Báo cáo khoa học: An active triple-catalytic hybrid enzyme engineered by linking cyclo-oxygenase isoform-1 to prostacyclin synthase that can constantly biosynthesize prostacyclin, the vascular protector pot

An active triple-catalytic hybrid enzyme engineered by linking cyclo-oxygenase isoform-1 to prostacyclin synthase that can constantly biosynthesize prostacyclin, the vascular protector Ke-He Ruan, Shui-Ping So, Vanessa Cervantes, Hanjing Wu*, Cori Wijaya and Rebecca R. Jentzen* Department of Pharmacological and Pharmaceutical Sciences, Center for Experimental Therapeutics and PharmacoInformatics, University of Houston, TX, USA Prostacyclin (prostaglandin I 2 , PGI 2 ) [1], which has strong antiplatelet aggregation and vasodilation prop- erties [1–4], and is synthesized from endothelial and vascular smooth muscle cells, has been identified as one of the most important vascular protectors against thrombosis and heart disease [5]. Recently, there have been many new studies that have confirmed the impor- tance of PGI 2 in vascular protection. For instance, it Keywords COX; cyclo-oxygenase; PG12; prostacyclin; prostaglandin 12 Correspondence K H. Ruan, Department of Pharmacological and Pharmaceutical Sciences, Center for Experimental Therapeutics and PharmacoInformatics, University of Houston, Room 521, Science & Research 2 Building, Houston, TX 77204-5037, USA Fax: +1 713 743 1884 Tel: +1 713 743 1771 E-mail: khruan@uh.edu *Present address The University of Texas Health Science Center, Houston, TX, USA (Received 15 July 2008, revised 23 September 2008, accepted 25 September 2008) doi:10.1111/j.1742-4658.2008.06703.x 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) Biochemistry 45, 14003–14011], which links cyclo-oxygenase isoform-2 (COX-2) to prostacy- clin (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 )-medi- ated thrombosis and vasoconstriction, both of which cause stroke, myo- cardial infarction, and hypertension. Here, we report another case of engineering of the Trip-cat enzyme, in which human cyclo-oxygenase iso- form-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 produc- tion 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 engi- neering 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. Abbreviations AA, arachidonic acid; COX, cyclo-oxygenase; COX-1, cyclo-oxygenase isoform-1; COX-2, cyclo-oxygenase isoform-2; ER, endoplasmic reticulum; FITC, fluorescein isothiocyanate; IP , PGI 2 receptor; PGE 2 , prostaglandin E 2 ; PGF 2 , prostaglandin F 2 ; PGG 2, prostaglandin G 2; PGH 2, prostaglandin H 2; PGI 2, prostaglandin I 2 (prostacyclin); PGIS, prostaglandin I 2 (prostacyclin) synthase; SLO, streptolysin-O; TM, transmembrane domain; TXA 2, thromboxane A 2; TXAS, thromboxane A 2 synthase. 5820 FEBS Journal 275 (2008) 5820–5829 ª 2008 The Authors Journal compilation ª 2008 FEBS was discovered that PGI 2 receptor (IP) -knockout mice showed an increase in thrombosis tendency [6]. Also, the suppression of PGI 2 biosynthesis by cyclo-oxygen- ase isoform-2 (COX-2) inhibitors was linked to increased rates of heart disease in human clinical trials [7]. Thus, increasing the biosynthesis of PGI 2 would be very useful for protection of the vascular system. It is known that the biosynthesis of prostanoids through the arachidonate– cyclo-oxygenase (COX) pathway occurs when arachidonic acid (AA) is first converted into prostaglandin G 2 (PGG 2 , catalytic step 1), and then to prostaglandin endoperoxide [prostaglandin H 2 (PGH 2 )] (catalytic step 2) by COX isoform-1 (COX-1) or COX-2 in cells [8]. The PGH 2 then serves as a com- mon substrate for downstream synthases, and is isom- erized to prostaglandin D 2 , prostaglandin E 2 (PGE 2 ), prostaglandin F 2 (PGF 2 ), and prostaglandin I 2 (PGI 2 ) or thromboxane A 2 (TXA 2 ) by individual synthases (catalytic step 3). The overproduction of TXA 2 , a pro- aggregatory and vasoconstricting mediator, has been identified as one of the key factors causing thrombosis, stroke, and heart disease [1,2]. PGI 2 is the primary AA metabolite in vascular walls, and has opposite biolo- gical properties to that of TXA 2 ; it therefore represents the most potent endogenous vascular protector, acting as an inhibitor of platelet aggregation and a strong vasodilator on vascular beds [9–12]. Specifically