EPITOPE MAPPING OF ANTIGLOMERULAR 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-GB M nephritis. These antibodies have been shown to be pathogenic. In thi s study the antibody specificity has been investigated in patients wit h Goodpasture's syndrome and from a patient with atypical anti-GBM ant ibodies, recognizing the alpha 1(IV)-chain only. Overlapping synthetic peptides, covering the complete NC1 domains of the alpha 1(IV)- and a lpha 3(IV)-chains were used in sandwich ELISA and competitive ELISA. N one of the Goodpasture sera showed reactivity to the synthetic peptide s. However, antibodies from the patient with atypical anti-GBM antibod ies 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 fou r last C-terminal amino acids of the alpha 1(IV)-chain, with the seque nce 1754-MRRT. The two arginine residues were found to be essential fo r antibody binding. Threonine is important, while methionine is of les s importance. These four amino acids are also determined to be the sma llest peptide that could inhibit the binding of the autoantibodies to the native alpha 1(IV)-chain. This study shows that overlapping peptid es can be used to map linear epitopes. However, for conformational epi topes 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 ha d a mild disease, while the Goodpasture patients with anti-alpha 3(IV) antibodies had a rapidly progressive disease.