Eukaryotic Initiation Factor 4D PURIFICATION FROM HUMAN RED BLOOD CELLS AND THE SEQUENCE OF AMINO ACIDS AROUND ITS SINGLE HYPUSINE RESIDUE*

Eukaryotic initiation factor 4D (eIF-4D) was puri- fied from human red blood cells by a simple 5-step procedure. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that most of the preparations of eIF-4D were composed of variable amounts of two closely migrating forms of the factor, each of which contained a single residue of the unique amino acid hypusine. The structural similarity of the two forms of human eIF-4D was evidenced by the indistinguishable patterns of radioactivity on peptide maps of tryptic digests prepared from radioiodinated samples. A pep- tide containing the single hypusine residue was readily isolated from a tryptic digest of human eIF-4D by virtue of its high positive charge and hydrophilic char-acter. Amino acid sequence determination on this pep- tide revealed the following primary structure around hypusine: Thr-Gly-hypusine-His-Gly-His-Ala-Lys. Eukaryotic initiation factor 4D (eIF-4D)’ was initially isolated from rabbit reticulocyte ribosomes (1) and was shown to stimulate the synthesis of methionyl-puromycin in incu-bations containing puromycin and the components necessary for 80 S initiation complex formation (1, 2). Factor eIF-4D

Eukaryotic initiation factor 4D (eIF-4D) was purified from human red blood cells by a simple 5-step procedure. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that most of the preparations of eIF-4D were composed of variable amounts of two closely migrating forms of the factor, each of which contained a single residue of the unique amino acid hypusine. The structural similarity of the two forms of human eIF-4D was evidenced by the indistinguishable patterns of radioactivity on peptide maps of tryptic digests prepared from radioiodinated samples. A peptide containing the single hypusine residue was readily isolated from a tryptic digest of human eIF-4D by virtue of its high positive charge and hydrophilic character. Amino acid sequence determination on this peptide revealed the following primary structure around hypusine: Thr-Gly-hypusine-His-Gly-His-Ala-Lys.
Eukaryotic initiation factor 4D (eIF-4D)' was initially isolated from rabbit reticulocyte ribosomes (1) and was shown to stimulate the synthesis of methionyl-puromycin in incubations containing puromycin and the components necessary for 80 S initiation complex formation (1,2). Factor eIF-4D does not appear to influence any of the reactions involved in the formation of the 80 S initiation complex (2, 3). However, there has not been sufficient testing to determine whether this factor is required for translation of natural mRNA (2, 3).
Despite some discrepancy in reported physical constants (1,4,5) for eIF-4D, this polypeptide appears to have a molecular weight of approximately 17,000 and a PI of about 5.1. It is unusual in that it is the only known cellular protein that contains the amino acid hypusine, the post-translational biosynthesis of which requires a structural contribution from the polyamine spermidine (5, 6). In this novel biosynthetic reaction, the intermediate deoxyhypusine is produced by transfer of the butylamine moiety of spermidine to the t-amino group of a specific lysine residue (7). Hypusine is formed by subsequent hydroxylation of this intermediate at a specific carbon in the butylamine portion of its side chain (8,9).
The unique presence of hypusine in eIF-4D has assisted us in isolating this protein from an abundant source, human red * 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.

5
To whom correspondence should be addressed. blood cells. The method for isolation is reported here, together with the sequence of amino acids that surrounds the single hypusine residue. During a number of preparations of this factor, we have observed co-purification of variable amounts of two closely related forms, each of which contains a single residue of hypusine. Multiple forms of several other mammalian initiation factors have been reported (10).

