Analysis of structure of proteasome-PCNA multiprotein conplex and its autoimmune response

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We have previously shown that a group of anti-PCNA monoclonal antibodies (mAbs) raised in our laboratory can react with PCNA bound to or interacting with other proteins associated with cell proliferation (PCNA complexes). In addition, we have found that proteasome is also interacting with PCNA. We purified the complexes by affinity chromatography using the mAbs, and also analyzed autoimmune response of proteins in the complex in lupus patients. We then found that 35% of SLE sera react with at least one component of the PCNA complex, which suggests that analysis of the targeted antigens may shed light on the mechanisms underlying autoimmune responses to proteins interacting with PCNA in SLE patients. To clarify this issue, we have identified elements of the PCNA complexes that are reactive to antibodies (Abs) in sera from SLE patients using 2-dimensional electrophoresis and ion-pair chromatography. Among the various elements of the complexes was a 37-kDa protein (PI 8.5) that specifically reacted with lupus sera, but not with sera from patients with other connective tissue diseases, and was later identified as glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Immunoblot analysis showed that lupus sera reactive with the 37-kDa protein specifically reacted with GAPDH, as did anti-GAPDH Abs purified from the sera. In addition, ELISAs revealed the presence of anti-GAPDH autoantibodies in 47% of lupus patients. Longitudinal analysis of the reactivity of lupus sera to PCNA complexes showed the autoimmune response to spread from GAPDH to other elements of PCNA complexes, and the presence of anti-GAPDH Abs was correlated with increased levels of serum PCNA. In addition, we have also found that lupus sera recognized proteasome activators as autoantigens, and there is a linkage of autoimmune response between PCNA and proteasome. Taken together, these findings suggest that "antige presentation" is playing a critical role to induce the autoimmune response to the PCNA complex.