Engineering of a novel hybrid enzyme: an anti-inflammatory drug target with triple catalytic activities directly converting arachidonic acid into the inflammatory prostaglandin E2

Abstract

Cyclooxygenase isoform-2 (COX-2) and microsomal prostaglandin E(2) synthase-1 (mPGES-1) are inducible enzymes that become up-regulated in inflammation and some cancers. It has been demonstrated that their coupling reaction of converting arachidonic acid (AA) into prostaglandin (PG) E(2) (PGE(2)) is responsible for inflammation and cancers. Understanding their coupling reactions at the molecular and cellular levels is a key step toward uncovering the pathological processes in inflammation. In this paper, we describe a structure-based enzyme engineering which produced a novel hybrid enzyme that mimics the coupling reactions of the inducible COX-2 and mPGES-1 in the native ER membrane. Based on the hypothesized membrane topologies and structures, the C-terminus of COX-2 was linked to the N-terminus of mPGES-1 through a transmembrane linker to form a hybrid enzyme, COX-2-10aa-mPGES-1. The engineered hybrid enzyme expressed in HEK293 cells exhibited strong triple-catalytic functions in the continuous conversion of AA into PGG(2) (catalytic-step 1), PGH(2) (catalytic-step 2) and PGE(2) (catalytic-step 3), a pro-inflammatory mediator. In addition, the hybrid enzyme was also able to directly convert dihomo-gamma-linolenic acid (DGLA) into PGG(1), PGH(1) and then PGE(1) (an anti-inflammatory mediator). The hybrid enzyme retained similar K(d) and V(max) values to that of the parent enzymes, suggesting that the configuration between COX-2 and mPGES-1 (through the transmembrane domain) could mimic the native conformation and membrane topologies of COX-2 and mPGES-1 in the cells. The results indicated that the quick coupling reaction between the native COX-2 and mPGES-1 (in converting AA into PGE(2)) occurred in a way so that both enzymes are localized near each other in a face-to-face orientation, where the COX-2 C-terminus faces the mPGES-1 N-terminus in the ER membrane. The COX-2-10aa-mPGES-1 hybrid enzyme engineering may be a novel approach in creating inflammation cell and animal models, which are particularly valuable targets for the next generation of NSAID screening.

Figures

Fig. 1

Comparison of the structural and topological differences between the hybrid enzymes linking COX-2 with PGIS and mPGES-1 in respect to their subcellular localization of the wild-type enzymes. The topological models of the PGIS (Ruan and Dogné, 2006; Ruan, 2004) and mPGES-1 (Jakobsson et al., 1999) in respect to the ER membrane were created from information of their respective references. (A) Model of the engineered COX-2-linker-PGIS linking the two enzymes through the C-terminus of COX-2 and the N-terminus of PGIS by a10 amino acid residues (10aa). (B) Model of the engineered COX-2-linker-mPGES-1 linking the two enzymes together through the C-terminus of COX-2 and N-terminus of mPGES-1 by a 10aa linker.

Fig. 2

Western blot analysis of the over-expressed recombinant proteins in HEK293 cells. The cells were grown for 24 h on 100 mm culture dishes until they were about 95% confluent and then transfected with the purified cDNA plasmid (pcDNA3.1(+)) using Lipofectamine 2000™ following the manufacturer's instructions (Invitrogen). For the co-transfection (lane 2), the cells were transfected with 12 µg of human COX-2 cDNA plasmid and 12 µg of human mPGES-1 cDNA plasmid. For the COX-2-10aa-mPGES-1 (lane 1) and pcDNA 3.1(+) (negative control, lane 3), 24 µg of each plasmid were used for each individual transfection. Approximately 48 h after transfection, the cells were harvested and used for western blot analysis. Equal amounts of protein (25 µg) were applied on a 7% SDS–polyacrylamide gel. Following electrophoresis, the protein was transferred to a nitrocellulose membrane which was probed with a mixture of rabbit anti-mPGES-1 and anti-COX-2 antibodies using a 1:500 dilution (Cayman, Ann Arbor, MI) and then stained with horseradish peroxidase-labeled goat-anti rabbit antibody using a Chemiluminescence kit (Amersham, England, UK). The numbers on the left represent the molecular mass (in kDa) of the proteins described.

