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J. Biol. Chem., Vol. 275, Issue 29, 21844-21849, July 21, 2000
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From the
Received for publication, March 2, 2000, and in revised form, April 24, 2000
A novel cytochrome P450, CYP4F8, was recently
cloned from human seminal vesicles. CYP4F8 was expressed in yeast.
Recombinant CYP4F8 oxygenated arachidonic acid to
(18R)-hydroxyarachidonate, whereas prostaglandin (PG)
D2, PGE1, PGE2,
PGF2 Human and primate seminal fluid contains conspicuous amounts of
prostaglandin (PG)1
E1, PGE2,
(19R)-hydroxy-PGE1, and
(19R)-hydroxy-PGE2 (1-4). PGs are formed by PGH
synthase of seminal vesicles, but the mechanism of biosynthesis of
(19R)-hydroxy-PGE has not been resolved. Many properties of
a tentative PG 19-hydroxylase can be deduced from analysis of PGE
compounds in semen. First, (19R)-hydroxy-PGE1 and (19R)-hydroxy-PGE2 are equally abundant.
18-Hydroxy-PGE and small amounts of It seems likely that 19-hydroxylation of PGs is catalyzed by cytochrome
P450 of seminal vesicles (4), but the enzyme has not been convincingly
demonstrated. Microsomes of primate and human seminal vesicles and
NADPH only slowly metabolized PGE2 to
19-hydroxy-PGE2, which differed from the biosynthesis
in vivo (1-4). In contrast, microsomes of monkey seminal
vesicles and NADPH rapidly metabolized arachidonic acid to
(18R)-HETE (10). This left us with an enigmatic microsomal
preparation, which rapidly catalyzed 18-hydroxylation of arachidonate
and only slowly catalyzed 19-hydroxylation of PGE2.
What is the biological function of seminal PGs in humans? Little can be
stated with absolute certainty. Seminal fluid contains an unprecedented
high concentration of PGs. Exposure of sperm to PGs is not required for
in vitro fertilization (11). It therefore seems likely that
these PGs contribute to fertility by ensuring maximum efficiency
in vivo, but the mechanism is unknown. PGs have a wide
spectrum of biological effects mediated through activation of
G-protein-coupled prostanoid receptors (12). Seminal PGE compounds may
have immunosuppressive actions in the female genital tract, induce
tolerance to sperm antigens, promote sperm survival, and contribute to
the acrosome reaction (3, 13, 14). Targeted disruption of PGH synthase
and prostanoid receptor genes has shown that PGs are of physiological
importance in rodent reproduction (12, 15, 16).
Knowledge of the biosynthesis of (19R)-hydroxy-PGE in male
genital organs might provide tools to study their role in reproduction. In pursuit of the tentative PG 19-hydroxylase, we recently performed a
systematic study of cytochrome P450 mRNA of human seminal vesicles (17). mRNA of a novel enzyme, CYP4F8, appeared to be abundantly expressed in all samples examined. The objective of the present study
was to identify the function of this novel human cytochrome P450.
Materials--
Arachidonic acid (99%), linoleic acid (99%),
bis(trimethylsilyl)trifluoroacetamide, and
(2S)-(+)-phenylpropionic acid (97%) were from Sigma.
(19R)-Hydroxy-PGE2, PGH1, and
PGH2 were obtained from Cayman Chemical Co., Inc. (Ann
Arbor, MI). PGH2 was also prepared as described (18),
stored in acetone at Expression of CYP4F8--
The coding region of CYP4F8 was
amplified with a sense primer
(5'-TTGGGATCCAAAATGTCGCTGCTGAGCCTGTCTTG-3') that contained a BamHI linker sequence, three A residues to increase the
expression efficiency (21), and 23 base pairs of the translation start site of CYP4F8 cDNA. The antisense primer
(5'-CTGGAATTCTCAGCCCAGGGGTTCTACTCGC-3') contained 19 base pairs of the
end of the coding sequence, the stop codon, and an EcoRI
linker sequence. Polymerase chain reaction was performed with
Pfu polymerase using a full-length cDNA clone of CYP4F8
(17) as template with an annealing temperature of 60 °C. The
polymerase chain reaction product was subcloned into the V60 yeast
expression vector at the BamHI and EcoRI sites. The expression of CYP4F8 was carried out with the S. cerevisiae strain W(R), which has been genetically modified to
also overexpress the yeast reductase (20). A galactose-inducible
promoter in the plasmid and in the yeast genome, respectively, was used
to control the expression. After transformation of the plasmid into the
W(R) yeast strain by a lithium acetate method, selection of clones was
achieved by growing the yeast in adenine- and uracil-deficient medium.
