The Biosynthesis of Hypusine - The Journal of Biological Chemistry

Vol. 259,No. 19, Issue of October 10,pp. 12123-12127 1984 Printed in L?S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY

The Biosynthesis of Hypusine (N”(4-Amino-2-hydroxybutyl)lysine) ALIGNMENT OF THE BUTYLAMINE SEGMENT AND SOURCE OF THE SECONDARY AMINO NITROGEN* (Received for publication, March 1, 1984)

Myung HeePark$, DanielJ. Liberato%, Alfred L. Yergeyt, andJ. E. Folk$ From the $National Institute of Dental Research and the §National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20205

The unusual amino acid hypusine is produced in a ing spermidine and lysine isotopically labeled at specific atoms as precursors for hypusine, we learned that carbons 5 single protein of mammalian cells by a novel posttranslational event in whicha lysine residue is conju- and 8 of spermidine’ are the sources of carbons 1 and 4, gated with the four-carbon moiety fromthe polyamine respectively, in the 4-amino-2-hydroxylbutyl moiety of hyspermidine to form an intermediate deoxyhypusine, pusine and that thenitrogen in the secondary amino group of and inwhich this intermediateis subsequently hydrox- hypusine originates from the c-position in the precursor lysine ylated. Specifically isotopically labeled precursors of residue. On the basis of these findings we conclude that a first hypusine were used to identify the biosynthetic origin step in hypusine biosynthesis involves transfer of the buof some of the atoms of hypusine and thus to provide tylamine segment from the secondary amine nitrogen of sperfurther insight into the mechanism of this in vivo chem- midine to the nitrogen at the c-position of protein-bound ical modification reaction. Radiolabel from [ 1,4-’H] lysine withformation of the unhydroxylated intermediate putrescine, [ 1,8-’H]spermidine, and [5-’H]spermidine entered hypusine during growth of Chinese hamster deoxyhypusine. ovary cells. The occurrenceof this label at positions 1 EXPERIMENTAL PROCEDURES* and 4, atposition 4, and at position 1, respectively, in the 4-amino-2-hydroxybutyl portion of hypusine reRESULTS vealed an alignment of atoms identical to that in the Incorporation of Radiolabelfrom Putrescine and Spermidine butylamine segment of spermidine. Growth of cells into Hypusine: Positions of Labeling-When Chinese hamster with [t-’5N]lysine as the source of lysine yielded hypusine enriched in “N, whereas only isotope-free hy- ovary cells are grown in the presence of [3H]putrescine or pusine was found in cells whose spermidine was re- [terminal methylene~-~H]spermidine and acid hydrolysates of placed duringgrowthby [4-16N]spermidine. These the trichloroacetic acid precipitates of these cells are examfindings are in accordance with a proposal that the ined by ion-exchange chromatography, the major radioactive first phaseof hypusine biosynthesis, the production of compound found is hypusine (4). With [5-3H]spermidineas a intermediate deoxyhypusine, occurs through transfer source of label, we find that Chinese hamster ovary cells of the butylamine moiety from spermidine to the t- contain most of their protein-bound radioactivity also in the amino nitrogenof protein-bound lysine. form of hypusine (data notshown). Thus, it follows that The techniqueof thermospray high-performance liq- tritium atomsfrom both positions 5 and 8 of spermidine’ (the uid chromatography/mass spectrometry providedpos1-and 4-positions of the butylamine part) enterhypusine. itive identification of l6N in hypusine through final A knowledge of the location of the radioactive atoms in separation andon-column direct analysisof this amino acid. Methods of preparation are given for spermidine hypusine produced during growth of cells with the specifically of high specific radioactivity, labeled specifically at radiolabeled spermidines should provide information about position 5 with ‘H, and for spermidine with15N at the the mechanism of the post-translationalsynthesis of this unusual amino acid. Oxidation of hypusine with periodate &position. cleaves the molecule between the vicinal amine and alcohol groups and, upon further oxidation with permanganate, the Investigation of the biosynthesis of hypusine has revealed that production of this unusual amino acid in mammalian cells occurs post-translationallyinoneprotein, eukaryotic initiation factor 4D (1-3). In this novel reaction, the fourcarbon moiety from the polyamine spermidine is covalently connected through secondary amine linkage with the side chain of lysine to form the intermediate deoxyhypusine ( N (4-aminobuty1)lysine) (1, 4). Deoxyhypusine is in turn hydroxylated to form hypusine (4). The presentwork wasundertaken inorder to obtain further information on themechanism of hypusine biosynthesis. Us* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The order of numbering of atoms in spermidine, first used by Tabor et al. (5), is fixed bythe requirement that thesecondary amino nitrogen be numbered as N-4 rather than N-5, because convention requires the lowest number for the secondary amino nitrogen. This numbering 1

