. Hyg.

Immunity to asexual erythrocytic stages of Plasmodium falciparum

Monoclonal antibody characterization of Plasmodium falciparum antigens

Am. J. Trop. Med. Hyg.

Cited by (64)

Toxoplasma secretory proteins and their roles in parasite cell cycle and infection

Secretory organelle trafficking in Toxoplasma gondii: A long story for a short travel

Compositional and expression analyses of the glideosome during the Plasmodium life cycle reveal an additional myosin light chain required for maximum motility

Toxoplasma Secretory Proteins and Their Roles in Cell Invasion and Intracellular Survival

Wherever i may roam: Protein and membrane trafficking in P. falciparum-infected red blood cells

Dissecting the apicomplexan rhoptry neck proteins