To achieve higher expression levels, the yeast cells were first grown
to high density with glucose as the main energy source; thereafter,
galactose was added to induce expression. In brief, the procedure was
as follows. Transformed yeast cells were grown to a density of
~30 × 106 cells/ml in SGI medium (containing per
liter: casamino acids 1 g, yeast nitrogen base 7 g, glucose 20 g,
tryptophan 20 mg). The cells were then diluted to ~2.5 × 106 cells/ml and grown for 22-24 h in YPGE medium
(containing per liter: yeast extract 10 g, bactopeptone 10 g, glucose 5 g, ethanol 16 g). Galactose (2%) was added, and the cells were
harvested 16 h later by centrifugation (22). The cell walls were
disrupted with glass beads, and the microsomal fraction was obtained by differential centrifugations at +4 °C (20,000 × g
for 10 min and 100,000 × g for 60 min). The pelleted
microsomes were homogenized in 0.05 M Tris-HCl, 20%
glycerol, and 1 mM EDTA (pH 7.4) and stored at Experimentation--
Yeast microsomes (50 µg, ~2 pmol of
CYP4F8) or microsomes of human seminal vesicles (1-2 mg/ml) were
incubated with 1 mM NADPH and 5-200 µM
substrate in a total volume of 100 µl of 0.1 M
KHPO4 and 2 mM EDTA (pH 7.4) for 45 s to
30 min at 37 °C. For GC-MS analysis, 25 pmol of CYP4F8 were used.
Substrates were added in <1 µl of solvent. The reactions were
terminated with 4 volumes of ethanol. Incubations with PGH2
were also terminated by addition of buffered SnCl2 (18).
The metabolites were extracted on a Sep-Pak C18 column with
~90% recovery (24).
Kinetics--
CYP4F8 (3 pmol in 0.3 ml) was preincubated with 5, 10, 50, and 200 µM U-44069 in triplicate for 2 min at
37 °C. NADPH (1 mM) was added, and the reaction was
stopped after 45 s. The biosynthesis of hydroxy metabolites of
U-44069 was quantified by LC-MS analysis using selective ion monitoring
of the carboxylate anion (m/z 365). 19-Hydroxy-PGB2 was added as an internal standard in some
experiments. Standard curves were constructed from known amounts of
U-44069 (selective ion monitoring of the carboxylate anion at
m/z 349). We assumed that U-44069 and its hydroxy
metabolites yielded the same response, as the increase in the hydroxy
metabolites corresponded to the decrease in U-44069.
Vmax and Km were calculated according to the Lineweaver-Burk plot. The rate of biosynthesis of 19- and 18-hydroxy metabolites of endoperoxide analogues, PGD2, PGE1, PGE2, PGF2 LC-MS Analysis--
Equipment for LC-MS analysis was as
described (24). The column contained octadecasilane silica (5 µm,
250 × 2 mm; Chromasil 5 C18 100 A, Phenomenex Inc.,
Torrance, CA) and was eluted at 0.2 ml/min. The mobile phase was
CH3OH/H2O/acetic acid (80:20:0.01) for analysis
of monohydroxy metabolites of arachidonic acid. Hydroxy-PGs and PGs
were analyzed by three different systems: a system with a linear
gradient from CH3OH/H2O/acetic acid
(60:40:0.01) to 100% methanol in 23 min and two isocratic systems
(CH3OH/H2O/acetic acid (60:40:0.01) and
CH3CN/H2O/acetic acid (35:65:0.01)). The effluent first passed a UV detector and was then subjected to negative
ion electrospray ionization in an ion trap mass spectrometer (LCQ,
ThermoQuest, San Jose, CA). The source voltage was 2.4 kV; the
capillary temperature was 230 °C; and the collision energy was set
to 30%. PGF1 GC-MS Analysis--
Methyl esters and trimethylsilyl ethers were
prepared as described (10). PGE compounds were dehydrated to PGB
compounds with 0.25 M KOH for 20 min (5). A gas
chromatograph (Varian 3100) with a nonpolar capillary column (30 m;
DB-5, J & W Scientific; film, 0.25 µm; diameter, 0.25 mm) was
connected to an ion trap mass spectrometer (ITS40, Finnigan MAT). The
gas chromatograph was programmed from 120 to 270 °C in 5 min and
from 270 to 294 °C in 8 min and then kept at 294 °C. C-values
(number of apparent carbons) were determined from the retention times
of fatty acid methyl esters (10, 25). Steric analysis of 18-HETE methyl esters was performed after hydrogenation by analysis of
(2S)-phenylpropionic acid derivatives of methyl
18-hydroxyeicosanoates (10).
