scholarly journals Cloning and sequencing of rat liver carboxylesterase ES-4 (microsomal palmitoyl-CoA hydrolase)

1996 ◽  
Vol 313 (3) ◽  
pp. 821-826 ◽  
Author(s):  
Mariette ROBBI ◽  
Emile VAN SCHAFTINGEN ◽  
Henri BEAUFAY
Keyword(s):  
Amino Acid ◽  
Rat Liver ◽  
Signal Peptide ◽  
Catalytic Properties ◽  
Amino Acid Residues ◽  
Cloning And Sequencing ◽  
Cos Cells ◽  
Coa Hydrolase ◽  
Naphthyl Acetate ◽  
Retention Signal

A cDNA which encodes a carboxylesterase of 561 amino acid residues including a cleavable signal peptide is described. The enzyme expressed in COS cells migrates during PAGE (SDS-, and non-denaturing) as a single prominent band in the region of liver ES-4. It ends in the C-terminal cell-retention signal -HNEL, which, in COS cells overexpressing the enzyme, appears to be slightly less efficient than the signals -HTEL and -HVEL of ES-3 and ES-10 respectively. Glycosylation is not essential for intracellular retention, but leads to a higher activity. As do many carboxylesterases, the enzyme expressed in COS cells hydrolyses o-nitrophenyl acetate and α-naphthyl acetate. It also hydrolyses acetanilide, although less efficiently than ES-3, and, distinctively, palmitoyl-CoA. In addition to the four canonical Cys residues of the carboxylesterases, it contains a fifth, unpaired Cys336, which apparently is not essential for the catalytic properties. Indeed, treatment with iodoacetamide or substitution of Cys336 by Phe does not markedly alter the activity of the enzyme on the various substrates. The predicted structure of ES-4 is highly homologous to that of two other recently cloned esterases which also end in -HNEL [Yan, Yang, Brady and Parkinson (1994) J. Biol. Chem. 269, 29688–29696; Yan, Yang and Parkinson (1995) Arch. Biochem. Biophys. 317, 222–234]. Together, these isoenzymes probably account for the closely spaced bands observed in the region of ES-4 in non-denaturing PAGE.

scholarly journals Rat liver β-glucuronidase. cDNA cloning, sequence comparisons and expression of a chimeric protein in COS cells

1988 ◽  
Vol 250 (2) ◽  
pp. 547-555 ◽  
Author(s):  
P P Powell ◽  
J W Kyle ◽  
R D Miller ◽  
J Pantano ◽  
J H Grubb ◽  
...  
Keyword(s):  
Amino Acid ◽  
Liver Enzyme ◽  
Rat Liver ◽  
Signal Sequence ◽  
Proteolytic Processing ◽  
Amino Acid Difference ◽  
Amino Acid Residues ◽  
Sequence Comparisons ◽  
Chimeric Clone ◽  
Cos Cells

A cDNA for rat liver beta-glucuronidase was isolated, its sequence determined and its expression after transfection into COS cells studied. The deduced amino acid sequence of the rat liver clone showed 77% homology with that from the cDNA for human placental beta-glucuronidase and 47% homology with that deduced from the cDNA for Escherichia coli beta-glucuronidase. Several differences were found between the cDNA from rat liver and that previously reported from rat preputial gland. Only one change leads to an amino acid difference in the mature enzyme. A chimeric clone was constructed by using a fragment encoding the first 18 amino acid residues of the signal sequence from the human placental cDNA clone and a fragment from the rat clone encoding four amino acid residues of the signal sequence, all 626 amino acid residues of the mature rat enzyme, and all of the 3′ untranslated region. After transfection into COS cells the chimeric clone expressed beta-glucuronidase activity that was specifically immunoprecipitated by antibody to rat beta-glucuronidase. The Mr value of 76,000 of the expressed gene product was characteristic of the glycosylated rat enzyme. It was proteolytically processed in COS cells to Mr 75,000 6 h after metabolic labelling. At least 50% of the expressed enzyme was secreted at 60 h post-transfection, but the secreted enzyme did not undergo proteolytic processing. These results provide evidence that the partial cDNA isolated from a rat liver library contains the complete coding sequence for the mature rat liver enzyme and that the chimeric signal sequence allows normal biosynthesis and processing of the transfected rat liver enzyme in COS cells.

scholarly journals Isolation and sequence analysis of a cDNA encoding rat liver α-l-fucosidase

