(Received for publication, November 13, 1995)
From the
Previous studies of the crystal structure of the ovine
prostaglandin endoperoxide H synthase-1 (PGHS-1)/S-flurbiprofen complex
(Picot, D., Loll, P. J., and Garavito, R. M.(1994) Nature 367,
243-249) suggest that the enzyme is associated with membranes
through a series of four amphipathic helices located between residues
70 and 117. We have used the photoactivatable, hydrophobic reagent
3-trifluoro-3-(m-[I]iodophenyl)diazirine
([
I]TID) which partitions into membranes and
other hydrophobic domains to determine which domains of microsomal
PGHS-1 are subject to photolabeling. After incubation of ovine
vesicular gland microsomes with [
I]TID, ovine
PGHS-1 was one of the major photolabeled proteins. Proteolytic cleavage
of labeled PGHS-1 at Arg
with trypsin established that
[
I]TID was incorporated into both the 33-kDa
tryptic peptide containing the amino terminus and the 38-kDa tryptic
peptide containing the carboxyl terminus. This pattern of photolabeling
was not affected by the presence of 20 mM glutathione,
indicating that the photolabeling observed for PGHS-1 was not due to
the presence of [
I]TID in the aqueous phase.
However, nonradioactive TID as well as two inhibitors, ibuprofen and
sulindac sulfide, which bind the cyclooxygenase active site of PGHS-1,
prevented the labeling of the 38-kDa carboxyl-terminal tryptic peptide.
These results suggest that [
I]TID can label
both the cyclooxygenase active site in the tryptic 38-kDa fragment and
a membrane binding domain located in the 33-kDa fragment. Cleavage of
photolabeled PGHS-1 with endoproteinase Lys-C yielded a peptide
containing residues 25-166 which was labeled with
[
I]TID. This peptide contains the putative
membrane binding domain of ovine PGHS-1. Our results provide
biochemical support for the concept developed from the crystal
structure that PGHS-1 binds to membranes via four amphipathic helices
located near the NH
terminus of the protein.
The prostaglandin endoperoxide H synthase (PGHS) ()isozymes catalyze the conversion of arachidonic acid to
prostaglandin endoperoxide H
, the committed step in the
biosynthetic pathway of prostaglandins and thromboxane(1) .
PGHS isozymes have two distinct activities: a cyclooxygenase activity,
which catalyzes the oxygenation of arachidonic acid to yield
PGG
, and a peroxidase activity, which reduces the
15-hydroperoxyl group of PGG
to form prostaglandin
endoperoxide H
(2, 3) . Two isoforms of
PGHS are known and are designated PGHS-1 and PGHS-2. PGHS-1 is the
original ``cyclooxygenase'' characterized from ovine
vesicular glands. PGHS-1 is expressed constitutively in most
tissues(4) . PGHS-2 is the more recently discovered isozyme
whose expression is inducible and has been implicated in
inflammation(4) . PGHS isozymes have considerable homology in
amino acid sequence and are thought to have similar
structures(5, 6, 7, 8, 9) .
Nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin,
ibuprofen, and sulindac sulfide inhibit both isoforms of PGHS by
binding the cyclooxygenase active site and blocking cyclooxygenase
activity(10, 11) . The peroxidase activity of the
isozymes is not appreciably affected by most NSAIDs consistent with the
idea that PGHS isozymes have distinct cyclooxygenase and peroxidase
active sites(12, 13) .
Both PGHS isozymes are
glycoproteins (14) which are associated with the membranes of
the endoplasmic reticulum (ER) and nuclear
envelope(15, 16) . The isozymes are both inserted
through the ER membrane via NH-terminal signal peptides,
which are cleaved in the mature
enzymes(5, 6, 7, 17) . Both isozymes
exhibit the characteristics of integral membrane proteins in that they
are associated with microsomal membranes and require detergent for
solubilization(2, 3, 17) . Additionally,
following purification, PGHS-1 can be reincorporated into liposomes,
indicating that the protein can bind to lipid bilayers
directly(18) . However, unlike other integral membrane
proteins, the PGHS isozymes do not appear to contain transmembrane
domains. This prediction is based on analysis of the crystal structure
of solubilized ovine PGHS-1(19) , and is supported by
biochemical studies showing that six different evenly spaced regions of
the isozymes are located in the lumen of the ER(20) .
An
interesting mechanism for the association of the PGHS isozymes has been
proposed by Garavito and co-workers based on examination of the crystal
structure of ovine PGHS-1(19, 21) . Residues
70-117 ()of ovine PGHS-1 contain four consecutive,
short amphipathic
-helices which form a hydrophobic face along one
side of the enzyme with the hydrophobic residues pointing away from the
body of the enzyme. This region of the enzyme is proposed to be a
membrane binding domain, associating with a single leaflet of the ER
membrane but without fully traversing it. Should this prediction hold
true, PGHS-1 will be the first monotopic membrane protein for which the
structure has been determined. However, there is currently no
biochemical evidence supporting this prediction.