Expression of CYP4F8 in Yeast--
The yield of CYP4F8 was 26-62
pmol/mg of microsomal proteins, and the yield of cytochrome P450
reductase was >10-fold higher (430-850 pmol/mg). The reduced
CYP4F8·CO complex had an absorption maximum at 449.6 nm. Control
microsomes did not contain appreciable amounts of cytochrome P450 and
did not metabolize any of the substrates discussed below.
Hydroxylation of Fatty Acids--
Recombinant CYP4F8 and NADPH
metabolized arachidonic acid to one major metabolite (Fig.
1A). This metabolite was
identified as 18-HETE by LC-MS analysis. The MS/MS analysis of the
carboxylate anion (m/z 319 Hydroxylation of PGs and Leukotriene
B4--
Recombinant CYP4F8 was incubated with
PGD2, PGE1, PGE2,
PGF2 Hydroxylation of PGH2 Analogues--
CYP4F8 was found
by LC-MS to metabolize U-44069 to two hydroxy metabolites at a ratio of
~6:1 (Fig. 2A). LC-MS
analysis was consistent with 19- and 18-hydroxy metabolites. GC-MS
analysis of the trimethylsilyl ether methyl ester derivative of the
main metabolite showed a mass spectrum at a C-value of 25.1 with a strong signal at m/z 117 (H3C-CH-O+-Si(CH3)3).
The C-value of the trimethylsilyl ether methyl ester derivative of
U-44069 was 22.8. The difference in the C-values of the metabolite and
the parent compound and the mass spectrum of the metabolite suggested
that it was 19-hydroxy-(9,11-epoxymethano)-PGH2 (25). The
minor metabolite showed a mass spectrum with a strong signal at
m/z 131 (H3C-CH2-CH-O+-Si(CH3)3)
at a C-value of 24.9 and was identified as
18-hydroxy-(9,11-epoxymethano)-PGH2 (25).
The hydroxylation of U-44069 at 37 °C was linear with time for at
least 6 min and linear with protein (0.1-1.6 mg/ml). Cytochrome b5 was without effect. Km and
Vmax values were estimated by a Lineweaver-Burk
plot. The apparent Km was ~7 µM, and
the Vmax ~260 pmol
min
CYP4F8 also efficiently metabolized U-46619 and U-51605 (Table
I). LC-MS analysis suggested that the main products of U-46619 were 19- and 18-hydroxy metabolites (Table II).
The structures were confirmed by GC-MS analysis (trimethylsilyl ether
methyl ester derivatives). The mass spectrum of the main metabolite
showed a characteristic signal at m/z 117 and had a C-value
of 25.6. The mass spectrum of the other metabolite showed a
characteristic signal at m/z 131 and had a slightly smaller
C-value of 25.4, whereas the C-value of U-46619 was 23.3. The two
metabolites were thus identified as
19-hydroxy-(11,9-epoxymethano)-PGH2 and
18-hydroxy-(11,9-epoxymethano)-PGH2 (25). The latter was
partially separated by HPLC into two equal components with identical
MS/MS spectra, conceivably stereoisomers at C-18, but this was not
further investigated. In U-51605, the endoperoxide oxygens are replaced
by a -N=N- group, and the hydroxy group at C-15 has been deleted. LC-MS
with triple MS (m/z 347 Hydroxylation of PGH1 and
PGH2--
Recombinant CYP4F8 was incubated with 10 µM PGH2 and 1 mM NADPH at
37 °C for 10 min, and the products were analyzed by LC-MS (Fig.
3). Under these conditions,
PGH2 will decompose in buffer to PGD2,
PGE2, and PGF2
The structure of the main hydroxy metabolite of PGH2 was
confirmed by GC-MS analysis. PGE2 compounds that were
formed during incubation with PGH2 and CYP4F8 were
converted to PGB2 compounds by alkali treatment. A polar
PGB2 metabolite was purified by HPLC and analyzed by GC-MS
(trimethylsilyl ether methyl ester derivative). The mass spectrum and
the C-value of 26.1 was identical to those reported for
19-hydroxy-PGB2 (4, 5).
