From the Department of Pharmacology, Mayo Clinic
Jacksonville, Jacksonville, Florida 32224, the § Department
of Neuroscience, The University of Pennsylvania, Philadelphia,
Pennsylvania 19010, and the ¶ Laboratory for Mass Spectrometry,
The Rockefeller University, New York, New York 10021
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ABSTRACT |
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The 4-kDa amyloid -Secretase activity is the final cleavage
event that releases the amyloid
peptide (A
) from the
-secretase cleaved carboxyl-terminal fragment of the amyloid
protein precursor (APP). No protease responsible for this highly
unusual, purportedly intramembranous, cleavage has been definitively
identified. We examined the substrate specificity of
-secretase by
mutating various residues within or adjacent to the transmembrane
domain of the APP and then analyzing A
production from cells
transfected with these mutant APPs by enzyme-linked immunosorbent assay
and mass spectrometry. A
production was also analyzed from a subset
of transmembrane domain APP mutants that showed dramatic shifts in
-secretase cleavage in the presence or absence of pepstatin, an
inhibitor of
-secretase activity. These studies demonstrate that
-secretase's cleavage specificity is primarily determined by
location of the
-secretase cleavage site of APP with respect to the
membrane, and that
-secretase activity is due to the action of
multiple proteases exhibiting both a pepstatin- sensitive activity and
a pepstatin-insensitive activity. Given that
-secretase is a major
therapeutic target in Alzheimer's disease these studies provide
important information with respect to the mechanism of A
production
that will direct efforts to isolate the
-secretases and potentially
to develop effective therapeutic inhibitors of pathologically relevant
-secretase activities.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
protein
(A
)1 that is invariably
deposited as amyloid in Alzheimer's disease (AD) is a normally
secreted proteolytic product of the amyloid
protein precursor (APP)
(1-3). Generation of A
from APP requires two proteolytic events,
one at the amino terminus referred to as
-secretase and one at the carboxyl terminus known as
-secretase (Fig.
1). To date, neither of the proteases
responsible for these activities has been definitively identified.
Comparison of the soluble A
secreted by cells, soluble A
in
cerebrospinal fluid, and insoluble A
isolated from the AD brain has
revealed that there are numerous A
species with extensive amino- and
carboxyl-terminal heterogeneity. The major A
species in both
conditioned cell culture media and human cerebrospinal fluid is
A
1-40 (~50-70%) although some A
1-42 (5-20%) is also present along with minor amounts of other peptides (e.g.
A
1-28, A
1-33, A
1-34, A
3-34, A
1-37, A
1-38, and
A
1-39) (4-6). The importance of the longer forms of A
, in
particular A
42, has been heightened by the fact that all of the
familial Alzheimer's disease (FAD) linked mutations that have been
analyzed result in an increase in the concentration of A
42 in a wide
variety of model systems (reviewed in Refs. 7 and 8). Biophysical studies have shown that the longer forms of A
aggregate at a much
faster rate and at lower concentrations than forms ending at A
40
suggesting that alterations in A
42 concentration, as occurs in FAD
linked forms of the disease, may account for the observation that the
longer forms of A
are often the initial species deposited in the
parenchyma of the AD and Down's syndrome brain (9-11).
View larger version (41K):
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Fig. 1.
Cleavage of APP to generate
A . The APP is cleaved at the amino
terminus of the A
sequence by an unknown protease referred to as
-secretase. This cleavage releases a large secreted derivative
referred to as sAPP
. Subsequent cleavage of the APP COOH-terminal
fragment (C99) by
-secretase generates A
. In the
-secretory pathway (not shown) the APP is cleaved within the A
region to generate a large secreted derivative referred to as sAPP
.
Subsequent cleavage of the APP COOH-terminal fragment (C83)
by
-secretase generates a truncated A
species referred to as
P3.
In addition to the - and
-secretase activities that generate
A
, a third proteolytic activity referred to as
-secretase cleaves
within the A
sequence at Lys16 to release a large
secreted derivative (12-14), thus precluding formation of full-length
A
. Following cleavage of APP at the extracellular/lumenal domain of
the APP by either
- or
-secretases,
-secretase cleavage of the
resulting COOH-terminal fragment results in the release of the p3 or
A
, respectively.
The findings that all of the FAD-linked mutations in APP and the
presenilin (PS) genes alter the concentration of A ending at
position 42 and, with the exception of the APPK670N,M671L
(NL) mutation, appear to act by altering
-secretase activity makes
understanding this activity pivotal to our knowledge of the disease
process. While previous studies have shown that
-secretase cleavage,
like most proteolytic cleavages, exhibits fairly rigid primary amino
acid sequence requirements (15), similar studies of the more complex
-secretase activity demonstrates fairly loose specificity (16, 17).
These studies, however, which either examine effects on total A
production (16) or effects of mutations at a single residue, A
43
(17), provide no compelling mechanisms for the observed alterations in
cleavage associated with FAD-linked mutations.
