(Received for publication, March 21, 1995; and in revised form, June 5, 1995)
From the
Deoxyhypusine synthase catalyzes the formation of deoxyhypusine,
the first step in hypusine biosynthesis. Amino acid sequences of five
tryptic peptides from rat deoxyhypusine synthase were found to match
partially the deduced amino acid sequence of the open reading frame of
gene YHR068w of Saccharomyces cerevisiae chromosome
VIII (AC:U00061). In order to determine whether the product of this
gene corresponds to yeast deoxyhypusine synthase, a 1.17-kilobase pair
cDNA with an identical nucleotide sequence to that of the YHR068w coding region was obtained from S. cerevisiae cDNA by
polymerase chain reaction and was expressed in Escherichia coli B strain BL21(DE3). The recombinant protein was found mostly in
the E. coli cytosol fraction and comprised
The biosynthesis of hypusine (N
Figure S1:
Scheme 1Deoxyhypusine synthase
reaction. The shadedareas indicate the 4-aminobutyl
moiety of spermidine that is transferred to the specific lysine of the
eIF-5A precursor.
Hypusine is
ubiquitous in eukaryotes and is found in some
archaebacteria(1, 8) . It does not occur in
eubacteria(8) . Several lines of evidence support the essential
role of hypusine in eIF-5A for eukaryotic cell
proliferation(9) . These include: (i) the lack of growth of
yeast in the absence of expression of the two eIF-5A genes (10, 11) ; (ii) the inability of the mutated eIF-5A
gene (Lys We have recently
purified deoxyhypusine synthase from rat testis(15) . The
partial amino acid sequences determined for five tryptic peptides from
the rat testis enzyme prompted us to search for similar sequences in
the molecular biology data banks. Although we found no protein with
similar sequences, the deduced amino acid sequence of one open reading
frame (387 amino acids) of the gene YHR068w in Saccharomyces cerevisiae chromosome VIII (AC:U00061) (16) yielded partial matches with the rat enzyme sequences. In
an effort to determine if the product of this gene corresponds to a
yeast deoxyhypusine synthase, we obtained from S. cerevisiae cDNA by PCR a 1.17-kilobase pair cDNA with the identical
nucleotide sequences as that of the entire YHR068w coding
region. The expression, purification, and characterization of the yeast
recombinant enzyme are the subject of this paper.
The deduced amino acid sequence of the open reading frame of
the yeast YHR068w gene (16) along with the fit of the
amino acid sequences of five tryptic peptides (P1-P5) from rat
deoxyhypusine synthase (15) is shown in Fig. 1. There is
a remarkable identity between P5 and residues 319-332 of the
yeast YHR068w gene product. By making the reasonable
assumption that P2 and P3 were originally joined by lysine in the rat
enzyme, a good match can be made for this extended peptide. The other
two peptides do not show as good a fit. Using primers based on the
nucleotide sequence of the yeast gene, PCR product was observed as a
single band after amplification of Quick Clone S. cerevisiae cDNA (Clontech). Two independent clones were found to have
nucleotide sequences (1,161 bases) identical to that deposited in the
GenBank for this gene, suggesting that we had obtained a full-length YHR068w cDNA, encoding a polypeptide of 387 amino acids, with
a calculated molecular mass of 42,892 Da. The cDNA clone, tentatively
identified as the cDNA for a yeast deoxyhypusine synthase, was
expressed in E. coli using pET-11a plasmid. As shown in Fig. 2A, deoxyhypusine synthase activity was detected
in the cell lysates and increased in a time-dependent manner up to 4 h
after induction by IPTG. SDS-PAGE of the cell lysates showed that a
43-kDa polypeptide was overexpressed (Fig. 2B), in
correspondence with the increase in enzyme activity. At 4 h, this
polypeptide amounted to
Figure 1:
Deduced amino acid sequence of the open
reading frame of YHR068w (AC:U00061) (top line) and
alignment of five tryptic peptides from rat testis deoxyhypusine
synthase (second line, underlined). The peptides are labeled P1-P5. The sequence of P1 is fitted with a gap of 10
residues.
