Volume 83, Issue 3, Pages 438-445 (March 2013) Precise mapping of the Goodpasture epitope(s) using phage display, site-directed mutagenesis, and surface plasmon resonance  Roberto Gozalbo-Rovira, Jesús Rodríguez-Díaz, Juan Saus, Javier Cervera  Kidney International  Volume 83, Issue 3, Pages 438-445 (March 2013) DOI: 10.1038/ki.2012.399 Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 1 Capture ELISA of cGP antibodies against phage display–selected peptides. Reactivity of phagotopes from the constrained library (c7c_1-8) and from the linear dodecapeptide library (12_1-3). cGP, circulating Goodpasture. Kidney International 2013 83, 438-445DOI: (10.1038/ki.2012.399) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 2 Quaternary organization of the α3,4,5(IV)NC1 hexamer. The hexamer is composed by two identical protomers containing one α3 chain (red), along with an α4 (blue) chain and an α5 (green) chain, respectively. The EA (yellow) and EB (purple) are depicted on the α3(IV)NCI surface. Residues proposed in this study to contribute to the GP epitope appear written and numbered in cyan. Kidney International 2013 83, 438-445DOI: (10.1038/ki.2012.399) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 3 Predicted residues from Pepsurf. The frequency of predicted residues from Pepsurf results using the selected phage display peptides is depicted on the amino-acid sequence of α3(IV)NC1. The residues involved in EA and EB regions are identified with blue and red bars, respectively. Kidney International 2013 83, 438-445DOI: (10.1038/ki.2012.399) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 4 Mutant M1–M6 forms of α2(IV)NC1. The higher bar (which would correspond with mutant M6) schematizes the α2(IV)NC1 molecule showing the 16 residues mutated (upper half of the bar) by their corresponding residues in α3(IV)NC1 (lower half of the bar). Thick lines in M1–M6 mutants represent the corresponding residues from the upper bar mutated in each type of α2(IV)NC1 mutant molecule. Kidney International 2013 83, 438-445DOI: (10.1038/ki.2012.399) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 5 ClustalW alignment of the sequences of α1,α2,α3(IV)NC1 domains. The EA and EB regions are gray shadowed. The nine amino acids previously described as epitope components, which are mutated in M1, are in bold (note that Ser31 is common between α2(IV)NC1 and α3(IV)NC1). Mutated residues in α2(IV)NC1 appear identified with the name of the chimera that contains it. Kidney International 2013 83, 438-445DOI: (10.1038/ki.2012.399) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 6 Chimera analysis. (a) SDS-polyacrylamide gel (PAGE) analysis of the chimeras. SDS-PAGE electrophoresis was performed in 12% gels under reducing conditions, and the proteins were Coomassie blue stained. (b) Immune detection of the chimeras. Ponceau red staining of nonreducing SDS-PAGE electrophoresis blotted onto nitrocellulose membranes (A). Protein immunodetection with α-FLAG antibody (B) or cGP antibodies (C). Kidney International 2013 83, 438-445DOI: (10.1038/ki.2012.399) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 7 ELISA of the reactivity of the cGP antibodies against the α3(IV)NC1 domain and different forms of the α2(IV)NC1 domain. The asterisks indicate significant differences P<0.01. Kidney International 2013 83, 438-445DOI: (10.1038/ki.2012.399) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 8 Surface plasmon resonance (SPR) analysis of the chimeras. SPR analysis of the effect of the mutations M1,4-6 on the affinity of cGP antibodies for the α2(IV)NC1 domain. (a) Affinity constant (KD) of the cGP antibodies. (b) Rmax value of binding. Kidney International 2013 83, 438-445DOI: (10.1038/ki.2012.399) Copyright © 2013 International Society of Nephrology Terms and Conditions