1 Proteolysis Research Group, School of Biochemistry and Molecular Biology,
University of Leeds, Leeds LS2 9JT, UK
2 Department of Pharmacology, University of Illinois at Chicago, Chicago, IL
60612, USA
* Author for correspondence (e-mail: bmbetp{at}bmb.leeds.ac.uk)
Accepted 22 April 2003
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Summary |
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Key words: Angiotensin-converting enzyme, Lipid rafts, Cholesterol, Glycosyl-phosphatidylinositol, Ectodomain shedding
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Introduction |
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Despite the fact that ACE is expressed as a type I integral membrane
protein, a soluble form of the enzyme exists in plasma and other body fluids
(Hooper, 1991). This soluble
form is derived from the membrane-bound form through the action of ACE
secretase (Oppong and Hooper,
1993
; Parvathy et al.,
1997
). This process of cleavage and secretion (often referred to
as shedding) appears to be an important and widely used post-translational
regulatory process (Hooper et al.,
1997
). A variety of structurally and functionally unrelated
cell-surface proteins are proteolytically cleaved from the membrane, including
tumour necrosis factor
, transforming growth factor
, and
L-selectin (reviewed by Hooper et al.,
1997
). The secretases that cleave these membrane proteins share
several properties including upregulation by phorbol esters and muscarinic
agonists, and inhibition by hydroxamate-based zinc metalloprotease inhibitors
such as batimastat. The ADAM (a disintegrin
and metalloprotease) family of proteins has been
implicated in the constitutive and regulated shedding of some of these
proteins (Black and White,
1998
; Schlondorff and Blobel,
1999
; Turner and Hooper,
1999
). However, the identity of the enzyme responsible for the
cell surface shedding of ACE remains to be determined.
Lipid rafts are regions of the plasma membrane rich in sphingomyelin,
glycosphingolipids, cholesterol and acylated proteins
(Hooper, 1999;
Simons and Toomre, 2000
).
Rafts are characterised by their relative insolubility at 4°C in certain
detergents such as Triton X-100 (Brown and
Rose, 1992
). Because of their high lipid-to-protein ratio, the
detergent-insoluble rafts float to a low density during buoyant sucrose
density gradient centrifugation in the presence of Triton X-100. The resulting
low-density, detergent-insoluble membrane fraction is enriched not only in
cholesterol and glycosphingolipids but also in certain proteins, including
multiple glycosylphosphatidylinositol (GPI)-anchored proteins
(Hooper and Turner, 1988
).
Lipid rafts have been implicated in a range of biological processes, including
intracellular trafficking, transmembrane signalling, lipid and protein
sorting, and regulated proteolysis (Brown
and London, 1998
; Wolozin,
2001
).
We therefore considered whether lateral segregation in lipid rafts could be
a mechanism to regulate the proteolytic shedding of a membrane protein. In
order to investigate this we expressed wild-type ACE (WT-ACE) and an ACE
construct with a GPI anchor attachment signal sequence replacing the
transmembrane and cytosolic domains (GPI-ACE)
(Marcic et al., 2000) in two
unrelated cell lines (Chinese hamster ovary (CHO) and human neuroblastoma
SH-SY5Y). Although only GPI-ACE was sequestered in lipid rafts, the shedding
of both constructs was stimulated to a similar extent by PMA or the muscarinic
agonist carbachol, and the inhibition profile for a range of hydroxamate-based
compounds for the shedding of WT-ACE and GPI-ACE was essentially identical.
These data show that the phorbol-ester-stimulated shedding of ACE does not
require the transmembrane or cytosolic regions of the protein. Furthermore,
the fact that shedding is neither enhanced nor inhibited when ACE is targeted
to lipid rafts by the addition of a GPI anchor also shows that lateral
segregation in the plane of the membrane is not involved in regulating the
shedding of ACE.
