©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Fatty Acid Substrate Specificities of Human Prostaglandin-endoperoxide H Synthase-1 and -2
FORMATION OF 12-HYDROXY-(9Z,13E/Z,15Z)-OCTADECATRIENOIC ACIDS FROM alpha-LINOLENIC ACID (*)

(Received for publication, May 3, 1995; and in revised form, June 1, 1995)

Odette Laneuville (1) Debra K. Breuer (1) Naxing Xu (1) Z. H. Huang (1) Douglas A. Gage (1) J. Throck Watson (1) Michel Lagarde (2) David L. DeWitt (1) William L. Smith (1)(§)

From the  (1)Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824 and the (2)Laboratoire de Chimie Biologique INSA-Lyon and Unité 352 INSERM, INSAL, B.406, 20 Avenue A. Einstein, 69621 Villeurbanne, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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, alpha-linolenic (Delta), -linolenic, and linoleic acids. Comparisons of k/Kvalues indicate that the order of efficiency of oxygenation is arachidonate > dihomo--linolenate > linoleate > alpha-linolenate for both isozymes; while the order of efficiency was the same for hPGHS-1 and hPGHS-2, alpha-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 alpha-linolenate were characterized. 13-Hydroxy-(9Z,11E)-octadecadienoic acid was found to be the main product formed from alpha-linoleate by both isozymes. The major products of oxygenation of alpha-linolenate were determined by mass spectrometry to be 12-hydroxy-(9Z,13E/Z,15Z)-octadecatrienoic acids. This result suggests that alpha-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.


INTRODUCTION

The biosynthesis of prostanoids depends upon the enzyme that catalyzes the committed step in the pathway: prostaglandin-endoperoxide H synthase (PGHS)(^1)(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(2) from arachidonic acid and (b) a peroxidase activity that catalyzes a 2-electron reduction of PGG(2) to PGH(2). The kinetic properties of the cyclooxygenase activities of the two isozymes are quite similar. PGHS-1 and PGHS-2 have similar V(max) and Kvalues 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 alpha-linolenic acid.


EXPERIMENTAL PROCEDURES

Materials

Arachidonic acid, eicosapentaenoic acid, dihomo--linolenic acid, linoleic acid, alpha-linolenic acid, -linolenic acid, 13-hydroxy-(9Z,11E,15Z)-octadecatrienoic acid (13-HOTrE), 13-HODE, (11R)-HETE, and 9-HODE were purchased from Cayman Chemical Co., Inc. [1-^14C]Linoleic acid (53 mCi/mmol), alpha-[1-^14C]linolenic acid (52 mCi/mmol), [1-^14C]arachidonic acid (40-60 mCi/mmol), and [1-^14C]eicosapentaenoic acid (40-60 mCi/mmol) were purchased from DuPont NEN. Hemoglobin, soybean lipoxygenase-1 (150,000 units/mg), aspirin, ricinoleic acid, and ricinelaidic acid were purchased from Sigma. Flurbiprofen was a gift from The Upjohn Co. Bis(trimethylsilyl)trifluoroacetamide (containing 1% trimethylchlorosilane) was from Regis Chemical Co. COS-1 cells were purchased from American Type Culture Collection (CRL-1650) and grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 8% calf serum, 2% fetal calf serum (Hyclone Laboratories), streptomycin (0.1 g/liter; Boehringer Mannheim), and penicillin G (0.1 g/liter; Sigma) in a water-saturated 5% CO(2) atmosphere at 37 °C. Other reagents were purchased from common commercial sources.

