scholarly journals Transcriptomic Profiling Reveals Extraordinary Diversity of Venom Peptides in Unexplored Predatory Gastropods of the Genus Clavus

2020 ◽  
Vol 12 (5) ◽  
pp. 684-700
Author(s):  
Aiping Lu ◽  
Maren Watkins ◽  
Qing Li ◽  
Samuel D Robinson ◽  
Gisela P Concepcion ◽  
...  
Keyword(s):  
Signal Sequence ◽  
Structural Similarity ◽  
Gene Families ◽  
Venom Gland ◽  
Mature Peptide ◽  
Secretion Signal ◽  
Living Species ◽  
Evolutionary Success ◽  
Peptide Toxins ◽  
Conus Venom

Abstract Predatory gastropods of the superfamily Conoidea number over 12,000 living species. The evolutionary success of this lineage can be explained by the ability of conoideans to produce complex venoms for hunting, defense, and competitive interactions. Whereas venoms of cone snails (family Conidae) have become increasingly well studied, the venoms of most other conoidean lineages remain largely uncharacterized. In the present study, we present the venom gland transcriptomes of two species of the genus Clavus that belong to the family Drilliidae. Venom gland transcriptomes of two specimens of Clavus canalicularis and two specimens of Clavus davidgilmouri were analyzed, leading to the identification of a total of 1,176 putative venom peptide toxins (drillipeptides). Based on the combined evidence of secretion signal sequence identity, entire precursor similarity search (BLAST), and the orthology inference, putative Clavus toxins were assigned to 158 different gene families. The majority of identified transcripts comprise signal, pro-, mature peptide, and post-regions, with a typically short (<50 amino acids) and cysteine-rich mature peptide region. Thus, drillipeptides are structurally similar to conotoxins. However, convincing homology with known groups of Conus toxins was only detected for very few toxin families. Among these are Clavus counterparts of Conus venom insulins (drillinsulins), porins (drilliporins), and highly diversified lectins (drillilectins). The short size of most drillipeptides and structural similarity to conotoxins were unexpected, given that most related conoidean gastropod families (Terebridae and Turridae) possess longer mature peptide regions. Our findings indicate that, similar to conotoxins, drillipeptides may represent a valuable resource for future pharmacological exploration.

Saccharomyces cerevisiae secretes and correctly processes human interferon hybrid proteins containing yeast invertase signal peptides

1986 ◽  
Vol 6 (5) ◽  
pp. 1812-1819
Author(s):  
C N Chang ◽  
M Matteucci ◽  
L J Perry ◽  
J J Wulf ◽  
C Y Chen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Signal Sequence ◽  
Phosphoglycerate Kinase ◽  
Yeast Cells ◽  
Signal Peptidase ◽  
Human Interferon ◽  
Expression Plasmid ◽  
Kinase Gene ◽  
Secretion Signal ◽  
Yeast Invertase

Synthetic oligonucleotides coding for the yeast invertase secretion signal peptide were fused to the gene for the mature form of human interferon (huIFN-alpha 2). Two plasmids (E3 and F2) were constructed. E3 contained the invertase signal codons in a reading frame with the mature huIFN-alpha 2 gene. F2 had a deletion of the codon for alanine at amino acid residue-5 in the invertase signal and an addition of a methionine codon located between the coding sequences for the invertase signal and mature huIFN-alpha 2. Both hybrid genes were located adjacent to the promoter from the 3-phosphoglycerate kinase gene on the multicopy yeast expression plasmid, YEp1PT. Yeast transformants containing these plasmids produced somewhat more IFN than did the same expression plasmid containing the IFN gene with its human secretion signal sequence. HuIFN-alpha 2, purified from the medium of yeast cells containing E3, was found to be processed at the correct site. The huIFN-alpha 2 made by plasmid F2 was found to be completely processed at the junction between the invertase signal (a variant) and the methionine of methionine-huIFN-alpha 2. These results strongly suggested that the invertase signal (or its variant) attached to huIFN was efficiently recognized by the presumed signal recognition particle and was cleaved by the signal peptidase in the yeast cells. These results also suggested that amino acid changes on the right side of the cleavage site did not necessarily prevent cleavage or secretion.

scholarly journals Cloning of fibA, Encoding an Immunogenic Subunit of the Fibril-Like Surface Structure ofPeptostreptococcus micros

