ABSTRACT
Autoantibodies directed against specific human aminoacyl-tRNA synthetases have been associated with a clinical picture including myositis, arthritis, interstitial lung disease and other features that has been referred to as the "anti-synthetase syndrome". Anti-asparaginyl-tRNA synthetase autoantibodies (anti-KS), the most recently described anti-synthetase autoantibodies, are directed against human cytosolic asparaginyl-tRNA synthetase and neutralize specifically its activity. Here we show that these antibodies recognize two epitopes on the human enzyme, an N-terminal epitope reactive in immunoblot experiments and a heat-labile epitope in the catalytic domain. In contrast to the well studied anti-Jo-1 autoantibodies anti-KS when bound to the synthetase increase the affinity of the synthetase for its tRNA substrate and prevent aminoacylation without interfering with the amino acid activation step.
Subject(s)
Amino Acyl-tRNA Synthetases/immunology , Amino Acyl-tRNA Synthetases/metabolism , Aspartate-tRNA Ligase , Autoantibodies/immunology , RNA, Transfer, Amino Acyl , RNA, Transfer, Asp/metabolism , Acylation/drug effects , Amino Acyl-tRNA Synthetases/antagonists & inhibitors , Amino Acyl-tRNA Synthetases/genetics , Antibody Specificity/immunology , Autoantibodies/pharmacology , Binding, Competitive , Catalytic Domain , Epitope Mapping , Epitopes/immunology , Humans , Immune Sera/immunology , Immune Sera/pharmacology , Molecular Sequence Data , Mutation , Neutralization Tests , RNA, Transfer, Asp/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
We have sequenced and expressed in Escherichia coli the gene encoding the seryl-tRNA synthetase from the pathogenic bacterium Staphylococcus aureus. The overexpressed and purified recombinant enzyme was able to aminoacylate unfractionated tRNA from E. coli. Its activity was not affected by antibodies raised against and inhibiting the E. coli seryl-tRNA synthetase.
Subject(s)
Genes, Bacterial , Serine-tRNA Ligase/genetics , Staphylococcus aureus/genetics , Amino Acid Sequence , Base Sequence , Escherichia coli/genetics , Immunoglobulin G/pharmacology , Molecular Sequence Data , Recombinant Proteins , Sequence Alignment , Sequence Analysis, DNA , Serine-tRNA Ligase/immunology , Staphylococcus aureus/enzymologyABSTRACT
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 M r 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.
