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.
OBJECTIVE
To isolate and characterize human nonpigmented ocular ciliary epithelial cDNA clones by screening a cDNA library constructed from the established human nonpigmented ciliary epithelial cell line (ODM-2) with sera raised against ciliary processes of human eye donors (cadavers).


METHODS
An ODM-2 cDNA expression library in lambda Uni-ZAP-XR vector was screened with sera of mouse anti-human ciliary processes. The cDNA inserts from plaque-purified clones were sequenced by the dideoxy-chain termination method with T3 and T7 polymerase. Sera were assayed by Western blot analysis and indirect immunofluorescence and were then compared with sera, enriched in anti-Ku antibodies, collected from patients with scleroderma polymyositis overlap syndrome.


RESULTS
Immunoscreening of 2.5 x 10(5) independent clones with sera induced experimentally in total tissue homogenates of human ciliary processes led to 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, were 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 clone, designated D2, failed to show any significantly similarity with previously known genes. Cell extracts from human ciliary processes and ODM-2 cells were analyzed by immunoblotting with anti-human ciliary process sera. The pattern of proteins immunodetected was compared with the profile of proteins immunodetected with human anti-Ku sera from a patient with scleroderma polymyositis overlap syndrome. These results demonstrated that sera from anti-human ciliary processes recognize a variety of antigens in ODM-2 cells and are strikingly rich in antibodies to the 80-kd subunit protein of Ku (p70/p80). Furthermore, indirect immunofluorescence analysis supported the observation that anti-human ciliary processes sera labeled nuclear antigens in a human ciliary epithelium 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 tissues and in ODM-2 cells.


CONCLUSIONS
The ocular ciliary epithelium induces a species-specific immune response in xenogenic animals. In particular, 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.
Hong
T J
TJ
Department of Opthalmology and Visual Science, Yale University School of Medicine, New Haven, Connecticut 06510.
Escribano
J
J
Coca-Prados
M
M
D000818:Animals; D034961:Antigens, Nuclear; D001324:Autoantigens / Q000032:analysis / Q000235:genetics*; D015153:Blotting, Western; D002460:Cell Line; D002924:Ciliary Body / Q000737:chemistry / Q000276:immunology*; D002999:Clone Cells; D004265:DNA Helicases; D018076:DNA, Complementary / Q000302:isolation & purification*; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology*; D000072200:Ku Autoantigen; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008875:Middle Aged; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000032:analysis / Q000235:genetics*; D010857:Pigment Epithelium of Eye / Q000737:chemistry / Q000276:immunology; D012333:RNA, Messenger / Q000378:metabolism; D014019:Tissue Donors
D034961:Antigens, Nuclear; D001324:Autoantigens; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D007074:Immunoglobulin G; D009687:Nuclear Proteins; D012333:RNA, Messenger; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen