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INTRODUCTION |
Aminoacyl-tRNA synthetases
(ARSs)1 catalyze ligation of
their cognate amino acids to specific tRNAs. Although basic
architecture of the core domain is well conserved among ARSs, unique
peptide extensions have been found in the N- or C-terminal ends of
metazoan enzymes (1-3). Although these extensions have been thought to be involved in heterologous molecular interactions, their functional significance is not yet understood.
A macromolecular protein complex consisting of at least nine different
ARSs has been found in higher eukaryotes (1-3). This multi-ARS complex
also contains three nonsynthetase components, p18, p38, and p43 whose
functions are not clear (4-7). Among these nonsynthetase components,
p43 has been proposed to be a precursor of a tumor-specific cytokine,
endothelial monocyte-activating polypeptide II (EMAPII) based on over
80% sequence identity between the two proteins (6). EMAPII was
originally identified in the culture medium of murine fibrosarcoma
cells induced by methylcholanthrene A (8). It triggers an acute
inflammatory response (9, 10) and is involved in development-related
apoptosis (11).
The precursor for EMAPII (pro-EMAPII) is processed at the Asp residue
of ASTD/S sequence to release the C-terminal cytokine domain of 23 kDa
(11). Its C-terminal domain shares homology with the C-terminal parts
of methionyl-tRNA synthetases of prokaryotes, archaea and nematode, and
also a yeast protein, Arc1p/G4p, which interacts with methionyl- and
glutamyl-tRNA synthetases. The N-terminal domain of pro-EMAPII does not
show homology to any known proteins, and its function has not been understood.
EMAPII is expressed in a wide range of cell lines and normal tissues
(12) and its mRNA level is unchanged during apoptosis (11) although
its production is induced by apoptosis. The present work was designed
to address whether pro-EMAPII is identical to p43 and to understand its
function in the normal cell. The results showed that pro-EMAPII is
associated with the N-terminal extension of human arginyl-tRNA
synthetase (RRS), facilitating aminoacylation reaction.
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EXPERIMENTAL PROCEDURES |
Expression and Purification of Recombinant tRNA Synthetases and
Pro-EMAPII--
Human pro-EMAPII is genetically separated into the N-
and C-terminal domains by proteolytic cleavage at Asp147.
The cDNA encoding the full-length pro-EMAPII was isolated from pM3382 by NdeI and
XhoI digestion and then used as a template to separately amplify the DNA encoding its N- and C-terminal domains by PCR using the
primer pairs of R1EF/S1ENB and R1ECF/S1EB (Table I). The PCR products
were digested and cloned into pET28a using EcoRI and
SalI. The DNA encoding the 72-amino acid N-terminal
extension of human RRS was also amplified by PCR using the primers of
R1RNF and S1RNB (Table I) and cloned into the EcoRI and
SalI sites of pET28a. The resulting clones were transformed
into Escherichia coli strain BL21-DE3, and the inserted
genes were induced at 0.1 mM IPTG. The cells expressing the
recombinant proteins were harvested, resuspended in 20 mM
KH2PO4, 500 mM NaCl (pH 7.8), and 2 mM 2-mercaptoethanol, and then lysed by ultrasonication.
After centrifugation of the lysate at 25,000 × g, the
supernatants were recovered and the recombinant proteins containing a
6-histidine tag were isolated by nickel affinity chromatography
according to the instructions of the manufacturer (Invitrogen).
The cDNAs encoding the full-length and N-terminal 72-amino acid
truncated (
N72) human RRS proteins were also amplified by PCR with
the primer pairs of R1RNF/S1RB and R1RTN/S1RB, respectively (Table I).
The resulting PCR products were cloned into pGEX4T-1 using the
EcoRI and SalI sites to express as the
glutathione S-transferase (GST) fusion proteins. Protein
extracts were prepared as described above, and the GST fusion proteins
were purified by glutathione affinity chromatography. The GST tag was
then removed by thrombin cleavage and the RRS proteins were further
purified according to the protocol of the manufacturer (Amersham
Pharmacia Biotech). The plasmid pM109 containing the full-length human
lysyl-tRNA synthetase (KRS) fused to a 6-histidine tag (13) was used to express the protein. The His-KRS fusion protein was purified using nickel affinity chromatography (CLONTECH).
