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Sci Rep
2025 Feb 04;151:4234. doi: 10.1038/s41598-025-88746-2.
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Characteristics of Plasmodium vivax apicomplexan amino acid transporter 8 (PvApiAT8) in the cationic amino acid transport.
Lee WJ
,
Mazigo E
,
Han JH
,
Cha SH
.
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Plasmodium vivax is the most widespread malaria parasite affecting humans, and its eradication is challenging due to the spread of drug-resistant parasites and their ability to remain in liver as a dormant stage. These parasites invade and multiply extensively within hepatocytes and erythrocytes in the host, relying on nutrient acquisition for their growth and replication. A promising new treatment aimed at targeting P. vivax involves blocking cationic amino acid uptake, which is a biological source of nutrients for the parasite. Novel Putative Transporter 1 (NPT1), identified as a cationic amino acid transporter in Apicomplexan, has a homologue in Plasmodium species known as apicomplexan amino acid transporter 8 (ApiAT8). This study focuses on P. vivax ApiAT8 to understand its precise role. PvApiAT8 was expressed in Xenopus laevis oocytes and shown to selectively uptake cationic amino acids. The uptake activity of [3H] L-arginine was shown to depend on PvApiAT8 expression time and substrate incubation time. PvApiAT8 was sodium-independent and functioned at pH levels between 6.5 and 8.5, with no efflux activity observed. Kinetic analysis showed saturable uptake for L-arginine consistent with Michaelis-Menten kinetics, with a Km of 1.5 ± 0.3 µM and a Vmax of 25.0 ± 4.8 pmol/oocyte/hr. Inhibition assays further confirmed its selectivity for cationic amino acids such as L-arginine, L-lysine, L-histidine, and L-ornithine. Sequence and structural analyses revealed a conserved binding pocket for cationic amino acids in Plasmodium species, distinct from that in Toxoplasma gondii NPT1. These findings highlight the potential of targeting PvApiAT8 in developing new treatments for P. vivax malaria.
Fig. 1. Schematic representation of PvApiAT8. (A) The predicted topology of PvApiAT8 (1–572 aa.) full-length reveals 12 transmembrane segments, with a short intracellular N-terminus (1–38 aa.) and C-terminus (549–572 aa.). The number of transmembrane domains and corresponding amino acid positions are indicated in parentheses. (B) The tertiary structure of PvApiAT8 is shown from Arg 38 (N-terminus, shown in blue) to Phe 547 (C-terminus, shown in red). A long intracellular loop spanning residues (250–354 aa.) characterized by low accuracy in the predicted structure, is represented by a connecting line. The horizontal bars illustrate the expected lipid bilayer boundaries for the 12 transmembrane domains. (C) The phylogenetic tree of ApiAT8 in Plasmodium species and Toxoplasma gondii was constructed using the MEGA11 software with the maximum likelihood method and 1,000 pseudo-replications, showing the evolutionary relationships among these species.
Fig. 2. PvApiAT8-mediated uptake of L-arginine. (A) The uptake rates of radiolabeled amino acids were measured in water-injected (control) oocytes and oocytes expressing PvApiAT8 over a 1-hour period. The concentrations of the incubated amino acids were as follows: [3H] L-arginine at 100 nM, [3H] L-glutamine at 100 nM, [3H] L-glycine at 100 nM, and [3H] L-leucine at 100 nM. (B) The uptake of 100 nM [3H] L-arginine in PvApiAT8-expressing oocytes was assessed at various expression times (0–72 h). (C) The uptake of 100 nM [3H] L-arginine in both control and PvApiAT8-expressing oocytes was measured at 15-minute intervals, extending up to 75 min. Significant differences are indicated by double asterisks (p < 0.01) and triple asterisks (p < 0.001). Data are presented as the mean ± S.E., n = 8–10.
Fig. 3. The transport properties of L-arginine via PvApiAT8. (A) The effect of extracellular cation ions on [3H] L-arginine uptake in PvApiAT8-expressing oocytes. The uptake rate of [3H] L-arginine (100 nM) was measured in the presence or absence of extracellular Na+. Extracellular Na+ was replaced with equimolar concentrations of lithium (Li+), choline, and NMDG (N-methyl-d-glucosamine). (B) Various extracellular pH conditions affected the uptake level of [3H] L-arginine (100 nM). Specifically, both low and high pH significantly reduced the uptake of [3H] L-arginine (100 nM) via PvApiAT8. Statistical analysis was performed using Student’s t-test to compare results with the pH 7.4 group, which represents neutral conditions. (C) The lack of a trans-stimulatory effect of L-arginine on PvApiAT8-mediated efflux of [3H] L-arginine was observed. Oocytes expressing PvApiAT8 were incubated with 100 nM [3H] L-arginine for 90 min, and washed oocytes were transferred to ND96 solution (control) or ND96 containing 10 µM or 100 µM unlabeled L-arginine. The efflux amount of L-arginine during 1 h was expressed as the percentage of the preloaded amount. All results are presented as mean ± S.E. (n = 6–8).
Fig. 4. Kinetics of PvApiAT8-mediated uptake of [3H] L-arginine and amino acid selectivity of PvApiAT8. (A) The uptake rates of [3H] L-arginine by control (water-injected) or PvApiAT8-expressing oocytes were measured for 1 h at varying concentrations (mean ± S.E.; n= 6–8). The inset shows a Lineweaver-Burk analysis of the concentration-dependent uptake of [3H] L-arginine. “υ” representing velocity and “S” representing the concentration of L-arginine. (B) Uptake assays with [3H] L-arginine were performed in the presence of various amino acids. Statistical analysis compared [3H] L-arginine uptake in PvApiAT8-expressing oocytes to both control (CTL) and cationic amino acids, as well as comparisons among the cationic amino acids. Significant differences are denoted by one asterisk (p < 0.05), two asterisks (p < 0.01), or three asterisks (p < 0.001).
Fig. 5. The binding mode of cationic amino acids in PvApiAT8. (A) The transmembrane region of PvApiAT8, shown as a surface representation, highlighting the N-terminal domain (NTD, light blue) and C-terminal domain (CTD, yellow), excluding the long loop spanning region (amino acids 250–354). The gray areas represent non-transmembrane regions. ECD refers to the extracellular domain, and ICD to the intracellular domain. (B) The surface view of the electric potential of PvApiAT8, with dash-dot lines circle indicating a negatively charged region that may bind cationic amino acids from the extracellular side. (C) The internal view of the electric potential of PvApiAT8, where the dash-dot lines circle show a cationic amino acid binding pocket with a strongly negatively charged region. The lower area also shows a negatively charged, channel-like structure. (D) The binding mode of L-arginine with PvApiAT8 is represented in a 3D model, and (E) in a 2D view. (F) The binding mode of L-lysine with PvApiAT8 is represented in a 3D model, and (G) in a 2D view. (H) The binding mode of L-histidine with PvApiAT8 is represented in a 3D model, and (I) in a 2D view.
