Memórias do Instituto Oswaldo Cruz Fundação Oswaldo Cruz, Fiocruz ISSN: 1678-8060 EISSN: 1678-8060 Vol. 95, Num. 3, 2000, pp. 345-352

Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 95(3) May/Jun. 2000, pp. 345-352

Plasmodium yoelii: Identification of a Gene Encoding a Putative ADP-ribosylation Factor-1 GTPase-activating Protein, PyAG1

Rémi Hienne⁺, Alain Rico, Daniel Parzy, Jean-Claude Doury

Unité de Parasitologie, Institut de Médecine Tropicale du Service de Santé des Armées, Parc du Pharo, BP 46, 13998 Marseille Armées, France

⁺Corresponding author. Fax: +33-4-9159.4477. E-mail: imtssa@gulliver.fr

Received 21 May 1999
Accepted 27 January 2000

Code Number: oc00057

The PyAG1 gene, identified by the screening of a Plasmodium yoelii genomic DNA library with a rhoptry-specific Mab, encodes a protein with a zinc finger structure immediately followed by the consensus sequence of the Arf GAP catalytic site. The serum of mice immunized with the recombinant protein recognized specifically the rhoptries of the late infected erythrocytic stages. Blast analysis using the Genbank database gave the highest scores with four proteins presenting an Arf1 GAP activity. If presenting also this activity, the PyAG1 protein could be involved in the regulation of the secreted protein vesicular transport and, consequently, in the rhoptry biogenesis.

Key words: Plasmodium yoelii - gene PyAG1 - immature rhoptries - vesicular transport

Rhoptries are located at the apical end of the invasive stages of all Apicomplexan parasites and have morphological characteristics of secretion organelles. Their contents are secreted into the host cell during invasion and play a critical role in the invasion process (Sam-Yellowe 1996). It has been proposed that rhoptries are formed de novo at the end of the asexual erythrocytic cycle by budding of secretory vesicles from the Golgi apparatus (GA), a process analogous to the secretory granules of mammalian cells (Porchet & Torpier 1977). Therefore, the rhoptry components must be synthesized every cycle and transported to the organelles via a secretory pathway involving the GA. The fact that this transport (Ogun & Holder 1994, Howard & Schmidt 1995) as well as the rhoptry maturation (Ward et al. 1997) can be blocked by brefeldin A (BFA) might indicate the intervention of coated vesicles the formation of which is regulated by the GTP-binding ADP-ribosylation factor (Arf) cycle (Becker & Melkonian 1996). Indeed, this fungal toxin maintains the Arf protein in an inactive form (Arf-GDP) by preventing the GDP/GTP exchange. This data and the microscopic observation of coated vesicles during apical organelles maturation (Bannister & Mitchell 1995) could imply that the GTP-Arf cycle plays an important role in the rhoptry biogenesis.

This short communication reports the cloning of a novel Plasmodium yoelii gene encoding a putative Arf1 GAP (GTPase-activating protein) which seems to be associated with the immature roptries of the 4-8 nucleus schizonts.

An EcoRI library of P. yoelii genomic DNA, in lambida-ExCell EcoRI/CIP (Amersham Pharmacia Biotech), was screened with a monoclonal antibody (Mab), named C5-10. This Mab belongs to a Mab library which specifically reacts, by immunofluorescence assay (IFA), with the P. yoelii rhoptries (Fig. 1A); an immunoelectron microscopy analysis confirmed this localization (Hienne et al. 1998). By immunoblot under reducing conditions (IB/R), the C5-10 Mab recognizes a major protein of 68 kDa and a minor doublet of 31/34 kDa (Fig. 2A).

From the genomic DNA library, a recombinant lambda phage, lambida-AT711, containing a 1011-bp insert, was isolated. This DNA insert presented an open reading frame of 885-bp but no initiation codon. To obtain the full-length sequence at the 5' end of the gene, we performed inverse PCR with three combinations of six oligonucleotides (C1, C2, C3, D1, D2, D3), using P. yoelii genomic DNA HindIII digests (Fig. 3). Two nested amplifications were carried out with the oligonucleotide pairs C3/D3 and C2/D2. The sequence, obtained with the oligonucleotide C1, contained a methionine codon as well as upstream stop codons in frame with the putative initiation codon. The complete nucleotide and derived amino acid sequences (Fig. 4) of this novel gene, named PyAG1, are available in the GenBankä data base under the accession number AF055920. To confirm the synthesis of this putative protein during Plasmodium erythrocytic cycle, we isolated poly(A)⁺RNA from late asexual stages of P. yoelii with Dynabeads kit (Dynal), after DNase treatment of the total RNA solution, and carried out RT PCR. The PCR-amplification and sequencing of cDNA, using the two oligonucleotides C4 and D4 (Fig. 3), demonstrated that PyAG1 gene is transcribed (data not shown).