2

3

4

5

6

7

8

H~N-CH~H~H-NH-CH~H-CHFCH-NH~ is used here. Thus, [terminal methylene~-~H]spermidine and [1,8-’H] spermidine are thesame compound. “Experimental Procedures” and Fig. 1 are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 84M-658, cite the authors, and include a check or money order for $1.60 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal thatis available from Waverly Press.

12123

12124

Biosynthesis of Hypusine

products formed are p-alanine, formic acid, and lysine (17) (Scheme 1).Tritium initially present in the 4-amino-2-hydroxybutyl portion of hypusine at the 1 carbon atom (designated by in Scheme 1) will appear after oxidation in formic acid or as tritiatedwater due to a tritium-hydrogen exchange from formaldehyde and in either case will be unbound to the ion-exchange column used for analysis. 3H at thecarbon atom contiguous with the primary amino group of the aminohydroxybutyl moiety (designated by * in Scheme 1)will befound after oxidation in p-alanine. The results given in Fig. 2 serve to identify the origin of several atoms in the 4-carbon segment of hypusine. The biosynthesis of spermidine in mammals occurs by transfer of a propylamine group to putrescine (for reviews, see Refs. 1820). Spermidine formed from [1,4-3H]putrescine must therefore be labeled in the 5- and 8-positions' (in the 1- and 4positions of its butylamine moiety). The datagiven in Fig. 2A show that atomsfrom both of these positions enter hypusine. in the hypusine side Fig. 2 B provides evidence that the atoms chain adjacent to the primary amino group originate from those at position 8 of spermidine. Consistent with this finding is the data given in Fig. 2C showing that hypusine that is labeled during growth with [5-4H]spermidine contains the label adjacent to its secondary amino group. Fig. 2 0 supplies evidence that the small amount of 3H that appears in palanine in the experiment of Fig. 2C arises as a result of relatively slow conversion of spermidine to putrescine and spermine, and resynthesis of spermidine with concurrent distribution of label into position 8. Inhibition of spermidine and sperminebiosynthesis by methylglyoxal bis(guany1hydrazone), which occurs as a consequence of lowered S-adenosylmethionine decarboxylase activity (21), prevents the radiolabel exchange. This is the case because decarboxylated Sadenosylmethionine is the source of the propylamine groups essential for spermidine and spermine biosynthesis. Origin of Secondary Amino Nitrogen of Hypusine: 15NLabeling-On the basis of our prior knowledge that lysine is the precursor amino acid for hypusine (4) and theabove assignment of atom positions inthe 4-carbon segment of this amino acid, we supposed that itssecondary amino nitrogen originates either from the t-amino group of protein-bound lysine or from the 4-position of spermidine. Experiments were therefore designed in which these proposed precursors containing the strategically located 'N atoms were examined as sources of label for the biosynthesis of ["N]hypusine. In these experiments, it was imperative that the cells be grown under conditionsthat would beexpected to yield levels of labeling in hypusine adequate for ready recognition by mass spectral analysis. This was easily accomplished in the case of the cells grown with ["N]lysine by providing high +

