First protein engineering centres meeting at CAPE/GBF on May 22-24, 1991. Foundation of INPEC (International Network of Protein Engineering Centres).
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Protein Engineering↗
Biomedical subjects
P Fromageot
Publications and source records attributed to P Fromageot.
Specific DNA binding by c-Myb: evidence for a double helix-turn-helix-related motif.
The c-Myb protein is a sequence-specific DNA binding protein that activates transcription in hematopoietic cells. Three imperfect repeats (R1, R2, and R3) that contain regularly spaced tryptophan residues form the DNA binding domain of c-Myb. A fragment of c-Myb that contained the R2 and R3 regions bound specifically to a DNA sequence recognized by c-Myb plus ten additional base pairs at the 3' end of the element. The R2R3 fragment was predicted to contain two consecutive helix-turn-helix (HTH) motifs with unconventional turns. Mutagenesis of amino acids in R2R3 at positions that correspond to DNA-contacting amino acids in other HTH-containing proteins abolished specific DNA binding without affecting nonspecific DNA interactions.
Amino Acid Sequence↗
Two polypeptide chains in yeast transcription factor tau interact with DNA.
Yeast transcription factor tau interacts with the A and B blocks of the intragenic promoter of tRNA genes. The structure of tau was investigated by identifying the polypeptide chains specifically complexed to the tRNA3Glu gene. Highly purified factor, obtained by an improved purification procedure, contained several polypeptide chains, four of which (Mr = 145,000, 135,000, 100,000 and 65,000) comigrated with tau-DNA complex by polyacrylamide gel electrophoresis. Antibodies raised against the 145- and 100-kDa components altered the migration of tau-DNA complexes in band shift assays and inhibited tRNA synthesis in a reconstituted transcription system. These components are immunologically unrelated proteins. By UV cross-linking to 32P-body-labeled tDNA followed by extensive DNase treatment, two polypeptides of the same size (145 and 100 kDa) were found to be radioactively labeled. Factor tau, therefore, appears to be a multisubunit DNA-binding protein with two distinct polypeptides contributing to DNA recognition. Limited proteolysis of tau generated a protease-resistant tau B (tau B) domain that binds solely to the B block. tau B-tDNA complexes were recognized by anti-145 IgG and contained a 120-kDa polypeptide that could originate from the 145-kDa component by proteolysis. These results strongly suggest that the 145-kDa polypeptide belongs to tau B and is responsible for B block binding.
Antigen-Antibody Reactions↗
Reversal of snake neurotoxin binding to mammalian acetylcholine receptor by specific antiserum.
Snake curaremimetic toxins are known to bind to the nicotinic acetylcholine receptor (AcChoR) [Changeux et al. (1970) Proc. Natl Acad. Sci. USA, 67, 1241-1247], thus blocking neuromuscular transmission, and producing respiratory failure in mammals. In the present paper we show that the toxic effects of Naja nigricollis toxin alpha to mammals can be efficiently reversed by toxin-alpha-specific antibodies. In vivo we observed that return to normal breathing in toxin-alpha-intoxicated and ventilated rats was 12 times faster after injection of specific antiserum or monoclonal antibody (M-alpha 1) as compared with control animals. Ex vivo we observed that return to normal contraction of a toxin-alpha-blocked phrenic nerve-hemidiaphragm preparation was 14 times more rapid after treatment with specific antiserum than after washings. In vitro we observed that antibodies accelerated the reversal of binding of [3H]toxin alpha to AcChoR prepared from rat diaphragm. The observation made in vitro furthermore indicates that antibodies are capable of destabilizing the [3H]toxin-AcChoR complex. A similar destabilization phenomenon occurs also in vivo, as inferred from measurements of receptor occupancy by [3H]toxin alpha in diaphragm of anaesthetized rats in the presence or absence of antibodies. The property of antibodies to reverse neurotoxin binding to AcChoR may be considered as a critical test for evaluation of the quality of a neurotoxin-specific antisera.
Animals↗
[Basic phospholipase from the venom of Naja nigricollis is cytotoxic].
The basic phospholipase A2 purified from the venom of Naja nigricollis (Institut Pasteur), possesses an intense cytotoxic activity toward fetal cells from FL strain. At a concentration equal to 1.6 x 10(-6) M, the PLA2 lyses 50% of the cells present in a suspension containing 3.5 x 10(6) cells per millilitre. Other PLA2 from various origins do not exhibit such cytotoxic activity.
Amnion↗
Role of tyrosine and tryptophan residues in the structure-activity relationships of a cardiotoxin from Naja nigricollis venom.
