p185, an immunodominant epitope, is an autoantigen mimotope

Abstract

An immunodominant peptide (p185(378-394)) derived from the c-erbB2 gene product, was recognized by an anti-DNA antibody, B3, and importantly by two classical DNA-binding proteins, Tgo polymerase and Pa-UDG. These reactivities were inhibited by DNA, confirming that the peptide mimicked DNA. BALB/c mice immunized with p185(378-394) developed significant titers of IgG anti-dsDNA antibodies. Screening of 39 human lupus sera revealed that 5% of these sera possessed reactivity toward p185(378-394). Representative mouse and human sera with anti-p185(378-394) reactivity bound intact p185, and this binding was inhibited by dsDNA. This is the first demonstration of a naturally occurring autoantigen mimotope. The present study identifies a potential antigenic stimulus that might trigger systemic lupus erythematosus in a subset of patients.

FIGURE 1.

a, quantitative comparative assessment as percentage of binding in relation to total binding to anti-human λ monoclonal antibody, of the recombinant Fab proteins in their native and hybrid forms, in ELISA, to an immunodominant peptide (p185(378–394)) derived from the extracellular domain of human c-erbB2. b, inhibition of the binding of the antibody BB to p185(378–394) by calf thymus dsDNA in ELISA. B and U, B3 and UK4, respectively; the first of the two letters that name the Fab construct denotes the light chain, and the second of the letters denotes the heavy chain.

FIGURE 2.

Binding of p185(378–394) peptide to the DNA repair enzyme Pa-UDG (a) or DNA polymerase Tgo (c) in its doubling dilutions starting from 80 μg/ml. Shown is inhibition of the binding of the peptide p185(378–394) used at 20 μg/ml to the enzymes Pa-UDG (b) or DNA polymerase Tgo (d) by three doubling concentrations of calf thymus dsDNA starting from 6.25 μg/ml (shown as 1:04).

FIGURE 3.

Binding of the sera from mice immunized with MAP + p185(378–394) peptide or MAP alone constituted in saline (a and c) or Freund's adjuvant (b and d), to dsDNA, ssDNA, or p185(378–394) peptide in traditional (a and b) or pull-down (dsDNA) (c and d) ELISAs. MAP + P, MAP + p185(378–394) peptide. Vertical bars represent the mean of (and S.D. is based on) five data points obtained for five mice.

FIGURE 4.

a, quantitative comparative assessment as a percentage of binding in relation to binding to p185(378–394) peptide of the sera from mice immunized with MAP + p185(378–394) peptide or MAP alone constituted in saline or Freund's adjuvant, to dsDNA, ssDNA, cardiolipin, or lysozyme in ELISAs. Shown is binding of four representative sera (two from each group of mice receiving MAP + p185(378–394) peptide in saline (b) or in Freund's adjuvant (c) in their doubling dilutions to dsDNA. Anti-peptide and anti-dsDNA activities of the four sera are plotted in decreasing order of their anti-peptide activities (d), indicating that the two activities were associated.

FIGURE 5.

Inhibition of the binding of the four representative sera, two from each group of mice receiving MAP + p185(378–394) peptide in Freund's adjuvant (a) or in saline (b), to peptide p185(378–394) by doubling concentrations of calf thymus dsDNA. Shown is binding of the IgG eluted in kidney extracts of the mice immunized with MAP + p185(378–394) peptide in Freund's adjuvant or in saline (c) to dsDNA.

FIGURE 6.

Binding of 39 human lupus sera and 20 control sera from healthy individuals in ELISA to p185(378–394) (a) or control peptide (p185(1238–1255)) (b). Serums number 23 and 31 react with p185(378–394). c, binding of a representative p185(378–394)-immunized mouse serum and the human serum number 23 with intact p185 molecule derived from SKBR-3 cells. The inhibition of the reactivity of the mouse and human sera to intact p185 molecule by dsDNA is shown in d.