increasing PGI 2 biosynthesis requires a highly efficient chain reaction between COX and PGI 2 synthase (PGIS), which consists of triple catalytic (Trip-cat) functions. Recently, we engineered a hybrid enzymatic protein with the ability to perform the Trip-cat functions by linking the inducible COX-2 to PGIS through a trans- membrane (TM) domain [13,14]. Here, we refer to this previously engineered enzyme as Trip-cat enzyme-2. Transient expression of active Trip-cat enzyme-2 in HEK293 and COS-7 cells has been demonstrated. However, there are concerns in using Trip-cat enzyme- 2 in vivo, because COX-2 has an inducible nature, has a lower capacity to be stably expressed, and may also lead to numerous pathological processes, such as cancers and inflammation. Given the nature of COX-1, a housekeeping enzyme that is consistently expressed in cells, we hypothesize that a Trip-cat enzyme, constructed by linking COX-1 to PGIS, is likely to demonstrate stable expression in cells and therefore lead to constant production of the vascular protective prostanoid PGI 2 . To test this hypothesis, in this article we report the construction of a new Trip-cat enzyme linking COX-1 to PGIS, which we call Trip-cat enzyme-1. Our studies have confirmed that Trip-cat enzyme-1 can be stably expressed in HEK293 cells and therefore lead to the generation of a cell line that con- stantly delivers the vascular protector PGI 2 . This study has provided a fundamental step towards specifically and stably upregulating PGI 2 biosynthesis in thera- peutic cells for the prevention and treatment of throm- bosis and heart disease. Results Design of a new-generation Trip-cat enzyme (COX-1 linked to PGIS) that directly converts AA to the vascular protector PGI 2 As described above, we recently invented an approach for engineering an active hybrid enzyme (Trip-cat enzyme-2), by linking human COX-2 to PGIS (COX-2– linker–PGIS), which demonstrated Trip-cat activities in converting AA to PGG 2 , PGH 2 , and finally PGI 2 [13,14] (Fig. 1). This finding provided great potential for specif- ically upregulating PGI 2 biosynthesis in ischemic tissues through the introduction of the Trip-cat enzyme-1 gene into these target tissues. On the other hand, there is the COX-1 enzyme, which is well known to have a similar function (coupling to PGIS to synthesize PGI 2 in vitro and in vivo) to that of COX-2. The housekeeping enzyme COX-1, which has less pathological impact, could be safer for gene and cell therapies than COX-2, which is involved in the pathological processes of PGI 2 PGH 2 PGG 2 3 rd Catalytic reaction 1 st Catalytic reaction 2 nd Catalytic reaction PGIS Substrate AA TM linker COX-1 Fig. 1. A model of the newly designed Trip-cat enzyme-1. Trip-cat enzyme-1 was created by linking COX-1 to PGIS through an opti- mized TM linker (10 amino acid residues) without alteration of the protein topologies in the ER membrane. The three catalytic sites in and reaction products of COX-1 and PGIS are shown. K H. Ruan et al. Prostacyclin-synthesizing protein with COX-1 and PGIS properties FEBS Journal 275 (2008) 5820–5829 ª 2008 The Authors Journal compilation ª 2008 FEBS 5821 inflammation and cancers, and shows inducible tran- sient expression. This suggested that the Trip-cat enzyme containing COX-1 (Fig. 1) may have better therapeutic potential than that containing COX-2 in terms of stable expression in cells and pathogenic prop- erties. Also, the X-ray crystal structure shows that the membrane orientation and the membrane anchor domain of COX-1 are similar to those of COX-2. This led us to design a single molecule containing the cDNA of human COX-1 and PGIS with a connecting TM lin- ker derived from human bovine rhodopsin [15] (Fig. 1). Cloning of Trip-cat enzyme-1 by linking COX-1 to PGIS A PCR approach was used to link the C-terminus of human COX-1 (NCBI GenBank ID: NM_080591) to human PGIS (NCBI GenBank ID: D38145) by a heli- cal linker with 10 residues (His-Ala-Ile-Met-Gly- Val-Ala-Phe-Thr-Trp) derived from human rhodopsin. The resultant cDNA sequence encoding the novel Trip-cat enzyme-1 (COX-1–10aa–PGIS) was then sub- cloned into the pcDNA3.1 vector for mammalian cell expression [13]. Note that the entire cDNA sequence of Trip-cat enzyme-1 encodes a single human protein sequence, which