Molecular and structural analysis of a continuous birch profilin epitope defined by a monoclonal antibody.
The interaction of a mouse monoclonal antibody (4A6) and birch profilin, a structurally well conserved actin- and phosphoinositide-binding protein and cross-reactive allergen, was characterized. In contrast to serum IgE from allergic patients, which shows cross-reactivity with most plants, monoclonal antibody 4A6 selectively reacted with tree pollen profilins. Using synthetic overlapping peptides, a continuous hexapeptide epitope was identified. The exchange of a single amino acid (Gln-47 --> Glu) within the epitope was found to abolish the binding of monoclonal antibody 4A6 to other plant profilins. The NMR analyses of the birch and the nonreactive timothy grass profilin peptides showed that the loss of binding was not due to major structural differences. Both peptides adopted extended conformations similar to that observed for the epitope in the x-ray crystal structure of the native birch profilin. Binding studies with peptides and birch profilin mutants generated by in vitro mutagenesis demonstrated that the change of Gln-47 to acidic amino acids (e.g. Glu or Asp) led to electrostatic repulsion of monoclonal antibody 4A6. In conclusion the molecular and structural analyses of the interaction of a monoclonal antibody with a continuous peptide epitope, recognized in a conformation similar to that displayed on the native protein, are presented.
Wiedemann
P
P
Institute of General and Experimental Pathology, University of Vienna, A-1090 Vienna, Austria.
Giehl
K
K
Almo
S C
SC
Fedorov
A A
AA
Girvin
M
M
Steinberger
P
P
Rüdiger
M
M
Ortner
M
M
Sippl
M
M
Dolecek
C
C
Kraft
D
D
Jockusch
B
B
Valenta
R
R
D000485:Allergens / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D003285:Contractile Proteins; D003429:Cross Reactions; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology; D005260:Female; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008807:Mice, Inbred BALB C; D008840:Microfilament Proteins / Q000276:immunology*; D008969:Molecular Sequence Data; D051304:Profilins; D011994:Recombinant Proteins / Q000276:immunology; D014197:Trees
D000485:Allergens; D000911:Antibodies, Monoclonal; D003285:Contractile Proteins; D000939:Epitopes; D008840:Microfilament Proteins; C495739:Pfn1 protein, mouse; D051304:Profilins; D011994:Recombinant Proteins