This site needs JavaScript to work properly. Please enable it to take advantage of the complete set of features!
Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

NIH NLM Logo
Log in
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Jul;9(13):3489-506.
doi: 10.1002/pmic.200800874.

Phosphoproteomic analysis of the human pathogen Trypanosoma cruzi at the epimastigote stage

Affiliations

Phosphoproteomic analysis of the human pathogen Trypanosoma cruzi at the epimastigote stage

Ernesto S Nakayasu et al. Proteomics. 2009 Jul.

Abstract

Trypanosoma cruzi is the etiologic agent of Chagas disease, which affects millions of people in Latin America and has become a public health concern in the United States and areas of Europe. The possibility that kinase inhibitors represent novel anti-parasitic agents is currently being explored. However, fundamental understanding of the cell-signaling networks requires the detailed analysis of the involved phosphorylated proteins. Here, we have performed a comprehensive MS-based phosphorylation mapping of phosphoproteins from T. cruzi epimastigote forms. Our LC-MS/MS, dual-stage fragmentation, and multistage activation analysis has identified 237 phosphopeptides from 119 distinct proteins. Furthermore, 220 phosphorylation sites were unambiguously mapped: 148 on serine, 57 on threonine, and 8 on tyrosine. In addition, immunoprecipitation and Western blotting analysis confirmed the presence of at least seven tyrosine-phosphorylated proteins in T. cruzi. The identified phosphoproteins were subjected to Gene Ontology, InterPro, and BLAST analysis, and categorized based on their role in cell structure, motility, transportation, metabolism, pathogenesis, DNA/RNA/protein turnover, and signaling. Taken together, our phosphoproteomic data provide new insights into the molecular mechanisms governed by protein kinases and phosphatases in T. cruzi. We discuss the potential roles of the identified phosphoproteins in parasite physiology and drug development.

PubMed Disclaimer

Conflict of interest statement

CONFLICT OF INTEREST

The authors have declared no conflict of interest.

Figures

Figure 1
Figure 1
Venn diagram of T. cruzi phosphopeptides identified by MS/MS, MS/MS/MS, and MSA analyses.
Figure 2
Figure 2
Gene ontology analysis. GO analysis was performed using Blast2GO algorithm. The representative categories for molecular function, cellular component, and biological process are shown in the graph. For the complete list, see Supplementary Tables 4 and 5.
Figure 3
Figure 3
(A) MS/MS spectrum of the tyrosine-phosphorylated peptide LSPSEPNVAYpICSR from glycogen synthase kinase 3 (GSK3). Matched b and y fragments are indicated. (B) Western blotting analysis of tyrosine phosphorylated proteins in T. cruzi. Epimastigote extracts were subjected to anti-phosphotyrosine immunoprecipitation and separation by 10% SDS-PAGE. After blocking with BSA, the membrane was probed with the anti-phosphotyrosine antibody, followed by detection with horseradish peroxidase conjugated anti-mouse IgG and chemiluminescent reagent. Arrows denote T. cruzi tyrosine phosphorylated proteins. Brackets denote immunoglobulin G heavy (IgG HC) and light chains (IgG LC).
Figure 3
Figure 3
(A) MS/MS spectrum of the tyrosine-phosphorylated peptide LSPSEPNVAYpICSR from glycogen synthase kinase 3 (GSK3). Matched b and y fragments are indicated. (B) Western blotting analysis of tyrosine phosphorylated proteins in T. cruzi. Epimastigote extracts were subjected to anti-phosphotyrosine immunoprecipitation and separation by 10% SDS-PAGE. After blocking with BSA, the membrane was probed with the anti-phosphotyrosine antibody, followed by detection with horseradish peroxidase conjugated anti-mouse IgG and chemiluminescent reagent. Arrows denote T. cruzi tyrosine phosphorylated proteins. Brackets denote immunoglobulin G heavy (IgG HC) and light chains (IgG LC).

References

    1. Barrett MP, Burchmore RJ, Stich A, Lazzari JO, et al. The trypanosomiases. Lancet. 2003;362:1469–1480. - PubMed
    1. Dias JC, Silveira AC, Schofield CJ. The impact of Chagas disease control in Latin America: a review. Mem Inst Oswaldo Cruz. 2002;97:603–612. - PubMed
    1. Bern C, Montgomery SP, Herwaldt BL, Rassi A, Jr, et al. Evaluation and treatment of Chagas disease in the United States: a systematic review. JAMA. 2007;298:2171–2181. - PubMed
    1. Piron M, Verges M, Munoz J, Casamitjana N, et al. Seroprevalence of Trypanosoma cruzi infection in at-risk blood donors in Catalonia (Spain) Transfusion. 2008;48:1862–1868. - PubMed
    1. Tyler KM, Engman DM. The life cycle of Trypanosoma cruzi revisited. Int J Parasitol. 2001;31:472–481. - PubMed

Publication types

MeSH terms

Full text links
Wiley full text link Wiley Free PMC article
Cite

AltStyle によって変換されたページ (->オリジナル) /