(Received for publication, May 3, 1995; and in revised form, June 1, 1995)
From the
Human prostaglandin-endoperoxide H synthase-1 and -2 (hPGHS-1
and hPGHS-2) were expressed by transient transfection of COS-1 cells.
Microsomes prepared from the transfected cells were used to measure the
rates of oxygenation of several 18- and 20-carbon polyunsaturated fatty
acid substrates including eicosapentaenoic, arachidonic,
dihomo--linolenic,
-linolenic (
),
-linolenic, and linoleic acids. Comparisons of k
/K
values
indicate that the order of efficiency of oxygenation is arachidonate
> dihomo-
-linolenate > linoleate >
-linolenate for
both isozymes; while the order of efficiency was the same for hPGHS-1
and hPGHS-2,
-linolenate was a particularly poor substrate for
hPGHS-1.
-Linolenate and eicosapentaenoate were poor substrates
for both isozymes, but in each case, these two fatty acids were better
substrates for hPGHS-2 than hPGHS-1. These studies of substrate
specificities are consistent with previous studies of the interactions
of PGHS isozymes with nonsteroidal anti-inflammatory drugs that have
indicated that the cyclooxygenase active site of PGHS-2 is somewhat
larger and more accommodating than that of PGHS-1. The major products
formed from linoleate and
-linolenate were characterized.
13-Hydroxy-(9Z,11E)-octadecadienoic acid was found to
be the main product formed from
-linoleate by both isozymes. The
major products of oxygenation of
-linolenate were determined by
mass spectrometry to be
12-hydroxy-(9Z,13E/Z,15Z)-octadecatrienoic
acids. This result suggests that
-linolenate is positioned in the
cyclooxygenase active site with a kink in the carbon chain such that
hydrogen abstraction occurs from the
5-position in contrast to
abstraction of the
8-hydrogen from other substrates.
The biosynthesis of prostanoids depends upon the enzyme that
catalyzes the committed step in the pathway: prostaglandin-endoperoxide
H synthase (PGHS)()(1) . Two isozymes of PGHS
(PGHS-1 and PGHS-2) have been described. The first isozyme, PGHS-1, was
initially purified (2, 3) and cloned from sheep
vesicular gland(4, 5, 6) , a tissue that is
highly enriched in this protein. cDNAs for PGHS-1 have since been
cloned from human(7) , rat(8) , and murine (9) sources. The processed form of PGHS-1 has 576 amino acids.
Ovine PGHS-1 is a hemoprotein(2, 3) , and the crystal
structure indicates that His-388 and His-207 serve as the proximal and
distal heme ligands, respectively (10) . Ovine PGHS-1 is also a
glycoprotein(11) , and Asn-69, Asn-144, and Asn-410 are sites
of N-glycosylation(12) . A number of other residues
including Tyr-385(13) , Ser-530(9) , and Arg-120 (10) have been shown to be located in the cyclooxygenase active
site of the enzyme. The crystal structure suggests that the
cyclooxygenase active site is in the form of a hydrophobic
channel(10) . Both the human and murine genes for PGHS-1 have
been characterized(14, 15) . The gene for PGHS-1
contains 11 exons spanning
22 kilobases. PGHS-1 is often referred
to as the ``constitutive'' form of PGHS and is expressed in
most tissues(16, 17, 18) .
The second
isozyme, PGHS-2, was originally detected as an immediate-early gene
product from chick embryo and murine fibroblasts (19, 20) . cDNA clones for PGHS-2 have since been
isolated from human (21, 22) and rat (8) sources. The deduced amino acid sequences are 61% identical
within a species, and those amino acids required for catalysis by
PGHS-1 (12, 13, 23, 24) are all
conserved in PGHS-2(1, 25) . Two major differences
exist between the two polypeptides: (a) PGHS-2 has a shorter
N-terminal signal peptide than PGHS-1(17, 26) , and (b) PGHS-2 contains a unique 18-amino acid insertion near its
C
terminus(8, 19, 20, 21, 22) ,
having an additional N-linked glycosylation site that is not
found in PGHS-1 (12) . PGHS-2 is encoded by a transcript of
4.5
kilobases(8, 19, 20, 21, 22) .
