Epitope mapping of anti-glomerular basement membrane (GBM) antibodies with synthetic peptides.
Autoantibodies to the non-collagenous (NC1) domain of the alpha 3(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 alpha 1(IV)-chain only. Overlapping synthetic peptides, covering the complete NC1 domains of the alpha 1(IV)- and alpha 3(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 alpha 1(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 alpha 1(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 alpha 1(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-alpha 1(IV) antibodies had a mild disease, while the Goodpasture patients with anti-alpha 3(IV) antibodies had a rapidly progressive disease.
Hellmark
T
T
Department of Nephrology, Lund University, Sweden.
Brunmark
C
C
Trojnar
J
J
Wieslander
J
J
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D019867:Anti-Glomerular Basement Membrane Disease / Q000276:immunology; D000918:Antibody Specificity; D001323:Autoantibodies / Q000737:chemistry*; D001485:Basement Membrane / Q000276:immunology; D003094:Collagen / Q000276:immunology; D018604:Epitope Mapping; D006801:Humans; D007678:Kidney Glomerulus / Q000276:immunology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry* / Q000276:immunology*
D000596:Amino Acids; D001323:Autoantibodies; D010446:Peptide Fragments; D003094:Collagen