EXPERIMENTAL PROCEDURES
Materials-Human red blood cells were purchased from the American Red Cross. Chinese hamster ovary cells WTB were kindly supplied by Dr. April Robbins (National Institutes of Health). lz5I (carrier-free) and [2,3-3H]putrescine.2HC1 (>15 Ci/mmol) were obtained from New England Nuclear. Other materials and reagents are described in earlier publications (6-8, 11, 12).
Methods-Ion exchange chromatographic determination of hypusine was carried out as described earlier (6) with the use of a Dionex D-400 analyzer, the three-buffer system, and fluorometric detection. In this system, the majority of neutral and acidic amino acids are eluted in the breakthrough volume.
Preparation from CHO cells of a radiolabeled eIF-4D fraction for use as a tracer in the preparation of the human red blood cell factor was carried out essentially as described earlier (9), except that a,adipyridyl was not included in the culture medium during growth of the cells. The 40-70% ammonium sulfate fraction prepared from the cell lysate and employed as the tracer was found to contain essentially all of its radioactivity in the form of hypusine in a single 17,000dalton protein. The specific radioactivity of hypusine in a typical preparation was found to be 5 X IO5 cpm/nmol.
Purification of eIF-4D from Human Erythrocytes-Indated or outdated red blood cells from citrate/phosphate/dextrose human blood were washed twice by suspension in 5 volumes of phosphate-buffered saline and centrifugation at 2000 X g for 5 min. The cells (600 ml of packed cells) were lysed in 2 liters of ice-cold water containing 1 mM dithiothreitol and 0.1 mM ethylenediaminetetraacetic acid. The hemolysate was centrifuged at 25,000 X g for 30 min to remove debris. This and all further operations were conductedat 0-4 "C. All solutions used in further steps of purification contained dithiothreitol and ethylenediaminetetraacetic acid at the concentrations given above. The supernatant was combined with the damp filter cake from 100 g of DEAE-cellulose powder (DE52, Reeve Angel) that had been preswollen with lysis solution. The mixture was gently stirred for 2 h after which the adsorbent was collected and washed on a Buchner funnel with 10 liters of 50 mM Tris acetate buffer, pH 6.8. The washed adsorbent was packed into a 5-cm-wide column and further rinsed with 1 liter of the same Tris acetate buffer containing 0.1 M KCl. Elution was conducted using this buffer, but with 0.35 M KC1. Those fractions containing radioactivity were combined.
Ammonium Sulfate Fractionation-Solid ammonium sulfate (0.243 g/ml) was added slowly with stirring. The precipitate collected after equilibration (30 min) and centrifugation (25,000 X g for 20 min) was discarded. To the supernatant was added solid ammonium sulfate (0.205 g/ml). The precipitate obtained was dissolved in 5-10 ml of 0.15 M ammonium acetate buffer, pH 7.8, and the small amount of insoluble material was removed by centrifugation at 25,000 X g.
Exclusion Chromatography on Sephacryl S-200-This solution, which contained about one-half of the original amount of radioactiv-ity, was applied to a column (2.6 X 90 cm) of Sephacryl S-200 superfine (Pharmacia) which had been equilibrated with the pH 7.8 ammonium acet,ate buffer. Elution was conducted with this buffer. The fractions containing radioactivity were pooled (Fig. lA), and the protein was collected by centrifugation after the addition of ammonium sulfate (0.413 g/ml). This protein was dissolved in about 2 ml of 0.05 M Tris acetate buffer, pH 7.9, and dialyzed against 2 changes of 1 liter of this buffer.

Ion Exchange Chromatography on Phosphocellulose-The dialyzed
protein solution was applied to a column (1.6 X 7 cm) of phosphocellulose (P-11, Whatman) that had been equilibrated with the same buffer. The column was washed with the buffer unt,il the absorbance dropped below 0.1 unit at which time elution was commenced using a 200-ml linear gradient of 0-0.3 M KC1 in the Tris buffer. Fractions containing radioactivity were combined (Fig. l B ) , the volume of the pooled material was reduced to less than 1 ml by ultrafiltration on a YM 5 filter (Amicon), and this solution of purified factor was stored frozen at -20 "C. A typical purification is summarized in Table I.