The organization of the PGHS-2 gene is very similar to that of the
PGHS-1 gene, but the PGHS-2 gene is considerably smaller (
8
kilobases)(27) . PGHS-2 is not expressed in most cells or
tissues(20) , but it can be induced in cells treated with
mitogens, cytokines, or tumor
promoters(8, 19, 20, 21, 22, 28, 29, 30, 31, 32, 33) .
Accordingly, PGHS-2 is often referred to as the ``inducible''
form of PGHS.
PGHS-1 and PGHS-2 both have two catalytic
activities(27, 34, 35) : (a) a
cyclooxygenase activity involved in forming PGG from
arachidonic acid and (b) a peroxidase activity that catalyzes
a 2-electron reduction of PGG
to PGH
. The
kinetic properties of the cyclooxygenase activities of the two isozymes
are quite similar. PGHS-1 and PGHS-2 have similar V
and K
values with
arachidonate(35, 36, 37) , both enzymes form
tyrosyl radicals (38) , both isozymes undergo suicide
inactivation(35, 36, 37) , and both enzymes
are inhibited by nonsteroidal anti-inflammatory
drugs(35, 36, 37, 39, 40, 41, 42, 43) .
Nonetheless, there are subtle differences between the active sites of
PGHS-1 and PGHS-2 as evidenced by their different affinities toward
nonsteroidal anti-inflammatory
drugs(35, 36, 37, 39, 40, 41, 42, 43) .
Nonsteroidal anti-inflammatory drugs compete with arachidonate for
binding to the cyclooxygenase active site of
PGHSs(35, 44, 45) . Other agents that are
competitive inhibitors of arachidonate oxygenation include various
fatty acid derivatives(46, 47, 48) . Some of
these fatty acids, including eicosapentaenoic acid(48) ,
dihomo-
-linolenic acid (49) , and linoleic
acid(50) , have previously been established to be substrates
for PGHS-1. However, there is little information on the roles of these
fatty acids as substrates for PGHS-2. Here, we describe studies in
which common 18- and 20-carbon fatty acids were compared for their
abilities to serve as substrates for both isozymes. In characterizing
the products of oxygenation, we identified
12-hydroxy-(9Z,13E/Z,15Z)-octadecatrienoic
acids (12-HOTrEs) as the major products of the oxygenation of
-linolenic acid.
Analysis by GC/EI-MS was carried out
on a Joel JMS AX505H double focusing mass spectrometer coupled with a
Hewlett-Packard 5890 gas chromatograph. GC separations employed a
DB5/MS column (30 m 0.32-mm (inner diameter) fused silica
capillary column with a 0.25-µm film coating; J & W Scientific,
Rancho Cordova, CA). GC conditions were as follows: injection port
temperature, 260 °C; initial column temperature, 100 °C for 2
min; and program rate, 20 °C/min to 300 °C. Direct (splitless)
injection was used. Helium gas flow was
1 ml/min. MS conditions
were as follows: interface temperature, 280 °C; and ion source
temperature, 150-200 °C. The scan rate of the mass
spectrometer was 50-600 Da in 1 s.
Additional GC/EI-MS and
GC-MS/MS experiments were performed on a Varian Saturn-I ion trap mass
spectrometer equipped with a waveboard option. The ion trap was
connected via a heated interface to a Varian 3400 gas chromatograph
with a 30 m 0.25-mm DB5/MS 0.25-µm film thickness capillary
column. Injections were made in the split mode (ratio of 30:1) with a
column head pressure of 10 p.s.i. using helium as the carrier gas,
producing a flow rate of
1 ml/min. The GC conditions and
temperature program employed were identical to those used for GC-MS.