could be used for therapeutics. Expression of the engineered Trip-cat enzyme-1 in HEK293 cells Despite the many similarities between human COX-1 and COX-2, there are several important differences. For example, it has been reported that the C-terminal Leu and the last six residues of COX-1 are important for the enzyme’s activity [16]. However, they are not identical to those of COX-2. Therefore, it was interest- ing to investigate whether the linkage (from the C-ter- minal Leu of COX-1 to the N-terminus of PGIS) in Trip-cat enzyme-1 would affect its expression, protein folding, and enzyme activity. Using the constructed pcDNA3.1 COX-1–10aa–PGIS plasmid, the recombi- nant COX-1–10aa–PGIS protein was successfully over- expressed in the HEK293 cell line, showing the correct molecular mass of approximately 130 kDa in western blot analysis (Fig. 2A, lane 1). This indicated that the linkage from the C-terminal Leu of COX-1 to the N-terminus of PGIS had no effect on Trip-cat enzyme expression. In addition, a comparison of the expression levels between COX-1–10aa–PGIS and COX-2–10aa– PGIS revealed that the transfected HEK293 cells expressed approximately three-fold more COX-1– 10aa–PGIS protein than COX-2–10aa–PGIS protein under identical conditions (Fig. 2A, lane 2). Subcellular localization of COX-1–10aa–PGIS To determine whether the linkage of the C-terminal Leu of COX-1 to PGIS had any effects on the sub- cellular localization of Trip-cat enzyme-1, HEK293 cells expressing the enzyme COX-1–10aa–PGIS were permeabilized and stained. Nonsignificant differences were observed in the endoplasmic reticulum (ER) staining patterns for the cells treated with streptolysin- O (SLO), which selectively permeabilized the cell membrane, and with saponin, which generally permea- bilized both the cell and the ER membranes (Fig. 2B). The results indicated that the modification of the link- age between the COX-1 Leu residue and the PGIS N-terminus had no significant effect on the subcellular localization of COX-1–10aa–PGIS in the cells. The idea that the PGIS domain is located on the cytoplas- mic side of the ER and that the COX-1 domain is located on the ER lumen for the overexpressed COX- 1–10aa–PGIS was also supported by immunostaining. Antibody against PGIS was used to stain the cells trea- ted with SLO or saponin, but antibody against COX-1 would only stain the cells treated with saponin (Fig. 2B). These data further confirmed that the 10 amino acid linkage between COX-1 to PGIS had no significant effects on the subcellular localization of COX-1 and PGIS in the ER membrane. Trip-cat activities of Trip-cat enzyme-1 in directly converting AA to the vascular protector PGI 2 The biological activities of HEK293 cells expressing the different eicosanoid-synthesizing enzymes that con- vert AA to PGI 2 were assayed by the addition of [ 14 C]AA. The resultant [ 14 C]eicosanoids, metabolized by the enzymes in the cells, were profiled by HPLC analysis (HPLC separation linked to an automatic scintillation analyzer; Fig. 3). The Trip-cat activities that occur during the conversion of [ 14 C]AA to [ 14 C]6- keto-PGF 1a (degraded PGI 2 ) require two individual enzymes, COX-1 and PGIS, in HEK293 cells (Fig. 3A), because neither COX-1 (Fig. 3B) nor PGIS (Fig. 3C) alone could produce [ 14 C]6-keto-PGF 1a from [ 14 C]AA in HEK293 cells. However, the cells express- ing Trip-cat enzyme-1 were able to integrate the Trip- cat activities of COX-1 and PGIS by converting the added [ 14 C]AA to the end-product, [ 14 C]6-keto-PGF 1a (Fig. 3D). It should be noted that in HEK293 cells expressing Trip-cat enzyme-1, most of the added [ 14 C]AA was converted to [ 14 C]6-keto-PGF 1a , with very low amounts of byproducts. In contrast, the cells coexpressing COX-1 and PGIS synthesized less PGI 2 and produced significant amounts of other unidentified Prostacyclin-synthesizing protein with COX-1 and PGIS properties K H. Ruan et al. 5822 FEBS Journal 275 (2008) 5820–5829 ª 2008 The Authors Journal compilation ª 2008 FEBS lipid molecules. These data clearly indicated that the enzymatic conversion of AA to PGI 2 is more efficient with Trip-cat enzyme-1 than with coexpressed individ- ual COX-1 and PGIS. Enzyme kinetics of Trip-cat enzyme-1 compared to those of its parent enzymes In cells coexpressing COX-1 