This paper is an attempt to localize the critical area determining toxicity in a snake cardiotoxin. Toxin gamma is a single-chain polypeptide of 60 amino acids, which has been isolated from the venom of the African spitting cobra, Naja nigricollis. Three aromatic residues, namely, Trp-11, Tyr-22, and Tyr-51, have been individually modified by chemical means. The structure of the native toxin and of each derivative has been carefully investigated by circular dichroism, fluorescence, proton magnetic resonance spectroscopy, and two specific monoclonal antibodies. None of the chemical modifications alters the overall structure of the toxin, which in all cases remains folded into three adjacent loops (I, II, and III) rich in beta-pleated sheet emerging from a small globular region containing four disulfide bridges. A number of subtle changes, however, have been detected in the structure of each derivative compared with that of the native toxin. In particular, nitration of Tyr-51 provoked a structural perturbation in the globular region. Nitration of Tyr-22 induces a more substantial change in the beta-sheet area of the molecule. Thus, the strong inter-ring NOE that is observed in the native toxin between Tyr-22 and Tyr-51 vanishes in the Tyr-22 derivative, and significant changes are observed in the globular region. In contrast, no alteration of the beta-sheet structure of loops II and III has been detected after modification of Trp-11. All changes observed for this derivative remain located in the vicinity of the indole side chain of Trp-11 in loop I. The biological consequences of the modifications were measured: the lethal potency in vivo in mice and the cytotoxic activities in vitro on FL-cells. Lethal activities correlate with cytotoxicity: Tyr-51 modified toxin is equally potent as native toxin, whereas Tyr-22 and Trp-11 derivatized toxins are characterized by substantially lesser activities, the Trp-11 derivatized toxin being the least potent. We conclude that (1) Tyr-51 is not involved in the functional site of the toxin, although it is in interaction with the core of the molecule, (2) Tyr-22 may play a dual structural and functional role, and (3) Trp-11 is in, or in close proximity to, the functional site of the toxin. These data indicate the importance of loop I in determining toxicity of the cardiotoxin.
Animals↗
A monoclonal antibody which recognized the functional site of snake neurotoxins and which neutralizes all short-chain variants.
We isolated a neurotoxin-specific monoclonal antibody (Mab) which is capable of recognizing and neutralizing all short-chain toxin variants that have been tested, including those with widely divergent sequences. The epitope incorporates the three invariant residues Lys-27, Trp-29 and Lys-47 which form part of the site by which the toxins bind to the nicotinic acetylcholine receptor. To our knowledge, this is the first Mab which possesses the universal capacity of neutralizing all natural variants of a toxic protein.
Amino Acid Sequence↗
Two neutralizing monoclonal antibodies specific for Naja nigricollis cardiotoxin: preparation, characterization and localization of the epitopes.
Two monoclonal antibodies have been raised against the native form of the potent cardiotoxin isolated from the venom of Naja nigricollis. The toxic action to mice as well as the depolarizing effect on muscle fibres in culture of the cardiotoxin are neutralized by the two immunoglobulins. Binding studies revealed that the radiolabelled toxin has a high affinity for both antibodies, the equilibrium dissociation constant values being equal to 0.2 and 0.4 nM. The epitopes that are recognized by the antibodies have been localized on the basis of competition experiments between the labelled toxin and a series of variants or a Trp-11 modified derivative, toward both antibodies. The data obtained indicate that the antibodies bind at topographically different antigenic sites. Knowing that the toxin is a single polypeptide chain folded in a structure that contains three adjacent loops emerging from a small globular region, it appears that one of the two antibodies binds on loop I, at a site which involves Trp-11 whereas the other binds at a site which involves one or both of loops II and III. Possible mechanisms of neutralization of the toxin by the antibodies are discussed.
Amino Acid Sequence↗
Isolation of structural genes for yeast RNA polymerases by immunological screening.
A lambda gt11 yeast genomic library was screened with antibodies directed against yeast RNA polymerases A, B, and C. Thirty-five individual recombinant phages that expressed proteins in Escherichia coli that were antigenically related to RNA polymerases A, B, or C were isolated by using 22 distinct antisera. Thus, all 22 genes for the RNA polymerase subunits were potentially cloned. In three cases (lambda A-43, lambda A-40, and lambda A-34.5), an antigenic protein was expressed in E. coli with the same molecular weight as the corresponding subunit. When lambda A-40 DNA was used to hybrid-select yeast mRNA, the protein translated in vitro was the expected size for the A-40 subunit, further supporting our isolation of the A-40 gene. However, mRNA hybrid selected by lambda A-27 DNA did not code for a protein of the correct size. The lengths of the mRNA that hybridized to phage lambda A-190 or lambda C-160 DNA on RNA blots were in agreement with the predicted sizes of the coding regions of the corresponding genes. As predicted by our previous immunological results, yeast DNA inserts of the lambda A-190 and lambda C-160 clones cross-hybridized to the B-220 subunit gene. The cloned genes for the RNA polymerase subunits will prove to be valuable tools for the study of the function, regulation, and genetics of the yeast RNA polymerases.
