title: Differential effects of CTLA-4 substitutions on the binding of human CD80 (B7-1) and CD86 (B7-2).
abstract: CTLA-4 expressed on activated T cells binds to CD80 (B7-1) and CD86 (B7-2) molecules present on APC with high avidity and appears to deliver a negative regulatory signal to the T cell. We have investigated the kinetics of CTLA-4 binding to CD80 and CD86, together with the effects of selected CTLA-4 mutations on binding activity. The dissociation constants (Kd) for binding of CTLA-4-Ig to CD80 and CD86 transfectants were 8.1 and 6.7 nM, respectively. Surface plasmon resonance was used to determine kinetic parameters of CTLA-4-Ig binding to CD80-Ig and CD86-Ig fusion proteins and revealed enhanced association (ka) and dissociation (kd) rate constants for CD86-Ig compared with CD80-Ig. Furthermore, CD80-Ig and CD86-Ig fusion molecules demonstrated variable abilities to cross-compete for binding to several modified forms of CTLA-4-Ig. Differential binding of CD80 and CD86 to CTLA-4 was further revealed by analysis of 10 discrete CTLA-4 mutants. Five single amino acid substitutions within the CTLA-4 MYPPPY domain exerted modest effects on CD80 binding, but each of these substitutions completely abrogated CD86 binding. In addition, substitutions just N-terminal of the MYPPPY region, and within the CDR1-like region of CTLA-4, eliminated both CD80 and CD86 binding. Hence, CD80 and CD86 bind with different association/dissociation kinetics to similar, but distinct, sites on CTLA-4.
authors.affiliation: Department of Immunology, G. D. Searle & Company, St. Louis, MO 63198, USA.
authors.forename: P A
authors.initials: PA
authors.lastname: Morton
authors.forename: X T
authors.initials: XT
authors.lastname: Fu
authors.forename: J A
authors.initials: JA
authors.lastname: Stewart
authors.forename: K S
authors.initials: KS
authors.lastname: Giacoletto
authors.forename: S L
authors.initials: SL
authors.lastname: White
authors.forename: C E
authors.initials: CE
authors.lastname: Leysath
authors.forename: R J
authors.initials: RJ
authors.lastname: Evans
authors.forename: J J
authors.initials: JJ
authors.lastname: Shieh
authors.forename: R W
authors.initials: RW
authors.lastname: Karr
mesh_terms: D000069594:Abatacept; D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000737:chemistry*; D000943:Antigens, Differentiation / Q000737:chemistry* / Q000235:genetics*; D018122:B7-1 Antigen / Q000737:chemistry*; D051940:B7-2 Antigen; D015374:Biosensing Techniques; D060908:CTLA-4 Antigen; D002460:Cell Line; D006801:Humans; D018796:Immunoconjugates; D007700:Kinetics; D008213:Lymphocyte Activation / Q000235:genetics / Q000276:immunology; D008562:Membrane Glycoproteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000276:immunology; D011869:Radioligand Assay; D015398:Signal Transduction / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology; D014162:Transfection
chemical_list: D015703:Antigens, CD; D000943:Antigens, Differentiation; D018122:B7-1 Antigen; D051940:B7-2 Antigen; C497964:CD86 protein, human; D060908:CTLA-4 Antigen; C556706:CTLA4 protein, human; D018796:Immunoconjugates; D008562:Membrane Glycoproteins; D000069594:Abatacept