Figure 2:
Induction of the yeast recombinant
deoxyhypusine synthase in E. coli by IPTG. Samples of cells
were removed after induction with IPTG for 1, 2, and 4 h. The cells in
each sample were lysed, and a portion containing
Figure 3:
Size exclusion chromatography and PAGE of
the yeast recombinant deoxyhypusine synthase. A, exclusion
chromatography on a Bio-Gel A 0.5-m column was carried out as described
under ``Experimental Procedures.'' A sample volume of 4.0 ml
containing 50 mg of protein from Step 2 was applied to the column. TGase, transglutaminase; Alb, albumin. B,
PAGE (non-denaturing) on a 10% precast gel; 4 µl from size
exclusion chromatography fractions 53, 55, 57, 59, and 61 (panelA) in lanes1-5, respectively; C, SDS-PAGE on a 10% precast gel; 4 µl from the same
fractions as in B; D, SDS-PAGE on a 10% precast gel
of purified deoxyhypusine synthase; 2 µl from peak fractions
70-74 (lanes 1-5, respectively) after Mono Q ion
exchange chromatography (Table 1, Step 4). Positions of molecular
mass standards are shown by arrows.
Figure 4:
Modification of eIF-5A precursor proteins
from several species by deoxyhypusine synthases from yeast and rat. The
deoxyhypusine synthase reaction was carried out as described under
``Experimental Procedures'' except that either the yeast
enzyme (A,
The identification of YHR068w in S. cerevisiae chromosome VIII as the gene for deoxyhypusine synthase provides
the first full amino acid sequence of deoxyhypusine synthase from any
source. The selection of this open reading frame as a candidate for a
yeast deoxyhypusine synthase gene was made on partial matching with the
amino acid sequences of five tryptic peptides from the purified rat
testis enzyme(15) . While this manuscript was in preparation,
the purification of a deoxyhypusine synthase from Neurospora crassa was reported(20) . The amino acid sequences of the four
tryptic peptides obtained from the N. crassa enzyme also show
a high degree of identity with the yeast enzyme. Deoxyhypusine
synthase exhibits a remarkable substrate specificity in its recognition
of a single lysine residue of one cellular protein, the eIF-5A
precursor. eIF-5A is highly conserved in a wide range of eukaryotic
species(1) , and the amino acid sequence identity is especially
high in the region surrounding the lysine residue that undergoes
modification to hypusine(1) . Furthermore, a large portion of
the substrate protein is required for recognition and modification by
deoxyhypusine synthase(13) . Nevertheless, the enzymes from rat
testis or yeast appear to recognize eIF-5A precursor proteins from
other species, even distantly related ones. The experiments of
Schwelberger et al. (21) and of Magdolen et
al. (11) provide evidence that the eIF-5A precursor
proteins from human, slime mold, and alfalfa can be modified in yeast
by the endogenous deoxyhypusine synthase and that these heterologous
proteins can functionally substitute for the yeast eIF-5A in support of
growth of this organism. The present results indeed show effective
modification of the yeast substrate proteins, the human eIF-5A
precursor, and those from CHO cells in vitro by the yeast
enzyme. The high conservation of the amino acid sequence of the eIF-5A
precursors from the diverse species may be the basis of the broad
species specificity. Yeast contains two forms of eIF-5A, designated
eIF-5Aa and eIF-5Ab here, which are derived from two distinct genes TIF51A and TIF51B(10) . Although the two
genes are reciprocally regulated by oxygen(22) , the two
proteins share 90% amino acid sequence identity (10) and appear
to be functionally indistinguishable in vitro(10) and in vivo(11, 21) . It is not known whether
yeast contains more than one form of deoxyhypusine synthase. Our
results, however, show that the recombinant enzyme can modify both of
the precursors with nearly equal efficiency. Recently we cloned a
full-length cDNA for human deoxyhypusine synthase. ( Despite obvious differences in primary structure between the yeast
recombinant and the rat testis enzymes, as well as the failure of a
polyclonal antibody to rat enzyme to recognize the yeast enzyme (data
not shown), the two enzymes exhibit certain similarities in physical
and enzymatic properties. Each appears to exist as a tetramer
(molecular mass, 160-170 kDa) of 43-kDa subunits. The
sedimentation equilibrium data obtained for the yeast recombinant
enzyme is evidence of the tetrameric structure and is consistent with a
high affinity of 43-kDa subunits for one another and with the stability
of the tetrameric complex. Each enzyme requires NAD for the catalytic
reaction; each displays a strict specificity for this nucleotide. They
also catalyze a NAD-dependent cleavage of spermidine in the absence of
protein substrate. The availability of the yeast cDNA clone and the
yeast recombinant enzyme should permit further studies on the structure
of this important en-zyme and on the mechanism of the complex reaction
that it catalyzes.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
20% of the
total soluble protein. The purified form of the expressed protein
effectively catalyzed the formation of deoxyhypusine in yeast eIF-5A
precursors as well as in human precursor and in those from Chinese
hamster ovary cells. The molecular mass of the enzyme was estimated to
be 172,000 ± 4,300 Da by equilibrium centrifugation. The mass of
its polypeptide subunit was determined to be
43,000 Da, in close
agreement with that calculated for the coding region of the YHRO68w gene. These findings show that this gene is a coding sequence for
yeast deoxyhypusine synthase and that the product of this gene exists
in a tetrameric form.
