Use of molecular modeling and site-directed mutagenesis to define the structural basis for the immune response to carbohydrate xenoantigens

Background: Natural antibodies directed at carbohydrates reject porcine xenografts. They are initially expressed in germline configuration and are encoded by a small number of structurally-related germline progenitors. The transplantation of genetically-modified pig organs prevents hyperacute rejection, but delayed graft rejection still occurs, partly due to humoral responses. IgV(H) genes encoding induced xenoantibodies are predominantly, not exclusively, derived from germline progenitors in the V(H)3 family. We have previously identified the immunoglobulin heavy chain genes encoding V(H)3 xenoantibodies in patients and primates. In this manuscript, we complete the structural analysis of induced xenoantibodies by identifying the IgV(H) genes encoding the small proportion of V(H)4 xenoantibodies and the germline progenitors encoding xenoantibody light chains. This information has been used to define the xenoantibody/carbohydrate binding site using computer-simulated modeling. Results: The VH4-59 gene encodes antibodies in the V(H)4 family that are induced in human patients mounting active xenoantibody responses. The light chain of xenoantibodies is encoded by DPK5 and HSIGKV134. The structural information obtained by sequencing analysis was used to create computer-simulated models. Key contact sites for xenoantibody/carbohydrate interaction for V(H)3 family xenoantibodies include amino acids in sites 31, 33, 50, 57, 58 and the CDR3 region of the IgV(H) gene. Site-directed mutagenesis indicates that mutations in predicted contact sites alter binding to carbohydrate xenoantigens. Computer-simulated modeling suggests that the CDR3 region directly influences binding. Conclusion: Xenoantibodies induced during early and delayed xenograft responses are predominantly encoded by genes in the V(H)3 family, with a small proportion encoded by V(H)4 germline progenitors. This restricted group can be identified by the unique canonical structure of the light chain, heavy chain and CDR3. Computer-simulated models depict this structure with accuracy, as confirmed by site-directed mutagenesis. Computer-simulated drug design using computer-simulated models may now be applied to develop new drugs that may enhance the survival of xenografted organs.