 |
INTRODUCTION |
Alzheimer's disease
(AD),1 a progressive
neurodegenerative disorder, is the most common cause of dementia in the
elderly. AD is characterized pathologically by the presence of senile
plaques and neurofibrillary tangles in the brains of affected
individuals (1). The predominant constituent of senile plaque is
40-43-amino acid
-amyloid peptides (A
), which are derived from
proteolytic processing of a type I integral membrane glycoprotein,
called the amyloid precursor protein (APP) (Ref. 2; reviewed in Ref. 3). Approximately 10% of all cases of AD, classified as familial early
onset AD (FAD) (age of onset <60 years), is caused by autosomal dominant inheritance of mutations in genes encoding APP (for a review,
see Ref. 4), presenilin 1 (PS1) (5) and presenilin 2 (PS2) (6-8).
Expression of FAD-linked APP mutations leads to increased production of
highly fibrillogenic A
species ending at residue 42 (or 43) (A
42)
(reviewed in Ref. 3). In addition, expression of FAD-linked PS1 and PS2
variants alters APP processing in a manner that leads to elevated
production of A
42 (9-14). Thus, genetic mutations in all the three
genes that cosegregate with FAD increase the production of the
pathogenic A
42 peptides. The role that PS plays in facilitating
proteolytic cleavage of APP at the
-secretase site is not known, but
PS1-deficient neurons exhibit selective defects in the production of
A
(15, 16).
PS1 and PS2 (PS) are homologous polytopic membrane proteins (17) that
are subject to endoproteolytic cleavage (12, 18-20). In previous
efforts, we documented that PS1-derived 28-kDa NH2-terminal (NTF) and 17-kDa COOH-terminal fragments (CTF) are the preponderant PS1-related species that accumulate in vivo (18). The PS NTF and CTF accumulate to 1:1 stoichiometry, and the absolute levels of
PS-derived fragments are established by a highly regulated and
saturable mechanism such that overproduction of human PS fragments led
to diminution of mouse PS fragments (18). In transfected cells
overexpressing PS, only a small fraction of full-length PS is converted
into stable fragments (t1/2 > 12 h); excess
full-length PS polypeptides are rapidly degraded (t1/2 ~ 1 h) (18, 21-25). Moreover, accumulation of PS1 and PS2 fragments are coordinately regulated, providing strong evidence that association with limiting cellular components regulates the abundance of PS fragments (21). More recently,
using in situ chemical cross-linking and
co-immunoprecipitation approaches, we demonstrated that the NTF and CTF
of either PS1 or PS2 can be coisolated (26). The accumulated data
support our view that the more stable endoproteolytic derivatives are the biologically functional forms of PS. Arguing against this notion,
Citron et al. (27) reported that expression of a truncated PS1 species corresponding to the human PS1 NTF with a FAD mutation failed to increase A
42 production; the authors suggested that the
pathogenic effect of mutant PS is elicited by the full-length molecule
and not the endoproteolytic derivatives.
In this report, we examine the assembly of PS1 and PS2 derivatives in
stable mouse N2a neuroblastoma cell lines expressing full-length PS,
recombinant PS1 NTF, or chimeric PS1/PS2 polypeptides. We demonstrate
that transgene-derived COOH-terminally truncated human PS polypeptides
that correspond to PS1 NTF (residues 1-298) fail to co-assemble with
endogenous PS1 CTF and are rapidly degraded. In cells co-expressing PS1
and PS2, processed fragments derived from full-length PS polypeptides
form assemblies consisting entirely of either PS1 NTF·CTF or PS2
NTF·CTF. Based on these results, we conclude that the association of
NH2 and COOH domains of the precursor PS polypeptide
precedes endoproteolytic cleavage and that PS1 and PS2 do not form
heteromultimeric assemblies. Interestingly, fragments derived from
proteolytic processing of a chimeric PS1/PS2 precursor polypeptide are
capable of forming stable heteromeric PS1 NTF·PS2 CTF assemblies.
Furthermore, expression of a FAD-linked M146L mutant PS1/PS2 chimera
elevated the production of A
42 peptides, indicating that the gain of
function properties associated with the FAD-linked mutation is
preserved in a PS1/PS2 chimera. Our results are consistent with a model
where stabilization of intramolecular interaction(s) between domains
within the NH2- and COOH-terminal regions of PS1 is a
prerequisite for endoproteolytic processing.
 |
MATERIALS AND METHODS |
Generation of Expression Plasmids--
Expression plasmids
encoding human PS1 (pCB6PS1), PS2 (pCB6PS2), FAD-linked PS variants,
and PS2 CTF have been described (10, 12, 18). A plasmid encoding a
polypeptide that corresponds to human PS1 NTF was generated as follows.
