ISOLATION OF CDNA CLONES ENCODING THE 80-KD SUBUNIT PROTEIN OF THE HUMAN AUTOANTIGEN KU (P70 P80) BY ANTISERA RAISED AGAINST CILIARY PROCESSES OF HUMAN EYE DONORS/

Purpose. To isolate and characterize human nonpigmented ocular ciliary epithelial cDNA clones by screening a cDNA library constructed from t he established human nonpigmented ciliary epithelial cell line (ODM-2) with sera raised against ciliary processes of human eye donors (cadav ers). Methods. An ODM-2 cDNA expression library in lambda Uni-ZAP-XR v ector was screened with sera of mouse anti-human ciliary processes. Th e cDNA inserts from plaque-purified clones were sequenced by the dideo xy-chain termination method with T3 and T7 polymerase. Sera were assay ed by Western blot analysis and indirect immunofluorescence and were t hen compared with sera, enriched in anti-Ku antibodies, collected from patients with scleroderma polymyositis overlap syndrome. Results. Imm unoscreening of 2.5 X 10(5) independent clones with sera induced exper imentally in total tissue homogenates of human ciliary processes led t o the isolation of four cDNA-positive clones. Three clones, designated A1, D1, and D3 and containing inserts measuring 1.4 kb to 1.8 kb, wer e found to be identical in DNA sequence analysis to that of the 80-kd subunit protein of the human autoantigen Ku (p70/p80). The fourth clon e, designated D2, failed to show any significant similarity with previ ously known genes. Cell extracts from human ciliary processes and ODM- 2 cells were analyzed by immunoblotting with anti-human ciliary proces s sera. The pattern of proteins immunodetected was compared with the p rofile of proteins immunodetected with human anti-Ku sera from a patie nt with scleroderma polymyositis overlap syndrome. These results demon strated that sera from anti-human ciliary processes recognize a variet y of antigens in ODM-2 cells and are strikingly rich in antibodies to the 80-kd subunit protein of Ku (p70/p80). Furthermore, indirect immun ofluorescence analysis supported the observation that anti-human cilia ry processes sera labeled nuclear antigens in a human ciliary epitheli um cell line and cross species in bovine ciliary epithelial cell, thus demonstrating the nuclear localization of Ku antigen. The mRNA in the human eye was found to be widely expressed in all tested ocular tissu es and in ODM-2 cells. Conclusions. The ocular ciliary epithelium indu ces a species-specific immune response in xenogenic animals. In partic ular, the human ciliary epithelium elicits a strong immune recognition of the 80-kd subunit protein of the human autoantigen Ku (p70/p80) in ODM-2 cells.