-(4-amino-2-hydroxybutyl)lysine) occurs in
the eIF-5A (
)precursor protein through a unique
posttranslational modification (reviewed in (1) ). In the first
step, the enzyme deoxyhypusine synthase catalyzes conversion of a
single lysine (Lys
in the human precursor) to
deoxyhypusine (N
-(4-aminobutyl)lysine) by
transfer of the butylamine moiety of the polyamine spermidine to the
-amino group of this lysine (2, 3, 4, 5) by an NAD-dependent
reaction (Fig. S1)(5, 6) . The second step
completes hypusine formation through catalytic hydroxylation of the
deoxyhypusine residue(2, 7) .
Arg
) to substitute for the
wild type gene in supporting growth of yeast(10) ; and (iii)
the arrest of proliferation of mammalian cells by inhibitors of
deoxyhypusine synthase(12) . In view of the importance of
hypusine in cell proliferation and the very narrow substrate
specificity of deoxyhypusine
synthesis(5, 13, 14) , this enzyme presents a
promising target for anti-proliferative therapy.
Materials
[1,8-H]Spermidine
HCl (15
Ci/mmol) was purchased from DuPont NEN. Oligonucleotide primers were
synthesized by the Midland Certified Reagent Company. pET-11a
expression vector and the host Escherichia coli B strain
BL21(DE3) were from Novagen; Vent polymerase and T4 DNA ligase from New
England Biolabs; restriction enzymes from Life Technologies, Inc.;
precast polyacrylamide gels and wide range protein standards (Mark 12)
from Novex; ec-eIF-5A, purified from E. coli lysates after
overexpression of the human eIF-5A cDNA as described(13) , was
kindly provided by Y. A. Joe of our laboratory.
Methods
Assay of Deoxyhypusine Synthase
The enzyme
activity was measured as described previously(5, 14) .
A typical reaction mixture contained, in total volumes of 20 µl,
0.2 M glycine NaOH buffer, pH 9.5, containing 1 mM
dithiothreitol, 25 mg of bovine serum albumin, 0.5 mM NAD, 7
µM [1,8-H]spermidine, 10
µM ec-eIF-5A, and enzyme. Incubations were at 37 °C
for 60 min. The radioactivity of [
H]deoxyhypusine
was measured after its ion exchange chromatographic separation from the
hydrolyzed protein fraction essentially as described
earlier(17) . One unit of activity is defined as the amount of
enzyme catalyzing the formation of 1 pmol of deoxyhypusine
h
.