An experiment with chemical reduction of products confirmed that
PGH2 was metabolized to 19-hydroxy-PGH2.
PGH2 was incubated with CYP4F8 for 2 min, and one-half of
the incubation was terminated with buffered SnCl2, whereas
the other half was terminated with ethanol. LC-MS analysis of the
sample reduced with SnCl2 showed two major peaks (Fig.
4A).
19-Hydroxy-PGF2
PGH1 was metabolized by CYP4F8 in the same way as
PGH2. The main metabolites decomposed in buffer to
19-hydroxy-PGE1 and 18-hydroxy-PGE1 (Table
II).
As shown in Table I, U-44069 and U-51605 appeared to be the best
substrates of CYP4F8. PGH2 was metabolized at a lower rate. However, PGH2 is unstable, and our in vitro
conditions may not mimic in vivo biosynthesis. It is
noteworthy that PGH2 and the stable endoperoxide analogues
were metabolized more efficiently than PGE2,
PGF2 Human Seminal Fluid--
18-Hydroxy-PGE compounds in pooled
seminal fluid (n = 12) were analyzed by LC-MS.
18-Hydroxy-PGE compounds were present in slightly lower relative
amounts in seminal fluid compared with those formed by hydroxylation of
PGH1 and PGH2 by CYP4F8 in vitro (Table
II). 18-HETE could not be detected in these samples by LC-MS.
Human Seminal Vesicles--
Microsomes of seminal vesicles
(n = 3) and NADPH metabolized U-44069 to 19- and
18-hydroxy metabolites at a ratio of ~6:1 as judged by LC-MS analysis
(Fig. 5A). U-44069, U-46619,
and U-51605 were metabolized to 19- and 18-hydroxy metabolites in the
same relative amounts as by CYP4F8 (Table II). To determine whether PGH2 was metabolized to 19-hydroxy-PGH2 by
microsomes of seminal vesicles, we repeated the experiments described
above with SnCl2. Treatment with buffered SnCl2
yielded significant formation of 19-hydroxy-PGF2
Arachidonic acid was metabolized to 18-HETE by CYP4F8 as described
above, and we confirmed that microsomes of human seminal vesicles and
NADPH also formed this metabolite (Fig. 5C). Small amounts
of 15-HETE (retention time of 23 min) and 11-HETE (retention time of 25 min) were also identified by LC-MS, but these HETEs can also be formed
by PGH synthase of seminal vesicles (27).
mRNA of a new enzyme, CYP4F8, was recently discovered in human
seminal vesicles (17). We have expressed CYP4F8 in yeast and report as
our main finding that recombinant CYP4F8 metabolizes PGH1
and PGH2 to 19-hydroxy-PGH1 and
19-hydroxy-PGH2, respectively. Three stable
PGH2 analogues were also substrates, whereas
PGD2, PGE1, PGE2, and
PGF2 9,11-Epoxymethano-PGH2 (U-44069) was the best substrate of
recombinant CYP4F8. U-44069 was converted to 19- and 18-hydroxy metabolites at a ratio of ~6:1, with a Km of ~7
µM and a Vmax of ~260 pmol
min To address the question of whether CYP4F8 is present in human seminal
vesicles, we compared the catalytic properties of microsomes and
recombinant CYP4F8. Both metabolized U-44069, U-46619, U-51605, and
arachidonic acid to virtually the same profile of 19- and 18-hydroxy
metabolites (Table II). Both preparations also oxygenated PGH2 to 19-hydroxy metabolites and to small amounts of
18-hydroxy metabolites. These observations suggest that CYP4F8 is
present in seminal vesicles. Human and primate semen contains >98% of the 19R-stereoisomers of 19-hydroxy-PGs (1-4). It remains
to be determined whether CYP4F8 oxidizes PGH2 to the
19R-stereoisomer of 19-hydroxy-PGH2. However, we
confirmed that CYP4F8 metabolizes arachidonate to >95% of the
18R-stereoisomer of 18-HETE, which is the stereoisomer
formed by microsomes of primate seminal vesicles (10).
PGH synthase-1 is an integral membrane protein with its catalytic
domains on the luminal side of the endoplasmic reticulum (28). The
substrate channel of PGH synthase-1 faces the endoplasmic reticulum.
Its entrance is surrounded by the membrane-binding surface, which does
not extend beyond one leaflet of the lipid bilayer. Human cytochromes
P450 are also integral membrane proteins, but their catalytic domains
are located on the cytosolic side of the endoplasmic reticulum (29).
