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:
*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. Kulmacz and Lee-Ho Wang for providing the origi-
nal wild-type cDNAs of human COX-1 and PGIS,
respectively. We also thank Anita Mohite for her assis-
tance with the anti platelet aggregation assays.
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