scholarly journals Molecular cloning, expression and characterization of a novel class glutathione S-transferase from the fungus Cunninghamella elegans

2002 ◽  
Vol 368 (2) ◽  
pp. 589-595 ◽  
Author(s):  
Chang-Jun CHA ◽  
Seong-Jae KIM ◽  
Yong-Hak KIM ◽  
Robin STINGLEY ◽  
Carl E. CERNIGLIA
Keyword(s):  
Amino Acid ◽  
Cdna Library ◽  
Fasciola Hepatica ◽  
Mrna Level ◽  
Amino Acid Level ◽  
Gene Probe ◽  
Cunninghamella Elegans ◽  
Glutathione S Transferase ◽  
Sequence Identity ◽  
Degenerate Oligonucleotide

The structural gene for glutathione S-transferase (CeGST1-1) in the fungus Cunninghamella elegans was cloned by screening a cDNA library using a degenerate oligonucleotide probe based on the N-terminal sequence of the purified protein. Open reading frame analysis indicated that the cegst1 gene encodes a protein of 210 amino acid residues. The deduced amino acid sequence showed 25% sequence identity with the sequence of the Pi-class GST from Danio rerio (zebrafish). Similarity was also shown with the Alpha-class GST from Fasciola hepatica (liver fluke; 23% identity), the Mu class from Mus musculus (22%) and the Sigma class from Ommastrephes sloani (squid; 21%). Further screening of a cDNA library with the cegst1 gene probe revealed the presence of another GST isoenzyme (CeGST2-2) in this fungus, which shows 84% sequence identity with CeGST1-1 at the amino acid level. Reverse transcription PCR revealed that cegst2 was also expressed at the mRNA level in the fungus C. elegans. Both cegst genes were overexpressed in Escherichia coli using the expression vector pQE51, displaying specific activities with 1-chloro-2,4-dinitrobenzene of 2.04 and 0.75μmol/min permg of protein respectively. Both enzymes exhibited a similar substrate specificity and inhibition profile, indicating that CeGST1-1 and CeGST2-2 belong to the same GST class. Mutagenesis analysis revealed that Tyr10 in the N-terminal region is essential for catalysis of CeGST1-1. We propose from these results that the CeGSTs are novel Gamma-class GSTs and designated as GSTG1-1 and GSTG2-2 respectively.

Salamander rods and cones contain distinct transducin alpha subunits

2000 ◽  
Vol 17 (6) ◽  
pp. 847-854 ◽  
Author(s):  
JAMES C. RYAN ◽  
SERGEY ZNOIKO ◽  
LIN XU ◽  
ROSALIE K. CROUCH ◽  
JIAN-XING MA
Keyword(s):  
Amino Acid ◽  
Blot Analysis ◽  
Cellular Localization ◽  
Amino Acid Level ◽  
Single Copy ◽  
Lower Vertebrate ◽  
Rods And Cones ◽  
Sequence Identity ◽  
Outer Segments ◽  
Cone Pigments

The mammalian retina is known to contain two distinct transducins that interact with their respective rod and cone pigments. However, there are no reports of a nonmammalian species having two distinct transducins. In the present study, we report the cloning and cellular localization of two transducin α subunits (Gαt) from the tiger salamander. Through degenerate polymerase chain reaction (PCR) and subsequent screening of a salamander retina cDNA library, we have identified two forms of Gαt. When compared to existing sequences in GenBank, the cloned subunits showed high similarity to rod and cone transducins. The salamander Gαt-1 has 91.2–93.7% amino acid sequence identity to mammalian rod Gαt subunits and 79.7–80.9% to mammalian cone Gαts. The salamander Gαt-2 has 86.2–87.9% sequence identity to mammalian cone Gαts and 78.9–80.9% to mammalian rod Gαts at the amino acid level. The Gαt-1 cDNA encodes 350 amino acids while the Gαt-2 cDNA encodes 354 residues, which is typical for rod and cone Gαts, respectively, and we thus identified the Gαt-1 as rod and Gαt-2 as cone Gαt. Sequences identified as effector binding sites and GTPase activity regions are highly conserved between the two subunits. Genomic Southern blot analysis showed that rod and cone Gαt subunits are both encoded by single-copy genes. Northern blot analysis identified retina-specific transcripts of 3.0 kb for rod Gαt and 2.6 kb for cone Gαt. Immunohistochemistry in the flat-mounted salamander retina demonstrated that rod Gαt is localized to rods, predominantly in the outer segments; similarly, cone Gαt is localized to cone outer segments. The results confirm that the two sequences encode rod and cone transducins and demonstrate that this lower vertebrate contains two distinct transducins that are localized specifically to rod and cone photoreceptors.