One of the more unusual aspects of -secretase cleavage is that based
on hydropathy plots the
-cleavage sites lie within the putative
transmembrane domain (TMD) of the APP (18). Several other proteins,
SREPB (19), Notch (20), and mitochondrial inner membrane proteins (21),
have been postulated to undergo such intramembranous cleavage. While
recent data for SREBP site two protease cleavage (22) and Notch (20),
indicate that the cleavage of these proteins occurs at hydrophobic
residues near the transmembrane junction, in no instance has the site
of cleavage and the location of the residues with respect to the
membrane at the time of cleavage been elucidated. Thus, the concept of intramembranous proteolysis remains controversial. To date, there is no
definitive evidence showing that a protease can cleave bonds buried
within a membrane.
As a first step toward defining the mechanism of -secretase
cleavage, we undertook a mutagenesis study in an attempt to define the
specificity and structural requirements that produce A
species of
varying lengths by using a large number of APP TMD mutants. The TMD
mutant APPs are shown in Fig. 2. These
mutations can be divided into several different categories. 1) Point
mutations at positions A
41 (I637X based on the APP695 sequence) and
A
43 (T639X) which correspond to the P1' positions for a
-secretase cleavage producing A
1-40 and A
1-42, respectively.
2) Deletion and insertion mutations designed to alter the localization
of the
-secretase cleavage site within the membrane (del and ins). 3) Point mutations that alter the putative membrane stop anchor signal
or increase the number of charged residues on the lumenal side of the
membrane and 4) replacement of residues carboxyl to the normal
-cleavage sites with alanine. All mutations were made in a
background of the APP695NL mutation in order to increase absolute
amounts of A
peptides generated without affecting
-cleavage as
this mutation increases activity at the
-secretase site without altering the relative amounts of either A
40 or A
42 (5,
23-25).
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EXPERIMENTAL PROCEDURES |
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Generation of Mutant APPs--
A two-step PCR based mutagenesis
strategy was employed to generate the various TMD domain mutants. In
the first step, the EcoRI/NotI fragment of
pcDNA3APP695NL was replaced with a PCR product generated from
wild-type APP695 using the forward primer APP+1803 and the reverse
primers 26 (5'-CATGCGGCCGCTCGTCTCTTGAACCCACATCTTCTGCA-3'),
39 (5'-CATGCGGCCGCTCGTCTCCAACACCGCCCACCATGAGT-3'), and
52
(5'-CATGCGGCCGCTCGTCTCTCAGCATCACCAAGGTGATGA-3'), respectively, to
generate the base constructs pcDNA3APP695NL
26, pcDNA3APP695NL
39, and pcDNA3APP695NL
52. These mutant
APPs incorporated a class IIa restriction site, BsmBI,
3' to A
26, A
39, and A
52. When cut with BsmBI and
NotI the BsmBI site is lost, leaving a 5' 4-bp
overhang at the 3' end, effectively truncating the APP. To produce the
various TMD mutants in this study, oligonucleotides incorporating the
various mutations were generated containing a BsmBI site at
their 5' end. PCR products were amplified using various mutant
oligonucleotides and a common reverse primer using wild type APP as
template. Subsequent cleavage of these products with BsmBI
and NotI followed by cloning into the appropriate base vectors (pcDNA3APP695NL
26, pcDNA3APP695NL
39, and
pcDNA3APP695NL
52) generated the desired mutants with no
additional base changes. All PCR reactions were carried out using the
High Fidelity PCR Kit from Boehringer Mannheim. All mutant cDNAs
were sequenced to ensure that no additional mutations were
incorporated. Sequences for all of the PCR primers used are available
upon request.
Transient Transfection of 293 T Cells and Media
Collection--
Cells were plated onto 6-well culture dishes (Corning)
and grown to 70-80% confluence in Dulbecco's modified Eagle's
medium (HyClone) supplemented with 10% fetal calf serum and 1%
penicillin/streptomycin solution (Life Technologies, Inc.). On the day
of transfection, culture media was changed to Opti-MEM (Life
Technologies, Inc.). After a short period of equilibration, the media
was again changed to fresh Opti-MEM (0.5 ml) and 1.5 µg of DNA and
4.0 µg of DOSPER liposomal transfection reagent (Boehringer Mannheim)
were added to each well (premixed) in a volume of 0.5 ml of additional
media. After overnight incubation, the media were discarded and
replaced with Dulbecco's modified Eagle's medium, 10% fetal calf
serum (1.0 ml/well). Twenty-four hours later, the media were collected and replaced with serum-free Dulbecco's modified Eagle's medium; complete protease inhibitor mixture (Boehringer Mannheim) was added to
each sample. The serum-free media were collected 24 h later, and
phenylmethylsulfonyl fluoride (to 1 mM) and EDTA (to 5 mM) added to each sample. In both cases, samples were
immediately centrifuged at high speed to pellet cellular debris,
transferred to clean Microfuge tubes, and frozen in aliquots at
80 °C until analyzed.
