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INTRODUCTION |
Angiotensin I-converting enzyme (ACE,1 EC
3.4.15.1) plays a key role in the control
of blood pressure and fluid and electrolyte homeostasis (1). It exists
primarily as a type I integral membrane glycoprotein, although a
soluble form is present under normal conditions in blood plasma and
other body fluids and is derived from the membrane-bound form through
proteolytic cleavage in the juxtamembrane stalk region (2). The
secretase responsible for the cleavage and secretion of ACE is a zinc
metalloproteinase located at the cell surface (3-6). It is inhibited
by hydroxamate-based compounds such as batimastat and displays a
remarkably similar inhibition profile to that of the
-secretase that
cleaves the amyloid precursor protein (7, 8). The precise site of
cleavage in ACE has been determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry following enzymic fragmentation of the purified protein (9). This analysis revealed that
the soluble somatic forms of human and porcine ACE were cleaved between
Arg1203 and Ser1204, some 27 residues on the
extracellular side of the transmembrane domain.
ACE is just one of several proteins that are post-translationally shed
from the membrane through the action of secretases (also called
sheddases or convertases) (10-12). Other proteins proteolytically
cleaved from the membrane include tumor necrosis factor-
,
transforming growth factor-
, amyloid precursor protein, and
L-selectin. The secretases that cleave and release such
proteins from the membrane have several properties in common including up-regulation by phorbol esters and inhibition by hydroxamate-based zinc metalloproteinase inhibitors. Numerous studies have investigated the sequence/structural requirements for recognition of an integral membrane protein by its cognate secretase (for example see Refs. 13-18). This usually has been investigated by mutating residues in the
extracellular juxtamembrane stalk region and monitoring their effect on
secretion of the expressed protein. Such studies, in which numerous
mutations failed to abrogate cleavage and secretion of the membrane
protein, have concluded that for each substrate protein there is a
single secretase with a relaxed sequence specificity and that the
critical parameter for cleavage efficiency is the relative conformation
(possibly
-helical) of the stalk region. However, an alternative
explanation for these observations is that the mutations in the
juxtamembrane stalk invoked the action of other distinct secretases.
The involvement of multiple secretases was suggested by Zhong et
al. (19) when numerous point mutations in the stalk region of the
amyloid precursor protein resulted in that protein being cleaved at
multiple sites, although there was no experimental evidence (inhibition
profile, cellular location) to support this suggestion.
Here we report that a single point mutation in the juxtamembrane stalk
region of ACE invokes the action of a mechanistically and spatially
distinct secretase. Asn631 in the single domain form of ACE
(20) (corresponding to Asn1196 in human somatic ACE), 7 residues N-terminal to the normal secretase cleavage site, was mutated
to Gln (Fig. 1). The mutant protein (ACENQ) was
transport-competent and enzymatically active, indicating that the
mutation has no general effects on the folding of ACE. Pulse-chase
analysis revealed that ACENQ was more readily cleaved and
secreted into the medium than the wild-type protein. Mass spectrometric
analysis of the secreted protein and temperature block and inhibitor
studies indicated that ACENQ was being cleaved between
Asn635 and Ser636 in the endoplasmic reticulum
(ER) by a serine protease and not between Arg638 and
Ser639 at the cell surface by the batimastat-sensitive
metallosecretase. In light of this observation, the conclusions of
earlier studies investigating the sequence requirements of various
secretases through analysis of the effect of mutations on the secretion
of the substrate protein require re-evaluation and should not be interpreted solely in the context of a single protease activity having
a relaxed specificity.
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EXPERIMENTAL PROCEDURES |
Construction of ACENQ--
The expression vector
pECE containing a C-terminal fragment of human ACE in which the
N-terminal signal peptide was fused with the C-terminal domain (pECE
hACE) (20) was used. In this construct the membrane-proximal stalk
region, transmembrane, and cytosolic domains are identical to those in
human somatic ACE. Asn631 (Fig.
