Epitope map and processing scheme for the 195,000-dalton surface glycoprotein of Plasmodium falciparum merozoites deduced from cloned overlapping segments of the gene.

Abstract

DNA fragments from human malaria parasites were cloned into Xgtll to produce a genomic DNA expression library. A pool of monoclonal antibodies (mAbs) recognizing three domains of the 195-kDa major merozoite surface glycoprotein (gp195) reacted with seven clones expressing malaria antigens. mAbs recognizing the 83-kDa product of gp195 reacted with the clones, but mAbs recognizing a glycosylated 45-kDa and a nonglycosylated 45-kDa domain did not.Restriction enzyme mapping revealed that the clones contained overlapping segments encoding about 70% of the gene beginning at the 5' end and ending at an EcoRI restriction enzyme site 3.3 kilobase pairs downstream. The mAbs recognizing the 83-kDa domain reacted differently with the clones, allowing the mapping of three epitopes, one of which was repetitive. Affinity-purified antibodies were selected from immune monkey serum with recombinant expression proteins adsorbed to nitrocellulose filters. When used to probe electrophoretic immunoblots of parasite extracts, these antigen-selected antibodies reacted with specific sets of processed products of gp195, including those associated with the 83-and the nonglycosylated 45-kDa domains. This information, combined with the mAb epitope map, allowed a tentative scheme for processing gp195 from the Camp strain to be proposed.Merozoites from Plasmodium falciparum malaria parasites have, as major components of their surface, a 195-kDa glycoprotein (gp195) (1) and polypeptides derived from this protein as a result of processing by proteases (2, 3). Processing of gp195 produces at least three smaller products found on the surface of merozoites (3) and has been observed in at least six P. falciparum strains (1,2,4,5). Processing occurs about the time merozoites are released from infected erythrocytes, and a series of transitory intermediates with molecular masses of about 153, 150, and 110 kDa appear (2). One of the final products has an Mr of 83 kDa (2, 4, 5) and possesses serotype-restricted epitopes (4, 5). Other products of 50 (1), 42, and 19 kDa (3) have been identified; the 50-kDa antigen is glycosylated (1) and may be homologous with the 42-kDa antigen. gp195 and its proteolytic products are recognized on intact merozoites by growth-inhibitory immune sera (6) and may be good candidates for vaccine development (1, 7-10).The molecular cloning and expression of parasite antigens in bacterial systems allows the preparation of sufficient amounts of pure antigen to conduct immunological and immunochemical studies. The cloning from cDNA of the complete structural gene encoding gp195 has been reported (11), and cDNA clones encoding 0.21 (7) and 1.1 (12) kilobase pairs (kb) of the gp195 gene have been reported as well. We report the cloning of a 3.6-kb portion of gp195 genomic DNA from the Camp strain and propose a scheme for processing the Camp strain protein, locating major proteolytic products and the positions of epitopes recognized by monoclonal antibodies (mAbs).MATERIALS AND METHODS Culture of P. falciparum. Camp strain...

Primary structure of the 175K Plasmodium falciparum erythrocyte binding antigen and identification of a peptide which elicits antibodies that inhibit malaria merozoite invasion.

“…These afffinity-purified antibodies were then used to probe blots of purified authentic EBA-175. Similarly, nitrocellulose filters saturated with 10 mM IPTG were used to make lifts of confluent plates (Lyon et al, 1986) of phage EBAI.8fI090. Affinity purification of A_otus monkey antibodies specific for the expressed protein on these filters was performed as described (Orlandi et al, 1990) and these antibodies were used to probe preparations of authentic EBA-175, as mentioned above.…”

“…Initially, affinity-purified monospecific antibodies to EBA-175 were used to screen a Camp strain genomic mung bean nuelease expression library in lambda gtll (Lyon, et al, 1986). Nitrocellulose filters (Scnieicher & SchueIl, Inc., Keene, NH) were saturated with I0 ram isopropyl-beta-thio-D-galactopyranoside (IPTG) (Sigma Chemical Company, St. Louis, MO), and duplicate plaque lifts of ~ 300,000 plaques plated at a density of 30,000 plaques per 150-ram Petri dish were made (Young and Davis, 1983).…”

Identification of T Cell Epitopes on the 33-kDa Fragment of Plasmodium yoelii Merozoite Surface Protein 1 and Their Antibody-Independent Protective Role in Immunity to Blood Stage Malaria

“…The primary processing at schizont rupture cleaves the precursor protein into major fragments of ϳ83 (MSP1 83 ), 30 (MSP1 30 ), 38 (MSP1 38 ), and 42 (MSP1 42 ) kDa. The fragments are found as a noncovalently associated complex held together on the free merozoite surface by the C-terminal membrane-bound 42-kDa fragment (2)(3)(4). At the time of merozoite invasion, MSP1 42 is cleaved into two products.…”

“…Merozoite surface protein 1 (MSP1) 3 is a high molecular mass (ϳ185-to 205-kDa) glycoprotein expressed on the surface of merozoites (2)(3)(4). It is a potential vaccine candidate because it is directly exposed and interacts with the host milieu during RBC invasion.…”

Evaluation of the Immunogenicity and Vaccine Potential of Recombinant Plasmodium falciparum Merozoite Surface Protein 8

“…MSP1 is an essential and abundant surface protein expressed during schizogony as an ϳ200-kDa precursor protein (30). Prior to the schizont rupture, MSP1 is proteolytically processed to produce the 83-, 30-, 38-, and 42-kDa fragments (31,36,38) that remain noncovalently associated along with MSP6, MSP7, and MSP9 (34,44,53) and tethered to the surface by the glycosylphosphatidylinositol (GPI)-anchored 42-kDa fragment (MSP1 42 ) (43,44). During invasion, MSP1 42 is further cleaved into MSP1 33 and MSP1 19 (25,26) to release the entire complex except for MSP1 19 that remains GPI anchored and is carried into the newly invaded red blood cell (RBC) (7,19).…”