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. 2014 Jul 11;289(28):19269-75.
doi: 10.1074/jbc.C114.571026. Epub 2014 Jun 4.

Secreted histidyl-tRNA synthetase splice variants elaborate major epitopes for autoantibodies in inflammatory myositis

Jie J Zhou  1 , Feng Wang  1 , Zhiwen Xu  1 , Wing-Sze Lo  1 , Ching-Fun Lau  1 , Kyle P Chiang  2 , Leslie A Nangle  2 , Melissa A Ashlock  2 , John D Mendlein  2 , Xiang-Lei Yang  3 , Mingjie Zhang  4 , Paul Schimmel  5
Affiliations

Secreted histidyl-tRNA synthetase splice variants elaborate major epitopes for autoantibodies in inflammatory myositis

Jie J Zhou et al. J Biol Chem. .

Abstract

Inflammatory and debilitating myositis and interstitial lung disease are commonly associated with autoantibodies (anti-Jo-1 antibodies) to cytoplasmic histidyl-tRNA synthetase (HisRS). Anti-Jo-1 antibodies from different disease-afflicted patients react mostly with spatially separated epitopes in the three-dimensional structure of human HisRS. We noted that two HisRS splice variants (SVs) include these spatially separated regions, but each SV lacks the HisRS catalytic domain. Despite the large deletions, the two SVs cross-react with a substantial population of anti-Jo-l antibodies from myositis patients. Moreover, expression of at least one of the SVs is up-regulated in dermatomyositis patients, and cell-based experiments show that both SVs and HisRS can be secreted. We suggest that, in patients with inflammatory myositis, anti-Jo-1 antibodies may have extracellular activity.

Keywords: Aminoacyl-tRNA Synthetase; Anti-Jo-1 Autoantibody; Autoimmune Disease; Dermatomyositis; Epitope Mapping; Immunology; Myositis; Secretion.

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Figures

FIGURE 1.
FIGURE 1.
Identification of transcript and protein for HisRSWHEP SV and up-regulation of HisRSWHEP transcript in muscle biopsies of DM patients. A, PCR was used to identify the mRNA encoding HisRSWHEP in human skeletal muscle. Locations of the primers used for PCR are indicated in the schematic. E, exon. B, electrophoretic analysis of the PCR. The upper fragment (red arrow) was amplified from the mRNA for HisRSWHEP, which is 122 nucleotides (nt) longer than the lower fragment amplified from the mRNA for HARS (black arrow). M, mass marker. C, schematic drawing of the intron 2 (I2) insertion in the mRNA for HisRSWHEP and location of the inserted nucleotides in HARS. Notably, the inserted sequence is flanked by canonical splicing signals (preceded by AG and followed by GT) and itself ends with AG. HisRSWHEP is encoded by 183 nucleotides and is translated into 60 aa, which encompass the WHEP domain of HisRS. CDS, coding sequence. D, schematic illustrations of HisRS and HisRSWHEP and their structures (Protein Data Bank code 4G84 for HisRS and code 1X59 for HisRSWHEP). One monomer of HisRS is shown in color (ABD in green and CD in blue), whereas the other monomer is shown in gray. Notably, HisRSWHEP is composed of the N-terminal WHEP domain (shown in red) of human HARS. E, distribution of the transcripts of HisRS and HisRSWHEP in 13 human tissues. Locations of the qPCR primers are indicated in the schematic. The expression levels were normalized to that of the HKG RPL9. The median value was taken as 1.0. Notably, the HisRSWHEP transcript is significantly higher in human lung compare with other tissues and is 3-fold above the median. The expression of the transcript for HARS was normally distributed in the various tissues, with expression levels <3 times that of the median value. F, HisRSWHEP protein was detected in the TCL of monocytic THP-1 cells, but not in that of human skeletal muscle cells (HSkMC). Expected running positions of the proteins are indicated by arrows. G, the transcript for HisRSWHEP is up-regulated in DM muscle biopsies (1.0 ± 0.1 in control (Con) versus 2.7 ± 0.2 in DM). The transcript for HisRS is also up-regulated in the DM samples (1.0 ± 0.01 in control versus 2.1 ± 0.3 in DM). The MXA gene serves as a positive control (1.0 ± 0.06 in control versus 82.8 ± 1.1 in DM). Locations of the qPCR primers are indicated in the schematic. Data are shown as means ± S.D. ***, p < 0.0001.
FIGURE 2.
FIGURE 2.
Anti-Jo-1 patient serum reacts mainly with the N-terminal WHEP domain and C-terminal ABD of human HisRS, and recombinant HisRS SVs are secreted from HEK293T cells and C2C12 myoblasts. A, illustration of depletion ELISAs. Details are provided under “Experimental Procedures.” B, reactivity of anti-Jo-1 Ab-positive patient serum against different HisRS recombinant proteins. Notably, apart from HisRS, anti-Jo-1 Ab-positive patient serum reacted mostly with HisRSWHEP and HisRSΔCD, with a significantly higher reactivity than with the recombinant CD or ABD. C, the two most reactive domains are far apart on the three-dimensional structure of HisRS (Protein Data Bank code 4G84). The C-terminal ABD (shown in green) and N-terminal WHEP domain (shown in red) are highlighted in the structure of HisRS. The N-terminal WHEP domain is not resolved in the structure of HisRS, but is resolved in that of HisRSΔCD (Protein Data Bank code 2LW7). D, the structures of HisRSΔCD and HisRSWHEP show that the two HisRS SVs contain the major anti-Jo-1 epitopes. E and F, recombinant HisRSWHEP, HisRSΔCD, and HisRS proteins (with a C-terminal Myc tag) were transiently expressed in HEK293T cells (E), and recombinant HisRSΔCD and HisRS proteins were transiently expressed in C2C12 myoblasts (F). Expressed proteins were detected in the TCLs with anti-Myc mAb. LDHB in the TCLs served as a loading control. The media were immunoprecipitated by anti-Myc polyclonal Ab and detected with anti-Myc mAb. Notably, all HisRS proteins were detected in the media. The bar graphs show that the LDH activities of all samples were below the detection limit of the assay (indicated by the red line), suggesting little cell damage. The results shown are representative of three separately conducted experiments.
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