title: An active triple-catalytic hybrid enzyme engineered by linking cyclo-oxygenase isoform-1 to prostacyclin synthase that can constantly biosynthesize prostacyclin, the vascular protector.
abstract: It remains a challenge to achieve the stable and long-term expression (in human cell lines) of a previously engineered hybrid enzyme [triple-catalytic (Trip-cat) enzyme-2; Ruan KH, Deng H & So SP (2006) Biochemistry45, 14003-14011], which links cyclo-oxygenase isoform-2 (COX-2) to prostacyclin (PGI(2)) synthase (PGIS) for the direct conversion of arachidonic acid into PGI(2) through the enzyme's Trip-cat functions. The stable upregulation of the biosynthesis of the vascular protector, PGI(2), in cells is an ideal model for the prevention and treatment of thromboxane A(2) (TXA(2))-mediated thrombosis and vasoconstriction, both of which cause stroke, myocardial infarction, and hypertension. Here, we report another case of engineering of the Trip-cat enzyme, in which human cyclo-oxygenase isoform-1, which has a different C-terminal sequence from COX-2, was linked to PGI(2) synthase and called Trip-cat enzyme-1. Transient expression of recombinant Trip-cat enzyme-1 in HEK293 cells led to 3-5-fold higher expression capacity and better PGI(2)-synthesizing activity as compared to that of the previously engineered Trip-cat enzyme-2. Furthermore, an HEK293 cell line that can stably express the active new Trip-cat enzyme-1 and constantly synthesize the bioactive PGI(2) was established by a screening approach. In addition, the stable HEK293 cell line, with constant production of PGI(2), revealed strong antiplatelet aggregation properties through its unique dual functions (increasing PGI(2) production while decreasing TXA(2) production) in TXA(2) synthase-rich plasma. This study has optimized engineering of the active Trip-cat enzyme, allowing it to become the first to stably upregulate PGI(2) biosynthesis in a human cell line, which provides a basis for developing a PGI(2)-producing therapeutic cell line for use against vascular diseases.
authors.affiliation: Department of Pharmacological and Pharmaceutical Sciences, Center for Experimental Therapeutics and PharmacoInformatics, University of Houston, TX, USA. khruan@uh.edu
authors.forename: Ke-He
authors.initials: KH
authors.lastname: Ruan
authors.forename: Shui-Ping
authors.initials: SP
authors.lastname: So
authors.forename: Vanessa
authors.initials: V
authors.lastname: Cervantes
authors.forename: Hanjing
authors.initials: H
authors.lastname: Wu
authors.forename: Cori
authors.initials: C
authors.lastname: Wijaya
authors.forename: Rebecca R
authors.initials: RR
authors.lastname: Jentzen
mesh_terms: D015121:6-Ketoprostaglandin F1 alpha / Q000378:metabolism; D016718:Arachidonic Acid / Q000378:metabolism / Q000494:pharmacology; D002384:Catalysis; D002460:Cell Line; D003001:Cloning, Molecular; D051545:Cyclooxygenase 1 / Q000235:genetics / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D011464:Epoprostenol / Q000096:biosynthesis* / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D019746:Intramolecular Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D010974:Platelet Aggregation / Q000187:drug effects; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013929:Thromboxane B2 / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D016718:Arachidonic Acid; D013929:Thromboxane B2; D015121:6-Ketoprostaglandin F1 alpha; D003577:Cytochrome P-450 Enzyme System; D011464:Epoprostenol; D051545:Cyclooxygenase 1; D019746:Intramolecular Oxidoreductases; C022461:prostacyclin synthetase