Cloning and characterization of two glutathione S-transferases from a DDT-resistant strain of Anopheles gambiae

Two cDNA species, aggst1-5 and aggst1-6, comprising the entire coding region of two distinct glutathione S-transferases (GSTs) have been isolated from a 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane (DDT) resistant strain (ZANDS) of Anopheles gambiae. The nucleotide sequences of these cDNA species share 80.2% identity and their derived amino acid sequences are 82.3% similar. They have been classified as insect class I GSTs on the basis of their high sequence similarity to class I GSTs from Drosophila melanogaster and Musca domestica and they are localized to a region of an An. gambiae chromosome known to contain further class I GSTs. The genes aggst1-5 and aggst1-6 were expressed at high levels in Escherichia coli and the recombinant GSTs were purified by affinity chromatography and characterized. Both agGST1-5 and agGST1-6 showed high activity with the substrates 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene but negligible activity with the mammalian theta class substrates, 1,2-epoxy-3-(4-nitrophenoxy)propane and p-nitrophenyl bromide. Despite their high level of sequence identity, agGST1-5 and agGST1-6 displayed different kinetic properties. Both enzymes were able to metabolize DDT and were localized to a subset of GSTs that, from earlier biochemical studies, are known to be involved in insecticide resistance in An. gambiae. This subset of enzymes is one of three in which the DDT metabolism levels are elevated in resistant insects.

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Grass Carp (Ctenopharyngodon idellus) NIMA-Related Kinase 6 Blocks dsRNA-Induced IFN I Response by Targeting IRF3

Frontiers in Immunology ◽

10.3389/fimmu.2020.597775 ◽

2021 ◽

Vol 11 ◽

Author(s):

Xiaowen Xu ◽

Meifeng Li ◽

Zeyuan Deng ◽

Jihuan Hu ◽

Zeyin Jiang ◽

...

Keyword(s):

Grass Carp ◽

Sequence Similarity ◽

Amino Acid Sequences ◽

Poly I:C ◽

Ctenopharyngodon Idellus ◽

Cell Viability Assay ◽

Antiviral Responses ◽

Cik Cells ◽

Viability Assay ◽

High Level

Accumulating evidence indicates that mammalian NIMA (never in mitosis, gene A)-related kinase 6 (NEK6) plays potential roles during the course of tumorigenesis, but little is known about NEK6 in lower vertebrates. Herein, we reported a mammalian ortholog of NEK6 in grass carp (Ctenopharyngodon idellus) (CiNEK6). Multiple alignment of amino acid sequences and phylogenetic analysis showed that CiNEK6 shares a high level of sequence similarity with its counterparts in birds. CiNEK6 was ubiquitously expressed in all tested tissues, and its expression level was increased under treatment with GCRV (dsRNA virus) or poly I:C (dsRNA analog). Q-PCR and dual-luciferase assays suggested that CiNEK6 overexpression suppressed IFN I activity in CIK cells treated with poly I:C. Knockdown of CiNEK6 resulted in a higher level of IFN I expression in CIK cells treated with poly I:C compared to those which received PBS. Interestingly, analysis of subcellular localization demonstrated that CiNEK6 protein scattered throughout the cytoplasm is gradually congregated together at the edges of karyotheca upon stimulation with poly I:C. Co-IP and co-localization assays suggested that CiNEK6 interacts with CiIRF3 after poly I:C challenge. In poly I:C-treated cells, the phosphorylation of CiIRF3 was increased by CiNEK6 knockdown, but was suppressed by CiNEK6 overexpression, suggesting that CiNEK6 decreases IFN I expression through inhibiting CiIRF3 activity. Cell viability assay, crystal violet staining, and detection of Vp5 also showed that CiNEK6 plays an inhibitory role in IRF3-mediated antiviral responses.