Pepstatin treatment was conducted by adding to the media (post-transfection) pepstatin A to a concentration of 100 µg/ml and Me2SO to 2%. Control cultures were treated with 2% Me2SO alone. Transfections and media collections were otherwise identical to untreated 293T cells.
sAPP Competitive ELISA-- To generate and purify recombinant human sAPP, a 6-histidine tag was placed in-frame near the amino terminus of human APP695 by cleavage with KpnI and in-frame insertion of a double-stranded DNA formed by annealing two oligonucleotides. After construction in the expression vector pAG3, the APP695nterm his was stably transfected into CHO cells. For purification, after 48 h serum-free media was collected and purified using Ni2+ affinity resin (Qiagen). To perform sAPP ELISAs, MaxiSorp 96-well immunoplates (Nunc) were coated overnight at 4 °C with 400 ng/ml purified recombinant sAPP in 100 µl of 0.1 M sodium carbonate buffer per well (pH 9.6). Plates were then blocked overnight at 4 °C with 1% Block Ace, 0.05% NaN3 in PBS (300 µl/well). Blocking solution was discarded, the plates washed twice with PBS, and the wells loaded with 40 µl of 1% bovine serum albumin in PBS (PBSB) and 10 µl of each sample, in duplicate. Standard samples, containing from 31.5 to 8000 ng/ml sAPP in PBSB, were also loaded in duplicate. The NH2-terminal APP antibody 207 was added (100 µl) to each well, diluted 1:32,000 in PBSB. Following overnight incubation at 4 °C, wells were washed twice with PBS, 0.05% Tween 20, twice with PBS, and then loaded with 100 µl of horseradish peroxidase-conjugated anti-goat IgG (1:5000 in PBSB; Rockland Immunochemicals). After a 4-h incubation at room temperature, plates were washed as above, then developed with 100 µl of TMB solution (Kirkegaard & Perry). The reaction was stopped after approximately 5 min with the addition of 100 µl of 6% phosphoric acid and the plates read at 450 nm. These conditions were determined empirically to optimize detection of sAPP in transfected cell lines in our laboratory, with typical expression levels placing sAPP concentrations in a linear region of the standard curve.
A Sandwich ELISAs and Normalization of A
Measurements--
For determination of A
concentrations we used 3 well characterized sandwich ELISA systems. Total A
was determined by
3160 capture and detection with either 4G8 or BNT77 (9, 26), and A
40
and 42 were determined by BAN50 capture and detection with either BA27
or BC05, respectively (5). To control for variance in transfection
efficiency and the amount of APP that is appropriately inserted in the
membrane and trafficked through the secretory pathway (thus APP
available for
-secretase cleavage) A
levels were normalized to
sAPP expression. This was accomplished by dividing the A
values
(fmol/ml) by the sAPP values (ng/ml) resulting in a normalized A
value (fmol/ng). The normalized A
value for each mutant was then
divided by the normalized A
value for APP695NL to give the %NL
A
.
Metabolic Labeling and Immunoprecipitation of sAPP and Full-length APP-- 48 h after transfection, 293T cells were labeled for 2 h with 100 µCi/ml [35S]methionine. Immunoprecipitation of full-length APP (flAPP) with Ab369W (27) from Triton X-100 cell lysates and sAPP in the 2-h conditioned media with antibody 207 were carried out as described previously (28, 29). Each analysis was performed in duplicate. Immunoprecipitated proteins were separated on 10% Tris-Tricine gels. PhosphorImaging analysis of the dried gels was performed. After subtracting the number of pixels of flAPP or sAPP present in the mock transfected (vector alone) sample from each of the experimental samples, the ratio of sAPP/flAPP was calculated by dividing the pixels of sAPP by the pixels of flAPP. sAPP/flAPP ratios were then compared with the ratio of sAPP/flAPP calculated for APP695NL. TMD mutants showing sAPP/flAPP ratios greater than 50% of APP695NL ratio were assumed to have preserved processing in the secretory pathway.
Mass Spectrometric A Analysis--
Serum-free media
(Dulbecco's modified Eagle's medium) collected for a 24-h period
beginning 24 h post-transfection was used for mass spectrometric
analysis. A
peptides were analyzed by immunoprecipitation/mass
spectrometric A
assay as described previously (4). The A
peptides
were immunoprecipitated from 1.0 ml of conditioned media using
monoclonal anti-A
antibody, 4G8 (Senetek, Maryland Heights, MO), and
protein G Plus/Protein A-agarose beads (Oncogene Science, Inc.,
Cambridge, MA) and analyzed using a matrix-assisted laser
desorption/ionization time of flight mass spectrometer (Voyager-DE STR
BioSpectrometry Workstation, PerSeptive Biosystem). Each mass spectrum
was averaged from 500 measurements and calibrated using bovine insulin
as the internal mass calibrant. For comparing the peptide levels
secreted by cells expressing different mutations and between the
treatments of Me2SO and pepstatin, synthetic A
(12-28)
peptide (10 nM) was used as internal standard.
