Molecular Cloning and Expression of Human Prostacyclin Synthase

Miyata, Akira; Hara, Shuntaro; Yokoyama, Chieko; Inoue, Hiroshi; Ullrich, Volker; Tanabe, Takayuki

doi:10.1006/bbrc.1994.1652

Cited by 102 publications

(64 citation statements)

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“…the expressed enzyme was too low to characterize the enzyme [6]. Thus, we first attempted to establish an efficient system for expressing the wild-type human PGI2 synthase with our cloned cDNA [7]. An expression vector (pCMV-7) with an insert of the entire protein-coding region of the cDNA for the enzyme (pCMV/PGISWT) was constructed and transfected into 293 cells.…”

Section: Resultsmentioning

confidence: 99%

“…A 1.6-kb fragment of the full-length human PGI2 synthase cDNA constructed by ligation of pHPGIS36 and pHPGIS135 [7] was inserted into the SalI and BamHI sites of the expression vector pCMV-7 to generate the plasmid pCMV/PGISWT. The mutants were prepared by 'overlap extension' mutagenesis [12] using threestep PCR amplification of the cDNA for human PGI2 synthase.…”

Section: Expression Vector and Mutagenesismentioning

confidence: 99%

“…PGI2 is formed via isomerization of PGH2 by PGI2 synthase (EC 5.3.99.4) in vascular endothelial cells and vascular smooth muscle cells [2]. We have suggested that PGI2 synthase is a hemoprotein of the cytochrome P450 type [3][4][5], and demonstrated by molecular cloning of PGI2 synthase cDNAs from bovine and human vascular endothelial cells that the enzyme belongs to a new family (CYPS) in the P450 superfamily [6][7][8]. As shown in Fig.…”

Section: Introductionmentioning

confidence: 99%

“…Abbreviations: PG, prostaglandin; PGI2, prostacyclin; PCR, polymerase chain reaction; DTT, dithiothreitol; BHT, butylated hydroxytoluene; PVDF, polyvinylidene difluoride; KPB, potassium phosphate buffer deduced primary structure of PGI2 synthase [7,9]. On the other hand, many cytochrome P450s generally catalyze the oxidation of a variety of hydrophobic substrates, but PGI2 synthase catalyzes the isomerization of PGH2.…”

Section: Introductionmentioning

confidence: 99%

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Site‐directed mutagenesis of human prostacyclin synthase: Alteration of Cys⁴⁴¹ of the Cys‐pocket, and Glu³⁴⁷ and Arg³⁵⁰ of the EXXR motif

et al. 1996

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The possible active site Cys 441 in the Cys-pocket and Glu 347 and Arg ~s° of the EXXR motif of the human prostacyclin synthase, which catalyzes the conversion of prostaglandin H2 to prostacyclin, were subjected to site-directed mutagenesis in order to understand the role of these residues in expressing the enzymatic activity. Five expression vectors encoding the mutant enzymes with a single replacement, Cys441Ala, Cys441Ser, Cys441His, Glu347Ala and Arg3S°Ala, as well as the wild-type enzyme were expressed in 293 cells. The microsomal fraction of the cells expressing the wild-type enzyme showed a specific activity of 96 nmol 6-keto-PGF1Jmin per mg protein. All of the mutant enzymes examined showed no detectable enzyme activity, although immunoblot analysis demonstrated that levels of all the expressed mutant enzymes were similar to that of the wild-type enzyme. These results indicated that the Cys 441 in the Cyspocket, and Glu 347 and Arg 35° of the EXXR motif of human prostacyclin synthase are important for expressing the enzymatic activity.

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Section: Resultsmentioning

confidence: 99%

Section: Expression Vector and Mutagenesismentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Site‐directed mutagenesis of human prostacyclin synthase: Alteration of Cys⁴⁴¹ of the Cys‐pocket, and Glu³⁴⁷ and Arg³⁵⁰ of the EXXR motif

et al. 1996

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“…# synthase (PGIS), a cytochrome P450, contains 500 amino acids with a calculated molecular mass of about 57 kDa [1][2][3] and is located in the endoplasmic-reticulum (ER) membrane [4][5][6]. PGIS converts prostaglandin H # (PGH # ) into prostaglandin I # (PGI # ), a potent inhibitor of platelet aggregation, vasoconstriction and leucocyte interactions with endothelium [7,8].…”

Section: Prostaglandin Imentioning

confidence: 99%

Substrate access channel topology in membrane-bound prostacyclin synthase

Deng¹,

Huang²,

So³

et al. 2002

Biochem. J.

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Results from our molecular-modelling and site-directed-mutagenesis studies of prostaglandin I # synthase (PGIS) have suggested that the large PGIS cytoplasmic domain is anchored to the endoplasmic reticulum (ER) membrane by the N-terminal segment in a way that orients the substrate access channel opening to face the membrane. To test this hypothesis we have explored the accessibility of the PGIS substrate channel opening to site-specific antibodies. The working three-dimensional PGIS model constructed by protein homology modelling was used to predict surface portions near the substrate access channel opening. Two peptides corresponding to the surface immediately near the opening [residues 66-75 (P66-75) and 95-116 (P95-116)], and two other peptides corresponding to the surface about 10-20 A H (1 A H l 0.1 nm) away from the opening [residues 366-382 (P366-382) and 472-482 (P472-482)] were used to prepare sitespecific antibodies. All four antipeptide antibodies specifically recognized the synthetic segments of human PGIS and recombinant PGIS, as shown by binding assays and Western-blot analysis. The site-specific antibodies were used to probe the

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Prostanoid Synthases

and¹,

Ullrich²

1999

Prostaglandins, Laukotrienes and Other Eicosanoids

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Molecular Cloning and Expression of Human Prostacyclin Synthase

Cited by 102 publications

References 0 publications

Site‐directed mutagenesis of human prostacyclin synthase: Alteration of Cys⁴⁴¹ of the Cys‐pocket, and Glu³⁴⁷ and Arg³⁵⁰ of the EXXR motif

Site‐directed mutagenesis of human prostacyclin synthase: Alteration of Cys⁴⁴¹ of the Cys‐pocket, and Glu³⁴⁷ and Arg³⁵⁰ of the EXXR motif

Substrate access channel topology in membrane-bound prostacyclin synthase

Prostanoid Synthases

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Molecular Cloning and Expression of Human Prostacyclin Synthase

Cited by 102 publications

References 0 publications

Site‐directed mutagenesis of human prostacyclin synthase: Alteration of Cys441 of the Cys‐pocket, and Glu347 and Arg350 of the EXXR motif

Site‐directed mutagenesis of human prostacyclin synthase: Alteration of Cys441 of the Cys‐pocket, and Glu347 and Arg350 of the EXXR motif

Substrate access channel topology in membrane-bound prostacyclin synthase

Prostanoid Synthases

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Resources

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Site‐directed mutagenesis of human prostacyclin synthase: Alteration of Cys⁴⁴¹ of the Cys‐pocket, and Glu³⁴⁷ and Arg³⁵⁰ of the EXXR motif

Site‐directed mutagenesis of human prostacyclin synthase: Alteration of Cys⁴⁴¹ of the Cys‐pocket, and Glu³⁴⁷ and Arg³⁵⁰ of the EXXR motif