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The vsp Locus of Mycoplasma bovis: Gene Organization and Structural Features

Journal of Bacteriology ◽

10.1128/jb.181.18.5734-5741.1999 ◽

1999 ◽

Vol 181 (18) ◽

pp. 5734-5741 ◽

Cited By ~ 67

Author(s):

Inessa Lysnyansky ◽

Konrad Sachse ◽

Ricardo Rosenbusch ◽

Sharon Levisohn ◽

David Yogev

Keyword(s):

High Frequency ◽

Sequence Similarity ◽

Membrane Surface ◽

Amino Acid Sequences ◽

High Rate ◽

Phenotypic Switching ◽

Upstream Region ◽

Mycoplasma Bovis ◽

High Sequence Similarity ◽

Polypeptide Structure

ABSTRACT Major lipoprotein antigens, known as variable membrane surface lipoproteins (Vsps), on the surface of the bovine pathogenMycoplasma bovis were shown to spontaneously undergo noncoordinate phase variation between ON and OFF expression states. The high rate of Vsp phenotypic switching was also shown to be linked with DNA rearrangements that occur at high frequency in the M. bovis chromosome (I. Lysnyansky, R. Rosengarten, and D. Yogev, J. Bacteriol. 178:5395–5401, 1996). In the present study, 13 single-copyvsp genes organized in a chromosomal cluster were identified and characterized. All vsp genes encode highly conserved N-terminal domains for membrane insertion and lipoprotein processing but divergent mature Vsp proteins. About 80% of eachvsp coding region is composed of reiterated coding sequences that create a periodic polypeptide structure. Eighteen distinct repetitive domains of different lengths and amino acid sequences are distributed within the products of the variousvsp genes that are subject to size variation due to spontaneous insertions or deletions of these periodic units. Some of these repeats were found to be present in only one Vsp family member, whereas other repeats recurred at variable locations in several Vsps. Each vsp gene is also 5′ linked to a highly homologous upstream region composed of two internal cassettes. The findings that rearrangement events are associated with Vsp phenotypic switching and that multiple regions of high sequence similarity are present upstream of the vsp genes and within the vsp coding regions suggest that modulation of the Vsp antigenic repertoire is determined by recombination processes that occur at a high frequency within the vsp locus of M. bovis.

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Optimized heterologous expression of the human zinc enzyme glyoxalase I

Biochemical Journal ◽

10.1042/bj3140463 ◽

1996 ◽

Vol 314 (2) ◽

pp. 463-467 ◽

Cited By ~ 26

Author(s):

Marianne ÖM ◽

Bengt MANNERVIK

Keyword(s):

Culture Medium ◽

Functional Characterization ◽

Zinc Salt ◽

Glyoxalase I ◽

Kinetic Properties ◽

Coding Region ◽

Expression Clone ◽

High Level ◽

Inhibition Studies

DNA coding for human glyoxalase I was isolated from a HeLa cell cDNA library by means of PCR. The deduced amino acid sequence differs from previously isolated sequences in that a glutamic acid replaces an alanine in position 111. This variant cDNA may represent the more acidic isoform of glyoxalase I originally identified at the protein level. An expression clone was constructed for high-level production of glyoxalase I in Escherichia coli. For optimal yield of the recombinant protein, silent random mutations were introduced in the cDNA coding region. Antisera against human glyoxalase I were used to select a high-level expression clone. This clone afforded 60 mg of purified enzyme per litre of culture medium. Addition of a zinc salt to the culture medium was essential to obtain an active enzyme and a stoicheiometric metal content. The functional characterization of the recombinant enzyme included determination of kinetic constants for methylglyoxal, phenylglyoxal and p-phenylphenylglyoxal, as well as inhibition studies. The kinetic properties of recombinant glyoxalase I were indistinguishable from those of the enzyme purified from human tissues.

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The “Green” Form I Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase from the Nonsulfur Purple BacteriumRhodobacter capsulatus

Journal of Bacteriology ◽

10.1128/jb.181.13.3935-3941.1999 ◽

1999 ◽

Vol 181 (13) ◽

pp. 3935-3941 ◽

Cited By ~ 12

Author(s):

Kempton M. Horken ◽

F. Robert Tabita

Keyword(s):