RESULTS
The single cellular protein that is radiolabeled when human peripheral lymphocytes or CHO cells are cultured in the

TABLE I Purification of eIF-4D from human red blood cells
The eIF-4D was purified as described in the text starting with 600 ml of packed erythrocytes. Protein concentration was measured by a published method (13) using bovine 7-globulin as a standard.

_ _ _~"
Total pro-Total Purification step tein  albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor, a-lactalbumin. Electrophoresis was performed on slab gels by the procedure of Laemmli (15). The acrylamide concentration of the running gels was 12.5%. Prior to application, the samples were made 2% in sodium dodecyl sulfate, 10% in glycerol, and 1% in dithiothreitol, and heated a t 100 "C for 3 min. Gels were stained with Coomassie Brilliant Blue.
presence of [2,3-'Hlputrescine or [terminal methylenes-'HI spermidine is eIF-4D (5, 6, 11). The labeling of this specific protein results from production of 1 residue of hypusine, the biosynthesis of which involves transfer of the radioactive butylamine moiety of the polyamine spermidine to the tamino group of a specific lysine residue (7). Since various mammalian forms of eIF-4D exhibit identical PI values and molecular weights (ll), we were able to utilize factor that was labeled biosynthetically in CHO cells as a tracer in the course of purification of eIF-4D from human red blood cells. The amount of labeled CHO cell eIF-4D added as tracer (about 2.0 nmol) provided only a small contribution to the total eIF-4D (between 275 and 550 nmo1/600 ml of packed red cells). The total content of eIF-4D at each step of purification was also estimated by fluorometric measurement of hypusine after its separation by ion exchange chromatography from acid hydrolysates. The close correlation found between the recovery of radioactivity and hypusine (data not shown) is strong evidence that eIF-4D is the only protein in human red blood cells that contains hypusine and that this erythrocyte eIF-4D, like the factor from other mammalian sources, contains a single residue of hypusine.
The majority of eIF-4D is found in the postribosomal fraction from lysate of rabbit reticulocytes (14). Its content in these cells was reported to be about 1440 nmo1/600 ml of packed cells (14). Our estimates of its level in rabbit reticulocytes calculated in several preparations (not described here) from recoveries of tracer radioactivity and amount of hypusine were in this same range. Although human erythrocytes con-   FIG. 4. Reverse phase high performance liquid chromatographic pattern of tryptic peptides from human red blood cell eIF-4D. Tr-yptic digestion of eIF-4D, preparation number 16 (0.5 mg/ml), was conducted with trypsin a t a level of 20 pg/ml using other conditions as outlined earlier (11). The water and buffer salt were removed by lyophilization and the residue was dissolved at %o the original volume in the starting mobile phase used for chromatography. Chromatography was performed on the digest from 0.2 mg of protein with the use of a 0.39-X 30-cm column of pBondapak CIS (Waters Associates) and a gradient as shown between 0.1% (v/v) trifluoroacetic acid and acetonitrile containing 0.1% (v/v) trifluoroacetic acid. The flow rate was 1 ml/min and monitoring was a t 210 nm with a full scale absorbance of 1.0. The hypusine-containingpeptide, denoted by the uertical arrow, was obtained in -70% yield as estimated from recovery of radioactivity.
tain only one-third to one-fifth the amount of this factor, they certainly offer a more plentiful and readily available source. Because isolated human erythrocytes contain little more than 1% ret.iculocytes, it seems likely that the erythrocytes are indeed the principal source of eIF-4D.
When our preparations of purified eIF-4D from human red blood cells were carefully examined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, we observed that only a few of the preparations displayed a single band staining with Coomassie Blue (lane A , Fig. 2). In the majority of preparations, an additional band of slightly slower mobility as indicated by the broken arrow, and which varied in int.ensity in different preparations, was seen (lanes B and C, Fig. 2).
The position of the more rapidly moving band as indicated by the solid arrow was observed to be identical with that of the single band of eIF-4D isolated from rabbit reticulocytes by the procedure given here (lane D, Fig. 2) as well as by that of eIF-4D purified from the rabbit reticulocyte ribosomal fraction by a different procedure (1) (a gift from Dr. Brian Safer, NIH) (gel not shown).
The Coomassie Blue-stained bands from several different preparations of red blood cell eIF-4D were excised from gels and washed well with 10% met,hanol. Treatment of each of the gel pieces with trypsin and separation of digestion products from the insoluble gel mat,erial was carried out as outlined earlier (11). Following hydrolysis of the digestion products with 6 N HCl, the amount of hypusine was est.imated fluorometrically after its separation by ion exchange chromatography. It was necessary to separate h-ypusine-containing peptide material from the gel pieces before acid hydrolysis in order to avoid formation from the gel constituents of compounds that caused false response in the fluorometric determination of hypusine. In those samples of red blood cell eIF-4D that displayed the two-band staining pat.tern, hypusine was found to be a component of the protein in each of the bands. In each sample, the content of h-ypusine in the protein from the two gel positions was approximately the same as judged from the band staining intensities and from the fluorescent intensities of the breakthrough peaks of amino acids in the chromatograms. Surprisingly, the radiolabeled CHO cell eIF-4D which was initially added as a tracer was found to co-migrate with only one of the protein component,s of those eIF-4D preparations that exhibited the two band pattern. Radioactivity

TABLE I1
Analysis of the hypusine-containingpptide from human red blood cell el%-4D Acid hydrolysis was performed in evacuated sealed tubes for 24 h with 4 M methanesulfonic acid containing 0.2% tryptamine at 115°C (16). Amino acid composition was estimated with the use of a Beckman amino acid analyzer 6300 using a single column system (17). Hypusine was determined on a separate sample by the method given under "Experimental Procedures." Microsequence analyses were carried out by automated Edman degradation on an Applied Biosystems gas-phase sequencer 470A in the presence of Polybrene (18) and by the manual dimethylaminoazobenzene isothiocyanate/phenyl isothiocyanate double coupling method (19  Since both proteins in the samples of purified eIF-4D were found to contain hypusine, it was of interest to examine further their structural relationships. The striking similarity in the patterns of radioactive peptides on maps prepared from tryptic digests of radioiodinated samples of the proteins from the two gel positions is seen in Fig. 3. The peptide denoted in each map by the broken arrow warrants special attention. This peptide occupies the approximate position of the hypusine-containing tryptic peptide from eIF-4D of several mammalian cells (11). The likelihood that it is indeed this peptide from each of the two protein components of human red blood cell eIF-4D becomes more evident with the knowledge that this peptide contains amino acid residues, i.e. 2 histidine residues (see below), that become radiolabeled under the experimental conditions used for radioiodination.
From the unique position occupied by the hypusine-containing peptide o n the tryptic maps of Fig. 3, it was speculated that this peptide is strongly positively charged and very hydrophilic and should be quite easily isolated in quantity from the bulk of tryptic peptides. This indeed proved to be the case. Fig. 4 shows the reverse phase high performance liquid chromatographic pattern of the tryptic peptides of red blood cell eIF-4D. The hypusine-containing peptide, designated by the vertical arrow, was eluted rapidly from the column, well separated from all other peptides. In Table I1 are given the amino acid composition of, and the sequence data on, this peptide. These results are in close agreement and allow us to postulate the sequence as: Thr-Gly-hypusine-His-Gly-His-Ala-Lys.

