Sequence and biochemical characterization of equilibrative nucleoside transporters from Crithidia fasciculata: seeking ligand binding residues

Eukaryotic cells acquire purines via de novo synthesis or salvage mechanisms. Unlike human cells, protozoan parasites from the Apicomplexan and Trypanosomatid lineages such as Plasmodium, Toxoplasma, Leishmania, Trypanosoma and Crithidia species lack de novo purine synthesis enzymes, and therefore rely on salvage of pre-formed purine bases for growth and proliferation, making purine salvage an attractive drug target. A key step in purine salvage is transport of purine nucleobases and nucleosides into the cell, and each species encodes a unique repertoire of transporter proteins from the equilibrative nucleoside transporter (ENT) family, each with its own ligand preference. No detailed structural information for ENT proteins yet exists, but residues within four of the eleven transmembrane domains (TMs) appear to be important for ligand selectivity. Recently we have described the pivotal role of a lysine residue within TM4 of the Crithidia fasciculata purine nucleoside transporter CfNT2 in ligand discrimination. Here we describe the cloning of additional ENT genes from C. fasciculata using partial genomic sequencing and molecular techniques, and biochemical characterization of the encoded proteins. The role of sequence variations among the C. fasiculata ENTs in determining ligand preference may shed additional light on residues and regions of ENTs that contribute to ligand binding