Substrate specificity of the Leishmania donovani myo-inositol transporter: critical role of inositol C-2, C-3 and C-5 hydroxyl groups

Abstract

Inositol is an essential precursor for the formation of glycosyl-phosphatidylinositol (GPI)-anchors found in the majority of surface molecules in trypanosomatids, in addition to its requirement for phoshatidylinositol signal transduction pathways. In Leishmania donovani, high-affinity inositol transport is catalyzed by the active myo-inositol/H+ transporter MIT, which is driven by a proton gradient across the parasite membrane. We have characterized the substrate specificity and pharmacology of L. donovani MIT in vitro and in promastigote cultures. High substrate specificity of myo-inositol transport was shown in competition studies with 14 different monosaccharides and MIT function was unaffected by the structurally similar pentose sugars or hexoses. L-Fucose and D-xylose, both inhibitors of the Na+-dependent inositol transport system in the human host, did not affect MIT transport function in the parasite. Competition studies with eight different inositol isomers revealed that proton bonds between the C-2, C-3 and C-5 hydroxyl groups of myo-inositol and the transporter protein played a critical role for substrate recognition, and the C-3 hydroxyl oxygen appears to act as an electron donor to form an H-bond with a positive charge of the MIT permease. The cytotoxic inositol analogue 3-fluoro-myo-inositol was recognized by MIT with similar affinity as myo-inositol and showed an IC50 value of 42 +/- 8 microM in L. donovani cultures. Finally, substrate affinities of MIT revealed apparent Km values of 84 +/- 8 microM for myo-inositol and 5.4 +/- 0.9 nM for H+, equal pH 8.27 + 0.08, suggesting that the L. donovani myo-inositol/H+ symporter is fully activated at physiological pH in the sandfly midgut or macrophage phagolysosome. We conclude that Leishmania MIT constitutes an attractive target for delivery of cytotoxic inositol analogues and differs significantly from the sodium-coupled myo-inositol transport system of the human host.