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Specific phosphorylation of the PfRh2b invasion ligand of Plasmodium falciparum.

ABSTRACT: Red blood cell invasion by the malaria parasite Plasmodium falciparum relies on a complex protein network that uses low and high affinity receptor-ligand interactions. Signal transduction through the action of specific kinases is a control mechanism for the orchestration of this process. In the present study we report on the phosphorylation of the CPD (cytoplasmic domain) of P. falciparum Rh2b (reticulocyte homologue protein 2b). First, we identified Ser3233 as the sole phospho-acceptor site in the CPD for in vitro phosphorylation by parasite extract. We provide several lines of evidence that this phosphorylation is mediated by PfCK2 (P. falciparum casein kinase 2): phosphorylation is cAMP independent, utilizes ATP as well as GTP as phosphate donors, is inhibited by heparin and tetrabromocinnamic acid, and is mediated by purified PfCK2. We raised a phospho-specific antibody and showed that Ser3233 phosphorylation occurs in the parasite prior to host cell egress. We analysed the spatiotemporal aspects of this phosphorylation using immunoprecipitated endogenous Rh2b and minigenes expressing the CPD either at the plasma or rhoptry membrane. Phosphorylation of Rh2b is not spatially restricted to either the plasma or rhoptry membrane and most probably occurs before Rh2b is translocated from the rhoptry neck to the plasma membrane.

SUBMITTER: Engelberg K

PROVIDER: S-EPMC3671792 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Specific phosphorylation of the PfRh2b invasion ligand of Plasmodium falciparum.

Engelberg Klemens K Paul Aditya S AS Prinz Boris B Kono Maya M Ching Wilhelm W Heincke Dorothee D Dobner Thomas T Spielmann Tobias T Duraisingh Manoj T MT Gilberger Tim-Wolf TW

The Biochemical journal 20130601 3

Red blood cell invasion by the malaria parasite Plasmodium falciparum relies on a complex protein network that uses low and high affinity receptor-ligand interactions. Signal transduction through the action of specific kinases is a control mechanism for the orchestration of this process. In the present study we report on the phosphorylation of the CPD (cytoplasmic domain) of P. falciparum Rh2b (reticulocyte homologue protein 2b). First, we identified Ser3233 as the sole phospho-acceptor site in ...[more]

PMID: 23544851

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Project description:Aldolase has been implicated as a protein coupling the actomyosin motor and cell surface adhesins involved in motility and host cell invasion in the human malaria parasite Plasmodium falciparum. It binds to the cytoplasmic domain (CTD) of type 1 membrane proteins of the thrombospondin-related anonymous protein (TRAP) family. Other type 1 membrane proteins located in the apical organelles of merozoites, the form of the parasite that invades red blood cells, including apical membrane antigen 1 (AMA1) and members of the erythrocyte binding ligand (EBL) and reticulocyte binding homologue (RH) protein families have been implicated in host cell binding and invasion. Using a direct binding method we confirm that TRAP and merozoite TRAP (MTRAP) bind aldolase and show that the interaction is mediated by more than just the C-terminal six amino acid residues identified previously. Single amino acid substitutions in the MTRAP CTD abolished binding to aldolase. The CTDs of AMA1 and members of the EBL and RH protein families also bound to aldolase. MTRAP competed with AMA1 and RH4 for binding to aldolase, indicating overlapping binding sites. MTRAP CTD was phosphorylated in vitro by both calcium dependent kinase 1 (CDPK1) and protein kinase A, and this modification increased the affinity of binding to aldolase by ten-fold. Phosphorylation of the CTD of members of the EBL and RH protein families also increased their affinity for aldolase in some cases. To examine whether or not MTRAP expressed in asexual blood stage parasites is phosphorylated, it was tagged with GFP, purified and analysed, however no phosphorylation was detected. We propose that CTD binding to aldolase may be dynamically modulated by phosphorylation, and there may be competition for aldolase binding between different CTDs. The use and efficiency of alternate invasion pathways may be determined by the affinity of adhesins and cell invasion proteins for aldolase, in addition to their host ligand specificity.

Project description:Studies of Plasmodium falciparum invasion pathways in field isolates have been limited. Red blood cell (RBC) invasion is a complex process involving two invasion protein families; Erythrocyte Binding-Like (EBL) and the Reticulocyte Binding-Like (PfRh) proteins, which are polymorphic and not fully characterized in field isolates. To determine the various P. falciparum invasion pathways used by parasite isolates from South America, we studied the invasion phenotypes in three regions: Colombia, Peru and Brazil. Additionally, polymorphisms in three members of the EBL (EBA-181, EBA-175 and EBL-1) and five members of the PfRh (PfRh1, PfRh2a, PfRh2b, PfRh4, PfRh5) families were determined. We found that most P. falciparum field isolates from Colombia and Peru invade RBCs through an atypical invasion pathway phenotypically characterized as resistant to all enzyme treatments (NrTrCr). Moreover, the invasion pathways and the ligand polymorphisms differed substantially among the Colombian and Brazilian isolates while the Peruvian isolates represent an amalgam of those present in the Colombian and Brazilian field isolates. The NrTrCr invasion profile was associated with the presence of the PfRh2a pepC variant, the PfRh5 variant 1 and EBA-181 RVNKN variant. The ebl and Pfrh expression levels in a field isolate displaying the NrTrCr profile also pointed to PfRh2a, PfRh5 and EBA-181 as being possibly the major players in this invasion pathway. Notably, our studies demonstrate the uniqueness of the Peruvian P. falciparum field isolates in terms of their invasion profiles and ligand polymorphisms, and present a unique opportunity for studying the ability of P. falciparum parasites to expand their invasion repertoire after being reintroduced to human populations. The present study is directly relevant to asexual blood stage vaccine design focused on invasion pathway proteins, suggesting that regional invasion variants and global geographical variation are likely to preclude a simple one size fits all type of vaccine.

Dataset Information

Specific phosphorylation of the PfRh2b invasion ligand of Plasmodium falciparum.

Publications

Specific phosphorylation of the PfRh2b invasion ligand of Plasmodium falciparum.

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