Identification and heterologous expression of a new dense granule protein (GRA7) from Toxoplasma gondii.
Immunoscreening of an expression library constructed with Toxoplasma gondii tachyzoite mRNA with sera from toxoplasmosis-positive humans has led to the identification of a new parasite antigen. Sequence analysis of the gene encoding this antigen allowed the calculation of the theoretical molecular mass (25,857 Da) and showed that the protein contains a putative signal sequence. The C-terminal region contains two hydrophobic regions, the last of which has the characteristics of a membrane-spanning domain. When the protein was heterologously expressed in E. coli and tested by Western blot, it reacted with the human sera originally used for screening. The new antigen also reacted with a monoclonal antibody raised against the entire parasite. Ultrastructural analysis showed that the protein is localized in the dense granules. After host cell invasion, the protein is secreted into the vacuolar network, the parasitophorous vacuole membrane, and into extensions protruding in the cytoplasm. Therefore, it is suggested to designate this new dense granule protein GRA7, following the established nomenclature for this protein family.
Jacobs
D
D
Innogenetics N.V., Gent, Belgium. dirkjac@innogenetics.be
Dubremetz
J F
JF
Loyens
A
A
Bosman
F
F
Saman
E
E
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000913:Antibodies, Protozoan / Q000276:immunology; D000953:Antigens, Protozoan / Q000032:analysis / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D002522:Chlorocebus aethiops; D003594:Cytoplasmic Granules / Q000737:chemistry*; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D017125:Genes, Protozoan; D006801:Humans; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis; D014122:Toxoplasma / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000648:ultrastructure; D014709:Vero Cells
D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D018076:DNA, Complementary; C112573:GRA7 protein, Toxoplasma gondii; D015800:Protozoan Proteins; D011994:Recombinant Proteins