Killer toxin from the yeast Saccharomyces cerevisiae : purification and properties

Note:Killer toxin from killer strains of Saccharomyces cerevisiae has been isolated from concentrates of extracellular medium by precipitation in polyethylene glycol and chromatography throughGlyceryl-controlled-pore glass in 4 M urea. Upon sodium dodecyl sulfate polyacrylamide gel electrophoresis the toxin exhibited a single band of protein with molecular weight of 11.000. Gel filtration indicated that the monomer protein vas the active unit. Amino acid analysis has been performed and indicated a molecular weight of 11 470. In the absence of dissociating conditions, toxin co-purified with mannan-protein. Two fractions containing aggregates of toxin and mannan protein have been purified. The mannan-protein components, 93% carbohydrate, were polydisperse with Mw - 200 000 and 370 000. The presence of concanavalin A during concentration of extracellular macromolecules inhibited the formation of aggregates containing active toxin. This implicated mannan in the binding of toxin.La toxine des souches 'killer' de Saccharomyces cerevisiae a été purifiée du médium extracellulaire par concentration, précipitation en présence de polyéthylène glycol, et chromatographie sur colonne de GLY-CPGl20 en présence de 4 M d'urée. D'après l'électrophorèse sur gel dodécyl sulfate de sodium (SDS), l'échantillon purifié semblait homogène. La bande protéique avait une mobilité correspondante à celle d'un polypeptide de 11 000 de poids moléculaire. D'après la filtration sur Sépharose CL-6B, la toxine agissante était une protéine monomère. La valeur du poids moléculaire évaluée par analyse chimique était 11 470. Dans les conditions de non dissociation, la toxine a été purifiée en même temps que mannoprotéine. Deux fractions qui contenaient des agrégats de la toxine et de la mannoprotéine ont été purifiées. Les mannoprotéines étaient à 93% d’hydrate de carbone, et avaient des poids moléculaire moyens (M ) de 200 000 et de 370 000. La présence de la concanavali A pendant la concentration des macromolécules extracellulaires a empêché la formation d'agrégats contenant la toxine active. Ceci suggère que la portion mannan était le site de liaison de la toxine

Comparison of the spatial placement of conserved residues in GrnA and GrnF.

<p>The GrnA (right) and GrnF (left) modules are shown in two orientations comparing the conformation around the common tryptophan (Trp21, GrnA and Trp22, GrnF) is shown in brown tube style. The space fill side chains are those which show module-specific high conservation. Residues conserved in all granulin modules are shown only as tube backbones coloured yellow for cysteine and black for those highly conserved in all granulin modules. The most variable residues are coloured grey. The colour coding for the space filling side chains follows the conventions in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133749#pone.0133749.g008" target="_blank">Fig 8</a>. Red side-chains are basic, dark blue residues are acidic.</p

Syntenic conservation between the genomic regions around <i>GrnC</i> gene from the coelacanth, Zebrafish short-form <i>Grn</i> genes and a human Chr7 region.

<p>The genomic context of the Coelacanth <i>GrnC</i> (<i>L_ChaC</i>) based on an Ensembl scaffold (vertical line) aligns with the short-form <i>Grn</i> genes of the zebrafish, and a region of human chromosome 7. Genes in color are orthologues. Gene names are as assigned in the NCBI databases (Grn1 and Grn2 are <i>D_rer1</i> and <i>D_rer2</i> in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133749#pone.0133749.g005" target="_blank">5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133749#pone.0133749.g006" target="_blank">6</a>). *This gene was unassigned by NCBI and was identified by pBlast analysis (Ensembl:ENSDARG00000035821, NCBI accession for translated protein XP_005159758.1). An additional predicted protein coding sequence for piggyBac transposable element-derived protein 3-like (XP_005159764.1, not shown) is contained within the DNA sequence for XP_005159758.1.</p

The Evolution of the Secreted Regulatory Protein Progranulin

<div><p>Progranulin is a secreted growth factor that is active in tumorigenesis, wound repair, and inflammation. Haploinsufficiency of the human progranulin gene, <i>GRN</i>, causes frontotemporal dementia. Progranulins are composed of chains of cysteine-rich granulin modules. Modules may be released from progranulin by proteolysis as 6kDa granulin polypeptides. Both intact progranulin and some of the granulin polypeptides are biologically active. The granulin module occurs in certain plant proteases and progranulins are present in early diverging metazoan clades such as the sponges, indicating their ancient evolutionary origin. There is only one <i>Grn</i> gene in mammalian genomes. More gene-rich <i>Grn</i> families occur in teleost fish with between 3 and 6 members per species including short-form <i>Grn</i>s that have no tetrapod counterparts. Our goals are to elucidate progranulin and granulin module evolution by investigating (i): the origins of metazoan progranulins (ii): the evolutionary relationships between the single <i>Grn</i> of tetrapods and the multiple <i>Grn</i> genes of fish (iii): the evolution of granulin module architectures of vertebrate progranulins (iv): the conservation of mammalian granulin polypeptide sequences and how the conserved granulin amino acid sequences map to the known three dimensional structures of granulin modules. We report that progranulin-like proteins are present in unicellular eukaryotes that are closely related to metazoa suggesting that progranulin is among the earliest extracellular regulatory proteins still employed by multicellular animals. From the genomes of the elephant shark and coelacanth we identified contemporary representatives of a precursor for short-from <i>Grn</i> genes of ray-finned fish that is lost in tetrapods. In vertebrate <i>Grn</i>s pathways of exon duplication resulted in a conserved module architecture at the amino-terminus that is frequently accompanied by an unusual pattern of tandem nearly identical module repeats near the carboxyl-terminus. Polypeptide sequence conservation of mammalian granulin modules identified potential structure-activity relationships that may be informative in designing progranulin based therapeutics.</p></div

Comparison of the spatial placement of conserved residues in GrnA and GrnF.

