Clinical manifestation of Plasmodium falciparum malaria is directly linked to the blood stage of the parasite life cycle. At the blood stage, circulating merozoites invade red blood cells (RBCs) through multiple receptor-ligand interactions that mediate a complex series of events in a period of approximately one minute. Certain strains of P. falciparum favor an invasion mechanism that depends on sialic acid residues on the host RBC surface, while other strains favor a sialic acid-independent mechanism to invade RBCs. Two putative non-glycosylated extracellular regions of human RBC band 3 termed, 5ABC and 6A, function as an invasion receptor binding the 42 kDa C-terminal processing product of P. falciparum merozoite surface protein-1 (MSP142) and its 19-kDa secondary processing product (MSP119) by a sialic acid-independent mechanism. Earlier, we reported screening of a P. falciparum cDNA library in a yeast two-hybrid system, which led to the identification of P. falciparum merozoite surface protein 9 (MSP9) as another parasite surface protein that interacts with the 5ABC receptor region of band 3. MSP9 formed a co-ligand complex with MSP142 and/or MSP119 in their native forms. Here, we present identification of a novel P. falciparum microneme protein from the previous cDNA library screen. Primary sequence analysis of a positive clone in the library screen revealed a cDNA fragment in the correct open reading frame of a novel P. falciparum gene of 1,221 bps, encoding a hypothetical protein of 406 amino acids. Further sequence analysis predicted it is a membrane protein transversing the lipid bilayer multiple times. Anti-peptide antibody raised against a predicted non-membrane domain recognized a single band at ~49 kDa (calculated mass, 46.9 kDa) from the P. falciparum protein extract by Western blotting. Protein co-localization studies that employed established markers for the merozoite surface, rhoptries, and micronemes by an indirect immunofluorescence assay suggested that the ~49 kDa protein was expressed in micronemes at trophozoite/schizont stages. Immunoelectron microscopy demonstrated that the 49 kDa protein is indeed localized primarily to micronemes in the merozoites. Based on our characterization, we have tentatively named this novel protein as microneme-associated membrane antigen 1 (MAMA1). Functional studies demonstrate that the MAMA1 antibody blocked RBC invasion by P. falciparum (3D7 strain) in vitro, in a concentration-dependent manner. Furthermore, MAMA1 presumably embedded in small vesicles present in the parasite protein extract bound to intact human RBCs, and the MAMA1-RBC binding was neuraminidase and trypsin resistant, but chymotrypsin sensitive. Native MAMA1 bound specifically to the 5ABC peptide of band 3 in our solution binding assay. Together, these results suggest that the novel microneme protein MAMA1 interacts with host band 3 and facilitates merozoite entry during RBC invasion.

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