title: Precise mapping of the Goodpasture epitope(s) using phage display, site-directed mutagenesis, and surface plasmon resonance.
abstract: Goodpasture disease is an autoimmune disorder mediated by circulating autoantibodies against the noncollagenous-1 (NC1) domain of the α3 chain of type IV collagen (α3(IV)NC1). The structure of Goodpasture epitope(s) has been previously mapped into two main binding regions (E(A) and E(B)) of the α3(IV)NC1 domain using a residue mutation approach on the highly related α1(IV)NC1 domain. Here we combined phage display and surface plasmon resonance technology to more precisely localize the pathogenic binding sites. Peptides mimicking the Goodpasture epitope(s) were used to identify residues involved in autoantibody binding and found involvement of eight residues previously unrecognized within and outside of the E(A) or E(B) regions. Residue involvement in pathogenic reactivity was confirmed by site-directed mutagenesis on a more divergent α2(IV)NC1 molecule. From a mutant (M1) of the α2(IV)NC1 molecule, harboring residues previously identified as belonging to the Goodpasture epitope, additional chimeras were generated on the bases of phage display findings. All these mutants were shown to display higher reactivity with circulating Goodpasture autoantibodies than the M1 mutant. Thus, our results more precisely define Goodpasture epitope determinants and open new avenues to delineate comprehensive autoantibody-blocking agents for therapeutics.
authors.affiliation: Centro de Investigación Príncipe Felipe (CIPF), Valencia, Spain.
authors.forename: Roberto
authors.initials: R
authors.lastname: Gozalbo-Rovira
authors.forename: Jesús
authors.initials: J
authors.lastname: Rodríguez-Díaz
authors.forename: Juan
authors.initials: J
authors.lastname: Saus
authors.forename: Javier
authors.initials: J
authors.lastname: Cervera
mesh_terms: D001323:Autoantibodies / Q000276:immunology; D001324:Autoantigens / Q000737:chemistry / Q000276:immunology*; D001666:Binding Sites, Antibody; D061505:Cell Surface Display Techniques / Q000379:methods*; D024141:Collagen Type IV / Q000737:chemistry / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping / Q000379:methods*; D006801:Humans; D008297:Male; D008875:Middle Aged; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D020349:Surface Plasmon Resonance / Q000379:methods*
chemical_list: D001323:Autoantibodies; D001324:Autoantigens; D024141:Collagen Type IV; C057112:type IV collagen alpha3 chain