and PGIS, the coordina- tion of COX-1 and PGIS in the ER membrane (for the biosynthesis of PGI 2 from AA) is very fast. Only 120 s were required for 50% of the maximum activity to be reached (Fig. 4A, triangles). The reaction was almost saturated after approximately 5 min. The amount of PGI 2 produced when the reaction was extended from 5 min to 15 min increased by only 5%. On the other hand, cells expressing the engineered Trip-cat enzyme-1 (Fig. 4A, closed circles) showed the same time-course pattern as that of the coexpressed wild-type COX-1 and PGIS. In addition, Trip-cat enzyme-1 also showed an identical dose-dependent response to that of the parent enzymes in the biosyn- thesis of PGI 2 (Fig. 4B). The K m and V max values for Trip-cat enzyme-1 were approximately 5 and 400 lm, respectively; these are almost identical to those of the coexpressed COX-1 and PGIS. This study has indi- cated that the expressed Trip-cat enzyme-1 in the cells has correct protein folding, subcellular localization and native enzymatic functions in a single folded protein, similar to to its parent enzymes. Establishing stable expression of Trip-cat enzyme-1 in cells Stable expression of the engineered Trip-cat enzyme-1 in cells is the basis for having the cells constantly pro- duce PGI 2 . In this study, an HEK293 cell line was used as the model for testing. After G418 screening for b a c d B A Fig. 2. (A) Western blot analysis for overexpressed COX-1–10aa–PGIS and COX-2–10aa–PGIS in HEK293 cells. HEK293 cells transiently trans- fected with cDNA of COX-1–10aa–PGIS (lane 1) or COX-2–10aa–PGIS (lane 2), or the pcDNA3.1 vector alone (lane 3), were solubilized and separated by 7% SDS ⁄ PAGE, and then transferred to a nitrocellulose membrane. The expressed Trip-cat enzymes were stained with antibody against PGIS. The molecular mass (130 kDa) of the engineered enzymes is indicated by an arrow. (B) Immunofluorescence micrographs of HEK293 cells. In brief, the cells were grown on coverslides and transfected with the cDNA plasmid(s) of COX-1–10aa–PGIS (row 1), cotrans- fected COX-1 and PGIS (row 2), or transfected with the pcDNA3.1 vector alone (row 3). The cells were permeabilized by SLO (columns a and b) or saponin (columns c and d), and then incubated with affinity-purified rabbit antibody against PGIS peptide (columns a and c) or mouse antibody against COX-1 (columns b and d) [13]. The bound antibodies were stained with FITC-labeled goat anti-(rabbit IgG) (columns a and c) or rhodamine-labeled goat anti-(mouse IgG) (columns b and d). The stained cells were then examined by fluorescence microscopy [13]. K H. Ruan et al. Prostacyclin-synthesizing protein with COX-1 and PGIS properties FEBS Journal 275 (2008) 5820–5829 ª 2008 The Authors Journal compilation ª 2008 FEBS 5823 the transiently transfected HEK293 cells containing the Trip-cat enzyme-1 cDNA, cells stably expressing Trip- cat enzyme-1 were successfully created, as indicated by the enzyme activity assays showing continuous [ 14 C]PGI 2 production after the addition of [ 14 C]AA (Fig. 5, black squares). However, the same cells trans- fected with COX-2–10aa–PGIS cDNA could only pro- duce PGI 2 for a few days (Fig. 5, open squares), due to a failure in the stable expression of Trip-cat enzyme-2. This study indicated that the engineered Trip-cat enzyme-1 most likely adopted the housekeep- ing properties of COX-1, which produced constant expression in the cells, whereas Trip-cat enzyme-2 mainly adopted the properties of inducible COX-2, which expressed the protein for only a short period of time. Antiplatelet aggregation The effects of HEK293 cells expressing COX-1–10aa– PGIS on antiplatelet aggregation were explored. It is known that platelets contain large amounts of COX-1 and thromboxane A 2 synthase (TXAS). When AA was added to the platelet-rich plasma, the platelets began to aggregate in minutes (Fig. 6A, line a). However, this aggregation was completely blocked in the presence of cells expressing COX-1–10aa–PGIS (Fig. 6A, line b). In contrast, the aggregation was only partially blocked in the presence of cells coexpressing COX-1 and PGIS (Fig. 6A, line c). This indicated that AA was not only converted into PGI 2 (by COX-1 and PGIS), to act against platelet aggregation, but also converted into TXA 2 , promoting