1) in this construct, which corresponds
to Asn1196 in human somatic ACE, was the template for
oligonucleotide-directed mutagenesis with the Quick
ChangeTM in vitro mutagenesis system
(Stratagene) using the following oligonucleotides: ACENQup
(5'-TGG CCG CAG TAC CAA TGG ACG CCG AAC-3') and ACENQdo
(5'-GTT CGG CGT CCA TTG GTA CTG CGG CCA-3'). The mutation of T to C was
confirmed by sequencing, and the plasmid obtained was denoted pECE
hACENQ.

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Fig. 1.
Schematic diagram of angiotensin I-converting
enzyme. The single domain form of human ACE has an N-terminal
signal peptide (diagonally hatched box) and a C-terminal
transmembrane domain (black box) followed by a short
cytoplasmic tail (20). The amino acid sequence of the juxtamembrane
stalk region is shown and numbered below the sequence according to this
single domain form of ACE. The batimastat-sensitive secretase cleavage
site ( ) and the DCI-sensitive cleavage site ( ) are
indicated, and part of the hydrophobic transmembrane domain is in
italics and underlined. The Asn residue (631)
that was mutated to Gln is in bold and
underlined.
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Cell Culture--
The neuronal cell line IMR-32 (21) was
cultured in Dulbecco's modified Eagle's medium/Ham's F-12
supplemented with 10% fetal bovine serum, penicillin (50 units/ml),
streptomycin (50 mg/ml), and 2 mM glutamate (all from Life
Technologies, Inc.). Cells were maintained at 37 °C in 5%
CO2 in air, grown to 70% confluence in 25-cm2
flasks, washed once with Opti-MEM, and then transiently transfected using LipofectAMINE and 8 µg of DNA diluted in 2 ml of Opti-MEM. After 5 h, 3 ml of growth medium was added to the flasks, and the
cells were incubated overnight. The LipofectAMINE/medium mix was then
replaced with fresh growth medium. After another 24 h the cells
were washed with Opti-MEM and incubated with either batimastat
(provided by Dr. G. Christie, SmithKline Beecham Pharmaceuticals, Harlow, U.K.) or 1,3-dichloroisocoumarin (DCI, Sigma) for 7 h. The
medium was then harvested and centrifuged at 1000 × g
to remove cell debris. Cells were then washed in phosphate-buffered
saline (PBS), scraped into PBS, centrifuged at 1000 × g for 5 min, and resuspended in PBS. After sonication and
centrifugation at 5000 × g for 20 min to remove
nuclei, the cell membranes were pelleted by centrifugation at
100,000 × g for 90 min.
Metabolic Labeling and Phase Separation in Triton
X-114--
Transiently transfected IMR-32 cells were labeled 48-60 h
post-transfection with 80 µCi of [35S]Met in Met-free
Dulbecco's modified Eagle's medium containing 2% fetal calf serum,
50 units/ml penicillin, and 50 mg/ml streptomycin (denoted Met-free
medium). In pulse-chase experiments, labeling was performed for 1 h at 37 °C followed by a chase with nonlabeled Met for different
periods of time. The labeled cells were rinsed twice with PBS prior to
harvesting. Cells were subjected to phase separation in Triton X-114
essentially as described by Wilson et al. (22).
Immunoprecipitation and SDS-Polyacrylamide Gel
Electrophoresis--
Cells were solubilized with 1 ml/dish cold lysis
buffer (25 mM Tris/HCl, pH 8.0, 50 mM NaCl,
0.5% Triton X-100, and 0.5% sodium deoxycholate), and the cell
extracts were centrifuged at 10,000 × g for 15 min to
remove nuclei and debris. Thereafter, the supernatants were incubated
with the rabbit anti-ACE polyclonal antibody (RP183) (23) and
precipitated with protein A-Sepharose. After immunoprecipitation, the
protein A-Sepharose beads were washed three times with washing buffer A
(0.5% Triton X-100 and 0.05% sodium deoxycholate in PBS) and three
times with washing buffer B (500 mM NaCl, 10 mM
EDTA, and 0.5% Triton X-100 in 125 mM Tris/HCl, pH 8.0)
prior to analysis of the samples by SDS-polyacrylamide gel
electrophoresis and fluorography as described previously (24). The
digestion of 35S-labeled immunoprecipitates with
endo-
-N-acetylglucosaminidase (Endo) H and Endo
F/glycopeptidase F was performed as previously described (24).