1999 ◽  
Vol 181 (8) ◽  
pp. 2485-2491 ◽  
Author(s):  
B. H. A. Kremer ◽  
J. J. E. Bijlsma ◽  
J. G. Kusters ◽  
J. de Graaff ◽  
T. J. M. van Steenbergen
Keyword(s):  
Amino Acid ◽  
Surface Structure ◽  
Signal Sequence ◽  
Open Reading Frames ◽  
Periodontal Pathogen ◽  
Immunogold Electron Microscopy ◽  
Expression Library ◽  
Gram Positive ◽  
Secretion Signal ◽  
Specific Serum

ABSTRACT Although we are currently unaware of its biological function, the fibril-like surface structure is a prominent characteristic of the rough (Rg) genotype of the gram-positive periodontal pathogenPeptostreptococcus micros. The smooth (Sm) type of this species as well as the smooth variant of the Rg type (RgSm) lack these structures on their surface. A fibril-specific serum, as determined by immunogold electron microscopy, was obtained through adsorption of a rabbit anti-Rg type serum with excess bacteria of the RgSm type. This serum recognized a 42-kDa protein, which was subjected to N-terminal sequencing. Both clones of a λTriplEx expression library that were selected by immunoscreening with the fibril-specific serum contained an open reading frame, designatedfibA, encoding a 393-amino-acid protein (FibA). The 15-residue N-terminal amino acid sequence of the 42-kDa antigen was present at positions 39 to 53 in FibA; from this we conclude that the mature FibA protein contains 355 amino acids, resulting in a predicted molecular mass of 41,368 Da. The putative 38-residue signal sequence of FibA strongly resembles other gram-positive secretion signal sequences. The C termini of FibA and two open reading frames directly upstream and downstream of fibA exhibited significant sequence homology to the C termini of a group of secreted and surface-located proteins of other gram-positive cocci that are all presumably involved in anchoring of the protein to carbohydrate structures. We conclude that FibA is a secreted and surface-located protein and as such is part of the fibril-like structures.

scholarly journals Sedolisins, a New Class of Secreted Proteases from Aspergillus fumigatus with Endoprotease or Tripeptidyl-Peptidase Activity at Acidic pHs

2006 ◽  
Vol 72 (3) ◽  
pp. 1739-1748 ◽  
Author(s):  
Utz Reichard ◽  
Barbara Léchenne ◽  
Abdul R. Asif ◽  
Frank Streit ◽  
Eric Grouzmann ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Signal Sequence ◽  
Gene Families ◽  
Peptidase Activity ◽  
New Class ◽  
Tripeptidyl Peptidase ◽  
Genes Encoding ◽  
Member Gene ◽  
Culture Supernatants ◽  
Hydrolysis Of

ABSTRACT The secreted proteolytic activity of Aspergillus fumigatus is of potential importance as a virulence factor and in the industrial hydrolysis of protein sources. The A. fumigatus genome contains sequences that could encode a five-member gene family that produces proteases in the sedolisin family (MEROPS S53). Four putative secreted sedolisins with a predicted 17- to 20-amino-acid signal sequence were identified and termed SedA to SedD. SedA produced heterologously in Pichia pastoris was an acidic endoprotease. Heterologously produced SedB, SedC, and SedD were tripeptidyl-peptidases (TPP) with a common specificity for tripeptide-p-nitroanilide substrates at acidic pHs. Purified SedB hydrolyzed the peptide Ala-Pro-Gly-Asp-Arg-Ile-Tyr-Val-His-Pro-Phe to Arg-Pro-Gly, Asp-Arg-Ile, and Tyr-Val-His-Pro-Phe, thereby confirming TPP activity of the enzyme. SedB, SedC, and SedD were detected by Western blotting in culture supernatants of A. fumigatus grown in a medium containing hemoglobin as the sole nitrogen source. A degradation product of SedA also was observed. A search for genes encoding sedolisin homologues in other fungal genomes indicates that sedolisin gene families are widespread among filamentous ascomycetes.

scholarly journals Lytic Activity of LysH5 Endolysin Secreted by Lactococcus lactis Using the Secretion Signal Sequence of Bacteriocin Lcn972