Preparation of Polyclonal Rabbit Antibody Specific to Human
Pro-EMAPII--
The purified recombinant human pro-EMAPII (500 µg)
was mixed with Freund's complete adjuvant at 1:1 volume ratio and then injected into two New Zealand White rabbits. Booster injections were
conducted three times at 1-week intervals using the same amount of the
protein mixed with the incomplete adjuvant at a 1:1 ratio. The rabbits
were sacrificed by cardiac puncture, and the antiserum was obtained.
The antibody was purified by protein A column chromatography.
Specificity and titer were determined by Western blotting.
Immunoprecipitation--
The purified N-terminal extension of
human RRS (10 µg) was mixed with each of the full-length, N- or
C-terminal domains of pro-EMAPII (10 µg each) at 4 °C overnight.
The polyclonal rabbit (20 µg) antibody raised against human
pro-EMAPII was then added to each of the mixtures and incubated on ice
for 4 h. The protein A-agarose suspension in 20 µl of 50 mM Tris-HCl (pH 7.5) and 25 mM NaCl was also
added, and incubation was continued at 4 °C for 5 h. The
mixture was centrifuged, and the agarose pellet was washed three times
with 400 µl of 50 mM Tris-HCl (pH 7.5) containing 25 mM NaCl and 0.01% Triton X-100. The agarose was treated
with 50 mM Tris-HCl (pH 6.8) containing 100 mM
dithiothreitol, 2% sodium dodecyl sulfate, 0.2% bromphenol blue, and
10% glycerol, and the solution was then boiled for 5 min to elute the
bound proteins. After centrifugation, the supernatant was loaded onto a
12% SDS-polyacrylamide gel. The proteins were separated by
electrophoresis and detected by Coomassie Blue staining.
Two-hybrid Assay--
Human proteins interacting with human
pro-EMAPII were screened by a yeast two-hybrid system (14). The
cDNA encoding the full-length pro-EMAPII was isolated by PCR using
the primers R1EF and S1EB (Table I) and
ligated next to the gene for LexA using the EcoRI and
SalI sites. The plasmid was transformed into yeast strain,
EGY48 (MAT, his3, trp1, ura3-52,
leu2::pLeu2-LexAop6/pSH 18-34
(LexAop-lacZ)). A human fetal brain cDNA library in
which the proteins are expressed as fusion proteins with the B42
transcriptional activator (CLONTECH) was used to
screen for proteins interacting with LexA-pro-EMAPII. The plasmids
containing human cDNAs were transformed into EGY48 expressing
LexA-pro-EMAPII. Interactions were detected by the induction of
reporter genes, LEU2 and LacZ, which resulted in
cell growth on leucine-depleted yeast synthetic media containing 2%
galactose and also formation of blue colonies on the yeast synthetic
media containing 0.2 mM X-gal, 2% galactose, and 2%
raffinose. The cDNAs encoding the N- and C-terminal domains of
pro-EMAPII were cleaved from the histidine tag construction using
EcoRI and SalI and religated into the pLexA
vector using the same sites.
Aminoacylation Assay--
Aminoacylation activity of the
purified human RRS was determined as described previously (15). The
reaction mixture contained 125 mM Tris acetate (pH 7.4),
0.2 mg/ml bovine serum albumin, 5 mM ATP, 4 mM
EDTA, 50 mM MgCl2 and 0.1 µCi/µl
[3H]arginine. Aminoacylation of human KRS was carried out
in a reaction mixture containing 50 mM HEPES (pH 7.5), 0.1 mg/ml BSA, 20 mM 2-mercaptoethanol, 4 mM ATP,
and 0.12 µCi/µl [3H]lysine. Human RRS and KRS were
pre-incubated on ice with the full-length, N- or C-terminal domain of
pro-EMAPII for 5 min and then added to their respective reaction
mixtures at a concentration of 0.14 nM. The reaction was
initiated by adding bovine liver total tRNA (0.34 µM).