Conditions for the ion trap mass spectrometer were as follows: manifold
temperature, 170 °C; electron energy, 70 eV; and partial pressure
of helium in the ion trap cavity,
1
10
torr. The ion trap mass spectrometer was scanned from m/z 50 to 450 in 1 s. The MS/MS parameters were as follows: isolation
window, 3 mass units; isolation time, 5 ms; excitation amplitude, 55 V;
and excitation time, 40 ms.
The initial rates of oxygenation of various C and C
polyunsaturated fatty acids (100
µM) by microsomes prepared from COS-1 cells expressing
either hPGHS-1 or hPGHS-2 were determined using an O
assay (Fig. 1). The data are expressed relative to activity with
arachidonate and are corrected for the fact that C
substrates incorporate 1 mol of O
/mol of fatty acid,
whereas C
substrates incorporate 2 (1.7-1.9) mol of
O
/mol of fatty acid. With both enzymes, the C
fatty acids arachidonate and dihomo-
-linolenate were the
best substrates. With hPGHS-1, all other substrates were oxygenated at
<15% of the rates observed with arachidonate. In contrast, hPGHS-2
utilized the C
fatty acid substrates at 30-75% of
the rates observed with arachidonate. Of the six fatty acids tested,
eicosapentaenoic acid was the poorest substrate for hPGHS-2 and the
next to poorest substrate for hPGHS-1. Further studies with
eicosapentaenoic acid are presented below. It should be emphasized that
the results depicted in Fig. 1are from rate measurements
performed at fatty acid substrate concentrations of 100
µM. The oxygenation of all fatty acids was completely
inhibited when 0.1 mM flurbiprofen was included in the
incubation mixture or when microsomes were preincubated at 37 °C
for 30 min with 1 mM aspirin (data not shown). (
)Thus, with all substrates, the oxygenation rates reported
in Fig. 1represent cyclooxygenase activities of hPGHS-1 or
hPGHS-2.
Figure 1:
Fatty acid substrate specificities of
hPGHS-1 and hPGHS-2 as determined by O electrode assays.
Microsomal membranes were prepared from COS-1 cells expressing either
hPGHS-1 or hPGHS-2 as described under ``Experimental
Procedures.'' Aliquots of the microsomal suspensions (
250
µg of protein) were added to assay mixtures, which included 100
µM concentrations of the indicated fatty acids. O
consumption was measured using an O
electrode assay.
Each value represents the mean ± S.D. of four determinations,
each from a minimum of two different experiments in which the rate for
each fatty acid was compared with that for arachidonate. In each
experiment, the rates were obtained with hPGHS-1 and hPGHS-2 using
arachidonate and then compared with the rates observed with other test
fatty acids. Values for initial rates were always within 10% of one
another for both hPGHS-1 and hPGHS-2 with arachidonate; the average
rate with arachidonate was 33 ± 4.0 nmol/min/mg of protein with
hPGHS-1 and 37 ± 4.6 nmol/min/mg of protein with hPGHS-2. 20:4, arachidonate; 20:5, eicosapentaenoate; 20:3, dihomo-
-linolenate; 18:2, linoleate;
-18:3,
-linolenate;
-18:3,
-linolenate.
K values were determined for
four of the fatty acids (Table 1); we were unable to obtain
consistent results for K
determinations
with eicosapentaenoic acid, and K
measurements were not performed with
-linolenic acid.
Using the experimentally determined K
values in combination with the relative velocities
determined with 100 µM substrate concentrations (Fig. 1), V
values were calculated (Table 1); as a measure of enzyme efficiencies, relative k
/K
values were
also calculated. For both hPGHS-1 and hPGHS-2, the fatty acid
substrates are utilized in the order of efficiency of arachidonate >
eicosatrienoate > linoleate >
-linolenate. However, in
comparing the apparent k
/K
values for the two isozymes for the different substrates, it
became obvious that
-linolenate was a particularly poor substrate
for PGHS-1.
