N-terminal processing of proteins exported by malaria parasites
Introduction
The asexual blood stage of the apicomplexan parasite Plasmodium falciparum is responsible for clinical manifestations associated with the most virulent form of malaria in humans [1]. As the parasite matures within a parasitophorous vacuole (PV) inside the red blood cell (RBC), it extensively modifies the RBC to make it amenable for nutrient uptake and to prevent clearance by the spleen [2], [3]. The parasite accomplishes this remodeling of the terminally differentiated RBC by exporting proteins across the parasite plasma membrane and the parasitophorous vacuolar membrane (PVM) to the red cell cytosol (RCC) or red cell membrane (RCM). A major example of a parasite-induced modification to the host RBC is the formation of knobs on the outer surface of the RCM [3]. These large protrusions consist of the parasite-encoded knob-associated histidine-rich protein (KAHRP) anchoring the immunovariant adhesin P. falciparum erythrocyte membrane protein 1 (PfEMP1) to the RBC cytoskeleton [4], [5], [6]. These protein complexes are implicated in the cytoadherance of infected RBCs to the host endothelium leading to their sequestration in the peripheral vasculature [7]. Therefore, the exported proteins involved in the formation of these knobs and the actual machinery involved in their transport are not only major virulence factors in severe disease pathology, they are also crucial to the survival of P. falciparum.
A highly conserved 5-amino acid motif (RxLxE/Q/D) termed the Plasmodium export element (PEXEL) or host-targeting signal was recently identified through bioinformatic approaches and experimentally verified to mediate the translocation of parasite-derived proteins across the PVM and into the host RBC [8], [9]. Site-directed mutagenesis of the conserved residues R, L, or E/Q/D with alanine abolished the export of green fluorescent protein (GFP) chimeras to the RCC. The discovery of the PEXEL has allowed for the in silico annotation of the P. falciparum “exportome” or “secretome” that has brought to light novel protein families exported by the parasite [10]. Conservation of the PEXEL motif and its corresponding pathway across the genus Plasmodium indicates the existence of common trafficking components in malaria parasites. The importance and uniqueness of this trafficking signal has generated considerable interest in the identification of the machinery that interacts with the PEXEL because of the potential for drug design.
With the discovery of this signal-mediated mechanism in P. falciparum, an updated model for the export of proteins to the host RBC involves recruitment into the parasite endoplasmic reticulum (ER) [11], [12], default secretion into the PV lumen [12], [13], [14], [15], and PEXEL-mediated translocation across the PVM [8], [9], [14], [15]. In other eukaryotic cells, proteins destined for export enter the secretory pathway by co-translational translocation across the ER membrane mediated by the recognition of a N-terminal signal peptide and concomitant cleavage of this signal peptide by the signal peptidase complex (SPC) [16]. It has been assumed that P. falciparum proteins exported to the host RBC have their ER-type signal peptides cleaved by the parasite SPC but this type of N-terminal processing has never been definitively characterized. Here, we show that exported parasite proteins do undergo N-terminal processing that involves cleavage and N-acetylation of the PEXEL. The two examples of PEXEL processing described here indicate that this N-terminal cleavage and acetylation are likely to occur in many soluble proteins exported by P. falciparum. Probing these N-terminal processing events with brefeldin A (BFA) reveal that PEXEL cleavage and N-acetylation occurs in the parasite ER. The PEXEL processing of a PV-trapped GFP chimera suggests a recognition event in the PV may be crucial for export beyond the PVM. The dissection of the PEXEL as a novel ER peptidase cleavage site and a classical N-acetyltransferase substrate sequence is also discussed.
Section snippets
In vitro culturing of P. falciparum
P. falciparum strain 3D7 and transfected parasites expressing the transgene KAHRP(-His)-GFP were obtained from the Malaria Research and Reference Reagent Resource Center (MR4) [12]. For clarification purposes, this construct has been renamed as KAHRP(1-60)-GFP. The transfected 3D7 line expressing the PfHRPIImyc transgene was kindly provided by Dr. Kasturi Haldar (Northwestern University, Chicago, IL) [17]. Parasites were cultured in human RBCs maintained at 2% hematocrit in RPMI 1640 medium
PEXEL of PfHRPII is cleaved and N-acetylated
To address whether exported Plasmodium proteins undergo N-terminal processing during trafficking to the host RBC, P. falciparium histidine-rich protein II (PfHRPII) was purified and its N-terminus was characterized. PfHRPII is a soluble parasite protein known to be exported to the RCC [13], [17], [25], [26]. RBCs infected with 3D7 parasites were harvested at the late trophozoite stage (∼33–36 h after invasion) because of the maximal expression and export of PfHRPII during this period [17]. The
Acknowledgements
We thank Dr. Sharleen Zhou and Dr. David S. King for Edman sequencing and mass spectrometry work. We also thank Dr. Thomas Akompong and Dr. Kasturi Haldar for providing the transfected parasite line expressing PfHRPIImyc. H.H.C was the recipient of a National Science Foundation Graduate Fellowship.
References (36)
- et al.
Cloning the P. falciparum gene encoding PfEMP1, a malarial variant antigen and adherence receptor on the surface of parasitized human erythrocytes
Cell
(1995) - et al.
Mapping the binding domains involved in the interaction between the Plasmodium falciparum knob-associated histidine-rich protein (KAHRP) and the cytoadherence ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1)
J Biol Chem
(1999) - et al.
Molecules on the surface of the Plasmodium falciparum infected erythrocyte and their role in malaria pathogenesis and immune evasion
Mol Biochem Parasitol
(2001) - et al.
Trans expression of a Plasmodium falciparum histidine-rich protein II (HRPII) reveals sorting of soluble proteins in the periphery of the host erythrocyte and disrupts transport to the malarial food vacuole
J Biol Chem
(2002) - et al.
High-level expression of soluble protein in Escherichia coli using a His6-tag and maltose-binding-protein double-affinity fusion system
Protein Expres Purif
(1997) - et al.
Stable transgene expression in Plasmodium falciparum
Mol Biochem Parasitol
(1997) - et al.
Fluorescence microscopy in three dimensions
Methods Cell Biol
(1989) - et al.
Common trafficking pathway for variant antigens destined for the surface of the Plasmodium falciparum-infected erythrocyte
Mol Biochem Parasitol
(2004) - et al.
Signal sequence cleavage of peptidyl-tRNA prior to release from the ribosome and translocon
J Biol Chem
(2004) - et al.
Function of the Plasmodium export element can be blocked by green fluorescent protein
Mol Biochem Parasitol
(2005)