Posttranslational glutamylation of alpha-tubulin.

The high degree of tubulin heterogeneity in neurons is controlled mainly at the posttranslational level. Several variants of alpha-tubulin can be posttranslationally labeled after incubation of cells with [3H]acetate or [3H]glutamate. Peptides carrying the radioactive moiety were purified by high-performance liquid chromatography. Amino acid analysis, Edman degradation sequencing, and mass spectrometric analysis of these peptides led to the characterization of a posttranslational modification consisting of the successive addition of glutamyl units on the gamma-carboxyl group of a glutamate residue (Glu445). This modification, localized within a region of alpha-tubulin that is important in the interactions of tubulin with microtubule-associated proteins and calcium, could play a role in regulating microtubule dynamics.

Polyglycylation of tubulin: a posttranslational modification in axonemal microtubules.

A posttranslational modification was detected in the carboxyl-terminal region of axonemal tubulin from Paramecium. Tubulin carboxyl-terminal peptides were isolated and analyzed by Edman degradation sequencing, mass spectrometry, and amino acid analysis. All of the peptides, derived from both alpha and beta tubulin subunits, were modified by polyglycylation, containing up to 34 glycyl units covalently bound to the gamma carboxyl group of glutamyl residues. This modification, present in one of the most stable microtubular systems, may influence microtubule stability or axoneme function, or both.

Selection and identification of proteins bound to DNA triple-helical structures by combination of 2D-electrophoresis and MALDI-TOF mass spectrometry.

Identification of proteins binding specifically to peculiar nucleic acid structures can lead to comprehension of their role in vivo and contribute to the discovery of structure-related gene regulation. This work was devoted to establishing a reliable procedure to select proteins on the basis of their interaction with a nucleic acid probe chosen to fold into a given structure. 2D-electrophoresis and mass spectrometry were combined for protein identification. We applied this procedure to select and identify triplex-binding activities in HeLa nuclear extracts. To achieve this, we used a panel of deoxyribonucleic probes adopting intramolecular triple-helices, varying in their primary sequence, structure or triple-helix motif. A limited number of spots was reproducibly revealed by South-western blotting. Spots of interest were localised among a complex population of (35)S-labelled proteins according to their (32)P-specific emission. Position of the same spots was extrapolated on a preparative gel coloured with Coomassie blue, allowing excision and purification of the corresponding proteins. The material was subjected to mass spectrometry upon trypsin digestion and MALDI-TOF peptide fingerprinting was used for research in databases: five of them were identified and found to belong to the hnRNP family (K, L, A2/B1, E1 and I). The identities of several of them were confirmed by comparing western and South-western blots on the same membrane using specific antibodies. The recognition specificity of most of these proteins is large, according to previous reports and our own experiments. It includes pyrimidine-rich DNA sequences in different contexts: single strand to a small extent, triplex and possibly other higher-order structures.

The relationships between the toxicity and the primary and secondary structures of elicitinlike protein elicitors secreted by the phytopathogenic fungus Pythium vexans.

Elicitins are toxic and signaling proteins secreted by Phytophthora spp. responsible for the incompatible reaction and systemic hypersensitive-like necroses of diverse plant species leading to resistance against fungal or bacterial plant pathogens. Such proteins were observed in the culture filtrate of another species of the Oomycete genus, Pythium vexans. Two alpha elicitinlike proteins were purified and sequenced. One of these novel elicitins (Vex2) exhibited a 100-residue sequence instead of 98 while the other (Vex1) had an N-glycosylation site, effectively glycosylated (equivalent of 16 hexose residues). In addition to the point mutations already observed in Phytophthora species, we found several novel amino acid changes. Furthermore, circular dichroism revealed some differences in their structure in solution compared with the Phytophthora elicitins that were correlated with specific point mutations. These sequences permitted the establishment of a phylogenic tree, suggesting that Pythium vexans is a species close to the Phytophthora genus. The toxicity of the Pythium vexans elictins to tobacco leaves was investigated and correlated with the occurrence of the carbohydrate moiety of one of the two isoforms, observed for the first time in an elicitin.

