How do two unrelated antibodies, HyHEL-10 and F9.13.7, recognize the same epitope of hen egg-white lysozyme?
The anti-hen egg-white lysozyme (HEWL) antibodies HyHEL-10 and F9.13.7 recognize a common epitope. The structures of the complexes differ, however, in the numbers of electrostatic and hydrogen-bond interactions and in the distributions of contacts between the light and heavy chains. The equilibria and kinetics characterizing the F9.13.7 complex formation were evaluated for both wild-type and mutant derivatives of HEWL to help to understand how the different contacts are effectively used in the complexes with the two antibodies. Three epitope hot spots, Y20, K96, and R73 (destabilization > 4 kcal/mole), were found by alanine scanning mutagenesis. The first two constitute two of the three hot spots in the HyHEL-10 complex. The hot spots of the HyHEL-10 paratope are centered on the HEWL epitope; whereas R73 (HEWL), the only important light-chain-contacting residue, is clearly separated from the other hot spots of the F9.13.7 complex. The larger number of epitope warm plus hot spots found in the F9.13.7 complex compared with that of HyHEL-10 shows that the specificity of the former is greater even though the K(D) value is 20-fold larger. Conservative mutations showed that the specificity enhancement is related to the greater number of functional polar and hydrogen bond interactions in the F9.13.7 complex. Alanine scanning mutagenesis would not have illuminated these distinctions. It is shown that the concept of antigen specificity, as defined by cross-reactivity with natural variant antigens, is flawed by phylogenetic bias, and that specificity can only be defined by the use of unbiased epitopes, which are conveniently accessed by site-directed mutagenesis.
Pons
Jaume
J
Department of Molecular and Cell Biology, University of California, Berkeley 94720, USA.
Stratton
Jennifer R
JR
Kirsch
Jack F
JF
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000915:Antibody Affinity / Q000235:genetics / Q000276:immunology; D000918:Antibody Specificity / Q000235:genetics / Q000276:immunology; D002645:Chickens / Q000276:immunology*; D017124:Conserved Sequence; D004527:Egg Proteins / Q000737:chemistry / Q000276:immunology*; D018604:Epitope Mapping; D000939:Epitopes; D009113:Muramidase / Q000737:chemistry / Q000276:immunology*; D009154:Mutation; D011485:Protein Binding / Q000235:genetics / Q000276:immunology
D000906:Antibodies; D004527:Egg Proteins; D000939:Epitopes; D009113:Muramidase