Proc. Nati. Acad. Sci. USA Vol. 78, No. 5, pp. 2869-2873, May. 1981 Biochemistry Identification of hypusine, an unusual amino acid, in a protein from human lymphocytes and of spermidine as its biosynthetic precursor [N6-(4-amino-2-hydroxybutyl)-2,6-diaminohexanoic acid or N'-(4-amino-2-hydroxybutyl)lysine/putrescine/polyamines/ posttranslational modification] MYUNG HEE PARK*, HERBERT L. COOPERt, AND J. E. FOLK* *National Institute of Dental Research, and the tNational Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205 Communicated byJohn M. Buchanan, February 9, 1981 ABSTRACT When normal human peripheral lymphocytes EXPERIMENTAL PROCEDURE are treated with mitogen and grown in the presence of Materials. [2,3-3H(N)]Putrescine-2HC1, [terminal, methyl- [3H]putrescine or [terminal methylenes-3H]spermidine, label is enes-3H(N)]spermidine-3HCl, and [3-aminopropyl-3-3H(N)]- incorporated predominantly into one cellular protein. The radio- spermine-4HCI were purchased from New England Nuclear. active constituent of this protein was identified as the unusual Their specific radioactivities were >20 Ci/mmol (1 Ci = 3.7 amino acid hypusine [Nt-4-amino-2-hydi-oxybutyl)lysine]. This X 10'° becquerels) and each was used without dilution of spe- was accomplished by isolation of the component from proteolytic digests or acid hydrolysates and comparison with authentic hy- cificradioactivity. Methylglyoxalbis(guanylhydrazone)-2HCl'H20 pusine by chromatography, conversion to the 2,4-dintitrophenyl (MGBG) was from Aldrich. Hypusine, isolated by T. Nakajima derivative, and oxidative degradation. The observed relationships and his colleagues and provided by them to John W. Daly of among intracellular levels oflabeled putrescine, polyamines, and the National Institutes of Health, was a gift from him to us. protein bound hypusine after growth of cells with the various la- Other materials and reagents have been described (1). beled amines and with or without an inhibitor of polyamine bio- Methods. Human peripheral lymphocytes from healthy nor- synthesis supply evidence that spermidine is the immediate amine mal donors were purified by a combination of nylon column precursor of hypusine and that the 4-amino-2-hydroxybutyl por- adsorption and density separation as described (5) and cultured tion ofhypusine derives from the butylamine moiety of spermidine. at a density of 106 cells per ml in RPMI 1640 containing glu- tamine (0.3 mg/ml), penicillin (50 international units/ml), streptomycin (50 international units/ml), 10 mM Hepes, pH While studying the role of polyamines as physiological sub- 7.4, and 10% autologous plasma. Growth was induced by ad- strates for transglutaminases, we observed an unidentified ra- dition of phytohemagglutinin (Wellcome, Beckenham, En- diolabeled material in proteolytic digests ofthe protein fraction gland) at 5 ,ug/ml the day after purification. Additions oflabeled from normal human peripheral lymphocytes that had been amines were made together with the mitogen at 5 ACi/ml ex- treated with mitogen in the presence of [3H]putrescine. This cept where noted otherwise. Cells were harvested by centrif- labeled basic component of lymphocyte protein was initially ugation for 10 min at 260 X g at 35°C. The cells were washed thought to be N'-(y-glutamyl.)spermidine because it chroma- twice with ice-cold phosphate-buffered saline (pH 7.4). The cell tographed in aposition close to thatofthis y-glutamylpolyamine pellet was suspended in a small volume ofphosphate-buffered in the ion exchange system used. It was subsequently recog- saline, and an equal volume of 20% trichloroacetic acid was nized and reported (1) as an unknown componentoflymphocyte added. Further details of preparation of trichloroacetic acid protein. This conclusion was based on its observed stability to precipitates and supernatant fractions are given elsewhere (1). acid hydrolysis. We now report that this compound, which is Enzymic digestions oftrichloroacetic acid precipitates were car- the major labeled component formed in the lymphocyte protein ried out as described (1). Acid hydrolysis was performed in 6 fraction during growth ofthe cells with eitherlabeled putrescine M HC1 at 106°C for 18 hr in sealed evacuated tubes. or labeled spermidine, is the unusual amino acid hypusine. The Ion exchange chromatographic separations were conducted present report provides evidence for the identity ofthis amino by using a Dionex D-400 analyzer with the three-buffer system acid and for the direct precursor role of spermidine in its bio- under conditions described (1). For measurement of radioac- synthesis. In addition, it is shown that hypusine occurs pre- tivity and other external analyses, collection