3-Trifluoro-3-(m-[I]iodophenyl)diazirine
([
I]TID) is a hydrophobic, photoactivatable
reagent which partitions into lipid bilayers making this reagent a
useful tool for determining regions of proteins which are
membrane-associated (22, 23, 24) . In order
to identify regions of PGHS-1 which are associated with the lipid
bilayer, we examined the photolabeling by
[
I]TID of PGHS-1 present in microsomal
membranes prepared from ovine seminal vesicles. Our results indicate
that [
I]TID photolabels a region of PGHS-1
which contains the putative membrane binding domain. We also found that
[
I]TID can photolabel the cyclooxygenase active
site of PGHS-1.
In some experiments, microsomes were incubated
with glutathione, ibuprofen, sulindac sulfide, or nonradioactive TID
for 10 min at 0 °C prior to the addition of
[I]TID. Incubations with
[
I]TID and photolabeling were then performed
using the procedure described above.
Figure 1:
Photolabeling of ovine PGHS-1 with
[I]TID. Microsomal membranes were prepared from
ovine vesicular glands as described in the text. Microsomal suspensions
were preincubated for 10 min at 0 °C in the absence (A, B, and C) or presence of 20 mM reduced
glutathione (D, E, and F) and then incubated
for 10 min at 0 °C with [
I]TID in low
light. Finally, the microsomal samples were photolabeled by irradiation
at 366 nm for 10 min at 0 °C. Microsomes were then collected by
centrifugation, resuspended, and solubilized, incubated in the absence
or presence of trypsin, and immunoprecipitated with anti-PGHS-1
antibodies. Samples were resolved on 15% SDS-PAGE gels. The gels were
subjected to silver staining (left panel) and exposed to film
for autoradiography (right panel). Lanes A and D, microsomal membranes; lanes B and E,
immunoprecipitated PGHS-1; lanes C and F, trypsin
treated, immunoprecipitated PGHS-1.
Following photolabeling of ovine
vesicular gland microsomes with [I]TID, PGHS-1
was one of the most prominently radiolabeled proteins (Fig. 1).
After tryptic digestion of photolabeled PGHS-1, both the 33-kDa
amino-terminal and the 38-kDa carboxyl-terminal peptides were found to
be labeled by [
I]TID. This result was
unexpected because the putative membrane binding domain of PGHS-1 is
located near the NH
terminus, and it was anticipated that
only the 33-kDa amino-terminal tryptic peptide would be labeled by
[
I]TID. To determine if the labeling of either
of the tryptic peptides was due to the presence of
[
I]TID in the aqueous phase, photolabeling was
performed in the presence of 20 mM reduced glutathione, which
scavenges any [
I]TID present in the aqueous
phase(22) . Reduced glutathione did not affect the
photolabeling of either tryptic peptide of PGHS-1 (Fig. 1),
indicating that the photolabeling was not due to the presence of
[
I]TID in the aqueous phase.
Figure 2:
Effect of nonradioactive TID on the
photolabeling of ovine PGHS-1 by [I]TID. Ovine
vesicular gland microsomes were preincubated in the presence or absence
of 100 µM nonradioactive TID prior to incubation and
photolabeling with [
I]TID. After photolabeling,
the microsomal samples were treated with trypsin and immunoprecipitated
as described in the text. Immunoprecipitated PGHS-1 and PGHS-1 tryptic
fragments were resolved on 15% SDS-PAGE gels and the gels exposed to
film for autoradiography. Lane A, no pretreatment; lane
B, 100 µM nonradioactive TID
pretreatment.
The cyclooxygenase active site of
PGHS-1 is a hydrophobic channel which contains segments of the peptide
chains found in both the 33-kDa amino-terminal tryptic peptide and in
the 38-kDa carboxyl-terminal tryptic peptide(19) . We reasoned
that the labeling of one or both of the tryptic peptides might result
from the binding of [I]TID within the
cyclooxygenase active site. A comparison of the structures of
[
I]TID and several NSAIDs suggest that a
hydrophobic compound such as [
I]TID could
occupy the cyclooxygenase active site (Fig. 3). Accordingly, the
ability of two NSAIDs, ibuprofen and sulindac sulfide, to compete for
labeling of PGHS-1 by [
I]TID was examined (Fig. 4). Incubation of microsomes with ibuprofen or sulindac
sulfide prior to photolabeling with [
I]TID did
affect the labeling pattern seen with ovine PGHS-1, decreasing somewhat
the intensities of labeling of the 33-kDa tryptic peptide and
essentially eliminating the labeling of the 38-kDa tryptic peptide.
Pretreatment of PGHS-1 with the NSAIDs did not affect the generation of
the 38- and 33-kDa peptides as determined by silver staining (data not
shown). These results suggest that much of the photolabeling of PGHS-1
by [
I]TID, particularly in the 38-kDa
carboxyl-terminal tryptic peptide, is caused by the binding of
[
I]TID within the cyclooxygenase active site.
Figure 3:
Structures of
[I]TID and several nonsteroidal
anti-inflammatory drugs.