scholarly journals Molecular Cloning and Expression of Cu/Zn-Containing Superoxide Dismutase from Fasciola hepatica

2000 ◽  
Vol 68 (7) ◽  
pp. 3941-3948 ◽  
Author(s):  
Tong-Soo Kim ◽  
Younghun Jung ◽  
Byoung-Kuk Na ◽  
Ki-Sun Kim ◽  
Pyung-Rim Chung
Keyword(s):  
Superoxide Dismutase ◽  
Amino Acid ◽  
Fasciola Hepatica ◽  
Human Subjects ◽  
Amino Acid Sequences ◽  
Cloning And Expression ◽  
Gene Fragment ◽  
Coding Region ◽  
Sod Activity ◽  
Degenerate Oligonucleotide

ABSTRACT The cytosolic superoxide dismutase (SOD) of Fasciola hepatica, a causative agent of fascioliasis, was purified and characterized. The enzyme consists of two identical subunits, each with an apparent molecular mass of 17.5 kDa. An analysis of the enzyme's primary structure and inhibition studies revealed that the enzyme is a copper/zinc-containing SOD (Cu/Zn-SOD). The enzyme activity was relatively stable in a broad pH range, from pH 7.0 to 10.0, and the enzyme showed maximum activity at pH 7.5. This enzyme also displayed strong antigenicity against sera of bovine and human subjects with fascioliasis. The SOD gene fragment was amplified by PCR with degenerate oligonucleotide primers derived from amino acid sequences conserved in the Cu/Zn-SODs of other organisms. An F. hepatica cDNA library was screened with the SOD gene fragment as a probe. As a result, a complete gene encoding the Cu/Zn-SOD was identified, and its nucleotide sequence was determined. The gene had an open reading frame of 438 bp and 146 deduced amino acids. Comparison of the deduced amino acid sequence of the enzyme with previously reported Cu/Zn-SOD amino acid sequences revealed considerably high homologies. The coding region of the F. hepatica Cu/Zn-SOD was cloned and expressed in Escherichia coli. Staining of native polyacrylamide gel for SOD activity of the expressed protein revealed SOD activity that was inactivated by potassium cyanide and hydrogen peroxide but not by sodium azide. This means that the presence of the recombinant fusion protein is indicative of Cu/Zn-SOD. The expressed protein also reacted with sera of bovine and human subjects with fascioliasis, but it did not react with sera of uninfected bovine and human subjects.

scholarly journals Molecular cloning, sequencing and expression of the cDNA of the mitochondrial form of phosphoenolpyruvate carboxykinase from human liver

1996 ◽  
Vol 315 (3) ◽  
pp. 807-814 ◽  
Author(s):  
Said MODARESSI ◽  
Bruno CHRIST ◽  
Jutta BRATKE ◽  
Stefan ZAHN ◽  
Tilman HEISE ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Human Liver ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Amino Acid Level ◽  
Cdna Probe ◽  
Rat Hepatocytes ◽  
Eukaryotic Expression ◽  
Amino Acid Sequences ◽  
Sequence Identity

