Identification and functional characterization of a novel arginine/ornithine transporter, a member of a cationic amino acid transporter subfamily in the Trypanosoma cruzi genome
Abstract Background Trypanosoma cruzi, the etiological agent of Chagas disease, is auxotrophic for arginine. It obtains this amino acid from the host through transporters expressed on the plasma membrane and on the membranes of intracellular compartments. A few cationic amino acid transporters have...
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| Format: | Article |
| Language: | English |
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BMC
2015-06-01
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| Series: | Parasites & Vectors |
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| Online Access: | https://doi.org/10.1186/s13071-015-0950-y |
| _version_ | 1797811765676867584 |
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| author | Cristina Henriques Megan P. Miller Marcos Catanho Técia Maria Ulisses de Carvalho Marco Aurélio Krieger Christian M. Probst Wanderley de Souza Wim Degrave Susan Gaye Amara |
| author_facet | Cristina Henriques Megan P. Miller Marcos Catanho Técia Maria Ulisses de Carvalho Marco Aurélio Krieger Christian M. Probst Wanderley de Souza Wim Degrave Susan Gaye Amara |
| author_sort | Cristina Henriques |
| collection | DOAJ |
| description | Abstract Background Trypanosoma cruzi, the etiological agent of Chagas disease, is auxotrophic for arginine. It obtains this amino acid from the host through transporters expressed on the plasma membrane and on the membranes of intracellular compartments. A few cationic amino acid transporters have been characterized at the molecular level, such as the novel intracellular arginine/ornithine transporter, TcCAT1.1, a member of the TcCAT subfamily that is composed of four almost identical open reading frames in the T. cruzi genome. Methods The functional characterization of the TcCAT1.1 isoform was performed in two heterologous expression systems. TcCAT subfamily expression was evaluated by real-time PCR in polysomal RNA fractions, and the cellular localization of TcCAT1.1 fused to EGFP was performed by confocal and immunoelectron microscopy. Results In the S. cerevisiae expression system, TcCAT1.1 showed high affinity for arginine (K m = 0.085 ± 0.04 mM) and low affinity for ornithine (K m = 1.7 ± 0.2 mM). Xenopus laevis oocytes expressing TcCAT1.1 showed a 7-fold increase in arginine uptake when they were pre-loaded with arginine, indicating that transport is enhanced by substrates on the trans side of the membrane (trans-stimulation). Oocytes that were pre-loaded with [3H]-arginine displayed a 16-fold higher efflux of [3H]-arginine compared with that of the control. Analysis of polysomal RNA fractions demonstrated that the expression of members of the arginine transporter TcCAT subfamily is upregulated under nutritional stress and that this upregulation precedes metacyclogenesis. To investigate the cellular localization of the transporter, EGFP was fused to TcCAT1.1, and fluorescence microscopy and immunocytochemistry revealed the intracellular labeling of vesicles in the anterior region, in a network of tubules and vesicles. Conclusions TcCAT1.1 is a novel arginine/ornithine transporter, an exchanger expressed in intracellular compartments that is physiologically involved in arginine homeostasis throughout the T. cruzi life cycle. The properties and estimated kinetic parameters of TcCAT1.1 can be extended to other members of the TcCAT subfamily. |
| first_indexed | 2024-03-13T07:28:32Z |
| format | Article |
| id | doaj.art-86598abaeb8a4edebc321127b288711a |
| institution | Directory Open Access Journal |
| issn | 1756-3305 |
| language | English |
| last_indexed | 2024-03-13T07:28:32Z |
| publishDate | 2015-06-01 |
| publisher | BMC |
| record_format | Article |
| series | Parasites & Vectors |