Expression of hPGHS-1 and hPGHS-2 cDNAs

The cDNAs coding for each of the human PGHS isozymes (1.8 kilobases) with unique SalI restriction sites in both the 5`- and 3`-untranslated sequences were subcloned in the expression vector pOSML as described earlier(37) . Transient transfections of COS-1 cells with pOSML-hPGHS-1 or pOSML-hPGHS-2 constructs were performed using the DEAE-dextran/chloroquine transfection method, also as reported previously(37) . Transfections were performed on COS-1 cells cultured in Dulbecco's modified Eagle's medium containing 8% calf serum and 2% fetal calf serum until near confluency. Approximately 16 h before transfections, the COS-1 cells were split 1:2. Forty hours following transfection, the cells were removed from the culture dishes by scraping them with a rubber policeman into 3 ml of phosphate-buffered saline. The cells were collected by centrifugation. Cells to be used for O(2) electrode assays of cyclooxygenase activities were resuspended in 0.1 M Tris-HCl, pH 7.4, and disrupted by sonication, and a microsomal membrane fraction was prepared. Membranes were isolated from cells transfected without DNA in an identical manner. For experiments involving characterization of products from radiolabeled fatty acids, the intact transfected cells (two culture dishes) were isolated and resuspended in 0.5 ml of Dulbecco's modified Eagle's medium and used directly.

Cyclooxygenase Activity

Cyclooxygenase activities were measured using microsomal membranes from sham-transfected COS-1 cells or COS-1 cells expressing either hPGHS-1 or hPGHS-2(37) . Microsomal membranes (5-10 mg of protein/ml of 0.1 M Tris-HCl, pH 7.4) were prepared and assayed on the same day. Cyclooxygenase assays were performed at 37 °C by monitoring the initial rate of O(2) uptake using an O(2) electrode (37) . Each assay mixture contained 3.0 ml of 0.1 M Tris-HCl pH 8.0, 1 mM phenol, 85 µg of hemoglobin, and 100 µM polyunsaturated fatty acid substrate. Reactions were initiated by adding 250 µg of microsomal protein in a volume of 50 µl to the assay mixture. K values for the different fatty acids were measured using concentrations of substrates between 2 and 100 µM. Inhibition of cyclooxygenase activity was measured by adding aliquots of microsomal suspensions of hPGHS-1 or hPGHS-2 to assay mixtures containing 100 µM fatty acid substrate and 10M flurbiprofen or by preincubating microsomes with 1 mM aspirin for 30 min at 37 °C prior to their assay.

Metabolism of Fatty Acids by Intact Transfected COS-1 Cells

[1-^14C]Linoleic acid or alpha-[1-^14C]linolenic acid (final concentration = 10 µM) was added to suspensions of transfected COS-1 cells, and the samples were incubated at 37 °C for 15 min. To terminate the reactions, the cells were centrifuged for 5 min at 1000 g, and the medium was removed. Residual protein from the medium was precipitated by adding 1 volume of ice-cold acetone. The supernatant containing the radioactive fatty acid metabolites and unreacted fatty acid was acidified with 1 volume of 0.1 M HCl and extracted with 6 volumes of chloroform. The organic phase was evaporated to dryness under a stream of N(2), resuspended in chloroform, and applied to a Silica Gel 60 thin-layer chromatography plate (VWR). The lipid products were separated by chromatography twice in diethyl ether/hexane/acetic acid (60:40:1, v/v/v)(51) . The thin-layer chromatography plates were exposed to x-ray film for 40 h, and autoradiographic bands corresponding to 9-HODE and 13-HODE were quantified by densitometry using a Visage 110 Image Analyzer. Radioactive bands were identified by comparison with authentic standards.

Preparation of 15-HPETE

15-HPETE was prepared according to Graff (52) with modifications. Soybean lipoxygenase-1 was diluted to 3000 units/ml in 0.1 M borate buffer, pH 9.0. The reaction was initiated by adding 0.8 µmol of arachidonic acid in a volume of 2.4 µl of ethanol to 1 ml of the diluted enzyme solution. The reaction was quenched by the addition of enough 1 M HCl (55 µl) to bring the pH to 3.0. The reaction products were extracted three times with 1 volume of ice-cold ether. The extracts were pooled and dried under a nitrogen stream. The products were resolved by thin-layer chromatography on Silica Gel G plates using hexane/ether/acetic acid (60:40:1, v/v/v) as the developing solvent at 4 °C. The region corresponding to 15-HPETE was scraped and extracted three times with diethyl ether and dried. The dried extracts were resuspended in ethanol. The concentration of 15-HPETE was determined by UV measurement at 235 nm ( = 3 10^4 mM cm). A typical yield using 3000 units of soybean lipoxygenase and 800 nmol of arachidonic acid was 300 nmol of 15-HPETE.

