Epitope mapping of anti-glomerular basement membrane (GBM) antibodies with synthetic peptides

Abstract

Autoantibodies to the non‐collagenous (NC1) domain of the α3(IV)‐chain of type IV collagen are found in sera from patients with anti‐GBM nephritis. These antibodies have been shown to be pathogenic. In this study the antibody specificity has been investigated in patients with Goodpasture’s syndrome and from a patient with atypical anti‐GBM antibodies, recognizing the α1(IV)‐chain only. Overlapping synthetic peptides, covering the complete NC1 domains of the α1(IV)‐ and α3(IV)‐chains were used in sandwich ELISA and competitive ELISA. None of the Goodpasture sera showed reactivity to the synthetic peptides. However, antibodies from the patient with atypical anti‐GBM antibodies recognized a 20 amino acid peptide from the α1(IV)‐chain. The reactive peptide was further narrowed down with glycine substitution of the different amino acids. We have localized the epitope to the four last C‐terminal amino acids of the α1(IV)‐chain, with the sequence 1754‐MRRT. The two arginine residues were found to be essential for antibody binding. Threonine is important, while methionine is of less importance. These four amino acids are also determined to be the smallest peptide that could inhibit the binding of the autoantibodies to the native α1(IV)‐chain. This study shows that overlapping peptides can be used to map linear epitopes. However, for conformational epitopes such as the Goodpasture epitope, other methods must be used. It would be prognostically important to know the fine specificity of anti‐GBM antibodies, since the patient with anti‐α1(IV) antibodies had a mild disease, while the Goodpasture patients with anti‐α3(IV) antibodies had a rapidly progressive disease.

Antibodies against Linear Epitopes on the Goodpasture Autoantigen and Kidney Injury

“…Levy et al (23) identified nine reactive regions on a3(IV)NC1, whereas their results were not confirmed by Hellmark et al (24). Given the different designs of overlapping peptides and the relatively small groups of patients, no conclusions could be drawn from these studies.…”

Immune Recognition of Glomerular Antigens

“…They bind a conformational epitope destroyed by denaturation or reduction of native α3(IV)NC1. The importance of the shape of the epitope presumably explains the different results reported from early studies of binding of patient sera to linear synthetic peptides [16, 17, 18]. Recently, studies in three laboratories on patient sera binding to α3(IV)NC1/α1(IV)NC1 recombinant chimeras have agreed that all patients have antibodies that recognize an amino-terminal epitope [19, 20, 21].…”

A Self T Cell Epitope Induces Autoantibody Response: Mechanism for Production of Antibodies to Diverse Glomerular Basement Membrane Antigens

“…However, a synthetic peptide encoding the B cell epitope failed to induce GN or anti-GBM Ab (11,25). Mapping linear epitopes or generating mAbs to native GBM with peptides was not successful, because the Abs reacted only with denatured GBM proteins or did not react with GBM at all (9,20,21,23,24). Using more sophisticated point mutation and other techniques, several studies demonstrated that the anti-GBM Abs are bound to threedimensional (3-D) conformation of native Ags (26 -29).…”