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RESULTS |
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Transmembrane Domain Mutations: Effects on Total A
Levels--
APP appears to be a substrate for
-secretase only after
it has been cleaved by
- or
-secretase (30). Because cleavage of
APP by these activities requires normal membrane insertion and
trafficking through the secretory pathway, we have only performed in
depth analyses of APP TMD mutants that showed preserved processing by
these activities and normalized A
production to sAPP levels. Processing in the secretory pathway was assessed by metabolic labeling
experiments and determination of the sAPP/flAPP ratio in comparison to
APP695NL as described under "Experimental Procedures" (data not
shown). To examine A
production in the mutants that showed preserved
processing in the secretory pathway, sAPP, total A
, A
1-40, and
A
1-42 were measured in the conditioned media by ELISA. Comparison
of normalized total A
shows that most mutants resulted in modest to
moderate decrements in A
production (Table I). Even strikingly non-conservative
mutations at residues 637 (A
41) or 639 (A
43) had relatively
modest effects on total A
production. Basic (Lys) or acidic (Glu)
amino acid substitutions at both sites, and substitutions of amino
acids with a large hydrophobic side chain (Phe and Trp) or even a
proline at position 637, did not prevent
-secretase cleavage. In
addition, production of A
was only modestly decreased by most
mutations at or near the lumenal transmembrane domain junction or by
deletion or insertion mutants. The notable exceptions being the large
decrements in A
production by del640-43, 624-626E, and ins625-631
mutants.
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Six mutants (I637R, T639P, 649-651E, 649-651D, del625-631, and
del640-647) showed dramatic decreases in A levels (<5% of APP695NL A
, data not shown). Pulse-chase analyses showed that these
mutations produced equivalent levels of full-length APP, but that the
sAPP/flAPP ratio was markedly diminished (<1% compared with APP695NL,
data not shown). Although the precise mechanism for the impaired
processing of these mutations is not definitively established by these
studies, based on previous studies (16) and our metabolic labeling data
it is likely that these mutants either alter membrane insertion or
prevent proper trafficking through the secretory pathway. In agreement
with this, one of these mutations (649-651E) has previously been shown
to result in a non-transmembrane, membrane-associated cell surface,
full-length APP (31).
Effects on Cleavage Site--
Comparison of the relative amounts
of A1-40 to A
1-42, measured, respectively, by BAN50/BA27 or
BAN50/BC05 ELISA, shows that many of these mutants dramatically alter
the relative amounts of the major A
species produced. These data are
expressed as the percent of total A
that A
1-40 and A
1-42
species represent (Table I). For the I637X mutants, detection of
A
1-42(43) species could be impaired as the 42(43) end specific BC05
detection antibody recognizes an epitope partially determined by the
residue at A
41; thus, altered A
42 peptides could be produced but
not detected (5). To illustrate how these data indicate shifts in
cleavage it is useful to look at the effects on %A
1-40 and
%A
1-42 in the ins625-628 mutant. Comparison of the %A
1-40
and %A
1-42 illustrates that the major shift in cleavage is a
reduction in processing at the A
40 cleavage site (2% of total
versus 51% of total in the APP695NL construct) while A
42
cleavage is preserved (4.6% of total versus 3.3% of total
in the APP695NL construct). These analyses also show that only 7% of
total A
is accounted for by A
1-40 and 1-42 versus
54% in the APP695NL construct, indicating that the vast majority of
A
peptides generated from this mutant are cleaved at a different
site or sites than the ones normally utilized. By these criteria,
almost all of the mutants show shifts in
-secretase cleavage. In the
last column of Table I, the statistically significant shifts in
cleavage for each of these mutations are summarized.
Mass Spectral Analysis--
In order to determine exactly how
cleavage is shifted by the TMD mutations, A produced from 293T cells
expressing APP695NL or select TMD mutant APPs was analyzed by
immunoprecipitation/mass spectrometric analysis (4). The monoclonal
antibody, 4G8, was used to immunoprecipitate A
from serum-free
conditioned media. This antibody recognizes A
17-24 (32), a region
of the A
not altered by the mutants used in this study, making it
highly unlikely that this analysis would be biased by selective
immunoprecipitation of different A
species. The molecular masses of
various A
peptides were measured in these analyses by using internal
mass calibrants, bovine insulin and A
12-28 peptide. These masses
were then used to identify A
peptides produced by the TMD mutant
APPs and infer the
-secretase cleavage sites as illustrated in Fig.
3. The relative peak intensity was used
to determine the relative abundance of A
peptides within each
spectra resulting from each TMD mutant APP. For clarity, all A
peptides are numbered according to the cleavage sites in wild type A
species; thus A
1-43 in del625-628 is a 39-amino acid A
peptide
derivative. Representative mass spectra for APP695NL, I637F, I637P, and
ins644-647 are shown in Fig. 3A.