In human liver, phosphoenolpyruvate carboxykinase (PCK; EC 4.1.1.32) is about equally distributed between cytosol and mitochondria in contrast with rat liver in which it is essentially a cytosolic enzyme. Recently, the isolation of the gene and cDNA of the human cytosolic enzyme has been reported [Ting, Burgess, Chamberlain, Keith, Falls and Meisler (1993) Genomics 16, 698–706; Stoffel, Xiang, Espinosa, Cox, Le Beau and Bell (1993) Hum. Mol. Genet. 2, 1–4]. It was the goal of this investigation to isolate the cDNA of the human mitochondrial form of hepatic PCK. A human liver cDNA library was screened with a rat cytosolic PCK cDNA probe comprising sequences from exons 2 to 9. A cDNA clone was isolated which had overall a 68% DNA sequence and a 70% deduced amino acid sequence identity with the human cytosolic PCK cDNA. Without the flanking 270 bases (=90 amino acids) each at the 5´ and 3´ end, the sequence identity was 73% on the DNA and 78% on the amino acid level. The isolated cDNA had an open reading frame of 1920 bp; it was 54 bp (equivalent to 18 amino acids) longer than that of human or rat cytosolic PCK cDNA. The isolated cDNA was cloned into the eukaryotic expression vector pcDNAI and transfected into human embryonal kidney cells HEK293; PCK activity was increased by 3-fold in the mitochondria, which normally contain 70% of total PCK activity, but not in the cytosol. The isolated cDNA was also transfected into cultured rat hepatocytes; again, PCK activity was enhanced by about 40-fold in the mitochondria, which normally possess only 10% of total PCK activity, but not in the cytosol. In the rat hepatocytes only the endogenous cytosolic PCK and not the transfected mitochondrial PCK was induced 3-fold with glucagon. Comparison of the amino acid sequences deduced from the isolated cDNA with human and rat cytosolic PCK showed that the additional 18 amino acids were located at the N-terminus of the protein and probably constitute a mitochondrial targeting signal. Northern-blot analyses revealed the human mitochondrial PCK mRNA to be 2.25 kb long, about 0.6 kb shorter than the mRNA of the cytosolic PCK. Primer extension experiments showed that the 5´-untranslated region of mitochondrial PCK mRNA was 134 nucleotides in length.

scholarly journals Cloning and expression of a new mammalian chaperonin gene from a multipotent hematopoietic progenitor clone

Blood ◽  
1994 ◽  
Vol 84 (7) ◽  
pp. 2171-2174
Author(s):  
X Xie ◽  
R Palacios
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cdna Library ◽  
Adenosine Triphosphate ◽  
Molecular Chaperones ◽  
Amino Acid Sequence Identity ◽  
Cloning And Expression ◽  
Hematopoietic Progenitor ◽  
Sequence Identity ◽  
T Complex

Molecular chaperones assist in the folding and assembly of proteins in cells. Although chaperonins have been shown in prokaryotes, mitochondria, and chloroplasts long-ago, a cytoplasmic heteromeric chaperonin complex was isolated only recently and found to contain at least five to six polypeptides, one of which was identified as the product of the T complex polypeptide-1 (TCP-1) gene. We have isolated and cloned a novel gene called A45 from a cDNA library constructed from poly (A)+ RNA of a multipotent hematopoietic progenitor clone. The A45 cDNA encodes a predicted polypeptide of M(r) 58,118 that exhibits 32% overall amino acid sequence identity to TCP-1 and contains the putative adenosine triphosphate-binding domain and two characteristic consensus regions that are conserved in all chaperonins. The A45 gene is expressed in hematopoietic precursors cells at a much higher level than in nonhematopoietic cells and tissues. We conclude that A45 represents a new member of the mammalian chaperonins that is involved in the folding and assembly of polypeptides.

scholarly journals Cloning and expression of a new mammalian chaperonin gene from a multipotent hematopoietic progenitor clone

Blood ◽  
1994 ◽  
Vol 84 (7) ◽  
pp. 2171-2174 ◽  
Author(s):  
X Xie ◽  
R Palacios
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cdna Library ◽  
Adenosine Triphosphate ◽  
Molecular Chaperones ◽  
Amino Acid Sequence Identity ◽  
Cloning And Expression ◽  
Hematopoietic Progenitor ◽  
Sequence Identity ◽  
T Complex

Abstract Molecular chaperones assist in the folding and assembly of proteins in cells. Although chaperonins have been shown in prokaryotes, mitochondria, and chloroplasts long-ago, a cytoplasmic heteromeric chaperonin complex was isolated only recently and found to contain at least five to six polypeptides, one of which was identified as the product of the T complex polypeptide-1 (TCP-1) gene. We have isolated and cloned a novel gene called A45 from a cDNA library constructed from poly (A)+ RNA of a multipotent hematopoietic progenitor clone. The A45 cDNA encodes a predicted polypeptide of M(r) 58,118 that exhibits 32% overall amino acid sequence identity to TCP-1 and contains the putative adenosine triphosphate-binding domain and two characteristic consensus regions that are conserved in all chaperonins. The A45 gene is expressed in hematopoietic precursors cells at a much higher level than in nonhematopoietic cells and tissues. We conclude that A45 represents a new member of the mammalian chaperonins that is involved in the folding and assembly of polypeptides.