PGH2 must cross the lipid bilayer to gain access to CYP4F8.
It is known that an important PGE synthase is also associated with the
same membrane system (30). PGE synthase might efficiently metabolize
PGH compounds, as non-enzymatic breakdown products, e.g. PGD
compounds, have not been detected in human seminal fluid. The rapid
biosynthesis of 19-hydroxy-PGE1 and
19-hydroxy-PGE2 in vivo suggests that PGH synthase, PGH 19-hydroxylase, and PGE synthase are closely linked (1,
2, 4). A proposed mechanism for biosynthesis of
(19R)-hydroxy-PGE2 in human seminal vesicles is
shown in Fig. 6.
There is a marked inter-individual variation in the amount of PGE and
19-hydroxy-PGE compounds in normal human seminal fluid (8, 9). This may
be due to variable expression of CYP4F8 or due to isoforms with reduced
enzyme activity. CYP4F8 and CYP4F3 are located on the CYP4F cluster on
chromosome 19p13.1 (17). CYP4F3 catalyzes Two thiolate hemoproteins, thromboxane synthase (CYP5A) of platelets
and prostacyclin synthase (CYP8A) of the vascular endothelium, can
rearrange PGH2 into thromboxane A2 and
prostacyclin, respectively (33, 34). These enzymes do not require NADPH
and differ from CYP4F8 in this respect. It is possible that
PGH2 might be a substrate of other cytochromes P450.
Recombinant CYP4A11 and microsomes from human kidneys hydroxylate
U-44069 at the In summary, our results suggest that CYP4F8 of human seminal vesicles
catalyzes We thank Drs. D. Pompon and P. Urban for the
generous gift of the S. cerevisiae W(R) strain and the
pYeDP60 expression vector.
*
This work was supported by Swedish Medical Research Council
Grant 06523, the Swedish Society for Medical Research, and the Swedish
Pharmaceutical Society.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be 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 Pharmaceutical
Biosciences, Uppsala Biomedical Centre, P. O. Box 591, Husargatan 3 (Entrance C 2), SE-751 24 Uppsala, Sweden. Tel.: 46-18-471-41-48; Fax:
46-18-55-29-36; E-mail: Johan.Bylund@farmbio.uu.se.
Published, JBC Papers in Press, May 1, 2000, DOI 10.1074/jbc.M001712200
2
J. Bylund and E. H. Oliw, unpublished observations.
The abbreviations used are:
PG, prostaglandin;
HETE, hydroxy-eicosatetraenoic acid;
GC-MS, gas chromatography-mass
spectrometry;
LC-MS, liquid chromatography-mass spectrometry;
MS/MS, tandem mass spectrometry;
HPLC, high pressure liquid
chromatography.
Identification of CYP4F8 in Human Seminal Vesicles as a
Prominent 19-Hydroxylase of Prostaglandin Endoperoxides*
§,
Division of Biochemical Pharmacology,
Department of Pharmaceutical Biosciences, Uppsala Biomedical Centre,
Uppsala University, SE-751 24 Uppsala, Sweden and the ¶ Division
of Molecular Toxicology, Institute of Environmental Medicine,
Karolinska Institute, SE-171 77 Stockholm, Sweden
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and leukotriene B4 appeared to be poor
substrates. Three stable PGH2 analogues, 9,11-epoxymethano-PGH2 (U-44069),
11,9-epoxymethano-PGH2 (U-46619), and
9,11-diazo-15-deoxy-PGH2 (U-51605) were rapidly metabolized by 
2- and 3-hydroxylation. U-44069 was oxygenated with a
Vmax of ~260 pmol
min
1 pmol P4501 and
a Km of ~7 µM. PGH2
decomposes mainly to PGE2 in buffer and to
PGF2 by reduction with SnCl2. CYP4F8
metabolized PGH2 to 19-hydroxy-PGH2, which
decomposed to 19-hydroxy-PGE2 in buffer and could be
reduced to 19-hydroxy-PGF2 with SnCl2. 18-Hydroxy metabolites were also formed (~17%). PGH1 was
metabolized to 19- and 18-hydroxy-PGH1 in the same way.
Microsomes of human seminal vesicles oxygenated arachidonate, U-44069,
U-46619, U-51605, and PGH2, similar to CYP4F8.