*

CHZ- CHZI NHZ

t

CH - CHZ- N - (CH214- CH - COOH I I I OH H NHz HI04 KMnO,

*

CHz-CHz-COOH I NH,

+

+ HCOOH

+ HzN-(CHz),-CHCOOH I NH2

SCHEME 1

B

C

D

FIG. 2. Distribution of radioactivity in oxidation products of hypusine isolated from Chinese hamster ovary cellsgrown with A , [ 1,4-'H]putrescine (H2NCSH2(CHa)2C'H2NH2), B, [1,8-'H]spermidine (HzNC'H~(CH~)ZNH(CH~)~C'H~NH~), C, [5-'H]spermidine (H2N(CH2)sNHC8H2(CH2)sNH2), and D, [B-'H]spermidine plus methylglyoxal bis(guany1hydrazone). Radioactive amine in each experiment was added to 10 dishes at the level of 10 pCi/ml of medium and at the time when the cells had covered 10-20%of the surface ofeach dish. The level of methylglyoxal . wasfor 24 h after bis(guany1hydrazone) in D was 100 p ~ Growth addition of labeled amine. Following acid hydrolysis of the protein fraction of combined cells from each experiment, 10 nmol ofsynthetic hypusine were added, and this amino acid was isolated as outlined using a column (0.7 X 13 cm (5 ml)) of Bio-Rex 70 resin. A portion M HC1 was oxidized of this hypusine (5-10% of total) in 0.1 ml of by treatment with 1-2 mg of HIO,. After 1.5 h at room temperature, KMnO, solution was added dropwiseuntil the purple color persisted. Measurements of radioactive components in unoxidized and oxidized samples were carried out by the use of the five-buffer ion-exchange system (10). Approximately the same amount of radioactivity was applied to the column in each experiment. Greater than 90% of the radioactivity in hypusine was accounted for in the oxidation products. Essentially all of the radioactivity was found as hypusine in unoxidized samples (1.5-3 X IO5 cpm total in each experiment).

atom per cent "N-labeled lysine as the sole source of this amino acid during growth. For cells grown with [15N]spermidine, it was first necessary essentially to deplete the cells of endogenous spermidine and then maintain a greatly reduced biosynthesis rate for this polyamine while supplying it in the medium in the form of the high atom per cent 15N-labeled compound. Depletion and reduced synthesis of spermidine were accomplished by the use of DL-2-difluoromethylornithine, a specific inhibitor of the enzyme ornithine decarboxylase (22). As reported for other cell types (23, 24), we found that, after24 h with the inhibitor (conditions of Fig. 3C), the Chinese hamster ovary cells were almost devoid of spermidine. Within 24 h after addition of [''Nlsperrnidine to the medium of the depleted cells, they were found to contain practically a normal amount of this polyamine, a result consistent with those reported previously for other cell types (23, 24). Fig. 3 summarizes the results of these experiments. The mass spectraobtained for hypusine from cells grown (A) under controlconditions, ( B ) in the presence of [e-"N]lysine, and (C) with [4-'5N]spermidine are similar. In each case several prominent fragments are observed in addition to the protonated parent ion. It is immediately apparent, however, that for hypusine isolated from cells grown with [t-"N]lysine all of the major ions, including the protonated parent ion, are shifted by addition of one mass unit (Fig. 3B). This finding provides the basis for a conclusion that the secondary amino nitrogen of hypusine derives from the nitrogen at the t position of protein-bound lysine.

12125

Biosynthesis of Hypusine T

:t,1

147

,171

l-lAlAId I 1 7 5 200

100

125

150

175

200

225 250100 125

m/z

mlz

C

2 2 5 2

100 150 125

175

200

225

250

mlz

FIG. 3. On-column positive ion mass spectra of hypusine isolated from Chinese hamster ovarycells grown withoutadded “N-labeled compounds (control,A), with [t-”N]D~-lysine( B ) ,and with [4-I6N] spermidine (C). In each experiment, cells were grown in 200 dishes and labeled compounds were added at the time when the cells had reached about 10-20% confluency (about 24 h after seeding). To 10 of the total200 dishes was added [3H]putrescine at the level of 5 pCi/ml of medium. This was done for the purpose of radiolabeling hypusine as anaid in its isolation. In A and C, growth was in the &-modifiedEagle’s mediumdescribed (4). Culture of cells in B was in RPMI 1640 medium (Select-Amine kit, Gibco), prepared free of lysine and supplemented with 5% dialyzed fetal calf serum, penicillin (50 IU/ml), streptomycin (50 gg/ml), and the “N-labeled lysine.2HCl at a level of 40 pg/ml. In C, the medium was made 4 mM in DL-2-difluoromethylornithineat the time of seeding and incubation was for 24 h before addition of labeled materials. The “N-labeled spermidine was employed at a level of 5.6 p ~ In. each experiment, growth was for 48 h after addition of labeled materials. Isolation of hypusine from the acid hydrolysates of the protein in cells combined from the 200 dishes (2-4 X lo9 cells) was performed on a column (1.5 X 17 cm (30 ml)) of Bio-Rex 70 resin. Approximately 20 nmol of hypusine were obtained in each experiment with a total recovery of about 70%, estimated on the basis of radioactivity. On-column injections of approximately 5 nmol were made. In addition to the protonated molecular ions at m/z 234 and 235, several prominentfragmentsare seen. The structures proposed for several of these are: m/z 216 and 217, [M + 1 - HzO]+; m/z 197 and 198, [M - 2 H ~ 0 ] + ;m/z 171 and 172, [HC=CHNH(CH2)4CHNHzr\ + and m/z 147 and 148, [HzN(CHZ)~CHNH&OOH+ 1]+. COOHI+ or