Activity Assays--
ACE activity in the medium and membrane
samples was determined by incubation with 5 mM
benzoyl-Gly-His-Leu in 0.1 M Tris/HCl, pH 8.3, 0.3 M NaCl, and 10 µM ZnCl2 at
37 °C. The released benzoyl-Gly was separated from the substrate and
quantitated by reverse phase HPLC as described previously (25).
Isolation and Mass Spectral Analysis of Secreted ACE--
The
medium was collected from IMR-32 cells transiently transfected with
either pECE hACE or pECE hACENQ, centrifuged at 3000 × g for 5 min and then concentrated in a stirred
ultrafiltration cell (Amicon) using a 10-kDa cut-off membrane. The
samples were then dialyzed overnight against 10 mM
Hepes/NaOH, 0.3 M KCl, and 0.1 mM
ZnCl2, pH 7.5. ACE was then isolated by affinity
chromatography on lisinopril-Sepharose as described previously
(26). The enzyme was eluted from the affinity column with 0.1 M sodium borate, pH 9.5, dialyzed, and concentrated using
Centricon 10-kDa cut-off filters (Vivascience, Cambridge, U.K.).
Purified secreted wtACE and ACENQ proteins were reduced and
protected with vinyl pyridine prior to digestion with endoproteinase
Lys-C. The total digest was analyzed directly by matrix-assisted laser
desorption ionization time-of-flight mass spectrometry, or the digest
was first fractionated by HPLC and the C-terminal peptide was
identified by automated N-terminal peptide sequencing, before mass
spectral analysis (27, 28).
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RESULTS |
ACENQ Is Secreted from the IMR-32 Cells--
IMR-32
cells, which previously have been shown to cleave and release ACE in a
batimastat-sensitive manner (8), were transfected with cDNA
encoding either wtACE or ACENQ. Both constructs were expressed in the cells as determined by metabolic labeling with [35S]Met followed by immunoprecipitation from the cell
lysate with the anti-ACE antibody (Fig.
2). wtACE was present in the medium, consistent with its release from the cell surface by the
batimastat-sensitive secretase (Fig. 2). ACENQ was also
detected in the medium from the cells, and indeed the relative ratio of
ACE protein in the medium compared with the cell lysate appeared
greater for the mutant as compared with the wild-type protein.
Deglycosylation with Endo F revealed that both wtACE and
ACENQ were N-glycosylated (Fig. 2).
Deglycosylation revealed that there was a distinct difference in size
between wtACE in the cell lysate as compared with that secreted into
the medium, consistent with removal of the C-terminal transmembrane and
cytosolic domains of the protein by the secretase (9). Interestingly,
such a size difference was not apparent between the cell lysate and
medium samples of ACENQ (Fig. 2).

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Fig. 2.
Secretion of ACE from IMR-32 cells.
IMR-32 cells were transfected with either wtACE or ACENQ
cDNA and biosynthetically labeled for 6 h with
[35S]Met. ACE was then immunoprecipitated from the cell
lysate (L) or medium (M) with the anti-ACE
antibody, and the immunoprecipitate was incubated in the absence ( )
or presence (+) of Endo F for 2 h at 37 °C. The samples were
analyzed on a 6% polyacrylamide SDS gel and visualized by
fluorography.
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ACENQ Is Secreted into the Medium Faster than Wild-type
ACE--
The rate at which ACENQ was secreted into the
medium compared with that of wild-type ACE was assessed by pulse-chase
labeling (Fig. 3). wtACE appeared in the
medium after 4 h of chase, consistent with previous reports using
other cell lines (27, 29). At 6 h of chase a considerable amount
of wtACE was still present in the cell lysate. In contrast,
ACENQ was detected in the medium from the cells after 30 min of chase. After 4 h of chase the majority of ACENQ
had been secreted from the cell and was no longer present in the lysate
sample. The apparent larger size of the medium form of the mutant
protein compared with that in the lysate is because of complex
glycosylation of the protein as it traffics along the secretory
pathway. The ACENQ detected in the cell lysate is
mannose-rich glycosylated as assessed by its complete sensitivity
toward Endo H, whereas the ACENQ species secreted into the
medium was Endo H-resistant, indicating that it has acquired
complex-type glycans in the Golgi apparatus (data not shown).

