Molecular Characterization of glucose transporters in Clonorchis sinensis

초록

Two full length complementary DNAs (cDNA) encoding glucose transporters of Clonorchis sinensis (CsGLUT and CsGLUT2) were isolated from adult C. sinensis cDNA library. The open reading frames of CsGLUT and CsGLUT2 cDNAs consists of 1653 and 1771 base pairs that encode a 550- and 496-amino acid residue proteins. Hydropathy analysis suggested that CsGLUT and CsGLUT2 possess 12 putative membrane spanning domains. As the first step we try to analyze the functional properties of CsGLUT. The Northern blot analysis result using poly(A)+RNA showed a strong band at ~2.1 kb for CsGLUT. When complementary RNA was expressed in Xenopus oocytes, CsGLUT mediated the transport of radiolabeled deoxy-D-glucose in a time dependent but sodium-independent manner. Dose dependency experimental results showed saturable kinetics and well fitted the Michaelis-Menten equation. Nonlinear regression analyses showed a Km value of 588.5 ± 53.0 ?M and a Vmaxvalue of 1500.0 ± 67.5 pmol/oocyte/30 min for [1,2-3H]2-deoxy-Dglucose. No trans-uptakes of bile acid (taurocholic acid) amino acids (tryptophan and arginine), or p-aminohippuric acid were observed. CsGLUT-mediated transport of deoxyglucose was significantly and concentration-dependently inhibited by radio-unlabeled deoxyglucose and D-glucose. 3-O-Methylglucose at 10 and 100 ?M inhibited deoxyglucose uptake by ~50 % without concentration dependence. No inhibitory effects by galactose, mannose, and fructose were observed. This work may contribute to the molecular biological study of carbohydrate metabolism and new drug development of C. sinensis.