MONOCLONAL ANTI-DIPEPTIDE ANTIBODIES CROSS-REACT WITH DETYROSINATED AND GLUTAMYLATED FORMS OF TUBULINS

Two monoclonal antibodies, GLU-1 and Al.6, raised against gamma-L-glut amyl-L-glutamic acid dipeptide (Glu-Glu) and Ca2+-dependent ATPase fro m Paramecium, respectively, recognized the dipeptide Glu-Glu sequence. Whereas the antibodies immunofluorescently stained very few, if any, cytoskeletal fibers in cultured mammalian cells, almost all interphase as well as mitotic spindle microtubules became visible after treatmen t of cells with carboxypeptidase A. Immunoblot analysis demonstrated i ntense cross-reaction of the antibodies to the alpha-tubulin subunit. alpha-Tubulin isotypes produced as fusion proteins in bacteria were la beled by both the antibodies only when the proteins did not contain a tyrosine residue at the C terminus, indicating that GLU-1 and Al.6 spe cifically recognize the detyrosinated form of alpha-tubulin. When micr otubule protein purified from brain was probed, not only alpha- but al so, to a lesser extent, beta-tubulin were revealed by the dipeptide an tibodies. A synthetic tripeptide YED containing one glutamyl group lin ked to the second residue of the peptide via the gamma position was al so recognized by; the antibodies. Since this peptide sequence correspo nds to the amino acid sequence of polyglutamyated class III beta isoty pe at amino acid position 437 to 439, it is suggested that GLU-1 and A l.6 are able to recognize the glutamylated form of P-tubulin. These re sults indicate that the C-terminal Glu-Glu sequence displays strong an tigenicity, and the antibodies recognize the sequence present in the C terminus of the detyrosinated form of alpha-tubulin and the glutamyl side chain of beta-tubulin. Particularly strong immunoreaction was det ected with ciliary and flagellar microtubules; thus, stable axonemal m icrotubules appear to be rich in post-translationally modified tubulin subunits. (C) 1995 Wiley-Liss, Inc.