1998 Oxford University Press
Nucleic Acids Research, 1998, Vol. 26, No. 2
Human cytosolic asparaginyl-tRNA synthetase: cDNA
sequence, functional expression in Escherichia coli and
characterization as human autoantigen
Mélanie Beaulande, Nicolas Tarbouriech and Michael Härtlein*
EMBL Grenoble Outstation, B.P. 156, F38042 Grenoble Cedex, France
Received September 23, 1997; Revised and Accepted November 21, 1997DDBJ/EMBL/GenBank accession no. AJ000334
The cDNA for human cytosolic asparaginyl-tRNA
synthetase (hsAsnRSc) has been cloned and
sequenced. The 1874 bp cDNA contains an open
reading frame encoding 548 amino acids with a
predicted Mr of 62 938. The protein sequence has 58 and
53% identity with the homologous enzymes from Brugia
malayi and Saccharomyces cerevisiae respectively. The
human enzyme was expressed in Escherichia coli as a
fusion protein with an N-terminal 4 kDa calmodulin-
binding peptide. A bacterial extract containing the
fusion protein catalyzed the aminoacylation reaction of
S.cerevisiae tRNA with [14C]asparagine at a 20-fold
efficiency level above the control value confirming that
this cDNA encodes a human AsnRS. The affinity
chromatography purified fusion protein efficiently
aminoacylated unfractionated calf liver and yeast tRNA
but not E.coli tRNA, suggesting that the recombinant
protein is the cytosolic AsnRS. Several human anti-
synthetase sera were tested for their ability to neutralize
hsAsnRSc activity. A human autoimmune serum (anti-
KS) neutralized hsAsnRSc activity and this reaction
was confirmed by western blot analysis. The human
asparaginyl-tRNA synthetase appears to be like the
alanyl- and histidyl-tRNA synthetases another example
of a human Class II aminoacyl-tRNA synthetase involved
in autoimmune reactions.
Aminoacyl-tRNA synthetases (aaRS) are enzymes involved in
protein biosynthesis catalyzing the specific attachment of amino
acids to their cognate tRNAs. Two classes of synthetases have
been defined, each of 10 members, based on their primary and
tertiary structures (1,2). Class II enzymes have three consensus
sequence motifs; motif 1 contributes to the dimer interface,
whereas motifs 2 and 3 are constituents of the catalytic site.
Sub-classification can be made of the class II enzymes based on
more extensive sequence and structural similarities (3). In higher
eukaryotes, nine aaRS of different specificities (not including
AsnRS) are associated within a multi-enzyme complex (4).
Asparaginyl-tRNA synthetase (AsnRS) is classified as a sub-class
IIb enzyme together with the aspartyl- and lysyl-enzymes on the
basis of similarities in their N-terminal extensions and the catalytic
domains (3,5–7). The three-dimensional structure of an AsnRS
determined in this laboratory (Thermus thermophilus AsnRS; 8)
further illustrates the strong structural homology between the three
class IIb synthetases.
Autoantibodies are found in many patients with polymyositis
or dermatomyositis. Some of these patients have antibodies raised
against aaRS, of which anti-Jo-1, directed at histidyl-tRNA
synthetase (HisRS) is by far the most common (9).
Below we describe the cDNA sequence of human cytosolic
AsnRS, the bacterial expression of the recombinant enzyme and
its activity assays with different sources of tRNA. Futhermore, we
report its reactivity with a human autoimmune serum. The
implication of the human cytoplasmic AsnRS in an autoimmune
disorder is an interesting property of this enzyme.
MATERIALS AND METHODS
unfractionated tRNAs were purchased from Boehringer Mannheim.
Oligonucleotides were supplied by Genosys. Autoimmune sera
(anti-HisRS, anti-AlaRS, anti-KS) were kindly provided by
Dr I.Targoff (Oklahoma Research Foundation) and Dr M.Hirakata
(University of Tokyo School of Medicine).
endonucleases, modification enzymes and
Cloning of hsAsnRSc cDNA
Molecular cloning methods were used according to Sambrook et
al. (10). Human Expressed Sequence Tag (EST) sequences
coding for peptides showing strong sequence similarities with
Brugia malayi AsnRS were aligned. Missing 5′ and 3′ regions
were amplified by PCR methods on human liver 5′ RACE-Ready
cDNA from Clontech. Thirty cycles of amplification were carried
out (20 s denaturation at 94?C, 30 s annealing at 60–68?C and
5 min elongation at 68?C). The complete cDNA was amplified
using the 5′ RACE-Ready cDNA with the oligonucleotide
(restriction sites are in bold and modified nucleotides are underlined)
creating a BamHI (and NdeI) restriction site for cloning the AsnRS
cDNA fragment into the pCal-n expression vector (Clontech)
and the oligonucleotide 5′-TCAGGTGATTTGAGATAGTTTTT-
*To whom correspondence should be addressed. Tel: +33 476 20 72 79; Fax: +33 476 20 71 99; Email: email@example.com
Nucleic Acids Research, 1998, Vol. 26, No. 2
Expression of hsAsnRS as a bacterial fusion protein and
The hsAsnRSc coding region was inserted into pCal-n vector (11)
as a BamHI–EcoRI fragment and transformed into the Escherichia
coli strain BL21 (DE3). Cells were grown in LB at 37?C to an A600
of 0.6, isopropyl-1thio-β-D-galactoside was added to a final
concentration of 0.2 mM and incubation at 23?C continued for a
further 3 h. Cells were lysed by lysozyme and sodium deoxycholate
treatment (12). The bacterial extract was applied to a 2.5 ml
calmodulin resin (13).
