Epitope mapping by screening of phage display libraries of a monoclonal antibody directed against the receptor binding domain of human alpha2-macroglobulin.
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.
Birkenmeier
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G
Institute of Biochemistry, Medical Faculty, University of Leipzig, Germany. birg@medizin.uni-leipzig.de
Osman
A A
AA
Kopperschläger
G
G
Mothes
T
T
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D000939:Epitopes / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D008961:Models, Structural; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019151:Peptide Library; D011487:Protein Conformation; D011971:Receptors, Immunologic / Q000737:chemistry* / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D000511:alpha-Macroglobulins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D019151:Peptide Library; D011971:Receptors, Immunologic; D000511:alpha-Macroglobulins