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Materials and Methods |
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Cell culture
The SH-SY5Y and CHO cells were cultured, respectively, in Dulbecco's
modified Eagle's medium (DMEM) or Ham's F12 supplemented with 10% foetal
bovine serum, penicillin (50 units/ml), streptomycin (50 mg/ml), and 2 mM
glutamate (all Gibco BRL, Paisley, UK). Cells were maintained at 37°C in
5% CO2 in air. For stable transfections, 30 µg DNA was
introduced to cells by electroporation and selection was performed in normal
growth medium containing 500 µg/ml of neomycin selection antibiotic.
Batimastat and all other hydroxamate-based inhibitor compounds were
synthesised at Glaxo SmithKline (Harlow, UK) and were used at 10 µM.
Structural details of these compounds have been published previously
(Parkin et al., 2002).
Carbachol and PMA (Sigma, Poole, UK) were used at 30 µM and 1 µM,
respectively. When the cells were confluent the medium was changed to OptiMEM
(Gibco BRL) and the cells were incubated for 7 hours at 37°C with the
indicated compounds. The medium was then harvested, concentrated and assayed
for ACE activity. For analysis of cell-associated ACE, cells were washed with
phosphate-buffered saline (PBS; 20 mM Na2HPO4, 2 mM
NaH2PO4, 0.15 M NaCl, pH 7.4) and scraped from the
flasks into PBS. Following centrifugation at 500 g for 5
minutes, the pelleted cells were lysed in 0.1 M Tris/HCl, 0.15 M NaCl, 1%
Triton X-100, 0.1% Nonidet P-40, pH 7.4.
Lipid raft isolation
The following procedures were all performed at 4°C. Harvested cells
were resuspended in 2 ml of Mes-buffered saline (MBS; 25 mM Mes/NaOH, 0.15 M
NaCl, pH 6.5) containing 0.5% Triton X-100 and homogenised by 15 passages
through a Luer 21 gauge needle. Unsolubilized cells were removed by
centrifugation at 500 g for 5 minutes and the supernatant was
adjusted to 40% (w/v) sucrose by the addition of an equal volume of 80% (w/v)
sucrose in MBS. An aliquot of the sample (1 ml) was then layered beneath a
discontinuous sucrose gradient consisting of 2 ml of 30% (w/v) sucrose and 2
ml of 5% (w/v) sucrose both in MBS. The tubes were then centrifuged overnight
at 140,000 g in an SW-55 rotor (Beckman Instruments). Sucrose
gradients were harvested from the base of the tubes in 0.5 ml fractions.
SDS-PAGE and immunoelectrophoretic blot analysis
Proteins were separated by SDS-PAGE on 7-17% polyacrylamide gradient gels
followed by transfer to Immobilon P poly(vinylidene difluoride) membranes as
previously described (Hooper and Turner,
1987). For the detection of ACE the rabbit polyclonal antibody
RP183 (Williams et al., 1992
)
was used at 1:2000 dilution. The monoclonal antibodies against ß-actin
and flotillin (BD Biosciences, Oxford, UK) were used at 1:5000 and 1:1000,
respectively, and the polyclonal antibody against caveolin (Affiniti Research
Products, Exeter, UK) was used at 1:4000. Bound antibodies were detected using
peroxidase-conjugated secondary antibodies in conjunction with the enhanced
chemiluminescence detection method (Amersham).
Enzyme and protein assays
ACE was assayed using BzGly-His-Leu as substrate, and the substrate and
reaction products were separated and quantified by reverse-phase HPLC as
described previously (Hooper and Turner,
1987). Protein was quantified using bicinchoninic acid in a
microtitre plate assay with BSA as standard
(Smith et al., 1985
).
Phosphatidylinositol-specific phospholipase C release of GPI-ACE from
cells
Phosphatidylinositol-specific phospholipase C (PI-PLC) purified from
Bacillus thuringiensis (Low,
1992) was diluted in 10 mM Hepes/NaOH, 150 mM NaCl, pH 7.4.
Diluted PI-PLC was filter-sterilized and added to CHO or SH-SY5Y cells at a
concentration of 0.17 mg/ml. Following a 7 hour incubation at 37°C the
conditioned medium was harvested and assayed for ACE activity as already
described.
