NONGLYCOSYLATED HUMAN B7-1(CD80) RETAINS THE CAPACITY TO BIND ITS COUNTER-RECEPTORS

Though the cell surface-associated costimulator B7-1(CD80) is known to be highly N-glycosylated, the functional significance of this N-glyco sylation has not been evaluated. Two experimental approaches were take n to assess the influence of N-glycosylation on human B7-1 function. F irst, stable K562 transfectants expressing human B7-1 were treated wit h the N-glycosylation inhibitor tunicamycin. This treatment reduced th e levels of B7-1 at the cell surface as judged by both indirect immuno fluorescence/flow cytometry and immunoprecipitation analyses. Signific antly, the non-glycosylated cell surface-associated B7-1 on tunicamyci n-treated cells retained the capacity to bind CTLA-4.1g, a soluble der ivative of the CTLA-4(CD152) counter-receptor. Second, experiments wer e performed with bacterially-produced non-glycosylated derivatives of human B7-1, comprising either the complete B7-1 extracellular domain ( hB7-1.ed) or the membrane-proximal IgC-homologue domain of B7-1 in iso lation (hB7-1.IgC). While the hB7-1.IgC derivative failed to bind to C TLA-4, the larger hB7-1.ed derivative associated with CTLA4.Ig in cell -free binding assays. Futhermore, recombinant hB7-1.ed effectively blo cked B7-1-mediated costimulation in an in vitro T cell proliferation a ssay, suggesting that this soluble non-glycosylated B7-1 derivative is capable of engaging CD28, the B7 counter-receptor implicated in T cel l activation. Taken together, these data indicate that the N-glycosyla tion of B7-1 is not required for its association with counter-receptor s, Moreover, the findings pave the way for the therapeutic use of reco mbinant bacterial B7-1 derivatives as competitive inhibitors of B7-med iated signals. (C) 1998 Federation of European Biochemical Societies.