Fig. 3

Immunofluorescence micrographs of ER staining pattern in HEK293 cells. The general procedures for the indirect immunostaining were described previously (Lin et al., 2000; Deng et al., 2002). In brief, the cells were grown on cover slides and transfected with the plasmid containing the cDNA of COX-2-10aa-mPGES-1 (1 and 2), COX-2 (3), mPGES-1 (4) or pcDNA vector (5) and (6). The cells were generally permeabilized by saponin, and then incubated with the affinity-purified rabbit anti-COX-2 peptide antibody (A) and mouse anti-mPGES antibody (B). The bound antibodies were stained by FITC-labeled goat-anti rabbit IgG (A) or Rhodamine-labeled goat anti-mouse IgG (B). The stained cells were examined by fluorescence microscopy.

Fig. 4

Determination of the triple catalytic activities of the recombinant proteins directly converting AA to PGE2 using an HPLC method for the HEK293 cells. The cells were transfected with the recombinant cDNAs of COX-2-10aa-mPGES-1 (A), COX-2 and mPGES-1 (a mixture of two plasmids), each containing one of the coding sequences (COX-2 or mPGES-1) (B), COX-2 (C), mPGES-1 (D) or pcDNA 3.1 vector (E). The untransfected cells were also used as a control (F). The cells (0.1 × 106) were washed three times, then 25 µl were suspended in 2.5 mM glutathione (GSH) buffer and then incubated with [14C]-AA (10 µM) in a total volume of 225 µl. After 2 min, the reaction was terminated by the addition of 2 mM SnCl2 and 0.1% TFA (200 µl each) and then centrifuged (6000 rpm, 5 min). The supernatant was separated by HPLC on a C18 column (4.5 × 250 mm) using 0.1% acetic acid containing 35% acetonitrile (buffer A) with a gradient of 35–100% acetonitrile. The [14C]-AA metabolites were determined by a liquid scintillation analyzer built in the HPLC system. The retention time of [14C]-PGE2 and [14C]-AA were calibrated by standards under the same conditions. The time represented above in the HPLC data was measured in minutes and CPM stands for counts per minute.

Fig. 5

Determination of the tri-catalytic activities of the recombinant proteins directly converting AA to PGE2 using enzyme immunoassay (EIA). Following the enzyme reactions (described in Fig. 3) for the reaction mixtures prepared from the cells transfected with the plasmid of the COX-2-10aa-mPGES-1 (A), the mixture of the plasmids of the COX-2 and mPGES-1 (B), or the pcDNA3.1 vector alone (C), the samples were diluted 100 times with PBS containing 0.1% bovine serum albumin (BSA), and then used for quantitative determination of PGE2 using an EIA kit following the instructions of the manufacturer (Cayman, Ann Arbor, MI). A PGE2 standard is shown in column (D).

Fig. 6

Determination of the triple catalytic activities of the recombinant proteins directly converting DGLA to PGE1 using an HPLC method for HEK293 cells. The [14C]-DGLA (10 µM) was added to the cells expressing COX-2-10aa-mPGES-1 (A). The [14C]-PGE1 standard produced is shown in (B). The methods used for assay and HPLC analysis were described in Fig. 4.

Fig. 7

(A) Time-course of the conversion of AA to PGE2 by the recombinant proteins. The conversion of AA to PGI2 by the HEK293 cells expressing COX-2-10aa-mPGES-1 (open circles) or those co-expressing COX-2 and mPGES-1 (closed squares) was performed with increasing times using the HPLC method as described in Fig. 4. The amount of [14C]-PGE2 produced at increasing reaction times were calculated and plotted as percentages of the added substrate, [14C]-AA (10 µM). (B) Enzyme kinetic properties of the expressed recombinant proteins. Increasing concentrations of [14C]-AA were added to the HEK293 cells expressing COX-2-10aa-mPGES-1 (open circles) or co-expressing COX-2 and mPGES-1 (closed squares) for 60 s. The reactions were terminated and then analyzed by HPLC. The conditions, including protein concentration and buffers used, were identical to that of Fig. 4.

Fig. 8

Using the hybrid enzyme as an anti-inflammatory drug target. The response of the hybrid enzyme as a NSAID target was tested by a cell-based assay using a COX-2 inhibitor. The cells transfected with COX-2-10aa-mPGES-1 cDNA (Fig. 2) were washed three times, then 25 µl were suspended in 2.5 mM GSH buffer in the absence (A) and presence (B) of a COX-2 inhibitor (NS-398, 10 µM) and then incubated with [14C]-AA in a total volume of 225 µl. The further assay and HPLC analysis steps followed the procedures described in Fig. 4.