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Epitope mapping by screening of phage display libraries of a monoclonal antibody directed against the receptor binding domain of human alpha2-macroglobulin

Epitope mapping by screening of phage display libraries of a monoclonal antibody directed against the receptor binding domain of human alpha2-macroglobulin

Febs Letters 416(2): 193-196

ISSN/ISBN: 0014-5793

PMID: 9369213

The human proteinase inhibitor, alpha-2-macroglobulin (alpha-2-M), inhibits a large number of proteinases. alpha-2-M-proteinase complexes are rapidly cleared from the circulation by binding to a cellular receptor (alpha-2-M-R/LRP) via the receptor binding domain (RBD) which is made up of a 20 kDa C-terminal stretch of the 180 kDa monomer of the inhibitor. A monoclonal antibody (mab alpha-1) has been described which reacts with the receptor-recognizable form of the inhibitor, the so called transformed alpha-2-M (alpha-2-Mt). By screening of a phage display library an epitope in the RBD of the inhibitor was identified that reacts with mab alpha-1. Out of 25 phage clones a heptapeptide sequence (S-x-1-x-2-D-x-3x-4-K) was obtained containing identical amino acids in three positions. A consensus peptide (S-R-S-D-P-P-K) was synthesized and found to displace alpha-2-Mt from binding to mab alpha-1 and to receptor. The specificity of competition was demonstrated by a reversed peptide and a control antibody. By structural comparison it was found that the consensus heptapeptide mimics a discontinuous conformationally constrained epitope present in the RBD of the inhibitor. This is the first report describing the detection of discontinuous epitopes by phage display using a short linear peptide.

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Accession: 008619548

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