(19R)-Hydroxy-PGE1 and
(19R)-hydroxy-PGE2 are the main prostaglandins
of human seminal fluid. We propose that they are formed by
CYP4F8-catalyzed 2-hydroxylation of PGH1 and
PGH2 in the seminal vesicles and isomerization to
(19R)-hydroxy-PGE by PGE synthase. CYP4F8 is the first
described hydroxylase with specificity and catalytic competence for
prostaglandin endoperoxides.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
18- and
19-PGE compounds are also present (5-7). The PG
19-hydroxylase will thus be expected to oxygenate C-19 and C-18,
whereas the 18- and 19-PGE compounds
might be formed by typical cytochrome P450-catalyzed desaturations
(5-8). Second, semen analysis suggests that PG 19-hydroxylase must be
closely linked to PGH synthase. The ratios of PGE1 to
(19R)-hydroxy-PGE1 and PGE2 to
(19R)-hydroxy-PGE2 change little in ejaculates
obtained at long or short time intervals (4). The ratios are
reproducible in each subject, but vary considerably between normal men
(4, 8-9). A majority can be defined as "rapid" hydroxylators with
PGE/(19R)-hydroxy-PGE ratios below 0.4 (8).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C, and checked for integrity by analysis
of products formed in buffer and by reduction with buffered
SnCl2 (18).
(15S)-Hydroxy-(9
,11-epoxymethano)prosta-(5Z,13E)-dienoic acid (U-44069),
(15S)-hydroxy-(11,9-epoxymethano)prosta-(5Z,13E)-dienoic acid (U-46619),
(9,11-diazo)prosta-(5Z,13E)-dienoic acid
(U-51605), and other PGs were from The Upjohn Co. (18S)-HETE
and (18R)-HETE were a generous gift of Dr. J. R. Falck
(Texas Southwestern Medical Center, Dallas, TX).
[1-14C]Arachidonic acid (56 Ci/mol), dNTPs, and T4 DNA
ligase were from Amersham Pharmacia Biotech (Solna, Sweden).
Pfu DNA polymerase was from Promega (Madison, WI), and
restriction enzymes were from New England Biolabs Inc. (Beverly, MA).
Cytochrome b5 was prepared as described (19).
Oligonucleotides were from Life Technologies, Inc. Human seminal
vesicles (n = 3) and seminal samples (n = 12) were obtained from the Karolinska Hospital (Stockholm, Sweden). Microsomes of human seminal vesicles were prepared as described (4).
The plasmid V60 (pYeDP60) and the Saccharomyces cerevisiae W(R) strain, which overexpresses the yeast NADPH-cytochrome P450 reductase under a galactose-inducible promoter, were kind gifts of Drs.
D. Pompon and P. Urban (Center de Génétique
Moléculaire, CNRS, Gif-sur-Yvette, France) (20).
80 °C.
Cytochrome P450 and cytochrome P450 reductase were measured as
described (22). Control microsomes were prepared from yeast transfected
with the V60 plasmid without an insert. Protein was determined as
described (22, 23).
, and
PGH2 by CYP4F8 (2 pmol; 1-5 min at 37 °C) was estimated
at a fixed substrate concentration (10 µM). Metabolite
formation was estimated by LC-MS using standard curves of parent
compounds or by percent conversion of substrate.
was used for tuning. Products were assayed by selective ion monitoring of the carboxylate anions and identified by
MS/MS analysis. These mass parameters were adjusted to the mass of the
carboxylate anions of the various compounds as they eluted from the
HPLC column.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
full scan) yielded a mass
spectrum that was identical to that of authentic 18-HETE.
Characteristic signals were noted at m/z 261 (319-58, loss
of CH3CH2CHO) and 217 (261-44, loss of CO2) (24). Steric analysis by GC-MS showed that the
metabolite consisted of >95% of the 18R-stereoisomer
(Fig. 1B). The mass spectra, which were obtained at
C-values of ~29.0 and ~29.5 for the S- and
R-stereoisomers, respectively, were identical to previous data (10). CYP4F8 was also incubated with linoleic acid. LC-MS analysis
(MS/MS 295 full scan) showed that the major metabolite had the same
mass spectrum as that of 16-hydroxylinoleic acid (24). Arachidonic and
linoleic acids were thus oxygenated to the very same metabolites
formed by monkey seminal vesicle microsomes (10, 26).
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Fig. 1.