Analysis by Gas Chromatography-Mass Spectrometry (GC-MS)

Products from the reaction of hPGHS-2 and alpha-[1-^14C]linolenic acid were first separated by thin-layer chromatography using ether/hexane/acetic acid (60:40:1) as a solvent(51) . The main product was extracted with 2 ml of methanol and dried under a N(2) stream. The material was redissolved in 2 ml of ethyl acetate and washed with 1 ml of water twice. The ethyl acetate fraction was taken to dryness. Derivatization for GC-MS and GC-MS/MS was accomplished by reacting the hydroxy fatty acids (0.1-5 µg) with 10-50 µl of bis(trimethylsilyl)trifluoroacetamide (containing 1% trimethylchlorosilane) at 50 °C for 10 min; 1-2 µl of the resulting silylated mixture was analyzed by GC-MS or GC-MS/MS. To establish the position of hydroxylation, we prepared and analyzed the hydrogenated derivative of the PGHS-2 product of alpha-linoleate. The enzymatic reaction products were first methylated with diazomethane and then subjected to hydrogenation under mild conditions (hydrogen gas bubbled through a methanolic solution containing platium oxide catalyst). Following trimethylsilylation, the products were analyzed by GC/EI-MS, as indicated below.

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.


RESULTS

The initial rates of oxygenation of various C and C(18) polyunsaturated fatty acids (100 µM) by microsomes prepared from COS-1 cells expressing either hPGHS-1 or hPGHS-2 were determined using an O(2) assay (Fig. 1). The data are expressed relative to activity with arachidonate and are corrected for the fact that C(18) substrates incorporate 1 mol of O(2)/mol of fatty acid, whereas C substrates incorporate 2 (1.7-1.9) mol of O(2)/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(18) 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). (^2)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(2) 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(2) consumption was measured using an O(2) 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; alpha-18:3, alpha-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(max) 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 > alpha-linolenate. However, in comparing the apparent k/K values for the two isozymes for the different substrates, it became obvious that alpha-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(2) electrode measurements (Fig. 1). We subsequently incubated [1-^14C]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(2), PGD(2), and 17-hydroxy-(5Z,8Z,10E)-heptadecatrienoic acid standards. COS-1 cells expressing hPGHS-1 converted 3.4 ± 0.1% of the [1-^14C]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-^14C]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-^14C]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-^14C]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(2) 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-^14C]linoleic acid or alpha-[1-^14C]linolenic acid incubated with COS-1 cells expressing either hPGHS-1 or hPGHS-2. [1-^14C]linoleic acid (10 µM) (A) or alpha-[1-^14C]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 alpha-[1-^14C]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(3)] 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 alpha-cleavage and elimination of heptadienal, C(6)H(9)CHO (Fig. 5). A weak but reproducible alpha-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 alpha-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. (^3)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 alpha-linolenic acid. alpha-[1-^14C]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 alpha-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 Delta-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 alpha-cleavage precursor m/z 225 through the loss of C(5)H from the terminal part of the chain (Fig. 6B). These results indicate that the Delta-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 alpha-cleavage fragments of selected TMS ether-TMS ester derivatives. A, MS/MS spectrum of the precursor ion at m/z 183 formed by alpha-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 alpha-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 alpha-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 alpha-linolenic acid, but to a lesser extent than hPGHS-2; only 10.9% of the initial alpha-[^14C]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.