A 252-base pair DNA fragment, which encodes PS1 amino acids 214-298
followed by a stop codon, was generated by polymerase chain reaction
using primers 5'-GCCTACATTACTGTTGCACTCC (195S) and 5'
CCGGATCCTACATATTCACCAACC and subsequent digestion with PflMI
and BamHI. After gel purification, this product was ligated
to PflM1-BamHI pCB6PS1 "vector" fragment, to generate
pBSPS1NTF. A plasmid encoding a chimeric PS1 NTF-PS2 CTF
polypeptide was generated as follows. A 235-base pair DNA fragment,
which encodes PS1 amino acids 214-269, was generated using primers
195S (see above) and 5'-GGCAACCAGCATTCGAAGTGGACCTTTCGG and subsequent
digestion with PflMI and BsmI. This product was incubated in a ligation mixture with an
Asp718I-PflMI fragment isolated from pBSPS1 (28)
and Asp718I-BsmI pBSPS2 "vector" fragment (18), to generate pBSPS1/PS2. Polymerase chain reaction-amplified sequences in pBSPS1NTF and pBSPS1/PS2 were confirmed by
sequencing using Sequenase (U.S. Biochemical Corp.), and the inserts
were subcloned into cytomegalovirus expression vectors to generate pCB6PS1NTF and pCDNAPS1/PS2, respectively. M146L
mutation was introduced into pCDNAPS1/PS2 by exchanging
Asp718I-XmnI fragment from a PS1M146L plasmid.
Cell Culture and Generation of Stable Cell Lines--
Mouse N2a
neuroblastoma cells were cultured in 1:1 Dulbecco's minimal essential
medium/OptiMEM (Life Technologies, Inc.) supplemented with 10% fetal
calf serum. Monkey COS-1 cells were cultured in Dulbecco's minimal
essential medium supplemented with 10% fetal calf serum. Stable N2a
cell lines expressing human PS1 and PS2 were described (18, 21). Stable
N2a lines expressing PS1 NTF or PS1/PS2 were generated by transfecting
0.5 µg of the respective expression plasmids (see above). Expression
of transgene-derived PS polypeptides in G418-resistant lines was
determined by Western blot analysis with PS1NT and
PS2Loop antisera (26). Cycloheximide was added to the culture medium
at a final concentration of 30 µg/ml to block protein synthesis in
some experiments.
Co-immunoprecipitation and Western Blot Analyses--
Detergent
lysates were prepared by lysing cells in cold PBS containing 0.25%
n-dodecyl
-D-maltoside (Sigma) as described previously (26). We obtained similar results when lysates were prepared
using buffers containing 1% digitonin (29). Aliquots of lysates were
incubated with 3 µl of antisera at 4 °C for 2 h. PS1 NTF and
CTF were immunoprecipitated using antisera PS1NT and
PS1Loop, respectively (18, 26). PS2 CTF was immunoprecipitated using
PS2Loop antiserum (21). A rat monoclonal antibody (mAb) specific for
human PS1 (30) (provided by Dr. Allan I. Levey, Emory University School
of Medicine, Atlanta) was used to immunoprecipitate transgene-derived
human PS1 NTF. To generate antibodies against the PS2
NH2-terminal region, New Zealand White rabbits were
immunized with a glutathione S-transferase fusion protein
containing residues 1-75 of human PS2. The specificity of the
resultant antiserum, termed PS2NT, for PS2 was established
by Western blot analysis of lysates prepared from stable N2a cell lines
expressing PS1 or PS2 (data not shown). As negative controls, we used
SOD1, an antiserum raised against human erythrocyte Cu/Zn superoxide
dismutase 1 (31), and normal rabbit serum (Sigma). Immunoprecipitates were collected using protein A-agarose, washed twice in lysis buffer,
and resuspended in Laemmli buffer. Proteins were eluted by incubation
at 37 °C for 15 min and analyzed by immunoblotting.
For Western blot analyses, cells were lysed in a buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 0.5% deoxycholate, 0.5% SDS,
and a protease inhibitor mixture (50 µg/ml pepstatin, 50 µg/ml
leupeptin, 10 µg/ml aprotinin, and 0.25 mM
phenylmethylsulfonyl fluoride). Lysates were briefly sonicated to
reduce viscosity and fractionated on SDS gels without heating. Membranes were incubated with PS1- or PS2-specific antisera (see above), and bound antibodies were detected using protein A-horseradish peroxidase (Sigma) followed by chemiluminescence detection (NEN Life
Science Products).
