cDNA cloning and partial characterization of amastigote specific surface protein from Trypanosoma cruzi.
Trypanosoma cruzi amastigote surface proteins are the target of both humoral and cell-mediated immune responses; however, few such molecules have been thoroughly studied. In order to study a T. cruzi amastigote-specific protein (SSP4), we used antibodies against the deglycosylated form of this molecule to clone cDNA. The selected cDNA clone (2070 bp) encodes for a 64 kDa protein product whose sequence analysis revealed no N-glycosylation signal. The DNA sequence showed high homology with a member of a previously reported dispersed repetitive gene family of T. cruzi. Antibodies against the recombinant protein reacted strongly with a 66 kDa protein and weakly with an 84 kDa protein in amastigote extracts. Immunoelectron microscopy studies showed that intracellular amastigotes express the native protein on their surfaces and flagellar pockets. The antibody label was also associated with an amorphous material present in the parasitic cavity and in direct contact with the parasite surface, which suggest that amastigotes are releasing this material. On cell-free amastigotes, the antibody showed strong decoration of the cell surface and labeling of intracellular vesicles. Immunofluorescence analysis showed that the superficial protein is expressed shortly after trypomastigotes begin to transform into amastigotes. Anti-recombinant protein antibodies recognized proteins of 100 kDa and 50-60 kDa in protein extracts of rat heart and skeletal muscle, respectively.
Olivas-Rubio
Marybell
M
Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del IPN, México DF 07360, Mexico. oliver@uady.mx
Hernández-Martínez
Salvador
S
Talamás-Rohana
Patricia
P
Tsutsumi
Victor
V
Reyes-López
Pedro A
PA
Rosales-Encina
José Luis
JL
D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D014355:Chagas Disease / Q000276:immunology / Q000378:metabolism / Q000469:parasitology / Q000473:pathology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005786:Gene Expression Regulation; D006801:Humans; D008018:Life Cycle Stages / Q000235:genetics; D008253:Macaca mulatta; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D014349:Trypanosoma cruzi / Q000502:physiology* / Q000648:ultrastructure
D000953:Antigens, Protozoan; D018076:DNA, Complementary; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; C501457:SSP4 protein, Trypanosoma cruzi