scholarly journals Global versus Local Regulatory Roles for Lrp-Related Proteins: Haemophilus influenzae as a Case Study

2001 ◽  
Vol 183 (13) ◽  
pp. 4004-4011 ◽  
Author(s):  
Devorah Friedberg ◽  
Michael Midkiff ◽  
Joseph M. Calvo
Keyword(s):  
Amino Acid ◽  
Haemophilus Influenzae ◽  
Regulatory Protein ◽  
Enteric Bacteria ◽  
Regulatory Role ◽  
Regulatory Function ◽  
Ecological Niches ◽  
E Coli ◽  
Sequence Identity ◽  
Related Proteins

ABSTRACT Lrp (leucine-responsive regulatory protein) plays a global regulatory role in Escherichia coli, affecting expression of dozens of operons. Numerous lrp-related genes have been identified in different bacteria and archaea, includingasnC, an E. coli gene that was the first reported member of this family. Pairwise comparisons of amino acid sequences of the corresponding proteins shows an average sequence identity of only 29% for the vast majority of comparisons. By contrast, Lrp-related proteins from enteric bacteria show more than 97% amino acid identity. Is the global regulatory role associated withE. coli Lrp limited to enteric bacteria? To probe this question we investigated LrfB, an Lrp-related protein fromHaemophilus influenzae that shares 75% sequence identity with E. coli Lrp (highest sequence identity among 42 sequences compared). A strain of H. influenzae having anlrfB null allele grew at the wild-type growth rate but with a filamentous morphology. A comparison of two-dimensional (2D) electrophoretic patterns of proteins from parent and mutant strains showed only two differences (comparable studies withlrp + and lrp E. coli strains by others showed 20 differences). The abundance of LrfB in H. influenzae, estimated by Western blotting experiments, was about 130 dimers per cell (compared to 3,000 dimers per E. colicell). LrfB expressed in E. coli replaced Lrp as a repressor of the lrp gene but acted only to a limited extent as an activator of the ilvIH operon. Thus, although LrfB resembles Lrp sufficiently to perform some of its functions, its low abundance is consonant with a more local role in regulating but a few genes, a view consistent with the results of the 2D electrophoretic analysis. We speculate that an Lrp having a global regulatory role evolved to help enteric bacteria adapt to their ecological niches and that it is unlikely that Lrp-related proteins in other organisms have a broad regulatory function.

scholarly journals Recognition Pattern of the Fasciola hepatica Excretome/Secretome during the Course of an Experimental Infection in Sheep by 2D Immunoproteomics

Pathogens ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 725
Author(s):  
David Becerro-Recio ◽  
Javier González-Miguel ◽  
Alberto Ucero ◽  
Javier Sotillo ◽  
Álvaro Martínez-Moreno ◽  
...  
Keyword(s):  
Experimental Infection ◽  
Fasciola Hepatica ◽  
Cathepsin L ◽  
Helminth Parasites ◽  
Glutathione S Transferase ◽  
Serum Samples ◽  
Secretory Products ◽  
Immunogenic Proteins ◽  
Host Parasite ◽  
First Time

Excretory/secretory products released by helminth parasites have been widely studied for their diagnostic utility, immunomodulatory properties, as well as for their use as vaccines. Due to their location at the host/parasite interface, the characterization of parasite secretions is important to unravel the molecular interactions governing the relationships between helminth parasites and their hosts. In this study, the excretory/secretory products from adult worms of the trematode Fasciola hepatica (FhES) were employed in a combination of two-dimensional electrophoresis, immunoblot and mass spectrometry, to analyze the immune response elicited in sheep during the course of an experimental infection. Ten different immunogenic proteins from FhES recognized by serum samples from infected sheep at 4, 8, and/or 12 weeks post-infection were identified. Among these, different isoforms of cathepsin L and B, peroxiredoxin, calmodulin, or glutathione S-transferase were recognized from the beginning to the end of the experimental infection, suggesting their potential role as immunomodulatory antigens. Furthermore, four FhES proteins (C2H2-type domain-containing protein, ferritin, superoxide dismutase, and globin-3) were identified for the first time as non-immunogenic proteins. These results may help to further understand host/parasite relationships in fasciolosis, and to identify potential diagnostic molecules and drug target candidates of F. hepatica.

scholarly journals Streptogramin B biosynthesis in Streptomyces pristinaespiralis and Streptomyces virginiae: molecular characterization of the last structural peptide synthetase gene.

