Functional characterization of Clonorchis sinensis choline transporter.

Clonorchis sinensis is commonly found in East Asian countries. Clonorchiasis is prevalent in these countries and can lead to various clinical symptoms. In this study, we used overlap extension polymerase chain reaction (PCR) and the Xenopus laevis oocyte expression system to isolate a cDNA encoding the choline transporter of C. sinensis (CsChT). We subsequently characterized recombinant CsChT. Expression of CsChT in X. laevis oocytes enabled efficient transport of radiolabeled choline, with no detectable uptake of arginine, α-ketoglutarate, p-aminohippurate, taurocholate, and estrone sulfate. Influx and efflux experiments showed that CsChT-mediated choline uptake was time- and sodium-dependent, with no exchange properties. Concentration-dependent analyses of revealed saturable kinetics consistent with the Michaelis-Menten equation, while nonlinear regression analyses revealed a Km value of 8.3 µM and a Vmax of 61.0 pmol/oocyte/h. These findings contribute to widen our understanding of CsChT transport properties and the cascade of choline metabolisms within C. sinensis.

Functional characterization of Plasmodium vivax hexose transporter 1.

Plasmodium vivax is the most widely distributed human malaria parasite. The eradication of vivax malaria remains challenging due to transmission of drug-resistant parasite and dormant liver form. Consequently, anti-malarial drugs with novel mechanisms of action are urgently demanded. Glucose uptake blocking strategy is suggested as a novel mode of action that leads to selective starvation in various species of malaria parasites. The role of hexose transporter 1 in Plasmodium species is glucose uptake, and its blocking strategies proved to successfully induce selective starvation. However, there is limited information on the glucose uptake properties via P. vivax hexose transporter 1 (PvHT1). Thus, we focused on the PvHT1 to precisely identify its properties of glucose uptake. The PvHT1 North Korean strain (PvHT1NK) expressed Xenopus laevis oocytes mediating the transport of [3H] deoxy-D-glucose (ddGlu) in an expression and incubation time-dependent manner without sodium dependency. Moreover, the PvHT1NK showed no exchange mode of glucose in efflux experiments and concentration-dependent results showed saturable kinetics following the Michaelis-Menten equation. Non-linear regression analysis revealed a Km value of 294.1 µM and a Vmax value of 1,060 pmol/oocyte/hr, and inhibition experiments showed a strong inhibitory effect by glucose, mannose, and ddGlu. Additionally, weak inhibition was observed with fructose and galactose. Comparison of amino acid sequence and tertiary structure between P. falciparum and P. vivax HT1 revealed a completely conserved residue in glucose binding pocket. This result supported that the glucose uptake properties are similar to P. falciparum, and PfHT1 inhibitor (compound 3361) works in P. vivax. These findings provide properties of glucose uptake via PvHT1NK for carbohydrate metabolism and support the approaches to vivax malaria drug development strategy targeting the PvHT1 for starving of the parasite.

Functional characterization of Clonorchis sinensis sodium-bile acid co-transporter (CsSBAT) as a steroid sulfate transporter.

Clonorchis sinensis (Cs) is a common trematode in Asian countries. Infection by Cs can result in many clinical symptoms. Here, a cDNA encoding a Cs apical sodium-dependent bile acid transporter (CsSBAT) was isolated from a Cs cDNA library, and functional characterization was performed using Xenopus laevis oocyte expression system. When expressed in Xenopus laevis oocytes, CsSBAT mediated the transport of radiolabeled estrone sulfate and dehydroepiandrosterone sulfate. No trans-uptake of carnitine, estradiol 17 ß-D glucuronide, prostaglandin E2, p-aminohippuric acid, α-ketoglutaric acid, and tetraethylammonium was observed. CsSBAT-mediated estrone sulfate uptake was in a time- and sodium-dependent manner. CsSBAT showed no exchange properties in efflux experiments. Concentration-dependent results showed saturable kinetics consistent with the Michaelis-Menten equation. Nonlinear regression analyses yielded a Km value of 0.3 ± 0.04 µM for [3H]estrone sulfate. CsSBAT-mediated estrone sulfate uptake was strongly inhibited by sulfate conjugates but not glucuronide conjugates. These findings contribute to our understanding of CsSBAT transport properties and the cascade of estrogen metabolite movement in Cs.