scholarly journals cDNA cloning and characterization of guinea-pig leukotriene B4 receptor

1999 ◽  
Vol 342 (1) ◽  
pp. 79-85 ◽  
Author(s):  
Kazuyuki MASUDA ◽  
Takehiko YOKOMIZO ◽  
Takashi IZUMI ◽  
Takao SHIMIZU
Keyword(s):  
Amino Acid ◽  
Guinea Pig ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Leukotriene B4 ◽  
Xenopus Laevis Oocytes ◽  
Amino Acid Residues ◽  
Ovary Cells ◽  
Hek 293 ◽  
Leukotriene B4 Receptor

The cDNA for leukotriene B4 (LTB4) receptor (BLT) was cloned from a guinea-pig leucocyte cDNA library. The cloned receptor cDNA encodes 348 amino acid residues and shares 73% identity with the amino acid sequence of human BLT. Northern blot analysis showed the highest expression of the receptor mRNA in leucocytes, followed by lung and spleen. The membrane fractions of HEK-293 and Cos-7 cells transfected with the cDNA showed specific LTB4-binding activities, with Kd values of 0.27 and 0.17 nM respectively. Xenopus laevis oocytes injected with the cRNA of guinea-pig BLT showed LTB4-induced Cl- currents, indicating that the cloned receptor is functional. LTB4 is metabolized to 20-hydroxy-LTB4 and then to 20-carboxy-LTB4, a transformation considered as a major inactivation pathway of the compound. Using the cloned receptor, we analysed the agonistic effects of LTB4 and these two metabolites. 20-Carboxy-LTB4 is a much weaker agonist, with a Kd value higher than that of LTB4 by three orders of magnitude, corresponding to a much weaker chemotactic activity. Although 20-hydroxy-LTB4 is as potent as LTB4 in inhibiting [3H]LTB4 binding and cAMP formation, it is less potent than LTB4 in the mobilization of intracellular Ca2+ and the chemotaxis of Chinese hamster ovary cells expressing the guinea-pig BLT. The present study demonstrated that although LTB4 and 20-hydroxy-LTB4 bind to the receptor with similar affinities, they do differ in activating intracellular signalling.

scholarly journals Functional analysis of Chinese hamster phosphatidylserine synthase 1 through systematic alanine mutagenesis

2004 ◽  
Vol 381 (3) ◽  
pp. 853-859 ◽  
Author(s):  
Tomoko OHSAWA ◽  
Masahiro NISHIJIMA ◽  
Osamu KUGE
Keyword(s):  
Amino Acid ◽  
Chinese Hamster Ovary ◽  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Intact Cells ◽  
Base Exchange ◽  
Ovary Cells

PtdSer (phosphatidylserine) synthesis in mammalian cells occurs through the exchange of L-serine with the base moieties of phosphatidylcholine and phosphatidylethanolamine, which is catalysed by PSS (PtdSer synthase) 1 and 2 respectively. PtdSer synthesis in intact cells and an isolated membrane fraction was inhibited by exogenous PtdSer, indicating that feedback control is involved in the regulation of PtdSer biosynthesis. PSS 1 and 2 are similar in amino acid sequence, with an identity of 32%; however, due to a lack of homology with other known enzymes, their amino acid sequences do not provide information on their catalytic and regulatory mechanisms. In the present study, to identify amino acid residues crucial for the activity and/or regulation of PSS 1, we systematically introduced mutations into a Chinese hamster PSS 1 cDNA clone; namely, each of the 66 polar amino acid residues common to PSS 2 was replaced with an alanine residue. On analysis of Chinese hamster ovary cells transfected with each of the alanine mutant clones, we identified eight amino acid residues (His-172, Glu-197, Glu-200, Asn-209, Glu-212, Asp-216, Asp-221 and Asn-226) as those crucial for the enzyme reaction or the maintenance of the correct structure required for serine base-exchange activity. Among these residues, Asn-209 was suggested to be involved in the recognition and/or binding of free L-serine. We also identified six amino acid residues (Arg-95, His-97, Cys-189, Arg-262, Gln-266 and Arg-336) as those important for regulation of PSS 1. In addition, we found that the alanine mutations at Tyr-111, Asp-166, Arg-184, Arg-323, and Glu-364 affected the production and/or stability of PSS 1 in Chinese hamster ovary cells.

scholarly journals Catalytic residues and a predicted structure of tetrahydrobiopterin-dependent alkylglycerol mono-oxygenase