Eicosapentaenoic acid was a poor substrate for both
hPGHS-1 and hPGHS-2 based on O electrode measurements (Fig. 1). We subsequently incubated
[1-
C]eicosapentaenoic acid (10 µM)
with intact COS-1 cells expressing hPGHS-1 or hPGHS-2 and used radio
thin-layer chromatography to quantitate the formation of products
migrating with PGF
, PGE
, PGD
,
and 17-hydroxy-(5Z,8Z,10E)-heptadecatrienoic
acid standards. COS-1 cells expressing hPGHS-1 converted 3.4 ±
0.1% of the [1-
C]eicosapentaenoic acid to
radioactive prostaglandins of the 3-series during a 15-min incubation;
when the incubation was performed in the presence of 5 µM 15-HPETE, the rate was 5.3 ± 0.3% conversion/15 min. The
rates obtained using [1-
C]eicosapentaenoic acid
and COS-1 cells expressing hPGHS-2 were 18.1 ± 1.7% in the
absence of 15-HPETE and 13.4 ± 1.0% in the presence of 5
µM 15-HPETE.
PGHS-1 from sheep vesicular gland was
shown previously to form 9-HODE and 13-HODE from linoleic acid, with
the major product being 9-HODE (50) . However, in our
experiments, the main oxygenation product formed when
[1-C]linoleic acid was incubated with COS-1
cells expressing either hPGHS-1 or hPGHS-2 migrated with 13-HODE on
thin-layer chromatography (Fig. 2A). For hPGHS-1, 1.7%
of the starting linoleate was converted to 9-HODE, while 8.9% was
converted to 13-HODE. With hPGHS-2, 4.0% of the initial
[1-
C]linoleic acid was converted to 9-HODE and
31.5% to 13-HODE. As predicted by the rates of oxygenation of linoleic
acid measured using an O
electrode (Fig. 1), hPGHS-2
produced
3 times more HODEs than hPGHS-1. The formation of
radioactive HODEs by hPGHS isozymes was completely inhibited by 0.1
mM flurbiprofen or by a preincubation of the isozymes with 1
mM aspirin.
Figure 2:
Products formed from
[1-C]linoleic acid or
-[1-
C]linolenic acid incubated with COS-1
cells expressing either hPGHS-1 or hPGHS-2.
[1-
C]linoleic acid (10 µM) (A) or
-[1-
C]linolenic acid (10
µM) (B) was added directly to intact transfected
COS-1 cells expressing either hPGHS-1 or hPGHS-2 as described under
``Experimental Procedures.'' After a 15-min incubation at 37
°C, the radioactive products in the supernatant were extracted,
separated by thin-layer chromatography, visualized by autoradiography,
and quantified by densitometry as described under ``Experimental
Procedures.'' Plotted in this figure is the percentage of total
extractable radioactivity comigrating with free fatty acid (FFA), 9-HODE, or 13-HODE. In B, material indicated
as comigrating with 13-HODE was subsequently identified by GC-MS and
GC-MS/MS to be primarily 12-HOTrE.
