Orientation of a human leukocyte interferon molecule on its cell surface receptor: carboxyl terminus remains accessible to a monoclonal antibody made against a synthetic interferon fragment.
An 125I-labeled monoclonal antibody made against a synthetic 56-residue fragment of human leukocyte interferon (IFN) alpha 1 recognizes human, Escherichia coli-derived IFN alpha A bound to the surface of Madin-Darby bovine kidney cells. A major fraction of the antibody recognizes IFN specifically bound to the cells, because the number of bound antibody molecules corresponds to the number of cell-bound IFN molecules (as measured with radiolabeled ligand) and because the fraction of the IFN unspecifically bound to the cells is less than 10% of the total bound IFN. A synthetic carboxyl-terminal 16-residue IFN peptide, though not inhibiting binding of IFN to cells, inhibits binding of antibody to IFN. A recombinant IFN alpha A molecule with a carboxyl-terminal 13-residue deletion, though still able to compete for binding of IFN to cells, is not recognized by the antibody. Scatchard plot analysis of the binding data revealed apparent dissociation constants of 6.0 x 10(-10) M for the antibody-IFN interaction and of 4.0 x 10(-11) M for the IFN-cell receptor interaction. The antibody inhibits the binding of IFN to cells only weakly and neutralizes the antiviral activity of the ligand only when in a large molar excess. We conclude that the carboxyl-terminal 10-16 residues that are predicted from the cloned IFN cDNAs and that are present in some natural IFNs are not involved in binding to cells but are antigenic and hence exposed on the molecules' surface. That the carboxyl terminus is not directly involved in binding to cells is consistent with the observation that some IFNs with carboxyl-terminal deletions are biologically active.
D000818:Animals; D000911:Antibodies, Monoclonal; D002417:Cattle; D002460:Cell Line; D002846:Chromatography, Affinity; D006801:Humans; D007370:Interferon Type I / Q000378:metabolism*; D007668:Kidney / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation; D011863:Radioimmunoassay; D011956:Receptors, Cell Surface / Q000378:metabolism*; D017471:Receptors, Interferon
D000911:Antibodies, Monoclonal; D007370:Interferon Type I; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D017471:Receptors, Interferon