Engineering of a Protein with Cyclooxygenase and Prostacyclin Synthase Activities That Converts Arachidonic Acid to Prostacyclin

Abstract

Prostacyclin (PGI2), a vascular protector with vasodilation and antithrombotic properties, is synthesized by coupling reactions of cyclooxygenase (COX, the first enzyme) with PGI2 synthase (PGIS, the second enzyme) using arachidonic acid (AA) as an initial substrate. The first COX product, prostaglandin H2 (PGH2) is also a command substrate for other prostanoid enzymes that produce distinct eicosanoids, such as thromboxane A2 (TXA2). The actions of TXA2 to cause vasoconstriction and platelet aggregation oppose the vasodilatory and anti-aggregatory effects of PGI2. Specifically upregulating PGI2 biosynthesis is an ideal model for the prevention and treatment of the TXA2-mediated thrombosis involved in strokes and myocardial infarctions. Here, we report that a single protein was constructed by linking COX-2 and PGIS together to form a new fusion enzyme through a transmembrane domain with 10 or 22 residues. The engineered protein expressed in HEK293 and COS-7 cells was able to continually convert AA to prostaglandin (PG) G2 (catalytic step 1), PGH2 (catalytic step 2), and PGI2 (catalytic step 3). The studies first demonstrate that a single protein with three catalytic functions could directly synthesize PGI2 from AA with a Km of approximately 3.2 microM. Specific upregulation of PGI2 biosynthesis through expression of the engineered single protein in the cells has shown strong activity in inhibiting platelet aggregation induced by AA in vitro, which creates a great potential for the fusion enzyme to be used as one of the new therapeutic interventions for strokes and heart attacks. The studies have also provided a model linking COX with its downstream enzymes to specifically regulate biosynthesis of eicosanoids which have potent biological functions.

A Covalent Linker Allows for Membrane Targeting of an Oxylipin Biosynthetic Complex

“…Similarly, prostaglandin biosynthetic pathways have branch points that must be navigated by the intermediates common to multiple prostaglandin products that induce fundamentally different, and often opposing, biological responses. In an effort to determine whether co-localization of enzymatic activities might impact the relative amounts of prostacyclin (PGI2) and thromboxane A2 produced, a bi-functional enzyme that contained both cyclooxygenase (required for the biosynthesis of both products) and PGI2 synthase was engineered (54). Indeed, cells expressing this construct produced elevated levels of PGI2 (54).…”

Elevated prostacyclin biosynthesis in mice impacts memory and anxiety-like behavior

“…CP-Tg mice overexpress the TriCat enzyme [20, 21], a hybridized enzyme linking COX-1 and PGIS via a transmembrane domain. Overexpression of this hybrid results in a three-fold increase in anabolism of PGI 2 in cell lines and shows an increased ability to efficiently convert AA into PGI 2 [22].…”

Identification of Tumorigenesis from the Specific Coupling of Cyclooxygenase-2 with Microsomal Prostaglandin E2 Synthase-1 in vivo

“…The COX-2 and mPGES-I were linked through a 10 amino acid (aa) sequence on the pcDNA 3.1(+) vector. The detailed method was given in our previous publication (16).…”

“…After incubation for 0.5-5 min the reaction was terminated by the addition of 0.2 ml 0.1% TFA (trifluoroacetic acid) and 0.2 ml 2 mM SnCI 2 • The details are described in a previous publication (16). The mixture was briefly vortexed for I min to ensure termination of the reaction.…”