Site-directed mutagenesis of lysine within the immunodominant autoepitope of PDC-E2.
The major autoantigens of PBC have been identified as the four closely related mitochondrial enzymes PDC-E2, BCKD-E2 OGDC-E2 and protein X. A major structural similarity of these enzymes is the presence of one or more lipoyl domains. The immunodominant epitope of each autoantigen has either been postulated or been demonstrated to be located within the lipoate binding region. However, it is not clear whether the binding of lipoic acid to the epitope is necessary for autoantibody recognition. To address this issue we have constructed by oligonucleotide site-directed mutatagenesis three mutants in the lipoyl domain of human PDC-E2. Because lipoic acid is covalently bound to the zeta-amino group of the lysine residue of PDC-E2, the mutants were designed to replace the lysine residue in the lipoyl domain with glutamine, a negatively charged amino acid; histidine, a positively charged amino acid; and tyrosine, an aromatic amino acid. Binding reactivity of sera from patients with PBC were analyzed by enzyme-linked immunosorbent assay, immunoblotting and specific absorption against each of the three mutants and control clones. All data were compared with parallel studies with a control recombinant clone, the liver-specific F alloantigen. We believe the recognition of the lipoyl domain is a reflection of the surface-exposed, hydrophilic and relatively mobile nature of this region of the autoantigen. Further studies on direct assay for the presence of lipoic acid will be needed to clarify these issues.
Leung
P S
PS
Division of Rheumatology, Allergy and Clinical Immunology, University of California, Davis 95616.
Iwayama
T
T
Coppel
R L
RL
Gershwin
M E
ME
D001324:Autoantigens / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D008105:Liver Cirrhosis, Biliary / Q000235:genetics / Q000276:immunology*; D008239:Lysine / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011768:Pyruvate Dehydrogenase Complex / Q000235:genetics*; D008063:Thioctic Acid / Q000235:genetics*
D001324:Autoantigens; D000939:Epitopes; D011768:Pyruvate Dehydrogenase Complex; D008063:Thioctic Acid; D004247:DNA; D008239:Lysine