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The mass spectrum of A produced from APP695NL shows that A
1-40
was the major A
species and that the minor A
species were A
1-42, A
1-39, A
1-38, and A
1-37. Based on comparison to
APP695NL, several of the different TMD mutations analyzed dramatically
shift the
-secretase cleavage site. Mass spectrometric A
analyses of APP695NL and TMD mutants are schematically summarized in Fig. 3B. The largest shifts in cleavage site utilization are seen
in the ins625-628 and del625-628 mutants, while less dramatic effects are seen with the del644-647 and ins644-647 mutations. G625K also increases long A
production, while G625P increases production of
shorter A
peptides. Introduction of Lys at position 637 or 639 has
only minor effects on cleavage, while substitution of Ala at 639 increases both long A
(A
1-42) and short A
(A
1-38) production. Pro or Phe substitutions at Ile637 dramatically
shift cleavage away from A
40. Finally, A
production is only
minimally affected by the 640-648A mutant. Notably, the mass spectral
data and the ELISA data are remarkably consistent with the exception
being that small amounts of A
1-42 detected by ELISA are not always
detected by mass spectrometric analysis (for example, in 640-648A) due
to the slightly lower detection efficiency of A
1-42 (4).
Pepstatin Treatment of Selected Mutants--
Given the loose
sequence specificity exhibited by -secretase, we postulated that the
-secretase activity responsible for generating the various A
species was unlikely to be due to a single protease. Therefore, we
treated cells transfected with several of the TMD mutants that shift
cleavage with pepstatin, an inhibitor of normal
-secretase activity.
Although it is not known whether pepstatin directly inhibits
-secretase, pepstatin treatment of cells transfected with APP
COOH-terminal fragments LC99, APP695wt, or APP695NL results both in
accumulation of COOH-terminal fragments and a decrease in A
production2 without
significantly altering sAPP secretion in either APP695wt or APP695NL
(Table II), consistent with it being an
inhibitor of
-secretase activity. Because cells are rather
impermeable to pepstatin, it is necessary to treat them with high
concentrations (100 µg/ml) in the presence of 2% Me2SO
in order to obtain effective intracellular concentrations (33). Again
sAPP, total A
, and A
1-40 and 1-42 were measured by ELISA from
conditioned media. Treatment of cells with vehicle alone and vehicle
with pepstatin had no overt toxic effect in the time course studied,
and although changes in sAPP levels were observed in individual
experiments in some mutants, none of these changes reached statistical
significance (Table II). Pepstatin treatment of cells transfected with
the APP695NL mutant decreased total A
and A
1-40 equally while
A
1-42 was less affected. The effects on three of the TMD domain
mutants: del625-628, G625P, and I637F were similar to the effect on
APP695NL. In contrast, pepstatin treatment did not alter A
production from the I637P mutant to any significant extent. The effects
of pepstatin on T639K, a mutant that had only subtle effects on
cleavage site utilization, were also distinct from APP695NL. A
1-40
was only modestly inhibited and A
1-42 was not inhibited. Although
A
1-40 produced from ins625-628 represents only a minor fraction of
the total A
produced, pepstatin actually increased the amount of A
1-40 produced from 15 to 50 fmol/ml.
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To definitively identify how pepstatin altered cleavage of these TMD
domain mutants, A secreted from treated and untreated cells was
analyzed by mass spectrometry (Fig. 4).
For APP695NL, G625P, I637F, and T639K, the relative peak heights of
each of the major A
species was similar before and after pepstatin
treatment, indicating that cleavage was inhibited equally at each site
(Fig. 4, data shown only for APP695NL). Consistent with the ELISA
analysis, the relative peak height of A
1-42 was not reduced as much
by pepstatin treatment in APP695NL and G625P. The effect of pepstatin treatment on del625-628 also revealed little difference in relative peak heights of the major A
species except that peaks corresponding to A
1-45 and A
1-46 were increased suggesting enhanced
utilization of minor cleavage sites (Fig. 4, G and
H). In the ins625-628 and I637P TMD mutants, there were
some marked shifts in the relative peak intensities of some A
species after pepstatin treatment. In ins625-628 the A
1-33 peak
was decreased more than the A
1-37 peak (Fig. 4, E and
F), while in I637P the relative peak intensity of A
1-37
decreased and the relative peak intensity of A
1-43 increased (Fig.
4, C and D). Thus, there appears to be
differential sensitivity of certain
-secretase cleavages to
pepstatin and increased utilization of alternative sites by
pepstatin-insensitive proteases in certain mutants when
pepstatin-sensitive cleavage is inhibited.
