The carboxy-terminal sequence Asp427-Glu432 of beta-tubulin plays an important function in axonemal motility.
Flagellar motility is the result of specific interactions between axonemal microtubular proteins and the dynein motors. Tubulin, the main component of microtubule, is a very polymorphic protein resulting from the expression of several isogenes and from the existence of various post-translational modifications. In order to characterize tubulin isoforms and tubulin domains that are important for flagellar movement, we prepared monoclonal antibodies against axonemal proteins from whole sea-urchin sperm tails. The monoclonal antibodies obtained were screened for their potency to inhibit demembranated-reactivated sperm models and for their monospecific immunoreactivity on immunoblot. Among the different antibodies we obtained, D66 reacted specifically with a subset of beta-tubulin isoforms. Limited proteolysis, HPLC, peptide sequencing, mass spectroscopy and immunoblotting experiments indicated that D66 recognized an epitope localized in the primary sequence Gln423-Glu435 of the C-terminal domain of Lytechinus pictus beta2-tubulin, and that this sequence belongs to class IVb. The use of synthetic peptides and immunoblotting analysis further narrowed the amino acids important for antibody recognition to Asp427-Glu432. Because the primary effect of this antibody on sperm motility is to decrease the flagellar beat frequency, we suggest that this sequence is involved in the tubulin-dynein head interaction.
Audebert
S
S
Urology Research Laboratory, McGill University, Royal Victoria Hospital, Montreal, Quebec, Canada.
White
D
D
Cosson
J
J
Huitorel
P
P
Eddé
B
B
Gagnon
C
C
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D012617:Sea Urchins; D017386:Sequence Homology, Amino Acid; D013081:Sperm Motility / Q000187:drug effects / Q000502:physiology*; D014404:Tubulin / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D014404:Tubulin