Background The presence of maternal autoantibodies directed against the amino acids (aa) 200–239 stretch of Ro52 (Ro52-p200) is associated with high risk of foetal CHB to develop.
Objectives To investigate the role of anti-Ro52 antibodies in the pathogenesis of CHB by identifying the epitope specificity of human anti-Ro52-p200 antibodies.
Methods Sera from 19 mothers of foetuses with CHB and 8 babies with CHB were analysed for binding to synthetic peptides representing native and mutated variants of human and rodent Ro52-p200 and overlapping peptides by ELISA. Competition experiments were performed to confirm specificity and affinity of the binding. Secondary structure of the peptides was analysed by circular dichroism spectroscopy (CD).
Results We first analysed autoantibody binding to human p200 and a highly overlapping peptide including aa 197-232 (p197), which demonstrated significantly higher reactivity towards p200 than to p197 (p<0.0001). The relevance of the C-terminal amino acids in p200 was confirmed using truncated human p200 peptides representing N-term, mid and C-terminal parts of the human p200 which demonstrated that the deletion of amino acids in the C-terminal of p200 completely abolished the binding to p200, while the deletion of amino acids in the N-terminal only slightly decreased the binding to p200. Furthermore, we took advantage of the fact that human sera do not bind rat p200 (r-p200), and generated peptides based on the rat p200 sequence, with selected crucial residues mutated into the human counterparts. Mutations in the C-terminal end including a glutamic acid substitution for an aspartic acid in position 233 localized in the C-terminal of the p200 peptide reestablished binding of sera to the peptide, while there was no gain of reactivity by substitutions in the N-terminal or mid part of the peptide. Analysis of the peptides by CD confirmed that introduction of the substitutions did not disrupt folding of the peptides compared to the r-p200 peptide. To unambiguously pinpoint the main contributing residues of the epitope we generated peptides representing an alanine scan with alanine substitutions of each residue from position 233 to 239 (pA233-pA239). Interestingly, we observed that the substitution of the aspartic acid 233 in the pA233 peptide abolished the antibody binding, while the other mutations did not affect binding of sera. The mutation at position 233 of the alanine scan substitutes the same aa mutated in position 233 of the rat-to-human mutated peptide, confirming the dominant role of this residue to the p200 epitope. Moreover, in competition experiment pre-incubation of the sera with the rat-to-human 233 mutated peptide blocked antibody reactivity to p200 to the same degree as the p200 peptide itself. The 233 aa is localized on the outer surface of the modeled peptide homodimer of the leucine zipper, suggesting that it is accessible for antibody binding.
Conclusions Our study suggests that the aspartic acid at aa residue position 233 of the Ro52-p200 peptide is crucial in forming the main epitope of the Ro52-p200 peptide bound by CHB-related human Ro52 antibodies. This specificity might be used as a tool to identify high risk pregnancies for CHB, and for identification of the cross-reactive target in the fetal heart bound by the maternal autoantibodies.
Disclosure of Interest: None Declared
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