platelet aggregation by the abundant TXAS in the platelets. In contrast, no effects were observed with the nontransfected, control HEK293 cells (Fig. 6A, line d). From these observations, it is clear that the engineered Trip-cat enzyme-1 has supe- rior antiplatelet aggregation activity to coexpressed COX-1 and PGIS. To test whether Trip-cat enzyme-1 can indirectly inhibit platelet aggregation induced by other factors, such as collagen (through non-COX pathways), it is necessary to compare the effects of HEK293 cells (expressing Trip-cat enzyme-1) on human platelets induced by collagen (Fig. 6B, bars 1 and 2) with those of the AA-induced platelets (Fig. 6B, bars 3 and 4). It is clear that cells expressing Trip-cat enzyme-1 could not only directly inhibit AA-induced platelet aggre- gation (Fig. 6B, bar 4), but also significantly inhibit collagen-induced platelet aggregation by up to 50% (Fig. 6B, bar 2). Competitively upregulating PGI 2 biosynthesis in the presence of platelets To further demonstrate the competitive upregulation of PGI 2 biosynthesis by COX-1–10aa–PGIS in the presence of TXAS, [ 14 C]AA was added to platelet-rich plasma containing endogenous COX-1 and TXAS, in the presence and absence of cells stably expressing CPM 0 100 200 300 400 A [ 14 C]-6-keto-PGF 1α [ 14 C]-AA 010203040 0 100 200 300 400 D [ 14 C]-6-keto-PGF 1α [ 14 C]-AA 0 10 20 30 40 0 100 200 300 400 C [ 14 C]-AA 0 100 200 300 400 B Non specific peak [ 14 C]-AA Fig. 3. Determination of the Trip-cat activi- ties of the fusion enzymes for directly con- verting AA to PGI 2 , using an isotope-HPLC method for HEK293 cells. Briefly, the cells ( 0.1 · 10 6 ) transfected with the cDNA(s) of both COX-1 and PGIS (A), COX-1 (B), PGIS (C) and COX-1–10aa–PGIS (D) were washed and then incubated with [ 14 C]AA (10 l M) for 5 min. The metabolized [ 14 C] eicosanoids produced from the [ 14 C]AA in the supernatant were analyzed by HPLC on a C18 column (4.5 · 250 mm) connected to a liquid scintillation analyzer. The total counts for the specific peaks in each assay are approximately: 400 counts in (A); 550 counts in (B); 600 counts in (C); and 750 counts in (D). Prostacyclin-synthesizing protein with COX-1 and PGIS properties K H. Ruan et al. 5824 FEBS Journal 275 (2008) 5820–5829 ª 2008 The Authors Journal compilation ª 2008 FEBS COX-1–10aa–PGIS or cells coexpressing COX-1 and PGIS. In the sample containing only platelet-rich plasma, the majority of the [ 14 C]AA was converted into [ 14 C]thromboxane B 2 (Fig. 7A), indicating the presence of endogenous COX-1 and TXAS in the plasma. However, when cells expressing COX-1–10aa– PGIS were added to the plasma, the major product shifted to [ 14 C]6-keto-PGF 1a (degraded PGI 2 ; Fig. 7B), which demonstrated that COX-1–10aa–PGIS could effectively compete with endogenous COX-1 and TXAS for the substrate, [ 14 C]AA. On the other hand, A B 400 300 250 200 150 100 50 0 0 12345 300 200 100 Activity (CPM) 0 0 200 400 600 Time (s) [ 14 C]-AA added (µM) 800 1000 Fig. 4. Comparison of the time course (A) and dose-dependent response (B) of HEK293 cells expressing Trip-cat enzyme-1 (closed circles) and coexpressing its parent enzymes, COX-1 and PGIS (tri- angles). The assay and HPLC analysis conditions used are described in the caption for Fig. 3. 0 102030405060 0 100 200 300 400 Time (days) [14C]-6-keto-PGF 1α produced (cpm) Fig. 5. Time course experiment for HEK293 cells expressing the recombinant Trip-cat enzymes. The cells transfected with the cDNA of Trip-cat enzyme-1 (black squares) or Trip-cat enzyme-2 (white squares) were selected by the G418 screening approach as described in Experimental procedures, and then taken for assay analysis at different days following the transfection. The assay con- ditions for the Trip-cat enzymes are described in the caption for Fig. 3 [13]. 