Non-glycosylated 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-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 x 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 x ed) or the membrane-proximal IgC-homologue domain of B7-1 in isolation (hB7-1 x IgC). While the hB7-1 x IgC derivative failed to bind to CTLA-4, the larger hB7-1 x ed derivative associated with CTLA-4 x Ig in cell-free binding assays. Futhermore, recombinant hB7-1 x 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.
Chen
A
A
Institute of Pathology, Case Western Reserve University, Cleveland, OH 44106, USA.
Meyerson
H J
HJ
Salvekar
A
A
Tykocinski
M L
ML
D000069594:Abatacept; D015703:Antigens, CD / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D000943:Antigens, Differentiation / Q000378:metabolism*; D018122:B7-1 Antigen / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D001665:Binding Sites; D060908:CTLA-4 Antigen; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D006031:Glycosylation; D006801:Humans; D018796:Immunoconjugates; D007141:Immunoglobulin Fc Fragments / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D014415:Tunicamycin / Q000494:pharmacology*
D015703:Antigens, CD; D000943:Antigens, Differentiation; D018122:B7-1 Antigen; D060908:CTLA-4 Antigen; C556706:CTLA4 protein, human; D017931:DNA Primers; D018796:Immunoconjugates; D007141:Immunoglobulin Fc Fragments; D011994:Recombinant Proteins; D014415:Tunicamycin; D000069594:Abatacept; D004121:Dimethyl Sulfoxide