Proteomic analysis of detergent-solubilized membrane proteins from insect-developmental forms of Trypanosoma cruzi.
The cell surface of Trypanosoma cruzi, the etiologic agent of Chagas disease, is covered by a dense layer of glycosylphosphatidylinositol (GPI)-anchored molecules. These molecules are involved in a variety of interactions between this parasite and its mammalian and insect hosts. Here, using the neutral detergent Triton X-114, we obtained fractions rich in GPI-anchored and other membrane proteins from insect developmental stages of T. cruzi. These fractions were analyzed by two-dimensional liquid chromatography coupled to tandem mass spectrometry (2D-LC-MS/MS), resulting in the identification of 98 proteins of metacyclic trypomastigotes and 280 of epimastigotes. Of those, approximately 65% (n=245) had predicted lipid post-translational modification sites (i.e., GPI-anchor, myristoylation, or prenylation), signal-anchor sequence, or transmembrane domains that could explain their solubility in detergent solution. The identification of some of these modified proteins was also validated by immunoblotting. We also present evidence that, in contrast to the noninfective proliferative epimastigote forms, the infective nonproliferative metacyclic trypomastigote forms express a large repertoire of surface glycoproteins, such as GP90 and GP82, which are involved in adhesion and invasion of host cells. Taken together, our results unequivocally show stage-specific protein profiles that appear to be related to the biology of each T. cruzi insect-derived developmental form.
Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, UNIFESP, Rua Botucatu, 862, CEP 04023-062, São Paulo, Brazil.
Esteban M
EM
Cordero
Ernesto S
ES
Nakayasu
Luciana G
LG
Gentil
Nobuko
N
Yoshida
Igor C
IC
Almeida
José Franco
JF
da Silveira
D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods; D019295:Computational Biology / Q000379:methods; D003902:Detergents / Q000494:pharmacology*; D006023:Glycoproteins / Q000737:chemistry; D017261:Glycosylphosphatidylinositols / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D008565:Membrane Proteins / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D017386:Sequence Homology, Amino Acid; D014349:Trypanosoma cruzi / Q000187:drug effects*
D003902:Detergents; D006023:Glycoproteins; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40