2012 ◽  
Vol 443 (1) ◽  
pp. 279-286 ◽  
Author(s):  
Katrin Watschinger ◽  
Julian E. Fuchs ◽  
Vladimir Yarov-Yarovoy ◽  
Markus A. Keller ◽  
Georg Golderer ◽  
...  
Keyword(s):  
Amino Acid ◽  
Chinese Hamster Ovary Cells ◽  
Active Form ◽  
Chinese Hamster ◽  
Fold Increase ◽  
Site Directed Mutagenesis ◽  
Fatty Aldehyde ◽  
Amino Acid Residues ◽  
Ovary Cells ◽  
Mutant Proteins

Alkylglycerol mono-oxygenase (EC 1.14.16.5) forms a third, distinct, class among tetrahydrobiopterin-dependent enzymes in addition to aromatic amino acid hydroxylases and nitric oxide synthases. Its protein sequence contains the fatty acid hydroxylase motif, a signature indicative of a di-iron centre, which contains eight conserved histidine residues. Membrane enzymes containing this motif, including alkylglycerol mono-oxygenase, are especially labile and so far have not been purified to homogeneity in active form. To obtain a first insight into structure–function relationships of this enzyme, we performed site-directed mutagenesis of 26 selected amino acid residues and expressed wild-type and mutant proteins containing a C-terminal Myc tag together with fatty aldehyde dehydrogenase in Chinese-hamster ovary cells. Among all of the acidic residues within the eight-histidine motif, only mutation of Glu137 to alanine led to an 18-fold increase in the Michaelis–Menten constant for tetrahydrobiopterin, suggesting a role in tetrahydrobiopterin interaction. A ninth additional histidine residue essential for activity was also identified. Nine membrane domains were predicted by four programs: ESKW, TMHMM, MEMSAT and Phobius. Prediction of a part of the structure using the Rosetta membrane ab initio method led to a plausible suggestion for a structure of the catalytic site of alkylglycerol mono-oxygenase.

Spontaneous splicing mutations at the dihydrofolate reductase locus in Chinese hamster ovary cells

1986 ◽  
Vol 6 (6) ◽  
pp. 1926-1935
Author(s):  
P J Mitchell ◽  
G Urlaub ◽  
L Chasin
Keyword(s):  
Amino Acid ◽  
Dihydrofolate Reductase ◽  
Splice Site ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Splice Sites ◽  
Ovary Cells ◽  
Splicing Mutations ◽  
Intron 4

We isolated and characterized three spontaneous mutants of Chinese hamster ovary cells that were deficient in dihydrofolate reductase activity. All three mutants contained no detectable enzyme activity and produced dihydrofolate reductase mRNA species that were shorter than those of the wild type by about 120 bases. Six exons are normally represented in this mRNA; exon 5 was missing in all three mutant mRNAs. Nuclease S1 analysis of the three mutants indicated that during the processing of the mutant RNA, exon 4 was spliced to exon 6. The three mutant genes were cloned, and the regions around exons 4 and 5 were sequenced. In one mutant, the GT dinucleotide at the 5' end of intron 5 had changed to CT. In a second mutant, the first base in exon 5 had changed from G to T. In a revertant of this mutant, this base was further mutated to A, a return to a purine. Approximately 25% of the mRNA molecules in the revertant were spliced correctly to produce an enzyme with one presumed amino acid change. In the third mutant, the AG at the 3' end of intron 4 had changed to AA. A mutation that partially reversed the mutant phenotype had changed the dinucleotide at the 5' end of intron 4 from GT to AT. The splicing pattern in this revertant was consistent with the use of cryptic donor and acceptor splice sites close to the original sites to produce an mRNA with three base changes and a protein with two amino acid changes. These mutations argue against a scanning model for the selection of splice site pairs and suggest that only a single splice site need be inactivated to bring about efficient exon skipping (a regulatory mechanism for some genes). The fact that all three mutants analyzed exhibited exon 5 splicing mutations indicates that these splice sites are hot spots for spontaneous mutation.

scholarly journals Molecular Cloning of a Functional Allatostatin Gut/Brain Receptor and an Allatostatin Preprohormone from the SilkwormBombyx mori

2001 ◽  
Vol 276 (50) ◽  
pp. 47052-47060 ◽  
Author(s):  
Thomas Secher ◽  
Camilla Lenz ◽  
Giuseppe Cazzamali ◽  
Gunnar Sørensen ◽  
Michael Williamson ◽  
...  
Keyword(s):  
Amino Acid ◽  
Molecular Cloning ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Peptide Hormone ◽  
Functional Expression ◽  
Bar Gene ◽  
Ovary Cells ◽  
Intron Phasing ◽  
Insect Gut