1989 ◽  
Vol 264 (3) ◽  
pp. 695-701 ◽  
Author(s):  
K J Fisher ◽  
N N Aronson
Keyword(s):  
Amino Acid ◽  
Rat Liver ◽  
Signal Peptide ◽  
Amino Acid Sequences ◽  
Cdna Clones ◽  
Amino Acid Residues ◽  
Expression Library ◽  
Cdna Insert ◽  
Untranslated Sequence ◽  
N Terminus

cDNA clones for alpha-L-fucosidase were isolated from a rat liver lambda gt11 expression library by using both monospecific polyclonal antibodies against the affinity-purified enzyme and biotinylated rat liver fucosidase cDNA sequences as probes. The largest clone, lambda FC9, contained a 1522 bp full-length cDNA insert (FC9) that encoded the 434-amino acid-residue subunit (Mr 50439) of rat liver alpha-L-fucosidase. A putative signal peptide 28 amino acid residues in length preceded the sequence for the mature protein. In addition, FC9 specified for 11 nucleotide residues of 5′ untranslated sequence, 78 nucleotide residues of 3′ untranslated sequence and a poly(A) tail. The deduced amino acid sequence from FC9 in conjunction with the experimentally determined N-terminus of the mature enzyme suggested that rat liver fucosidase did not contain a pro-segment. However, there was the possibility of limited N-terminal processing (one to five amino acid residues) having occurred after removal of the predicted signal peptide. Amino acid sequences deduced from FC9 were co-linear with amino acid sequences measured at the N-terminus of purified fucosidase and on two of its CNBr-cleavage peptides. An unusual aspect of rat liver alpha-L-fucosidase protein structure obtained from the FC9 data was its high content of tryptophan (6%). The coding sequence from FC9 showed 82% sequence identity with that from a previously reported incomplete human fucosidase sequence [O'Brien, Willems, Fukushima, de Wet, Darby, DiCioccio, Fowler & Shows, (1987) Enzyme 38, 45-53].

Cloning, Expression, and Characterization of Mouse Tissue Factor Pathway Inhibitor (TFPI)

1998 ◽  
Vol 79 (02) ◽  
pp. 306-309 ◽  
Author(s):  
Dougald Monroe ◽  
Julie Oliver ◽  
Darla Liles ◽  
Harold Roberts ◽  
Jen-Yea Chang
Keyword(s):  
Amino Acid ◽  
Tissue Factor ◽  
Signal Peptide ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Xa ◽  
Protein Sequences ◽  
Cloning And Expression ◽  
Mouse Tissue ◽  
Amino Acid Residues ◽  
Tissue Factor Pathway

SummaryTissue factor pathway inhibitor (TFPI) acts to regulate the initiation of coagulation by first inhibiting factor Xa. The complex of factor Xa/ TFPI then inhibits the factor VIIa/tissue factor complex. The cDNA sequences of TFPI from several different species have been previously reported. A high level of similarity is present among TFPIs at the molecular level (DNA and protein sequences) as well as in biochemical function (inhibition of factor Xa, VIIa/tissue factor). In this report, we used a PCR-based screening method to clone cDNA for full length TFPI from a mouse macrophage cDNA library. Both cDNA and predicted protein sequences show significant homology to the other reported TFPI sequences, especially to that of rat. Mouse TFPI has a signal peptide of 28 amino acid residues followed by the mature protein (in which the signal peptide is removed) which has 278 amino acid residues. Mouse TFPI, like that of other species, consists of three tandem Kunitz type domains. Recombinant mouse TFPI was expressed in the human kidney cell line 293 and purified for functional assays. When using human clotting factors to investigate the inhibition spectrum of mouse TFPI, it was shown that, in addition to human factor Xa, mouse TFPI inhibits human factors VIIa, IXa, as well as factor XIa. Cloning and expression of the mouse TFPI gene will offer useful information and material for coagulation studies performed in a mouse model system.

Peroxisome targeting signal of rat liver acyl-coenzyme A oxidase resides at the carboxy terminus

1989 ◽  
Vol 9 (1) ◽  
pp. 83-91
Author(s):  
S Miyazawa ◽  
T Osumi ◽  
T Hashimoto ◽  
K Ohno ◽  
S Miura ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rat Liver ◽  
Coenzyme A ◽  
Terminal Region ◽  
Proteinase K ◽  
Amino Acid Residues ◽  
C Terminus ◽  
Targeting Signal ◽  
Acyl Coenzyme A

To identify the topogenic signal of peroxisomal acyl-coenzyme A oxidase (AOX) of rat liver, we carried out in vitro import experiments with mutant polypeptides of the enzyme. Full-length AOX and polypeptides that were truncated at the N-terminal region were efficiently imported into peroxisomes, as determined by resistance to externally added proteinase K. Polypeptides carrying internal deletions in the C-terminal region exhibited much lower import activities. Polypeptides that were truncated or mutated at the extreme C terminus were totally import negative. When the five amino acid residues at the extreme C terminus were attached to some of the import-negative polypeptides, the import activities were rescued. Moreover, the C-terminal 199 and 70 amino acid residues of AOX directed fusion proteins with two bacterial enzymes to peroxisomes. These results are interpreted to mean that the peroxisome targeting signal of AOX residues at the C terminus and the five or fewer residues at the extreme terminus have an obligatory function in targeting. The C-terminal internal region also has an important role for efficient import, possibly through a conformational effect.