| spelling | doaj.art-86598abaeb8a4edebc321127b288711a2023-06-04T11:14:43ZengBMCParasites & Vectors1756-33052015-06-018111810.1186/s13071-015-0950-yIdentification and functional characterization of a novel arginine/ornithine transporter, a member of a cationic amino acid transporter subfamily in the Trypanosoma cruzi genomeCristina Henriques0Megan P. Miller1Marcos Catanho2Técia Maria Ulisses de Carvalho3Marco Aurélio Krieger4Christian M. Probst5Wanderley de Souza6Wim Degrave7Susan Gaye Amara8Fundação Oswaldo Cruz, Fiocruz-Mato Grosso do SulDepartment of Neurobiology, University of Pittsburgh School of MedicineFiocruz, Instituto Oswaldo Cruz, Laboratório de Genômica Funcional e BioinformáticaInstituto de Biofísica Carlos Chagas Filho-UFRJ, CCS-Bloco G-Laboratório de Ultraestrutura Celular Hertha MeyerInstituto Carlos Chagas-ICC-FIOCRUZInstituto Carlos Chagas-ICC-FIOCRUZInstituto de Biofísica Carlos Chagas Filho-UFRJ, CCS-Bloco G-Laboratório de Ultraestrutura Celular Hertha MeyerFiocruz, Instituto Oswaldo Cruz, Laboratório de Genômica Funcional e BioinformáticaNational Institute of Mental Health, NIH Building 10 Center DriverAbstract Background Trypanosoma cruzi, the etiological agent of Chagas disease, is auxotrophic for arginine. It obtains this amino acid from the host through transporters expressed on the plasma membrane and on the membranes of intracellular compartments. A few cationic amino acid transporters have been characterized at the molecular level, such as the novel intracellular arginine/ornithine transporter, TcCAT1.1, a member of the TcCAT subfamily that is composed of four almost identical open reading frames in the T. cruzi genome. Methods The functional characterization of the TcCAT1.1 isoform was performed in two heterologous expression systems. TcCAT subfamily expression was evaluated by real-time PCR in polysomal RNA fractions, and the cellular localization of TcCAT1.1 fused to EGFP was performed by confocal and immunoelectron microscopy. Results In the S. cerevisiae expression system, TcCAT1.1 showed high affinity for arginine (K m = 0.085 ± 0.04 mM) and low affinity for ornithine (K m = 1.7 ± 0.2 mM). Xenopus laevis oocytes expressing TcCAT1.1 showed a 7-fold increase in arginine uptake when they were pre-loaded with arginine, indicating that transport is enhanced by substrates on the trans side of the membrane (trans-stimulation). Oocytes that were pre-loaded with [3H]-arginine displayed a 16-fold higher efflux of [3H]-arginine compared with that of the control. Analysis of polysomal RNA fractions demonstrated that the expression of members of the arginine transporter TcCAT subfamily is upregulated under nutritional stress and that this upregulation precedes metacyclogenesis. To investigate the cellular localization of the transporter, EGFP was fused to TcCAT1.1, and fluorescence microscopy and immunocytochemistry revealed the intracellular labeling of vesicles in the anterior region, in a network of tubules and vesicles. Conclusions TcCAT1.1 is a novel arginine/ornithine transporter, an exchanger expressed in intracellular compartments that is physiologically involved in arginine homeostasis throughout the T. cruzi life cycle. The properties and estimated kinetic parameters of TcCAT1.1 can be extended to other members of the TcCAT subfamily.https://doi.org/10.1186/s13071-015-0950-yArginineOrnithineTransporterProtozoanTrypanosomatidsT. cruzi |
| spellingShingle | Cristina Henriques Megan P. Miller Marcos Catanho Técia Maria Ulisses de Carvalho Marco Aurélio Krieger Christian M. Probst Wanderley de Souza Wim Degrave Susan Gaye Amara Identification and functional characterization of a novel arginine/ornithine transporter, a member of a cationic amino acid transporter subfamily in the Trypanosoma cruzi genome Parasites & Vectors Arginine Ornithine Transporter Protozoan Trypanosomatids T. cruzi |
| title | Identification and functional characterization of a novel arginine/ornithine transporter, a member of a cationic amino acid transporter subfamily in the Trypanosoma cruzi genome |
| title_full | Identification and functional characterization of a novel arginine/ornithine transporter, a member of a cationic amino acid transporter subfamily in the Trypanosoma cruzi genome |
| title_fullStr | Identification and functional characterization of a novel arginine/ornithine transporter, a member of a cationic amino acid transporter subfamily in the Trypanosoma cruzi genome |
| title_full_unstemmed | Identification and functional characterization of a novel arginine/ornithine transporter, a member of a cationic amino acid transporter subfamily in the Trypanosoma cruzi genome |
| title_short | Identification and functional characterization of a novel arginine/ornithine transporter, a member of a cationic amino acid transporter subfamily in the Trypanosoma cruzi genome |
| title_sort | identification and functional characterization of a novel arginine ornithine transporter a member of a cationic amino acid transporter subfamily in the trypanosoma cruzi genome |
| topic | Arginine Ornithine Transporter Protozoan Trypanosomatids T. cruzi |
| url | https://doi.org/10.1186/s13071-015-0950-y |
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