The pathogenicity of T cell epitopes on human Goodpasture antigen and its critical amino acid motif

Abstract

Goodpasture antigen, the non-collagenous domain of α3 chain of type IV collagen [α3(IV)NC1], is the target antigen of anti-glomerular basement membrane (GBM) antibodies. The pathogenicity of T cell epitopes is not elucidated clearly. In this study, we aim to define the nephritogenic T cell epitopes and its critical amino acid residues. Twenty-four overlapping linear peptides were synthesized covering the whole sequence of human α3(IV)NC1. Wistar-Kyoto rats were immunized with linear peptides, and experimental autoimmune glomerulonephritis was evaluated. Critical amino acid was identified by the loss of nephritogenic function after each amino acid substitution by alanine. Of the 24 peptides, P14 (α3_127-148 ) could induce 90.5% (19/21) of WKY rats developing anti-GBM glomerulonephritis with proteinuria, elevated serum urea and creatinine, IgG linear deposit on GBM and substantial (in average 82.4 ± 5.6%) crescent formation in glomeruli. Lymphocytes of immunized rats proliferated in response to α3_127-148 and α3(IV)NC1 in vitro. Sera of these rats recognized α3_127-148 and later on together with intact human α3(IV)NC1. Antibodies towards α3_127-148 and intact α3(IV)NC1 could also be detected from the kidney elutes. These antibodies showed no cross-reaction with each other, which implies intramolecular epitope spreading during disease progress. After sequential amino acid substitution, the α3_127-148 with substitution of tryptophan₁₃₆ , isoleucine₁₃₇ , leucine₁₃₉ or tryptophan₁₄₀ lost its nephritogenicity. Human α3_127-148 is a nephritogenic T cell epitope in WKY rats, with the critical amino acids as W₁₃₆ I₁₃₇ xL₁₃₉ W₁₄₀ . These findings might facilitate future investigation on microbial aetiology and potential specific immunotherapy of anti-GBM disease.

Keywords: Goodpasture antigen; T cell; anti-GBM disease; epitope; experimental autoimmune glomerulonephritis.