0 20 40 60 80 100 4 3 2 1 Platelet aggregation (%) 100 012345 Time (min) 80 60 40 20 Platelet aggregation (%) 0 –20 AA added B C D A A B Fig. 6. (A) Effects of Trip-cat enzyme-1 on antiplatelet aggregation. The platelet-rich plasma was incubated with 100 l M AA at 37 °Cin the presence of NaCl ⁄ P i (a), HEK293 cells expressing Trip-cat enzyme-1 (b), HEK293 cells coexpressing individual COX-1 and PGIS (c), and nontransfected HEK293 cells (d). The number of HEK293 cells used for the experiments was approximately 0.2 · 10 6 per assay. The addition of AA to the platelets is indicated by an arrow. (B) Comparison of the effects of HEK293 cells expressing Trip-cat enzyme-1 on platelet aggregation stimulated by collagen and AA. The platelet-rich plasma, prepared from fresh human blood, was incubated with 100 l M of collagen (bars 1 and 2) or AA (bars 3 and 4) at 37 °C in the presence of NaCl ⁄ P i (bars 1 and 3) or HEK293 cells (0.5 · 10 6 ) expressing Trip-cat enzyme-1 (bars 2 and 4). Five minutes after the initiation of the experiment, the levels of platelet aggregation were recorded and plotted; n =3. K H. Ruan et al. Prostacyclin-synthesizing protein with COX-1 and PGIS properties FEBS Journal 275 (2008) 5820–5829 ª 2008 The Authors Journal compilation ª 2008 FEBS 5825 addition of cells coexpressing COX-1 and PGIS led to only partial conversion of [ 14 C]AA to [ 14 C]PGI 2 (Fig. 7C). These results are consistent with the obser- vations from the platelet aggregation assay described above. Discussion COX-1 is a housekeeping enzyme that is constantly expressed in tissues to maintain the physiological functions of the organs. However, COX-2 is an induc- ible enzyme and is related to the pathological processes of cancer cells and inflammation [6,7]. From the point of view of therapeutic potential, it should be safer to use the Trip-cat enzyme constructed with COX-1 rather than with COX-2 for upregulating PGI 2 biosyn- thesis in vivo. Thus, the successful engineering of the active COX-1–10aa–PGIS represents an advance in our COX-based enzyme engineering, and provides a basis for developing a novel therapeutic approach against thrombosis and ischemic diseases. It should also be noted that the COX-2-based Trip-cat enzyme could not be simply replaced by the COX-1-based Trip-cat enzyme, because it is known that the mecha- nisms for the upregulation of COX-1 and COX-2 activities in vivo are different. For instance, in a situa- tion where PGI 2 is only required for a short time in the circulation, the COX-2 based Trip-cat enzyme could be preferable. It is known that the C-terminal amino acid sequence of human COX-1 is different from that of human COX-2 [16]. The crystal structures of the COX-1 C-ter- minal domain are not available yet. Therefore, it remains a challenge to clearly define its orientation with respect to the ER membrane, which may affect ER retention and anchoring, as well as enzyme cata- lytic functions. Active Trip-cat enzyme-1 was prepared by linking the human COX-1 C-terminus to the PGIS N-terminus through a 10 residue TM linker. The fact that this linkage did not affect COX-1 catalytic func- tion is consistent with earlier studies, in which COX-1 activity was not affected by elongation of the C-termi- nal segment [16]. The linkage also configured the COX-1 C-terminus on the membrane of the ER lumen in Trip-cat enzyme-1 (Fig. 1). Our data (Fig. 2B) clearly indicate that catalytic activity and ER anchor- ing were not affected by this configuration. This implies that the C-terminus is likely to be located close to the ER membrane in native COX-1. Whether the C-terminal structure is related to COX-1 stable expres- sion in cells remains a challenging question to be explored. Recently, Smith’s group reported that the recombi- nant COX-1 (t 1 ⁄ 2 > 24 h), expressed in HEK293 cells, happens to be more stable than COX-2 (t 1 ⁄ 2 approxi- mately 5 h), and found that a unique 19 amino acid cassette in the C-terminal region of COX-2 facilitates degradation of the expressed COX-2 in the cells [17]. Without the 19 amino acid cassette in the COX-1 sequence, the expressed COX-1 maintains a higher expression level and activity level in the cells than COX-2. This finding has provided a partial explana- tion for the improved activity and stable expression of 50 A B C 40 30 20 10 0 50 40 30 20 10 0 500 400 300 200 100 0 30 20 10 0 30 20 10 0 302010 Time (min) [ 14 C]-TXB 2 CPM [ 14 C]-6-keto PGF 1α α [ 14 C]-6-keto PGF 1 α [ 14 C]-TXB 2 0 Fig. 7. HPLC analysis of the profiles of [ 14 C]AA metabolized by platelets in blood in the absence and presence of HEK293 cells. [ 14 C]AA (10 lM) was incubated with 100 lL of fresh blood in the absence (A) and the presence (B) of HEK293 cells (0.1 · 10 6 ) expressing COX-1–10aa–PGIS, or coexpressing