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DISCUSSION |
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APP Transmembrane Domain Mutants Shift -Secretase
Cleavage--
An unanticipated result of this analysis is that many
TMD mutations alter the major sites of
-secretase cleavage. The most striking finding resulting from the analyses of the mutations, ins625-628 and del625-628, is that the major
-secretase cleavage site appears to be determined by the length of the TMD lumenal to the
normal
-secretase sites. In wild type APP, the major
-secretase site carboxyl to A
40 lies 12 amino acids from the lysine at A
28 (KA
28) that is predicted to delineate the lumenal TMD boundary. In
these mutations, the normal
-site is shifted either 4 amino acids
(del625-628) closer or farther away (ins625-628) relative to KA
28.
Although the primary sequence surrounding the normal
-site is
unaltered, the major cleavage shifts to the 13th amino acid
(A
37) from KA
28 in ins625-628 or the 11th amino acid
(A
43) from KA
28 in del625-628. Only a minor portion of A
produced from either mutant is cleaved at the V-I bond at A
40 (14%
in del625-628 and 2% in ins625-628). Mutations altering charge at
the presumptive lumenal border of the transmembrane domain (625-626K,
624-626E, and 624-626D) had a similar effect as the del625-628
mutant increasing production of longer A
peptides. These mutants
might be predicted to decrease the length of the TMD proximal to the
-secretase site by several amino acids resulting in increased
cleavage at sites distal to A
40.
The insertion and deletion mutants designed to alter the length of the
TMD distal to the normal -cleavage sites had much subtler effects.
The major effect of the ins644-647 mutation was to decrease cleavage
at A
40 and increase cleavage at A
38. The major effect of the
del644-647 mutant was to modestly decrease A
1-42 production. This
is similar to the effect seen in the I637K and T639K mutants, where
A
1-42 production is decreased. In all three cases, increased
positive charge has been placed closer to the normal A
42 cleavage
site. Replacement of residues 640-648 with alanine (640-648A) had
only a minor effect on A
production shifting cleavage away from the
A
40 site (28% A
1-40) indicating that this region is not as
crucial in determining the membrane positioning of the
-secretase
sites in comparison to the region amino to the
-secretase cleavage
site. This finding is similar to a previous report showing that
substitution of APP residues 635-642 or 636-653 with the
corresponding residues of the epidermal growth factor receptor TMD did
not significantly impair A
production (16). Nevertheless,
alterations in this region do result in subtle, but important, shifts
of
-cleavage. All FAD-linked mutations in this region, V641I, V641F,
V641G, and I640V (based on the APP695 sequence), replace hydrophobic
residues in the transmembrane domain with other hydrophobic residues
and each results in increases in A
42 (5, 17, 34; reviewed in Refs. 7
and 8). In this study, a similar substitution, T639A, increased A
42
cleavage. This result is similar to a recent report demonstrating that
hydrophobic substitutions at A
43 increased A
42 production
relative to A
40 (17). However, that study only looked at ratios of
A
42:A
40; thus, alternative cleavages would have been missed, and
it is unclear from the data presented whether the increase in the ratio is due to a decrease in A
40 production or an increase in A
42 production. Of the mutations on the carboxyl side of the
-site, only
del640-643 had dramatic effects on A
, significantly decreasing total A
production and markedly increasing %A
1-42 production. This mutant's more dramatic effect compared with deletions or insertions downstream could be explained by the fact that this region
may represent the end of the APP TMD (16). Taken together, these data
indicate that mutations in the lumenal portion of the TMD have effects
on cleavage that are much more profound than mutations which alter
charge or TMD length on the cytoplasmic side of the normal
-secretase site.
-Secretase Is Not a Single Proteolytic Activity--
Our data
shed some light on the proteolytic mechanism responsible for generation
of A
peptides of various lengths. The finding that the del625-628
preferentially secretes an A
species of 39 amino acids ending at
A
43 is evidence against a mechanism in which a single endoprotease
cleavage occurs carboxyl to the major
-sites followed by trimming by
carboxypeptidase. However, for any given mutation, we cannot rule out
the possibility that shorter A
species are generated through
carboxypeptidase degradation of longer A
peptides. Furthermore, the
differential effect of pepstatin on select TMD mutants is most
consistent with the action of at least two and possibly more
proteolytic activities. This differential inhibition is most clearly
demonstrated in the I637P mutant. This mutant is readily processed into
A
but is almost completely pepstatin insensitive. Mass spectral
analysis indicates that the
-secretase that generates A
1-43 in
the I637P mutant is pepstatin-resistant, while a different
-secretase that cleaves this mutant at A
1-37 is
pepstatin-sensitive. When cleavage is inhibited at A
37 by pepstatin
a corresponding increase is seen in the cleavage at A
43, resulting
in almost identical amounts of total A
secreted. Similarly, other
TMD domain mutants as well as APP695NL exhibit both a
pepstatin-sensitive and a pepstatin-resistant
-secretase activity.