Quantification of A
by Two-site ELISAs--
Monkey COS-1
cells were transfected by the DEAE-dextran method with cDNAs
encoding PS1, PS2, or PS1/PS2 polypeptides and cDNAs encoding
either full-length APP or the COOH-terminal 100 amino acids of APP as
described (12, 32). Conditioned media were collected 48 h after
transfection, and secreted A
species ending at 40 or 42/43 were
quantified using BNT77/BA27 or BC05 two-site ELISAs (33-35) as
described previously (12, 32). Data obtained from full-length APP or
APP C100 cDNA transfections were combined for analysis. To
facilitate comparison between media from cells transfected with
different cDNA, the ratio of A
42/total A
was calculated for
each sample and statistically examined using analysis of variance
followed by Fisher LSD test and expressed as mean ± S.E.
 |
RESULTS |
Transgene-derived Human PS1 NTF Fails to Assemble with Endogenous
PS1 CTF--
In previous studies, we and others reported that the NTF
and CTF derived from endoproteolysis of PS1 (or PS2) are noncovalently associated to each other in vivo (26, 36, 37). To further characterize the NTF and CTF assemblies, we generated stable mouse N2a
neuroblastoma cell lines transfected with a plasmid that encodes human
PS1 residues 1-298 (analogous to NTF). Expression of transgene-derived human PS1 NTF was assessed by Western blotting using PS1NT,
a polyclonal PS1 antiserum that recognizes epitopes in the
NH2 terminus of PS1 (26). Transgene-derived human PS1 NTF
was readily detected in several independent stable N2a cell lines (Fig.
1A, top,
lanes 3-5). Consistent with previous reports
(10, 11, 18, 21), the recombinant human PS1 NTF exhibited accelerated
migration relative to the mouse PS1 NTF on SDS-polyacrylamide gel
electrophoresis (lane 1) and co-migrated with the
human NTF derived from full-length human PS1 (lane
2). Previously, we reported that the expression of human
full-length PS1 in mouse N2a cells resulted in a decrease in the levels
of murine PS1-derived NTF and CTF (21). To examine whether the
expression of recombinant human NTF abrogated accumulation of
endogenous murine PS1 derivatives, we performed Western blots using
PS1Loop, an antiserum that reacts with epitopes in the hydrophilic
loop domain of PS1 (18). The steady-state levels of mouse PS1 CTF in
three independent N2a lines examined were essentially identical to N2a
cells transfected with empty vector (21) (Fig. 1A,
bottom), indicating that human NTF did not interfere with
the accumulation of mouse CTF.

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 1.
Characterization of transgene-derived human
NTF expressed in stably transfected mouse N2a cells. A,
detergent lysates prepared from stable N2a cell lines transfected with
empty vector (lane 1), full-length PS1 cDNA
(lane 2), or human PS1 NTF cDNA
(lanes 3-5) were fractionated by
SDS-polyacrylamide gel electrophoresis and analyzed by immunoblotting
with PS1NT (top) or PS1Loop
(bottom). Full-length human PS1 (FL), human
(Hu), and murine (Mo) PS1 NTF and CTF are
indicated. B, stable N2a cells expressing human PS1 NTF were
incubated in culture medium containing cycloheximide (30 µg/ml) for
the intervals indicated. Detergent lysates prepared after the
incubation period were analyzed by immunoblotting with
PS1NT (top) or PS1Loop (bottom).
C, stable N2a cells expressing human PS1 NTF were lysed
under nondenaturing conditions, and lysates were used to
co-immunoprecipitate PS1 NTF using polyclonal PS1NT
antiserum, a PS1 mAb (which reacts specifically with human PS1
NH2-terminal epitopes), or control Myc and SOD1 antibodies.