Immunocytochemistry
CHO cells were cultured on coverslips and then fixed in 3% (w/v)
paraformaldehyde prior to blocking in 5% (v/v) goat serum in Tris-buffered
saline (TBS; 25 mM Tris, 0.15 M NaCl, pH 7.2). ACE was detected using either
polyclonal ACE antibody RP183 or RH179 (both at 1:1000) followed by 1:200
dilutions of goat anti-rabbit secondary antibodies conjugated to either FITC
or Texas Red (Stratech Scientific, Soham, UK). The cells were visualised using
the Deltavision deconvolution microscope system (Applied Precision,
Washington, USA) with an Olympus IX70 inverted microscope base. In the Triton
X-100 extraction experiment, cells were incubated for 20 minutes at 4°C in
TBS containing 2% (v/v) Triton X-100 prior to paraformaldehyde fixation. In
the filipin treatment experiment, cells were incubated with either PMA (1
µM) or PMA (1 µM) and filipin complex (10 nM) (Sigma, Poole, UK) for 1
hour at 37°C prior to paraformaldehyde fixation.
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Results |
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|
|
GPI-ACE is released from cells by exogenous PI-PLC
To confirm the GPI-anchored nature of GPI-ACE, cells were incubated with
bacterial PI-PLC and the conditioned medium was harvested and assayed for ACE
activity (Table 1). The release
of WT-ACE from both CHO and SH-SY5Y cells in the presence of PI-PLC was
indistinguishable from that which occurred in controls when PI-PLC was omitted
from the incubations. In contrast, the amount of GPI-ACE released by PI-PLC
from CHO cells was increased 6.9-fold over control cells incubated in the
absence of PI-PLC and the release from SH-SY5Y cells was increased
14.6-fold.
|
GPI-ACE is sequestered in lipid rafts
In order to determine whether GPI-ACE was localised in lipid rafts, cells
were solubilized in Triton X-100 at 4°C and rafts isolated by buoyant
sucrose density-gradient centrifugation as described in Materials and Methods.
In both CHO and SH-SY5Y cells most of the total cellular protein was
effectively solubilized and subsequently located in fractions 1-3 of the
sucrose gradients (Fig. 3A). A
lesser amount of protein (between 21 and 28% of the total) was located in a
high-density detergent-insoluble pellet at the base of the centrifuge tube
(fraction 0). The assay technique employed was not sensitive enough to detect
protein in the low-density raft region of the gradients (fractions 4-6). The
position of lipid rafts in the sucrose gradients prepared from CHO cells was
determined by immunoblotting with an antibody against caveolin
(Lisanti et al., 1993). The
majority of caveolin (WT-ACE cells, 87.5%; GPI-ACE cells, 66.5%) was located
in fractions 4-6 of the sucrose gradients
(Fig. 3B). Neuronal cells do
not express caveolin (Gorodinsky and
Harris, 1995
; Parkin et al.,
1997
), therefore, an antibody against flotillin
(Bickel et al., 1997
) was used
to determine the position of lipid rafts in sucrose gradients prepared from
SH-SY5Y cells. Like caveolin in CHO cells, the majority of flotillin (WT-ACE
cells, 75.0%; GPI-ACE cells, 65.5%) in SH-SY5Y cells was located in fractions
4-6 (Fig. 3B). The sucrose
gradient fractions were also assayed for ACE
(Fig. 3C). In CHO cells the
majority (94.3%) of WT-ACE was excluded from lipid rafts whilst the same
construct was completely excluded from rafts in SH-SY5Y cells. In contrast,
52.8% and 55.0% of GPI-ACE activity was located in lipid rafts isolated from
CHO and SH-SY5Y cells, respectively.