I 1

r

1

“*a

OH

hypusine at the position adjacent to the secondary amino nitrogen. This is nicely confirmedby the results given in Fig. We postulate, based on the results of the two experiments 2, C and D, in which it is shown that 3H on carbon 5 of detailed in Figs. 2 and 3 of this paper, that transfer of the spermidine is the source of label at this position in hypusine. butylamine moietyfrom the secondary amino nitrogen of Whether the difference observed in 3H retention at the two spermidine to the €-amino group of protein-bound lysine is positions of hypusine from cells grown with [1,4-3H]putresone of the steps in the post-translational synthesis of hypusine cine (Fig. 2A) is significant is not known at present. Double in mammals. Oxidative cleavage of spermidine between car- labeling experiments should resolvethis question and provide bon 3 and nitrogen 4 has been reported by an enzyme in rat a further degree of knowledge concerning this novel reaction. liver (25). Although an oxidative cleavage reactiin thatoccurs The initial proposals that spermidine is the immediate between nitrogen 4 and carbon 5 of spermidine is observedin amine precursor of hypusine and thatentrance of radioactivplants (26), up until present there has been no indication of ity into thisamino acid fromputrescine occurs rapidly because fragmentation in mammals of spermidine between nitrogen 4 putrescine israpidly converted to spermidine were based and carbon 5 (27). It is indeed possible that butylamine entirely on the results of studies with inhibitors of enzymes transfer from spermidine in mammalian systems is reserved involved in polyamine synthesis (1).The data given in Fig. 2 solely for hypusine biosynthesis. Some interest has been di- provide irrefutable evidence that spermidine and not putresrected toward the fate of the portion of spermidine that cine is the direct source of the butylamine moiety used in remains after butylamine transfer. To date,however, we have hypusine biosynthesis. This is apparent in the finding that obtained no evidence for accumulation in cells of 1,3-diami- 3H atoms on the carbons at positions 5 and 8 of spermidine nopropane or of any derivative thereof. enter hypusine at single and separate positions. Hypusine isolated from cellsgrown in the presence of [1,4Our earlier conclusion that protein-bound lysineis the 3H]putrescineand thusderived from [5,8-3H]spermidine (see amino acid precursor of hypusine is given strong support by “Results”) contains 3H in both positions 1 and 4 of its 4- the results shown in Fig. 3. In serving to define the t-amino amino-2-hydroxybutyl moiety (Fig. 2A). This followsfrom group of lysine as the source of secondary amino nitrogen in the fact that, after oxidation of the labeledhypusine,3H hypusine, these findings greatly extend our prior knowledge appears both unbound to theion-exchange columnand in 0- that a portion of the carbon side chain of lysine enters alanine. Of interest is the finding that 3H is retained in hypusine (4). DISCUSSION

12126

Biosynthesis of Hypusine

The present findings, along with earlier knowledge of the mechanism of hypusine biosynthesis, provide a firm basis upon which to conduct a search for the enzymes involved in this post-translational event, and, subsequently, an investigation of their cellular regulations. It was shown earlier that hydroxylation of the intermediate deoxyhypusine occurs as the last step in hypusine biosynthesis (4). It is not likely, however, that this stepis rate limiting, because the intermediate does not accumulate during normal growth of cells (4). We are interested to learn whether the production of biological activity of eukaryotic initiation factor 4D is controlled by the enzyme(s) that catalyzes butylamine transfer from spermidine. #

