1 Laboratory for Neuronal Cell Biology, Center for Human Genetics,
Gasthuisberg/KULeuven and Flanders Interuniversity Institute for Biotechnology
(VIB), Herestraat 49, 3000 Leuven, Belgium
2 Max-Planck-Institute for Brain Research, Department of Neurochemistry, D-60584
Frankfurt, Germany
Author for correspondence (e-mail:
bart.destrooper{at}med.kuleuven.ac.be)
Accepted 25 November 2002
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Nicastrin, Presenilin, -Secretase, Alzheimer's disease, Glycosylation
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It is clear that -secretase processing is a prototype example of a
very general biological phenomenon and further understanding of its molecular
cell biology is therefore of major importance. Questions concerning the
specificity and regulation of the cleavage process or the subcellular
compartment in which the
-secretase operates need to be addressed in
more detail. In the current manuscript, we focus on the relationship between
two established
-secretase components: PS and nicastrin. The absolute
requirement for PS in the
-secretase process was established some years
ago (De Strooper et al., 1998
;
Herreman et al., 2000
;
Zhang et al., 2000
) and has
not convincingly been challenged since then
(Nyabi et al., 2002
). An
important issue is whether the two aspartate residues in transmembrane domains
6 and 7 constitute the active catalytic site of the
-secretase complex
(Wolfe et al., 1999
). This
claim found considerable support both in the observation that several known
-secretase inhibitors bind to PS directly
(Esler et al., 2000
;
Li et al., 2000b
;
Seiffert et al., 2000
) and in
the recent identification of proteolytically active, remote homologues of PS
(Ponting et al., 2002
;
Weihofen et al., 2002
). The
fact that substrates like Notch and APP are co-precipitated with PS
(Esler et al., 2002
;
Ray et al., 1999b
) seems to
contradict the hypothesis that PS mediates their cleavage. However, this can
probably be explained by the intramolecular separation between the
substrate-binding site and the putative catalytic site in PS
(Annaert et al., 2001
),
implying that binding and cleavage are two separate events and that a
conformational change is needed to bring the substrate towards the catalytic
site. This could explain why substrate and putative protease remain in complex
for a certain period of time (Annaert et
al., 2001
). In any case, PSs are not capable of cleaving APP
substrates without additional cofactors. For instance, while PSs are
abundantly present in the endoplasmic reticulum (ER), the intermediate
compartment and the cis-Golgi (Annaert et
al., 1999
; Culvenor et al.,
1997
; Lah et al.,
1997
), they do not efficiently cleave an APP substrate that is
specifically retained in these compartments
(Cupers et al., 2001a
;
Maltese et al., 2001
). Only
after addition of brefeldin A are the APP substrates proteolyzed, indicating
that other proteins or specific post-translational modifications of the
-secretase complex in the Golgi apparatus are needed to trigger this
proteolytic activity (Cupers et al.,
2001a
). This dissociation between PS subcellular localization and
-secretase activity has been previously called the `spatial paradox'
(Annaert and De Strooper,
1999
). Nicastrin was shown to co-purify biochemically with PS
(Yu et al., 2000
) and to
interact with the
-secretase substrates APP
(Yu et al., 2000
) and Notch
(Chen et al., 2001
). By
contrast, the crucial functional role of nicastrin in
-secretase
cleavage of Notch has been convincingly demonstrated recently by genetic and
biochemical means in Drosophila
(Chung and Struhl, 2001
;
Hu et al., 2002
;
Lopez-Schier and St Johnston,
2002
). These experiments also indicated that nicastrin is needed
to stabilize PS protein expression (Hu et
al., 2002
; Lopez-Schier and St
Johnston, 2002
). Although the experiments indicated at first
glance that the effects of nicastrin inactivation on Notch cleavage and
signalling could be indirect via the destabilizing effect on PSs, other
possibilities are not excluded. Indeed, normal PS expression was maintained in
nicastrin-deficient cells under certain conditions, while Notch signalling
remained perturbed (Lopez-Schier and St
Johnston, 2002
).
