(Received for publication, June 5, 1995)
From the
Deoxyhypusine synthase catalyzes the first step in the
post-translational formation of hypusine (N-(4-amino-2-hydroxybutyl)lysine). cDNA
clones encoding deoxyhypusine synthase were isolated from a human HeLa
cell library. Full-length cDNA clones encoding a 369-amino acid protein
(calculated molecular mass of 40,970 Da) and a shorter cDNA clone that
would potentially encode a protein with an internal deletion of 56
amino acids (Asp
-Ser
) were isolated.
The deduced amino acid sequence of the human enzyme shows a high degree
of identity to that of yeast deoxyhypusine synthase and to the known
sequences of tryptic peptides from the rat and Neurospora crassa enzymes. The recombinant enzyme formed upon expression in Escherichia coli effectively catalyzed deoxyhypusine
synthesis. Variant human recombinant proteins with (i) a truncation of
48 or 97 NH
-terminal amino acids, (ii) a truncation of 39
COOH-terminal amino acids, or (iii) an internal deletion
(Asp
-Ser
) were inactive. A chimeric
protein consisting of the complete human sequence and 16 amino acids of
the yeast sequence (Gln
-Asn
, not
present in the human enzyme) inserted between Glu
and
Gln
exhibited moderate activity.
The unusual amino acid hypusine (N-(4-amino-2-hydroxybutyl)lysine) is formed
post-translationally in a single cellular protein, eukaryotic
translation initiation factor 5A (eIF-5A) (
)(1, 2, 3) . Hypusine
biosynthesis involves two enzymatic steps. In the first step,
deoxyhypusine synthase catalyzes the NAD-dependent transfer of the
butylamine moiety of spermidine to the
-amino group of a specific
lysine residue of the eIF-5A precursor protein to form the intermediate
deoxyhypusine (N
-(4-aminobutyl)lysine)
residue(1, 4, 5, 6, 7) . In
the second step, mediated by deoxyhypusine hydroxylase, the conversion
of the deoxyhypusine residue to the hypusine residue (8) completes eIF-5A maturation.
Hypusine is found in all eukaryotes and in some archaebacteria, but not in eubacteria(1, 9) . The amino acid sequence of the hypusine-containing protein, eIF-5A, is highly conserved, especially in the region surrounding the hypusine residue, suggesting an important fundamental function for this protein throughout eukaryotic evolution. In spite of its in vitro activity in the stimulation of methionyl puromycin synthesis and tentative identification as an initiation factor in eukaryotic protein synthesis(10, 11) , its true cellular function is unknown(12, 13) . However, hypusine and eIF-5A appear to be vital for cell proliferation in eukaryotes(12) . The correlation between a reduction in hypusine formation and growth arrest in spermidine-depleted mammalian cells (14) and the requirements for expression of eIF-5A precursor protein and its hypusine modification for yeast viability (15, 16) strongly support this notion. Furthermore, inhibitors of the hypusine biosynthetic enzymes exert antiproliferative effects(17, 18, 19) . In this regard, deoxyhypusine synthase presents a potential target for intervention in cell proliferation. A detailed knowledge of the amino acid sequence and properties of this enzyme should contribute to attaining more specific inhibitors of this enzyme and perhaps to better control of hyperproliferative diseases.
We have recently purified deoxyhypusine synthase from rat testis, determined the amino acid sequences of several tryptic peptides, and raised polyclonal antibodies to this enzyme(20) . Cross-reactivity of anti-rat deoxyhypusine synthase with the human enzyme (from HeLa cells) was utilized to facilitate the cloning of cDNAs encoding human deoxyhypusine synthase.
In this study, we report the molecular cloning, expression, purification, and characterization of human recombinant deoxyhypusine synthase. Studies of deletion and insertion mutations, designed on the basis of a comparison of the amino acid sequences of the human and yeast (21) enzymes, provide insight into the structure-function relationship of the enzyme.
Figure 1: Nucleotide sequence of a cDNA encoding full-length human deoxyhypusine synthase. The nucleotide sequence of clone hDS-13 is denoted in lower-case letters and the deduced amino acids in upper-case letters. The numbers on the left designate the nucleotides and the numbers on the right the amino acids from positions 1 to 369 in the open reading frame. The putative stop codon is indicated (*). The nucleotide sequences that are absent from clone hDS-1a are underlined.