Structure of tubulin C-terminal domain obtained by subtilisin treatment. The major alpha and beta tubulin isotypes from pig brain are glutamylated.

Limited subtilisin digestion of the tubulin alpha, beta heterodimer has been used in this work to reduce the total number of tubulin isotypes from 20 for native to 9 for subtilisin-cleaved tubulin. This indicates that the major part of tubulin heterogeneity is located at the C-terminus of the molecule. The C-terminal peptides of both alpha and beta subunits of tubulin were purified by anion-exchange HPLC. Combined use of Edman degradation chemistry and mass spectrometry on the isolated peptides shows that subtilisin cleavage occurs at position Asp-438 and His-406 of alpha and Gln-433 and His-396 of beta tubulin chains. Quantitative analysis of our data show that cleavage at positions His-406 (alpha) and His-396 (beta) occurs with a low efficiency and indicates that the major isotypes of pig brain tubulin are modified by sequential attachment of 1 to 5 glutamic acid residues at positions Glu-445 or -435 of alpha and beta tubulin, respectively.

Class I and IVa beta-tubulin isotypes expressed in adult mouse brain are glutamylated.

Several types of post-translational modifications contribute to the high level of tubulin heterogeneity in the brain. An important modification is glutamylation of the major brain-specific isotypes, such as class Ia/b of alpha-tubulin and classes II and III of beta-tubulin. Here we describe experiments to determine if additional, minor tubulin isotypes, expressed in adult mouse brain, could also be glutamylated. Purified tubulin from adult mouse brain was cleaved with thermolysin. Proteolytically released carboxy-terminal peptides of both alpha- and beta-tubulin were isolated by sequential anion exchange and reverse-phase column-chromatography. Anionic peptides were then characterized by amino acid sequencing and mass spectrometry. We show that brain-specific class IVa and constitutive class I beta-tubulin isotypes can be glutamylated, at Glu434 and Glu441, respectively.

A 15 kDa proteolipid found in mediatophore preparations from Torpedo electric organ presents high sequence homology with the bovine chromaffin granule protonophore.

Upon SDS PAGE of isolated mediatophore, an acetylcholine-translocating protein, a doublet at 15 kDa was identified. Amino acid sequencing after CNBr cleavage gave a 17 residue-long peptide completely homologous with a sequence of the proton-translocating proteolipid from bovine chromaffin granules. A 51-mer oligodeoxynucleotide corresponding to this sequence was used to screen a library of electric lobe cDNAs constructed in lambda Zap II. A positive recombinant clone was isolated and found to encode the complete sequence of a 15.5 kDa protein highly homologous to the bovine chromaffin or yeast vacuolar ATPase proteolipid. In vitro translation of sense RNA transcripts of the clone indeed yielded a single 15 kDa proteolipid. Northern blot analysis showed that the 1.3 kb mRNA encoding this protein is significantly expressed in nervous tissues but not in electric organ or liver of Torpedo marmorata.

mRNP3 and mRNP4 are phosphorylatable by casein kinase II in Xenopus oocytes, but phosphorylation does not modify RNA-binding affinity.

mRNP3 and mRNP4 (also called FRGY2) are two mRNA-binding proteins which are major constituents of the maternal RNA storage particles of Xenopus laevis oocytes. The phosphorylation of mRNP3-4 has been implicated in the regulation of mRNA masking. In this study, we have investigated their phosphorylation by casein kinase II and its consequence on their affinity for RNA. Comparison of the phosphopeptide map of mRNP3-4 phosphorylated in vivo with that obtained after phosphorylation in vitro by purified Xenopus laevis casein kinase II strongly suggests that casein kinase II is responsible for the in vivo phosphorylation of mRNP3-4 in oocytes. The phosphorylation occurs on a serine residue in a central domain of the proteins. The affinity of mRNP3-4 for RNA substrates remained unchanged after the treatment with casein kinase II or calf intestine phosphatase in vitro. This suggests that phosphorylation of these proteins does not regulate their interaction with RNA but rather controls their interactions with other proteins.

