Derbyshire ET et al. (2008), Identification, expression and characterisation...

XB-ART-38152

Mol Biochem Parasitol 2008 Oct 01;1612:124-9. doi: 10.1016/j.molbiopara.2008.06.010.

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Identification, expression and characterisation of a Babesia bovis hexose transporter.

Derbyshire ET , Franssen FJ , de Vries E , Morin C , Woodrow CJ , Krishna S , Staines HM .

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Babesia are tick-transmitted haemoprotozoan parasites that infect cattle, with an estimated 500 million at risk worldwide. Here, two predicted hexose transporters (BboHT1 and 2) have been identified within the Babesia bovis genome. BboHT1, having 40% and 47% amino acid sequence similarity compared with the human (GLUT1) and Plasmodium falciparum (PfHT) hexose transporters, respectively, is the only one that could be characterised functionally after expression in Xenopus laevis oocytes. Radiotracer studies on BboHT1 showed that it is a saturable, Na(+)-independent, stereo-specific hexose transporter, with a K(m) value for glucose of 0.84+/-0.54 mM (mean+/-SEM). Using D-glucose analogues, hydroxyl positions at O-4 and O-6 have been identified as important for ligand binding to BboHT1. D-glucose transport was inhibited maximally by cytochalasin B (50 microM). A long-chain O-3 hexose derivative (compound 3361) that selectively inhibits PfHT also inhibited relatively potently BboHT1, with an apparent K(i) value of 4.1+/-0.9 microM (mean+/-SEM). Compound 3361 did not inhibit B. bovis proliferation in in vitro growth assays but inhibited invasion of glucose-depleted bovine erythrocytes. Taken together with results of inhibition studies with cytochalasin B and beta-glucogallin, these data provide new insights into glucose metabolism of erythrocytic-stage Babesia infections.

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Species referenced: Xenopus laevis
Genes referenced: slc2a1

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	Fig. 1. Alignments of transmembrane helices V and VII from GLUT1 with PfHT, BboHT1 (BbHT1) and BboHT2 (BbHT2). Bold letters indicate conserved residues compared with GLUT1 and shading indicates residues at the exofacial binding site of GLUT1, defined as being accessible to p-chloromercuribenzene-sulphonic acid if mutated to a cysteine residue.
	Fig. 2. Concentration-dependence of the influx of d-glucose in oocytes injected with BboHT1 mRNA. All values presented were first corrected for the uptake of d-glucose into water-injected controls. Data are averaged from three experiments, each on oocytes from a different toad, and are shown as means ± SEM.
	Fig. 3. Temperature-dependence of the influx of d-glucose in oocytes injected with BboHT1 mRNA. Inset: Arrhenius plot constructed from the data in the main figure. All values presented were first corrected for the uptake of d-glucose into water-injected controls. The extracellular d-glucose concentration was 38 μM. Data are averaged from three experiments, each on oocytes from a different toad, and are shown as means ± SEM.
	Fig. 4. Effect of modulators on the influx of d-glucose in oocytes injected with BboHT1 mRNA. All values were first corrected for the uptake of d-glucose into water-injected controls and are presented as a percentage of paired experiments performed in the absence of any modulators. The extracellular d-glucose concentration was 38 μM. Data are averaged from three experiments, each on oocytes from a different toad, and are shown as means ± SEM. Control, influx in oocytes injected with BboHT1 mRNA in the absence of modulators; CytoB, cytochalasin B (50 μM); Phdz, phloridzin (50 μM); Phrt, phloretin (50 μM); No Na+, sodium-free Barth's medium where Na+ was replaced with equimolar choline chloride; l-Gluc, l-glucose (10 mM); d-Fruct, d-fructose (10 mM); 1-DOG, 1-deoxy-d-glucose (10 mM); 2-DOG, 2-deoxy-d-glucose (10 mM); d-Mann, d-mannitol (10 mM); 3-DOG; 3-deoxy-d-glucose (10 mM); 3-O-MG, 3-O-methyl-d-glucose (10 mM); d-Galac, d-galactose (10 mM); 6-DOG, 6-deoxy-d-glucose (10 mM).
	Fig. 5. Effect of compound 3361 on the influx of d-glucose in oocytes injected with BboHT1 mRNA. All values were first corrected for the uptake of d-glucose into water-injected controls and are presented as a percentage of paired experiments performed in the absence of compound 3361. The extracellular d-glucose concentration was 38 μM. Data are averaged from four experiments, each on oocytes from a different toad, and are shown as means ± SEM.
	Fig. 6. Effect of compound 3361 (circles), cytochalasin B (squares) and β-glucogallin (triangles) on the in vitro growth of B. bovis parasites (A) and B. bovis merozoite invasion into normal (closed symbols) and glucose-depleted (open symbols) bovine erythrocytes (B). For growth assays, in vitro cultures were established at an initial parasitaemia of 0.3% and the final parasitaemia was determined after 48 h of growth and expressed as a percentage of control experiments performed in the absence of inhibitors. For invasion assays, the parasitaemia was determined 2 h after initiating invasion and expressed as a percentage of control experiments performed in the absence of inhibitors (100% invasion normally represented a parasitaemia between 0.5% and 1% after 2 h). Data are averaged from three experiments and are shown as means ± SEM.

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