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.

Figures

Figure 1

A model of the newly designed Trip-cat Enzyme-1. The Trip-cat Enzyme-1 was created by linking COX-1 to PGIS through an optimized TM linker (10 amino acid residues) without alteration of the protein topologies in the ER membrane. The three catalytic sites and reaction products in COX-1 and PGIS enzymes are shown.

Figure 2

(i).Western blot analysis for the over-expressed COX-1-10aa-PGIS and COX-2-10aa-PGIS in HEK293 cells. The HEK293 cells transiently transfected with the cDNA of COX-1-10aa-PGIS (lane 1), COX-2-10aa-PGIS (lane 2) or the pcDNA 3.1 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 by anti-PGIS antibody. The molecular weight (130 kDa) of the engineered enzymes is indicated with an arrow. (ii). Immunofluorescence micrographs of HEK293 cells. In brief, the cells were grown on cover-slides and transfected with the cDNA plasmid(s) of the COX-1-10aa-PGIS (row 1), co-transfected COX-1 and PGIS (row 2) or the pcDNA 3.1 vector alone (row 3). The cells were permeabilized by SLO (panels A and B) or saponin (panels C and D) and then incubated with the affinity-purified rabbit anti-PGIS peptide antibody (A and C) or mouse anti-COX-1 antibody (B and D) [13]. The bound antibodies were stained by FITC-labeled goat-anti rabbit IgG (A and C) or rhodamine-labeled goat anti-mouse IgG (B and D). The stained cells were then examined by fluorescence microscopy [13].

Figure 3

Determination of the triple-catalytic activities of the fusion enzymes for directly converting AA to PGI2, using an isotope-HPLC method for HEK293 cells. Briefly, the cells (~0.1 × 106) 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 [14C]-AA (10 µM) for five min. The metabolized [14C]-eicosanoids from the [14C]-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.

Figure 4

Comparison of the time course (A) and dose-dependent response (B) of the HEK293 cells expressing Trip-cat Enzyme-1 (closed circles) and co-expressing its parent enzymes, COX-1 and PGIS (triangles). The assay and HPLC analysis conditions used are described in Fig. 3.

Figure 5

Time course experiment for the HEK293 cells expressing the recombinant Trip-cat Enzymes. The cells transfected with the cDNA of the Trip-cat Enzyme-1 (black squares) or the Trip-cat Enzyme-2 (white squares) were selected by the G418 screening approach as described in the Experimental Procedures and then taken for assay analysis at different days following the transfection. The assay conditions for the Tri-cat enzymes are described in Figure 3 [13].

Figure 6

(i) Effects of the Trip-cat Enzyme-1 in anti-platelet aggregation. The platelet-rich plasma was incubated with 100 µM AA at 37°C in the presence of PBS (A), the HEK293 cells expressing Trip-cat Enzyme-1 (B), HEK293 cells co-expressing individual COX-1 and PGIS (C), and non-transfected HEK293 cells (D). The amount of HEK293 cells used for the experiments were approximately 0.2 × 106 per assay. The addition of AA to the platelets is indicated with an arrow. (ii). Comparison of the effects of the HEK293 cells expressing the Trip-cat Enzyme-1 on the platelet aggregation stimulated by collagen and AA. The platelet-rich plasma, prepared from fresh human blood, was incubated with 100 µM of collagen (bars 1 and 2) or AA (bars 3 and 4) at 37 °C in the presence of PBS (lanes 1 and 3) or HEK293 cells (0.5 × 106) expressing the Trip-cat Enzyme-1 (lanes 2 and 4). After five minutes from the initiation of the experiment, the levels of platelet aggregation were recorded and plotted, where n=3.

Figure 7

HPLC analysis for the profiles of the [14C]-AA metabolized by platelets in blood in the absence and presence of the HEK293 cells. [14C]-AA (10 µM) was incubated with 100 µL of fresh blood in the absence (A) and the presence of HEK293 cells (0.1 × 106) expressing COX-1-10aa-PGIS (B) or co-expressing individual COX-1 and PGIS (C) for five min. The metabolized [14C]-eicosanoids from the [14C]-AA in the supernatant were analyzed by the HPLC system as described in Figure 3.