Non-glycosylated human B7-1(CD80) retains the capacity to bind its counter-receptors1This work was supported in part by grants from the National Institutes of Health P01 DK-38181, R01 AI-31044, and R01 CA-74958.1

Abstract

AbstractThough the cell surface-associated costimulator B7-1(CD80) is known to be highly N-glycosylated, the functional significance of this N-glycosylation has not been evaluated. Two experimental approaches were taken to assess the influence of N-glycosylation on human B7-1 function. First, stable K562 transfectants expressing human B7-1 were treated with the N-glycosylation inhibitor tunicamycin. This treatment reduced the levels of B7-1 at the cell surface as judged by both indirect immunofluorescence/flow cytometry and immunoprecipitation analyses. Significantly, the non-glycosylated cell surface-associated B7-1 on tunicamycin-treated cells retained the capacity to bind CTLA-4·Ig, a soluble derivative of the CTLA-4(CD152) counter-receptor. Second, experiments were 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 isolation (hB7-1·IgC). While the hB7-1·IgC derivative failed to bind to CTLA-4, the larger hB7-1·ed derivative associated with CTLA-4·Ig in cell-free binding assays. Futhermore, recombinant hB7-1·ed effectively blocked B7-1-mediated costimulation in an in vitro T cell proliferation assay, suggesting that this soluble non-glycosylated B7-1 derivative is capable of engaging CD28, the B7 counter-receptor implicated in T cell activation. Taken together, these data indicate that the N-glycosylation of B7-1 is not required for its association with counter-receptors. Moreover, the findings pave the way for the therapeutic use of recombinant bacterial B7-1 derivatives as competitive inhibitors of B7-mediated signals