Figure 2: Comparison of amino acid sequences of deoxyhypusine synthases from different species. The amino acid sequences deduced from human and yeast cDNAs are compared with the partial amino acid sequences determined for tryptic peptides from the rat testis enzyme (20) (underlined) and the Neurospora enzyme (25) (italic). To allow for maximal alignment of the human and yeast sequences, gaps have been introduced (. . . . ). The amino acid residue numbers for the human and yeast sequences are indicated on the right. The vertical lines between residues denote identity, while the dots indicate similarity (generated by the program GCG ``BestFit'' (Genetics Computer Group, Madison, WI) using the local homology algorithm of Smith and Waterman(26) ).
A comparison
of the primary structure of human deoxyhypusine synthase with those of
other species is shown in Fig. 2. The deduced amino acid
sequence of the human enzyme is 58% identical and 73% similar to that
of the yeast enzyme(21) . Three regions of high sequence
identity exist, encompassing residues 98-188, 201-248, and
277-330 of the human enzyme. In addition, the amino acid
sequences determined for several tryptic peptides from the rat testis
enzyme (20) or the Neurospora crassa enzyme (25) also are highly similar to the corresponding regions of
the human and yeast enzymes. It is interesting to note that the human
enzyme is shorter than the yeast enzyme. No counterpart sequence for 16
amino acids (Gln-Asn
) of the yeast
enzyme is present in the human enzyme.
Figure 3:
Northern blot analysis. Total RNAs
(25 µg) isolated from HeLa or human trophoblast cells (A) and normal human keratinocytes or tumor cell lines derived
from head and neck cancers or dysplasias (B) were subjected to
electrophoresis on a 0.66 M formaldehyde, 1% agarose gel and
transferred to a nitrocellulose membrane by the method of Sambrook et al.(27) . Both prehybridization and hybridization
were conducted at 42 °C in 6
SSC containing 50% formamide,
5
Denhardt's solution, 100 µg/ml yeast tRNA, and 0.1%
SDS. After overnight hybridization with a cDNA probe, which was
generated by PCR of hDS-1a cDNA using primers Pr5-2 and Pr3-5 and
labeled with
P, the membranes were washed at 23 °C for
10-20 min three times with 2
SSC containing 0.1% SDS and
once with 1
SSC containing 0.1% SDS and then washed at
55-60 °C for 20 min three times with 0.2
SSC
containing 0.1% SDS. In A, 144 JAR is a human trophoblast cell
line. In B, T 45(28) , HN 30(29) , HN
22(29) , HN 8 (29) , and SCC 25 (30) are
derived from oral squamous cell carcinomas; DOK (31) from
premalignant keratinocytes; and NHEK (Clonetics Corp.) from normal
human epidermal keratinocytes (source or reference given in
parentheses). Kb, kilobases.
Figure 4:
Ion-exchange chromatography of human
recombinant deoxyhypusine synthase on Mono Q. Protein (144 mg) from
step 3 of Table 3was applied to a Mono Q HR 10/10 column as
described under ``Experimental Procedures.'' After washing
the column for 10 min with buffer A, a salt gradient of 0-0.6 M KCl in buffer A was applied, and fractions of 2 ml (1 min)
were collected. A, absorbance at 280 nm, conductivity, and
enzymatic activity; B, the Coomassie Blue-stained pattern of
an SDS-polyacrylamide gel (10%). Lane1, crude E.
coli lysate; lane2, starting material before
Mono Q chromatography; lane3, fraction 41 (2
µl); lane4, fraction 43 (2 µl); lane5, standard marker proteins. All samples were heated in
-mercaptoethanol-containing SDS sample buffer prior to
electrophoresis. The position of the human recombinant deoxyhypusine
synthase protein is indicated by an arrowhead.