individual COX-1 and PGIS (C), for 5 min. The metabolized [ 14 C]eicosanoids produced from the [ 14 C]AA in the supernatant were analyzed by the HPLC system as described in the caption for Fig. 3. Prostacyclin-synthesizing protein with COX-1 and PGIS properties K H. Ruan et al. 5826 FEBS Journal 275 (2008) 5820–5829 ª 2008 The Authors Journal compilation ª 2008 FEBS the Trip-cat enzyme-1 derived from COX-1. In addi- tion, 26S proteosome inhibitors retarded COX-2 degra- dation but not that of COX-1 in cells [16]. This indicated that COX-2 is easily degraded in cells, which could be another key factor that would lead to more difficulty in achieving stable expression of COX-2– 10aa–PGIS in cells. However, the exact involvement of gene regulation in the different expression levels of the COX-1- and COX-2-derived Trip-cat enzymes remains to be further characterized. One of the major difficulties in using membrane pro- tein as a therapeutic agent is the limited number of options currently available for solubilizing and purify- ing the protein. Nonionic detergent is commonly used for solubilizing and purifying the membrane proteins, but is not suitable for experiments requiring admission of the membrane protein in vivo. One way to deliver the membrane protein in vivo is to introduce engi- neered cells that specifically overexpress the target pro- tein (COX-1–10aa–PGIS, Trip-cat enzyme-1). The successful establishment of an HEK293 cell line that can stably overexpress Trip-cat enzyme-1 and con- stantly produce active PGI 2 , while demonstrating strong antiplatelet aggregation properties, has pro- vided a model for generating a therapeutic cell line for potential therapeutic use of Trip-cat enzyme-1 in vivo. Antiplatelet aggregation assays provide an important method for confirmation of the antithrombotic benefits of the newly engineered Trip-cat enzyme-1. Human platelet cells are rich in COX-1 and TXAS. Following the release of AA from the cell membrane (via stimuli on the platelets), the majority of the AA is converted to TXA 2 by the coupling reaction of COX-1 and TXAS. The resultant TXA 2 binds to its receptor on the surface of the platelet and causes platelet aggre- gation. The inhibition of platelet aggregation by HEK293 cells stably expressing Trip-cat enzyme-1 (Fig. 6A) strongly indicates that: (a) expressed Trip-cat enzyme-1 was able to compete with endogenous COX- 1 and use AA as a substrate; (v) PGH 2 produced by Trip-cat enzyme-1 was readily available to the PGIS active site, even in the presence of TXAS, which com- petitively binds to PGH 2 ; and (c) the immediate increase in PGI 2 production by Trip-cat enzyme-1 reduced the amount of PGH 2 available for TXAS to produce TXA 2 (Fig. 7), which further prevented plate- let aggregation. These factors led Trip-cat enzyme-1 to possess dual functions: increasing PGI 2 biosynthesis and reducing TXA 2 biosynthesis, which could be a unique and novel antithrombosis and anti-ischemic approach that has not yet been available thus far. In addition, Trip-cat enzyme-1 also showed significant activity in inhibiting non-AA-induced aggregation (Fig. 7B), such as that of collagen. This indicates that HEK293 cells stably expressing Trip-cat enzyme-1 could use endogenous AA in the plasma, released from the platelets, to produce PGI 2 which then acts against platelet aggregation. Furthermore, this suggests the therapeutic potential of Trip-cat enzyme-1 in antiplat- elet aggregation through cell delivery. Experimental procedures Materials The HEK293 cell line was purchased from ATCC (Manas- sas, VA, USA). Medium for culturing the cell lines was purchased from Invitrogen (Carlsbad, CA, USA). [ 14 C]AA was purchased from Amersham (Piscataway, NJ, USA). Goat anti-(rabbit IgG)–fluorescein isothiocyanate (FITC) conjugate, saponin, SLO, Triton X-100 and triethylenedi- amine were purchased from Sigma (St Louis, MO, USA). Mowiol 4-88 was purchased from Calbiochem (San Diego, CA, USA). Cell culture HEK293 cells were cultured in a 100 mm cell culture dish with high-glucose DMEM (containing 10% fetal bovine serum and antibiotic and antimycotic), and were grown at 37 °C in a humidified 5% CO 2 incubator. Engineered cDNA plasmids with single genes encoding the human COX-1 and PGIS sequences The sequence of COX-1 linked to PGIS