Reactivity of anti-proliferating cell nuclear antigen (PCNA) murine monoclonal antibodies and human autoantibodies to the PCNA multiprotein complexes involved in cell proliferation.
Proliferating cell nuclear Ag (PCNA) occurs as a component of multiprotein complexes during cell proliferation. We found the complexes to react with murine anti-PCNA mAbs, but not with anti-PCNA Abs in lupus sera. The complexes were purified from rabbit thymus extract by affinity chromatography using anti-PCNA mAbs (TOB7, TO17, and TO30) and analyzed by ELISA, immunoprecipitation, immunoblotting, and HPLC gel filtration. That PCNA was complexed with other proteins was demonstrated by its copurification with a group of proteins excluded by an HPLC G3000 SW column. Although immunoblot analysis showed the mAbs to react exclusively with the 34-kDa PCNA polypeptide, they nonetheless immunoprecipitated the same group of proteins, confirming the interaction of the isolated PCNA with other proteins. Anti-PCNA sera, including AK, which reacts with biologically functional sites on PCNA, did not react with complexed PCNA, but did react with it once it was dissociated from the complexes. PCNA complexes in turn reacted with murine anti-DNA mAbs, as well as with Abs against p21, replication protein A, DNA helicase II, cyclin-dependent kinases 4 and 5, and topoisomerase I. These findings suggest that the PCNA complexes purified using anti-PCNA mAbs comprise the "protein machinery" for DNA replication and cell cycle regulation. They also suggest that anti-PCNA mAbs are useful tools with which to characterize the protein-protein interactions within PCNA complexes, as well as the autoimmune responses to proteins interacting with PCNA, which may shed light on the mechanisms of autoantibody production in lupus patients.
Department of Internal Medicine and Rheumatology, Juntendo University School of Medicine, Tokyo, Japan. tyoshi@med.juntendo.ac.jp
Y
Y
Takasaki
T
T
Kogure
K
K
Takeuchi
K
K
Kaneda
T
T
Yano
K
K
Hirokawa
S
S
Hirose
T
T
Shirai
H
H
Hashimoto
D000818:Animals; D000911:Antibodies, Monoclonal / Q000097:blood / Q000378:metabolism*; D000937:Antigen-Antibody Reactions; D001323:Autoantibodies / Q000378:metabolism*; D002455:Cell Division / Q000276:immunology; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D006133:Growth Substances / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D006825:Hybridomas; D007106:Immune Sera / Q000378:metabolism; D015151:Immunoblotting; D008180:Lupus Erythematosus, Systemic / Q000276:immunology; D046911:Macromolecular Substances; D051379:Mice; D011233:Precipitin Tests; D018809:Proliferating Cell Nuclear Antigen / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D013949:Thymus Extracts / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D006133:Growth Substances; D007106:Immune Sera; D046911:Macromolecular Substances; D018809:Proliferating Cell Nuclear Antigen; D013949:Thymus Extracts