DISCUSSION
The procedure for isolation of eIF-4D polypeptide outlined here provides a large-fold purification in good yield from a plentiful human source. The procedure is novel in that specifically biosynthetically radiolabeled factor, prepared in cultured cells from another mammalian source, was employed to follow steps in purification. The decision to purify using this means of assay was based on our earlier evidence for very similar physical properties and a large degree of homology in this protein from various mammalian sources (11). In light of the complexity of the biological assay for eIF-4D, and because of the lack of knowledge as to its specific physiological function, the simple radiotracer procedure seemed appropriate for isolation of this hypusine-containing factor. Indeed, this purified material should be most useful for physical and chemical studies.
Antibodies prepared to the isolated factor should provide a means for comprehensive study of the physiological role of eIF-4D. Together with knowledge of the primary structure of this factor, the first information on which is included in this report, should come some understanding of the function of the unique hypusine residue, and, ultimately, the importance of this post-translational modification to which the polyamine spermidine contributes a structural part.
It is tempting to speculate that the highly positively charged hydrophilic region of sequence around hypusine bears a special role in interactions between eIF-4D and ribosomes, nucleic acids, and/or acidic domains in other proteins. Certainly, knowledge of this sequence confirms our earlier suggestions that eIF-4D contains 1 single residue of hypusine (5,ll). It supplies a glimmer of the basis for specificity of the biosynthesis of hypusine and provides a preliminary pattern for construction of oligonucleotide probes, and of peptide substrates and inhibitors for the enzymes that promote this unique post-translational event.
Biosynthetic labeling of hypusine in eIF-4D has provided strong evidence for a single form of eIF-4D in cultured cells (5,6). Therefore, the finding of varying amounts of two forms of eIF-4D in human red blood cell preparations was surprising. However, multiple molecular weight forms of certain other initiation factors have been isolated from rabbit reticulocytes (10). The relative amounts of these forms were reported to vary considerably from one preparation to another, probably as a result of limited proteolysis in vivo and/or during isolation of the factors. In our preparations, the radiolabeled tracer from CHO cells does not change during the isolation procedure and radioactivity is consistently found associated with one form only of the human red cell eIF-4D. There are several possible causes for this finding. (i) The native form of human eIF-4D is identical with the tracer CHO cell eIF-4D. I n vivo or during storage of the red cells there is partial conversion to the second form, but, after addition of tracer to cell lysate, and during the subsequent purification, there is no further change. (ii) The native form of red cell eIF-4D displays the identical migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as tracer factor. However, because they originate from separate mammalian species, the two factors contain one or more differences in their primary structures. During storage and/or isolation, the red blood cell eIF-4D is partially converted to a new slower migrating form, whereas the CHO cell radioactive tracer eIF-4D is not. This differential change occurs because the chemical modification that causes this conversion is limited to a region of primary structure that exists exclusively in the red blood cell factor. (iii) The native form of human eIF-4D is that which migrates more slowly than the radioactive tracer. In uiuo, during storage, and/or during purification, there is a partial or complete conversion to a form that migrates with the tracer eIF-4D. (iv) Finally, there may exist two genetically distinct forms of human eIF-4D.
At present we have no strong reason to favor one of these causes for association of tracer radioactivity with one form only of red blood cell eIF-4D. We have seen no consistent relationship between the ratios of the two forms in eIF-4D preparations and the times or conditions of storage of whole human blood or red cells. It is interesting that after treatment of a sample of the red blood cell factor that displays one band only with HzOz and examination by sodium dodecyl sulfatepolyacrylamide gel electrophoresis, several additional Coomassie Blue-staining components are seen, one of which migrates close to the slower moving form seen in other preparations (not shown). A similar nonenzymatic oxidative modification of purified Escherichia coli glutamine synthetase has been reported (21). Consistent with a possible oxidative change in red cell eIF-4D and suggestive of a conversion in the direction of the slower migrating component is the finding of a substantially higher level of methionine in preparation number 16, which displays a single component that migrates with tracer, than in preparation number 18 in which there is a majority of the slower moving form. Whether a conversion of one form of eIF-4D to another that may occur in red blood cells in vivo or during aging or storage is of biological significance in terms of factor inactivation or degradation is not known. This, together with the structural differences in the two forms of human red blood cell eIF-4D, is under investigation.