The resulting immunoprecipitates and total lysates (corresponding to
of the volume used for immunoprecipitations) were probed
with PS1NT (top panels) or PS1Loop
(bottom panels). PS1 NTF and CTF are indicated by
arrowheads. The slower migrating PS1Loop-reactive band
probably represents the phosphorylated form of PS1 CTF (36, 43). The
high molecular weight PS1NT-reactive species (lane
5) represent nonspecific aggregation of overexpressed human
NTF. The small decrease in the CTF signal in lane
5 is not reproducible and is seen only in samples with
aggregated human NTF.
|
|
Recently, Steiner and colleagues (25) reported that recombinant human
PS1 NTF expressed in stably transfected human 293 cells undergoes rapid
degradation mediated by the proteasome. While endogenous NTF and CTF
were detected in a stable human 293 cell line expressing recombinant
human NTF, it could not be determined whether high level expression of
recombinant human NTF affected endogenous NTF because of the
comigration of endogenous and exogenous NTFs (25, 27). We addressed
this issue in our stable N2a cell lines expressing human PS1 NTF. A
PS1NT-reactive species co-migrating with the mouse NTF was
visible in lysates of stable N2a cells expressing low levels of
recombinant human NTF (Fig. 1A, top, compare
lanes 1 and 3). However, the mouse NTF
and the recombinant human NTF were not sufficiently resolved on
SDS-polyacrylamide gel electrophoresis to convincingly demonstrate
whether or not the expression of human NTF influenced the accumulation
of mouse NTF. Therefore, we used an alternate strategy that takes
advantage of the findings that while the recombinant human NTF is short lived (25), endogenous PS-derived NTF has a half-life of several hours
(22, 23). We incubated stable N2a cell line PS1298.6, which
expresses high levels of human NTF (Fig. 1B, lane
1), with cycloheximide to arrest protein synthesis and
examined the decay of human NTF over a 6-h interval. Immunoblot of cell
lysates collected at various time points revealed that the faster
migrating PS1NT-reactive species, which corresponds to
recombinant human NTF, degraded rapidly (t1/2 ~ 1.5 h) after the addition of cycloheximide. The slower
migrating PS1NT-reactive species, which corresponds to mouse PS1 NTF was readily visible after 1 h of cycloheximide
treatment and was the only remaining PS1 NTF after 4 h of
cycloheximide treatment (Fig. 1B, top). Reprobing
of the blots using
PS1Loop antiserum revealed that the levels of
mouse CTF remained unchanged during the 6-h cycloheximide treatment
(Fig. 1B, bottom). Similar results were obtained
in cell lines expressing low or high levels of human PS1 NTF (data not
shown). These results provide evidence for the presence of stable pools
of endogenous NTF in N2a cell lines expressing high levels of
recombinant human NTF. Furthermore, the short half-life of
transgene-derived human NTF in our stable N2a cell lines is entirely
consistent with previous studies (25). From these results, we conclude
that the expression of transgene-derived human NTF failed to interfere
with the endoproteolysis of endogenous murine PS1, or the accumulation
of mouse PS1 derivatives.
We then examined whether the transgene-derived human NTF associated
with endogenous mouse PS1 CTF. Previous studies demonstrated that after
repeated immunodepletion of PS1 NTF·CTF complexes, only recombinant
human NTF remained in the lysates of human 293 cells expressing human
PS1 NTF (25). We were interested in directly testing whether we could
isolate complexes containing human PS1 NTF and mouse PS1 CTF using a
human PS1-specific antibody. To distinguish between the
transgene-derived human NTF and endogenous mouse NTF, we used a rat mAb
that reacts specifically with the human PS1 NH2 terminus
(21, 30). Stable N2a cells expressing human PS1 NTF were lysed under
nondenaturing conditions, and aliquots of the lysates were incubated
with either the anti-human PS1 mAb or polyclonal PS1NT
antiserum (which reacts with both human and murine PS1). The
immunoprecipitates were first probed using PS1NT to confirm
successful immunoprecipitation of PS1 NTF by both antibodies (Fig.
1C, top panels, lanes
1 and 3). As expected, control
immunoprecipitations performed using monoclonal anti-Myc antibody,
9E10, and polyclonal anti-superoxide dismutase antibody, SOD1, failed
to isolate PS1 NTF (lanes 2 and 4).
Reprobing of the blots using
PS1Loop antiserum revealed the presence
of co-purified mouse PS1 CTF in immunoprecipitates prepared using
PS1NT antiserum (Fig. 1C, bottom
panels, lane 3), but not by using
anti-human PS1 mAb (lane 1). These results
demonstrate that transgene-derived recombinant human NTF could not
assemble into complexes containing the endogenous murine PS1 CTF.
Cells Co-expressing PS1 and PS2 Do Not Form heteromeric PS1·PS2
Assemblies--
Both PS1 and PS2 have been reported to form
heteromeric assemblies and migrate as high molecular weight complexes
on size exclusion columns and velocity density gradients (29, 36, 37).
