HUMAN CYTOSOLIC ASPARAGINYL-TRANSFER-RNA SYNTHETASE - CDNA SEQUENCE, FUNCTIONAL EXPRESSION IN ESCHERICHIA-COLI AND CHARACTERIZATION AS HUMAN AUTOANTIGEN

The cDNA for human cytosolic asparaginyl-tRNA synthetase (hsAsnRSc) ha s 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 pr otein sequence has 58 and 53% identity with the homologous enzymes fro m Brugia malayi and Saccharomyces cerevisiae respectively. The human e nzyme was expressed in Escherichia coli as a fusion protein with an N- terminal 4 kDa calmodulin-binding peptide. A bacterial extract contain ing the fusion protein catalyzed the aminoacylation reaction of S.cere visiae tRNA with [C-14]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 aminoacyl ated unfractionated calf liver and yeast tRNA but not E.coli tRNA, sug gesting that the recombinant protein is the cytosolic AsnRS. Several h uman antisynthetase sera were tested for their ability to neutralize h sAsnRSc activity. A human autoimmune serum (anti-KS) neutralized hsAsn RSc activity and this reaction was confirmed by western blot analysis. The human asparaginyl-tRNA synthetase appears to be like the alanyl-a nd histidyl-tRNA synthetases another example of a human Class II amino acyl-tRNA synthetase involved in autoimmune reactions.