The cockroach-type or A-type allatostatins are inhibitory insect neuropeptides with the C-terminal sequence Tyr/Phe-X-Phe-Gly-Leu-NH2. Here, we have cloned an A-type allatostatin receptor from the silkwormBombyx mori(BAR). BAR is 361 amino acid residues long, has seven transmembrane domains, shows 60% amino acid residue identity with the firstDrosophilaallatostatin receptor (DAR-1), and 48% identity with the secondDrosophilaallatostatin receptor (DAR-2). The BAR gene has two introns and three exons. These two introns coincide with and have the same intron phasing as two introns in the DAR-1 and DAR-2 genes, showing that the three receptors are not only structurally but also evolutionarily related. Furthermore, we have cloned aBombyxallatostatin preprohormone that contains eight different A-type allatostatins. Chinese hamster ovary cells permanently transfected with BAR DNA react on the addition of 4 × 10−9mBombyxA-type allatostatins with a second messenger cascade (measured as bioluminescence), showing that BAR is a functional A-type allatostatin receptor. Southern blots suggest thatBombyxhas at least one other BAR-related gene in addition to the BAR gene described in this paper. Northern blots and quantitative reverse transcriptase-polymerase chain reaction of different larval tissues show that BAR mRNA is mainly expressed in the gut and to a much lesser extent in the brain. To our knowledge, this is the first report on the molecular cloning and functional expression of an insect gut/brain peptide hormone receptor.

scholarly journals Human PIG-U and Yeast Cdc91p Are the Fifth Subunit of GPI Transamidase That Attaches GPI-Anchors to Proteins

2003 ◽  
Vol 14 (5) ◽  
pp. 1780-1789 ◽  
Author(s):  
Yeongjin Hong ◽  
Kazuhito Ohishi ◽  
Ji Young Kang ◽  
Satoshi Tanaka ◽  
Norimitsu Inoue ◽  
...  
Keyword(s):  
Amino Acid ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Amino Acid Identity ◽  
Complementation Group ◽  
Long Chain Fatty Acid ◽  
Ovary Cells ◽  
Eukaryotic Proteins ◽  
Gpi Transamidase

Many eukaryotic proteins are anchored to the cell surface via glycosylphosphatidylinositol (GPI), which is posttranslationally attached to the carboxyl-terminus by GPI transamidase. The mammalian GPI transamidase is a complex of at least four subunits, GPI8, GAA1, PIG-S, and PIG-T. Here, we report Chinese hamster ovary cells representing a new complementation group of GPI-anchored protein-deficient mutants, class U. The class U cells accumulated mature and immature GPI and did not have in vitro GPI transamidase activity. We cloned the gene responsible, termed PIG-U, that encoded a 435-amino-acid hydrophobic protein. The GPI transamidase complex affinity-purified from cells expressing epitope-tagged-GPI8 contained PIG-U and four other known components. Cells lacking PIG-U formed complexes of the four other components normally but had no ability to cleave the GPI attachment signal peptide. Saccharomyces cerevisiae Cdc91p, with 28% amino acid identity to PIG-U, partially restored GPI-anchored proteins on the surface of class U cells. PIG-U and Cdc91p have a functionally important short region with similarity to a region conserved in long-chain fatty acid elongases. Taken together, PIG-U and the yeast orthologue Cdc91p are the fifth component of GPI transamidase that may be involved in the recognition of either the GPI attachment signal or the lipid portion of GPI.

scholarly journals Multiple Signal Transduction Pathways through Two Prostaglandin E Receptor EP3 Subtype Isoforms Expressed in Human Uterus

2000 ◽  
Vol 85 (11) ◽  
pp. 4315-4322 ◽  
Author(s):  
Masato Kotani ◽  
Issei Tanaka ◽  
Yoshihiro Ogawa ◽  
Takayoshi Suganami ◽  
Tsunekazu Matsumoto ◽  
...  
Keyword(s):  
Amino Acid ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Mitogen Activated Protein Kinase ◽  
Human Myometrium ◽  
Prostaglandin E ◽  
Messenger Rnas ◽  
Amino Acid Residues ◽  
Human Uterus ◽  
Rt Pcr

