Plasmodium vivax merozoite surface proteins-3beta and-3gamma share structural similarities with P. vivax merozoite surface protein-3alpha and define a new gene family.

The genes encoding two merozoite surface proteins of Plasmodium vivax that are related to PvMSP3 [1] are reported. One of these genes was identified within P. vivax lambdagt11 clone 5.4, which was selected by immunoscreening with a Saimiri monkey antiserum. The insert DNA of this clone was used as a probe to isolate the complete gene from a P. vivax lambdaDASH genomic (g) DNA library. Antibodies to recombinant 5.4 and subsequent fusion proteins produce a pattern of circumferential surface fluorescence by indirect immunofluorescence assays (IFA) on segmented schizonts and free intact merozoites, and recognize a 125 kDa protein via western immunoblots. The gene, however, encodes a protein with a calculated size of 75677 Da, and 3' and 5' RACE analyses were employed to confirm the size of the gene and its coding region. The second related P. vivax gene was isolated by hybridization of a fragment of an orthologous P. knowlesi gene. The encoded proteins of all three related P. vivax genes have putative signal peptides, large central domains that contain >20% alanine residues bound by charged regions, are predicted to form alpha-helices with heptad repeat coiled-coil structures, and do not have a hydrophobic region that could anchor them to the surface of the merozoite. Although the overall identity in amino acid alignment among the three encoded proteins is low (<40%), the shared predicted structural features and motifs indicate that they are members of an intra-species family, which we are designating as the PvMSP-3 family with the reported members being Pvmsp-3alpha, Pvmsp-3beta, and Pvmsp-3gamma. We further demonstrate that this family also includes related proteins from P. knowlesi and P. falciparum.

Plasmodium vivax merozoite surface protein-3 contains coiled-coil motifs in an alanine-rich central domain.

Plasmodium merozoites are covered with a palisade layer of proteins that are arranged as organized bundles or appear as protruding spikes by electron microscopy. Here we present a third Plasmodium vivax merozoite surface protein, PvMSP-3, which is associated with but not anchored in the merozoite membrane. Serum from a P. vivax immune squirrel monkey was used to screen a lambdagt11 P. vivax genomic DNA (gDNA) library. Plaque-selected antibodies from clone no. 6.1, and rabbit antisera against its encoded protein, produced a pattern in immunofluorescence assays (IFAs) that is consistent with a localization at the surface of mature schizonts and free merozoites. Specific antisera also agglutinated merozoites and recognized a protein of 150 000 Da by SDS-PAGE. The complete msp-3 gene and flanking sequences were cloned from a P. vivax lambda Dash II gDNA library and also partly characterized by RACE (rapid amplification of cDNA ends). The immediate upstream sequence contains non-coding repeats and a putative protein encoding open reading frame (ORF), which are also present on the msp-3 5'RACE gene product. Pvmsp-3 encodes a protein with a calculated mass of 89 573 Da, which has a potential signal peptide and a major central alanine-rich domain (31%) that exhibits largely alpha-helical secondary structure and is flanked by charged regions. The protein does not have a putative transmembrane domain or a consensus sequence for a glycosylphosphatidylinositol (GPI) anchor modification. However, the alanine-rich domain has heptad repeats that are predicted to form coiled-coil tertiary structures, which mediate protein-protein interactions. PvMSP-3 is structurally related to P. falciparum MSP-3 and the 140000 Da MSP of P. knowlesi. Characterization of PvMSP-3, thus, also begins to define a new interspecies family of evolutionarily related Plasmodium merozoite proteins.