Reaction samples were taken at 1-min intervals and spotted on filter
discs presoaked with 5% trichloroacetic acid. After 1 min, the filter
discs were added to ice-cold 5% trichloroacetic acid and washed three
times with fresh 5% trichloroacetic acid at 4 °C. The radioactivity
adsorbed to the filters was quantitated by liquid scintillation
counting. Reactions were also carried out at different concentrations
of pro-EMAPII for kinetic analysis.
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RESULTS |
Screening of Proteins Interacting with Human Pro-EMAPII--
To
investigate the function of pro-EMAPII and its relationship to p43, we
screened for protein(s) interacting with human pro-EMAPII using a yeast
two-hybrid system (16, 17). The 312-amino acid polypeptide of human
pro-EMAPII was fused to LexA (DNA-binding domain), and this fusion
protein was used as a bait. Human proteins fused to B42
(transcriptional activator) were screened, and interaction between the
two fusion proteins was detected by the induction of the reporter
genes, LEU2 and LacZ, in a yeast host strain
(14).
Approximately 300,000 cDNA clones of human fetal brain were
screened to identify proteins interacting with pro-EMAPII. The N-terminal 58-amino acid region of human RRS was selected as one of the
six positive clones interacting with pro-EMAPII (data not shown). In
the present work, we focused on the interaction between pro-EMAPII and
RRS. The N-terminal 72-amino acid peptide region is only found in human
(18) and hamster RRS proteins (19). We conducted deletion analysis to
determine the peptide regions of pro-EMAPII and RRS responsible for the
interaction. The peptides from Gln15 to Tyr53
and from Ser38 to Asn72 were able to interact
with pro-EMAPII, suggesting that the residues from Gln15 to
Ser38 are responsible for the interaction (Fig.
1). The N-terminal domain of pro-EMAPII
showed the interaction with RRS but its C-terminal cytokine domain did
not (Fig. 1).

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Fig. 1.
Interaction of pro-EMAPII with arginyl-tRNA
synthetase. The peptide regions of pro-EMAPII and RRS responsible
for the interaction were mapped by two-hybrid analysis. The positive
interactions were determined by cell growth on leucine-depleted yeast
synthetic media (14). Three peptide fragments in the N-terminal
extension of RRS (28) were tested for the interaction with pro-EMAPII.
Amino acids commonly present in the two interacting peptides are shown
in large letters. The peptides of Met1-Lys30
and Leu41-Asn67 were predicted to form
-helices (underlined). Human pro-EMAPII was divided into
the N- and C-terminal domains at Asp147. The N-terminal
domain (gray box) showed the interaction with the N-terminal
extension of RRS. RRS-N indicates the N-terminal 72-amino
acid region. F, N, and C represent the
full-length, N- and C-terminal domains of pro-EMAPII,
respectively.
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Interaction between the N-terminal extension of RRS and pro-EMAPII was
also tested by co-immunoprecipitation. The full-length, N- and
C-terminal domains of pro-EMAPII and the 72-amino acid N-terminal
extension of RRS were all expressed as His-tag fusion proteins and were
purified by nickel affinity chromatography (Fig. 2). The purified N-terminal peptide of
RRS was mixed with each of the isolated full-length, N- and C-terminal
pro-EMAPII in separate reactions. Polyclonal rabbit antibody raised
against pro-EMAPII was then added to the mixture and precipitated with
protein A-agarose. The proteins in the precipitate were dissolved and
separated on an SDS-polyacrylamide gel. The N-terminal peptide of RRS
was co-precipitated with the full-length or N-terminal domains of
pro-EMAPII but not with its C-terminal domain (Fig. 2). These results
further confirmed that the N-terminal domain of pro-EMAPII interacts
with the N-terminal extension of RRS as initially identified by the two
hybrid analysis (Fig. 1).

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Fig. 2.
