The signal sequence and C-terminal hydrophobic domain are required for localization of the sexual stage antigen pgs28 to the surface of P. gallinaceum ookinetes.
The Pgs28 protein is a major surface antigen of the sexual stages of Plasmodium gallinaceum the zygotes and the ookinetes. The protein contains conserved motifs, namely an N-terminal signal sequence, four epidermal growth factor-like repeats and a C-terminal hydrophobic domain that serves as a signal for glycosylphosphatidylinositol (GPI)--anchor modification. In this study, we define the protein motifs required for the surface localization of Pgs28 in ookinetes. using transient transfection combined with immunofluorescence and confocal microscopy. Pgs28 fused to the green fluorescent protein (Pgs28-GFP) is expressed in zygotes, intermediate retort forms and ookinetes. Mutational analyses of Pgs28 coding regions reveal that deletions of the signal sequence and the C-terminal domain result in intracellular retention of the fusion protein. Therefore, the signal sequence and C-terminal domain are required for cell surface localization. Additionally, the Pgs28-GFP fusion proteins are shed from the surface of live ookinetes, suggesting that Pgs28 may be involved in interactions with the cells of the mosquito midgut or during motility.
Patankar
S
S
Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA 02115, USA.
Fujioka
H
H
Wirth
D F
DF
D000818:Animals; D000953:Antigens, Protozoan; D005455:Fluorescent Antibody Technique; D017353:Gene Deletion; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D016253:Microscopy, Immunoelectron; D009154:Mutation; D015388:Organelles / Q000378:metabolism; D010964:Plasmodium gallinaceum / Q000235:genetics / Q000254:growth & development / Q000378:metabolism / Q000502:physiology*; D021382:Protein Sorting Signals / Q000502:physiology*; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection
D000953:Antigens, Protozoan; D008164:Luminescent Proteins; C079421:Pgs28 protein, Plasmodium gallinaceum; D021382:Protein Sorting Signals; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins