Major and minor epitopes on the self antigen mouse cytochrome c mapped by site-directed mutagenesis.
Variants of rat (mouse) cytochrome c, prepared by site-directed mutagenesis or represented by closely-related cytochromes c from different species, were employed to map the functional boundaries of a number of mouse monoclonal antibodies (mAb) specific for the major antigenic region on the self antigen (Ag) around residue 62 and the minor antigenic region around residue 44. The recombinant mouse cytochromes c tested were, unlike the tissue-derived Ag, trimethylated at position 72, and included the wild-type which was acetylated at the amino terminus, a variant that was unacetylated at the amino terminus, and variants with the following single amino acid residue replacements: V11I (valine to isoleucine at position 11), Q12M, A15S, A44P, F46Y, D50A, T58I and G89E. Of these, only the A44P variant affected the binding of mAb to the region previously localized to the vicinity of residue 44, thus confirming that assignment. Loss of the acetyl group at the amino terminus affected the binding of most of the mAb to the region around residue 62. The other mutations had little, if any, affect on mAb binding. The epitopes of mAb binding the region around residue 62 were shown in this study to have similar functional boundaries. This site on the self Ag, which encompasses at least three discontinuous segments of the polypeptide chain, is comparable in size to epitopes on other protein Ag that have been mapped by X-ray crystallography and is similar to an epitope in the corresponding region of the foreign Ag, horse cytochrome c, that has been mapped by hydrogen-deuterium exchange. In addition to the mAb binding the regions around residues 44 and 62, a third group of mouse cytochrome c-specific mAb known to be broadly reactive with mammalian cytochromes c and that represents a minor portion of the mAb was tested for binding the site-directed mutants of mouse cytochrome c. None of these mAb were affected by the mutations, indicating the presence of at least one more antigenic region on the self Ag in an area not encompassed by these mutations that is structurally highly conserved.
Minnerath
J M
JM
Department of Microbiology, University of Minnesota Medical School, Minneapolis 55455, USA.
Crump
B L
BL
Margoliash
E
E
Jemmerson
R
R
D000595:Amino Acid Sequence; D000818:Animals; D010856:Columbidae; D003574:Cytochrome c Group / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D018604:Epitope Mapping; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D006825:Hybridomas; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011817:Rabbits; D016415:Sequence Alignment
D003574:Cytochrome c Group; D000939:Epitopes