Genetic Manipulation ◽

Sequence Similarity ◽

Large Subunit ◽

Small Subunit ◽

Amino Acid Sequences ◽

Kinetic Properties ◽

Phylogenetic Groups ◽

Bisphosphate Carboxylase ◽

Related Organism ◽

Green Form

ABSTRACT Form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) of the Calvin-Benson-Bassham cycle may be divided into two broad phylogenetic groups, referred to as red-like and green-like, based on deduced large subunit amino acid sequences. Unlike the form I enzyme from the closely related organism Rhodobacter sphaeroides, the form I RubisCO from R. capsulatus is a member of the green-like group and closely resembles the enzyme from certain chemoautotrophic proteobacteria and cyanobacteria. As the enzymatic properties of this type of RubisCO have not been well studied in a system that offers facile genetic manipulation, we purified theR. capsulatus form I enzyme and determined its basic kinetic properties. The enzyme exhibited an extremely low substrate specificity factor, which is congruent with its previously determined sequence similarity to form I enzymes from chemoautotrophs and cyanobacteria. The enzymological results reported here are thus strongly supportive of the previously suggested horizontal gene transfer that most likely occurred between a green-like RubisCO-containing bacterium and a predecessor to R. capsulatus. Expression results from hybrid and chimeric enzyme plasmid constructs, made with large and small subunit genes fromR. capsulatus and R. sphaeroides, also supported the unrelatedness of these two enzymes and were consistent with the recently proposed phylogenetic placement of R. capsulatus form I RubisCO. The R. capsulatus form I enzyme was found to be subject to a time-dependent fallover in activity and possessed a high affinity for CO2, unlike the closely similar cyanobacterial RubisCO, which does not exhibit fallover and possesses an extremely low affinity for CO2. These latter results suggest definite approaches to elucidate the molecular basis for fallover and CO2 affinity.

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Isolation of Cysteine-Rich Peptides from Citrullus colocynthis

Biomolecules ◽

10.3390/biom10091326 ◽

2020 ◽

Vol 10 (9) ◽

pp. 1326

Author(s):

Behzad Shahin-Kaleybar ◽

Ali Niazi ◽

Alireza Afsharifar ◽

Ghorbanali Nematzadeh ◽

Reza Yousefi ◽

...

Keyword(s):

Trypsin Inhibitor ◽

High Performance ◽

De Novo ◽

Sequence Similarity ◽

In Silico Analysis ◽

Reversed Phase ◽

Amino Acid Sequences ◽

Peptide Fragments ◽

Citrullus Colocynthis ◽

High Sequence Similarity

The plant Citrullus colocynthis, a member of the squash (Cucurbitaceae) family, has a long history in traditional medicine. Based on the ancient knowledge about the healing properties of herbal preparations, plant-derived small molecules, e.g., salicylic acid, or quinine, have been integral to modern drug discovery. Additionally, many plant families, such as Cucurbitaceae, are known as a rich source for cysteine-rich peptides, which are gaining importance as valuable pharmaceuticals. In this study, we characterized the C. colocynthis peptidome using chemical modification of cysteine residues, and mass shift analysis via matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. We identified the presence of at least 23 cysteine-rich peptides in this plant, and eight novel peptides, named citcol-1 to -8, with a molecular weight between ~3650 and 4160 Da, were purified using reversed-phase high performance liquid chromatography (HPLC), and their amino acid sequences were determined by de novo assignment of b- and y-ion series of proteolytic peptide fragments. In silico analysis of citcol peptides revealed a high sequence similarity to trypsin inhibitor peptides from Cucumis sativus, Momordica cochinchinensis, Momordica macrophylla and Momordica sphaeroidea. Using genome/transcriptome mining it was possible to identify precursor sequences of this peptide family in related Cucurbitaceae species that cluster into trypsin inhibitor and antimicrobial peptides. Based on our analysis, the presence or absence of a crucial Arg/Lys residue at the putative P1 position may be used to classify these common cysteine-rich peptides by functional properties. Despite sequence homology and the common classification into the inhibitor cysteine knot family, these peptides appear to have diverse and additional bioactivities yet to be revealed.

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Identification of a novel class of insect glutathione S-transferases involved in resistance to DDT in the malaria vector Anopheles gambiae

Biochemical Journal ◽

10.1042/bj3590295 ◽

2001 ◽

Vol 359 (2) ◽

pp. 295-304 ◽

Cited By ~ 137

Author(s):

Hilary RANSON ◽

Louise ROSSITER ◽

Federica ORTELLI ◽

Betty JENSEN ◽

Xuelan WANG ◽

...