Cysteinyl-tRNA synthetase from Saccharomyces cerevisiae. Purification, characterization and assignment to the genomic sequence YNL247w.

Cysteinyl-tRNA synthetase (CRS) from Saccharomyces cerevisiae was purified 2300-fold with a yield of 33%, to a high specific activity (kcat4.3 s-1 at 25 degrees C for the aminoacylation of yeast tRNACys). SDS-PAGE revealed a single polypeptide corresponding to a molecular mass of 86 kDa. Polyclonal antibodies to the purified protein inactivated CRS activity and detected only one polypeptide of 86 kDa in a yeast extract subjected to SDS-PAGE followed by immunoblotting. In contrast to bacterial CRS which is a monomer of about 50 kDa, the native yeast enzyme behaved as a dimer, as assessed by gel filtration and cross-linking. Its subunit molecular mass is in good agreement with the value of 87.5 kDa calculated for the protein encoded by the yeast genomic sequence YNL247w. The latter was previously tentatively assigned to CRS, based on limited sequence similarities to the corresponding enzyme from other sources. Determination of the amino acid sequence of internal polypeptides derived from the purified yeast enzyme confirmed this assignment. Alignment of the primary sequences of prokaryotic and yeast CRS reveals that the larger size of the latter is accounted for mostly by several insertions within the sequence.

Protein processing and morphogenesis of secretory granules in Paramecium.

The ciliated protozoan Paramecium provides a model system for the study of regulated secretion, featuring architecturally complex secretory storage granules-trichocysts-docked at the plasma membrane, ready to respond to an exocytotic stimulus. The trichocysts are characterized by crystalline contents that confer upon the organelle a defined shape which can be altered by single gene mutation. The crystalline trichocyst contents are built up from a heterogeneous set of small acidic polypeptides generated by proteolytic maturation of a family of precursor molecules, suggesting an important role for protein processing in this system. We have recently shown that the primary defect in several secretory mutants lacking functional trichocysts is in intracellular trafficking rather than protein processing. However, analysis of how these defects lead to altered trichocyst shape supports the notion that the protein processing is essential for morphogenesis. Preliminary results of a cloning project reveal that an extensive multigene family (approximately 100 genes) codes for the trichocyst matrix proteins. Deduced amino acid sequences of putative processing sites indicate that (at least) two distinct processing reactions are probably involved in the maturation of these proteins, and allow us to speculate that each reaction may control a key event of trichocyst biogenesis.

Treatment with crystalline ultra-pure urea reduces the aggregation of integral membrane proteins without inhibiting N-terminal sequencing.

We have demonstrated that N-terminal sequencing can be performed successfully despite boiling protein samples in the presence of urea under precise conditions, before loading them onto SDS-PAGE and transfer to polyvinylidene difluoride membrane. Using myoglobin as a test protein, we found that its ability to undergo N-terminal sequencing was not affected by the presence of urea provided "ultra-pure" urea was used. Consistent with this result, we verified that urea did not carbamylate myoglobin since its molecular mass was measured by mass spectrometry after electroelution of the protein band from the gel. These observations are useful for the study of integral membrane proteins, in particular to study their topology from proteolysis experiments, since heating in the presence of urea before SDS-PAGE reduces membrane protein aggregation [Soulié, S., Mo/ller, J.V., Falson, P., and le Maire, M. (1996) Anal. Biochem. 236, 363-364]. We show that the sequencing yield of a hydrophobic peptide from reticulum Ca2+-ATPase was more than doubled in the presence of urea in accord with the quantification of the Coomassie Blue staining of the gel and of the amount present on the polyvinylidene difluoride membrane. For three peptides of the gastric H+K+-ATPase, the sequencing yield after urea treatment increased almost threefold.