Construction of a Plasmid Encoding Full-length Yeast
Deoxyhypusine Synthase and Expression in E. coli
On the
basis of the nucleotide sequence in the open reading frame of the gene YHR068w in S. cerevisiae chromosome VIII, primers for
PCR representing the 5`-end (43-mer,
CTTCCAGTATGCTCATATGTCCGATATCAACGAAAAACTCCCA) and the 3`-end
region (45-mer, CTTCCAGTATGGATCCTCAATTCTTAACTTTTTTGATTGGTTTAC)
were synthesized with a built-in NdeI site (5`-end primer) and
a BamHI site (3`-end primer) (underlined) to facilitate
cloning into the expression vector pET-11a. PCR was performed in a
thermal cycler (Perkin-Elmer) using yeast Quick Clone cDNA (Clontech)
as a template. Conditions for PCR were: denaturation at 94 °C for
30 s, annealing at 55 °C for 30 s, extension reaction at 72 °C
for 1.5 min for 35 cycles, and a final extension reaction at 72 °C
for 10 min. Agarose gel electrophoretic analysis of the PCR product
showed only one band of 1.2 kilobase pair. The PCR product was
cleaved with NdeI and BamHI, ligated to the insertion
site of the vector pET-11a, and introduced into the BL21(DE3) strain of E. coli. The cDNA clones containing an insert of the correct
size were sequenced by the dideoxy-mediated chain termination procedure
with Sequenase (version 2.0, U. S. Biochemical Corp.) using
double-stranded DNA as a template. The recombinant strains obtained by
transformation were grown in Luria-Bertani medium, supplemented with 50
µg/ml ampicillin. When the cell density in the culture reached an
OD of 0.6 at 600 nm, IPTG was added to a final concentration of 1
mM. The cells were harvested by centrifugation (5,000
g for 5 min) after 4 h of induction.
Purification of S. cerevisiae Deoxyhypusine Synthase
Expressed in BL21(DE3) Strain
The induced E. coli cells
(14 g) were suspended in 150 ml of Buffer A (20 mM Tris
acetate, pH 8.0, 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride) and thrice sonicated for a 1-min
interval at a 70-watts setting, and the supernatant was collected after
centrifugation for 30 min at 15,000 g. The supernatant
was applied to a fast flow Q-Sepharose (Pharmacia Biotech Inc.) column
(5
6 cm) previously equilibrated with Buffer A, the column was
washed with Buffer A until the effluent showed A
< 0.1, and a 1-liter linear gradient of 0-0.5 M NaCl in Buffer A was applied. The fractions containing enzyme
activity, eluting between 0.23 and 0.27 M NaCl, were pooled,
concentrated to
4.0 ml by ultrafiltration (Amicon), applied to a
Bio-Gel A 0.5-m (200-400 mesh, Bio-Rad) column (2.5
96
cm) previously equilibrated with Buffer B (0.05 M citrate
buffer, pH 6.0, 0.07 M NaCl), and eluted with Buffer B. The
fractions containing enzyme activity were pooled, concentrated by
ultrafiltration, dialyzed against Buffer A for 1 h, and applied to a
Mono Q (5/5) column. The adsorbed enzyme was eluted with a 90-ml linear
gradient of 0.1-0.5 M NaCl in Buffer A. PAGE analysis of
the purified recombinant enzyme showed a single band in the presence or
absence of SDS.
Preparation of the Yeast Recombinant eIF-5A Precursors,
ec-eIF-5Aa and ec-eIF-5Ab
S. cerevisiae contains two
eIF-5A genes, TIF51A and TIF51B(10) . The
products of these two genes, ec-eIF-5Aa and ec-eIF-5Ab, respectively,
were produced by a method similar to that described above for
preparation of the recombinant deoxyhypusine synthase. The cDNA for
each of the yeast eIF-5A precursors was obtained by PCR amplification
of Quick Clone S. cerevisiae cDNA (Clontech) using a set of
two primers containing the underlined NdeI site and BamHI site as follows: 5`-end primer
(CTTCCAGTATGCTCATATGTCTGACGAAGAACATACCTTTGAAACTG) and 3`-end
primer (CTTCCAGTATGGATCCTTAATCGGTTCTAGCAGCTTCCTTGAAG) for the
eIF-5Aa cDNA and 5`-end primer
(CTTCCAGTATGCTCATATGTCTGACGAAGAACACACCTTTGAAAATG) and the
3`-end primer (CTTCCAGTATGGATCCCTAATCAGATCTTGGAGCTTCCTTGAAG)
for the eIF-5Ab cDNA, respectively. The PCR product after cleavage with NdeI and BamHI was inserted into the vector pET-11a
and used for transformation of BL21(DE3) cells. Expression of the
eIF-5A precursor proteins in the selected transformants was induced
with IPTG (1 mM) for 4 h as described above. The lysate
supernatant of IPTG-induced cells (14 g of packed cells from a
10-liter culture) was applied to a fast flow S-Sepharose (Pharmacia)
column (5
4 cm) equilibrated with Buffer C (50 mM Tris
acetate, pH 6.0, 1 mM EDTA, 5 mM dithiothreitol, and
1 mM phenylmethylsulfonyl fluoride). After a wash of the
column with 200 ml of Buffer C containing 0.25 M KCl, the
eIF-5A precursor proteins were eluted with Buffer C containing 1 M KCl. The fractions containing the eIF-5A precursors were
pooled. After ammonium sulfate fractionation (40-75% saturation)
of this pool and subsequent dialysis,
20 mg of each of the eIF-5A
precursor proteins was obtained in >90% purity. In the transformant
containing yeast eIF-5Aa cDNA, two forms of the eIF-5A precursor
protein, exhibiting apparent molecular masses of 21 and 19 kDa,
respectively, were detected on SDS-PAGE. The 19-kDa form, presumably
generated by proteolytic cleavage of a 10-amino-acid fragment of the
intact protein(18) , copurified with the intact form upon ion
exchange chromatography on an S-Sepharose or Mono-Q column (data not
shown). From the recombinant strain transformed with yeast eIF-5Ab
cDNA, one form of eIF-5A precursor with an apparent molecular mass of
20 kDa was isolated.
