Merozoite surface protein 8 of Plasmodium falciparum contains two epidermal growth factor-like domains

By motif searching of the unfinished sequences in the Malaria Genome Sequen cing Project databases we have identified a novel EGF-like domain-containin g protein of Plasmodium falciparum. The sequence lies within a single open reading frame of 1791 bp and is predicted to encode a polypeptide of 597 am ino acids. There are hydrophobic regions at the extreme N- and C-termini, w hich could represent secretory signal peptide and GPI attachment sites, res pectively. Similar to MSP1, there are two EGF-like domains located neat the C-terminus. RT-PCR analysis of the novel gene shows that it is transcribed in asexual stages of the malaria parasite, We have expressed portions of t he protein as recombinant GST fusions in Escherichia coil and raised antise ra in rabbits. Antibodies to the EGF-like domains of the novel protein are highly specific and do not cross-react with the EGF-like domains of MSP1, M SP4 or MSP5 expressed as GST fusion proteins. Antiserum raised to the most C-terminal region of the protein reacts with four bands of 98, 50, 25 and 1 9 kDa in P. falciparum parasite lysates whereas antisera to the N-terminal fusion proteins recognise the 98 and 50 kDa bands, suggesting that the nove l protein may undergo processing in a similar way to MSPI. Immunoblot analy sis of stage-specific parasite samples reveals that the protein is present throughout the parasite asexual life cycle and in isolated merozoites, with the smaller fragments present in ring stage parasites. The protein partiti ons in the detergent-enriched phase after Triton X-114 fractionation and is localized to the surfaces of trophozoites, schizonts and free merozoites b y indirect immunofluorescence. Antisera to the C-terminus stain the surface of rings, whereas antisera to the N-terminus do not, suggesting that a fra gment of the protein is carried into the developing ring stage parasite. Ba sed on the accepted nomenclature in the field we designate this protein MSP 8. We have shown that the MSP8 fusion proteins are in a conformation that c an be recognised by human immune sera and that there is very limited divers ity in the MSP8 gene sequences from various P. falciparum laboratory isolat es. MSP8 shows significant similarity to the recently reported sequence of the protective P. yoelii merozoite surface protein pypAg-2 [Burns JM, Belk CC, Dunn PD. Infect Immun 2000;68:6189-95.] suggesting that the two protein s are homologues. Taken together, these findings suggest that MSP8/pypAg-2 may play an important role in the process of red cell invasion and is a pot ential malaria vaccine candidate. (C) 2001 Elsevier Science B.V. All rights reserved.