However, it is not clear whether PS1 derivatives form heteromeric complexes with PS2 derivatives. To address this issue, we used a stable
N2a cell line transfected with human full-length PS2 cDNA. As we
reported previously (21), high level expression of human PS2 in mouse
N2a cells results in the diminution of mouse PS1 derivatives (Fig.
2A, lane
6). Therefore, we chose a cell line that expresses low
levels of human PS2 (lane 2) for the present studies; PS1 derivatives are only partially replaced in this cell line
(compare lanes 4 and 5). We performed
co-immunoprecipitation analyses using PS1 and PS2 antibodies: PS1
derivatives were immunoprecipitated using antibodies PS1NT
and
PS1Loop; PS2 NTF and CTF were immunoprecipitated using
PS2NT and
PS2Loop, respectively. In parallel, polyclonal SOD1 antiserum and normal rabbit serum were used in control
immunoprecipitations. The immunoprecipitates were analyzed by
sequential immunoblotting using PS1 and PS2 antisera. As expected,
PS1NT and
PS1Loop antisera co-immunoprecipitated PS1 CTF
and NTF, respectively (Fig. 2B, top
panels, lanes 1 and 5), but
failed to co-purify PS2 derivatives (Fig. 2B,
bottom panels, lanes 1 and
4). Furthermore, PS2NT and
PS2Loop antisera
co-immunoprecipitated PS2 CTF and NTF, respectively (Fig.
2B, bottom panels, lanes
2 and 6), but failed to co-purify PS1 derivatives
(Fig. 2B, top panels, lanes
2 and 6). These results demonstrate that the
derivatives resulting from endoproteolysis of PS1 and PS2 form either
PS1 NTF·PS1 CTF or PS2 NTF·PS2 CTF assemblies but do not form mixed
complexes containing the derivatives from both of the precursor
polypeptides.

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 2.
PS1 and PS2 derivatives do not form mixed
complexes. A, detergent lysates prepared from cells
transfected with empty vector or PS2 cDNA were analyzed by
immunoblotting with PS2Loop (lanes 1-3) or a
mixture of PS1NT and PS1Loop (lanes
4-6). Full-length human PS2 (FL) and
endoproteolytic PS1 and PS2 derivatives (arrowheads) are
indicated. B, stable N2a cells expressing low levels of
human PS2 were lysed under nondenaturing conditions, and lysates were
used for co-immunoprecipitation analyses using PS1NT,
PS1Loop (PS1L), PS2 NT, or PS2Loop
(PS2L) antibodies as indicated. SOD1 and normal rabbit serum
(NRS) were used as negative controls. After fractionation by
SDS-polyacrylamide gel electrophoresis and transfer to membranes, blots
containing NTF immunoprecipitates were sequentially probed with
PS1Loop (top panels, lanes
1-4) and PS2Loop antibodies (bottom
panels, lanes 1-4). Blots containing
CTF immunoprecipitates were sequentially probed with PS1NT
(top panels, lanes 5-8)
and PS1NT antibodies (bottom panels,
lanes 5-8).
|
|
The Endoproteolytic Derivatives from a PS1/PS2 Chimeric Polypeptide
Can Form Functional PS1/PS2 Assemblies--
PS1 and PS2 have similar
protein structures, share a high degree of amino acid sequence
identity, and undergo endoproteolytic cleavage, and the resulting
fragments derived from each precursor associate with each other.
Despite these similarities, our results indicate that fragments derived
from endoproteolytic cleavage of PS1 and PS2 fail to form mixed
complexes. The absence of complexes containing PS1 NTF and PS2 CTF or
PS2 NTF and PS1 NTF strongly suggests that the noncovalent association
between the NH2- and COOH-terminal structural domains of
the precursor polypeptide may precede endoproteolytic cleavage. If this
prediction were correct, one would expect that endoproteolysis of a
chimeric polypeptide containing PS1 NTF and PS2 CTF should result in
the generation of processed PS1 NTF and PS2 CTF derivatives that can be
co-immunoprecipitated. To test this hypothesis, we generated stable N2a
cell lines transfected with an expression plasmid encoding a chimeric
polypeptide corresponding to amino acids 1-280 of human PS1 fused to
amino acids 287-448 of human PS2. Expression of the PS1/PS2 chimera
was assessed in several cell lines by Western blotting using
PS1NT and
PS2Loop antisera (data not shown). The
chimeric polypeptide was endoproteolytically processed to generate
fragments corresponding to PS1 NTF and PS2 CTF, and in cell lines
expressing high levels of the PS1/PS2 chimera, endogenous PS1
derivatives were replaced (Fig.
