Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Parasites Hosts Dis
2024 Nov 22;624:450-460. doi: 10.3347/PHD.24051.
Show Gene links
Show Anatomy links
Functional characterization of glucose transporter 4 involved in glucose uptake in Clonorchis sinensis.
Jun H
,
Mazigo E
,
Lee WJ
,
Park YK
,
Han JH
,
Cha SH
.
???displayArticle.abstract???
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.
Fig. 1. CsGTP4-mediated uptake of deoxy-D-glucose. The uptake rates of the radiolabeled compounds were measured in water-injected (Control, white bar) and CsGTP4-expressing (black bar) oocytes for 1 h (mean±SE, n=8–10). The concentration of the injected substrate was as follows: [3H] deoxy-D-glucose, 100 μM; [3H] arginine, 100 nM; [14C] α-ketoglutarate, 5 μM; [14C] p-aminohippurate, 10 μM; [3H] taurocholate, 200 nM; and [14C] carnitine, 40 nM. Significant differences were calculated using the student’s t-test. **P<0.01.
Fig. 2. Time dependency of deoxy-D-glucose transport via CsGTP4. (A) The uptake of 100 μM [3H] deoxy-D-glucose in the CsGTP4-expressing or water-injected (control) oocytes was measured at the indicated CsGTP4 expression times. (B) The uptake of 100 μM [3H] deoxy-D-glucose in control (open circle) and CsGTP4-expressing (closed circle) oocytes was measured for an incubation period of 120 min at 10–30 min intervals. All results are represented as mean±SE (n=6–8). Significant differences in D-glucose uptake levels between the control and CsGTP4-expressing oocytes were calculated using the student’s t-test. **P<0.01.
Fig. 3. Glucose transport properties of CsGTP4. (A) Effect of extracellular cation on [3H] deoxy-D-glucose uptake in oocytes expressing CsGTP4. The uptake rate of [3H] deoxy-D-glucose (100 μM) was measured in the presence or absence of extracellular Na+. Extracellular Na+ was replaced with equimolar concentrations of choline and lithium-ion. The results are represented as the mean±SE (n=6–8). N.S.,not significant. **P<0.01. (B) The trans-stimulatory effect and concentration dependence of the CsGTP4-mediated uptake of [3H] deoxy-D-glucose. A lack of a trans-stimulatory effect of glucose on the CsGTP4-mediated glucose efflux was observed. Oocytes expressing CsGTP4 were incubated with 100 μM [3H] deoxy-D-glucose for 1 h and transferred to ND96 solution with or without (control) 1 mM or 10 mM unlabeled glucose. The efflux amount of glucose within 1 h was shown as the percentage of the preloaded amount. (C) Various extracellular pH conditions affected the [3H] deoxy-D-glucose (100 μM) uptake level. Specifically, low and high pH significantly reduced [3H] deoxy-D-glucose (100 μM) uptake via CsGTP4. Statistical analysis was performed using the student’s t-test to compare the results with the pH 7.4 group, representing neutral conditions *P<0.05. (D) The saturation of CsGTP4-mediated uptake of [3H] deoxy-D-glucose. The uptake rates of [3H] deoxy-D-glucose by the control (water-injected) or CsGTP4-expressing oocytes for 1 h were measured at variable concentrations (mean±S.E.; n=6–8). Inset, Lineweaver–Burk analysis of the concentration-dependent uptake of [3H] deoxy-D-glucose. V, velocity; S, concentration of D-glucose.
Fig. 4. Active analogue and substrate binding model on CsGTP4. (A) Uptake assays were carried out with [3H] deoxy-D-glucose using various concentrations of sugar derivates, including glucose (control), galactose, mannose, fructose, and 3-O-methylglucose. Significant differences were calculated using the student’s t-test. *P<0.05. (B) The coordination of D-glucose in CsGTP4 is depicted in the structure, with core residues for glucose binding. The number of carbons is represented as numeric circles, and the interactions between the 2 molecules are illustrated as yellow dotted lines with predicted distances (Å). The tertiary structure of CsGTP4 shows putative D-glucose-binding residues, including Gln 146, Gln 267, Gln 268, Asn 273, and Gln 404, depicted with their side chains.