title: Monoclonal antibody screening of a phage-displayed random peptide library reveals mimotopes of chemokine receptor CCR5: implications for the tertiary structure of the receptor and for an N-terminal binding site for HIV-1 gp120.
abstract: The chemokine receptor CCR5 contains seven transmembrane-spanning domains. It binds chemokines and acts as co-receptor for macrophage (m)-tropic (or R5) strains of HIV-1. Monoclonal antibodies (mAb) to CCR5, 3A9 and 5C7, were used for biopanning a nonapeptide cysteine (C)-constrained phage-displayed random peptide library to ascertain contact residues and define tertiary structures of possible epitopes on CCR5. Reactivity of antibodies with phagotopes was established by enzyme-linked immunosorbent assay (ELISA). mAb 3A9 identified a phagotope C-HASIYDFGS-C (3A9 / 1), and 5C7 most frequently identified C-PHWLRDLRV-C (5C7 / 1). Corresponding peptides were synthesized. Phagotopes and synthetic peptides reacted in ELISA with corresponding antibodies and synthetic peptides inhibited antibody binding to the phagotopes. Reactivity by immunofluorescence of 3A9 with CCR5 was strongly inhibited by the corresponding peptide. Both mAb 3A9 and 5C7 reacted similarly with phagotopes and the corresponding peptide selected by the alternative mAb. The sequences of peptide inserts of phagotopes could be aligned as mimotopes of the sequence of CCR5. For phage 3A9 / 1, the motif SIYD aligned to residues at the N terminus and FG to residues on the first extracellular loop; for 5C7 / 1, residues at the N terminus, first extracellular loop, and possibly the third extracellular loop could be aligned and so would contribute to the mimotope. The synthetic peptides corresponding to the isolated phagotopes showed a CD4-dependent reactivity with gp120 of a primary, m-tropic HIV-1 isolate. Thus reactivity of antibodies raised to CCR5 against phage-displayed peptides defined mimotopes that reflect binding sites for these antibodies and reveal a part of the gp120 binding sites on CCR5.
authors.affiliation: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia.
authors.forename: C
authors.initials: C
authors.lastname: Königs
authors.forename: M J
authors.initials: MJ
authors.lastname: Rowley
authors.forename: P
authors.initials: P
authors.lastname: Thompson
authors.forename: M A
authors.initials: MA
authors.lastname: Myers
authors.forename: M
authors.initials: M
authors.lastname: Scealy
authors.forename: J M
authors.initials: JM
authors.lastname: Davies
authors.forename: L
authors.initials: L
authors.lastname: Wu
authors.forename: U
authors.initials: U
authors.lastname: Dietrich
authors.forename: C R
authors.initials: CR
authors.lastname: Mackay
authors.forename: I R
authors.initials: IR
authors.lastname: Mackay
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000915:Antibody Affinity; D000918:Antibody Specificity; D001402:B-Lymphocytes; D001667:Binding, Competitive; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D015699:HIV Envelope Protein gp120 / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D018716:Molecular Mimicry; D019151:Peptide Library; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D019713:Receptors, CCR5 / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D015699:HIV Envelope Protein gp120; D019151:Peptide Library; D010455:Peptides; D019713:Receptors, CCR5