title: Identification of critical residues of linear B cell epitope on Goodpasture autoantigen.
abstract: BACKGROUND
  The autoantigen of anti-glomerular basement membrane (GBM) disease has been identified as the non-collagenous domain 1 of α3 chain of type IV collagen, α3(IV)NC1. Our previous study revealed a peptide on α3(IV)NC1 as a major linear epitope for B cells and potentially nephrogenic, designated as P14 (α3129-150). This peptide has also been proven to be the epitope of auto-reactive T cells in anti-GBM patients. This study was aimed to further characterize the critical motif of P14.
  
  
  METHODS
  16 patients with anti-GBM disease and positive anti-P14 antibodies were enrolled. A set of truncated and alanine substituted peptides derived from P14 were synthesized. Circulating antibodies against the peptides were detected by enzyme linked immunosorbent assay (ELISA).
  
  
  RESULTS
  We found that all sera with anti-P14 antibodies reacted with the 13-mer sequence in the C-terminus of P14 (P14c) exclusively. The level of antibodies against P14 was highly correlated with the level of antibodies against P14c (r=0.970, P<0.001). P14c was the core immunogenic region and the amino acid sequence (ISLWKGFSFIMFT) was highly hydrophobic. Each amino acid residue in P14c was sequentially replaced by alanine. Three residues of glycine142, phenylalanine143, and phenylalanine145 were identified crucial for antibody binding based on the remarkable decline (P<0.001) of antibody reaction after each residue replacement.
  
  
  CONCLUSIONS
  We defined GFxF (α3142, 143,145) as the critical motif of P14. It may provide some clues for understanding the etiology of anti-GBM disease.
authors: Renal Division, Department of Medicine, Peking University First Hospital; Institute of Nephrology, Peking University; Key Laboratory of Renal Disease, Ministry of Health of China; Key Laboratory of CKD Prevention and Treatment, Ministry of Education of China, Pekin, PR China.
authors: Xiao-yu
authors: XY
authors: Jia
authors: Renal Division, Department of Medicine, Peking University First Hospital; Institute of Nephrology, Peking University; Key Laboratory of Renal Disease, Ministry of Health of China; Key Laboratory of CKD Prevention and Treatment, Ministry of Education of China, Pekin, PR China.
authors: Zhao
authors: Z
authors: Cui
authors: Renal Division, Department of Medicine, Peking University First Hospital; Institute of Nephrology, Peking University; Key Laboratory of Renal Disease, Ministry of Health of China; Key Laboratory of CKD Prevention and Treatment, Ministry of Education of China, Pekin, PR China.
authors: Jian-nan
authors: JN
authors: Li
authors: Renal Division, Department of Medicine, Peking University First Hospital; Institute of Nephrology, Peking University; Key Laboratory of Renal Disease, Ministry of Health of China; Key Laboratory of CKD Prevention and Treatment, Ministry of Education of China, Pekin, PR China.
authors: Shui-yi
authors: SY
authors: Hu
authors: Renal Division, Department of Medicine, Peking University First Hospital; Institute of Nephrology, Peking University; Key Laboratory of Renal Disease, Ministry of Health of China; Key Laboratory of CKD Prevention and Treatment, Ministry of Education of China, Pekin, PR China; Peking-Tsinghua Center for Life Sciences, Peking, PR China.
authors: Ming-hui
authors: MH
authors: Zhao
mesh_terms: D000328:Adult; D000368:Aged; D019867:Anti-Glomerular Basement Membrane Disease / Q000276:immunology*; D000918:Antibody Specificity; D001323:Autoantibodies / Q000737:chemistry*; D001324:Autoantigens / Q000737:chemistry*; D024141:Collagen Type IV / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D018985:Epitopes, B-Lymphocyte / Q000737:chemistry*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D010455:Peptides / Q000737:chemistry; D017434:Protein Structure, Tertiary
chemical_list: D001323:Autoantibodies; D001324:Autoantigens; D024141:Collagen Type IV; D018985:Epitopes, B-Lymphocyte; D010455:Peptides; C051951:antiglomerular basement membrane antibody; C057112:type IV collagen alpha3 chain