Keyword(s):

Gene Family ◽

Anopheles Gambiae ◽

Malaria Vector ◽

Sequence Similarity ◽

Mrna Levels ◽

Class Iii ◽

Glutathione S Transferase ◽

Close Proximity ◽

Genes Encoding ◽

Glutathione S Transferases

The sequence and cytological location of five Anopheles gambiae glutathione S-transferase (GST) genes are described. Three of these genes, aggst1-8, aggst1-9 and aggst1-10, belong to the insect class I family and are located on chromosome 2R, in close proximity to previously described members of this gene family. The remaining two genes, aggst3-1 and aggst3-2, have a low sequence similarity to either of the two previously recognized classes of insect GSTs and this prompted a re-evaluation of the classification of insect GST enzymes. We provide evidence for seven possible classes of insect protein with GST-like subunits. Four of these contain sequences with significant similarities to mammalian GSTs. The largest novel insect GST class, class III, contains functional GST enzymes including two of the A. gambiae GSTs described in this report and GSTs from Drosophila melanogaster, Musca domestica, Manduca sexta and Plutella xylostella. The genes encoding the class III GST of A. gambiae map to a region of the genome on chromosome 3R that contains a major DDT [1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane] resistance gene, suggesting that this gene family is involved in GST-based resistance in this important malaria vector. In further support of their role in resistance, we show that the mRNA levels of aggst3-2 are approx. 5-fold higher in a DDT resistant strain than in the susceptible strain and demonstrate that recombinant AgGST3-2 has very high DDT dehydrochlorinase activity.

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Metalloproteinase-like, disintegrin-like, cysteine-rich proteins MDC2 and MDC3: novel human cellular disintegrins highly expressed in the brain

Biochemical Journal ◽

10.1042/bj3340093 ◽

1998 ◽

Vol 334 (1) ◽

pp. 93-98 ◽

Cited By ~ 55

Author(s):

Koji SAGANE ◽

Yukio OHYA ◽

Yoshikazu HASEGAWA ◽

Isao TANAKA

Keyword(s):

Snake Venom ◽

Sequence Similarity ◽

Amino Acid Sequences ◽

Cytogenet Cell ◽

Binding Motif ◽

High Sequence Similarity ◽

Cell Matrix ◽

A Disintegrin And Metalloproteinase ◽

Cell Matrix Interactions ◽

The Brain

Cellular disintegrins are a family of membrane-anchored proteins structurally related to snake venom disintegrins, and are potential regulators of cell–cell and cell–matrix interactions. The members of this protein family are also called ADAMs (a disintegrin and metalloproteinase) or MDC proteins (metalloproteinase-like disintegrin-like cysteine-rich), because they all contain disintegrin-like and metalloproteinase-like domains. In this paper, we report the cloning and sequence analysis of two novel additional members of this family, which we have termed MDC2 and MDC3. The deduced amino acid sequences reveal that the two proteins possess typical cellular disintegrin structures [that is, pro-, metalloproteinase-like, disintegrin-like, cysteine-rich, epidermal growth factor-like, transmembrane, and cytoplasmic domains] and exhibit high sequence similarity with human MDC/ADAM11 protein [Katagiri, Harada, Emi and Nakamura (1995) Cytogenet. Cell Genet. 68, 39–44]. A zinc-binding motif, which is critical for proteinase activity, is disrupted in the metalloproteinase-like domain of MDC2 and MDC3, as well as MDC/ADAM11. In the disintegrin-like domain of snake venom short disintegrins, the RDG-containing loops are critical for integrin binding. These three MDCs do not contain the RDG sequences, but the corresponding loops in these proteins are similar to each other. Northern blot analysis revealed that the mRNAs of MDC2, MDC3 and MDC/ADAM11 are highly expressed in the brain. These findings suggest that these proteins may function as integrin ligands in the brain.