|
In order to confirm that GPI-ACE was localised in lipid rafts, CHO cells expressing either WT-ACE or GPI-ACE were subjected to immunocytochemistry as described in the Materials and Methods using an anti-ACE polyclonal primary antibody (Fig. 4). WT-ACE (Fig. 4A) exhibited a relatively diffuse staining pattern at the cell surface whilst GPI-ACE (Fig. 4B) had a much more punctate staining pattern consistent with a raft localisation. When the cells were incubated with 2% (v/v) Triton X-100 prior to fixation, the level of fluorescence in WT-ACE expressing cells was dramatically reduced (Fig. 4C) consistent with this construct residing in detergent-soluble regions of the plasma membrane. In contrast, GPI-ACE staining remained punctate even when cells were pre-treated with Triton X-100 (Fig. 4D) consistent with its localisation in detergent-resistant rafts.
|
WT-ACE and GPI-ACE shedding is stimulated by activators of protein
kinase C
The ability of activators of the protein kinase C signalling cascade to
stimulate WT-ACE and GPI-ACE shedding from CHO and SH-SY5Y cells was assessed
(Fig. 5). When CHO cells
expressing WT-ACE were incubated with the protein kinase C agonist PMA the
release of ACE into the medium was stimulated 2.0-fold
(Fig. 5A). The
hydroxamate-based zinc metalloprotease inhibitor batimastat prevented the
PMA-induced release of WT-ACE into the medium. Similarly, the release of
GPI-ACE from CHO cells was stimulated 2.8-fold upon PMA treatment and this
PMA-induced release was completely inhibited by batimastat.
|
SH-SY5Y cells express muscarinic receptors at the cell surface, which can
be activated by agonists such as carbachol. The resultant signalling cascade
leads to the activation of protein kinase C
(Canet-Aviles et al., 2002).
The ability of carbachol to stimulate WT-ACE and GPI-ACE release from SH-SY5Y
cells was assessed (Fig. 5B). When SH-SY5Y cells expressing WT-ACE were incubated with carbachol the release
of ACE into the medium was stimulated 2.3-fold
(Fig. 5B). Batimastat prevented
the carbachol-induced release of WT-ACE into the medium. Similarly, the
release of GPI-ACE from SH-SY5Y cells was stimulated 2.0-fold upon carbachol
treatment and this carbachol-induced release was inhibited by batimastat.
Regulated shedding of WT-ACE and GPI-ACE is inhibited in an identical
manner by a range of hydroxamate-based metalloprotease inhibitors
CHO cells expressing either WT-ACE or GPI-ACE were co-incubated with PMA
and a range of hydroxamate-based zinc metalloprotease inhibitors that we have
characterised previously against the ACE secretase
(Parkin et al., 2002). The
medium was then harvested and samples assayed for ACE activity. The results
(Fig. 6) show that the
inhibition profile of these seven compounds for the regulated shedding of
WT-ACE and GPI-ACE was essentially identical.
|
Filipin treatment does not alter the shedding of GPI-ACE
Although the majority of GPI-ACE was located in the lipid raft fractions of
the sucrose density gradients, approximately 40% of the enzyme activity was
recovered in the detergent-soluble fractions
(Fig. 3C). We considered the
possibility that this detergent-soluble pool of GPI-ACE may be shed more
effectively than the lipid raft pool such that, although a substantial
proportion of GPI-ACE was sequestered in lipid rafts, the overall shedding of
GPI-ACE in comparison to WT-ACE shedding was not altered. In order to assess
this, CHO cells transfected with either GPI-ACE or WT-ACE were incubated with
PMA in the absence or presence of the lipid raft-disrupting compound, filipin.
When filipin was used to disrupt rafts the diffuse staining pattern of WT-ACE
at the cell surface did not change (Fig.
7A,C). In contrast, the punctate distribution of GPI-ACE
(Fig. 7B) changed to a more
diffuse staining pattern (Fig.
7D) consistent with filipin disrupting the lipid rafts. However,
the PMA-regulated shedding of GPI-ACE from CHO cells remained unchanged
whether or not the cells were co-incubated with filipin
(Fig. 7E).
|
PMA does not alter the membrane compartmentalisation of WT-ACE and
GPI-ACE
Although GPI-ACE is present in rafts
(Fig. 3C and
Fig. 4B), we considered the
possibility that upon PMA stimulation of the cells, the protein might move
laterally into non-raft regions of the membrane where it is subsequently
cleaved. In order to address this, CHO cells transfected with either WT-ACE or
GPI-ACE were incubated with or without PMA. In order to retain ACE in the cell
membrane the incubations were all performed in the presence of batimastat.