DISCUSSION

Prostanoid synthesis is believed to require the mobilization of fatty acids from lipid precursors via one or more phospholipases A(2)(25) . Three different phospholipases A(2) have been identified as being involved in different situations in prostanoid formation, including cytosolic Ca-dependent (56, 57) and -independent (58) phospholipases A(2) and nonpancreatic type II phospholipase A(2)(59) . Only with the cytosolic Ca-dependent phospholipase A(2) 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.

Arachidonic Acid

It is well documented that PGHS-1 can utilize arachidonate as a substrate in vivo as the major prostanoids present in plasma are of the 2-series(60) . This present study as well as previous investigations establish that PGHS-1 and PGHS-2 use arachidonate with comparable efficiencies(35, 36, 37, 61) ; in fact, the turnover numbers of purified hPGHS-1 and hPGHS-2 with arachidonate are about the same(36) .

Dihomo--linolenic Acid

The major prostanoids in seminal fluid are of the 1-series(60) , indicating that dihomo--linolenic acid can also serve as a PGHS substrate in vivo. Moreover, in sheep vesicular gland, the major C fatty acid is dihomo--linolenic acid, not arachidonic acid(62) , and this tissue has the highest known concentration of PGHS-1(1, 2, 3) . Pioneering work on prostanoid biosynthesis was performed using seminal vesicles as a source of enzyme and dihomo--linolenic acid as the prostanoid precursor(49, 50) . Our present results indicate that hPGHS-2 can use dihomo--linolenic acid with an efficiency even slightly greater than PGHS-1. It should also be noted that these results are consistent with those of earlier investigations indicating that the K for dihomo--linolenate is somewhat higher than that for arachidonate(63, 64) .

Eicosapentaenoic Acid

When added exogenously to either human platelets or purified PGHS-1, eicosapentaenoic acid is converted, albeit in relatively small amounts, to prostanoid metabolites(48, 65) . Similar results were obtained in our studies with COS-1 cells expressing PGHS-1 and with microsomal preparations from these cells. A small enhancement in product formation was observed upon the addition of 15-HPETE, consistent with findings that oxygenation of eicosapentaenoic acid by PGHS-1 is more sensitive than arachidonate oxygenation to hydroperoxide concentrations(48, 65) . The rates of oxygenation of eicosapentaenoic acid were at least 3 times greater with intact cell and microsomal preparations of PGHS-2 than with comparable preparations of PGHS-1. However, in contrast to the results observed with COS-1 cells expressing PGHS-1, 15-HPETE did not increase the oxygenation of eicosapentaenoic acid by COS-1 cells expressing PGHS-2; in fact, a small decrease was observed. Although eicosapentaenoic acid appears to be a better substrate for PGHS-2 than PGHS-1, it is not obvious from our studies or earlier results that this fatty acid would be used as a substrate in vivo. It should be noted, however, that eicosapentaenoic acid does compete effectively with arachidonate for binding to PGHS-1 (46, 48) and thus may be a potent inhibitor of the oxygenation of arachidonate in intact cells.

Linoleic Acid

Although linoleate is used less efficiently by PGHS-1 and PGHS-2 than either arachidonate or dihomo--linolenate, the kinetic constants obtained with this C(18) substrate do overlap with those of the 6-C substrates, suggesting that linoleic could be a substrate for both PGHS-1 and PGHS-2 in vivo. Indeed, thrombin treatment of human umbilical vein endothelial cells releases linoleate from endogenous lipids, 9- and 13-HODE formation occurs in conjunction with the linoleate release, and synthesis of 9- and 13-HODEs by endothelial cells is attenuated by cyclooxygenase inhibitors(66) .

alpha-Linolenic Acid

alpha-Linolenate is an exceedingly poor substrate for PGHS-1, with an apparent k/K value that is 0.2% of that obtained with arachidonate (Table 1). In contrast, with PGHS-2, the k/K value for alpha-linolenate is 8% of that for arachidonate and 53% of the value for alpha-linoleate. These results lead us to speculate that alpha-linolenate could serve as a substrate for PGHS-2 but not PGHS-1 in vivo. The major products formed from alpha-linolenate are 12-hydroxy-(9Z,13E/Z,15Z)-octadecatrienoic acids. The major product formed upon incubation of alpha-linolenate acid with 15-lipoxygenase is 13-HODTrE(67) . Thus, alpha-linolenate may be used to discriminate between cyclooxygenase and 15-lipoxygenase pathways.

