Cytochrome P450IID6 recognized by LKMl antibody is not exposed on the surface of hepatocytes

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Abstract
LKM1 autoantibody, directed againsl P450IID6, is accepted as a marker of a particular type of autoimmune hepatitis, but its role in the pathogenesisof the disease is controversial. Localisation of P450IID6 on the cell surface of rat hepatocytes was previously reported, suggesting that membranebound P450IID6 could be the target of LKM 1 antibodies, thus allowing immune lysis of hepatocytes. The objective of the present study was to determine, using various methods, the cell localization of P450IID6 in human and rat hepatocytes. Incubation of rat and human hepalocytes with LKMI‐positive serum showed slight, if any. cell membrane staining using immunofluorcscencc. immunoperoxidase and immunoelectron microscopic sttidies. No staining of the plasma membrane of human hepatocytes was observed when incubations were earried out with imniunoaffinity‐purificd antibody directed against peptide254–271, the main epitope of P450IID6 recognized by all LKMl sera tested. Chinese hamsler ovary cells, transfected with the complete P450IID6 cDNA and incubated with the supernatant from a B cell lymphoblastoid cell line prepared with the lymphocytes of a LKMI‐positive patient, did not show any staining of the cell surface by immunofluorescence. Incubation of rat microsomal fraction vesicles with LKMl‐positive serum, followed by prolein A‐gold immunoelectron microscopy, displayed a staining of almost all vesicles, confirming that P450IID6 is present on the cyloplasmic side of the microsomal membrane, which makes it unable to be expressed on the ceil surface even if it were transported from the endoplasmic reticulum (ER). Sulpho NHS Biolin labelling of rat hepatocyte cell membranes did nol show the presence of a 50‐kD molecule that could have reacted with LKMl antibody. DNA sequencing of exon 1 of the CYP2D6 gene of a patient positive for LKM l antibody did not show any difference from that of the normal published sequence of the gene. This does not favour an alteration of the NH2 terminal sequence of the P450IID6 molecule that could explain a translocation of the molecule to the luminal side of the ER, allowing its expression on the ceil surface. These results indicate that, in all likelihood. P450IID6 molecule is not present on the cell surface of normal rat and human hepatocytes. Other mechanisms than antibody‐mediated cell lysis directed against membrane P450IID6 antigenic determinants must be found to account for the destruction of hepatocytes observed in this disease.

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