Surface expression of the conserved ribosomal protein P0 on parasite and other cells

Abstract

Invasion-blocking antibodies against the ribosomal phosphoprotein P0 of the human malarial parasite Plasmodium falciparum (PfP0) have been identified through a differential immunoscreen using immune and patient sera from malaria endemic regions of Eastern India [1]. Purified IgG from rabbit sera, raised against different domains of the PfP0 protein, show no effect on the intraerythrocytic stages of Plasmodium falciparum in culture, but inhibit the invasion of erythrocytes by merozoites in a concentration dependent manner [2,3]. The same antibodies against the PfP0 protein protect mice against challenge with a virulent strain of Plasmodium yoelii [4]. We have recently demonstrated this protein to be present on the surface of merozoites and gametocytes of Plasmodium [3,4]. The phosphoprotein P0 is highly conserved across eukaryotic organisms [5], and is related to the family of phosphoproteins P1 and P2, due to the highly homologous carboxy-terminal 22 amino acid domain [6]. The ribosomal function of P0 is mediated through a pentameric P12·P0·P22 complex, which forms the stalk of the large ribosomal subunit at the GTPase center [7]. P0 interacts with the elongation factor eEF2 [8], and is essential for the ribosomal activity and cell viability in yeast [9]. Through deletion analysis of the P0 protein, the ribosomal function has been mapped to amino acid position 185–230 in yeast P0 [10]. Since P0 is a very conserved protein [5], we wondered whether the surface expression of P0 is an exclusive property of Plasmodium cells, or if it is a general property of the P0 protein in other organisms as well. The presence of this protein on the host cell surface will have serious implications with the usage of this protein in a malaria vaccine. This question also has a direct bearing on the mechanism of translocation of this protein to the surface, as to whether it is a unique phenomenon for Plasmodium. Therefore, we decided to investigate the presence of this protein on different cell types. In this paper we report the localization of P0 protein on the surface of another Apicomplexan parasite Toxoplasma, yeast and mammalian cell lines using mono-specific cross-reactive anti-PfP0 antibodies. In order to test for cross-reactivity of anti-PfP0 sera with different cell types, Western Blot analysis was carried out with total protein extract of these cell types. Anti-PfP0 antisera were raised in rabbits against recombinant GST-fusion proteins, PfP0N (amino acid 17–61) and PfP0C (amino acid 61–316), and purified as described earlier [3]. Specific single band reactivities of 38, 33, 34 and 38 kD proteins were observed with Toxoplasma gondii, yeast (Saccharomyces cere isiae, strain EG103), Chinese Hamster Ovary (CHO) cells and human leukocyte protein preparations respectively, using Abbre iations: GST, glutathione-S-transferase; IPTG, isopropylthio-galactosidase; PfP0, Plasmodium falciparum phosphoriboprotein P0; PfP0N, amino-terminal domain of PfP0; PfP0C, carboxy-terminal domain of PfP0. * Corresponding author. Tel: +91-22-215-2971/2979 x 2570; fax: +91-22-215-2110/2181. E-mail address: sharma@tifr.res.in (S. Sharma). 1 Present address: Lecturer, Agricultural Science Unit, Indian Statistical Institute, 203, B.T. Road, Calcutta 700 035, India.