Isolation and structure-functional characterization of phage display library-derived mimotopes of noxiustoxin, a neurotoxin of the scorpion Centruroides noxius Hoffmann

Noxiustoxin (NTX) is a short-chain toxin from the venom of the scorpion Cen truroides noxius Hoffmann, whose molecular structure and physiological effe cts have been characterized in detail, whereas the antigenic properties of this and other K+ channel-blocking toxins are poorly studied. A monoclonal antibody against NTX, BNTX18, able to inhibit the binding of NTX to rat bra in synaptosomes, was used in the present study for selecting immunoreactive peptides, mimotopes, from a 12mer and a 7mer phage library. The peptides w ere characterized immunologically and used for mapping the epitope on NTX. In total, 75 phage clones carrying 43 different peptides were analyzed of w hich 42 clones carrying 17 different peptides, twelve 12mer and five 7mer p eptides, presented a single consensus motif: Leu(Ile, Val)-Tyr(Phe, Trp, Le u)-Gly-Met(Ala). All but three of the peptides containing this motif were r eactive with selected mAb BNTX18 in a dot-blot assay of which eight were cl early positive in ELISA and exhibited in competition-inhibition assay the a ntibody binding specificity of the NTX epitope recognized by BNTX18. The tw o most reactive mimotopes injected into mice showed the ability to induce a ntibodies reacting with NTX, thus, to mimic the epitope of NTX antigenicall y. Sequence comparison and the analysis of the three-dimensional structure of NTX led to the proposal that residues Glu19-Leu20-Tyr21-Gly22 and the hy drophobic part of the side chain of Lys18 form the C-terminal part of the e pitope. Due to the frequent presence of residues Pro, Leu, Thr, Arg, and Gl n in the N-terminal part of the mimotopes, corresponding homologous residue s in the N-terminal proximity of the partial epitope may be part of an addi tional more hydrophilic epitope element. (C) 2001 Elsevier Science Ltd. All rights reserved.