The Functional Epitope Study Based On The Spatial Structure Of Antigen And Antibody

An epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells. Following this, antigen can trigger a immune response.The interaction between antibody and its antigen is the heart of the immune response. A series of research works showed that the precise localization of an epitope can be essential in the development of biomedical applications such as rationally designed vaccines, designed and screening novel drugs and immuno-therapeutics.In the past years, several experimental techniques have been developed for mapping antibody interacting residues in antigen that includes identification of interacting residues from 3-D antibody-antigen complex structure. One of the popular approaches is overlapping peptide synthesis covering the entire antigen sequence, which identifies mainly sequential epitopes. However, Mapping of antibody interacting residues was hampered by the costly and time taking process of 3D structure determination.With the large increase of solved antibody–antigen co-crystal structures in the protein data bank (PDB), it is now possible to perform a large-scale analysis to define epitope characteristics and reveal new aspects of immunological molecular recognition.In the present work, we chose C5a,TNF as research targets, and with their antibodies (TGMB(i.e. anti-TNF functional antibody),F20 and P29(i.e. anti-C5a functional antibody)) in possession of intellectual property to study the interaction between the antigen and antibody and determine the binding domains. According to the crystal structure of antigen and theoretical modeling structure of antibody fragment, the interaction mode between the antigen and antibody was studied theoretically. The key residue in antigen identified by antibody was predicted and tested by mutant experiment. Some details of this research were shown as following: 1. The binding domains between TNF and TGMB were determined theoretically .The functional antigen epitopes were predicted and tested by mutant experiment.As an important cytokine, Tumor Neurous Factor-α(TNF-α) involves in many physiological function. However, aberrant production of TNF-αhas been discovered to mediate or be involved in many pathological processes including rheumatoid arthritis (RA), Crohn's disease (CD), etc. Neutralization of TNF-αhas become an effective therapeutic strategy for these diseases.In this work , based on computer-guided homology modeling and conformation optimization method, the 3-D structure of TGMB-Fv fragment was constructed and minimized. Using molecular docking and dynamics simulation method, the 3-D stable complex structure of TNF and TGMB-Fv fragment was obtained. Considering the orientation property, surface electrostatic distribution, residues chemical-physical property and intermolecular hydrogen bond, the binding mode and functional antigen epitopes were predicted and mutants were designed rationally. Further experimental results showed the same results as the theoretical predictions.2. The binding domains between C5a and F20 or P29 were determined theoretically. The functional antigen epitopes were predicted and tested by mutant experiment.Among the complement activation products, C5a is one of the most potent inflammatory peptides with a broad spectrum of functions. C5a causes an oxidative burst in neutrophils and enhances phagocytosis and release of granule enzymes. Inhibition of C5a by antibodies has been demonstrated to dramatically improve survival in various sepsis models in mice and rats.The structural basis of C5a mediated bioactivity and C5a antibody mediated neutralization are of interesting to be investigated. In this study, with computer-guided modeling method, the 3-D theoretical structure of F20 or P29 fragment was constructed. Using the crystal structure of C5a, the 3-D complex structure of C5a and F20 or P29 fragment was modeled with molecular docking method. Based on distance geometry method and intermolecular interaction theory, the key residue K68 or D31,R37in C5a identified by F20 or P29 was predicted. The mutant experimental results showed that the residue Lys68 or D31,R37was the critical residue of C5a for it`s bioactivity and F20 or P29 binding activity. In summary, we confirmed the residue D143 was the critical residue of TNF for it`s bioactivity and TGMB binding activity and residue D31,R37 or K68 was the critical residue of C5a for it`s bioactivity and P29 or F20 binding activity. The present study shed new light on the structural basis of C5a or TNF mediated bioactivity. The identification of the critical residue will provide useful information for C5a or TNF targeted therapeutic intervention.