<p>The GrnA (right) and GrnF (left) modules are shown in two orientations comparing the conformation around the common tryptophan (Trp21, GrnA and Trp22, GrnF) is shown in brown tube style. The space fill side chains are those which show module-specific high conservation. Residues conserved in all granulin modules are shown only as tube backbones coloured yellow for cysteine and black for those highly conserved in all granulin modules. The most variable residues are coloured grey. The colour coding for the space filling side chains follows the conventions in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133749#pone.0133749.g008" target="_blank">Fig 8</a>. Red side-chains are basic, dark blue residues are acidic.</p

Mammalian progranulin sequences analyzed and their NCBI or Ensembl accession codes.

<p>* See text for information on manually predicted and amended sequences.</p><p>Mammalian progranulin sequences analyzed and their NCBI or Ensembl accession codes.</p

The protein and gene structure of a mammalian progranulin.

<p>(A). Progranulin is composed of multiple repeats of a highly conserved 12-cysteine granulin motif [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133749#pone.0133749.ref001" target="_blank">1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133749#pone.0133749.ref039" target="_blank">39</a>]. Mammalian progranulins possess 7.5 granulin modules labeled A to G (circles) as well as a half-granulin motif called paragranulin (half circle, p) that contains the N-terminal six cyteines of the full granulin motif. The modules are separated by short joining sequences and can be released as individual granulin peptides of about 6kDa by proteolysis of PGRN (B) Each granulin module is encoded by two exons [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133749#pone.0133749.ref040" target="_blank">40</a>]. These are referred to as N-type exons (red), C-type exons (blue) that encode the first six N-terminal and last six C-terminal cysteines respectively or CN exons (green) that span the final C-terminally located six cysteines and the first N-terminally located six cysteines of adjacent granulin modules. The corresponding granulin polypeptide modules are bracketed. (C) The spatial conformation of carp granulin, determined by 2-dimension nuclear magnetic resonance spectroscopy [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133749#pone.0133749.ref041" target="_blank">41</a>] reveals that the granulin modules adopt a compact fold of beta turns (red and green ribbons) with the 6 disulfide bridges shown as yellow bonds.</p

Interspecies variability within mammalian granulin modules.

<p>(A) To calculate distance relationships between mammalian consensus granulin modules the aligned consensus sequences for the 7 full modules in mammalian progranulin were analyzed with PROTDIST followed by FITCH. The JTT matrix was used, and global rearrangements applied. Branch lengths in parentheses result from removing the 12 conserved Cys residue positions from the analysis. (B) To determine the degrees of sequence variability for each granulin module the <b>s</b>equence distances between 37 species were calculated for each module using the Phylip Protdist program (JTT matrix). The data are represented by a box plot. The median divides the box with green down to the 1st quartile and purple up to the 3rd quartile. The background grey bars encompass all distances including outliers. (C). Diversity at the nucleotide level was determined by calculating the pairwise distances between DNA sequences from 37 species for each half module using DNAdist with the LogDet method. The means and standard deviations are plotted for each N-half, including the paragranulin, p, and each C-half in a way which shows their relationship to the modular structure of the progranulin and to its underlying exon structure.</p

C-half module relationships illustrated by a DNA-based maximum likelihood tree.

<p>The <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133749#pone.0133749.g005" target="_blank">Fig 5</a> legend provides most of the explanation. In addition, by analogy with labelling of paragranulins, a C-half with no paired N-half is labelled "q". The small form from Atlantic cod, G_morC, has the module form gpp, but if the second position stop codon in coding exon 4 is suppressed, the read completes an otherwise good C-half. This was included in the analysis and the virtual module called G_morC_2sup. Thus the third G_morC N-half module is properly a second paragranulin, G_morC_p2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133749#pone.0133749.g005" target="_blank">Fig 5</a>, but is also the N-half of the G_morC_2sup virtual module.</p

N-half module relationships illustrated by a DNA-based maximum likelihood tree.

<p>Letters and numbers are used to label some sub-trees and groups of sub-trees to facilitate reference from the text. Abbreviations for species and the specific Grn gene if there two or more are shown in the “species” boxes in the figure. In half-module labelling, this abbreviation is followed by a number based on the sequential numbering of whole modules encoded within the gene. For visual clarity, a "z" follows the number of the last module in a long-form of progranulin. When a repetitive module sequence or a lack of data make the number of modules uncertain, as in the lancelet (B_flo), the last letters of the alphabet replace numbering for the last sequential modules. N-half modules which are unpaired with C-half modules, paragranulins, are labeled "p", and followed by a number if there is more than one in the progranulin. An asterisk (*) indicates a 5-Cys form of half-module. (See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133749#pone.0133749.g006" target="_blank">Fig 6</a> for a note on the cod module nomenclature). Bipartition support values of ≥20 are included. These were based upon data for trees from which rogue taxa had been pruned. The rogue taxa are indicated by red branches, and the support values for their placement in this topology are in red.</p