Aminoacylation assay and kinetic parameters
The aminoacylation reaction assay was as previously described
(14) in the presence of 1.12 µM tRNAAsn of unfractionated tRNA
from E.coli MRE600, Saccharomyces cerevisiae or calf liver ; the
determination of asparagine acceptance activity in unfractionated
tRNA from E.coli was performed with an E.coli protein extract,
that in unfractionated S.cerevisiae and calf liver tRNA with
hsAsnRSc fusion protein. The concentration of recombinant
human AsnRS was 33 nM.
AsnRS (66 nM) was preincubated for 10 min on ice with the
various sera (1:10 dilution of the sera donated by Drs Targoff and
Hirakata). After preincubation the aminoacylation activity was
determined using calf liver tRNA. In the aminoacylation reaction
the sera are present in a 1:100 dilution.
Detection of the recombinant hsAsnRSc by western blot
using autoimmune serum (anti-KS)
Protein samples were separated electrophoretically on a 12%
SDS–polyacrylamide gel and transferred to a Immobilon-P
membrane for western blot analysis (15). The immunological
reactivity of the recombinant hsAsnRSc was tested against 5.0 µl
human anti-KS serum. [35S]protein A (16.7 mM, 600 Ci/mmol;
Amersham) was used to detect specific AsnRS–antibody
interactions by autoradiography (Fig. 2) after 16 h exposure to
Biomax film (Kodak).
Cloning and sequencing of the human AsnRS cDNA (EMBL
Human EST sequences coding for peptides which show strong
sequence similarities with B.malayi AsnRS were aligned to a
1302 bp fragment. The assembled cDNA sequence comprises
1874 bp with a large predicted open reading frame of 1644 bp.
This encodes a protein of 548 amino acids with a predicted Mr of
62 938. Sequence alignment of several bacterial and eukaryotic
AsnRSs indicates that the human enzyme is composed of three
characteristic domains; an N-terminal extension, typical for
eukaryotic AsnRS, followed by a putative β-barrel domain
probably involved in tRNAAsn anticodon recognition and a
catalytic domain containing the three Class II specific motifs
Bacterial expression and purification of the recombinant
The recombinant protein comprises an N-terminal 4 kDa
Calmodulin Binding Peptide (CBP) fusion tag coupled to the
AsnRS. Figure 2 shows the SDS–PAGE analysis of the AsnRS
fusion protein in an unfractionated bacterial extract (lane 3) and
its purified form (lane 4). The apparent molecular weight of the
fusion protein is in agreement with the predicted molecular
weight of AsnRS (63 + 4 kDa CBP).
Unfractionated bacterial extracts were assayed for their ability to
catalyze the aminoacylation of S.cerevisiae tRNA with
[14C]asparagine; these extracts had 20-fold greater aminoacylation
activity with S.cerevisiae tRNA relative to E.coli extracts carrying
only the pCal-n vector.
Bacterial extracts were loaded on a calmodulin column in the
presence of calcium. EGTA eluted fractions were collected and
analyzed by western blot methods for the presence of E.coli
AsnRS contamination using a rabbit anti-E.coli AsnRS serum
(data not shown).
Aminoacylation activity of the recombinant human AsnRS
using tRNA from different origins
The purified AsnRS fusion protein was tested for its enzymatic
activity with tRNA substrates of different origins i.e. E.coli,
S.cerevisiae and calf liver at the same relative concentration of
tRNAAsn. Figure 3 shows that calf liver and S.cerevisiae tRNAs
are both efficient substrates for the human enzyme. For both
tRNAs similar plateau values are reached although the initial rate
is somewhat higher for the calf liver tRNA (0.15 pmol/s–1
compared to 0.09 pmol/s–1 for S.cerevisiae tRNA).
Neutralization of AsnRS activity by a human autoimmune
The AsnRS fusion protein was preincubated with the different
autoimmune sera (anti-KS, anti-AlaRS and anti-HisRS ) and two
control sera. After preincubation, residual aminoacylation activity
was determined. Only the anti-KS serum neutralized the human
AsnRS activity significantly with an inhibition of 98%. The other
anti-synthetase sera (anti-HisRS and anti-AlaRS) did not neutralize
significantly the enzyme activity (<4% of inhibition).
