Defining the requirements for an antibody epitope on influenza virus neuraminidase: how tolerant are protein epitopes?
To determine the conformational requirements for antibody recognition and extent of flexibility within a protein epitope, a chimeric influenza A virus neuraminidase (NA) has been constructed in which five discontinuous polypeptide segments from a subtype N9 NA, which comprise the monoclonal antibody NC41 epitope, have been grafted onto a subtype N2 NA. The resulting chimeric NA was expressed, assembled as a tetramer, and transported to the cell surface, but was not recognized by NC41 in immunoprecipitation experiments or by surface immunofluorescence. Although the N2 and N9 protein folds are identical and this chimera contains all the antibody contacts as defined by the crystal structure of the complex, NC41 binding was not achieved. Modeling studies suggest that at least one polypeptide segment is displaced from its normal position which would account for the observed lack of enzyme activity as well as lack of antibody binding. This implies that in addition to the specific critical interactions between NA and Fab residues required for antibody binding, the overall arrangement of amino acids within an epitope must be in a specific orientation that is necessary for initial antibody recognition.
Nuss
J M
JM
Department of Microbiology, University of Alabama at Birmingham 35294.
Air
G M
GM
D000595:Amino Acid Sequence; D000914:Antibodies, Viral / Q000378:metabolism*; D001483:Base Sequence; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology; D009980:Influenza A virus / Q000201:enzymology* / Q000276:immunology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009439:Neuraminidase / Q000737:chemistry / Q000276:immunology*; D011487:Protein Conformation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology
D000914:Antibodies, Viral; D000939:Epitopes; D011993:Recombinant Fusion Proteins; D009439:Neuraminidase