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.
Posgrado en Ciencias Genómicas, Universidad Autónoma de la Ciudad de México, San Lorenzo # 290, Col. Del Valle, CP 03100, Mexico City, Mexico.
Bertha Isabel
BI
Carvajal-Gamez
Laura Itzel
LI
Quintas-Granados
Rossana
R
Arroyo
Guillermo
G
Mendoza-Hernández
Maria Elizbeth
ME
Alvarez-Sánchez
D015153:Blotting, Western; D015180:Electrophoresis, Gel, Two-Dimensional; D006031:Glycosylation; D007526:Isoelectric Point; D010448:Peptide Initiation Factors / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*; D014246:Trichomonas vaginalis / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*; D000097574:Eukaryotic Translation Initiation Factor 5A
D010448:Peptide Initiation Factors; D016601:RNA-Binding Proteins; D012694:Serine; D014443:Tyrosine; D000097574:Eukaryotic Translation Initiation Factor 5A