Immunoreactivity of the anti-KS serum in a western blot
Since only the anti-KS serum produced significant inhibition of
AsnRS activity the interaction of this serum with recombinant
protein was examined by western blot analysis. Samples of
bacterial extract from the overproducing strain containing
recombinant synthetase and a control strain containing only the
pCal-n vector together with purified human AsnRS fusion protein
were loaded on an SDS–polyacrylamide gel. After electro-
phoresis, the proteins were transferred to a nylon membrane and
incubated with human anti-KS serum. Antigen–antibody
interactions were detected using 35S-labeled protein A. Figure 2
shows that the human anti-KS serum specifically interacts with
the human AsnRS both in the bacterial extract and in purified
Nucleic Acids Research, 1994, Vol. 22, No. 1
Nucleic Acids Research, 1998, Vol. 26, No. 2
Figure 1. Multiple alignment between prokaryotic and eukaryotic AsnRS sequences. The program PILEUP was used (GCG package, University of Wisconsin). The
origins are (accession number in the SwissProt or EMBL data banks are indicated in parenthesis): Homo sapiens, hsAsnRS (AJ000334); Brugia malayi, bmAsnRS
(P10723); Saccharomyces cerevisiae scAsnRS (P38707); Thermus thermophilus, ttAsnRS (X91009). The position where the residues are strictly conserved in this
alignment are in bold type. The Class II specific motifs are indicated by #. The N-terminal extensions characteristic for eukaryotic AsnRS sequences are boxed. Dashed
lines indicate the putative β-barrel domain most probably involved in tRNA anticodon recognition.
We have isolated the cDNA coding for the complete human
AsnRS. This provides the first example of a mammalian AsnRS
sequence. The sequence exhibits a high degree of similarity with
the two other known eukaryotic AsnRSs: a 58% amino acid
identity with the AsnRS from B.malayi and a 53% identity with
that from S.cerevisiae. Based on the following observations we
conclude that the sequence we have determined is that of human
cytosolic AsnRS: (i) the absence of a mitochondrial import signal,
(ii) strong sequence similarities to the cytosolic AsnRSs from
B.malayi and S.cerevisiae and weaker similarities with bacterial
enzymes, (iii) estimated molecular weight and calculated
isoeletric point is typical for a cytosolic AsnRS, (iv) calf liver and
S.cerevisiae tRNA are significantly better substrates than E.coli
Despite a similar degree of overall sequence identity of human
tRNA compared to tRNA from E.coli (62%) or from S.cerevisiae
(65%), E.coli tRNA is poorly aminoacylated by the hsAsnRSc
fusion protein in contrast to its S.cerevisiae counterpart. This
could be due to one base insertion into the D-loop of the
eukaryotic tRNAAsn at position 21 (16).
Some eukaryotic synthetases are involved in pathological
conditions (9). Patients with systemic autoimmune diseases make
specific autoantibodies that are directed against self structures.
According to one hypothesis, these autoantibodies arise through an
immune response to foreign antigens such as infectious agents that
share, by molecular mimicry, common structures with host proteins.
Autoantibodies are found in most patients with polymyositis or
dermatomyositis and 35–40% of these patients have myositis-
specific antibodies. 25–30% of these patients have antibodies raised
against aminoacyl-tRNA synthetases, of which anti-Jo-1, directed at
histidyl-tRNA synthetase (HisRS) is by far the most common (9).
Of the several autoimmune sera tested for their capacity to
neutralize the hsAsnRSc activity, only the anti-KS auto immune
Nucleic Acids Research, 1998, Vol. 26, No. 2 Download full-text
Figure 2. Expression in E.coli, purification and immunological reactivity with
the anti-KS serum of the recombinant hsAsnRSc. (A) Coomassie-brilliant blue
stained SDS gel (12% polyacrylamide). Lane 1, molecular mass marker; lane
2, bacterial control extract; lane 3, bacterial extract containing the recombinant
CBP tagged protein; lane 4, purified hsAsnRSc. (B) Autoradiography of the
western blot performed with the SDS gel showing the immunoreactivity of
hsAsnRSc with anti-KS serum.
serum isolated by Dr M.Hirakata was able to neutralize the activity
of the recombinant hsAsnRSc. The other anti-synthetase sera
(anti-AlaRS and anti-HisRS) did not show any significant inhibition.
Besides its neutralizing activity, the anti-KS serum was also able to
recognize the recombinant AsnRS fusion protein on an immunoblot.
It has been shown that anti-Jo-1 antibodies recognize multiple
conformation-dependent and independent epitopes on human HisRS
and that auto-epitopes vary among different myositis patients (17).
Furthermore, it has been demonstrated that the substrates ATP and
histidine act as competitive inhibitors for the formation of the
synthetase-anti-Jo-1 antibody complex, whereas the tRNA acts in a
non-competitive way (18). The human AsnRS has yet to be
characterized for this complex formation.
We would like to thank Dr Reuben Leberman and Darcy Birse for
their critical review of the manuscript, Drs Hirakata and Targoff
for the generous gifts of human sera.
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Figure 3. Asparaginyl-tRNA synthetase activity of the recombinant human
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enzyme (33 nM) of [14C]asparagine (pmol) into unfractionated tRNA from
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