EPITOPE MAPPING BY SCREENING OF PHAGE DISPLAY LIBRARIES OF A MONOCLONAL-ANTIBODY DIRECTED AGAINST THE RECEPTOR-BINDING DOMAIN OF HUMAN ALPHA-2-MACROGLOBULIN

The human proteinase inhibitor, alpha 2-macroglobulin (alpha 2-M), inh ibits a large number of proteinases. alpha 2-M-proteinase complexes ar e rapidly cleared from the circulation by binding to a cellular recept or (alpha 2-M-R/LRP) via the receptor binding domain (RBD) which is ma de up of a 20 kDa C-terminal stretch of the 180 kDa monomer of the inh ibitor. A monoclonal antibody (mab alpha-1) has been described which r eacts with the receptor-recognizable form of the inhibitor, the so cal led transformed alpha 2-M(alpha 2-Mt). By screening of a phage display library an epitope in the RED of the inhibitor was identified that re acts with mab alpha-1. Out of 25 phage clones a heptapeptide sequence (S-x(1)-x(2)-D-x(3)-x(4)-K) was obtained containing identical amino ac ids in three positions. A consensus peptide (S-R-S-D-P-P-K) was synthe sized and found to displace alpha 2-Mt from binding to mab alpha-1 and to receptor. The specificity of competition was demonstrated by a rev ersed peptide and a control antibody, By structural comparison it was found that the consensus heptapeptide mimics a discontinuous conformat ionally constrained epitope present in the RED of the inhibitor. This is the first report describing the detection of discontinuous epitopes by phage display using a short linear peptide. (C) 1997 Federation of European Biochemical Societies.