Leishmania are the protozoan parasites from the Trypanosomatidae family, which are responsible for the series of important tropical diseases. These ancient eukaryotes are also characterised by unique molecular features such as polycistronic transcription and trans-splicing. The spliced leader RNA donates the 5' cap structure to all mRNAs, which consists of a hypermethylated cap-4 structure (m⁷Gpppm₂^6,6AmpAmpCmpm³Um). We were interested to understand how these hypermethylations affect the interaction between cap-4 and cap-binding proteins.

We identified in the Leishmania major genome database four homologues of the eukaryote initiation factor 4E (denoted LeishIF4E-1 to -4), which are distributed in the cytoplasm. Homology modelling based on the known crystal structure of the mouse protein indicated that the cap-binding pocket was conserved in each of the four proteins. However, in LeishIF4E-3 the position that corresponds to the human Trp56 is occupied by metionine and in LeishIF4E-4 by tyrosine residue. The positively charged amino acids are conserved only in part. By using of Trp fluorescence titration assays we determined the binding constants of all four proteins with cap-4 and its analogs. The highest binding affinity for cap-4 is observed for LeishIF4E-4 (K_as~ 0.8 mM^-1). However, this isoform as well as LeishIF4E-1 binds cap-4 and m⁷GTP comparably well. The clear preferential binding to cap-4 is shown for LeishIF4-2, which binds cap-4 eight-fold stronger than m⁷GTP. An opposite effect is observed for LeishIF4E-3 which binds specifically m⁷GTP. These different binding preferences of the eIF4E isoforms suggest that they may be involved with different functions in Leishmania.

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1. Binding affinity of human 4EHP for mRNA cap analogues