Glucose acquisition is essential for the long-term survival of adult Clonorchis sinensis within the host bile duct. Although several glucose transporter-related proteins have been identified in C. sinensis, the molecular and functional properties of facilitative glucose transport proteins, including the protein designated as glucose transporter protein 5 (CsGTP5) in this study, have not been characterized. In this study, CsGTP5 was cloned from adult C. sinensis and functionally analyzed using the Xenopus laevis oocyte expression system. The CsGTP5 open reading frame comprised 1,575 bp and encoded a 524-amino-acid protein with 12 predicted transmembrane domains, consistent with the topology of facilitative glucose transporter-like proteins. CsGTP5-expressing oocytes showed significantly increased uptake of [3 H] deoxy-D-glucose compared with water-injected control oocytes, whereas no significant uptake was observed for other tested substrates, including arginine, α-ketoglutarate, p-aminohippurate, taurocholate, and tetraethylammonium. CsGTP5-mediated [3 H] deoxy-D-glucose uptake increased in a time-dependent manner and was not affected by replacement of extracellular Na+ with Li+ or choline, indicating sodium-independent transport. Uptake was saturable, with an apparent Km of 4.0 mM and a Vmax of 300.0 pmol/oocyte/h. Competition assays showed that deoxy-D-glucose and glucose strongly inhibited CsGTP5-mediated uptake, whereas other monosaccharides had little effect. Molecular docking analysis further supported the predicted glucose-recognition capacity of CsGTP5. These findings demonstrate that CsGTP5 mediates sodium-independent deoxy-D-glucose uptake in a heterologous oocyte expression system and provide a basis for further investigation of its physiological role in C. sinensis.

Clonorchis sinensis, which causes clonorchiasis, is prevalent in East Asian countries and poses notable health risks, including bile duct complications. Although praziquantel is the primary treatment for the disease, the emerging resistance among trematodes highlights the need for alternative strategies. Understanding the nutrient uptake mechanisms in trematodes, including C. sinensis, is crucial for developing future effective treatments. This study aimed to characterize the function of C. sinensis glucose transporter 4 (CsGTP4) and determine its role in nutrient uptake employing synthesized cDNA of adult C. sinensis worms. The functional characterization of CsGTP4 involved injecting its cRNA into Xenopus laevis oocytes and analyzing the deoxy-D-glucose uptake levels. The results demonstrated that deoxy-D-glucose uptake depended on the deoxy-D-glucose incubation and CsGTP4 expression time, but not sodium-dependent. The concentration-dependent uptake followed the Michaelis–Menten equation, with a Km value of 2.7 mM and a Vmax value of 476 pmol/oocyte/h based on the Lineweaver–Burk analysis. No uptake of radiolabeled α-ketoglutarate, p-aminohippurate, taurocholate, arginine, or carnitine was observed. The uptake of deoxy-D-glucose by CsGTP4 was significantly inhibited by unlabeled glucose and galactose in a concentration-dependent manner. It was significantly inhibited under strongly acidic and basic conditions. These insights into the glucose uptake kinetics and pH dependency of CsGTP4 provide a deeper understanding of nutrient acquisition in trematodes. This study contributes to the development of novel antiparasitic agents, addressing a considerable socioeconomic challenge in affected regions.