Glycosylation of the alpha and beta tubulin by sialyloligosaccharides

To examine whether alpha and beta tubulin are glycoproteins, we used a pyridylamino labeling method and a monoclonal antibody, SG3-1, raised against NeuAcalpha2-3Gal structure. Alpha and beta tubulin from both pig brain and HeLa cells were positive for the SG3-1 antibody by immunoblot assay. Sialidase treatment reduced the reactivity of the SG3-1 antibody to alpha and beta tubulin molecules. N-linked oligosaccharide analysis also showed that alpha and beta tubulin are glycosylated. Moreover, immunofluorescence analysis showed that the filamentous structure recognized by the SG3-1 antibody was overlapped with microtubules, especially in the vicinity of the nucleus. These results indicate that alpha and beta tubulin are glycosylated with sialyloligosaccharides.     

Purification and characterisation of bovine brain protein kinase C isotypes alpha, beta and gamma

Several polyclonal antisera specific for each of the protein kinase C isotypes alpha, beta 1, beta 2 and gamma have been generated and used to monitor the purification and subsequent separation of these polypeptides. A simple protocol has been developed for the efficient co-purification of these isotypes from bovine brain. The separation of the alpha, beta 1, beta 2 and gamma isotypes has been monitored using the antibodies and pools containing pure alpha, beta 1, and gamma forms have been produced. These isotypes have been characterised for activator dependence and substrate specificity. The results indicate that while the isotypes have similar requirements for magnesium, calcium, ATP and phosphatidylserine, they differ in their dependence on phorbol esters and diacylglycerols. The isotypes also differ in their range of substrate specificities. The implications of these results are discussed.     

Preparation of a monoclonal antibody specific for the class IV isotype of beta-tubulin. Purification and assembly of alpha beta II, alpha beta III, and alpha beta IV tubulin dimers from bovine brain.

Tubulin, the 100-kDa subunit protein of microtubules, is a heterodimer of two 50-kDa subunits, alpha and beta. Both alpha and beta subunits exist as numerous isotypic forms. There are four isotypes of beta-tubulin in bovine brain tubulin preparations; their designations and relative abundances in these preparations are as follows: beta I, 3%; beta II, 58%; beta III, 25%; and beta IV, 13%. We have previously reported the preparation of monoclonal antibodies specific for beta II and beta III (Banerjee, A., Roach, M. C., Wall, K. A., Lopata, M. A., Cleveland, D. W., and Luduena, R. F. (1988) J. Biol. Chem. 263, 3029-3034; Banerjee, A., Roach, M. C., Trcka, P., and Luduena, R. F. (1990) J. Biol. Chem. 265, 1794-1799). We here report the preparation of a monoclonal antibody specific for beta IV. By using this antibody together with those specific for beta II and beta III, we have prepared isotypically pure tubulin dimers with the composition alpha beta II, alpha beta III, and alpha beta IV. We have found that, in the presence of microtubule-associated proteins, all three dimers assemble into microtubules considerably faster and to a greater extent than does unfractionated tubulin. More assembly was noted with alpha beta II and alpha beta III than with alpha beta IV. When assembly is measured in the presence of taxol (10 microM), little difference is seen among the isotypically purified dimers or between them and unfractionated tubulin. These results indicate that the assembly properties of a tubulin preparation are influenced by its isotypic composition and raise the possibility that the structural differences among tubulin isotypes may have functional significance.     

The molecular karyotype of Leishmania major and mapping of alpha and beta tubulin gene families to multiple unlinked chromosomal loci.

The arrangement of tubulin genes in the genome of the protozoan parasite Leishmania major was studied by genomic Southern blot analysis and mapping of genes to chromosomes fractionated by pulsed field gradient gel (PFG) electrophoresis. alpha-tubulin genes exist as a tandem array of 2.4 kb PstI fragments. beta-tubulin genes are found as a tandem array of 3.9 kb AvaI or PvuI fragments, but additional genes are also found on other genomic DNA fragments. Chromosome-sized DNA molecules released from promastigotes of L. major were fractionated into at least 17 chromosome bands of approximate size 400-4000 kb by PFG gel electrophoresis. Some bands may be present in non-equimolar amounts suggesting that there may be more than 17 chromosomes. All alpha-tubulin genes were localized to a single band (chromosome 7). beta-tubulin genes were localized to four bands (chromosomes 6, 10, 16 and 17). This shows that the alpha- and beta- tubulin gene families are unlinked in L. major. There is a single chromosomal locus for the alpha-tubulin tandem array whereas beta-tubulin genes exist both as a tandem array and as dispersed genes at four chromosomal loci.     