Fatty Acid Substrates and the Cyclooxygenase Active Site

Analysis of the crystal structure of the ovine PGHS-1bulletflurbiprofen complex suggests (a) that the cyclooxygenase active site is in the form of a hydrophobic pocket within the core of the enzyme and (b) that a conserved active-site arginine residue (Arg-120 of ovine PGHS-1) near the open end of this pocket serves as the anchoring counterion for the carboxylate group of polyunsaturated fatty acid substrates(10) . Recent studies in our laboratory of substitutions of Arg-120 in ovine PGHS-1 by site-directed mutagenesis are consistent with this latter hypothesis. (^4)As discussed in the Introduction, the properties of PGHS-1 and PGHS-2 are quite similar, suggesting that the cyclooxygenase active sites of the two isozymes have homologous structures.

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(18) 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 alpha-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 alpha-linolenate to the active-site arginine^2 and the existence of a kink in the carbon chain similar to that observed with C substrates; the binding of alpha-linolenate in a kinked conformation within the cyclooxygenase active site would position the 5-allylic hydrogen of alpha-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) .


FOOTNOTES

*
This work was supported by National Institutes of Health Grants DK42509 and DK22042 (to W. L. S.) and Grant GM40713 (to D. L. D.). Mass spectrometry performed in the Michigan State University Mass Spectrometry Facility was funded in part by Grant RR-00480-25 from the Biotechnology Research Technology Program of the National Center for Research Resources of the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, 513 Biochemistry Bldg., Michigan State University, East Lansing, MI 48824. Tel.: 517-353-8680; Fax: 517-353-9334; smithww{at}pilot.msu.edu.

(^1)
The abbreviations used are: PGHS, prostaglandin-endoperoxide H synthase; hPGHS, human PGHS; PG, prostaglandin; 12-HOTrE, 12-hydroxy-(9Z,13E/Z,15Z)-octadecatrienoic acid; 13-HOTrE, 13-hydroxy-(9Z,11E,15Z)-octadecatrienoic acid; 9-HODE, 9-hydroxy-(10E, 12Z)-octadecadienoic acid; 13-HODE, 13-hydroxy-(9Z,11E)-octadecadienoic acid; (11R)-HETE, (11R)-hydroxy-(5Z,8Z,12E,14Z)-eicosatetraenoic acid; 15-HPETE, (15S)-hydroperoxy-(5Z,8Z,11Z,13E)-eicosatetraenoic acid; GC-MS, gas chromatography-mass spectrometry; MS/MS, tandem mass spectrometry; EI-MS, electron impact mass spectrometry; TMS, trimethylsilyl.

(^2)
Treatment of microsomes or whole cells containing human PGHS-2 with 500 µM aspirin for 30 min at 37 °C consistently converts PGHS-2 to a form that catalyzes the formation of 15-HETE from arachidonate. Higher aspirin concentrations and/or longer treatments lead to complete inactivation of the cyclooxygenase activity of PGHS-2. Under the conditions employed in the experiments, no cyclooxygenase activity was observed with PGHS-2 treated with 1 mM aspirin for 30 min.

(^3)
Diagnostic ions were observed at m/z 355 (M - 31), 301 ((M - 85), loss of (CH(2))(5)CH(3)), 272 (loss of CH(3)(CH(2))(5)CHO followed by a rearrangement of the trimethylsilyl to the carbomethoxy group) (69), and 187 ((M - 199), loss of (CH(2))COOCH(3)).

(^4)
An Arg-120 Asn mutant of PGHS-1 has a 1000-fold higher K for arachidonate than native PGHS-1 and does not use alpha-linolenate as a substrate (D. Bhattacharyya, M. Lecomte, and W. L. Smith, unpublished results).


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