SUMMARY: Five adherence-inhibiting monoclonal antibodies (mAbs) were used for topological mapping of the binding sites of the 169 kDa membrane-integrated adhesin of . Antibody binding sites were characterized using overlapping synthetic octapeptides. Three regions of the protein seem to be involved in adherence: the N-terminal region [N-reg, epitopes beginning at amino acid (aa) 1 to aa 14 and aa 231 to aa 238, respectively]; a domain (D1) approximately in the middle of the molecule (beginning at aa 851 to aa 858 and aa 921 to aa 928); and a domain (D2) closer to the C-terminus (beginning at aa 1303 to aa 1310, aa 1391 to aa 1398 and aa 1407 to aa 1414). Each of the mAbs P1.26 and P1.62 reacted with two primary amino acid sequences. Both antibodies bound to the D1 region, but mAb P1.62 showed additional binding to a sequence (aa 231 to aa 238) near the N-terminus, and mAb P1.26 reacted with a second epitope in the D2 domain (aa 1303 to aa 1310). Such dual binding by the two antibodies suggests that in the native protein the epitopes are composed of two sequences which are located on two different sites of the molecule (D1/N-reg and D1/D2, respectively). In a competitive ELISA test using native cells as antigen, both the mAb directed to the N-terminus (aa 1 to aa 14) and mAbs directed to those epitopes nearest to the C-terminus (aa 1391 to aa 1398 and aa 1407 to aa 1414) were able to prevent binding of mAbs P1.26 and P1.62 to the native cells. The results of epitope mapping and inhibition tests suggest a close spatial relationship of the domains N-reg, D1 and D2 in forming a functionally active adherence binding site. Furthermore, the outermost epitopes of the domains involved seem to be located on top of folded loops of the protein molecule.


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