Antibodies, Fungal↗
A general upstream binding factor for genes of the yeast translational apparatus.
Fractionation of yeast extracts on heparin-agarose revealed the presence of a DNA footprinting activity that interacted specifically with the 5'-upstream region of TEF1 and TEF2 genes coding for the protein synthesis elongation factor EF-1 alpha, and of the ribosomal protein gene RP51A. The protected regions encompassed the conserved sequences 'HOMOL1' (AACATC TA CG T A G CA) or RPG-box (ACCCATACATT TA) previously detected 200-400 bp upstream of most of the yeast ribosomal protein genes examined. Two types of protein-DNA complexes were separated by a gel electrophoresis retardation assay. Complex 1, formed on TEF1, TEF2 and RP51A 5'-flanking region, was correlated with the protection of a 25-bp sequence. Complex 2, formed on TEF2 or RP51A probes at higher protein concentrations, corresponded to an extended footprint of 35-40 bp. The migration characteristics of the protein-DNA complexes and competition experiments indicated that the same component(s) interacted with the three different promoters. It is suggested that this DNA factor(s) is required for activation and coordinated regulation of the whole family of genes coding for the translational apparatus.
Base Sequence↗
Yeast RNA polymerase C and its subunits. Specific antibodies as structural and functional probes.
Yeast RNA polymerase C purified by a simple large scale method was resolved into multiple components by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Specific antibodies directed against each polypeptide chain were prepared in rabbits and used as structural and functional probes. With minor exceptions, each antibody recognized specifically the corresponding polypeptide by blot-immunodetection. Cross-reactions with purified RNA polymerases A and B confirmed our previous description of the subunits shared by the three nuclear RNA polymerases. Immunoadsorption of RNA polymerase C at different stages of purification using antibodies to subunits C160 and C128 yielded the same collection of polypeptides as found in the purified enzyme: C160, C128, C82, C53, C40, C37, C34, C31, C27, C25, C23, C19, C14.5, C12.5, and C10. Subunit-specific antibodies were used to probe the activity of RNA polymerase C in a specific, reconstituted transcription system as well as on a nonspecific template. Transcription of the tRNAGlu3 gene in vitro was inhibited when RNA polymerase C was preincubated with antibodies directed to C128, C82, C53, C34, C23, or C19. Antibodies to C82, C53, and C34 were much less inhibitory in the nonspecific assay. Inhibition by anti-C128 or anti-C23 was relieved by preincubation of enzyme C with plasmid DNA prior to antibody addition. These results are discussed in terms of the participation of these polypeptides to the active enzyme molecule, and of their possible role in DNA binding or transcription factor recognition.
Antibodies↗
Cloning, nucleotide sequence, and expression of one of two genes coding for yeast elongation factor 1 alpha.
One gene coding for yeast cytoplasmic elongation factor 1 alpha (EF-1 alpha) was isolated by colony hybridization using a cDNA probe prepared from purified EF-1 alpha mRNA. A recombinant plasmid, pLB1, with a 6-kilobase yeast DNA insert, was found by hybrid selection and translation experiments to carry the entire gene. The nucleotide sequence of the gene with its 5'- and 3'-flanking regions was determined. The 5' and 3' ends of EF-1 alpha mRNA were localized by the S1 nuclease mapping technique. The cloned gene, called TEF1, encodes a protein of 458 amino acids (Mr = 50,071) in a single, uninterrupted reading frame. The amino acid sequence shows a strong homology with several domains of Artemia salina EF-1 alpha cytoplasmic factor, as evidenced by diagonal dot matrix analysis. Protein sequence homology is comparatively much lower with the yeast mitochondrial elongation factor. S1 nuclease mapping of the mRNA, hybridization analysis of chromosomal DNA using intragenic or extragenic DNA probes, and gene disruption experiments demonstrated the existence of two genes coding for the cytoplasmic elongation factor EF-1 alpha/haploid genome. The presence of an intact chromosomal TEF1 gene is not essential for growth of haploid yeast cells.
Base Sequence↗
Elongation factor 1 alpha from Saccharomyces cerevisiae. Rapid large-scale purification and molecular characterization.