?-Galactoside soluble lectin from human brain: complete amino acid sequence.

A soluble lectin from human brain, specific for ?-galactoside-containing glycoconjugates, has been sequenced and compared with a similar protein purified from human placenta. The sequence was established from the analysis of the peptides obtained by chemical and proteolytic cleavage. The brain lectin has a complete homology with that of placenta. This and similar results observed in other mammals strongly suggest that there is only one gene, per species, encoding for this protein.

Primary structure of two isoforms of the vitellogenesis inhibiting hormone from the lobster Homarus americanus.

The amino acid sequence of two isoforms of the Vitellogenesis Inhibiting Hormone from the lobster Homarus americanus (one biologically active and one inactive in a heterologous bioassay) has been established by gas-phase microsequencing and fast-atom bombardment mass spectrometry. These two isoforms, isolated from sinus glands display the same sequence of 77 amino acid residues (m.w.: 9135 Da) and have a free N-terminus. Structurally related to Crustacean Hyperglycemic Hormone and Molt Inhibiting Hormone, the Vitellogenesis Inhibiting Hormone of the lobster clearly appears as an original member of the newly described family of neuropeptides, so far proper to crustaceans, which are involved in the control of major physiological functions.

Frog prodermorphin expressed in mammalian cells is partly converted to the hydroxyproline containing precursor.

Using recombinant vaccinia virus, we have expressed in mammalian cells the cDNA coding for the precursor of dermorphin, a D-alanine containing opioid peptide from the skin of the South American frog Phyllomedusa sauvagei. HeLa cells and AtT-20 cells produced prodermorphin where proline-6 of dermorphin was partly hydroxylated. This was demonstrated by digesting the partially purified precursors with trypsin and carboxypeptidase B. After immunoprecipitation and separation by HPLC, two decapeptides were detected which differed by the presence of proline or hydroxy-proline at position 6. This demonstrates that HeLa cells as well as AtT-20 cells can perform the post-translational conversion of certain proline residues to hydroxyproline in a foreign hormone precursor expressed in these cells.

Purification of arginase from Aspergillus nidulans.

Arginase (EC 3.5.3.1) of Aspergillus nidulans, the enzyme which enables the fungus to use arginine as the sole nitrogen source was purified to homogeneity. Molecular mass of the purified arginase subunit is 40 kDa and is similar to that reported for the Neurospora crassa (38.3 kDa) and Saccharomyces cerevisiae (39 kDa) enzymes. The native molecular mass of arginase is 125 kDa. The subunit/native molecular mass ratio suggests a trimeric form of the protein. The arginase protein was cleaved and partially sequenced. Two out of the six polypeptides sequenced show a high degree of homology to conserved domains in arginases from other species.

Lysozyme fragmentation induced by gamma-radiolysis.

Irradiation of lysozyme in frozen states in the absence of oxygen induces specific fragmentation at defined sites along the backbone chain. This paper localizes radio-fragmentation sites by two methods. First, N-terminal sequencing of radiolysis fragments after separation by SDS-polyacrylamide gel electrophoresis and estimation of their molecular masses. Secondly, after purification of radiolysis fragments by reverse phase-HPLC and determination of their molecular mass by electro-spray-ionization mass-spectrometric analysis, combined to N-terminal sequencing and total amino acid analysis. Evidence for the breakage of the peptide bond itself (CO-NH) is given, with radio-fragmentation sites mostly found at the surface of irradiated lysozyme in solvent exposed loops and turns.

A metabolic prototype for eliminating tryptophan from the genetic code.