1997 ◽  
Vol 41 (9) ◽  
pp. 1904-1909 ◽  
Author(s):  
V de Crécy-Lagard ◽  
W Saurin ◽  
D Thibaut ◽  
P Gil ◽  
L Naudin ◽  
...  
Keyword(s):  
Amino Acid ◽  
Acid Activation ◽  
Activation Domains ◽  
Amino Acid Activation ◽  
Streptomyces Virginiae ◽  
Streptomyces Pristinaespiralis ◽  
Degenerate Oligonucleotide ◽  
Peptide Synthetase ◽  
Different Origins

Streptomyces pristinaespiralis and S. virginiae both produce closely related hexadepsipeptide antibiotics of the streptogramin B family. Pristinamycins I and virginiamycins S differ only in the fifth incorporated precursor, di(mono)methylated amine and phenylalanine, respectively. By using degenerate oligonucleotide probes derived from internal sequences of the purified S. pristinaespiralis SnbD and SnbE proteins, the genes from two streptogramin B producers, S. pristinaespiralis and S. virginiae, encoding the peptide synthetase involved in the activation and incorporation of the last four precursors (proline, 4-dimethylparaaminophenylalanine [for pristinamycin I(A)] or phenylalanine [for virginiamycin S], pipecolic acid, and phenylglycine) were cloned. Analysis of the sequence revealed that SnbD and SnbE are encoded by a unique snbDE gene. SnbDE (4,849 amino acids [aa]) contains four amino acid activation domains, four condensation domains, an N-methylation domain, and a C-terminal thioesterase domain. Comparison of the sequences of 55 amino acid-activating modules from different origins confirmed that these sequences contain enough information for the performance of legitimate predictions of their substrate specificity. Partial sequencing (1,993 aa) of the SnbDE protein of S. virginiae allowed comparison of the proline and aromatic acid activation domains of the two species and the identification of coupled frameshift mutations.

scholarly journals The transient receptor potential, TRP4, cation channel is a novel member of the family of calmodulin binding proteins

2001 ◽  
Vol 355 (3) ◽  
pp. 663-670 ◽  
Author(s):  
Claudia TROST ◽  
Christiane BERGS ◽  
Nina HIMMERKUS ◽  
Veit FLOCKERZI
Keyword(s):  
Amino Acid ◽  
Ion Channels ◽  
Transient Receptor Potential ◽  
Direct Interaction ◽  
Receptor Potential ◽  
Amino Acid Residues ◽  
Glutathione S Transferase ◽  
Calmodulin Binding ◽  
Transient Receptor

The mammalian gene products, transient receptor potential (trp)1 to trp7, are related to the Drosophila TRP and TRP-like ion channels, and are candidate proteins underlying agonist-activated Ca2+-permeable ion channels. Recently, the TRP4 protein has been shown to be part of native store-operated Ca2+-permeable channels. These channels, most likely, are composed of other proteins in addition to TRP4. In the present paper we report the direct interaction of TRP4 and calmodulin (CaM) by: (1) retention of in vitro translated TRP4 and of TRP4 protein solubilized from bovine adrenal cortex by CaM–Sepharose in the presence of Ca2+, and (2) TRP4–glutathione S-transferase pull-down experiments. Two domains of TRP4, amino acid residues 688–759 and 786–848, were identified as being able to interact with CaM. The binding of CaM to both domains occurred only in the presence of Ca2+ concentrations above 10µM, with half maximal binding occurring at 16.6µM (domain 1) and 27.9µM Ca2+ (domain 2). Synthetic peptides, encompassing the two putative CaM binding sites within these domains and covering amino acid residues 694–728 and 829–853, interacted directly with dansyl–CaM with apparent Kd values of 94–189nM. These results indicate that TRP4/Ca2+-CaM are parts of a signalling complex involved in agonist-induced Ca2+ entry.

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