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

A monoclonal antibody (ID5) raised against the synthetic tetradecapeptide corresponding to the C-terminal region of detyrosinated alpha-tubulin showed an unexpected cross-reactivity with beta-tubulin from pig brain tissue. The specificity and the minimal epitope requirements of ID5 were characterized by competitive enzyme-linked immunosorbent assay (ELISA) and spot blots using a series of synthetic peptides and the natural peptides of beta-tubulin and detyrosinated alpha-tubulin from brain. The epitope of ID5 is comprised of the carboxyterminal sequence -XEE carrying the terminal alpha-carboxylate group with X being a variable residue. All linkages in the epitope involve alpha-peptide bonds. This epitope is provided by the detyrosinated alpha-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 show that except for the first gamma-peptide linkage the alpha-peptide bond is the preferred linkage type in the tubulin polyglutamyl side chains.