2012 ◽  
Vol 78 (9) ◽  
pp. 3469-3472 ◽  
Author(s):  
Lorena Rodríguez-Rubio ◽  
Dolores Gutiérrez ◽  
Beatriz Martínez ◽  
Ana Rodríguez ◽  
Pilar García
Keyword(s):  
Staphylococcus Aureus ◽  
Lactococcus Lactis ◽  
Signal Peptide ◽  
Signal Sequence ◽  
Content Type ◽  
Inducible Promoters ◽  
Secretion Signal ◽  
Cell Extracts ◽  
Culture Supernatants ◽  
Secretion Signal Sequence

ABSTRACTBacteriophage endolysins have an interesting potential as antimicrobials. The endolysin LysH5, encoded byStaphylococcus aureusphage vB_SauS-phi-IPLA88, was expressed and secreted inLactococcus lactisusing the signal peptide of bacteriocin lactococcin 972 and lactococcal constitutive and inducible promoters. Up to 80 U/mg of extracellular active endolysin was detected in culture supernatants, but most of the protein (up to 323 U/mg) remained in the cell extracts.

scholarly journals Comparative venom gland transcriptomics ofNaja kaouthia(monocled cobra) from Malaysia and Thailand: elucidating geographical venom variation and insights into sequence novelty

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3142 ◽  
Author(s):  
Kae Yi Tan ◽  
Choo Hock Tan ◽  
Lawan Chanhome ◽  
Nget Hong Tan
Keyword(s):  
Expression Patterns ◽  
Gene Families ◽  
Venom Gland ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Sequence Alignments ◽  
Illumina Hiseq ◽  
Multiple Sequence ◽  
Toxin Genes ◽  
Transcriptional Pattern

BackgroundThe monocled cobra (Naja kaouthia) is a medically important venomous snake in Southeast Asia. Its venom has been shown to vary geographically in relation to venom composition and neurotoxic activity, indicating vast diversity of the toxin genes within the species. To investigate the polygenic trait of the venom and its locale-specific variation, we profiled and compared the venom gland transcriptomes ofN. kaouthiafrom Malaysia (NK-M) and Thailand (NK-T) applying next-generation sequencing (NGS) technology.MethodsThe transcriptomes were sequenced on the Illumina HiSeq platform, assembled and followed by transcript clustering and annotations for gene expression and function. Pairwise or multiple sequence alignments were conducted on the toxin genes expressed. Substitution rates were studied for the major toxins co-expressed in NK-M and NK-T.Results and discussionThe toxin transcripts showed high redundancy (41–82% of the total mRNA expression) and comprised 23 gene families expressed in NK-M and NK-T, respectively (22 gene families were co-expressed). Among the venom genes, three-finger toxins (3FTxs) predominated in the expression, with multiple sequences noted. Comparative analysis and selection study revealed that 3FTxs are genetically conserved between the geographical specimens whilst demonstrating distinct differential expression patterns, implying gene up-regulation for selected principal toxins, or alternatively, enhanced transcript degradation or lack of transcription of certain traits. One of the striking features that elucidates the inter-geographical venom variation is the up-regulation of α-neurotoxins (constitutes ∼80.0% of toxin’s fragments per kilobase of exon model per million mapped reads (FPKM)), particularly the long-chain α-elapitoxin-Nk2a (48.3%) in NK-T but only 1.7% was noted in NK-M. Instead, short neurotoxin isoforms were up-regulated in NK-M (46.4%). Another distinct transcriptional pattern observed is the exclusively and abundantly expressed cytotoxin CTX-3 in NK-T. The findings suggested correlation with the geographical variation in proteome and toxicity of the venom, and support the call for optimising antivenom production and use in the region. Besides, the current study uncovered full and partial sequences of numerous toxin genes fromN. kaouthiawhich have not been reported hitherto; these includeN. kaouthia-specificl-amino acid oxidase (LAAO), snake venom serine protease (SVSP), cystatin, acetylcholinesterase (AChE), hyaluronidase (HYA), waprin, phospholipase B (PLB), aminopeptidase (AP), neprilysin, etc. Taken together, the findings further enrich the snake toxin database and provide deeper insights into the genetic diversity of cobra venom toxins.

scholarly journals Improved Production of Recombinant Myrosinase in Pichia pastoris

