Monoclonal antibody ID5: epitope characterization and minimal requirementsfor the recognition of polyglutamylated alpha- and beta-tubulin

A monoclonal antibody (ID5) raised against the synthetic tetradecapeptide c orresponding to the C-terminal region of detyrosinated alpha-tubulin showed an unexpected cross-reactivity with beta-tubulin from pig brain tissue. Th e specificity and the minimal epitope requirements of IDS were characterize d by competitive enzyme-linked immunosorbent assay (ELISA) and spot blots u sing a series of synthetic peptides and the natural peptides of beta-tubuli n and detyrosinated alpha-tubulin from brain. The epitope of ID5 is compris ed of the carboxyterminal sequence -XEE carrying the terminal alpha-carboxy late group with X being a variable residue. All linkages in the epitope inv olve alpha-peptide bonds, This epitope is provided by the detyrosinated alp ha-tubulin main chain and the polyglutamyl side chains of both brain alpha- and beta-tubulins. Affinity purification of beta-tubulin peptides and mass spectrometric characterization reveal that peptides carrying three to nine glutamyl residues in the side chain are recognized by ID5, These results s how: that except for the first gamma-peptide linkage the alpha-peptide bond is the preferred linkage type in the tubulin polyglutamyl side chains.