title: Identification of conserved amino acids in murine B7-1IgV domain critical for CTLA4/CD28:B7 interaction by site-directed mutagenesis: a novel structural model of the binding site.
abstract: The B7: CD28/CTLA4 interaction plays a major role in T cell responses. Immune intervention targeted at this interaction has demonstrated a vast potential in enhancing tumor immunity and blocking autoimmunity and transplant rejection. However, the structural basis for this interaction is unclear. While we and others have performed site-directed mutagenesis to define amino acids involved in binding CD28 and CTLA4, these residues are localized in different regions, and it is unlikely for all of them to be directly involved. In addition, the effect of the mutations on the overall conformation of B7 has not been systematically evaluated. In this study, we have carried out site-directed mutagenesis to define the amino acids within B7-1 IgV-like domain which participate B7:CD28/CTLA4 interaction. Four anti-B7-1 mAbs that recognize three independent antigenic epitopes on B7-1 were used to monitor the effect of mutations on the overall conformation of B7-1. Of the five mutations in the IgV domain that we have produced, D113 > A appears to interfere with cell surface expression and/or overall conformation of B7-1. while four others do not significantly affect the overall conformation and cell surface expression of B7-1. Among them, G115 > A and Y91 > A eliminated B7-1 binding to both CD28Ig and CTLA4Ig; our previously reported mutants L109 > A and W88 > A selectively affect the B7-1 binding to either CD28Ig or CTLA4Ig. Structural modeling of B7-1 based on the structure of immunoglobulin revealed that these four and other previously identified critical amino acids in both IgV- and IgC-like domains can form a localized structure.
authors.affiliation: Department of Pathology, Skirball Institute for Molecular Biomedical Research, New York University Medical Center, NY 10016, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Guo
authors.forename: Y
authors.initials: Y
authors.lastname: Wu
authors.forename: X
authors.initials: X
authors.lastname: Kong
authors.forename: Y
authors.initials: Y
authors.lastname: Liu
mesh_terms: D000069594:Abatacept; D000595:Amino Acid Sequence / Q000235:genetics*; D000818:Animals; D015703:Antigens, CD; D000943:Antigens, Differentiation / Q000378:metabolism / Q000502:physiology*; D018122:B7-1 Antigen / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D001665:Binding Sites / Q000235:genetics; D018106:CD28 Antigens / Q000378:metabolism / Q000502:physiology*; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D060908:CTLA-4 Antigen; D002460:Cell Line; D017124:Conserved Sequence / Q000235:genetics*; D000939:Epitopes / Q000235:genetics; D018796:Immunoconjugates; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics*; D008213:Lymphocyte Activation; D051379:Mice; D008810:Mice, Inbred C57BL; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D017434:Protein Structure, Tertiary
chemical_list: D015703:Antigens, CD; D000943:Antigens, Differentiation; D018122:B7-1 Antigen; D018106:CD28 Antigens; D060908:CTLA-4 Antigen; C556707:Ctla4 protein, mouse; D000939:Epitopes; D018796:Immunoconjugates; D007135:Immunoglobulin Variable Region; D000069594:Abatacept