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

Abstract

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.

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

“…Recent studies have shown PCNA to be involved in numerous cellular functions associated with cell proliferation and to directly bind to or interact with various proteins, as listed in Table 1 (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)24). Our previous study (24) revealed that the PCNA multiprotein complexes purified by mAb to PCNA contained components of multiprotein machinery such as the DNA replication fork (Figure 1) (12,13,21,22) and a quaternary PCNA/ p21/CDK/cyclin complex reported to play a key role in the regulation of the cell cycle (14,17).…”

“…Our previous study (24) revealed that the PCNA multiprotein complexes purified by mAb to PCNA contained components of multiprotein machinery such as the DNA replication fork (Figure 1) (12,13,21,22) and a quaternary PCNA/ p21/CDK/cyclin complex reported to play a key role in the regulation of the cell cycle (14,17).…”

“…The beads were then washed once with 1M NaCl/0.01M phosphate buffer (pH 7.4) and either IP buffer or phosphate buffered saline (PBS). The proteins bound to the beads were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by autoradiography as previously described (24).…”

“…In contrast, anti-PCNA-positive sera (including serum AK, which reacts with biologically functional sites on PCNA) (25)(26)(27) do not react with complexed protein but did react with monomeric and trimeric forms that are free from other proteins (24). In addition, PCNA complexes purified from rabbit thymus extract (RTE) by affinity chromatography using anti-PCNA-positive mAb were shown to also react with murine anti-DNA mAb, as well as with antibodies against p21, replication protein A (RPA), DNA helicase II (NDH II), cyclin-dependent protein kinase 4 (CDK4), CDK5, and topoisomerase I (topo I) (24). These findings indicate that the PCNA complexes comprise a substantial part of the "protein machinery" for DNA replication and cell cycle regulation and can be simply purified using anti-PCNA mAb, comparing with the methods previously reported by Loor et al and by Applegren et al (21,22).…”

“…We recently demonstrated that when PCNA is complexed with other proteins associated with cell proliferation (PCNA complex), it can still react with a group of anti-PCNA monoclonal antibodies (mAb) raised in our laboratory (TOB7, TO17, and TO30) (23,24). In contrast, anti-PCNA-positive sera (including serum AK, which reacts with biologically functional sites on PCNA) (25)(26)(27) do not react with complexed protein but did react with monomeric and trimeric forms that are free from other proteins (24).…”