of effluent was dominantly in one protein of lymphocytes. made directly from the column without mixing with fluoro- Hypusine was discovered by Nakajima and coworkers (2), metric reagent. who isolated the free amino acid from homogenates of bovine Radioactivity was measured in Hydrofluor counting fluid brain and determined its structure as N8-(4-amino-2-hydroxy- (National Diagnostics, Somerville, NJ) by using a liquid scin- butyl)lysine. In addition to its detection in free form in a number tillation spectrometer. of organs of several mammals (3), hypusine was found to be a constituent ofanimal proteins (4). Attempts to find any specific protein abundant in this amino acid were, however, without RESULTS success (4). Identification of Hypusine in the Protein Fraction of Lym- phocytes. Peak I of Fig. 1 shows the position in ion exchange The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- Abbreviations: MGBG, methylglyoxal bis(guanylhydrazone); DNP, ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 2,4-dinitrophenyl. 2869 Downloaded by guest on September 23, 2021 2870 Biochemistry: Park et al. Proc. Natl. Acad. Sci. USA 78 (1981) chromatography of the major radioactive compound formed by acid hydrolysis ofthe protein fraction from lymphocytes grown I in the presence of [3H]putrescine. A peak of radioactivity is 7.5F found in the same position after chromatography ofan acid hy- 0 III 2 drolysate ofthe protein from cells grown with [terminal meth- x ylenes-3H]spermidine in place of [3H]putrescine. Exhaustive proteolytic digests prepared from the protein fractions of cells 5.0F I 0 I' grown with each of the labeled amines show patterns of radio- . II" I activity similar to that shown in Fig. 1. Typical chromatographic 1._ I % co patterns of radioactivity in proteolytic digests are given else- 10 % 1638 1 where (see figure 2 ofref. 1). Peaks II and III ofFig. 1 are those 1764 of standard [3H]N'-(y-glutamyl)spermidine and N8-(y-gluta- I/ I/ Timesec myl)spermidine, respectively, chromatographed in this system. Comparison of the elution profiles of the labeled compounds II oL with those of standard nonlabeled hypusine, N'-(y- glutamyl)spermidine, and N8-(y-glutamyl)spermidine (Fig. 1, 0 1600 1700 1800 Inset; elution times of 1638 sec, 1694 sec, and 1764 sec, re- Time, sec spectively) shows that the acid-stable labeled compound from FIG. 1. Ion exchange chromatographic separation of the labeled lymphocyte protein chromatographs in the position ofhypusine acid-stable component of lymphocyte protein (I) from N'-(-gluta- in this system. myl)spermidine (II) and N-(t-glutamyl~spermidine (HI). The protein fraction was prepared from lymphocytes (1.5 x 107) that had been in- Table 1 summarizes the results ofthin-layer chromatography cubated with mitogen and [3H]putrescine for 24 hr. The chromato- ofthe 2,4-dinitrophenyl (DNP) derivatives ofhypusine and the graphic conditions are those given in the inset to figure 2 of ref. 1. acid-stable radioactive component oflymphocyte protein. That Fractions were collected every 24 sec. Preparation of the 3H-labeled the two derivatives chromatograph in identical positions in sev- y-glutamylspermidines was carried out as described (1), except for the eral use of3H-labeled polramine. (Inset) Recorded elution ofstandard non- solvent systems is substantial evidence for the identity of labeled hypusine, N -, and N'-(y-glutamyl)spermidines, respectively, the lymphocyte component as hypusine. in the same chromatographic system with use of automatic fluoro- Further evidence for the identity of this component was ob- metric detection. tained from oxidation experiments, the results ofwhich are also given in Table 1. Nakajima and coworkers (2) showed that ox- Identification of Spermidine as the Precursor of Hypusine. idation of hypusine with periodate cleaves the molecule at the The data shown in Fig. 2 provide evidence that spermidine is vicinal amino alcohol group and that the products obtained after the direct precursor of the 4-amino-2-hydroxybutyl portion of further treatment with are formic lymphocyte hypusine. Examination of the distribution of label permanganate /-alanine, in the cellular amine fractions from lymphocytes shows that, acid, and lysine: after 24 hr, more than halfof the label in this fraction from cells CH2CH2CH CH2NH(CH2)4 CH COOH grown with [3H]putrescine is in the form of spermidine and I I spermine (experiment 1). This fraction from cells supplied with NH2 OH NH2 labeled spermidine, on the other hand, contains most of the label as spermidine and spermine and very little in the form of HO04 putrescine (experiment 3). This observation, together with the fact that both labeled putrescine and labeled spermidine are