Precise mapping of the Goodpasture epitope(s) using phage display, site-directed mutagenesis, and surface plasmon resonance

Goodpasture disease is an autoimmune disorder mediated by circulating autoantibodies against the noncollagenous-1 (NC1) domain of the alpha 3 chain of type IV collagen (alpha 3(IV)NC1). The structure of Goodpasture epitope(s) has been previously mapped into two main binding regions (E-A and E-B) of the alpha 3(IV)NC1 domain using a residue mutation approach on the highly related alpha 1(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 sitedirected mutagenesis on a more divergent alpha 2(IV)NC1 molecule. From a mutant (M1) of the alpha 2(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. Kidney International (2013) 83, 438-445; doi:10.1038/ki.2012.399; published online 19 December 2012