Isolation and characterization of cDNA encoding the 80-kDa subunit protein of the human autoantigen Ku (p70/p80) recognized by autoantibodies from patients with scleroderma-polymyositis overlap syndrome (DNA-binding protein/molecular cloning/autoimmune disease/"leucine zipper")

Anti-Ku (p7O/p80) autoantibodies in patients with scleroderma-polymyositis overlap syndrome recognize a 70-kDa/80-kDa protein heterodimer which binds to terminal regions of double-stranded DNA. In the present study, we isolated full-length cDNAs that encode the 80-kDa Ku subunit. Initial screening of a human spleen cDNA library with anti-Ku antibodies yielded a cDNA of 1.0 kilobase (kb) (termed K71) encoding a portion of the 80-kDa Ku polypeptide (identification based on immunological criteria). In RNA blots, this cDNA hybridized with two mRNAs of 3.4 and 2.6 kb. In rescreening of a cDNA library constructed from simian virus 40-trans- formed human fibroblast mRNA with the K71 cDNA as a hybridization probe, three positive clones were isolated, and that bearing the longest insert (termed Ku8O-6) was selected for further characterization. In vitro transcription and translation experiments produced an immunoprecipitable polypeptide which comigrated with the 80-kDa Ku subunit. The Ku8O-6 cDNA proved to be 3304 nucleotides in length, with an addi- tional poly(A) tail, closely approximating the size of the larger mRNA. It contains a single long open reading frame encoding 732 amino acids (Mr = 82,713).- The putative polypeptide has a high content of acidic amino acids and a region with periodic repeat of leucine in every seventh position which may form the "leucine zipper" structure. In genomic DNA blots, probes derived from the opposite ends of cDNA Ku8O-6 hybridized with several nonoverlapping restriction fragments from human leukocyte DNA, indicating that the gene encoding the 80-kDa Ku polypeptide is divided into several exons by intervening sequences. Various autoantibodies to cellular constituents are produced in patients with autoimmune diseases. Autoantibodies to the