IDENTIFICATION OF A SUBSTRATE SITE FOR TRANSGLUTAMINASES ON THE HUMAN PROTEIN-SYNTHESIS INITIATION-FACTOR 5A

Protein synthesis initiation factor 5A (eIF-5A) from human erythrocytes was found to be a substrate for both plasma transglutaminase (Factor XIIIa) and guinea pig liver transglutaminase (GPLTG). When purified eIF-5A was incubated with GPLTG or Factor XIIIa in the presence of succinylated beta-casein, a covalent complex was identified. By isolating and analysing the product of the transglutaminases (TGases) reaction, the site of modification on eIF-SA has been identified as the unique amino acid hypusine. The complex beta-casein eIF-5A was enzymically digested with proteinases and the predicted covalent cross-link of gamma-glutamyl-<(omega)over bar>-hypusine was isolated from the digests by ion-exchange chromatography and purified by reversed-phase h.p.l.c. Acid hydrolysis of the purified dipeptide yielded equimolar amounts of hypusine and glutamic acid, Furthermore, fast atom bombardment m.s. analysis confirmed the isomer assignment to be gamma-glutamyl-(omega)over bar>-hypusine. These data indicate that hypusine-50 of the eIF-5A chain functions as acyl acceptor substrate for TGases, and reveal that eIF-5A may be cross-linked to intracellular proteins by TGases, Because the precise function of eIF-5A is still unknown, our results appear particularly stimulating in the light of the recent finding of a new biological role for this protein as a cellular factor binding specifically to the human immunodeficiency virus-1 Rev activation domain [Ruhl, Himmelspach, Bahr, Hammerschmid, Jaksche, WoIff; Auschauer, Farrington, Probst, Bevec and Hauber (1993) J. Cell Biol. 123, 1309-1320].