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Fig. 3.
Transport kinetics of ACE. After
transfection with either wtACE or ACENQ cDNA, IMR-32
cells were biosynthetically labeled with [35S]Met for 30 min and chased with cold medium as indicated. ACE was then
immunoprecipitated from the cell lysate (L) or medium
(M) with the anti-ACE antibody, and the immunoprecipitated
samples were analyzed by SDS-polyacrylamide gel electrophoresis
followed by fluorography.
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ACENQ Is Not Cleaved by a Batimastat-sensitive
Secretase--
The ability of the secretase inhibitor batimastat to
block the release of wtACE and ACENQ from the IMR-32 cells
was assessed (Table I). As compared with
untreated cells, batimastat significantly inhibited (81%) the
release of wtACE into the medium with a concomitant increase in the
amount of activity detected in the membrane fraction, consistent with
previous results (8). Surprisingly, no ACE activity could be detected
in the membranes from the cells expressing ACENQ, and
batimastat failed to significantly inhibit the release of this
mutant form of the protein into the medium. No ACE activity was
detected in the total cellular membrane fraction from the untreated
cells expressing ACENQ even after an eight-times longer incubation period with the substrate benzoyl-Gly-His-Leu (data not
shown). Incubation of the cells expressing ACENQ with the serine protease inhibitor DCI reduced the secretion of the protein into
the medium by 75% with a concomitant increase in activity detected in
the membrane sample. Interestingly, DCI seemed to stimulate the release
of wtACE into the cell medium with a concomitant decrease in the
membrane fraction. This is consistent with a previous observation (27),
although the mechanism responsible for this increased shedding is not
known.
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Table I
Effect of inhibitors on the shedding of ACE and ACENQ
IMR-32 cells transfected with either wtACE or ACENQ cDNA
were incubated for 7 h in the absence of inhibitor or in the
presence of either 20 µM batimastat or 100 µM DCI. The medium was then harvested, and membranes were
prepared as described under "Experimental Procedures," and the
samples assayed for ACE activity with benzoyl-Gly-His-Leu as substrate.
Results are the mean ± S.E. of three experiments.
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ACENQ Is Cleaved in the ER--
The failure to detect
ACENQ enzymic activity in the total cellular membrane
fraction (Table I) suggested that this mutant form of ACE may be
cleaved soon after synthesis in the ER. To investigate this, IMR-32
cells expressing ACENQ were incubated at 15 °C to
prevent transport of the proteins beyond the ER (Fig. 4A). ACENQ was
susceptible to deglycosylation with Endo H, indicating that it was in
the high mannose form. Phase separation in Triton X-114 was used to
determine whether ACENQ retained in the ER by the
temperature block lacked the hydrophobic membrane-anchoring domain
(Fig. 4B). Although a significant amount of newly
synthesized ACENQ was detected in the detergent-rich
pellet, possibly because the 15 °C treatment impaired the activity
of the DCI-sensitive protease, a significant amount of a smaller form
was also present in the detergent-poor supernatant, consistent with
removal of the C-terminal membrane-anchoring and cytosolic domains by
an activity within the ER.

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Fig. 4.
Analysis of the cellular compartment of
ACENQ cleavage. After transfection with
ACENQ cDNA, IMR-32 cells were biosynthetically labeled
with [35S]Met at 15 °C for 4 h. A,
cells were lysed in lysis buffer, and ACE was immunoprecipitated with
the anti-ACE antibody prior to incubation in the absence ( ) or
presence (+) of Endo H for 2 h at 37 °C. B, the
cells were lysed in the presence of Triton X-114 followed by
centrifugation at 13,000 × g. Supernatant
(S) and pellet (P) fractions were
immunoprecipitated separately with the anti-ACE antibody and analyzed
by SDS-polyacrylamide gel electrophoresis followed by
fluorography.