Cells were then harvested and lipid rafts were prepared in the presence of
batimastat as described in Materials and Methods. The resultant sucrose
gradient fractions were assayed for ACE activity and the results show that
there was no change in the distribution in rafts of either WT-ACE
(Fig. 8A) or GPI-ACE
(Fig. 8B) when cells were
incubated in the presence of PMA.
|
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Discussion |
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The basal shedding of GPI-ACE from SH-SY5Y cells was indistinguishable from
that of WT-ACE. However, whereas the total GPI-ACE activity in CHO cell
lysates was approximately half that of the WT-ACE activity
(Fig. 2C) the basal shedding of
the former protein (Table 1)
was not significantly different from that of WT-ACE. At first sight these data
would seem to imply that the basal shedding of GPI-ACE from CHO cells was more
efficient than that of WT-ACE. However, as the amount of the proteins at the
cell surface was not determined we cannot rule out that the apparent increase
in the basal shedding of GPI-ACE is due to enhanced trafficking of the protein
to the surface of the CHO cells where the secretase acts
(Parvathy et al., 1999).
Upon incubation of the CHO or SH-SY5Y cells with PMA or carbachol the
shedding of WT-ACE was enhanced 2.0- and 2.3-fold, respectively
(Fig. 5) in agreement with
previously published data relating to the shedding of human somatic ACE from
CHO cells (Beldent et al.,
1993) and rabbit testicular ACE from mouse epithelial cells
(Ramchandran et al., 1994
).
Surprisingly, the shedding of GPI-ACE from CHO and SH-SY5Y cells was also
stimulated 2.0- and 2.8-fold by PMA and carbachol, respectively
(Fig. 5). One possibility is
that this enhanced secretion of GPI-ACE upon PMA or carbachol treatment was
due to the release of a pre-existing intracellular pool of cleaved ACE.
However, multiple lines of evidence argue against this possibility. First, an
ACE mutant lacking any form of membrane anchorage does not exhibit enhanced
secretion from CHO cells upon phorbol ester treatment
(Beldent et al., 1993
). Second,
the entire ACE content of GPI-ACE expressing cells in the present study was
shown, by temperature-induced phase separation in Triton X-114, to be
hydrophobic, possessing the membrane-anchoring domain (data not shown). Third,
the PMA/carbachol-induced shedding of GPI-ACE from CHO and SH-SY5Y cells was
prevented by multiple hydroxamate-based compounds which have previously been
identified as ACE secretase inhibitors
(Parkin et al., 2002
).
Therefore, it is clear that treatment of cells expressing GPI-ACE with PMA or
carbachol results in enhanced de novo secretase-mediated ACE release.
ACE secretase requires an accessible stalk region in ACE of at least 11
residues in length and a minimum distance of 3 residues from the proximal
extracellular domain and 8 residues from the transmembrane domain
(Ehlers et al., 1996). These
distances are essentially preserved in GPI-ACE, as there are 14 residues
between the Arg-Ser bond cleaved by the secretase
(Woodman et al., 2000
) and the
site of GPI attachment on the Ser residue in the Ser-Ala-Ala (
,
+ 1,
+ 2) motif of carboxypeptidase M
(Tan et al., 2003
)
(Fig. 1). Also, deletion of 17
residues from the end of the transmembrane domain and into the juxtamembrane
stalk, up to the same Arg residue to which the carboxypeptidase M GPI signal
was attached, did not alter the site of cleavage by the secretase in CHO cells
(Ehlers et al., 1996
). Thus
there is no obvious alteration to either the sequence or structure of the
juxtamembrane stalk that should prevent the normal secretase from cleaving
GPI-ACE. This was essentially confirmed by examining the effect of a range of
hydroxamate-based zinc metalloprotease inhibitors on the shedding of GPI-ACE
and WT-ACE (Fig. 6). Using
these compounds we were unable to distinguish between the shedding of the two
constructs indicating that both WT-ACE and GPI-ACE are probably released from
the cell surface by the same secretase.
