Abstract 2068: Sera with Autoantibodies to the MJ Antigen React with NXP2
Authors
Ira N. Targoff1, Edward P. Trieu2, Mauricio Levy-Neto2, Noreen Fertig3, Chester V. Oddis3.
Background
Of MSAs (myositis specific antibodies), some are primarily associated with dermatomyositis (DM), such as anti-Mi-2 and the recently identified anti-p155. Preliminary reports describe another DM-associated Ab, anti-MJ, seen in 17.5% of 80 juvenile DM (JDM) patients. It is distinct from anticaDM140 and anti-p155, and shows a 140 kd protein with no associated nucleic acid by immunoprecipitation (IP). Dr. Oddis and colleagues sought molecular characterization of the MJ antigen to facilitate antibody detection and study.
Methods
Sera showing a 140 kd protein by IP were confirmed MJ-positive by IP-blotting from K562 cells. Test sera were used for the IP step since some anti-MJ-positive sera did not react when used for the blotting step. Prototype serum MJ (MJ1) was used to confirm anti-MJ in sera MJ2 and MJ3. MJ2 was used to screen a K562 cDNA-expression library. MJ antigen was immunoaffinity-purified using MJ2 serum, and subjected to SDSPAGE. The excised 140 kd band was digested with trypsin, followed by HPLC and peptide analysis by mass spectrometry. Rabbit antiserum was developed by immunization with a 19-mer 4 component MAP peptide derived from the NXP-2 amino acid sequence.
Results
Two cDNA clones were identified, and plaques from each consistently reacted with MJ1 and three other anti-MJ sera, but not with 12 anti-MJ negative myositis sera or blot-negative anti-MJ-positive sera. The ~2.2 kb cDNA of both encoded a portion of NXP-2, a nuclear matrix protein, including most of the coiled coil region. The calculated NXP-2 MW is 107.1 kd, but anti-MJ antibody eluted from the plaque proteins of the clones reacted with the 140 kd protein in anti-MJ IP’s. Mascot analysis of the mass spectrometry of the 140 kd band gave a high significance score for protein KIAA0136 (human MORC family CW-type zinc finger 3), which was identical with NXP-2. By IP-blotting, rabbit anti-NXP-2-peptide blotted the 140kd band immunoprecipitated by MJ2 and MJ3 sera, while pre-immunized control serum and rabbit antiserum to a MAP peptide of p155 antigen were negative. Rabbit antiserum also reacted with IP’s of MJ1 prototype, and two other MJ-positive sera, but did not react by blot with anti-p155 IP’s (although p155 antigen can show a 140kd band), nor with IP’s of 15 JDM sera that were IP-blot negative for anti-MJ.
Conclusion
These studies demonstrate that anti-MJ sera react with NXP-2, which has RNA-binding and nuclear matrix binding domains. NXP-2 has recently been reported to be a SUMO target and to have a possible role in SUMO-mediated transcriptional repression, which is interesting because Abs to SUMO 1 activating enzyme have
been found in DM, and the p155 and Mi-2 antigens may also be involved in transcriptional regulation.
Editorial Comment
This new DM-associated Ab, anti-MJ, is distinct from anti-caDM140 and anti-p155, and shows a 140 kd protein with no associated nucleic acid by immunoprecipitation (IP). Anti-MJ sera react with NXP-2, a recently reported SUMO target. This finding may help to further our knowledge of pathogenesis and may prove to be helpful in establishing a phenotype-antibody correlation, as has occurred in many myositis syndromes.