Figure 4:
Effects of ibuprofen and sulindac sulfide
on the photolabeling of ovine PGHS-1 by [I]TID.
Ovine vesicular gland microsomes were preincubated in the presence or
absence of 100 µM ibuprofen or 100 µM sulindac sulfide prior to incubation and photolabeling with
[
I]TID. After photolabeling, the microsomal
samples were treated with trypsin and immunoprecipitated as described
in the text. Immunoprecipitated PGHS-1 and PGHS-1 tryptic fragments
were resolved on 15% SDS-PAGE gels, and the gels were silver stained
and exposed to film for autoradiography. Lane A, no drug
pretreatment; lane B, 100 µM ibuprofen
pretreatment; and lane C, 100 µM sulindac sulfide
pretreatment.
Figure 5:
Cleavage of I-photolabeled
ovine PGHS-1 with endoproteinase Lys-C. Ovine vesicular gland
microsomes were incubated with [
I]TID and
photolabeled as described in the legend to Fig. 1. PGHS-1 was
immunoprecipitated from solubilized microsomes, denatured, and
subjected to exhaustive digestion with endoproteinase Lys-C as
described in the text. Samples were resolved on a 15% SDS-PAGE gel and
transferred to a nitrocellulose filter. Western blotting (left
panel) was performed on the filter using a rabbit anti-PGHS-1
antibody raised against a peptide corresponding to residues 25-35
of ovine PGHS-1 (20) . After waiting several hours for the
chemiluminescence to fade, the filter was exposed to film for
autoradiography (right panel). Lane A, photolabeled
microsomes; lane B, endoproteinase Lys-C digest of
immunoprecipitated, photolabeled PGHS-1.
[I]TID has been a useful tool for
identifying transmembrane domains of
proteins(22, 23, 24) .
[
I]TID partitions efficiently into membrane
lipid bilayers, and photolabeling of proteins with
[
I]TID occurs predominately in domains which
are in direct contact with membranes(22) . There is, however, a
precedent for the incorporation of [
I]TID into
regions of a protein which are not in contact with the lipid bilayer.
Photolabeling of the nicotinic acetylcholine receptor with
[
I]TID could be partially inhibited by receptor
agonists and antagonists apparently because these agents exclude
[
I]TID from the receptor binding
site(31) . In order to distinguish between the photolabeling of
membrane-associated regions of the nicotinic acetylcholine receptor and
photolabeling of the receptor binding site,
[
I]TID photolabeling was performed in the
presence of excess non-radioactive
TID(24, 31, 32) . The reasoning behind these
experiments was that nonradioactive TID would compete with
[
I]TID for saturable binding sites, whereas
nonradioactive TID could not compete for photolabeling of
membrane-associated domains because the membranes could not be
saturated with TID at submillimolar
levels(24, 31, 32) .
We have found that
[I]TID is a useful probe for analyzing the
structure of ovine PGHS-1. The photolabeling of ovine PGHS-1 is similar
to that of the nicotinic acetylcholine receptor in that PGHS-1 was
photolabeled by [
I]TID in both a nonspecific
manner consistent with the association of regions of the enzyme with
the ER membrane and in a specific manner consistent with the occupation
of a hydrophobic pocket in the enzyme by
[
I]TID. Our data suggest that a region of ovine
PGHS-1 contained in the NH
-terminal 277 amino acids is
associated with the ER membrane and that the COOH-terminal half of the
enzyme does not contain a membrane-associated region. Additionally, a
peptide composed of residues 25-166 of ovine PGHS-1 is
photolabeled by [
I]TID, although we have not
proven unequivocally that the labeling occurs specifically in the
putative membrane binding domain encompassing residues 70-117.
Our data are consistent with the prediction that residues 70-117
form a novel membrane binding domain (19, 21) .
Examination of peptides photolabeled by [
I]TID
in the presence of nonradioactive TID and NSAIDs by amino acid
sequencing will provide a valuable test of this prediction and should
allow for the determination of which amino acids of PGHS are important
for membrane association.
The ability of both nonradioactive TID,
ibuprofen, and sulindac sulfide to block photolabeling of the
COOH-terminal half of PGHS-1 by [I]TID suggests
that [
I]TID can photolabel the cyclooxygenase
active site of PGHS-1. Thus, [
I]TID may also be
useful for identifying amino acids which are exposed in the hydrophobic
cyclooxygenase pocket. Furthermore, because
[
I]TID labeling can be performed with
membrane-associated enzyme, this labeling procedure may be used with
the membrane-bound enzyme to test predictions made from the crystal
structure of detergent-solubilized PGHS-1. Finally,
[
I]TID may prove to be a valuable tool for
comparing structural differences between the cyclooxygenase active
sites of PGHS-1 and -2. Recent studies have indicated that isozyme
specific NSAIDs can be
developed(10, 11, 33, 34) , and
comparison of the labeling of the active sites may reveal some of the
differences in the structures of the active sites of the isozymes which
lead to NSAID selectivities(33, 34, 35) .