Analysis of 18-HETE formed from arachidonic
acid by CYP4F8. A, LC-MS analysis with selective ion
monitoring of two ions, m/z 319 for HETEs and epoxides of
arachidonic acid and m/z 337 for dihydroxyeicosatrienoic
acids. The trace shows the combined ion intensities. The
main peak (marked 18-HETE) was identified by MS/MS (MS/MS
319 full scan). The mobile phase was MeOH/H2O/acetic
acid (80:20:0.01). B, steric analysis of 18-HETE methyl
ester by GC-MS after hydrogenation as the
(2S)-phenylpropionic acid derivative (selective ion
monitoring at m/z 325) (10).
, and leukotriene B4, and the formation
of hydroxylated metabolites was analyzed by LC-MS with MS/MS. These
compounds were all poor substrates, and metabolites could not be
identified in many experiments (Table I).
However, using a high substrate concentration (100 µM)
and long incubation times, small amounts of 19-hydroxy metabolites of
PGE2 and PGF2 could be identified by LC-MS
analysis and by comparison with authentic standards. Cytochrome
b5 at the same concentration as CYP4F8 did not
augment the oxygenation of PGE1 and PGE2.
Hydroxylation of different substrates by CYP4F8
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Fig. 2.
LC-MS analysis of hydroxy metabolites of
U-44069 formed by recombinant CYP4F8. A, the
metabolites were analyzed by selective ion monitoring of the
carboxylate anions at m/z 365, and the ion intensity is
shown. The mobile phase was CH3OH/H2O/acetic
acid (60:40:0.01). The first eluting peak (marked
19-OH-U-44069) was found by both LC-MS and GC-MS to contain
the 19-hydroxy metabolite of 9,11-epoxymethano-PGH2, and
the second peak (marked 18-OH-U-44069) was found to contain
the 18-hydroxy metabolite. B, shown are the results of the
biosynthesis of hydroxy metabolites of U-44069 at different substrate
concentrations. Data are the means ± S.D. of triplicate
determinations. The inset shows a Lineweaver-Burk plot of
1/[S (µM)] versus 1/[V (pmol
min 1 pmol
P4501)].
1 pmol P4501
(Fig. 2B).
m/z 319 full
scan) suggested that CYP4F8 metabolized U-51605 to 19- and 18-hydroxy
metabolites (Table II). U-51605 and its 19- and 18-hydroxy metabolites
were too unstable for GC-MS analysis.
Formation of 19- and 18-hydroxy metabolites of stable PGH2
analogues, PGH1, and PGH2 by CYP4F8 and by microsomes
of human seminal vesicles in vitro and a comparison with 19- and
18-hydroxy-PGE of human seminal fluid
as shown in Fig. 3 and to
(12S)-hydroxyheptadecatrienoic acid (data not shown) (18).
Hydroxy metabolites of PGH2 will decompose to hydroxy
metabolites of PGs in the same way. The first eluting product (peak I)
was identified as 19-hydroxy-PGF2 by its MS/MS spectrum
(MS/MS 369 full scan). Peak II mainly contained
19-hydroxy-PGE2 (MS/MS 367 full scan), but trace
amounts of 19-hydroxy-PGD2 eluted on the right shoulder of
the peak as judged by MS/MS analysis (due to the relative intensity at
m/z 251). The formation of 19-hydroxy-PGE2 was
confirmed by GC-MS analysis as described below.
18-Hydroxy-PGE2 was identified in peak III by MS/MS
analysis, which showed a characteristic signal at m/z 273 (loss of 2H2O and CH3CH2CHO).
PGF2, PGE2, and PGD2 eluted
after 18, 24, and 29 min, respectively.
View larger version (18K):
[in a new window]
Fig. 3.
LC-MS analysis of PG compounds formed during
incubation of CYP4F8 with PGH2. A, CYP4F8
was incubated with PGH2. PGH2 and its
metabolites were then allowed to decompose in buffer, and formation of
hydroxy-PGs and PGs was analyzed by LC-MS. MS/MS analysis suggested
that peak I contained 19-hydroxy-PGF2 , that peak II
contained 19-hydroxy-PGE2 (with trace amounts of
19-hydroxy-PGD2 on the right shoulder), and that peak III
contained 18-hydroxy-PGE2. PGF2,
PGE2, and PGD2 eluted as marked.
was present in peak I, and
19-hydroxy-PGE2 and 18-hydroxy-PGF2 were
present in peak II. In contrast, the second sample yielded
19-hydroxy-PGE2 as the main product due to decomposition of
19-hydroxy-PGH2 in aqueous ethanol (Fig. 4B).