Other Methods
Matrix-assisted laser desorption
mass spectrometry was carried out using a Kratos Kompact MALDI-3
spectrometer (Kratos, Ltd., Manchester, UK). Sedimentation equilibrium
analysis was performed at 20 °C with a 5-mm column height of sample
(0.34 mg/ml yeast enzyme) using a Beckman XL-A analytical
ultracentrifuge.
20% of the total soluble protein of the
lysate.
0.1 µg of
protein was analyzed for deoxyhypusine synthase activity (A);
another portion containing
10 µg of protein was used for
SDS-PAGE on 10% gel to follow the expression of recombinant 43-kDa
polypeptide (B). Protein bands were visualized by Coomassie
Blue R-250 staining. The 43-kDa protein is denoted by the arrowhead on the right. The positions of molecular mass standards
are shown on the left.
Purification of Yeast Recombinant Deoxyhypusine
Synthase
A four-step purification procedure is summarized in Table 1. A fast flow Q-Sepharose anion exchange chromatographic
step of the cell lysate supernatant fraction allowed 3-fold
purification with 90% recovery of total enzyme activity. Upon size
exclusion chromatography (Step 3), the enzyme (
90% purity) eluted
in a single symmetrical peak (Fig. 3A). Analysis of the
peak fractions by PAGE under non-denaturing conditions showed
correspondence between the staining intensity of the major band and
enzyme activity (Fig. 3B). Upon SDS-PAGE of the same
fractions after their treatment with SDS and dithiothreitol, a
predominant band at 43 kDa corresponded with the enzyme activity (Fig. 3C). Minor impurities from the gel filtration
step were removed in the final step of purification, ion exchange
chromatography on a Mono Q (5/5) column (Table 1, Step 4), as
evidenced by SDS-PAGE (Fig. 3D). The overall protocol
yielded pure yeast recombinant deoxyhypusine synthase with an average
recovery of
50% and a specific activity of 0.7-1.1
10
units/mg of protein (Table 1).
Enzymatic Properties
The YHR068w gene
product expressed in E. coli catalyzes the formation of
deoxyhypusine in the human eIF-5A precursor protein in the presence of
spermidine and NAD. Its specific enzymatic activity is comparable with
that of the deoxyhypusine synthase purified from rat testis (0.8
10
units/mg of protein)(15) . Thus this
gene product is defined as a deoxyhypusine synthase. Like the rat
enzyme, the yeast recombinant enzyme displays a strict specificity for
NAD. NADP, FAD, and FMN cannot substitute for NAD (data not shown). Its
requirement for the precursor protein is also specific. No
[
H]deoxyhypusine was formed in any E. coli cytosol protein or in albumin after incubation with enzyme,
[
H]spermidine, and NAD in the absence of the
eIF-5A precursor protein. However, the yeast enzyme recognized the
eIF-5A precursor proteins from yeast and other species (Fig. 4).
