Identification of a substrate site for transglutaminases on the human protein synthesis initiation factor 5A

Abstract

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-5A has been identified as the unique amino acid hypusine. The complex beta-casein.eIF-5A was enzymatically digested with proteinases and the predicted covalent cross-link of gamma-glutamyl-omega-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-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, Wolff, Auschauer, Farrington, Probst, Bevec and Hauber (1993) J. Cell Biol. 123, 1309-1320].

The spermidine analogue GC7 (N1-guanyl-1,7-diamineoheptane) induces autophagy through a mechanism not involving the hypusination of eIF5A

“…Future studies should clarify by which mechanism GC7 is able to promote autophagy. In keeping with this assumption it is interesting to note that hypusine of the eIF-5A chain functions as an acyl acceptor substrate for transglutaminases (Beninati et al 1995). Considering the structural similarity between hypusine molecule and GC7, it would be interesting to study whether this polyamine analogue can act as a substrate of Type 2 transglutaminase transamidating activity which has been shown to play an important role in the recruitment of ubiquitinated proteins into the autophagosomes (D'Eletto et al 2009).…”

Structural organization of the human eukaryotic initiation factor 5A precursor and its site-directed variant Lys50 ? Arg

“…This intermediate is not accumulated in cells but is immediately hydroxylated at C-2 of the incoming 4-aminobutyl moiety to form hypusine (Abbruzzese et al, 1985(Abbruzzese et al, , 1986(Abbruzzese et al, , 1988b. More recently it was found that eIF-5A undergoes a post-translational modification catalyzed by transglutaminase (Beninati et al, 1995).…”

Role of the FAD-dependent polyamine oxidase in the selective formation of N1,N8-bis(γ- glutamyl)spermidine protein cross-links

“…In order to modify the intracellular level of a radioactive polyamine reaching a suitable specific radioactivity, it may be useful to perform the incubation of the cells with the polyamine whose conversion rate is lower. A mixture of two radiolabelled polyamines has proved to be applicable for this type of incubation study [23].…”