Characterization of proliferating cell nuclear antigen recognized by autoantibodies in lupus sera.

Takasaki, Yoshinari; Fishwild, Dianne M.; Tan, Eng M.

doi:10.1084/jem.159.4.981

Cited by 183 publications

(84 citation statements)

References 18 publications

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“…DID assays were conducted on plates containing 0.4% agarose (Sea Kem; FMC Bioproducts, Rockland, ME) and 0.01% sodium azide in PBS (2,4). Precipitation reactions were allowed to develop for 48 hours at room temperature.…”

Section: Methodsmentioning

confidence: 99%

“…Purified PCNAs were transferred electrophoretically onto nitrocellulose filters (Bio-Rad), as previously described (2,29). For immunochemical detection of proteins, the filters were first blocked for 24 hours in 3% bovine serum albumin (BSA) in 0.1% Tween-PBS (T-PBS) and then incubated for 2 hours with anti-PCNA-positive mAb in T-PBS (2.5 mg/ml) or with sera obtained from patients (anti-PCNApositive sera, anti-Sm, and U1 RNP serum YT; anti-topo I serum YM) (1:200 dilution in T-PBS).…”

Section: Methodsmentioning

confidence: 99%

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Autoimmune responses to proliferating cell nuclear antigen multiprotein complexes involved in cell proliferation are strongly associated with their structure and biologic function in patients with systemic lupus erythematosus

Kogure

Takasaki

Takeuchi

et al. 2002

Arthritis & Rheumatism

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Objective. To analyze the reaction of lupus sera with proliferating cell nuclear antigen (PCNA) multiprotein complexes (PCNA complexes), which are part of the protein machinery involved in cell proliferation.Methods. PCNA complexes were purified from rabbit thymus extract by affinity chromatography using anti-PCNA monoclonal antibodies (TOB7, TO17, and TO30); monomeric and trimeric PCNA forms (AK-PCNA) were purified using anti-PCNA serum AK. The reactions to these antigens of 10 anti-PCNA-positive and 40 anti-PCNA-negative sera selected from 560 lupus patients were tested by immunoblotting, immunoprecipitation, and enzyme-linked immunosorbent assays (ELISAs).Results. With one exception (serum OK), anti-PCNA-positive sera reacted exclusively with only the 34-kd polypeptide. In contrast, 14 of 40 anti-PCNAnegative sera reacted with multiple proteins within PCNA complexes. Most anti-PCNA-positive sera probably recognize as epitopes the binding sites for other proteins on PCNA, which are likely hidden when PCNA is complexed with other proteins. As a consequence, only serum OK reacted with the PCNA complex in a series of ELISAs. Using AK-PCNA as a competitive inhibitor, it was determined that serum OK reacts with both the 58-kd polypeptide and the 34-kd PCNA within complexes. Together with the results of a longitudinal analysis, these results suggest that the immune system of patient OK likely recognized the complexed PCNA protein, after which the autoimmune response spread to other elements of the complexes.Conclusion. Intermolecular-intrastructural help, leading to the spread of autoimmune response from PCNA to other proteins associated with its biologic function, plays a crucial role in the induction of the autoimmune response seen in lupus patients.Autoantibodies against proliferating cell nuclear antigen (PCNA) were first observed in a patient with systemic lupus erythematosus (SLE) (1). Using anti-PCNA antibodies from SLE patients as a probe, our group subsequently determined that PCNA is a 34-kd intranuclear polypeptide that exists mainly as a homotrimer (2,3), and that expression of PCNA is increased in the late G1 and early S phases of the cell cycle, immediately before DNA synthesis (4). These findings suggested an involvement of PCNA in DNA replication, and, indeed, PCNA was later identified as an auxiliary protein of DNA polymerase ␦ (Pol-␦), playing an essential role in DNA replication and repair (5-9).

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Autoimmune responses to proliferating cell nuclear antigen multiprotein complexes involved in cell proliferation are strongly associated with their structure and biologic function in patients with systemic lupus erythematosus

Kogure

Takasaki

Takeuchi

et al. 2002

Arthritis & Rheumatism

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“…Different anti-PCNA antibodies were used: a well defined autoantiserum obtained from a patient (AK) with systemic lupus erythematosus (SLE) (21,24,40,42) and three monoclonal antibodies (moabs), 19F4 (IgG1) (311, TOB7 (IgG,) (411, and PClO (IgG,,) (43). The AK serum, 19F4, and TOB7 antibodies were kindly supplied by Dr. E.M. Tan, La Jolla, CA.…”

Section: Antibodiesmentioning

confidence: 99%

Flow cytometric analysis of proliferation associated nuclear antigens using washless staining of unfixed cells

Landberg

Roos

1992

Cytometry

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The expression of different proliferation associated nuclear antigens was analyzed using a washless double-staining method and flow cytometry. It is a simple and rapid two-step procedure which can be performed on low cell numbers. A series of hematopoietic cell lines and fresh lymphoma cells were tested and the methodology was found to be applicable to a number of nuclear antigens (PCNA, Ki-67, p105, MPM-2, fibrillarin). For PCNA, the detectability was dependent on the type of antibody used. The immunofluorescence pattern observed by microscopy was altered for antigens stained by the washless technique in comparison with the pattern obtained with fixed cells. With the washless method, detailed cell cycle analysis could be obtained by dual parameter analysis of PCNA and Ki-67.

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“…When we purified and characterized this Ag from cell extracts, we found it to be a 34-kDa intranuclear polypeptide, existing mainly as a homotrimer (2,3) whose expression increased in the late G 1 to S phases of the cell cycle immediately before DNA synthesis (4). These findings suggested an involvement of PCNA in DNA replication, and, indeed, PCNA was later identified as an auxiliary protein of DNA polymerase (Pol)-␦, playing an essential role in DNA replication and repair (5)(6)(7)(8)(9).…”

mentioning

confidence: 99%

“…Autoantibodies against PCNA isolated from the sera of lupus patients have frequently been used to analyze the cellular functions of PCNA (2,4,(17)(18)(19). However, sera from lupus patients often contain other autoantibodies, and it has been difficult to obtain sufficient amounts of monospecific sera for PCNA because of its low incidence in lupus patients (1).…”

mentioning

confidence: 99%

Reactivity of Anti-Proliferating Cell Nuclear Antigen (PCNA) Murine Monoclonal Antibodies and Human Autoantibodies to the PCNA Multiprotein Complexes Involved in Cell Proliferation

Takasaki

Kogure

Takeuchi

et al. 2001

The Journal of Immunology

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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.

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Characterization of proliferating cell nuclear antigen recognized by autoantibodies in lupus sera.

Cited by 183 publications

References 18 publications

Autoimmune responses to proliferating cell nuclear antigen multiprotein complexes involved in cell proliferation are strongly associated with their structure and biologic function in patients with systemic lupus erythematosus

Autoimmune responses to proliferating cell nuclear antigen multiprotein complexes involved in cell proliferation are strongly associated with their structure and biologic function in patients with systemic lupus erythematosus

Flow cytometric analysis of proliferation associated nuclear antigens using washless staining of unfixed cells

Reactivity of Anti-Proliferating Cell Nuclear Antigen (PCNA) Murine Monoclonal Antibodies and Human Autoantibodies to the PCNA Multiprotein Complexes Involved in Cell Proliferation

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