Previous reports based on differential inhibition of A40 and A
42
cleavage have indicated that these A
species may be generated by
distinct proteases. Unfortunately, interpretation of these previous
studies has been difficult. The compound, MDL 21760, used in several
studies could have altered trafficking of APP as it markedly increased
sAPP (35, 36). In other studies, reporting an A
1-40 inhibitory
effect of calpain 1 inhibitor, no controls for either APP synthesis or
sAPP production were reported (37, 38). In fact, only one potential
-secretase inhibitor, a substrate-based difluoroketone compound,
altered
-secretase activity without apparent alteration of other
secretase activity (39). In this study, pepstatin had no toxic effects
and no significant effect on sAPP production from APP695NL; yet, it was
a less effective inhibitor of A
1-42 (~35% of control) production
than A
1-40 (~20% control).
The differential inhibition observed at A40 and A
42 sites could
be due to a number of mechanisms. Since A
1-42 has previously been
shown to be specifically generated in the endoplasmic reticulum (although endoplasmic reticulum-derived A
1-42 is not secreted), it
is possible that this difference simply reflects a varying degree of
organelle penetrance (a similar argument could be made for any of the
other published inhibitors) (40-42). Alternatively, this differential
inhibition could be viewed as additional evidence that multiple
proteases generate both A
40 and A
42, but that pepstatin-sensitive
cleavage is responsible for a higher percentage of activity at the
A
40 site. The finding that differential inhibition of A
1-40 and
A
1-42 was constant among several of the TMD mutants which alter
cleavage site preference (del625-628, G625P, I637F), but not the T639K
mutant supports this notion of multiple proteases as it is most
consistent with enhancing the A
40 and A
42 cleavages by a
pepstatin-insensitive protease. Based on our results, we would predict
that for wild type substrate a pepstatin-sensitive protease cleaves
both A
40 and A
42, with preferential specificity for cleavage at
A
40 and that additional pepstatin-insensitive proteases account for
~20% of the cleavage events at A
40 and ~35% of the cleavage
events at A
42. Thus, when the major A
40 cleavage site is
"protected" (see below) from cleavage in the del625-628 mutant
(14% A
1-40 versus 51% in APP695NL, Table II) and
shifted to A
1-42(43) (48% versus 3.3% in APP695NL,
Table II), a remarkably similar degree of inhibition is seen on both species following pepstatin treatment. A final mechanism in which a
single pepstatin-sensitive protease cleaves both sites but is differentially inhibited could also be considered. This mechanism, however, is not consistent with known models of proteolytic inhibition by pepstatin and would be an unprecedented mechanism of action for a
protease inhibitor that acts in a competitive fashion.
Implications for Models of -Secretase Cleavage: the Length of
the Lumenal Portion of the TMD Is the Prime Determinant of
Cleavage--
Until the
-secretases are definitively identified and
the location of the residues at the time of cleavage defined, the issue of intramembranous proteolysis is likely to remain controversial. Unless
-secretases are completely novel proteases that can cleave proteins in the hydrophobic environment of the membrane, mechanisms must exist that permit membrane-embedded regions of polypeptides to
transfer from the lipid bilayer into a hydrophilic proteinaceous environment that supports proteolysis. This could occur in one of two
fashions. First, as has been generally proposed,
-secretase activity
may be due to the action of proteases or proteolytic complexes that are
capable of creating a pore or hydrophilic pocket within the membrane.
Based on our data, in this model the interaction of the protease with
the TMD domain of the APP lumenal to the
-secretase site would
appear to be a critical factor in determining cleavage (see Fig.
5A). A second possibility is
that these cleavages require translocation of the cleavage sites out of
the membrane (Fig. 5B). One of the similarities between the
-secretase-like cleavages in APP, Notch and SREBP, is that the
potentially intramembranous cleavage occurs only after an initial
cleavage or cleavages that remove significant portions of the protein
amino-terminal to the intramembranous cleavage site. Thus, it is
possible that the sites are normally protected by the membrane prior to
the primary lumenal cleavage of the holoprotein. Once the lumenal
domain is cleaved, the protein either assumes a different conformation
or is actively translocated resulting in exposure of the
intramembranous site to the cytoplasm where it can be cleaved. Such a
cut-expose-cut model for the APP is illustrated in Fig. 5B.
In this model one could envision slippage or translocation of the
intramembranous site in either direction; for APP we propose that the
-site is exposed to proteases that are associated directly or
indirectly with the membrane and have active sites facing the
cytoplasm. This model, while subject to some potential thermodynamic
penalties associated with translocation of the APP TMD, is consistent
with our data showing that length of the TMD lumenal to the
-cleavage site has more profound effects on
-secretase cleavage
than mutations carboxyl to the normal
-sites, offers a very simple
explanation as to why these sites are resistant to proteolysis in the
intact holoprotein, is consistent with another model of intramembranous cleavage (21), and does not implicate an unprecedented type of
proteases in this cleavage.
