Identification and subcellular localization of TcHIP, a putative Golgi zDHHC palmitoyl transferase of Trypanosoma cruzi

Abstract

Protein palmitoylation is a post-translational modification that contributes to determining protein localization and function. Palmitoylation has been described in trypanosomatid protozoa, but no zDHHC palmitoyl transferase has been identified in Trypanosoma cruzi, the etiological agent of Chagas disease in Latin America. In this study we identify and show the subcellular localization of TcHIP (Tc00.1047053508199.50), a putative T. cruzi zDHHC palmitoyl transferase. Analysis of the deduced protein sequence indicates that it contains ankyrin repeats (Ank and Ank2) and the zDHHC conserved domain, typical of zDHHC palmitoyl transferases. A TcHIP polyclonal antiserum obtained from mice immunized with the purified recombinant protein was used to study the presence and subcellular localization of the native enzyme. In western blots this antiserum recognized a protein of about 95 kDa, consistent with the predicted molecular mass of TcHIP (95.4 kDa), in whole extracts of T. cruzi epimastigotes, metacyclic trypomastigotes and intracellular amastigotes. Immunolocalization by confocal microscopy showed TcHIP labeling at the Golgi complex, co-localizing with the T. cruzi Golgi marker TcRab7-GFP. Transfectant T. cruzi epimastigotes containing a construct encoding TcHIP fused to proteins A and C (TcHIP/AC) were obtained. In western blotting experiments, the TcHIP polyclonal antiserum recognized both native and TcHIP/AC proteins in extracts of the transfectants. Confocal microscopy showed co-localization of native TcHIP with TcHIP/AC. These findings demonstrate the presence of a putative zDHHC palmitoyl transferase (TcHIP) containing ankyrin and zDHHC domains in different developmental forms of T. cruzi, and its association with the Golgi complex.

mAb CZP-315.D9: An Antirecombinant Cruzipain Monoclonal Antibody That Specifically Labels the Reservosomes ofTrypanosoma cruziEpimastigotes

“…TcHIP is a marker of the Golgi apparatus of T. cruzi [24]. Incubation of TcHIP/AC-transfected epimastigotes with both anti-protein-A tag and anti-TcCruzipain polyclonal sera revealed colocalization of protein A and TcCruzipain in a single spot at the anterior end of the cells, close to the kinetoplast, in a region corresponding to the Golgi complex.…”

“…We further analyzed colocalization of native cruzipain in the Golgi apparatus in transfected epimastigotes expressing the T. cruzi Golgi marker TcHIP/AC [24]. Three-day-old culture transfected epimastigotes were washed twice in PBS, fixed for 30 min with 4% paraformaldehyde, and incubated for one hour at 37°C with anti-TcCruzipain mouse polyclonal serum (1 : 500) and rabbit anti-protein A antibody (1 : 40,000).…”

Treatment of Trypanosoma cruzi with 2-bromopalmitate alters morphology, endocytosis, differentiation and infectivity

“…gDNA was extracted from three-day-old culture epimastigotes by a phenol-chloroform method [33]. TcPAT1 (TcHIP) primers [26] were also used for PCR. Amplifications were confirmed by 1.0% agarose gel electrophoresis.…”

“…A putative PAT has been identified in this protozoan (TcHIP/TcPAT1), localized in the Golgi complex of different life stages [26] and other nine could be overexpressed in epimastigotes, being mostly located at the anterior end of the parasites [27]. However, other still unidentified proteins should be also palmitoylated in T. cruzi , and palmitoylation is probably involved in diverse biological functions.…”

Origem, evolução e relações filogenéticas de homólogos de prolina racemase em espécies de Trypanosoma.

“…O gene TvPRAC, que codifica a prolina racemase em T. vivax, um parasita que se multiplica extracelularmente na corrente sanguínea, em compartimentos teciduais e no Sistema Nervoso Central (Batista et al, 2013;Galiza et al, 2011;Whitelaw et al, 1988), foi descrito como um gene de cópia única sem peptídeo sinal . A PRAC de T. vivax mantém características e parâmetros cinéticos comparáveis às da PRAC de T. cruzi e exibe também atividade mitogênica sobre células B, esta última caracterizada em experimentos in vitro e in vivo utilizando proteínas recombinantes.…”