Identification of plasma membrane autoantigens in autoimmune hepatitis type 1 using a proteomics tool

Autoimmune hepatitis (AIH) is a liver disease with circulating autoantibodies predominantly directed against widely held cellular components. Because AIH is a liver-specific disease, autoantibodies against plasma membrane antigens may be involved in its pathogenesis and have been reported; however, no definite identification has been described. We thus investigated the fine specificity of anti-hepatocyte plasma membrane autoantibodies in type 1 AIH (AIH-1) using a proteomic tool. A plasma membrane-enriched fraction was validated using enzymatic activity and western blot analysis experiments. Sera from AIH-1 patients (n = 65) and from 90 controls, that is, healthy blood donors (n = 40) and patients with systemic diseases (n = 20) or other liver diseases (n = 30), were studied by immunoblot performed with plasma membrane proteins resolved by either sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or 2-dimensional (2D) electrophoresis. Proteins contained in the immunoreactive spots were identified by sequences provided by ion-trap mass spectrometry. Hepatocytes probed with sera were also studied using confocal immunofluorescence and immunoelectron microscopy. The more prominent bands stained by patient sera were located at 38 kDa, 48, 50, 52 kDa, 62 kDa, 70 kDa, and a 95-kDa double band. Six proteins with known potential plasma membrane expression were identified: liver arginase (38 kDa), cytokeratins (CK) 8 and 18 (48-52 kDa), heat shock proteins (HSP) of 60, 70, 90 kDa, and valosin-containing protein (VCP) of 92 kDa. The presence of anti-membrane antibodies was confirmed by immunofluorescence and immunoelectron microscopy.

Overall, our data demonstrate that liver arginase, CK 8/18, HSP 60, HSP 70, HSP 90, and VCP represent potential candidate targets on liver membrane for autoantibodies in AIH-1.