We set out to reduce the chemical constitution of a living organism to 19 amino acids. A strain was constructed for reassigning the tryptophan codon UGG to histidine and eliminating tryptophan from Escherichia coli. Histidine codons in the gene for an essential enzyme were replaced with tryptophan codons and the restoration of catalytic activity by missense suppressor His-tRNA bearing a CCA anticodon was selected. We used automated cultivation to assess the stability of this genetic construct during evolution. Histidine to tryptophan mutation at codon 30 in the transketolase gene from yeast and its cognate suppressor tRNA were stably propagated in a tktAB deletant of E. coli over 2500 generations. The ratio of histidine misincorporation at tryptophan sites in the proteome increased from 0.0007 to 0.03 over 300 days of continuous culture. This result demonstrated that the genetic code can be forced to evolve by permanent metabolic selection.

On the use of polyethylenimine as a carrier for protein sequencing: comparison with polybrene.

In the gas-liquid phase automated protein sequencer, polyethylenimine was used as a hydrophilic entrapping polymer. Glass fiber filters soaked in 0.3% solution of polyethylenimine were used. Sperm whale myoglobin, beta-lactoglobulin, and several peptides with basic or acidic pI were sequenced. Loads from 20 to 26,000 pmol were tested. Initial and repetitive yields compare favorably to those obtained with polybrene-coated glass fiber filters. Recovery of individual amino acids shows that none gave a particularly low yield, in contrast with the low recovery of Arg, Trp, His, Glu, and Asp when polybrene was used. The usual artefacts were greatly diminished and even disappeared as in the case of N,N'-diphenylurea. Substitution of polyethylenimine for polybrene sped up the analysis because the precycling employed to condition polybrene-coated glass fiber filters was no longer necessary. In conclusion polyethylenimine appears superior to polybrene for sequencing protein and peptides.

Characterization of hyperglycemic and molt-inhibiting activity from sinus glands of the penaeid shrimp Penaeus vannamei.

To design a homologous bioassay for the molt-inhibiting hormone and the crustacean hyperglycemic hormone of the shrimp Penaeus vannamei, the effect of sinus gland homogenate (SGh), in vitro, on ecdysteroid production by Y-organs (YOs), and the effect of the injection of SGh, in vivo, on the glycemia of shrimps have been investigated. Addition of SGh to incubation medium of shrimp YOs dose dependently reduced, within a few hours, ecdysteroid release into the medium. Moreover, inhibition by SGh decreases drastically in YOs from animals in late premolt stages, when there is maximal ecdysteroid production. Injection of SGh into shrimps evokes a hyperglycemic response maximal after 2 hr. Immunoadsorption of SGh with an anti-Homarus americanus cHHA antiserum inhibited both biological activities of the homogenate. After fractionation of acidic sinus gland extract by RP-HPLC, the maximal response in both bioassays was associated with the major UV absorbent peak, which was also the major immunoreactive peak when tested by ELISA with the anti-lobster cHHA. After a further purification step, the molecular mass of the bioactive and immunoreactive peptide was found to be 8627 +/- 0.3 Da by electrospray ionization mass spectrometry. The amino acid sequence of the first 38 residues of this peptide was established by gas-phase microsequencing. This sequence shows 55% homology with the first 38 residues of the lobster cHHA.

Specific Ser-Pro phosphorylation by the RNA-recognition motif containing kinase KIS.

We present here a first appraisal of the phosphorylation site specificity of KIS (for 'kinase interacting with stathmin'), a novel mammalian kinase that has the unique feature among kinases to possess an RNP type RNA-recognition motif (RRM). In vitro kinase assays using various standard substrates revealed that KIS has a narrow specificity, with myelin basic protein (MBP) and synapsin I being the best in vitro substrates among those tested. Mass spectrometry and peptide sequencing allowed us to identify serine 164 of MBP as the unique site phosphorylated by KIS. Phosphorylation of synthetic peptides indicated the importance of the proline residue at position +1. We also identified a tryptic peptide of synapsin I phosphorylated by KIS and containing a phosphorylatable Ser-Pro motif. Altogether, our results suggest that KIS preferentially phosphorylates proline directed residues but has a specificity different from that of MAP kinases and cdks.