The major product formed upon incubation of
-[1-
C]linolenic acid with COS-1 cells
expressing hPGHS-2 comigrated with 13-HODE during thin-layer
chromatography and accounted for 57.4% of the radioactivity (Fig. 2B). This material was isolated from the
thin-layer plate and analyzed by GC-MS (electron impact ionization)
after derivatization to the corresponding O-trimethylsilyl
ether and ester of the fatty acid. Two major chromatographic peaks (Fig. 3, peaks a and b) with retention times
of 9 min, 35 s and 9 min, 40 s, respectively, were observed. Both
compounds represented by the chromatographic peaks exhibit nearly
identical mass spectra (Fig. 4) with
[M-CH
]
ions at m/z 423,
suggesting that these two peaks represent a pair of isomers of a
hydroxylated octadecatrienoic acid. Fig. 5shows the proposed
formation of three important fragment ions observed in the mass
spectra. The base peaks at m/z 183 correspond to a fragment
ion containing one O-TMS group linked to a 7-carbon diene
unit, which dictated the location of the oxygen at C-12 in the two
isomers (Fig. 5). Another diagnostic fragment ion at m/z 328 can be attributed to migration of a TMS radical from the
oxygen atom at C-12 to the acid carbonyl site (53, 54) with concurrent
-cleavage and
elimination of heptadienal, C
H
CHO (Fig. 5). A weak but reproducible
-cleavage fragment was
detected at m/z 357 (Fig. 4), which serves as
additional evidence for 12-OH substitution. To establish unequivocally
the position of hydroxylation, we prepared and analyzed the
hydrogenated derivative of the PGHS-2 product of
-linoleate, which
was extracted from the thin-layer chromatogram, methylated,
hydrogenated, silylated, and subjected to GC-MS analysis. The mass
spectrum matched that of the corresponding derivative of
12-hydroxystearate. (
)All these observations unequivocally
established the hydroxyl substitution position and suggested the
presence of two double bonds in the portion of the chain between C-12
and C-17, with the third double bond likely at C-9.
Figure 3:
Reconstructed total ion current (TIC) chromatogram and mass chromatogram for m/z 183
from GC-MS analysis of the TMS derivatives of reaction products formed
during incubation of human PGHS-2 with -linolenic acid.
-[1-
C]Linolenic acid (10 µM)
was incubated with transfected COS-1 cells expressing hPGHS-2, and the
radioactive products were separated by thin-layer chromatography as
described in the legend to Fig. 2. The radioactive material
cochromatographing with 13-HODE standard was eluted from the thin-layer
plate, derivatized, and analyzed by GC-MS as described under
``Experimental Procedures.'' Compounds represented by the
major peaks a and b were further characterized by
GC-MS/MS. R.T., retention time.
Figure 4: EI mass spectrum of 12-HOTrE TMS esters. Shown is the EI mass spectrum of peak a from the total ion current chromatogram in Fig. 3. Peak b from the reconstructed total ion current chromatogram gave an identical EI mass spectrum.
Figure 5: Proposed fragmentation scheme for 12-HOTrE TMS esters.
Analysis by
GC-MS/MS was used to determine the location of the double bonds (Fig. 6). In one experiment, the -cleavage fragment ion at m/z 183 was selected as the precursor, which upon collisional
activation yielded a major product ion at m/z 155 (Fig. 6A). The latter was formed by a specific
elimination involving hydrogen transfer cleavage occurring at the
terminal vinylic position, with elimination of a 2-carbon unit
(C-17-C-18) from the tail. Loss of the ethylene neutral would not
be observed if there were a
-double bond. This
interpretation was further supported by an MS/MS experiment with the
analogous hydroxylated fatty acid, 11-HETE. In the latter case, vinylic
elimination involving hydrogen transfer operates in the same way to
produce m/z 155 from the
-cleavage precursor m/z 225 through the loss of C
H
from the
terminal part of the chain (Fig. 6B). These results
indicate that the
-double bond was not modified and
therefore that a shift of a double bond from C-12 to C-13 occurred when
the oxygen was introduced to the methylene-interrupted unsaturated
system at C-12. The formation of a new C-13, C-15 diene is supported by
the UV absorption maximum at 231 nm corresponding to the presence of a
conjugated diene chromophore(55) .
Figure 6:
MS/MS of -cleavage fragments of
selected TMS ether-TMS ester derivatives. A, MS/MS spectrum of
the precursor ion at m/z 183 formed by
-cleavage to the O-TMS group of 12-HOTrE TMS esters; B, MS/MS spectrum
of the analogous precursor ion at m/z 225 formed by
-cleavage to the O-TMS group of (11R)-HETE TMS
ester.