3A). Thus, the metabolism of
the PS1/PS2 chimera was similar to that of PS1 or PS2, and the
accumulation of the resulting derivatives (PS1 NTF and PS2 CTF) was
similar to that of the fragments derived from full-length PS1 or PS2.
Next, we performed co-immunoprecipitation studies to assess whether the
fragments derived from endoproteolytic processing of the PS1/PS2
chimera could be co-immunoprecipitated. For these studies, we chose a
stable cell line in which the endogenous mouse PS1 derivatives are
incompletely replaced. Nondenaturing lysates were subject to
immunoprecipitation with PS1 and PS2 antibodies. As expected,
PS1NT antiserum co-immunoprecipitated endogenous murine
PS1-derived CTF (Fig. 3B, top panels,
lane 1). Interestingly, reprobing of the blot
using
PS2Loop antiserum revealed that PS1NT antiserum
also co-purified PS2 CTF (bottom panels,
lane 1). Furthermore,
PS1Loop antiserum
co-precipitated the slower migrating mouse PS1 NTF, whereas
PS2Loop
antiserum co-precipitated the faster migrating human PS1 NTF
(top panels, lanes 7 and
8, respectively). Because endogenous murine PS2 NTF is not
detectable in total lysates prepared from the PS1/PS2 cell line used
for this experiment (bottom panel,
lane 12), we failed to detect any signal for PS2
CTF or NTF in co-precipitations (bottom panels,
lanes 2 and 8, respectively). These
results demonstrate that the PS1 NTF and PS2 CTF are capable of forming
mixed complexes when derived from cleavage of a chimeric polypeptide
and strongly support our view that interaction between NH2-
and COOH-terminal regions of a precursor PS polypeptide precedes endoproteolysis.

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 3.
PS1 NTF and PS2 CTF derived from a chimeric
precursor can be co-precipitated. A, detergent lysates
prepared from stable N2a cells transfected with an empty vector or
cDNA encoding a chimeric PS1/PS2 polypeptide (PS1/2)
were analyzed by immunoblotting using PS1NT
(lanes 1 and 2) and PS2Loop
antibodies (lanes 3 and 4). The
positions of mouse (Mo) and human (Hu) PS1 NTF
and PS2 CTF are indicated. B, stable N2a cells expressing
PS1/PS2 chimera were lysed under nondenaturing conditions, and lysates
were used for co-immunoprecipitation analyses as described in the
legend to Fig. 2. Total lysate corresponding to of the
volume of lysate used for immunoprecipitation and an aliquot of stable
PS2 cell line were also fractionated on SDS gels as markers for PS2 NTF
and CTF. Note that the ~30-kDa band in the PS2NT blot
indicated by an asterisk (bottom
panel, lane 8) is the PS1 NTF signal
that remained due to the incomplete stripping of PS1NT
antibody.
|
|
Next we examined whether the chimeric PS1 NTF·PS2 CTF assembly is
stable. To address this issue, we incubated cells expressing PS1/PS2
chimera with cycloheximide to block protein synthesis. In parallel, we
incubated cells expressing full-length PS1 or PS2 with cycloheximide.
Detergent lysates prepared at the end of the incubation period were
analyzed by immunoblotting using PS1 and PS2 antisera. As expected from
previous studies (21-24), in stable N2a cells expressing PS1 the
full-length PS1 molecules were rapidly degraded, while the NTF and CTF
were stable (Fig. 4A). Similar
results were obtained in stable cell lines expressing PS2 (data not
shown). Analysis of lysates from stable N2a cells expressing the
PS1/PS2 chimera revealed that full-length PS1/PS2 polypeptides were
also rapidly degraded, while the endoproteolytic fragments derived from
the chimera remained stable over a 6-h incubation period in the
presence of cycloheximide (Fig. 4A). From these results, we
conclude that endoproteolytic fragments derived from the processing of
PS1/PS2 chimera are long lived.

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 4.