As a proportion of GPI-ACE was not localised in the detergent-insoluble
rafts, we considered the possibility that this non-raft pool of GPI-ACE may be
preferentially shed over that localised in the rafts. However, disruption of
the rafts with the cholesterol-binding agent filipin did not lead to an
increase in the shedding of GPI-ACE (Fig.
7), and we could obtain no evidence for the lateral movement of
GPI-ACE from the detergent-insoluble rafts into the detergent-soluble regions
of the membrane upon PMA stimulation of the cells
(Fig. 8). Collectively these
data show that sequestration of ACE in lipid rafts does not regulate its
ectodomain shedding. Following on from this, our data also imply that the
secretase is present in both raft and non-raft domains of the plasma membrane
where it can act on available substrate, although the lack of any effect on
the shedding process may indicate that the levels of available secretase are
not the rate-limiting factor in this process. Although the identity of the
secretase that sheds ACE remains unknown, it has the properties of a member of
the ADAM family of membrane-bound zinc metalloproteases
(Hooper et al., 1999), and in
this context it is interesting to note that ADAM10 has been localised to both
raft and non-raft domains following buoyant sucrose density gradient
centrifugation in the presence of Triton X-100
(Kojro et al., 2001
).
It has been suggested that upon cell activation attachments between the
cytoskeleton and the cytoplasmic domains of the transmembrane substrates (and
their cognate secretases) change, co-clustering the transmembrane protein and
its secretase and allowing them to interact
(Werb and Yan, 1998). In
addition, it has been proposed that activation of the protein kinase C
signalling cascade could modify the cytoplasmic domain of the substrate
protein causing a conformational change in it which makes the secretase
cleavage site more accessible (Werb and
Yan, 1998
). Our results argue against both of these as possible
mechanisms whereby the shedding of ACE is increased upon activation of protein
kinase C. GPI-ACE, which lacks a cytoplasmic domain, is clearly not capable of
direct interaction with the cytoskeleton as the GPI anchor only interacts with
the outer leaflet of the bilayer and yet ACE shedding is still enhanced upon
incubation of cells with PMA or carbachol
(Fig. 5). Thus the cytosolic
domain of ACE is not required for the regulated shedding of its ectodomain or
involved in regulating this process. It has recently been demonstrated
(Kohlstedt et al., 2002
) that
protein kinase CK2 phosphorylates the cytoplasmic tail of ACE and retains it
in the plasma membrane, thus reducing the basal shedding of the protein. The
authors also demonstrated that PMA was still able to stimulate the shedding of
a form of ACE in which the phosphorylatable serine residue at position 1270
had been mutated to alanine. In support of the present study, the authors
concluded that regulated ACE shedding could be attributed to the activation of
a secretase whereas the basal shedding of the enzyme was regulated by the
CK2-mediated phosphorylation of the cytoplasmic tail.
The role of the transmembrane domain of a substrate protein in regulating
shedding has been investigated by deleting the whole of this region
(Cheng and Flanagan, 1994;
Deng et al., 1996
). However,
the resultant constructs were not membrane-anchored and/or trafficked to the
cell-surface where shedding occurs
(Parvathy et al., 1999
). In
the present study we overcame this problem by replacing the transmembrane
domain of ACE with an alternative membrane-anchoring domain, a GPI anchor. The
resultant construct was membrane-anchored and located at the cell surface
similarly to WT-ACE (Table 1)
(Marcic et al., 2000
), and was
similarly subject to ectodomain shedding, indicating unequivocally that the
transmembrane domain is not involved in the shedding process.
In conclusion, we have shown using a GPI-anchored form of ACE that sequestration of the protein in lipid rafts does not regulate its shedding, and that neither the cytoplasmic nor the transmembrane domains are required for the shedding of ACE via activation of the protein kinase C signalling cascade.
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Acknowledgments |
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