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

FEBS Lett. 1997 Oct 20;416(2):193-6. doi: 10.1016/s0014-5793(97)01201-5.

Abstract

The human proteinase inhibitor, alpha2-macroglobulin (a2-M), inhibits a large number of proteinases. Alpha2-M-proteinase complexes are rapidly cleared from the circulation by binding to a cellular receptor (alpha2-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 alpha2-M (a2-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-x1-x2-D-x3-x4-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 alpha2-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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibodies, Monoclonal*
  • Binding Sites
  • Binding, Competitive
  • Cattle
  • Epitopes / chemistry*
  • Humans
  • Kinetics
  • Low Density Lipoprotein Receptor-Related Protein-1
  • Models, Structural
  • Molecular Sequence Data
  • Molecular Weight
  • Peptide Library*
  • Protein Conformation
  • Receptors, Immunologic / chemistry*
  • Receptors, Immunologic / metabolism*
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • alpha-Macroglobulins / chemistry*
  • alpha-Macroglobulins / immunology
  • alpha-Macroglobulins / metabolism*

Substances

  • Antibodies, Monoclonal
  • Epitopes
  • Low Density Lipoprotein Receptor-Related Protein-1
  • Peptide Library
  • Receptors, Immunologic
  • alpha-Macroglobulins