Epitopic characterization of neuropeptide Y (NPY) by alanine-scanning mutagenesis

Characterization of the epitopic structures of neuropeptide Y (NPY) has been studied by alanine-scanning mutagenesis, based on our previous investigation of a panel of six murine anti-NPY IgM monoclonal antibodies. To evaluate the structural requirement for these anti-NPY IgM antibodies, recognition variants of the native sequences of the NPY fragment (19-36) were prepared by single alanine substitutions in residues 22 and 25-36. Their binding to these antibodies was examined by competitive inhibition assays. The results demonstrated that the epitopic structures are largely confined to residues 25-36 of NPY and the C-terminal residues of NPY are essential for these anti-NPY IgM antibodies recognition. It emphasizes the notion that even small regions of a protein consisting of as few as 15 residues (22-36) can exhibit multiple epitopic structures. In several anti-NPY IgM antibodies, pairs of residues on opposite faces of the alpha-helix interact with the antibody site, which indicates that the antibody site consists either of a cavity or a deep groove either of which encompasses the alpha-helical segment sufficiently to allow simultaneous contact with most of the residues of this segment.