Bcl-2–dependent oxidation of pyruvate dehydrogenase-E2, a primary biliary cirrhosis autoantigen, during apoptosis
Joseph A. Odin, … , Nicholas F. LaRusso, Antony Rosen
Joseph A. Odin, … , Nicholas F. LaRusso, Antony Rosen
Published July 15, 2001
Citation Information: J Clin Invest. 2001;108(2):223-232. https://doi.org/10.1172/JCI10716.
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Bcl-2–dependent oxidation of pyruvate dehydrogenase-E2, a primary biliary cirrhosis autoantigen, during apoptosis

Abstract

The close association between autoantibodies against pyruvate dehydrogenase-E2 (PDC-E2), a ubiquitous mitochondrial protein, and primary biliary cirrhosis (PBC) is unexplained. Many autoantigens are selectively modified during apoptosis, which has focused attention on apoptotic cells as a potential source of “neo-antigens” responsible for activating autoreactive lymphocytes. Since increased apoptosis of bile duct epithelial cells (cholangiocytes) is evident in patients with PBC, we evaluated the effect of apoptosis on PDC-E2. Autoantibody recognition of PDC-E2 by immunofluorescence persisted in apoptotic cholangiocytes and appeared unchanged by immunoblot analysis. PDC-E2 was neither cleaved by caspases nor concentrated into surface blebs in apoptotic cells. In other cell types, autoantibody recognition of PDC-E2, as assessed by immunofluorescence, was abrogated after apoptosis, although expression levels of PDC-E2 appeared unchanged when examined by immunoblot analysis. Both overexpression of Bcl-2 and depletion of glutathione before inducing apoptosis prevented this loss of autoantibody recognition, suggesting that glutathiolation, rather than degradation or loss, of PDC-E2 was responsible for the loss of immunofluorescence signal. We postulate that apoptotic cholangiocytes, unlike other apoptotic cell types, are a potential source of immunogenic PDC-E2 in patients with PBC.

Authors

Joseph A. Odin, Robert C. Huebert, Livia Casciola-Rosen, Nicholas F. LaRusso, Antony Rosen

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Figure 7

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Direct oxidation of PDC-E2 by oxidized glutathione abrogates recognition...
Direct oxidation of PDC-E2 by oxidized glutathione abrogates recognition of PDC-E2 by PBC patient sera. (a) Apoptotic NRC lysate was treated with SDS, boiled, and then sequentially treated with 5 mM DTT, 10 mM GSSG, and 25 mM DTT. Strong blotting of PDC-E2 was detected in lysate treated with 5 mM DTT (lane 1). Blotting of PDC-E2 was absent after addition of GSSG to the DTT-treated lysate (lane 2), but subsequent treatment with additional DTT restored detection of PDC-E2 (lane 3). (b) The difference between recognition of oxidized versus reduced PDC-E2 by PBC patient autoantibodies was quantified by immunoblotting lysate treated with 10 mM GSSG vs. 5 mM DTT. A representative blot is shown. (c) The oxidative state of PDC-E2 does not significantly affect its transfer to nitrocellulose. PDC-E2 was immunoprecipitated using PBC patient sera from lysate of HeLa cells incubated with [35S]-methionine to label proteins. Half the immunoprecipitate was treated with DTT and the other half with GSSG before SDS-PAGE was performed. The autoradiogram performed after transfer to nitrocellulose showed that similar amounts of both reduced and oxidized PDC-E2 were transferred. Results using two different PBC patient sera are shown (lanes 1 and 2). (d) The expression level of Bcl-2 correlates with persistence of PDC-E2 staining following apoptosis. Eighty micrograms of lysate protein was loaded per lane and immunoblotted with an mAb specific for human Bcl-2. High levels of Bcl-2 were only detected in the Bcl-2–transfected HeLa (lane 1) and HSG cell lysates (lane 2).