This gene has an open reading frame of 888-bp in length which encodes a hydrophilic protein of 296 amino acids (33 kDa). This protein presents, at its N-terminus, two interesting motifs: a zinc finger element (spanning residues 22-45) of the form [C-(X)₂-C-(X)₁₆-C-(X)₂ -C] (with C, cysteine; X, any amino acid) immediately followed by the consensus sequence of the Arf GAP catalytic site (Scheffzek et al. 1998) (spanning residues 47-53) of the form [s-h-H-R-x-h-x] (with s, glycine or alanine; h, hydrophobic amino acid; H, histidine; R, arginine; x, any amino acid).

A phylogenetic analysis by sequencing with the D4 and C3 oligonucleotides, using genomic DNA of rodent (P. yoelii nigeriensis, P. berghei, P. chabaudi adami, P. vinckei petteri) and human (P. falciparum Palo Alto and 3D7) plasmodial species, revealed an important preservation of this interesting region (Fig. 5, Table IA). This observation was confirmed by the sequencing of the 285 first nucleotides of the P. falciparum homologous gene (Ma href="385405.html">Fig. 5 and Table IB).

The PyAG1 gene product, expressed as glutathione S-transferase fusion protein (GST-PyAG1) in Escherichia coli (pGEX-3X plasmid/GST Gene Fusion System, Amersham Pharmacia Biotech), was recognized by the Acm C5-10, using IB/R (Fig. 2C). By IFA, the serum of female BALB/c mice (Charles River, France), immunized with the recombinant protein, recognized specifically red blood cells infected by P. yoelii young schizonts (4-8 nuclei), with a rhoptry-like labelling pattern (Fig. 1B). An immunoelectron microscopy study will be required to confirm this ultrastructural localization. By IB/R, this polyclonal antibody confirmed the presence of the PyAG1 gene product in a reduced antigenic extract of P. yoelii mature erythrocytic stages (Fig. 2B).

Blast analysis using the Genbank™ database gave the highest homology scores with four proteins presenting the same two interesting motifs in a similar position and an Arf1 GAP activity: Arf1 GAP of Arabidopsis thaliana (Genbank accession number AC004684), Drosophila melanogaster (Genbank accession number AF011427), Rattus norvegicus (Cukierman et al. 1995), and Gcs1 of Saccharomyces cerevisiae (Ireland et al. 1994, Poon et al. 1996) (Fig. 6, Table II). The structural homology with these proteins and the presence of the consensus sequence of the Arf GAPs catalytic site allowed us to hypothetize that the PyAG1 gene product may possess an Arf1 GAP activity. This activity steps in the Arf-GTP cycle by catalysing the GTP hydrolysis and, consequently, the transport vesicle uncoating, indispensable step for the membrane fusion between the vesicles and the target membrane.

The specific labelling of the immature rhoptries with polyclonal anti-PyAG1 serum corroborates this putative activity. Indeed, at first schizont stages, the parasites present immature rhoptries with low density (1.12 g.ml^-1) on sucrose gradient, even though the rhoptries have a significantly greater density in sucrose (1.16 g.ml^-1) at the mature schizonts, consequence of the accumulation of rhoptry proteins probably transported through coated vesicles (Jaikaria et al. 1993).

Therefore, the PyAG1 protein may interfere with the regulation of the secreted proteins vesicular transport and, consequently, with the biogenesis of the secreting organelles like rhoptries. The identification of such an activity supports the presence of a classical eukaryotic transport pathway involving coated vesicules in malarial parasite which has been suggested by BFA-inhibition experiences (Crary & Haldar 1992, Benting et al. 1994, Das et al. 1994, Hinterberg et al. 1994, Ogun & Holder 1995, Howard & Schmidt 1995) and P. falciparum Arf or Arl (ADP-ribosylation factor-like) characterization (Stafford et al. 1996, Lee et al. 1997, Truong et al. 1997).

Through this preliminary study, we have identified a new element of the intracellular protein transport between plasmodial organelles. Due to its putative regulator activity on the secreting organelles biogenesis, this protein could become a new target with a view to inhibit the parasite development.

ACKNOWLEDGEMENTS

To Drs P Falanga, Y Boulard, and Prof. I Landau for the providing of parasite strains and species.

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This work is supported by grants from GDR "CNRS-DGA/DSP".

Copyright 2000 Fundacao Oswaldo Cruz - Fiocruz

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