Acknowledgment-We thank Dr. Gerald L. Mechanic for a sample of 4-p-nitrobenzamidobutyraldehydeused in the early stages of our synthetic work. REFERENCES 1. Park, M.H.,Cooper, H. L., and Folk, J. E. (1981)Proc. Natl. Acud. Sci. U. S. A. 78,2869-2873 2. Cooper, H. L., Park, M. H., and Folk, J. E. (1982)Cell 29, 791797 3. Cooper, H. L., Park, M. H., Folk, J. E., Safer, B., and Braverman, R. (1983)Proc. Natl. Acud. Sci. U. S. A. 80, 1854-1857 4. Park, M. H., Cooper, H. L., and Folk, J. E. (1982)J . Biol. Chem. 257,7217-7222 5. Tabor, H., Tabor, C. W., and DeMeis, L. (1971)Methods Enzymol. 17B, 829-833 6. Paz, M. A., Henson, E., Rombauer, R., Abrash, L., Blumenfeld, 0. O., and Gallop, P. M. (1970)Biochemistry 9,2123-2127 7. Aspinall, S. R.(1940)J. Am. Chem. SOC.62, 2160-2162 8. Shiba, T., Akiyama, H., Umeda, I., Okada, S., and Wakamiya, T. (1982)Bull. Chem. SOC.Jpn. 55,899-903

9. Borch, R. F., Bernstein, M. D., and Durst, H. D. (1971)J. Am. Chem. Soc. 93,2897-2904 10. Folk, J . E., Park, M. H., Chung, S. I., Schrode, J. Lester, E. P., and Cooper, H. L. (1980)J. Biol. Chem. 255,3695-3700 11. Jackson, E. I. (1956)J. Org. Chem. 21,1374-1375 12. Tabor, H.,Rosenthal, S. M., and Tabor, C. W. (1958)J. Biol. Chem. 233,907-914 13. Blakley, C. R., Carmody, J. J., and Vestal, M. L. (1980)J . Am. 102,5931-5933 Chem. SOC. 14. De, N. C., Mittelman, A., Dutta, S. P., Edmonds, C. G., Jenkins, E. E., McCloskey, J. A., Blakley, C. R., Vestal, M.L., and Chheda, G . B. (1981)J. Carbohydr. Nucleosides Nucleotides8, 363-389 15. Blakley, C.R., and Vestal, M. L. (1983)Anal. Chem. 55, 750754 16. Liberato, D. J., Fenselan, C. C., Vestal, M. L., and Yergey, A. L. (1983)Anal. Chem. 55,1741-1744 17. Shiba, T., Mizote, H., Kaneko, T., Nakajima, T., Kakimoto, Y., and Sano, I. (1971)Biochim. Biophys. Acta244,523-531 18. Tabor, H., and Tabor, C. W. (1972)Adu. Enzymol. 36, 203-268 19. Williams-Ashman, H.G., Janne, J., Coopoc,G.L., Geroch, M. E., and Schenone, A. (1972)Adu. Enzyme Regul. 10,225-245 20. Morris, D. R.,and Fillingame R. H. (1974)Annu. Rev. Biochem. 43,305-325 21. Corti, A., Dave, C., Williams-Ashman, H. G., Mihich, E., and Schenone, A. (1974)Biochem. J. 139,351-357 22. Metcalf, B. W., Bey, P., Danzin, C., Jung, M. J., Casara, P., and Vevert, J. P. (1978)3.Am. Chem. SOC. 100,2551-2553 23. Mamont, P. S., Duchesne, M.-C., Joder-Ohlenbusch, A.-M., and Grove, J. (1978) in Enzyme-Actiuated Irreuersible Inhibitors (Seiler, N., Jung, M. J., and Koch-Weser, J., eds) pp. 43-54, Elsevier, Amsterdam 24. Pegg, A. E., Tang, K.-C., and Coward, J. K. (1982)Biochemistry 21,5082-5089 25. Holtta, E. (1977)Biochemistry 16,91-100 26. Suzuki, O., Matsumoto, T., Oya, M., Katsumata, Y., and Samejima, K. (1981)Anal. Biochem. 115,72-77 27. Smith, T. A. (1983)Methods Enzymol. 94,311-314

12127

Biosynthe sis of Hypusine

100 -

> m

f

c

L w

5

5 w

a

n Y

u

0

1

2

3

4

5

TIME (rninl

FIG. 1. Reconstructed total ion current chromatogramshowing separation of Iysine (0.25 nmol) ( I ) , hypusine (6 nmol) (2),and spermidine (1.5 nmol) (3)by thermospray high-performance liquid chromatography/mass spectrometry. Separation was accomplished with the use of the isocratic mobile phase 0.1 M ammonium acetate, pH 6.75,a t a flow rate of 1 ml/min. The sample was introduced in a totalvolume of 20 cl of mobile phase.