Conformational properties of the beta(400-436) and beta(400-445) C-terminal peptides of porcine brain tubulin.

Two peptides from the C-terminal region of the major beta-tubulin isotype (400-436 and 400-445) that include the critical areas for interaction with MAP2 and tau were examined to determine their conformations in aqueous solution. Despite a high theoretical potential for alpha-helix formation, CD spectroscopy showed that these peptides consisted primarily of random coil with some reverse turn. This was unaffected by the presence of counterions to the negatively charged side chains (Ca2+, Mg2+), but did change when the side-chain charges were neutralized by lowering the pH; under these conditions, the alpha-helix content of the longer peptide rose to 25% and the C-terminal truncated peptide to 15%. The peptides also adopt alpha-helical structure in the presence of trifluoroethanol, the truncated peptide again attaining a lower maximum percentage. The beta(400-445) peptide was also studied by 1-D and 2-D NMR techniques. The results indicate that at pH 5.6 or 7 in an aqueous solution the peptide is extremely flexible and lacks regular secondary structure, consistent with the CD results. Both peptides inhibited microtubule-associated protein-stimulated tubulin assembly, with the longer peptide being about 4 times as inhibitory as the smaller peptide. Neither was inhibitory in the absence of microtubule-associated proteins, indicating that interaction with this species was necessary for inhibition. The greater activity of the longer peptide could be due to the extra negative charges in this peptide and/or the greater tendency of this peptide to form an alpha-helical structure under the appropriate conditions.     

Complexes of rat alpha 1-macroglobulin and subtilisin are endocytosed by parenchymal liver cells.

Rat alpha 1-macroglobulin was isolated from plasma. Gel electrophoresis of the denatured and reduced protein showed two bands, with Mr values of 163 000 and 37 000. The large subunit contained an autolytic site. This subunit was also split after reaction of the macroglobulin with trypsin. Electron microscopy showed that the macroglobulin changed towards a more compact conformation after reaction with this proteinase. Subtilisin, or alpha 1-macroglobulin, was labelled with a sucrose-containing radio-iodinated group that stays in lysosomes after endocytosis and breakdown of the tagged protein. After intravenous injection into rats, alpha 1-macroglobulin was cleared from plasma with first-order kinetics, showing a half-life of about 9 h, whereas complexes of alpha 1-macroglobulin and subtilisin were cleared with half-lives of only 3 min. Liver contained about 60% of the label at 30 min after injection of complexes. About 90% of the liver radioactivity was found in parenchymal cells isolated after perfusion of the liver with a collagenase solution. Subcellular fractionation indicated a lysosomal localization of the complexes. We conclude that endocytosis by parenchymal liver cells is the major cause of the rapid clearance of alpha 1-macroglobulin-proteinase complexes from plasma.     

Helicity of alpha(404-451) and beta(394-445) tubulin C-terminal recombinant peptides

