Monoclonal anti-dipeptide antibodies cross-react with detyrosinated and glutamylated forms of tubulins.
Two monoclonal antibodies, GLU-1 and A1.6, raised against gamma-L-glutamyl-L-glutamic acid dipeptide (Glu-Glu) and Ca(2+)-dependent ATPase from 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 treatment of cells with carboxypeptidase A. Immunoblot analysis demonstrated intense cross-reaction of the antibodies to the alpha-tubulin subunit. alpha-Tubulin isotypes produced as fusion proteins in bacteria were labeled by both the antibodies only when the proteins did not contain a tyrosine residue at the C terminus, indicating that GLU-1 and A1.6 specifically recognize the detyrosinated form of alpha-tubulin. When microtubule protein purified from brain was probed, not only alpha-but also, to a lesser extent, beta-tubulin were revealed by the dipeptide antibodies. A synthetic tripeptide YED containing one glutamyl group linked to the second residue of the peptide via the gamma position was also recognized by the antibodies. Since this peptide sequence corresponds to the amino acid sequence of polyglutamyated class III beta isotype at amino acid position 437 to 439, it is suggested that GLU-1 and A1.6 are able to recognize the glutamylated form of beta-tubulin. These results indicate that the C-terminal Glu-Glu sequence displays strong antigenicity, 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 detected with ciliary and flagellar microtubules; thus, stable axonemal microtubules appear to be rich in post-translationally modified tubulin subunits.
Kuriyama
R
R
Department of Cell Biology and Neuroanatomy, University of Minnesota, Minneapolis 55455, USA.
Levin
A
A
Nelson
D
D
Madl
J
J
Frankfurter
A
A
Kimble
M
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry*; D016466:CHO Cells / Q000737:chemistry; D002417:Cattle; D002923:Cilia / Q000737:chemistry; D006224:Cricetinae; D003429:Cross Reactions; D004151:Dipeptides / Q000276:immunology*; D000939:Epitopes / Q000737:chemistry; D005407:Flagella / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D018698:Glutamic Acid / Q000737:chemistry*; D008297:Male; D051379:Mice; D008870:Microtubules / Q000737:chemistry; D012617:Sea Urchins; D013082:Sperm Tail / Q000737:chemistry; D014404:Tubulin / Q000276:immunology*; D014443:Tyrosine / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D004151:Dipeptides; D000939:Epitopes; D014404:Tubulin; D018698:Glutamic Acid; D014443:Tyrosine