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Baculovirus expression of the 11 mycoreovirus-1 genome segments and identification of the guanylyltransferase-encoding segment

Journal of General Virology ◽

10.1099/vir.0.82318-0 ◽

2007 ◽

Vol 88 (1) ◽

pp. 342-350 ◽

Cited By ~ 37

Author(s):

S. Supyani ◽

Bradley I. Hillman ◽

Nobuhiro Suzuki

Keyword(s):

Active Site ◽

Sequence Similarity ◽

Expression System ◽

Amino Acid Sequences ◽

Site Directed Mutagenesis ◽

Sequence Alignments ◽

High Sequence Similarity ◽

Chestnut Blight Fungus ◽

A Genome ◽

Hypovirulent Strain

The type member Mycoreovirus 1 (MyRV-1) of a newly described genus, Mycoreovirus, isolated from a hypovirulent strain 9B21 of the chestnut blight fungus, has a genome composed of 11 dsRNA segments (S1–S11). All of the segments have single ORFs on their capped, positive-sense strands. Infection of insect cells by baculovirus recombinants carrying full-length cDNAs of S1–S11 resulted in overexpression of protein products of the expected sizes, based on their deduced amino acid sequences. This expression system was utilized to identify the S3-encoded protein (VP3) as a guanylyltransferase by an autoguanylylation assay, in which only VP3 was radiolabelled with [α-32P]GTP. A series of progressive N-terminal and C-terminal deletion mutants was made to localize the autoguanylylation-active site of VP3 to aa residues 133–667. Within this region, a sequence stretch (aa 170–250) with relatively high sequence similarity to homologues of two other mycoreoviruses and two coltiviruses was identified. Site-directed mutagenesis of conserved aa residues revealed that H233, H242, Y243, F244 and F246, but not K172 or K202, play critical roles in guanylyltransferase activities. Together with broader sequence alignments of ‘turreted’ reoviruses, these results supported the a/vxxHx8Hyf/lvf motif, originally noted for orthoreovirus and aquareoviruses, as an active site for guanylyltransferases of viruses within the Orthoreovirus, Aquareovirus, Cypovirus, Oryzavirus, Fijivirus, Coltivirus and Mycoreovirus genera, as well as for the proposed Dinovernavirus genus.

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Bovine cytosolic IMP/GMP-specific 5′-nucleotidase: cloning and expression of active enzyme in Escherichia coli

Biochemical Journal ◽

10.1042/bj3280483 ◽

1997 ◽

Vol 328 (2) ◽

pp. 483-487 ◽

Cited By ~ 33

Author(s):

Simone ALLEGRINI ◽

Rossana PESI ◽

Maria Grazia TOZZI ◽

J. Carol FIOL ◽

B. Robert JOHNSON ◽

...

Keyword(s):

Cdna Sequence ◽

Amino Acid Sequences ◽

Cloning And Expression ◽

Kinetic Properties ◽

Coding Region ◽

Northern Blots ◽

Sds Page ◽

Calf Thymus ◽

Rapid Amplification ◽

First Time

A cDNA coding for bovine cytosolic IMP/GMP-specific 5ʹ-nucleotidase endowed with phosphotransferase activity was cloned from calf thymus RNA, by 5ʹ and 3ʹ rapid amplification of cDNA ends protocols (5ʹ and 3ʹ RACE). Two products were isolated: a 5ʹ RACE 1.6 kb fragment and a 3ʹ RACE 2.0 kb fragment, with an overlapping region of 505 bp, leading to a total length of approx. 2951 bp. The similarity in the coding region to that of the human 5ʹ-nucleotidase cDNA sequence [Oka, Matsumoto, Hosokawa and Inoue (1994) Biochem. Biophys. Res. Commun. 205, 917-922], indirectly identified as a 5ʹ-nucleotidase, was 94% and the deduced amino acid sequences were 99.5% identical. The bovine cDNA sequence included the sequences codifying for six peptides obtained from 5ʹ-nucleotidase/phosphotransferase purified from calf thymus. Northern blots of human mRNA species from different tissues showed a 3.6 kb mRNA expressed at equal levels in most tissues. The cDNA was cloned into a pET-28c expression vector and the protein obtained after induction had a molecular mass of 61 kDa under SDS/PAGE. It exhibited both 5ʹ-nucleotidase and phosphotransferase activity, as well as immunological and kinetic properties similar to those of the enzyme purified from calf thymus. This is the first time that a fully active recombinant 5ʹnucleotidase has been described.

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