Analysis of PS1/PS2 stability and the
influence of mutant PS1/PS2 on A 42
production. A, stable N2a cells expressing human PS1 or
PS1/PS2 chimera were incubated in culture medium containing
cycloheximide (30 µg/ml) for the intervals indicated. Detergent
lysates prepared from stable PS1 cells were analyzed by immunoblotting
with PS1NT and PS1Loop (top three
panels); lysates from PS1/PS2 cells were blotted with
PS1NT and PS2Loop (bottom three
panels). Only the regions of gels corresponding to the
full-length polypeptide (FL) and endoproteolytic derivatives
(NTF and CTF) are shown. B, COS cells transfected with PS1,
PS2, or PS1/PS2 cDNA along with cDNA encoding full-length APP
or APP C100 cDNA (see "Materials and Methods"). The levels of
A x-40 and A x-42 secreted from doubly transfected cells were
quantified by two-site ELISAs. Mean values ± S.E. from two
independent experiments are shown. *, p = 0.0012; **,
p < 0.001, relative to values from cells transfected
with the corresponding wild-type cDNA.
|
|
Finally, we examined whether the PS1/PS2 chimera could influence APP
processing and elevate the production of A
42 peptides. For these
studies, we constructed expression plasmids encoding PS1/PS2 chimeric
polypeptide that harbors the FAD-linked M146L substitution. COS cells
were co-transfected with plasmids encoding wild-type or mutant PS1/PS2,
and APP (or C100; see "Materials and Methods"). In parallel, we
also transfected cells with plasmids encoding wild-type or mutant
full-length PS1 or PS2. The levels of secreted A
x-40 and x-42
peptides in conditioned media of transfected cells were quantified
using a two-site ELISA assay as described (33-35). To account for
variations in the levels of A
40 and A
42 due to differences in
transfection efficiencies of different expression plasmids, we
calculated the ratio A
x-42/total A
for each expression construct
and compared the relative ratios (32, 27). Results of these analyses
are presented in Fig. 4B. As reported previously (10, 12,
13), expression of mutant PS1 (A
x-42/total A
ratio PS1 wild-type
was 10.30 ± 0.7 versus PS1 M146L, 18.16 ± 1.2)
or PS2 (wild-type was 14.51 ± 0.8 versus N141I,
26.72 ± 1.8) elevated the secretion of A
42 peptides. The
levels of A
42 in cells transfected with PS1/PS2 chimera lacking
FAD-linked mutation were comparable with that of wild-type PS2. In
contrast, expression of the PS1/PS2 chimera harboring the FAD-linked
M146L mutation markedly elevated the levels of A
42 in the
conditioned medium (wild-type PS1/PS2 was 14.37 ± 1.8 versus M146L PS1/PS2, 23.16 ± 1.9). Thus, we argue
that chimeric PS1/PS2 polypeptides harboring FAD-linked PS1 M146L are
effective in influencing A
42 production. Collectively, these results
document that the endoproteolytic fragments derived from PS1/PS2
chimera can co-assemble into stable heteromeric assemblies, and PS1/PS2 polypeptides harboring FAD-linked mutation were capable of elevating the production of A
42 peptides.
 |
DISCUSSION |
Our understanding of the nature of the functional PS "unit" is
still very incomplete. Knowledge about the subunit stoichiometry is
essential for further studies on the structure and function of the PS
complex. In the present study, we exclusively focused on the
composition and stoichiometry of PS NTF and CTF complexes immunoprecipitated from stable mouse neuroblastoma cells that express
recombinant human PS1 NTF, full-length PS2, or a chimeric PS1/PS2
polypeptide. These studies demonstrate that transgene-derived recombinant PS1 NTF neither influenced the metabolism of endogenous PS1
polypeptides nor associated with endogenous PS1-derived CTF. PS1 and
PS2 fragments derived from the cleavage of co-expressed full-length PS1
and PS2 do not form mixed heteromeric assemblies. In contrast,
endoproteolysis of a PS1/PS2 chimera generates stable PS1 NTF·PS2 CTF
assemblies. The results of our studies provide an important insight: an
intramolecular association(s) between domains of the PS NTF and CTF is
established prior to endoproteolytic cleavage.
In the present study, by examining the metabolism of a PS1/PS2 chimera
we provide evidence that intramolecular association(s) between
structural domains located within NH2- and COOH-terminal halves of PS precedes endoproteolytic cleavage. Our conclusion is
consistent with previous gel filtration chromatography and velocity
gradient analyses, which revealed that full-length PS and the
PS-derived NTF and CTF are components of high molecular weight
complexes (29, 36, 37). Independently, two studies demonstrated that
complexes containing PS1 NTF and CTF were larger than the complexes
containing full-length PS1 (29, 37). Interestingly, a FAD-linked PS1
deletion mutant that fails to undergo endoproteolytic cleavage (18, 38)
was found to be in a high molecular weight complex, similar in size to
that of the cleaved fragments, suggesting that cleavage was not
necessary for the assembly of stable PS complexes (37). Nevertheless,
attempts to bypass the requirement for endoproteolysis by co-expressing
truncated PS2 polypeptides, which correspond to FAD-linked mutant NTF
and CTF, did not lead to elevated A
42 production (32). Together with
our present results, these studies indicate that the formation of a
functional PS complex may be an ordered and stepwise process in which
newly synthesized PS polypeptides establish intramolecular
association(s) during the process of folding and subsequently bind to
other proteins expressed at limiting levels that "stabilize" the PS
complex; properly folded and "mature" PS molecules that have
associated with limiting accessory molecules are then cleaved (or not
in the case of PS1 lacking exon 9-encoded residues). The mechanisms involved in facilitating PS endoproteolysis are not known.