The human eIF-5A precursor protein and the two eIF-5A precursors from
CHO cells, PI and PII(19) , were modified by the yeast enzyme
as were the yeast precursor proteins (Fig. 4A). In the
reaction mixture containing [
H]spermidine, no
significant difference was observed in the degree of labeling of the
two bands of yeast ec-eIF-5Aa proteins and the yeast ec-eIF-5Ab protein (Fig. 4A). Interestingly, when the same panel of
protein substrates was tested with the rat testis enzyme (Fig. 4B), labeling also occurred in all cases but was
much weaker with the precursor proteins from CHO cells than with the
human or the yeast precursors. Table 2lists the kinetic
constants of the yeast enzyme for the three substrates, spermidine,
NAD, and eIF-5A precursor, obtained using three different eIF-5A
precursors. The kinetic parameters for spermidine and NAD do not appear
to vary significantly whether the protein substrate used was human or
either of the yeast proteins. When the variable substrate was eIF-5A
precursor, the K
and V values
for the two yeast proteins were found to be similar, in accordance with
the results of Fig. 4A. Upon comparison of these
kinetic parameters with those of the rat enzyme(5) , the most
striking difference observed is the
20-fold higher value of K
for NAD with the yeast enzyme. In the
absence of the protein substrates, the yeast recombinant enzyme
catalyzes the NAD-dependent hydrolysis of spermidine to
1,3-diaminopropane and
-pyrroline, as has been shown
to occur with the rat testis enzyme(5) .
0.2 µg) or the rat enzyme (B,
0.2 µg) and the substrate proteins (
2 µg) from three
species, yeast, human, and CHO cells, were used in the assay. One
portion (15 µl) of the reaction mixture (total of 20 µl) was
used for SDS-PAGE, and the other portion (5 µl) was used for
determination of [
H]deoxyhypusine formed. The
amounts of radioactivity of [
H]deoxyhypusine in
the samples were: panelA, lane2,
44,922 cpm; lane3, 52,440 cpm; lane4, 49,248 cpm; lane5, 22,068 cpm; lane6, 45,966 cpm; panelB, lane2, 71,100 cpm; lane3, 86,745
cpm; lane4, 92,175 cpm; lane5,
9,198 cpm; lane6, 8,718 cpm. Abbreviations used are: STD, standard; h5A, human eIF-5A precursor,
ec-eIF-5A; y5Aa, yeast ec-eIF-5Aa; y5Ab, yeast
ec-eIF-5Ab; CHOPI and CHOPII, eIF-5A precursors
isolated from DL-
-difluoromethylornithine (DFMO)-treated
CHO cells (19) .
Structural Properties
Values for the molecular
mass of the recombinant polypeptide determined by SDS-PAGE (43 kDa) and
by matrix-assisted laser-desorption mass spectrometry (42,998 ±
100 Da) are in close agreement with a value of 42,891.2 Da calculated
for the coding region of the YHR068w cDNA. The enzyme migrated
as a single band upon electrophoresis under non-denaturing conditions (Fig. 3B) and eluted from an exclusion chromatography
column as a single symmetrical peak corresponding to a mass size of 165
kDa (Fig. 3A). Analysis of equilibrium sedimentation
data, using a calculated partial specific volume of 0.718
cm/g, gave a weight average molecular mass of 172,000
± 4,300 Da. A value of 3.3
10
M
was obtained for K
for formation of tetramer and a change
of standard free energy
G
=
-22.2 ± 0.1 kcal mol
. The association
appears to be strongly cooperative, since no molecular species other
than monomer and tetramer could be observed. The mean dissociation
constant per monomer may be calculated to be 3.12
10
M. These results indicate that the
tetrameric form is the dominant species under physiologic conditions.
)The
deduced amino acid sequences of the human and the yeast enzymes show
58% identity. Thus, conservation of deoxyhypusine synthase, as well as
of its substrate protein, may contribute to the maintenance of the
specificity of hypusine synthesis and of the functionality of eIF-5A.
-D-galactopyranoside; CHO, Chinese
hamster ovary.
We are indebted to Marc S. Lewis, NCRR, NIH, for the
ultracentrifugation analysis, to Henry M. Fales and Edward A.
Sokoloski, NHLBI, NIH, for the mass spectrometry, and to Yong Sok Kim,
NHLBI, NIH, for insightful comments and discussions throughout the
course of this work.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.