We have investigated the solution conformation of the functionally relevant C-terminal extremes of alpha- and beta-tubulin, employing the model recombinant peptides RL52alpha3 and RL33beta6, which correspond to the amino acid sequences 404-451(end) and 394-445(end) of the main vertebrate isotypes of alpha- and beta-tubulin, respectively, and synthetic peptides with the alpha-tubulin(430-443) and beta-tubulin(412-431) internal sequences. Alpha(404-451) and beta(394-445) are monomeric in neutral aqueous solution (as indicated by sedimentation equilibrium), and have circular dichroism (CD) spectra characteristic of nearly disordered conformation, consistent with low scores in peptide helicity prediction. Limited proteolysis of beta(394-445) with subtilisin, instead of giving extensive degradation, resulted in main cleavages at positions Thr409-Glu410 and Tyr422-Gln423-Gln424, defining the proteolysis resistant segment 410-422, which corresponds to the central part of the predicted beta-tubulin C-terminal helix. Both recombinant peptides inhibited microtubule assembly, probably due to sequestration of the microtubule stabilizing associated proteins. Trifluoroethanol (TFE)-induced markedly helical CD spectra in alpha(404-451) and beta(394-445). A substantial part of the helicity of beta(394-445) was found to be in the CD spectrum of the shorter peptide beta(412-431) with TFE. Two-dimensional 1H-NMR parameters (nonsequential nuclear Overhauser effects (NOE) and conformational C alphaH shifts) in 30% TFE permitted to conclude that about 25% of alpha(404-451) and 40% of beta(394-451) form well-defined helices encompassing residues 418-432 and 408-431, respectively, flanked by disordered N- and C-segments. The side chains of beta(394-451) residues Leu418, Val419, Ser420, Tyr422, Tyr425, and Gln426 are well defined in structure calculations from the NOE distance constraints. The apolar faces of the helix in both alpha and beta chains share a characteristic sequence of conserved residues Ala,Met(+4),Leu(+7),Tyr(+11). The helical segment of alpha(404-451) is the same as that described in the electron crystallographic model structure of alphabeta-tubulin, while in beta(394-451) it extends for nine residues more, supporting the possibility of a functional coil --> helix transition at the C-terminus of beta-tubulin. These peptides may be employed to construct model complexes with microtubule associated protein binding sites.     

During Drosophila spermatogenesis beta 1, beta 2 and beta 3 tubulin isotypes are cell-type specifically expressed but have the potential to coassemble into the axoneme of transgenic flies

alpha and beta Tubulins exist in a number of different isotypes with distinct expression patterns during development. We have shown by immunofluorescent staining that beta 1, beta 2 and beta 3 tubulins are distributed very specifically in the testes of Drosophila. beta 3 Tubulin is present exclusively in cytoplasmic microtubules of cells somatic in origin, while the beta 1 isotype is localized in the somatic cells and in early germ cells of both the microtubules of the cytoskeleton as well as in the mitotic spindle. In contrast, beta 2 tubulin is present in all microtubular arrays (cytoskeleton, meiotic spindles, axoneme) of germ cells from meiotic prophase onward, though not detectable in somatic cells. Thus, a switch of beta tubulin isotypes from beta 1 to beta 2 occurs during male germ cell differentiation. This switch is also observed in the distantly related species Drosophila hydei. By fusing beta 1 or beta 3 amino acid coding regions to the control region of the beta 2 tubulin gene and performing germ line transformation experiments, we have examined the copolymerization properties of the different tubulin isotypes. Neither beta 1 nor beta 3 are detectable in the axoneme in the wild-type situation. Analysis of transgenic flies carrying beta 2-beta 1 fusion genes or beta 2-beta 3 fusion genes revealed that both beta 1 and beta 3 tubulin isotypes have the potential to co-incorporate with beta 2 tubulin into microtubules of the sperm axoneme. Male flies homozygous for the fusion genes (beta 2-beta 1 or beta 2-beta 3) remain fertile, despite the mixture of beta tubulin isotypes in the axoneme.     

Differential sorting of beta tubulin isotypes into colchicine-stable microtubules during neuronal and muscle differentiation of embryonal carcinoma cells.

Pluripotent P19 embryonal carcinoma (EC) cells were differentiated along the neuronal and muscle pathways. Comparisons of class I, II, III, and IV beta tubulin isotypes in total and colchicine-stable microtubule (MT) arrays from uncommitted EC, neuronal, and muscle cells were made by immunoblotting and by indirect immunofluorescence microscopy. In undifferentiated EC cells the relative amounts of these four isotypes are the same in both the total and stable MT populations. Subcellular sorting of beta tubulin isotypes was demonstrated in both neuronal and muscle differentiated cells. During neuronal differentiation, class II beta tubulin is preferentially incorporated into the colchicine-stable MTs while class III beta tubulin is preferentially found in the colchicine-labile MTs. The subcellular sorting of class II into stable MTs correlates with the increased staining of MAP 1B, and with the expression of MAP 2C and tau. Although muscle differentiated cells express class II beta tubulin, stable MTs in these cells do not preferentially incorporate this isotype but instead show increased incorporation of class IV beta tubulin. Muscle cells do not show high levels of MAP 1B and do not express MAP 2C or tau. These results are consistent with the hypothesis that a subcellular sorting of tubulin isotypes is the result of a complex interaction between tubulin isotypes and MT-associated proteins.     