Immunoprecipitation of pro-EMAPII and
RRS. The 72-amino acid N-terminal extension of RRS and the
full-length, N- and C-terminal domains of pro-EMAPII were expressed as
His-tag fusion proteins and purified by nickel affinity chromatography.
Each of the pro-EMAPII derivatives was mixed with the RRS peptide.
Subsequently, anti-pro-EMAPII antibody was added to each mixture, and
protein complexes were precipitated with protein A agarose. The
precipitated proteins were separated by SDS-polyacrylamide gel
electrophoresis and detected by Coomassie Blue staining. IgG (heavy
chain) is shown as marked, and protein sizes are indicated in
kDa.
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Pro-EMAPII Stimulates the Catalytic Activity of RRS--
The
functional significance of the interaction between RRS and pro-EMAPII
was further investigated. We tested whether the aminoacylation activity
of RRS was affected by interaction with pro-EMAPII. The full-length and
N-terminal 72-amino acid truncated (
N72) RRS were expressed as
GST-fusion proteins. The fused GST was removed by proteolytic cleavage,
and the purified full-length and N-terminal truncated RRS proteins were
used for the enzyme assay (Fig. 3).

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Fig. 3.
Purification of the full and N-terminal
truncated RRS. The full-length and 72-amino acid N-terminal
truncated RRS ( N72) were expressed as GST fusion proteins. The GST
tag was cleaved, and the two forms of RRS were purified. Marker sizes
are shown in kDa.
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The reaction catalyzed by tRNA synthetases proceeds in two steps. The
first step is activation of the amino acid by reaction with ATP, and
the second step involves transfer of the activated amino acid to the
cognate tRNAs. Aminoacylation activity of the full-length RRS was
enhanced approximately 2.5-fold in the presence of pro-EMAPII (Fig.
4, left bars). Since
arginine-dependent [32P]pyrophosphate-ATP
exchange assay showed that the adenylation step of RRS was not affected
by addition of pro-EMAPII (data not shown), the activity enhancement
probably results from the second step of the reaction. Activity
stimulation was not detected when the separated N- or C-terminal domain
of pro-EMAPII was added, indicating that the full-length pro-EMAPII is
necessary for the effect (Fig. 4, left bars). The truncated
RRS retained aminoacylation activity comparable with the wild-type
enzyme, suggesting that the N-terminal extension is not essential for
the enzyme activity (Fig. 4, middle bars). However, the
activity of this mutant was not increased by pro-EMAPII, indicating
that interaction of pro-EMAPII with the N-terminal extension of RRS is
essential for the stimulatory effect (Fig. 4, middle bars).
To investigate whether the stimulatory effect of pro-EMAPII is specific
for RRS, we employed human lysyl-tRNA synthetase (KRS) which does not
appear to interact with p43 (7). The aminoacylation activities of KRS
were measured in the absence and presence of pro-EMAPII. KRS activity
was not affected by the addition of pro-EMAPII, suggesting that
activity stimulation is specific to RRS (Fig. 4, right
bars).

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Fig. 4.
Stimulation of aminoacylation activity of RRS
by interaction with pro-EMAPII. Aminoacylation activities of the
full-length and N-terminal truncated RRSs were determined in the
absence and presence of the full-length, N- or C-terminal domains of
pro-EMAPII. The activity of the full-length KRS was also determined in
the absence and presence of the full-length pro-EMAPII. The activities
of the full-length RRS without pro-EMAPII were normalized to 100%, and
other activities were compared accordingly. The KRS activities with and
without pro-EMAPII were also compared. The experiments were repeated
three times. F, N, and C represent the
full-length, N- and C-terminal domains of pro-EMAPII,
respectively.
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Kinetic analyses on the aminoacylation of RRS were carried out at
different concentrations of pro-EMAPII to understand how pro-EMAPII
enhances the RRS activity. The activity enhancement reached a maximum
at a 2-fold molar excess of pro-EMAPII to RRS and further addition of
pro-EMAPII resulted in gradual decrease in the reaction rate (Fig.
