EPITOPE AND MIMOTOPE FOR AN ANTIBODY TO THE NA,K-ATPASE

The epitope of a monoclonal antibody specific for the alpha 2 isoform of the Na,K-ATPase was determined and its accessibility in native enzy me was examined. Protein fragmentation with N-chlorosuccinimide, formi c acid, trypsin, and leucine aminopeptidase indicated binding near the Na,K-ATPase N-terminus but did not unambiguously delineate the extent of the epitope. The ability of the antibody to bind to denatured enzy me made it a good candidate for screening a random peptide library dis played on M13 phage, but the consensus sequence that emerged was not f ound in the Na,K-ATPase. Full-length cDNA for the Na,K-ATPase was rand omly fragmented and cloned into beta-galactosidase to create a lambda gt11 expression library; screening with the antibody yielded a set of overlaps spanning 23 amino acids at the N-terminus. Chimeras of Na,K-A TPase alpha 1 and alpha 2 narrowed down the epitope to 14-19 amino aci ds. The antibody did not recognize fusion proteins constructed with sh orter segments of this epitope. It did recognize a fusion protein cont aining the M13 library consensus sequence, however, indicating that th is sequence, which is rich in proline and hydrophobic amino acids (FPP NFLFPPPP), was a mimotope. The natural epitope, unique to the Na,K-ATP ase alpha 2 isoform, was GREYSPAATTAENG. Reconstitution of antibody bi nding in a foreign context such as M13 PIII protein or beta-galactosid ase thus required a relatively large number of amino acids, indicating that antibody mapping approaches must allow for epitopes of significa nt size. The epitope was accessible in native enzyme and exposed on th e cytoplasmic side, documenting the surface exposure of a stretch of a mino acids at the N-terminus, where the Na, K-ATPase isoforms differ m ost.