Proteomic Analysis of Detergent-Solubilized Membrane Proteins from Insect-Developmental Forms of Trypanosoma cruzi

Abstract

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 twodimensional 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 posttranslational 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 insectderived developmental form.

TcTASV-C, a Protein Family in Trypanosoma cruzi that Is Predominantly Trypomastigote-Stage Specific and Secreted to the Medium

“…On the other hand, 5 TcTASV-A genes were identified in the trypomastigote proteome, in agreement to our evidences of the intracellular localization of TcTASV-A in trypomastigotes (unpublished observations). The proteomes of metacyclic trypomastigotes and epimastigotes –designed to preferentially identify membrane-bound and/or hydrophobic proteins- failed to find any TcTASV peptide [64], in line with results presented here that showed a minimum expression (or absence) of TcTASV-C in insect-derived parasite-stages.…”

The Repetitive Cytoskeletal Protein H49 of Trypanosoma cruzi Is a Calpain-Like Protein Located at the Flagellum Attachment Zone

“…This could be due to the translocation of cytoplasmic calpains to the plasma membrane after cellular stimulation. Ennes-Vidal et al (2011) showed by ultrastructural immunolabeling that calcium-dependent cysteine peptidases are mainly located at the cytoplasm of epimastigotes [50].Recently, we identified seven calpain-like proteins including two cytoskeletal associated proteins CAP5.5 in the plasma membrane of T. cruzi epimastigotes and metacyclic trypomastigotes [51]. Six of these calpain-like proteins contain N-terminal fatty acid acylation motifs, indicating an association with cellular membranes.…”

Structural Features Affecting Trafficking, Processing, and Secretion of Trypanosoma cruzi Mucins

“…Purification of GPI-anchored Proteins-Pellets containing 1.5 ϫ 10 8 parasites were homogenized in 2 ml of GPI buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2% Triton X-114 (TX-114), 1 mM PMSF and a protease inhibitor mixture (Sigma)) on ice for 1 h and the homogenate processed as described (37). Briefly, the homogenate was centrifuged at 8,800 ϫ g for 10 min at 0°C, and the supernatant (S1) was stored at Ϫ20°C for 24 h. The pellet (P1) was washed with 1 ml of buffer A (10 mM TrisHCl, pH 7.4, 150 mM NaCl, 0.06% TX-114, 1 mM PMSF), sonicated, and resuspended in denaturing loading buffer containing 6 M urea.…”