Release of C-terminal tyrosine from tubulin and microtubules at steady state.

Microtubule protein preparations purified by cycles of assembly-disassembly contain the enzyme tubulinyltyrosine carboxypeptidase (TTCPase). Using these preparations, containing tubulinyl[14C]tyrosine, we studied the release of [14C]tyrosine from assembled and non-assembled tubulin under steady-state conditions. It was found that both states of aggregation were detyrosinated at similar rates by the action of the endogenous TTCPase. However, practically no release of [14C]tyrosine from the non-assembled tubulin pool was found when microtubules were previously eliminated from the incubation mixture. These results indicated that non-assembled tubulin requires to interact with microtubules to be detyrosinated. This interaction seems to occur through the incorporation of dimers into microtubules, since when the capability of tubulin to incorporate into microtubules was diminished by binding of colchicine a concomitant decrease in the rate of release of tyrosine was observed. When detyrosination was accelerated by increasing the concentration of TTCPase relative to the microtubule protein concentration, microtubules were found to be detyrosinated faster than was non-assembled tubulin. Using exogenous TTCPase in an incubation system in which the formation of microtubules was not allowed, tubulinyl[14C]tyrosine and tubulinyl[14C]tyrosine-colchicine complex were shown to have similar capabilities to act as substrates for this enzyme. Free colchicine was shown not to affect the activity of TTCPase.     

beta 1-Integrin-mediated glioma cell adhesion and free radical-induced apoptosis are regulated by binding to a C-terminal domain of PG-M/versican.

Integrins are cell-surface glycoproteins that mediate cell activities, including tissue morphogenesis, development, immune response, and cancer, through interaction with extracellular proteins. Here we report a novel means by which integrin signaling and functions are regulated. In pull-down assays and immunoprecipitation, beta(1)-integrin bound to the C-terminal domain of PG-M/versican, an extracellular chondroitin sulfate proteoglycan. This was confirmed by cell-surface binding assays. Binding was calcium- and manganese-dependent. Upon native gel electrophoresis, beta(1)-integrin comigrated with the C-terminal domain of PG-M/versican. The interaction of beta(1)-integrin with the C-terminal domain of PG-M/versican activated focal adhesion kinase, enhanced integrin expression, and promoted cell adhesion. As a result, cells expressing the C-terminal domain of PG-M/versican were resistant to free radical-induced apoptosis. As the PG-M/versican peptide used in this study does not contain the RGD consensus-binding motif for integrins, the mechanism of the observed binding represents an entirely new function.     

Active-site properties of the oxidized and reduced C-terminal domain of DsbD obtained by NMR spectroscopy

The periplasmic C-terminal domain of the Escherichia coli DsbD protein (cDsbD) has a thioredoxin fold. The two cysteine residues in the CXXC motif serve as the reductant for the disulfide bond of the N-terminal domain which can in turn act as a reductant for various periplasmic partners. The resulting disulfide bond in cDsbD is reduced via an unknown mechanism by the transmembrane helical domain of the protein. We show by NMR analysis of (13)C, (15)N-labelled cDsbD that the protein is rigid, is stable to extremes of pH and undergoes only localized conformational changes in the vicinity of the CXXC motif, and in adjacent regions of secondary structure, upon undergoing the reduced/oxidized transition. pK(a) values have been determined, using 2D NMR, for the N-terminal cysteine of the CXXC motif, Cys461, as well as for other active-site residues. It is demonstrated using site-directed mutagenesis that the negative charges of the side-chains of Asp455 and Glu468 in the active site contribute to the unusually high pK(a) value, 10.5, of Cys461. This value is higher than expected from knowledge of the reduction potential of cDsbD. In a double mutant of cDsbD, D455N/E468Q, the pK(a) value of Cys461 is lowered to 8.6, a value close to that expected for an unperturbed cysteine residue. The pK(a) value of the second cysteine in wild-type cDsbD, Cys464, is significantly higher than the maximum pH value that was studied (pH 12.2).     