scholarly journals Septal Pore Cap Protein SPC18, Isolated from the Basidiomycetous Fungus Rhizoctonia solani, Also Resides in Pore Plugs

2008 ◽  
Vol 7 (10) ◽  
pp. 1865-1873 ◽  
Author(s):  
Kenneth G. A. van Driel ◽  
Arend F. van Peer ◽  
Jan Grijpstra ◽  
Han A. B. Wösten ◽  
Arie J. Verkleij ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Amino Acid ◽  
Rhizoctonia Solani ◽  
Filamentous Fungi ◽  
Signal Peptide ◽  
Biochemical Composition ◽  
Amino Acid Residues ◽  
Terminal Sequence ◽  
Basidiomycetous Fungus ◽  
Central Pore

ABSTRACT The hyphae of filamentous fungi are compartmentalized by septa that have a central pore. The fungal septa and septum-associated structures play an important role in maintaining cellular and intrahyphal homeostasis. The dolipore septa in the higher Basidiomycota (i.e., Agaricomycotina) are associated with septal pore caps. Although the ultrastructure of the septal pore caps has been studied extensively, neither the biochemical composition nor the function of these organelles is known. Here, we report the identification of the glycoprotein SPC18 that was found in the septal pore cap-enriched fraction of the basidiomycetous fungus Rhizoctonia solani. Based on its N-terminal sequence, the SPC18 gene was isolated. SPC18 encodes a protein of 158 amino acid residues, which contains a hydrophobic signal peptide for targeting to the endoplasmic reticulum and has an N-glycosylation motif. Immunolocalization showed that SPC18 is present in the septal pore caps. Surprisingly, we also observed SPC18 being localized in some plugs. The data reported here strongly support the hypothesis that septal pore caps are derived from endoplasmic reticulum and are involved in dolipore plugging and, thus, contribute to hyphal homeostasis in basidiomycetous fungi.

scholarly journals Cloning and Sequence Analysis of a New Cellulase Gene Encoding CelK, a Major Cellulosome Component of Clostridium thermocellum: Evidence for Gene Duplication and Recombination

1999 ◽  
Vol 181 (17) ◽  
pp. 5288-5295 ◽  
Author(s):  
Irina Kataeva ◽  
Xin-Liang Li ◽  
Huizhong Chen ◽  
Sang-Ki Choi ◽  
Lars G. Ljungdahl
Keyword(s):  
Amino Acid ◽  
Gene Duplication ◽  
Signal Peptide ◽  
Clostridium Thermocellum ◽  
Catalytic Domain ◽  
Glycosyl Hydrolase ◽  
Size Difference ◽  
Amino Acid Residues ◽  
Cellulase Gene ◽  
Original Activity

ABSTRACT The cellulolytic and hemicellulolytic complex of Clostridium thermocellum, termed cellulosome, consists of up to 26 polypeptides, of which at least 17 have been sequenced. They include 12 cellulases, 3 xylanases, 1 lichenase, and CipA, a scaffolding polypeptide. We report here a new cellulase gene, celK, coding for CelK, a 98-kDa major component of the cellulosome. The gene has an open reading frame (ORF) of 2,685 nucleotides coding for a polypeptide of 895 amino acid residues with a calculated mass of 100,552 Da. A signal peptide of 27 amino acid residues is cut off during secretion, resulting in a mature enzyme of 97,572 Da. The nucleotide sequence is highly similar to that of cbhA(V. V. Zverlov et al., J. Bacteriol. 180:3091–3099, 1998), having an ORF of 3,690 bp coding for the 1,230-amino-acid-residue CbhA of the same bacterium. Homologous regions of the two genes are 86.5 and 84.3% identical without deletion or insertion on the nucleotide and amino acid levels, respectively. Both have domain structures consisting of a signal peptide, a family IV cellulose binding domain (CBD), a family 9 glycosyl hydrolase domain, and a dockerin domain. A striking distinction between the two polypeptides is that there is a 330-amino-acid insertion in CbhA between the catalytic domain and the dockerin domain containing a fibronectin type 3-like domain and family III CBD. This insertion, missing in CelK, is responsible for the size difference between CelK and CbhA. Upstream and downstream flanking sequences of the two genes show no homology. The data indicate thatcelK and cbhA in the genome of C. thermocellum have evolved through gene duplication and recombination of domain coding sequences. celK without a dockerin domain was expressed in Escherichia coli and purified. The enzyme had pH and temperature optima at 6.0 and 65°C, respectively. It hydrolyzedp-nitrophenyl-β-d-cellobioside with aKm and a V max of 1.67 μM and 15.1 U/mg, respectively. Cellobiose was a strong inhibitor of CelK activity, with a Ki of 0.29 mM. The enzyme was thermostable, after 200 h of incubation at 60°C, 97% of the original activity remained. Properties of the enzyme indicated that it is a cellobiohydrolase.

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