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-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].
Beninati
S
S
Department of Biology, II University of Rome Tor Vergata, Italy.
Nicolini
L
L
Jakus
J
J
Passeggio
A
A
Abbruzzese
A
A
D000818:Animals; D001665:Binding Sites; D002364:Caseins / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000737:chemistry; D018698:Glutamic Acid / Q000378:metabolism; D006168:Guinea Pigs; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008239:Lysine / Q000031:analogs & derivatives / Q000378:metabolism; D010448:Peptide Initiation Factors / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins; D011503:Transglutaminases / Q000378:metabolism*; D000097574:Eukaryotic Translation Initiation Factor 5A
D002364:Caseins; D018698:Glutamic Acid; D008239:Lysine; D010448:Peptide Initiation Factors; D016601:RNA-Binding Proteins; D011503:Transglutaminases; D000097574:Eukaryotic Translation Initiation Factor 5A; C100028:hypusine