ABSTRACT
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 Mrof 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.
Restriction endonucleases, modification enzymes and 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).
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 primer 5'-CCGGATCCCATATGGTGCTAGCAGAGCTGT-3' (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'-TCAGGTGATTTGAGATAGTTTTTATGG-3'.
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-[beta]-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).
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.
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).
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 [beta]-barrel domain probably involved in tRNAAsn anticodon recognition and a catalytic domain containing the three Class II specific motifs (Fig. 1).
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).
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).
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).
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 electrophoresis, 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 form.
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 tRNA.
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 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.
Nucleic Acids Research
Pages
Introduction
Materials And Methods
Cloning of hsAsnRSc cDNA
Expression of hsAsnRS as a bacterial fusion protein and purification
Aminoacylation assay and kinetic parameters
Neutralization assay
Detection of the recombinant hsAsnRSc by western blot using autoimmune serum (anti-KS)
Results
Cloning and sequencing of the human AsnRS cDNA (EMBL database: Aj000334)
Bacterial expression and purification of the recombinant enzyme
Aminoacylation activity of the recombinant human AsnRS using tRNA from different origins
Neutralization of AsnRS activity by a human autoimmune serum
Immunoreactivity of the anti-KS serum in a western blot experiment
Discussion
Acknowledgements
References
REFERENCES
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