Qiu-Hua Gu1, Xiao-Yu Jia1, Jian-Nan Li1, Fang-Jin Chen2, Zhao Cui3, Ming-Hui Zhao4. 1. Renal Division, Peking University First Hospital, Beijing 100034, PR China; Institute of Nephrology, Peking University, Beijing 100034, PR China; Key Laboratory of Renal Disease, Ministry of Health of China, Beijing 100034, PR China; Key Laboratory of CKD Prevention and Treatment, Ministry of Education of China, Beijing 100034, PR China. 2. State Key Laboratory for Structural Chemistry of Unstable and Stable Species, BNLMS, College of Chemistry and Molecular Engineering and Center for Theoretical Biology, Peking University, Beijing 100871, PR China. 3. Renal Division, Peking University First Hospital, Beijing 100034, PR China; Institute of Nephrology, Peking University, Beijing 100034, PR China; Key Laboratory of Renal Disease, Ministry of Health of China, Beijing 100034, PR China; Key Laboratory of CKD Prevention and Treatment, Ministry of Education of China, Beijing 100034, PR China. Electronic address: cuizhao@bjmu.edu.cn. 4. Renal Division, Peking University First Hospital, Beijing 100034, PR China; Institute of Nephrology, Peking University, Beijing 100034, PR China; Key Laboratory of Renal Disease, Ministry of Health of China, Beijing 100034, PR China; Key Laboratory of CKD Prevention and Treatment, Ministry of Education of China, Beijing 100034, PR China; Peking-Tsinghua Center for Life Sciences, Beijing 100871, PR China.
Abstract
BACKGROUND: Anti-GBM disease is caused by autoimmunity to Goodpasture antigen on α3(IV)NC1 and had strong associations with HLA-DRB1*1501. Previous studies identified α3127-148 (P14: TDIPPCPHGWISLWKGFSFIMF) as a T cell epitope. The present study was aimed to investigate the binding capacity of P14 to HLA-DRB1*1501 and the critical amino acids for this binding. METHODS: A line of EBV-transformed human B cells homozygous for HLA-DRB1*1501 was used to detect the binding capacity of peptides to HLA-DRB1*1501 using flow cytometry analysis. P14 was sequentially truncated into 8 peptides with 15 amino acids to identify the core binding motif. A set of alanine substituted peptides of P14-2 was then synthesized to identify its critical residues for binding to HLA-DRB1*1501. The structure of HLA-DR2b-Peptide-TCR complex was constructed by modeling to analyze the interaction of each amino acids of P14-2 with the HLA-DR2b molecule. RESULTS: P14 could bind to HLA-DRB1*1501 expressed on B cell surface. The N-terminus of P14 was the core binding motif and the truncated peptide P14-2 (DIPPCPHGWISLWKG) 128-142 had the strongest binding capacity. After sequential amino acid substitution, we found the binding capacity of P14-2 was completely lost by the substitution of cysteine (C) 132 and significantly decreased by the substitution of tryptophan (W) 136, lysine (K) 141, or glycine (G) 142, but still at a high level. The modeling showed that (C) 132 had a strong interaction with pocket 4 on the β chain of DR2b. Thus, C132, W 136, K141, and G142 were defined as the critical amino acid residues for the binding capacity of P14 to HLA-DRB1*1501. CONCLUSION: We identified α3128-142 (DIPPCPHGWISLWKG) as the core binding motif of P14 to HLA-DRB1*1501 molecule. And the critical amino acid residues for this binding were further defined as C132, W 136, K 141, and G 142.
BACKGROUND: Anti-GBM disease is caused by autoimmunity to Goodpasture antigen on α3(IV)NC1 and had strong associations with HLA-DRB1*1501. Previous studies identified α3127-148 (P14: TDIPPCPHGWISLWKGFSFIMF) as a T cell epitope. The present study was aimed to investigate the binding capacity of P14 to HLA-DRB1*1501 and the critical amino acids for this binding. METHODS: A line of EBV-transformed human B cells homozygous for HLA-DRB1*1501 was used to detect the binding capacity of peptides to HLA-DRB1*1501 using flow cytometry analysis. P14 was sequentially truncated into 8 peptides with 15 amino acids to identify the core binding motif. A set of alanine substituted peptides of P14-2 was then synthesized to identify its critical residues for binding to HLA-DRB1*1501. The structure of HLA-DR2b-Peptide-TCR complex was constructed by modeling to analyze the interaction of each amino acids of P14-2 with the HLA-DR2b molecule. RESULTS:P14 could bind to HLA-DRB1*1501 expressed on B cell surface. The N-terminus of P14 was the core binding motif and the truncated peptide P14-2 (DIPPCPHGWISLWKG) 128-142 had the strongest binding capacity. After sequential amino acid substitution, we found the binding capacity of P14-2 was completely lost by the substitution of cysteine (C) 132 and significantly decreased by the substitution of tryptophan (W) 136, lysine (K) 141, or glycine (G) 142, but still at a high level. The modeling showed that (C) 132 had a strong interaction with pocket 4 on the β chain of DR2b. Thus, C132, W 136, K141, and G142 were defined as the critical amino acid residues for the binding capacity of P14 to HLA-DRB1*1501. CONCLUSION: We identified α3128-142 (DIPPCPHGWISLWKG) as the core binding motif of P14 to HLA-DRB1*1501 molecule. And the critical amino acid residues for this binding were further defined as C132, W 136, K 141, and G 142.