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.
Deng
Y J
YJ
Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia 19104, USA.
Andrews
G C
GC
Karush
F
F
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001667:Binding, Competitive; D000939:Epitopes / Q000737:chemistry / Q000235:genetics*; D006825:Hybridomas / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009478:Neuropeptide Y / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D009478:Neuropeptide Y; D000409:Alanine