Identification and functional characterization of a novel arginine/ornithine transporter, a member of a cationic amino acid transporter subfamily in the Trypanosoma cruzi genome

Particle and Fibre Toxicology

Susan Gaye Amara²  Wim Degrave³  Wanderley de Souza⁶  Christian M. Probst⁵  Marco Aurélio Krieger⁵  Técia Maria Ulisses de Carvalho⁴  Marcos Catanho³  Megan P. Miller²  Cristina Henriques¹ 

[1] Nucleo de Biologia Estrutural e Biomagens, Universidade Federal do Rio de Janeiro-CENABIO, Rio de Janeiro, RJ, Brazil;Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh 15260, PA, USA;Fiocruz, Instituto Oswaldo Cruz, Laboratório de Genômica Funcional e Bioinformática, Av. Brasil 4365, Manguinhos, Rio de Janeiro, 21040-900, RJ, Brazil;Instituto de Biofísica Carlos Chagas Filho-UFRJ, CCS-Bloco G-Laboratório de Ultraestrutura Celular Hertha Meyer, Rio de Janeiro 21949-900, RJ, Brazil;Instituto Carlos Chagas-ICC-FIOCRUZ, Curitiba 81350-010, PR, Brazil;Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Biomagens-INBEB, Rio de Janeiro, Brazil

关键词: Parasite;    T. cruzi;    Trypanosomatids;    Protozoan;    Transporter;    Ornithine;    Arginine;   

Others  :  1224140
DOI  :  10.1186/s13071-015-0950-y

 received in 2014-09-16, accepted in 2015-06-13,  发布年份 2015

【 摘 要 】

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 [ ³ H]-arginine displayed a 16-fold higher efflux of [ ³ H]-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.

【 授权许可】

   
2015 Henriques et al.

【 图 表 】

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