Site-directed mutagenesis of lysine within the immunodominant autoepitope of PDC-E2

Patrick S Leung, Takashi Iwayama, Ross L. Coppel, M. Eric Gershwin

Research output: Contribution to journalArticle

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Abstract

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 ξ-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.

Original languageEnglish (US)
Pages (from-to)1321-1328
Number of pages8
JournalHepatology
Volume12
Issue number6
StatePublished - Dec 1990

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Thioctic Acid
Autoantigens
Site-Directed Mutagenesis
Lysine
dihydrolipoamide succinyltransferase
Clone Cells
Amino Acids
Immunodominant Epitopes
Aromatic Amino Acids
Isoantigens
Enzymes
Glutamine
Immunoblotting
Histidine
Oligonucleotides
Autoantibodies
Tyrosine
Epitopes
Enzyme-Linked Immunosorbent Assay
Liver

ASJC Scopus subject areas

  • Hepatology

Cite this

Site-directed mutagenesis of lysine within the immunodominant autoepitope of PDC-E2. / Leung, Patrick S; Iwayama, Takashi; Coppel, Ross L.; Gershwin, M. Eric.

In: Hepatology, Vol. 12, No. 6, 12.1990, p. 1321-1328.

Research output: Contribution to journalArticle

Leung, Patrick S ; Iwayama, Takashi ; Coppel, Ross L. ; Gershwin, M. Eric. / Site-directed mutagenesis of lysine within the immunodominant autoepitope of PDC-E2. In: Hepatology. 1990 ; Vol. 12, No. 6. pp. 1321-1328.
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