Translation initiation factor eIF-5A, the hypusine-containing protein, is phosphorylated on serine and tyrosine and O-glycosylated in Trichomonas vaginalis.

The eukaryotic translation factor eIF-5A is highly conserved throughout eukaryotes and undergoes an unusual polyamine-dependent post-translational modification called hypusination. Trichomonas vaginalis has two tveif-5a genes (tveif-5a1 and tveif-5a2), each encoding a 19-kDa protein. In this report, we describe the detection of two forms with different isoelectric points (5.2 and 5.5) that correspond to the precursor and mature TveIF-5A, respectively. In addition, we demonstrated that only the mature form of TveIF-5A is phosphorylated and glycosylated via two-dimensional gel electrophoresis-western blot (2DE-WB) assays using anti-phosphoserine and anti-phosphotyrosine antibodies and the SNA, ConA and MAA lectins. Interestingly, when the protozoa were grown in 1,4-diamino-2-butanone (DAB), an inhibitor of putrescine biosynthesis, and transferred to medium containing exogenous putrescine, a new spot with an isoelectric point of 5.3 was observed, presumably corresponding to a phosphorylated intermediate or deoxyhypusine form. Our data indicate that, in T. vaginalis, phosphorylations and glycosylations are necessary to obtain the mature TveIF-5A, and we confirm the identity of the precursor, intermediate and mature forms of TveIF-5A by mass spectrometry analysis.