2021 ◽  
Vol 22 (21) ◽  
pp. 11889
Author(s):  
Zuzana Rosenbergová ◽  
Zuzana Hegyi ◽  
Miroslav Ferko ◽  
Natália Andelová ◽  
Martin Rebroš
Keyword(s):  
Pichia Pastoris ◽  
Signal Sequence ◽  
Specific Productivity ◽  
Endoplasmic Reticulum Membrane ◽  
Volumetric Activity ◽  
Secretion Signal ◽  
Plant Enzymes ◽  
Optimum Ph ◽  
Mating Factor ◽  
Native Signal Sequence

The effect of the deletion of a 57 bp native signal sequence, which transports the nascent protein through the endoplasmic reticulum membrane in plants, on improved AtTGG1 plant myrosinase production in Pichia pastoris was studied. Myrosinase was extracellularly produced in a 3-liter laboratory fermenter using α-mating factor as the secretion signal. After the deletion of the native signal sequence, both the specific productivity (164.8 U/L/h) and volumetric activity (27 U/mL) increased more than 40-fold compared to the expression of myrosinase containing its native signal sequence in combination with α-mating factor. The deletion of the native signal sequence resulted in slight changes in myrosinase properties: the optimum pH shifted from 6.5 to 7.0 and the maximal activating concentration of ascorbic acid increased from 1 mM to 1.5 mM. Kinetic parameters toward sinigrin were determined: 0.249 mM (Km) and 435.7 U/mg (Vmax). These results could be applied to the expression of other plant enzymes.

scholarly journals Recombinant cephalosporin-acid synthesase: optimisation of expression in E.coli cells, immobilisation and application for biocatalytic cefazolin synthesis

2015 ◽  
Vol 61 (5) ◽  
pp. 646-651
Author(s):  
M.A. Eldarov ◽  
A.V. Sklyarenko ◽  
M.V. Dumina ◽  
N.V. Medvedeva ◽  
A.A. Jgoun ◽  
...  
Keyword(s):  
Signal Sequence ◽  
Erwinia Carotovora ◽  
Growth Media ◽  
Media Composition ◽  
Specific Enzyme ◽  
Secretion Signal ◽  
Mild Conditions ◽  
Escherichia Coli Cells ◽  
Effective Synthesis ◽  
Efficient Expression

Cephalosporin acid synthetase (CASA) is responsible for specific to synthesis of cephalosporin-acids, its expression in Escherichia coli cells is accompanied by accumulation of unprocessed insoluble precursor. In order to optimize conditions of recombinant CASA production we have studied the effects of several parameters of strain cultivation, including growth media composition, temperature, and inoculation dose. Also plasmids for production of CASA variants with the signal sequence of Erwinia carotovora L-asparaginase (ansCASA) and “leaderless” CASA were created in search of more efficient expression constructs. Removal of the N-terminal secretion signal sequence reduced the production of functionally active CASA more than 10-fold and inhibited strain growth. Insertion of the L-asparaginase signal sequence increased the specific enzyme activity in the resultant recombinant strain. The ansCASA producing strain was used to develop the method of immobilization of the recombinant enzyme on an epoxy-activated macroporous acrylic support. The resultant biocatalyst performed effective synthesis of cefazolin from 3-[(5-methyl-1,3,4-thiadiazol-2-il)-thiomethyl]-7- aminocephalosporanic acid (MMTD-7-ACA) and methyl ester of 1(H)-tetrazolilacetic acid (МETzAA), under mild conditions a transformation level of MMTD-7-ACA to cefazolin of 95% is reached.

scholarly journals Assimilation of Cellooligosaccharides by a Cell Surface-Engineered Yeast Expressing β-Glucosidase and Carboxymethylcellulase from Aspergillus aculeatus

1998 ◽  
Vol 64 (12) ◽  
pp. 4857-4861 ◽  
Author(s):  
Toshiyuki Murai ◽  
Mitsuyoshi Ueda ◽  
Takashi Kawaguchi ◽  
Motoo Arai ◽  
Atsuo Tanaka
Keyword(s):  
Cell Surface ◽  
Rhizopus Oryzae ◽  
Signal Sequence ◽  
Hydrolytic Enzymes ◽  
Water Soluble ◽  
Aspergillus Aculeatus ◽  
Cellulosic Materials ◽  
Secretion Signal ◽  
Engineered Yeast ◽  
Secretion Signal Sequence

