IDENTIFICATION OF A SUBSTRATE SITE FOR TRANSGLUTAMINASES ON THE HUMAN PROTEIN-SYNTHESIS INITIATION-FACTOR 5A
IDENTIFICATION OF A SUBSTRATE SITE FOR TRANSGLUTAMINASES ON THE HUMAN PROTEIN-SYNTHESIS INITIATION-FACTOR 5A
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DOI：
10.1042/bj3050725
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发表时间：
1995-02-01
影响因子：
4.1
通讯作者：
ABBRUZZESE, A
中科院分区：
文献类型：
--
作者：
BENINATI, S;NICOLINI, L;ABBRUZZESE, A
人红细胞蛋白质合成起始因子5A(EIF-5A)是血浆转谷氨酰胺酶(FXIIIA)和豚鼠肝脏转谷氨酰胺酶(GPLTG)的底物。当纯化的eIF-5A与GPLTG或因子XIIIA在琥珀酸化的β-酪蛋白存在下孵育时，鉴定出一种共价复合体。通过对转谷氨酰胺酶(TGase)反应产物的分离和分析，确定了eIF-SA的修饰部位为唯一的氨基酸--苏氨酸。将复合β-酪蛋白eIF-5A用酶消化，用离子交换层析分离得到预测的γ-谷氨酰亚硫氨酸的共价交联物，并用反相H.P.L.C对其进行纯化。纯化的二肽经酸解后得到等摩尔数的亚精氨酸和谷氨酸，并且快原子轰击M.S.分析证实了异构体的归属是γ-谷氨酰基-(Omega)，而不是bar>；-亚硫氨酸。这些数据表明，eIF-5A链的hythusine-50可作为TGase的酰基受体底物，并揭示eIF-5A可能通过TGase与细胞内蛋白交联，因为eIF-5A的确切功能尚不清楚，鉴于最近发现该蛋白作为一种新的生物因子与人类免疫缺陷病毒-1 Rev激活结构域特异结合[Ruhl，Himmelspach，Babr，Hammerschid，Jaksche，WoIff；Auschauer，Farrington，Probst，Bevec和Hauber(1993)J.Cell Biol]，我们的结果似乎特别刺激。123,1309-1320]。
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--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].