Analysis of recombinant merozoite surface protein-1 of Plasmodium falciparum expressed in mammalian cells.
Synthetic chimeric DNA constructs with a reduced A + T content coding for full-length merozoite surface protein-1 of Plasmodium falciparum (MSP1) and three fragments thereof were expressed in HeLa cells. To target the recombinant proteins to the surface of the host cell the DNA sequences coding for the N-terminal signal sequence and for the putative C-terminal recognition/attachment signal for the glycosyl-phosphatidyl-inositol (GPI)-anchor of MSP1 were replaced by the respective DNA sequences of the human decay-accelerating-factor (DAF). The full-length recombinant protein, hu-MSP1-DAF, was stably expressed and recognised by monoclonal antibodies that bind to the N-terminus or the C-terminus of the native protein, respectively. Its apparent molecular mass is higher as compared to the native protein and it is post-translationally modified by attachment of N-glycans whereas native MSP1 is not glycosylated. Immunofluorescence images of intact cells show a clear surface staining. After permeabilization hu-MSP1-DAF can be detected in the cytosol as well. As judged by protease treatment of intact cells 25% of recombinant MSP1 is located on the surface. This fraction of hu-MSP1-DAF can be cleaved off the cell membrane by phosphatidylinositol-specific phospholipase C indicating that the protein is indeed bound to the cell membrane via a GPI-anchor. Human erythrocytes do not adhere to the surface of mammalian cells expressing either of the constructs made in this study.
Burghaus
P A
PA
Zentrum für Molekulare Biologie Heidelberg, Germany. burghaus@mailer.uni-marburg.de
Gerold
P
P
Pan
W
W
Schwarz
R T
RT
Lingelbach
K
K
Bujard
H
H
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D018958:CD55 Antigens / Q000235:genetics* / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D020066:Merozoite Surface Protein 1 / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D010963:Plasmodium falciparum / Q000737:chemistry* / Q000235:genetics / Q000254:growth & development; D011233:Precipitin Tests; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D014357:Trypsin
D018958:CD55 Antigens; D017261:Glycosylphosphatidylinositols; D020066:Merozoite Surface Protein 1; D011994:Recombinant Proteins; D014357:Trypsin