Characterization of a high-affinity monoclonal antibody to calcineurin whose epitope defines a new structural domain of calcineurin A.
Monoclonal antibodies have been raised against native calcineurin using conventional in vivo immunization and hybridoma procedures. The relatively high affinity of nonimmune IgG for the two subunits of calcineurin resulted in large nonspecific binding values for immunoassays of native, dissociated and denatured calcineurin, which complicated the antibody screening. Monoclonal aCn5, a high-affinity IgG1 that exhibits specific binding, was characterized. Other calmodulin-binding proteins tested were not recognized by aCn5. Simple binding properties were exhibited in solid-phase experiments, Kd = 26 (+/- 4) pM, but the stoichiometry was low. The loss of immunoreactivity after denaturation of calcineurin indicated that the aCn5 epitope is of the assembled topographic, not segmental, type. The epitope was located to the A subunit and affinity was unaffected by the presence of calcineurin B. The epitope remained intact after proteolytic removal of the amino-terminal 20 residues of calcineurin A essential for phosphatase activity, and the carboxyl-terminal inhibitory and calmodulin-binding domains. The calmodulin-binding peptide derived from calcineurin, cA8, was not recognized by aCn5. Addition of Ca2+, Mn2+, Ni2+, chelators or dithiothreitol did not influence the affinity of aCn5 for the holoenzyme. Phosphatase activity of calcineurin, in the presence and absence of calmodulin and after removal of the inhibitory domain, was little affected by aCn5. Thus, the aCn5 epitope defines a previously unidentified structural domain of calcineurin A located in a region of the proteolytically resistant core that is topologically distinct from the catalytic, inhibitory, calmodulin-binding and calcineurin-B-binding domains, and not functionally connected with calcineurin B or the putative metal-binding domain(s).
Hubbard
M J
MJ
Laboratory of Biochemistry, National Cancer Institute, Bethesda, MD 20892.
Klee
C B
CB
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D000915:Antibody Affinity; D000918:Antibody Specificity; D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000276:immunology* / Q000378:metabolism; D000939:Epitopes / Q000276:immunology*; D005260:Female; D015151:Immunoblotting; D007074:Immunoglobulin G / Q000276:immunology; D015592:Immunoradiometric Assay; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D010749:Phosphoprotein Phosphatases / Q000276:immunology* / Q000378:metabolism; D011863:Radioimmunoassay; D051381:Rats
D000911:Antibodies, Monoclonal; D002148:Calmodulin-Binding Proteins; D000939:Epitopes; D007074:Immunoglobulin G; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases