RESUMO
A monoclonal antibody (GT335) directed against polyglutamylated tubulin was obtained by immunization with a synthetic peptide which mimics the structure of the polyglutamylated site of alpha-tubulin. This peptide corresponds to the C-terminal sequence Glu441-Gly448 and was chemically modified by the addition of two glutamyl units at Glu445. The specificity of GT335 was assayed by direct and competitive enzyme-linked immunosorbent assay (ELISA) against tubulin and several synthetic peptides differing either by the structure of the added polyglutamyl chain or by their amino acid sequence. Further characterization was carried out by immunoblotting detection after one- or two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The epitope appears to be formed by at least two constituents: a basic motif of monoglutamylation which is retained in the polyglutamylated forms independent of their degree of glutamylation, and some elements of the polypeptide chain close to the site of glutamylation. Given the specificity of GT335 and the delineation of its epitope, our results indicate that, in addition to alpha and beta' (class III)-tubulin, other beta-tubulin isotypes are also glutamylated. This antibody has been used to analyze the cell and tissue distributions of glutamylated tubulin. In mouse brain extracts, GT335 reacts strongly with alpha-tubulin and, to a lesser extent, with beta' (class III) and beta-tubulin. The same reactivity is also observed with cultured neurons whereas astroglial cells exhibit only low levels of glutamylated tubulin. In non-nervous mouse tissues such as spleen, lung or testis, glutamylation was shown to involve only beta-tubulin, but at far lower levels than in brain.
Assuntos
Anticorpos Monoclonais , Glutamatos/metabolismo , Tubulina (Proteína)/análise , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos/imunologia , Astrócitos/química , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Imuno-Histoquímica , Pulmão/química , Masculino , Camundongos , Dados de Sequência Molecular , Neurônios/química , Baço/química , Testículo/químicaRESUMO
The production and identification of a monoclonal antibody, 111 B52 C2, raised against fragments obtained after limited proteolysis of purified tubulin is described. The recognized epitope is located on the aminoterminal domain of the alpha-tubulin subunit and differs from the antigenic sites reacting with the presently existing panel of available monoclonal antibodies. This monoclonal antibody thus constitutes a potentially useful tool to explore interactions between tubulin and other specific ligands.
Assuntos
Anticorpos Monoclonais , Epitopos/análise , Tubulina (Proteína)/análise , Animais , Anticorpos Monoclonais/imunologia , Sítios de Ligação , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Immunoblotting , Camundongos , Dodecilsulfato de Sódio , Tubulina (Proteína)/imunologiaRESUMO
Tubulin from the central nervous system of the Quail (Coturnix coturnix japonica) was analysed by two-dimensional gel electrophoresis. Either in soluble extracts or in tubulin enriched fractions, tubulin of forebrain, cerebellum and spinal cord is resolved into 3 components alpha, beta' and beta, characterized by their electrophoretic coordinates (alpha: pI = 5.4, MW = 54 kd, beta: pI = 5.2, MW = 52 kd; beta': pI = de mapping, by comparison with mouse brain alpha, beta and beta'-tubulin components. With highly resolutional isoelectric focusing 6 alpha isotubulins and 14 beta isotubulins are found in enriched-tubulin fractions of Quail forebrain and cerebellum.
Assuntos
Sistema Nervoso Central/análise , Coturnix/genética , Codorniz/genética , Tubulina (Proteína)/análise , Animais , Eletroforese , Focalização Isoelétrica , Polimorfismo Genético , Tubulina (Proteína)/genéticaRESUMO
Polyglutamylation, a posttranslational modification which consists of the sequential addition of one to six glutamyl units in the carboxy-terminal domain of both tubulin subunits, is a major event in neurons. Its structure has been investigated by using monoreactive polyclonal antibodies directed against distinct glutamylation motifs, ie alpha- and gamma-linkages between glutamyl units. It is shown that, beside alpha-linkages previously characterized, gamma-linkages also occur in glutamyl chains of brain tubulin. The co-existence of these two basic motifs leads to a conception of the polyglutamyl chain with a very sophisticated structure which could, through its complexity, help the microtubule to reach its structure and fulfil its functions.