Carbohydrates contribute to the interactions between cockroach allergen Bla g 2 and a monoclonal antibody.
The crystal structure of a murine mAb, 4C3, that binds to the C-terminal lobe of the cockroach allergen Bla g 2 has been solved at 1.8 Å resolution. Binding of 4C3 involves different types of molecular interactions with its epitope compared with those with the mAb 7C11, which binds to the N-terminal lobe of Bla g 2. We found that the 4C3 surface epitope on Bla g 2 includes a carbohydrate moiety attached to Asn(268) and that a large number of Ag-Ab contacts are mediated by water molecules and ions, most likely zinc. Ab binding experiments conducted with an enzymatically deglycosylated Bla g 2 and a N268Q mutant showed that the carbohydrate contributes, without being essential, to the Bla g 2-4C3 mAb interaction. Inhibition of IgE Ab binding by the mAb 4C3 shows a correlation of the structurally defined epitope with reactivity with human IgE. Site-directed mutagenesis of the 4C3 mAb epitope confirmed that the amino acids Lys(251), Glu(233), and Ile(199) are important for the recognition of Bla g 2 by the 4C3 mAb. The results show the relevance of x-ray crystallographic studies of allergen-Ab complexes to identify conformational epitopes that define the antigenic surface of Bla g 2.
Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, MD 21702, USA.
Mi
M
Li
Alla
A
Gustchina
Jill
J
Glesner
Sabina
S
Wünschmann
Lisa D
LD
Vailes
Martin D
MD
Chapman
Anna
A
Pomés
Alexander
A
Wlodawer
D000117:Acetylglucosamine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000485:Allergens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism*; D000937:Antigen-Antibody Reactions; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003058:Cockroaches / Q000276:immunology*; D018360:Crystallography, X-Ray; D006031:Glycosylation; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000276:immunology; D011487:Protein Conformation
D000485:Allergens; D000911:Antibodies, Monoclonal; D016282:Aspartic Acid Endopeptidases; C094699:allergen Bla g 2; D000117:Acetylglucosamine