Identification of the discontinuous epitope in human complement protein C9 recognized by anti-melittin antibodies.
Polyclonal rabbit antibodies against melittin recognize human C protein C9 and retard C9-mediated hemolysis. Human C9 contains a tetrameric and a pentameric sequence (amino acids 293-296 and 528-532, respectively) that together match a continuous segment in the melittin sequence, i.e., residues 8-16. It has been suggested that the tetrameric and the pentameric regions on C9 form a discontinuous epitope on folded C9 that mimics the structure of melittin. To further test this hypothesis, antibodies to C9-sequence-specific peptides were prepared. Peptides containing either the homologous tetrameric or the homologous pentameric sequence together with short stretches of the respective amino- and carboxyl-terminal flanking regions were synthesized, as well as a composite peptide predicted to resemble the discontinuous epitope as a linear, nine-amino acid sequence. Direct and competitive binding assays demonstrated that the tetrameric and the pentameric sequences are part of the epitope on human C9 that is recognized by anti-melittin IgG. However, only antibodies directed against the complete epitope are capable of inhibiting hemolysis. Because neither anti-tetramer nor anti-pentamer antibodies affect hemolysis whereas anti-melittin and anti-composite antibodies do, we propose that human C9 changes conformation around a hinge located between residues 296 and 528 and that the latter two antibodies inhibit unfolding required for membrane insertion and subsequent hemolysis.
Laine
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Department of Comparative and Experimental Pathology, University of Florida, Gainesville 32610.
Esser
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D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology* / Q000502:physiology; D000937:Antigen-Antibody Reactions; D001514:Bee Venoms / Q000276:immunology*; D003186:Complement C9 / Q000032:analysis* / Q000276:immunology; D000939:Epitopes / Q000032:analysis* / Q000276:immunology; D006801:Humans; D008555:Melitten / Q000276:immunology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000276:immunology; D010449:Peptide Mapping; D011487:Protein Conformation; D011817:Rabbits; D012689:Sequence Homology, Nucleic Acid
D000906:Antibodies; D001514:Bee Venoms; D003186:Complement C9; D000939:Epitopes; D010446:Peptide Fragments; D008555:Melitten