A motif in human histidyl-tRNA synthetase which is shared among several aminoacyl-tRNA synthetases is a coiled-coil that is essential for enzymatic activity and contains the major autoantigenic epitope.

N Raben,P Plotz,R Leff,J Dohlman,P Mcphie,V Sridhar,C Hyde,R Nichols

doi:10.1016/s0021-9258(19)51078-x

Abstract

In myositis, disease-specific autoantibodies may be directed against an aminoacyl-tRNA synthetase, usually histidyl-tRNA synthetase. To explore the basis for this phenomenon, we have made recombinant histidyl-tRNA synthetase in the baculovirus system. It was enzymatically active and recognized by human autoantibodies. A truncated protein lacking the first 60 amino acids was inactive as an antigen and as an enzyme. This region is within the first two exons, is predicted to have a coiled-coil configuration, and is found in some other synthetases but not in Escherichia coli or yeast histidyl-tRNA synthetase. Circular dichroism showed that the peptides from this region (amino acids 1-60 and 1-47) have the predicted high alpha-helical content, but smaller fragments (1-30, 14-45, and 31-60) do not. The peptides with a high alpha-helical content could inhibit autoantibodies almost completely, whereas the smaller peptides were unable to do so. The amino acid sequence of this coiled-coil domain in human histidyl-tRNA synthetase resembles the sequence of the extended this coiled-coil arm near the NH2 terminus of bacterial seryl-tRNA synthetase as well as similar regions in some eukaryotic aminoacyl-tRNA synthetases, raising the possibility that this domain serves a similar tRNA-stabilizing role and has been preserved from a common ancestor.

Highlights

In myositis, disease-specific autoantibodmieasy bedi- bodies precede the clinical illness, and the immune response rected against an aminoacyl-tRNA synthetase, usually bears the hallmarkosf a typical secondary immune response(8, histidyl-tRNA synthetase.To explore the basis for this 9), but why these particular proteins are selected as targets phenomenon, we have made recombinant histidyl-tRNAremains obscure
We present here studies of recombithe NH, terminus of bacterial seryl-tRNA synthetase as nant Histidyl-tRNA synthetase (HRS)-hu and describe both the structure and well as similar regions in some eukaryotic aminoacyl- the immunologic role of its NH,terminal domain
The abbreviations used are: HRS, histidyl-tRNA synthetase;HRS- HRS-hu was obtained as a product of PCR amplificationof a full-length hu, recombinant whole H R S (rHRS)-hu, rHRS-hu-M,human histidyl-tRNA synthetase,recombi- cDNA synthesized by reverse transcription from total RNA isolated nant HRS-hu, and mutated rHRS-hu; PCR, polymerase chain reaction; from the Hep G2 cell line [21].A truncated fragment lacking the se

Summary

MATERIALS AND METHODS

The sample was chromatographed tion, the antibodies in the sera from nine myositis patients were first on a 1.6 cm x 10 cm hydroxylapatite column (W,Prissm ceramic, 20 titrated against the antigen (cytoplasm or purified rHRS-hu) at 5 ngl pm)using a linear gradient with 500 m~ KH,PO,, pH 7.0, containing 1 well to determine the optimal concentrations of the reagents. Fractions were pre-incubated with the peptides (0.025, 0.25,0.5,or 1 pg/well) or were analyzed using SDS-PAGE, and fractions containing rHRS-hu rHRS-hu or rHRS-hu-M (0.02-0.2 pg/well) for min at room temperwere pooled and buffer exchanged into 25 mM bis-Tris propaneiNaOH, ature and added to the ELISA plates coated with rHRS-hu. The sample was applied to a Pharmacia Mono Q 10/10 HR column and eluted with a linear

RESULTS

DISCUSSION

54 NKLKNLCSKTIGEKMKKKEAVGDDESVPENVL

Methods

Findings