Characterization of the DNA-binding protein antigen Ku recognized by autoantibodies from patients with rheumatic disorders.

Abstract

We have characterized the biochemical nature of the KU protein, the antigen recognized by autoantibodies from certain patients with scleroderma-polymyositis overlap syndrome.From extracts of HeLa cells labeled with [32P]orthophosphate, anti-Ku antibodies precipitated a high molecular weight nucleic acid identified as DNA because of sensitivity to DNase I and resistance to RNase.From extracts of cells labeled with ["%I methionine, these antibodies precipitated two polypeptides of 70,000 and 80,000 Da.These proteins were purified using immunoaffinity column chromatography.In immunoblots most sera containing anti-Ku antibodies recognized both Ku proteins but one serum bound only to the 70,000-Da subunit.When nucleosomal segments of chromatin were used as antigen, anti-Ku antibodies precipitated dinucleosomes and larger forms of chromatin but not mononucleosomes.Thus, the Ku antigen is a novel DNA-binding protein that is at least partially exposed on nucleosomal segments of chromatin.Autoantibodies to a broad range of cellular constituents are found in patients with various rheumatic diseases (1).These antibodies have proved to be useful probes for studies of the structure and function of many different intracellular macromolecules for which animal antisera are difficult to prepare.For example, they provide a useful approach to defining the complex array of highly conserved proteins found within chromatin (2) or as components of ribonucleoprotein particles Recently, we described an autoantibody termed anti-Ku that was found in about half of Japanese patients with scleroderma-polymyositis overlap syndrome (9).This antibody was originally detected in immunodiffusion experiments using saline extracts of calf thymus as antigen.It was sensitive to trypsin but resistant to ribonuclease and deoxyribonuclease, and in gel filtration it migrated with an apparent molecular weight of 300,000.In the present study we have defined the physical nature of the Ku antigen and identified it as a DNAbinding protein. (3-8).Health Service (AM 32549 to J. A. H.