Requirement of multiple phage displayed peptide libraries for optimal mapping of a conformational antibody epitope on CCR5.
In the absence of information from crystallography, conformational epitopes can often be discerned by antibody screening of phage displayed random peptide libraries. However the context in which the peptide is displayed, and the number of copies displayed in the library, can influence results and interpretations. Here, the monoclonal antibodies 3A9 specific for the transmembrane chemokine receptor CCR5, and CII-C1 specific for type II collagen, were used to screen multiple phage-displayed peptide libraries in which peptides were displayed in either the pIII or pVIII coat proteins. ELISA was used to test for reactivity and cross-inhibitory activity of isolated phage clones. Based on sequences of reactive phage inserts, epitope motifs were initially inferred from a molecular model of CCR5 and subsequently confirmed experimentally using mutagenesis to alanine. For each mAb, phage sequences from pIII biopannings were more diverse than from pVIII biopannings. Notably, sequences from either biopanning were cross-inhibitory despite a lack of linear sequence homology. For CCR5, residues 88H and 94W in the first loop of CCR5 were identified by pIII biopannings, and 7S9IYD11 at the N-terminus by pVIII biopannings. Thus conformational epitopes can be identified using phage display, but optimal mapping of complex epitopes can require the use of multiple peptide libraries.
O'Connor
Karen H
KH
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, 3800, Australia.
Königs
Christoph
C
Rowley
Merrill J
MJ
Irving
James A
JA
Wijeyewickrema
Lakshmi C
LC
Pustowka
Anette
A
Dietrich
Ursula
U
Mackay
Ian R
IR
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology; D018604:Epitope Mapping / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D017433:Protein Structure, Secondary; D019713:Receptors, CCR5 / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*
D000911:Antibodies, Monoclonal; D019151:Peptide Library; D019713:Receptors, CCR5