Identification and specificity of a cDNA encoding the 70 KD mitochondrial antigen recognized in primary biliary cirrhosis

M. Eric Gershwin, I. R. Mackay, A. Sturgess, R. L. Coppel

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Abstract

Mitochondrial autoantibodies are characteristic of the disease primary biliary cirrhosis (PBC), but the immunoreactive mitochondrial antigens have not been defined. We used a rat liver cDNA library in λgt 11-Amp3 to clone a 1370-base pair insert that coded for a polypeptide reactive with PBC sera. This insert was subcloned for expression into pBTA224, a plasmid vector in the same reading frame as λ-Amp3. A positive clone, designated pRMIT, that expressed a fused polypeptide of 160 kd, was recognized by 25 of 25 sera from patients with PBC and none of 96 sera from normal persons or patients with systemic lupus erythematosus, rheumatoid arthritis, or chronic active hepatitis. This fused polypeptide was shown to correspond with the 70 kd mitochondrial autoantigen by several experiments. First, lysates of pRMIT in J101 absorbed out the 70 kd reactivity of PBC sera when probed against fractionated placental mitochondria. Second, affinity-purified antisera reactive with the fused polypeptide also reacted with the 70 kd mitochondrial antigen. Third, such affinity-purified antisera produced the characteristic anti-mitochondrial pattern of immunofluorescence on tissue sections. Finally, immunization of BALB/c mice with the fused polypeptide elicited antibodies to mitochondria. These murine antibodies reacted with the 70 kd mitochondrial protein and also produced typical mitochondrial immunofluorescence on tissue sections. The nucleotide and amino acid sequence of the recombinant protein, which encodes for approximately a 48 kd protein, showed no significant homologies with known proteins, and there were no homologies with mitochondrial genomic DNA. The availability of a recombinant form of the 70 kd mitochondrial autoantigen will allow several definitive questions to be addressed in PBC, including identification of B cell epitopes, T cell recognition, and a model of PBC in mice.

Original languageEnglish (US)
Pages (from-to)3525-3531
Number of pages7
JournalJournal of Immunology
Volume138
Issue number10
StatePublished - 1987

ASJC Scopus subject areas

  • Immunology

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