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In either model proposed above, it is apparent that the prime
determinant of -secretase cleavage is the length of the
transmembrane domain proximal to the
-secretase cleavage site. In an
intramembranous cleavage model, we would predict that the position of
the active site of the protease is relatively fixed within the membrane
and that the protease recognizes determinants in the APP TMD lumenal to
the normal cleavage site. Thus, varying the length of the lumenal TMD
alters the residues that contact the active site. In contrast, the
membrane serves as the delineating factor in the cut-expose-cut model,
residues buried within the membrane are protected from cleavage while
exposed residues are cleaved.
Because at least two, and possibly more, proteolytic activities appear
to contribute to -secretase cleavage, defining the substrate
specificity is problematic until the secretases are definitively
identified. Nevertheless, it is almost certain that any individual
-secretase would exhibit at least some sequence specificity as even
nonspecific proteases such as proteinase K preferentially cleave
certain substrates (43, 44). Consistent with this, altering sequence at
the normal
-cleavage site has dramatic effects on recognition of the
normal cleavage site by the major pepstatin-sensitive
-secretase
activity which results in either increased cleavage by a
pepstatin-sensitive protease at sites other than A
40 and A
42,
increased cleavage by a pepstatin-insensitive protease at normally
utilized sites, increased cleavage by a pepstatin insensitive protease
at A
40 and A
42, or a combination of these alterations.
Multiple Cellular Factors Could Influence -Secretase
Cleavage--
If
-secretase cleavage is primarily dependent upon
the location of the
-secretase cleavage site with respect to the
membrane or active site of a protease within the membrane, then it is
likely that a number of cellular factors could influence this cleavage including membrane thickness or composition and interaction with other
proteins. PSs, which are important regulators of
-secretase activity
(reviewed in Refs. 7 and 8), could influence
-secretase cleavage of
APP by altering trafficking of APP to different cellular microdomains
where cleavage would be influenced by membrane thickness, by directly
interacting with APP carboxyl-terminal fragments and positioning them
within the membrane, by translocating the
-site out of the membrane,
or by altering the position of
-secretase with respect to the APP
transmembrane domain. Alternatively, the possibility that PSs, which do
not resemble any known protease, could in fact be
-secretase has not
been excluded, in which case altered interaction between PS and APP
could provide a simple explanation for shifts in cleavage induced by
FAD-linked PS mutants and would be entirely consistent with decreased
production of A
from PS 1 knockout mice (45).
Conclusions--
-Secretase appears to represent a membrane
protein secretase defined as a proteolytic activity that cleaves a
proteolytically sensitive region of a transmembrane protein resulting
in secretion of the proximal portion of the cleaved protein (46).
Compared with other secretase activities,
-secretase activity
appears to be distinct in that it does not cleave its substrate within an extracellular/lumenal "stalk" like region proximal to the
membrane, but rather within a hydrophobic region purportedly within the TMD of the APP COOH-terminal fragment. The demonstration in this study
that the
-secretase is not a single proteolytic activity and that
the membrane plays a critical role in determining
-secretase cleavage of APP will be important in developing strategies for isolation of the various
-secretase activities, which remain a major
therapeutic target in AD. Additional studies will be needed to
determine whether the pathological shifts in A
cleavage are caused
by the alterations in the major pepstatin-sensitive
-secretase activities or by additional proteases, which might play a role in
pathogenic processing. Given that the common effect of all early onset
FAD-linked mutations is to modestly increase long A
production, this
study offers important insight into how various A
peptides are
generated and suggests possible mechanisms whereby various FAD-linked
mutations might shift
-secretase cleavage. Because other
biologically important cleavage events in SREBP and Notch may be
intramembranous, it will be important to perform similar studies on
such proteins to determine if similar, atypical, proteolytic mechanisms
are responsible for such cleavages. Together, these studies should also
offer additional insights into the important question of how
transmembrane domains, in general, are degraded.
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ACKNOWLEDGEMENTS |
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We thank T. Dechert, J. Shockely, D. Yager, and T. Smith for excellent technical assistance. Antibodies 207 and 369W were kindly provided by B. Greenberg and S. Gandy, respectively. BC05, BAN50, and BA27 were gifts of Takeda industries.
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FOOTNOTES |
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* This work was supported by a Beeson Award from American Federation for Aging Research (to T. G.) and National Institutes of Health/National Institute of Aging Grant AG-16065 (to R. W.).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. Tel.:
904-953-2538; Fax: 904-953-7370; E-mail: tgolde{at}mayo.edu.
2 C. Eckman, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are:
A, amyloid
protein;
APP, amyloid
protein precursor;
AD, Alzheimer's disease;
FAD, familial Alzheimer's disease;
NL, APPK670N,M671L
mutation (numbering based on APP770 isoform);
TMD, transmembrane
domain;
PS, presenilin;
Me2SO, dimethyl sulfoxide;
KA
28, lysine at A
28;
ELISA, enzyme-linked immunosorbent assay;
PBS, phosphate-buffered saline;
PBSB, phosphate-buffered saline with bovine
serum albumin;
flAPP, full-length APP;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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REFERENCES |
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