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ACENQ Is Cleaved at a Different Bond in the
Juxtamembrane Stalk to wtACE--
The cleaved forms of both wtACE and
ACENQ were purified from the conditioned medium of
transfected cells by chromatography on lisinopril-Sepharose
(25). The purified proteins were digested with endoproteinase Lys-C and
subjected to matrix-assisted laser desorption ionization time-of-flight
mass spectrometry. Mass spectrometric analysis of wtACE secreted from
the IMR-32 cells revealed a peak at m/z 1690.8 (Table II), identical to the calculated
m/z for the peptide
Leu625-Arg638. This is consistent with the
normal secretase cleavage site between Arg638 and
Ser639 and is in agreement with the site of cleavage seen
in ACE secreted from other cells and in serum (9). In contrast,
analysis of the HPLC fractionated Lys-C digest of ACENQ
revealed a major peak at m/z 1406.3 (Table II),
which likely represented the peptide Leu625-Asn635 (m/z
1406.3, calculated m/z 1390.5: the increase in
mass is caused by the oxidation of Trp632). The identity of
this peptide was confirmed by partial N-terminal sequencing. Thus, the
major site of cleavage was at the
Asn635-Ser636 bond, three residues on the
N-terminal side of the normal Arg638 and Ser639
cleavage site (see Fig. 1). There was also evidence for secondary trimming of the C terminus of the soluble form of ACENQ to
Trp632.
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DISCUSSION |
The Asn to Gln point mutation in the juxtamembrane stalk of ACE
clearly results in more efficient cleavage and secretion of the
protein. At first sight this could be attributed to the mutation making
ACENQ a better substrate for the batimastat-sensitive cell surface metallosecretase. However, closer inspection clearly shows that
the shedding of ACENQ is caused by the action of a
spatially and mechanistically distinct secretase. The serine protease
cleaving ACENQ is acting in the ER and cleaves ACE at a
different bond in the stalk region to the normal secretase.
Interestingly a mutant form of ACE, ACE-JGL, in which the stalk region
had been replaced with a Ser/Thr-rich sequence that was partially
O-glycosylated, was observed to be secreted from Chinese
hamster ovary cells more rapidly than wtACE, and its shedding was not
blocked by the hydroxamic acid-based compound TAPI (27). Similar
to ACENQ, the shedding of ACE-JGL was blocked by DCI;
however, it was not determined whether cleavage of this mutant occurred
intracellularly. The major serine proteases in the secretory pathway
are the family of proprotein convertases, including furin, which are
involved in the proteolytic processing of a variety of secreted and
membrane-bound proteins (30). However, this family of proteases shows a
specificity for cleaving after dibasic or monobasic sequences, and
the cleavage site in ACENQ does not fit this
specificity (see Fig. 1). Also, the active forms of the proprotein
convertases are located primarily in the trans-Golgi network rather
than in the ER.
The effect of the Asn631 to Gln mutation invoking a
distinct secretase is in contrast to a recent report in which a
different point mutation in the juxtamembrane stalk of somatic ACE,
associated with a variation in the levels of soluble ACE in plasma,
resulted in enhanced cleavage by a metallosecretase (31). In that case mutation of Pro1199 in human somatic ACE (equivalent to
Pro634 in the ACE construct used in the present study; see
Fig. 1) to Leu resulted in more efficient cleavage/secretion of the
mutant protein. Although the cleavage of the P1199L mutant was
blocked by the hydroxamate-based metallosecretase inhibitor compound 3, the precise site of cleavage was not determined. Those authors, Eyries
et al. (31), proposed that a local conformational
modification caused by the Pro to Leu mutation leads to better
accessibility of the stalk region to the normal ACE secretase,
resulting in the enhancement of the cleavage/secretion process.
Secondary structure predictions of wild-type ACE using the on-line
JPred server indicated that the region from Leu620
to Asp657 is predicted to be a flexible loop, bounded by an
-helix on the N-terminal side and the transmembrane
-helix on the
C-terminal side. The mutation of Asn631 to Gln had no
affect on the secondary structure prediction, suggesting that the
observed difference in cleavage of ACENQ is not caused by a
dramatic change in secondary structure. However, it is possible that
the mutation promotes binding to the serine protease either directly
(Gln is a larger side chain and may be able to make more favorable
contacts with the enzyme) or indirectly (by allowing the loop to adopt
a more favorable conformation for binding).