Small amounts of 18-hydroxy-PGE2 were present in peak III
in both chromatograms (Fig. 4B).
View larger version (19K):
[in a new window]
Fig. 4.
Identification of 18- and
19-hydroxy-PGH2 as CYP4F8 metabolites of PGH2
by reduction to 18- and
19-hydroxy-PGF2 . One-half of
the incubation was terminated with buffered SnCl2, and the
other half with ethanol. The metabolites were analyzed by selective ion
monitoring of the interval m/z 367-369 with MS/MS analysis
of hydroxy-PGE2 (MS/MS 367 full scan) and
hydroxy-PGF2 (MS/MS 369 full scan). A,
products formed after termination with SnCl2; B,
products formed after termination with ethanol. The LC-MS analysis
suggested that peak I contained 19-hydroxy-PGF2, that
peak II contained mainly 19-hydroxy-PGE2 (and
18-hydroxy-PGF2 in A), and that peak III
contained 18-hydroxy-PGE2. The mobile phase was
CH3CN/H2O/acetic acid (35:65:0.01).
, PGD2, and PGE1.
,
whereas 19-hydroxy-PGE2 was the main product formed in
aqueous ethanol (Fig. 5B). Small amounts of
18-hydroxy-PGE2 were detected in some experiments. The
yield of 19-hydroxy metabolites of PGH2 was rather poor.
Microsomal fractions of seminal vesicles may contain PGE synthase and
other PGH2-metabolizing enzymes. For example,
PGH2 was also transformed to 6-keto-PGF1 in some experiments. As previously reported, microsomes of human seminal
vesicles only slowly convert PGE1 and PGE2 to
their 19-hydroxy metabolites (4). Attempts to compare the biosynthesis
of 19-hydroxy-PGE2 from PGE2 with the
biosynthesis of 19-hydroxy-PGH2 from PGH2 were unsuccessful due to the low metabolism of PGE2.
View larger version (19K):
[in a new window]
Fig. 5.
LC-MS analysis of metabolites of U-44069,
PGH2, and arachidonic acid formed by microsomes of human
seminal vesicles. A, metabolites of U-44069 assayed by
selective ion monitoring at m/z 365. The main peak contained
the 19-hydroxy metabolite of U-44069, and the minor peak contained the
18-hydroxy metabolite. B, partial chromatograms of
metabolites formed from PGH2 (single reaction monitoring,
MS/MS 367-369). Left, analysis of one-half of the
incubation, which was terminated with buffered SnCl2;
right, analysis after termination with ethanol. Peak I
contained 19-hydroxy-PGF2 , and peak II contained
19-hydroxy-PGE2. C, metabolites of arachidonic
acid. The main peak contained 18-HETE. See Fig. 1 for experimental
details.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
were metabolized poorly (Table I). Our results
suggest that PGH1 and PGH2 are endogenous
substrates for CYP4F8, which thus can be named PGH 19-hydroxylase.
CYP4F8 is the first described hydroxylase with pronounced specificity for prostaglandin endoperoxides.
1 pmol P4501.
This Vmax value is remarkably high for a
mammalian cytochrome P450 (20, 22).
9,11-Diazo-15-deoxy-PGH2 (U-51605), PGH1,
PGH2, and 11,9-epoxymethano-PGH2 (U-46619) were
also good substrates, although there was a remarkable difference
between U-44069 and U-46619 (Table I). PGH1 and
PGH2 and their hydroxy metabolites decompose in buffer with
a half-life of ~5 min at 37 °C (18), which decreases the
metabolism by CYP4F8 and complicates the analysis of the products.
View larger version (21K):
[in a new window]
Fig. 6.
Summary of the proposed mechanism for
biosynthesis of (19R)-hydroxy-PGE2 in
human seminal vesicles.
-hydroxylation of
leukotriene B4 and was recently found to consist of
distinct isoforms that were regulated by alternative promoter usage and
exon splicing (31, 32). Whether isoforms of CYP4F8 exist is now under investigation.
-side
chain.2 The
NADPH-dependent metabolism of PGH2 in the
kidney and other tissues merits further investigation.
2-hydroxylation of PGH1 and PGH2,
which will lead to biosynthesis of the two main PGs of human seminal
fluid, (19R)-hydroxy-PGE1 and
(19R)-hydroxy-PGE2. CYP4F8 is the first described enzyme with both specificity and kinetic competence for
hydroxylation of PGH1 and PGH2.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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