The position of the third
double bond was confirmed by an MS/MS experiment using the TMS
migration fragment ion at m/z 328 as the precursor. The MS/MS
of m/z 328 for both 12-hydroxyoctadecatrienoic acid and
ricinoleic acid showed similar product ion spectra (data not shown),
thus suggesting that modification of the double bond at C-9 in the
-linolenic acid substrate is quite unlikely. Therefore, the
structure of the hydroxylated linolenic acid is
12-hydroxy-(9Z,13E/Z,15Z)-octadecatrienoic
acid. Because both peaks a and b (Fig. 3) displayed identical
results in the MS/MS experiments, we conclude that these two components
represent two geometrical isomers of the 12-hydroxylated
octadecatrienoic compounds with opposite configurations of the C-13
double bond. Although authentic standards of the individual isomers are
not available, when the trimethylsilyl derivative of 13-HOTrE was
subjected to GC-MS analysis immediately preceding the analysis of the
12-hydroxy-(9Z,13E/Z,15Z)-octadecatrienoic
acids, 96% of the 13-HOTrE chromatographed as a single peak. These
results suggest that both the cis- and trans-isomers
of 12-hydroxy-(9,13,15)-octadecatrienoic acid are enzymatic products.
hPGHS-1 also seems to form this monohydroxylated fatty acid from
-linolenic acid, but to a lesser extent than hPGHS-2; only 10.9%
of the initial
-[
C]linolenic acid added was
converted to this product by hPGHS-1 as compared with 57.4% for hPGHS-2 (Fig. 2B). However, no attempt was made to confirm the
structure of the product formed via the action of hPGHS-1.
Prostanoid synthesis is believed to require the mobilization
of fatty acids from lipid precursors via one or more phospholipases
A(25) . Three different phospholipases A
have been identified as being involved in different situations in
prostanoid formation, including cytosolic
Ca
-dependent (56, 57) and
-independent (58) phospholipases A
and
nonpancreatic type II phospholipase A
(59) . Only
with the cytosolic Ca
-dependent phospholipase A
is there evidence of a marked selectivity toward different fatty
acyl groups(56) . Thus, it is likely that, at least under some
conditions, PGHS-1 and PGHS-2 will be exposed to a substrate pool
composed of a mixture of different polyunsaturated fatty acids. The
overall goal of this study was to determine and compare the
efficiencies with which the two PGHS isozymes utilize the most common
6- and
3-polyunsaturated fatty acids as substrates and to
rationalize that information in terms of the potential oxygenation of
various fatty acid substrates by intact cells.
With
arachidonate, dihomo--linolenate, or eicosapentaenoate, all of
which are converted to prostanoids, PGHSs hold the substrates in an
L-shaped conformation in which a kink is present by virtue of rotation
about the C-9-C-10 bond(1, 49) . At the same, we
presume that the carboxylate group is anchored by an active-site
arginine. Hydrogen abstraction from the
8-allylic position occurs
with all these substrates. For linoleate, allylic hydrogen abstraction
can only occur from the
8-position. If the carboxylate group of
linoleate is anchored in the cyclooxygenase site by the active-site
arginine, then linoleate, which is a C
substrate, must be
in a linear conformation without a kink in order that the
8-position can neighbor the site of hydrogen abstraction in the
active site. The formation of 12-HOTrE from
-linolenate most
likely results from hydrogen abstraction from the
5-position.
This observation can be rationalized on the basis of binding of the
carboxylate group of
-linolenate to the active-site arginine
and the existence of a kink in the carbon chain similar to that
observed with C
substrates; the binding of
-linolenate in a kinked conformation within the cyclooxygenase
active site would position the
5-allylic hydrogen of
-linolenate near the site of hydrogen abstraction. The appropriate
positioning of allylic methylene groups has previously been used to
explain the positional specificities of lipoxygenases(68) .