through a 10 amino acid linker (COX-1–10aa–PGIS, Trip-cat enzyme-1) was generated by a PCR approach and subcloning proce- dures provided by the vector company (Invitrogen). The procedures have been previously described [13]. Transient and stable expression of the Trip-cat enzymes in cells Recombinant Trip-cat enzyme-1 and Trip-cat enzyme-2 were expressed in HEK293 cells using the pcDNA3.1 vector. Briefly, the cells were grown and transfected with the purified cDNA of the recombinant protein by the Lipofecta- mine 2000 method [13], following the manufacturer’s instruc- tions (Invitrogen). For transient expression, the cells were harvested approximately 48 h after transfection for further enzyme assays and western blot analysis. For stable expres- sion, the transfected cells were cultured in the presence of geneticin (G418 screening) for several weeks, following the manufacturer’s instructions (Invitrogen). The cells stably expressing Trip-cat enzyme-1 and Trip-cat enzyme-2 were identified by enzyme assay and western blot analysis. K H. Ruan et al. Prostacyclin-synthesizing protein with COX-1 and PGIS properties FEBS Journal 275 (2008) 5820–5829 ª 2008 The Authors Journal compilation ª 2008 FEBS 5827 Enzyme activity determination for the Trip-cat enzymes using the HPLC method To determine the activities of the synthases that converted AA to PGI 2 through the Trip-cat functions, different con- centrations of [ 14 C]AA (0.2–17.5 lm) were added to HEK293 cells either expressing Trip-cat enzyme-1 or coex- pressing COX-1 and PGIS, or to the nontransfected cells, in a total reaction volume of 100 lL. After 10 s to 15 min of incubation, the reactions were terminated by adding 200 lL of the solvent containing 0.1% acetic acid and 35% acetonitrile (solvent A). After centrifugation (8000 g for 5 min), the supernatant was injected into a C18 column (Varian Microsorb-MV 100-5, 4.6 · 250 mm), using sol- vent A with a gradient from 35% to 100% of acetonitrile for 45 min at a flow rate of 1.0 mLÆmin )1 . The 14 C-labeled AA metabolites, including 6-keto-PGF 1a (degraded PGI 2 ), were monitored directly with a flow scintillation analyzer (Packard 150TR). Immunofluorescence staining The stable ⁄ transiently transfected HEK293 cells either expressing Trip-cat enzyme-1, coexpressing COX-1 and PGIS, or expressing the vector (pcDNA 3.1) alone, were cultured on a coverglass. The cells were then washed with NaCl⁄ P i , and incubated either with 0.5 mU of SLO for 10 min or with 1% saponin for 20 min. The cells were then incubated with the primary anti- body (10 lgÆmL )1 , affinity-purified antibody against human PGIS or antibody against mouse COX-1) for 1 h. After being washed with NaCl ⁄ P i , the cells were incu- bated with the FITC- or rhodamine-labeled second- ary antibodies [13,18] and viewed under a fluorescence microscope. Antiplatelet aggregation assays A sample of fresh blood was collected using a collection tube with 3.2% sodium citrate for anticoagulation, and then centrifuged (150 g for 10 min) to separate the plasma from the red blood cells. A total of 450 lLof this platelet-rich plasma was placed inside the 37 °C incu- bator of an aggregometer (Chrono-Log) for 3 min. The nontransfected HEK293 cells, as well as those transfected with the recombinant cDNAs of COX-1–10aa–PGIS (Trip-cat enzyme-1) or coexpressed COX-1 and PGIS, were added to different tubes containing platelet-rich plasma. The sample was then treated with 500 lgÆmL )1 AA, while inside the platelet aggregometer’s incubator, to initiate the aggregation process. Readings by the anti- coagulant analyzer were obtained, indicating the amount of platelet aggregation inhibited by each of the treated samples. Acknowledgements This work was supported by NIH Grants HL56712 and HL79389 (to Ke-He Ruan). In addition, we thank R. 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An active triple-catalytic hybrid enzyme engineered by linking cyclo-oxygenase isoform-1 to prostacyclin synthase that can constantly biosynthesize prostacyclin, the vascular protector Ke-He. 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. gene into these target tissues. On the other hand, there is the COX-1 enzyme, which is well known to have a similar function (coupling to PGIS to synthesize PGI 2 in vitro and in vivo) to that