The residue mutated in the present study, Asn631, lies in a
potential N-glycosylation sequon (Asn-Trp-Thr-Pro), raising
the possibility that this residue is glycosylated such that its
mutation to Gln prevents glycosylation and exposes a site, that is
normally obscured by the glycan chain, to cleavage by a serine protease
within the ER. However, this explanation seems unlikely on four counts.
1) Trp in the middle position of the N-glycosylation sequon
has been shown to severely reduce the efficiency of glycosylation of
the preceding Asn, whereas a Pro following the sequon prevents
N-glycosylation altogether (32, 33). 2) Consistent with
this, there is no evidence for glycosylation of this Asn residue in the
mature form of either human somatic or testicular ACE (Refs. 27 and 34 and Table II). 3) In rabbit ACE this potential glycosylation site is
absent, yet the enzyme is cleaved by a hydroxamic acid-sensitive metallosecretase (6). 4) Attempts to mimic the effect of the Asn to Gln
mutation in wild-type ACE by incubation of cells with tunicamycin,
which blocks formation of the core dolichol-linked oligosaccharide thus
preventing N-glycosylation from taking place, failed to lead
to secretion of wtACE in a batimastat-insensitive manner (data not shown).
An interesting feature of ACENQ is the dramatic increase in
its transport kinetics and maturation rate along the secretory pathway
as compared with the membrane-bound wild-type protein. In fact,
attainment of complex glycans on ACENQ occurs at a rate that is almost three orders of magnitude greater than that of wtACE. In
general it is unlikely that nonanchoring of a protein leads per
se to a higher efficiency in its intracellular transport relative
to its anchored counterpart. This view is supported by several examples
of proteins from which the transmembrane domains have been eliminated.
Membrane-bound intestinal sucrase-isomaltase, for instance, exhibits
similar transport kinetics as its soluble anchorless isoform (35).
Likewise, deletion of the transmembrane domain in the neurotrophin
receptor has no implications on the intracellular transport of the
mutant protein (36). Furthermore, there is no general rule that
anchorless proteins are transported more rapidly along the secretory
pathway than membrane-bound ones. Indeed, with certain proteins the
reverse seems to be the case. For example, deletion of the
membrane-anchoring domain from the murine prion protein results in the
secreted form of the protein reaching the cell surface at a 4-fold
slower rate than the wild-type protein (37), and the soluble form of
the trypanosome variant surface glycoprotein is also trafficked at a
slower rate than the membrane-bound form (38).
The main rate-limiting step in the transport of membrane and secretory
proteins from the ER to the Golgi apparatus is their acquisition to a
correct folding and transport-competent quaternary structure in the ER
(for a review see Ref. 39). In this respect, oligomerization in the ER
constitutes for many proteins a crucial criterion before they exit this
organelle. This event has been shown to involve the transmembrane
domains (40-45) (for a review see Ref. 46). Wild-type membrane-bound
ACE does not dimerize in the ER (47), thus excluding a possible
influence of dimerization and the transmembrane domain on its transport
kinetics as compared with soluble ACE. One may speculate that the
delayed intracellular transport of membrane-bound ACE versus
ACENQ is caused by the existence of a structural motif in
the cytosolic tail that promotes an interaction of ACE with soluble
and/or membrane-bound factors in the cytosol and thus regulates its
transport kinetics. As such, cleavage of ACENQ in the ER
eliminates this motif and results in a more efficiently transported protein.
In conclusion, in previous studies the results of mutations at or near
the secretase cleavage site in a membrane protein have usually been
interpreted in the context of a single protease activity having a
relaxed sequence specificity. However, the results of the present study
reveal another explanation: such mutations may invoke the action of
other distinct proteases. In the light of this and in the absence of
such additional data, the conclusions of earlier studies investigating
the sequence requirements of various secretases through analysis of the
effect of mutations on the secretion of the substrate protein must be
interpreted with caution and require re-evaluation.