title: Two monoclonal antibodies to precisely the same epitope of type II collagen select non-crossreactive phage clones by phage display: implications for autoimmunity and molecular mimicry.
abstract: Two monoclonal antibodies (mAb) CB268 and CII-C1 to type II collagen (CII) react with precisely the same conformational epitope constituted by the residues ARGLT on the three chains of the CII triple helix. The antibodies share structural similarity, with most differences in the complementarity determining region 3 of the heavy chain (HCDR3). The fine reactivity of these mAbs was investigated by screening two nonameric phage-displayed random peptide libraries. For each mAb, there were phage clones (phagotopes) that reacted strongly by ELISA only with the selecting mAb, and inhibited binding to CII only for that mAb, not the alternate mAb. Nonetheless, a synthetic peptide RRLPFGSQM corresponding to an insert from a highly reactive CII-C1-selected phagotope, which was unreactive (and non-inhibitory) with CB268, inhibited the reactivity of CB268 with CII. Most phage-displayed peptides contained a motif in the first part of the molecule that consisted of two basic residues adjacent to at least one hydrophobic residue (e.g. RRL or LRR), but the second portion of the peptides differed for the two mAbs. We predict that conserved CDR sequences interact with the basic-basic-hydrophobic motif, whereas non-conserved amino acids in the binding sites (especially HCDR3) interact with unique peptide sequences and limit cross-reactivity. The observation that two mAbs can react identically with a single epitope on one antigen (CII), but show no cross-reactivity when tested against a second (phagotope) indicates that microorganisms could exhibit mimics capable of initiating autoimmunity without this being evident from conventional assays.
authors.affiliation: Department of Biochemistry and Molecular Biology, Box 13D, Monash University, Clayton 3800, Australia.
authors.forename: Yuekang
authors.initials: Y
authors.lastname: Xu
authors.forename: Paul A
authors.initials: PA
authors.lastname: Ramsland
authors.forename: Janet M
authors.initials: JM
authors.lastname: Davies
authors.forename: Marita
authors.initials: M
authors.lastname: Scealy
authors.forename: Kutty Selva
authors.initials: KS
authors.lastname: Nandakumar
authors.forename: Rikard
authors.initials: R
authors.lastname: Holmdahl
authors.forename: Merrill J
authors.initials: MJ
authors.lastname: Rowley
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D015551:Autoimmunity; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D002417:Cattle; D024043:Collagen Type II / Q000737:chemistry / Q000276:immunology*; D016384:Consensus Sequence; D003429:Cross Reactions; D018360:Crystallography, X-Ray; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D057927:Hydrophobic and Hydrophilic Interactions; D051379:Mice; D008811:Mice, Inbred DBA; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019151:Peptide Library; D011487:Protein Conformation
chemical_list: D000911:Antibodies, Monoclonal; D024043:Collagen Type II; D000939:Epitopes; D019151:Peptide Library