Cytoplasmic elongation factor 1 alpha (EF-1 alpha) was purified to homogeneity from the yeast Saccharomyces cerevisiae using a large-scale procedure. The three steps of purification used were batch adsorption on phosphocellulose, phosphocellulose chromatography and, as the last step, GDP-Sepharose or Biorex column chromatography. The protein is very basic (pI = 9.2) and has an apparent molecular mass of 49 kDa, as determined by polyacrylamide gel electrophoresis using denaturing conditions. It is one of the most abundant proteins in yeast (about 5% of total soluble protein), as shown by two-dimensional gel electrophoresis and by immunological titration. A strong immunological and structural homology was found between yeast EF-1 alpha and elongation factors from other sources. Common immunological features were found between yeast and wheat germ EF-1 alpha. Tryptic hydrolysis of yeast EF-1 alpha in the presence of 25% glycerol generated a large trypsin-resistant polypeptide (Mr = 43,000) which had the same NH2-terminal sequence as the proteolyzed product from rabbit reticulocyte, Artemia salina EF-1 alpha and Escherichia coli EF-Tu. Completed DNA sequence determination of one structural gene for yeast EF-1 alpha confirmed a remarkable conservation of several protein sequence domains in yeast and animal EF-1 alpha (Cottrelle, P., Thiele, D., Price, V., Memet, S., Micouin, J.Y., Marck, C., Buhler, J.M. Sentenac, A., and Fromageot, P. (1985) J. Biol. Chem. 260, 3090-3096).
Electrophoresis, Polyacrylamide Gel↗
Either one of the two yeast EF-1 alpha genes is required for cell viability.
Two genes, TEF1 and TEF2, encode the protein elongation factor EF-1 alpha in the yeast Saccharomyces cerevisiae. We have generated yeast haploid strains containing either TEF1 or TEF2 interrupted by insertion of a large piece of foreign DNA. Cells which contain either one functional copy of the EF-1 alpha genes are viable. In contrast, attempts to isolate a yeast haploid strain with both TEF1 and TEF2 inactivated have failed suggesting that the double gene disruption is a lethal event.
Genes↗
Further evidence showing that neurotoxin-acetylcholine receptor dissociation is accelerated by monoclonal neurotoxin-specific immunoglobulin.
We have demonstrated that the dissociation of Naja nigricollis alpha-toxin from the two acetylcholine receptor sites [Weber and Changeux, Molec. Pharmac. 10, 1-14 (1974); Rousselet et al., Eur. J. Biochem. 140, 31-37 (1984)], is markedly accelerated by a monoclonal neurotoxin-specific antibody. The dissociation of the toxin occurs in a biphasic manner in the presence of a 900 molar excess of immunoglobulin (with respect to toxin concn). The progress curves are characterized by first-order kinetics. Under these conditions the maximal dissociation rate is achieved as further rate enhancement cannot be induced by exposure to an increased immunoglobulin level. In contrast when a toxin-immunoglobulin complex is incubated with a large excess of receptor, the dissociation kinetics of the complex are not enhanced. The data fit a kinetic model which implicates the existence of a transient ternary complex involving the receptor, the toxin and the antibody.
Animals↗
Cloning and sequence analysis of the cDNA encoding a snake neurotoxin precursor.
A recombinant plasmid has been constructed containing a sequence of 186 nucleotides encoding a potent neurotoxin found in the venom of the sea-snake Laticauda semifasciata and designated as erabutoxin a. This sequence is flanked, in the upstream region, by a sequence of 60 nucleotides encoding a hydrophobic peptide fragment presumably involved in the secretion process of the neurotoxin. The sequence coding for the toxin ends with a termination codon which is followed by a 3'-untranslated sequence of approximately 240 nucleotides (excluding the poly(A) tract).
Amino Acid Sequence↗
C-terminal amidation of neuropeptides. Gly-Lys-Arg extension an efficient precursor of C-terminal amide.
Biosynthesis of the C-terminal carboxamide group of peptide hormones was studied using comparatively pGlu-His-Pro-Gly and Glu-His-Pro-Gly-Lys-Arg as putative precursors of the tripeptide, thyroliberin (TRH). Rat hypothalamus granules were found to contain an amide group forming activity which converts both peptide substrates into TRH. Comparison of the rate of conversion of the two substrates indicated that the C-terminal dibasic extension favored a 10-fold increase in the production of amidated peptide. It is suggested that this type of structure may be present in the putative biosynthetic precursor of TRH and that it may provide a better substrate for the enzyme(s) involved in C-terminal amidation.
Animals↗
Isolation of a class C transcription factor which forms a stable complex with tRNA genes.
A yeast extract was fractionated to resolve the factors involved in the transcription of yeast tRNA genes. An in vitro transcription system was reconstituted with two separate protein fractions and purified RNA polymerase C (III). Optimal conditions for tRNA synthesis have been determined. One essential component, termed tau factor, was partially purified by conventional chromatographic methods on heparin-agarose and DEAE-Sephadex; it sedimented as a large macromolecule in glycerol gradients (mol. wt. approximately 300 000). tau factor was found to form a stable complex with the tRNA gene in the absence of other transcriptional components. Complex formation is very fast, is not temperature dependent between 10 degrees C and 25 degrees C and does not require divalent cations. The factor-DNA complex is stable for at least 30 min at high salt concentration (0.1 M ammonium sulfate). These results indicate that gene recognition by a specific factor is a primary event in tRNA synthesis.
DNA Polymerase III↗