Hypusine is ubiquitous in all eukaryotic species, and its
biosynthetic pathway appears to be highly conserved. This study shows
that the human enzyme shares similar properties with deoxyhypusine
synthases from diverse species, e.g. rat(7, 20) , N. crassa(25) , and Saccharomyces cerevisiae(21) , in the catalysis of a
complex post-translational modification reaction. Primary structure is
highly conserved in the major portion of human and yeast deoxyhypusine
synthase molecules, i.e between Thr and
Ile
of the human sequence. The known sequences of
peptides from the rat (20) and Neurospora(25) enzymes also show a marked similarity to the primary
structures of the human and yeast enzymes in this region. The
maintenance of protein structure in these enzymes may have been
mandated for the recognition of their three substrates, spermidine,
NAD, and especially the highly conserved eIF-5A precursor, and, in
turn, may have served to preserve a vital cellular function of eIF-5A.
Despite the strict specificity of deoxyhypusine synthases from all
species for this single cellular protein, cross-species reactivities
between the enzymes and the eIF-5A precursor proteins are observed. As
previously shown (21) for the yeast and rat enzymes, human
deoxyhypusine synthase also catalyzes the modification of heterologous
protein substrates, namely eIF-5A precursors from Chinese hamster ovary
cells and yeast (data not shown).
It is intriguing that the initial
clone, hDS-1a, isolated by means of immunological screening, has an
internal deletion that results in the loss of 56 amino acids
(Asp-Ser
). It was unclear whether
this in-frame deletion reflects natural cellular processing of the
deoxyhypusine synthase gene transcript or whether it arose as an
artifact of expression library construction. Since no other clone with
the same deletion was detected, we sought to determine the existence of
a natural mRNA(s) with this deletion. Using different sets of primers
flanking the deletion region, PCR amplification of a HeLa cDNA mixture
(HeLa QuickClone cDNA (CLONTECH) or cDNA prepared by a reverse
transcriptase reaction of total RNA from HeLa cells) as template thus
far has provided no clear indication of the existence of a natural
transcript with the corresponding deletion (data not shown).
Determination of the genomic structure of deoxyhypusine synthase might
offer insight as to whether such a transcript can be generated by
alternative splicing.
A mutant protein expressed from hDS m3, lacking Asp-Ser
(corresponding to the internal coding region missing in hDS-1a),
displayed no detectable deoxyhypusine synthase activity. This lack of
activity is not surprising since the deletion removed a highly
conserved region of the protein (Fig. 2) and strongly indicates
that this region is critical for enzyme activity. The mutant proteins
expressed from clones hDS m4, hDS m5, and hDS m6 (Table 2), with truncations of 48 or 97
NH
-terminal amino acids (Met
-Ala
or Met
-Cys
) or of 39 COOH-terminal
amino acids (Asp
-Asp
), respectively,
were also devoid of enzymatic activity, suggesting that, although not
highly conserved, these regions also contain amino acid residues
important for the proper conformation of the enzyme, necessary for
binding substrates and/or for catalysis.
The most interesting
difference between the primary structures of the human and yeast
enzymes is the absence of a human sequence matching
Gln-Asn
in the yeast enzyme. Although
this gap (indicated by . . . . in Fig. 2) is located in the
middle of a highly conserved region, the human recombinant
deoxyhypusine synthase exhibits as high a specific activity as the
yeast enzyme. A chimeric protein with the 16-amino acid yeast sequence
inserted between Glu
and Gln
of the human
enzyme exhibited moderate activity (Table 2). Since the insertion
of this partial yeast sequence into the human enzyme did not result in
a dramatic increase in its K
value for NAD, one
cannot attribute the large differences in affinities toward NAD
specifically to this region of the molecule. On the other hand, it is
interesting that this insertion does have a significantly detrimental
effect on the human enzyme activity.
Evidence for a vital role of hypusine in eukaryotic cell proliferation has led to proposed targeting of the catalytic steps in hypusine production as potential methods for antiproliferative therapy(1, 12, 18, 19, 32) . Promising results on the intervention in growth of mammalian cells, including various human tumor cell lines, have been obtained with inhibitors developed against the rat testis deoxyhypusine synthase(17, 19) . Although some details concerning the steps in the biosynthesis of deoxyhypusine have been revealed(1) , little is known about the precise mechanism of the reaction. The availability of a human cDNA clone and its encoded protein should enable us to determine structural features of the active site and of the substrate binding domains as well as the residues involved in catalysis. A better understanding of the enzyme structure and the reaction mechanism will facilitate the development of potent and specific inhibitors of the human enzyme and pave the way to improved means of cellular regulation through the inhibition of hypusine biosynthesis.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L39068[GenBank].