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Anti-liver-kidney microsome antibody type 1 recognizes human cytochrome P450 db1
M Gueguen1, A M Yamamoto, O Bernard
1INSERM U 56, Hôpital de Bicêtre, Le Kremlin-Bicêtre, France.
Insights
Autoimmune hepatitis in children is linked to anti-liver-kidney microsome antibody type 1 (LKM1) targeting cytochrome P450 db1. This finding enables a specific diagnostic ELISA test for the disease.
Area of Science:
- Immunology
- Hepatology
- Biochemistry
Background:
- Autoimmune hepatitis (AIH) is a chronic liver disease of unknown etiology.
- Anti-liver-kidney microsome antibody type 1 (LKM1) is a serological marker associated with a specific subtype of AIH in children.
- LKM1 autoantibodies recognize a 50 kDa protein, identified as rat liver cytochromes P450 db1 and db2.
Purpose of the Study:
- To identify the specific human liver antigen recognized by LKM1 autoantibodies.
- To investigate the potential role of human cytochrome P450 db1 in LKM1-positive autoimmune hepatitis.
- To develop a diagnostic tool for LKM1-positive autoimmune hepatitis.
Main Methods:
- Screening of a human liver cDNA expression library using rat P450 db1 cDNA as a probe.
- Restriction mapping to identify homologous human cDNA clones.
- Immunoblot analysis using purified fusion protein to assess antibody reactivity.
- Development of an Enzyme-Linked Immunosorbent Assay (ELISA) for diagnostic purposes.
Main Results:
- Two human cDNA clones identical to human P450 db1 were identified.
- The fusion protein derived from a human P450 db1 clone was specifically recognized by LKM1 autoantibodies.
- An ELISA test utilizing this fusion protein demonstrated high specificity for sera from children with LKM1-positive autoimmune hepatitis.
Conclusions:
- The primary liver autoantigen recognized by LKM1 antibodies is human cytochrome P450 db1.
- Mutations in the human P450 db1 gene may lead to its presentation as an autoantigen in autoimmune hepatitis.
- A specific and simple diagnostic ELISA test for LKM1-positive autoimmune hepatitis has been developed.
Abstract:
Anti-liver-kidney microsome antibody type 1 (LKM1), present in the sera of a group of children with autoimmune hepatitis, was recently shown to recognize a 50 kDa protein identified as rat liver cytochromes P450 db1 and db2. High homology between these two members of the rat P450 IID subfamily and human P450 db1 suggested that anti-LKM1 antibody is directed against this human protein. To test this hypothesis, a human liver cDNA expression library in phage lambda GT-11 was screened using rat P450 db1 cDNA as a probe. Two human cDNA clones were found to be identical to human P450 db1 by restriction mapping. Immunoblot analysis using as antigen, the purified fusion protein from one of the human cDNA clones showed that only anti-LKM1 with anti-50 kDa reactivity recognized the fusion protein. This fusion protein was further used to develop an ELISA test that was shown to be specific for sera of children with this disease. These results: 1) identify the human liver antigen recognized by anti-LKM1 auto-antibodies as cytochrome P450 db1, 2) allow to speculate that mutation on the human P450 db1 gene could alter its expression in the hepatocyte and make it auto-antigenic, 3) provide a simple and specific diagnostic test for this disease.