Structure and conformational changes in the C-terminal domain of the beta2-adrenoceptor: insights from fluorescence resonance energy transfer studies

The C terminus of the beta(2)-adrenoceptor (AR) interacts with G protein-coupled receptor kinases and arrestins in an agonist-dependent manner, suggesting that conformational changes induced by ligands in the transmembrane domains are transmitted to the C terminus. We used fluorescence resonance energy transfer (FRET) to examine ligand-induced structural changes in the distance between two positions on the beta(2)-AR C terminus and cysteine 265 (Cys-265) at the cytoplasmic end of transmembrane domain 6. The donor fluorophore FlAsH (Fluorescein Arsenical Helix binder) was attached to a CCPGCC motif introduced at position 351-356 in the proximal C terminus or at the distal C terminus. An acceptor fluorophore, Alexa Fluor 568, was attached to Cys-265. FRET analyses revealed that the average distances between Cys-265 and the proximal and distal FlAsH sites were 57 and 62A(,) respectively. These relatively large distances suggest that the C terminus is in an extended, relatively unstructured conformation. Nevertheless, we observed ligand-specific changes in FRET. All ligands induced an increase in FRET between the proximal C-terminal FlAsH site and Cys-265. Ligands that have been shown to induce arrestin-dependent ERK activation, including the catecholamine agonists and the inverse agonist ICI118551, led to a decrease in FRET between the distal FlAsH site and Cys-265, whereas other ligands had no effect or induced a small increase in FRET. Taken together the results provide new insight into the structure of the C terminus of the beta(2)-AR as well as ligand-induced conformational changes that may be relevant to arrestin-dependent regulation and signaling.     

Identification of rat brain polysomes synthesizing the brain specific enolase (14.3.2 protein), S100 protein and alpha and beta tubulin subunits

Polysomes prepared from frozen rat brain powder were fractionated by centrifugation in a sucrose gradient. Individual fractions were used to program a reticulocyte lysate in a run-off reaction. The products of cell-free synthesis were assayed for the brain-specific enolase (14.3.2 protein) and S100 protein by immunoprecipitation with specific antisera and for tubulin by two-dimensional electrophoresis in polyacrylamide slab gels. The relative synthesis of these proteins by unfractionated free brain polysomes were 0.1 per cent, 0.05 per cent and 0.7 per cent respectively. After centrifugation in a sucrose gradient polysomes synthesizing S100 protein were separated from those synthesizing the other two markers. There was a threefold enrichment in the specific messenger RNA activity for each of the three proteins studied in their respective peak fractions of polysomes.     

Structure and function of the regulatory C-terminal HRDC domain from Deinococcus radiodurans RecQ

RecQ helicases are critical for maintaining genome integrity in organisms ranging from bacteria to humans by participating in a complex network of DNA metabolic pathways. Their diverse cellular functions require specialization and coordination of multiple protein domains that integrate catalytic functions with DNA–protein and protein–protein interactions. The RecQ helicase from Deinococcus radiodurans (DrRecQ) is unusual among RecQ family members in that it has evolved to utilize three ‘Helicase and RNaseD C-terminal’ (HRDC) domains to regulate its activity. In this report, we describe the high-resolution structure of the C-terminal-most HRDC domain of DrRecQ. The structure reveals unusual electrostatic surface features that distinguish it from other HRDC domains. Mutation of individual residues in these regions affects the DNA binding affinity of DrRecQ and its ability to unwind a partial duplex DNA substrate. Taken together, the results suggest the unusual electrostatic surface features of the DrRecQ HRDC domain may be important for inter-domain interactions that regulate structure-specific DNA binding and help direct DrRecQ to specific recombination/repair sites.     

Structure of arterivirus nsp4. The smallest chymotrypsin-like proteinase with an alpha/beta C-terminal extension and alternate conformations of the oxyanion hole

Arteriviruses are enveloped, positive-stranded RNA viruses and include pathogens of major economic concern to the swine- and horse-breeding industries. The arterivirus replicase gene encodes two large precursor polyproteins that are processed by the viral main proteinase nonstructural protein 4 (nsp4). The three-dimensional structure of the 21-kDa nsp4 from the arterivirus prototype equine arteritis virus has been determined to 2.0 A resolution. Nsp4 adopts the smallest known chymotrypsin-like fold with a canonical catalytic triad of Ser-120, His-39, and Asp-65, as well as a novel alpha/beta C-terminal extension domain that may play a role in mediating protein-protein interactions. In different copies of nsp4 in the asymmetric unit, the oxyanion hole adopts either a collapsed inactive conformation or the standard active conformation, which may be a novel way of regulating proteolytic activity.