Molecular analysis of a major antigenic region of the 240-kD protein of Mi-2 autoantigen.
Anti-Mi-2 autoantibody is strongly associated with dermatomyositis and found in sera of 20% of patients. Mi-2 antigen contains at least eight components and previous evidence suggested that the 240-kD protein was the antigenic component for at least some sera. In this study, anti-M-2 patient sera were used to screen human thymocyte and HeLa cell lambda gt11 expression libraries, and two clones from each had plaques specifically reactive with anti-Mi-2 sera. Studies with affinity-purified antibody supported the identification of the clones. All of 44 anti-Mi-2 sera reacted with the plaques, but none of 44 control sera reacted significantly. The cDNAs were identical, and full sequencing of one revealed an open reading frame spanning a 1,054-bp insert. Rescreening the library with the cDNA yielded a 1,589-bp cDNA that continued the open reading frame. The Mi-2 cDNA hybridized to a single 7.5-8.0 kb mRNA of HeLa cells, by Northern blot. Rabbit antiserum directed at a portion of the cDNA product reacted with HeLa 240-kD Mi-2 protein. The sequence was notable for four potential zinc-fingers and several charged regions. The protein encoded by the cDNA produced in vitro reacted with only one of five of the Mi-2 sera. These findings indicate that the Mi-2 240 kD is a novel protein that is antigenic for all Mi-2 sera, and strongly suggests that a major common epitope is conformational in nature.
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Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, USA.
Nilasena
D S
DS
O'Brien
C A
CA
Frank
M B
MB
Targoff
I N
IN
D000251:Adenosine Triphosphatases; D000595:Amino Acid Sequence; D000818:Animals; D001323:Autoantibodies / Q000276:immunology; D001324:Autoantigens / Q000235:genetics*; D001483:Base Sequence; D004265:DNA Helicases; D018076:DNA, Complementary / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D003882:Dermatomyositis / Q000276:immunology*; D000939:Epitopes; D006367:HeLa Cells; D006801:Humans; D008297:Male; D056245:Mi-2 Nucleosome Remodeling and Deacetylase Complex; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011817:Rabbits; D016335:Zinc Fingers
D001323:Autoantibodies; D001324:Autoantigens; C115323:CHD4 protein, human; D018076:DNA, Complementary; D000939:Epitopes; D056245:Mi-2 Nucleosome Remodeling and Deacetylase Complex; D000251:Adenosine Triphosphatases; D004265:DNA Helicases