5, left panel). A
Lineweaver-Burk plot of the reaction showed that the apparent
Km of RRS with respect to tRNA was reduced by the
addition of pro-EMAPII, whereas its kcat value
was not changed (Fig. 5, right panel). Excess pro-EMAPII probably binds to the tRNA substrate and lowers its effective concentration.

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Fig. 5.
Kinetic analysis of the RRS aminoacylation
reaction at different concentrations of pro-EMAPII. Aminoacylation
reactions of RRS were carried out at different concentrations of
pro-EMAPII. Left, the relative reaction rates of RRS were
plotted against the molar ratio of pro-EMAPII and RRS.
Right, the effect of pro-EMAPII on the reaction was analyzed
by a Lineweaver-Burk plot. Total bovine liver tRNA was added to the
reaction from 42 to 336 nM. The reactions were repeated
three times.
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DISCUSSION |
Pro-EMAPII (8) and p43 (6) have been independently isolated from
different organisms. In this work, we found that pro-EMAPII interacts
with RRS (Figs. 1 and 2). Previous cross-linking and genetic
experiments showed the linkage of p43 and RRS (7, 20). Thus, all of
these results support that p43 and pro-EMAPII are responsible for
similar functions within the cell.
The full-length pro-EMAPII was required for the activity enhancement of
RRS although the N-terminal domain of pro-EMAPII was sufficient for the
direct interaction with pro-EMAPII (Fig. 4). It was previously shown
that the C-terminal domain of pro-EMAPII contains tRNA binding activity
(6). The kinetic analyses showed that pro-EMAPII affected only the
apparent Km value to tRNA and not
kcat of the enzyme (Fig. 5). Probably, tRNA
recruited to the C-terminal domain of pro-EMAPII is delivered to the
active site of RRS. Although the activity of RRS was enhanced about
2.5-fold by pro-EMAPII under our experimental conditions, its effect
may be more significant in vivo because RRS present in the
multi-protein complex would have limited accessibility to tRNA
Mammalian RRS exists in two forms differing by the N-terminal extension
(15). The larger RRS containing the N-terminal extension is found in
the multi-synthetase complex, whereas the smaller RRS exists in a free
form (18, 19). The complex-associated larger RRS showed a 7-fold higher
Km for the tRNA substrate than the complex-free RRS,
whereas other kinetic properties were similar (15). Perhaps, the higher
Km value of the complex-associated RRS for the tRNA
substrate requires compensation by an active delivery of the tRNA
substrate. In the case of RRS, the delivery of tRNA appears to be
mediated by a trans-acting factor, pro-EMAPII. This
mechanism is also reminiscent of yeast Arc1p, which forms a complex
with methionyl-tRNA synthetase and stimulates its aminoacylation activity (21).
ARSs have developed different ways to modulate their catalytic
activities and the efficiency of protein synthesis. For example, the
N-terminal extension of rat aspartyl-tRNA synthetase facilitates the
release of aminoacylated tRNA to elongation factor (22, 23), and the
aminoacylation reaction of rabbit valyl-tRNA synthetase is enhanced by
interaction with elongation factor EF-1H (24). The N-terminal extension
of yeast glutaminyl-tRNA synthetase promotes specific recognition of
its cognate tRNA (25), and the C-terminal appendix of E. coli methionyl-tRNA synthetase helps to dock its cognate tRNA to
the active site (26). Whereas all of these functions are exerted by the
peptide extensions connected in cis to the catalytic domains
of ARSs, yeast Arc1p and mammalian pro-EMAPII are
trans-acting factors. These factors may have more functional flexibility than the cis-acting peptide extensions because
they can easily dissociate from the ARS and interact with cellular molecules for other physiological roles. Human tyrosyl-tRNA synthetase was recently shown to be secreted from apoptotic tumor cells and is cleaved to release the two distinct cytokine domains (27). Interestingly, the released C-terminal domain is homologous to EMAPII.
These results along with our data suggest that protein synthesis and
apoptosis are functionally coordinated via novel domains covalently or
noncovalently linked to ARSs.