It is likely that both protein stabilization and endoproteolytic
cleavage may rely on proper folding of the synthetic PS polypeptides. The lack of an intact COOH terminus, for example, might prevent proper
folding and/or association with PS binding proteins and thereby lead to
rapid degradation. In this regard, previously we showed that
COOH-terminally truncated PS polypeptides, which lack the last two
transmembrane domains and the cytoplasmic tail, failed to undergo
endoproteolysis and were rapidly degraded (21). Our present results
confirm studies by Steiner et al. (25) that recombinant
transgene-derived PS1 NTF is not incorporated into complexes containing
endogenous PS1 CTF and is rapidly degraded. In recent studies, it was
reported that recombinant PS1 NTF with a FAD mutation failed to
increase A
42 production, even after stabilization of recombinant NTF
by incubation with proteasome inhibitors (25, 27). Based on the
recombinant NTF studies, it was suggested that full-length PS
molecules, and not the endoproteolytic derivatives, were the functional
PS units in vivo (27). It is known that proper assembly
contributes to the structural maturation as well as the function of
oligomeric membrane proteins. Thus, our demonstration that exogenously
expressed human NTF failed to assemble with endogenous CTF provides
direct evidence that the recombinant NTFs were not incorporated into PS
complexes. We argue that the transgene-derived NTF molecules are
structurally quite different from NTF·CTF assemblies derived from
full-length PS polypeptides; hence, their inability to increase A
42
production cannot be interpreted in a way that provides any meaningful
information regarding the functional form of PS.
The outcome of co-expression of full-length and truncated membrane
receptors has been investigated for several membrane proteins. In
several instances, expression of truncated polypeptides disrupted the
functional assembly and activity of endogenous proteins by a dominant
negative mechanism (39). For example, expression of fragments derived
from the extracellular NH2-terminal domains of
-,
-,
and
-subunits of the acetylcholine receptor blocked the assembly and
cell surface expression of functional acetylcholine receptors (40). In
analogous studies, co-expression of truncated and full-length
gonadotropin-releasing hormone receptors resulted in the inhibition of
gonadotropin-releasing hormone signaling via the full-length receptor,
probably because of specific interactions between the two receptor
proteins resulting in impaired maturation of the full-length receptor
(41). In contrast to these studies, our analysis of stable cell lines
expressing transgene-derived human PS1 NTF shows that the expression of
PS1 NTF did not have any dominant negative inhibitory effect on the
cleavage of endogenous full-length PS1 or on the stable accumulation of
endogenous PS1-derived fragments. Moreover, we and others showed that
transgene-derived PS1 NTF cannot be co-isolated with endogenous
PS1-derived CTF, excluding any direct physical interaction between
truncated PS molecules and endogenous PS1. Together, these results
strongly suggest that the COOH terminus of PS might provide information essential for protein folding and/or interaction with proteins that
interact with PS. In this regard, a calcium-binding protein, termed
calsenilin, has been shown to bind to the COOH-terminal 40 residues of
PS (42).
As described above, PS1 and PS2 fragments derived from cleavage of
co-expressed full-length PS1 and PS2 neither form mixed heteromeric
assemblies nor exchange subunits to result in complexes containing
PS1-NTF·PS2 CTF or PS2 NTF·PS1 CTF. Yet, overexpression of human
PS1 resulted in diminution of endogenous PS2 derivatives (21). In
studies presented here, we demonstrate that a chimeric polypeptide
consisting of PS1 NTF and PS2 CTF undergoes endoproteolysis, and the
resulting derivatives accumulate to saturable levels. Furthermore,
expression of chimeric PS1/PS2 polypeptides harboring the FAD-linked
M146L mutation resulted in the elevated production of A
42. Together,
these findings indicate that PS1 and PS2 share common pathways of
processing and likely interact with similar factors that modulate
42
processing of APP. Future efforts to identity of individual polypeptide
components of the oligomeric PS complex will be required to define the
multiple steps involved in the regulation of assembly of the functional
PS "unit".