PGE2 is known to induce uterine contraction by increasing intracellular Ca2+. In the present study, to investigate other functions of PGE2 in human uterus, two EP3 isoforms were isolated by the RT-PCR method using human uterus polyadenylated ribonucleic acid (RNA). These EP3 isoforms, named EP3-V and EP3-VI, are composed of 402 and 393 amino acid residues, respectively, which are unique compared with EP3 isoforms of other species. Their N-terminal 359 amino acid residues are identical to those of previously reported human EP3 isoforms, whereas the two isoforms contained a novel amino acid sequence in their C-terminal tails. The dissociation constant values of EP3-V and EP3-VI for PGE2 were 3.9 and 1.4 nmol/L, respectively, which were consistent with those of previously reported EP3 isoforms. Signaling experiments revealed that M&B28767, an EP3 agonist, not only inhibited forskolin-induced cAMP concentrations, but also activated mitogen-activated protein kinase in Chinese hamster ovary cells stably expressing EP3-V and EP3-VI. These responses were abolished by treatment with pertussis toxin. In addition, M&B28767 increased cAMP concentrations in EP3-VI-expressing cells, whereas it did not in EP3-V-expressing cells. M&B28767 did not stimulate phosphoinositide turnover in EP3-V- or EP3-VI-expressing cells. EP3-V and EP3-VI messenger RNAs (mRNAs) were detected abundantly in human uterus, whereas weak, but substantial, bands were detected in the lung and kidney in RT-PCR specific for each mRNA. In situ hybridization revealed EP3-V and EP3-VI mRNAs in the human myometrium, but not in the endometrium. The present study suggests that EP3-V and EP3-VI are possibly involved in the proliferation of cells in human myometrium.

α1C (CaV1.2) L-type calcium channel mediates mechanosensitive calcium regulation

2002 ◽  
Vol 283 (3) ◽  
pp. C1001-C1008 ◽  
Author(s):  
Greg L. Lyford ◽  
Peter R. Strege ◽  
Allan Shepard ◽  
Yijun Ou ◽  
Leonid Ermilov ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Calcium Channel ◽  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Calcium Entry ◽  
Intestinal Smooth Muscle ◽  
Ovary Cells ◽  
Hek 293 ◽  
Rich Domain

Smooth muscle exhibits mechanosensitivity independent of neural input, suggesting that mechanosensitive pathways reside within smooth muscle cells. The native L-type calcium current recorded from human intestinal smooth muscle is modulated by stretch. To define mechanosensitive mechanisms involved in the regulation of smooth muscle calcium entry, we cloned the α1C L-type calcium channel subunit (CaV1.2) from human intestinal smooth muscle and expressed the channel in a heterologous system. This channel subunit retained mechanosensitivity when expressed alone or coexpressed with a β2 calcium channel subunit in HEK-293 or Chinese hamster ovary cells. The heterologously expressed human cardiac α1C splice form also demonstrated mechanosensitivity. Inhibition of kinase signaling did not affect mechanosensitivity of the native channel. Truncation of the α1C COOH terminus, which contains an inhibitory domain and a proline-rich domain thought to mediate mechanosensitive signaling from integrins, did not disrupt mechanosensitivity of the expressed channel. These data demonstrate mechanical regulation of calcium entry through molecularly identified L-type calcium channels in mammalian cells and suggest that the mechanosensitivity resides within the pore forming α1C-subunit.

AMINO ACID CHANGES AT LYS-158 THAT ALTER THE SENSITIVITY OF SCUPA TO CLEAVAGE BY PLASMIN

1987 ◽  
Author(s):  
G F VOVIS ◽  
J MAO ◽  
R BROEZE ◽  
D ABERCROMBIE ◽  
P PUMA ◽  
...  
Keyword(s):  
Amino Acid ◽  
Fibrinolytic Activity ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Amino Acid Position ◽  
Peptide Bond ◽  
Site Directed Mutagenesis ◽  
Chromogenic Substrate ◽  
Amidolytic Activity ◽  
Ovary Cells

Plasmin converts Scu-PA to two-chain urokinase by hydrolyzing the lys-158/ile-159 peptide bond. Using site directed mutagenesis, the codon at amino acid position 158 was changed from one that codes for lysine to one that codes for either alanine, glutamic acid, or methionine. These DNA constructions were expressed and amplified in Chinese hamster ovary cells. The resulting protein products were isolated and characterized ip vitro. Under conditions where Scu-PA is completely converted by plasmin to two chain urokinase, none of these derivatives were cleaved by plasmin. However, all of these molecules, including Scu-PA, were cleaved by thrombin. These derivatives exhibited low amidolytic activity as determined by using the chromogenic substrate S2444 and low fibrinolytic activity as determined by using fibrin plates. All three derivatives activated either glu-plasminogen or lys-plasminogen to plasmin ip vitro but at rates significantly slower than that seen with Scu-PA.

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