Localization of binding sites for carboxyl terminal specific anti-rhodopsin monoclonal antibodies using synthetic peptides

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Abstract
The binding sites for 4 monoclonal antibodies, rho 1D4, rho 3C2, rho 3A6 and rho 1C5, were localized within the C-terminal region of bovine rhodopsin: Asp18''-Glu-Ala16''-Ser-Thr-Val12''-Ser-Lys-Thr-Glu8''-Thr-Ser-Gln-Val4''-Ala-Pro-Ala1''. Antibody binding sites were localized by using synthetic C-terminal peptides in conjunction with solid-phase competitive inhibition assays and limited proteolytic digestion of rhodopsin in conjunction with electrophoretic immunoblotting techniques. Binding of the rho 1D4 and rho 3C2 antibodies to immobilized rhodopsin was inhibited with peptides of length 1''-8'' and longer. Antibody rho 1D4 binding was not inhibited by peptides 2''-13'' or 3''-18'', indicating that the C-terminal alanine residue of rhodopsin was required. Similar competitive inhibition studies indicated that the antibody rho 3A6 required peptides of length 1''-12'' and longer whereas rho 1C5 required peptide 1''-18''. Peptide 3''-18'' was as effective as 1''-18'' in inhibiting rho 3A6 binding to rhodopsin, but replacement of glutamic acid in position 8'' with glutamine abolished competition. This substitution had little effect on the binding of antibody rho 1C5. Thus, Glu8'' was essential for rho 3A6 binding but not for the binding of the rho 1C5 antibody. Cleavage of the 7 amino acid C-terminus from rhodopsin and further cleavage to F1 (MW 25,000) and F2 (MW 12,000) fragments with Staphylococcus aureus V8 protease abolished binding of rho 1D4 antibody to the membrane-bound rhodopsin fragments. Antibodies rho 3A6 and rho 1C5, however, were found to bind both rhodopsin lacking the 7 amino acid C-terminus and the corresponding F2 fragment. These results indicate that a peptide longer than 1''-6'' is required for antibodies rho 1D4 and rho 3C2 binding, 8''-12'' for antibody rho 3A6 binding, and 9''-17'' or 9''-18'' for antibody rho 1C5. Since longer peptides and rhodopsin are more effective competitors, other factors such as conformation also affect binding reactivity. These studies in conjunction with related studies on the binding of these antibodies to membrane-bound and detergent solubilized bleached and unbleached rhodopsin indicate that the carboxyl-terminal 1''-18'' segment of rhodopsin is highly accessible to these immunological probes and relatively insensitive to the state of bleaching and solubilization of rhodopsin. The cross-reactivity of these monoclonal antibodies as well as monoclonal antibodies against other regions of bovine rhodopsin was also studied. The C-terminal segment (as specified by the rho 1D4 and rho 3C2 antibodies), the F1-F2 linking region, and the N-terminal region of rhodopsin are highly conserved in pig, dog, cat, rat, rabbit and frog rhodopsins. Segments farther in from the C-terminus as specified by the rho 3A6 and rho 1C5 antibodies are more variable.

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