Defining the minimal domain of the Plasmodium falciparum protein MESA involved in the interaction with the red cell membrane skeletal protein 4.1.
During part of its life cycle, the protozoan parasite Plasmodium falciparum lives within the human red blood cell and modifies both the structural and functional properties of the red cell. It does this by synthesizing a number of polypeptides that it transports into the red cell cytoplasm and to the red cell membrane. One of these transported proteins, MESA (mature parasite-infected erythrocyte surface antigen), is anchored to the red cell membrane by noncovalent interaction with erythrocyte protein 4.1. We have utilized a combination of in vitro transcription and translation and a membrane binding assay to identify the protein sequence involved in anchoring MESA to the membrane. Labeled fragments of different regions of the MESA protein were evaluated for their ability to bind to inside-out vesicle membrane preparations of human red cells. Binding was dependent on the presence of red cell membrane proteins and was abolished either by trypsin treatment or by selective depletion of membrane proteins. Binding was specific and could be inhibited by the addition of competing protein, with an IC50 of (6.3 +/- 1.2) x 10(-7) M, indicative of a moderate affinity interaction. Fractionation studies demonstrated that binding fragments interacted most efficiently with membrane protein fractions that had been enriched in protein 4.1. Binding inhibition experiments using synthetic peptides identified the binding domain of MESA for protein 4.1 as a 19-residue sequence near the amino terminus of MESA, a region capable of forming an amphipathic helix.
Bennett
B J
BJ
Walter and Eliza Hall Institute of Medical Research, Victoria 3050, Australia.
Mohandas
N
N
Coppel
R L
RL
D000818:Animals; D003598:Cytoskeletal Proteins; D003599:Cytoskeleton / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000378:metabolism*; D009479:Neuropeptides; D010963:Plasmodium falciparum / Q000378:metabolism*; D015800:Protozoan Proteins / Q000378:metabolism*
D003598:Cytoskeletal Proteins; C079081:MESA protein, Plasmodium falciparum; D008565:Membrane Proteins; D009479:Neuropeptides; D015800:Protozoan Proteins; C028841:erythrocyte membrane band 4.1 protein; C120557:erythrocyte membrane protein band 4.1-like 1