ABSTRACT Since Saccharomyces cerevisiae lacks the cellulase complexes that hydrolyze cellulosic materials, which are abundant in the world, two types of hydrolytic enzymes involved in the degradation of cellulosic materials to glucose were genetically co-immobilized on its cell surface for direct utilization of cellulosic materials, one of the final goals of our studies. The genes encoding FI-carboxymethylcellulase (CMCase) and β-glucosidase from the fungusAspergillus aculeatus were individually fused with the gene encoding the C-terminal half (320 amino acid residues from the C terminus) of yeast α-agglutinin and introduced into S. cerevisiae. The delivery of CMCase and β-glucosidase to the cell surface was carried out by the secretion signal sequence of the native signal sequence of CMCase and by the secretion signal sequence of glucoamylase from Rhizopus oryzae for β-glucosidase, respectively. The genes were expressed by the glyceraldehyde-3-phosphate dehydrogenase promoter from S. cerevisiae. The CMCase and β-glucosidase activities were detected in the cell pellet fraction, not in the culture supernatant. The display of CMCase and β-glucosidase proteins on the cell surface was confirmed by immunofluorescence microscopy. The cells displaying these cellulases could grow on cellobiose or water-soluble cellooligosaccharides as the sole carbon source. The degradation and assimilation of cellooligosaccharides were confirmed by thin-layer chromatography. This result showed that the cell surface-engineered yeast with these enzymes can be endowed with the ability to assimilate cellooligosaccharides. This is the first step in the assimilation of cellulosic materials by S. cerevisiae expressing heterologous cellulase genes.

scholarly journals Biochemical and structural investigation of rutinosidase from Aspergillus oryzae

2020 ◽  
Author(s):  
Koki Makabe ◽  
Ruka Hirota ◽  
Yoshihito Shiono ◽  
Yoshikazu Tanaka ◽  
Takuya Koseki
Keyword(s):  
Crystal Structure ◽  
Aspergillus Oryzae ◽  
Structural Investigation ◽  
Signal Sequence ◽  
Structural Similarity ◽  
Industrial Applications ◽  
Flavonoid Glycoside ◽  
Flavonoid Glycosides ◽  
High Catalytic Activity ◽  
Tim Barrel

The rutinosidase-encoding gene Aorut has been expressed in Pichia pastoris with its native signal sequence from Aspergillus oryzae. Biochemical and structural investigation of the purified recombinant mature AoRut, designated as rAoRutM, was performed in this study. A 1.7 Å resolution crystal structure of rAoRutM was determined, which is an essential step forward in the utilization of AoRut as a potential catalyst. The crystal structure of rAoRutM was represented by a (β/α)8 TIM barrel fold with structural similarity to rutinosidase from Aspergillus niger (AnRut) and an exo-β (1, 3)-glucanase from Candida albicans. The crystal structure revealed that the catalytic site was located in a deep cleft, similar to AnRut, and internal cavities and water molecules were also present. Purified rAoRutM hydrolyzed not only 7-O-linked and 3-O-linked flavonoid rutinosides, but also 7-O-linked and 3-O-linked flavonoid glucosides. rAoRutM displayed high catalytic activity toward quercetin 3-O-linked substrates such as rutin and isoquercitrin, rather than the 7-O-linked substrate, quercetin-7-O-glucoside. Unexpectedly, purified rAoRutM exhibited increased thermostability after treatment with endo-β-N-acetylglucosaminidase H. Circular dichroism (CD) spectra of purified intact rAoRutM and the enzyme after N-deglycosylation showed a typical α-helical CD profile, however, the molar ellipticity values of the peaks at 208 nm and 212 nm varied. The Km and kcat values for the substrates modified by rutinose were higher than those for substrates modified by β-D-glucose. Importance Flavonoid glycosides constitute a class of secondary metabolites widely distributed in nature. These compounds are involved in the bitter taste or clouding in plant-based foods or beverages, respectively. Flavonoid glycoside degradation can proceed through two alternative enzymatic pathways: one that is mediated by monoglycosidases, and the other catalyzed by a diglycosidase. The present study on the biochemical and structural investigation of A. oryzae rutinosidase provides a potential biocatalyst for industrial applications of flavonoids.

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