title: Use of molecular modeling and site-directed mutagenesis to define the structural basis for the immune response to carbohydrate xenoantigens.
abstract: 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. IgVH genes encoding induced xenoantibodies are predominantly, not exclusively, derived from germline progenitors in the VH3 family. We have previously identified the immunoglobulin heavy chain genes encoding VH3 xenoantibodies in patients and primates. In this manuscript, we complete the structural analysis of induced xenoantibodies by identifying the IgVH genes encoding the small proportion of VH4 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 VH4 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 VH3 family xenoantibodies include amino acids in sites 31, 33, 50, 57, 58 and the CDR3 region of the IgVH 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.
  
  
  CONCLUSIONS
  Xenoantibodies induced during early and delayed xenograft responses are predominantly encoded by genes in the VH3 family, with a small proportion encoded by VH4 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.
authors.affiliation: Department of Cardiothoracic Surgery, Saban Research Institute of the Children's Hospital of Los Angeles, University of Southern California Keck School of Medicine, Los Angeles, CA 90027 USA. mkearns@chla.usc.edu
authors.forename: Mary
authors.initials: M
authors.lastname: Kearns-Jonker
authors.forename: Natasha
authors.initials: N
authors.lastname: Barteneva
authors.forename: Robert
authors.initials: R
authors.lastname: Mencel
authors.forename: Namath
authors.initials: N
authors.lastname: Hussain
authors.forename: Irina
authors.initials: I
authors.lastname: Shulkin
authors.forename: Alan
authors.initials: A
authors.lastname: Xu
authors.forename: Margaret
authors.initials: M
authors.lastname: Yew
authors.forename: Donald V
authors.initials: DV
authors.lastname: Cramer
mesh_terms: D000818:Animals; D000910:Antibodies, Heterophile / Q000737:chemistry* / Q000235:genetics; D015478:Antigens, Heterophile / Q000737:chemistry* / Q000276:immunology; D002241:Carbohydrates / Q000737:chemistry* / Q000276:immunology; D002478:Cells, Cultured; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013552:Swine
chemical_list: D000910:Antibodies, Heterophile; D015478:Antigens, Heterophile; D002241:Carbohydrates; D000939:Epitopes