In the current manuscript, we have investigated the subcellular
distribution and the post-translational modifications of nicastrin in neurons
and fibroblasts. We have studied the effects of PS deficiency on the cell
biology of nicastrin and have analyzed the role of nicastrin glycosylation and
PS association in -secretase activity.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
HEK293 cells stably transfected with APP695/Sw were kindly provided by C.
Haass (Adolf-Butenandt-Institut, Munich, Germany). HEK293 cells were
transiently transfected with plasmids encoding mNotchE or NICD using
fugene (Roche), according to the instructions of the manufacturer.
Antibodies
A new antibody, B59.2, was raised against the C-terminal 15 amino acids of
nicastrin coupled to keyhole limpet hemocyanin (Imject, Pierce). Rabbit
polyclonal antibody (pAb) B12.6 against APP
(De Strooper et al., 1995),
B19.2 against PS1 N-terminal fragment (NTF)
(De Strooper et al., 1997
) and
B32.1 against PS1 C-terminal fragment (CTF)
(Annaert et al., 1999
) have
been characterized before. Antibodies against calnexin and human PS1 CTF (mAb
5.2) were kindly provided by A. Helenius (University of Zurich, Switzerland)
and B. Cordell (Scios, Sunnyvale, California). Antibodies against BIP and
nicastrin were obtained from Stressgen and Chemicon. Monoclonal antibody (mAb)
WO2, recognizing the C-terminus of the Aß sequence was from Abeta GmbH
(Heidelberg). For some applications, pAb B19.2 and B59.2 were biotinylated
according to the instructions of the manufacturer (Pierce).
Deglycosylation of nicastrin
MEFs/neurons were harvested in PBS supplemented with 5 mM EDTA, 1 µg/ml
pepstatin A and 100 U/ml aprotinin. After centrifugation (800
g, 10 minutes), cells were lysed in either 100 mM phosphate
buffer pH 5.7 for endoglycosidase H (endoH)- or O-glycosidase treatment or in
phosphate buffer pH 7.4 for N-glycosidase F treatment. Phosphate buffer was
supplemented with 0.1% SDS, 0.5% Triton X-100, 0.5% ß-mercapto-ethanol
and protease inhibitors to optimize enzyme activities. In the case of
neuraminidase treatment (1 mU/20 µl, Roche) cells were lysed in 20 mM
phosphate buffer pH 7.4. The cell lysate was incubated on ice for 20 minutes
and centrifuged at 20,800 g for 15 minutes. After 10 minutes
incubation at 70°C, cell lysates were incubated overnight with endoH (1
U/20 µl, Roche), N-glycosidase F (1 U/20 µl, Roche) or O-glycosidase (1
mU/20 µl, Roche). Samples were separated by SDS-PAGE on pre-casted 7%
Tris-Acetate gels (Nupage, Invitrogen, Life Technologies) and blotted.
Affinity-purified nicastrin pAb B59.2 was used at a dilution of 1/3000 in
blocking buffer (Tris-buffered saline, 0.1% Tween, 5% nonfat dry milk). For
detection, HRP-coupled secondary antibodies (BioRad) were used followed by
chemiluminescence detection (Renaissance, Perkin Elmer).
Surface biotinylation
For cell-surface biotinylation, cells at 80% confluency were incubated in
PBS pH 8-8.5 containing 0.5 mg/ml NHS-SS-biotin (Pierce) for 30 minutes at
4°C on a rocking platform. After quenching with 100 mM glycine and 0.5%
BSA, cells were extracted in DIP buffer (1% Triton X-100, 1% sodium
deoxycholate and 0.1% SDS in 150 mM NaCl and 50 mM Tris-HCl pH 7.4). After
centrifugation (20,800 g, 10 minutes), cleared cell extracts
were incubated overnight (4°C) with streptavidin beads (Pierce). Bound
material was eluted with 25 µl Nupage sample buffer (Invitrogen),
electrophoresed on pre-casted 4-12% Bis-Tris Nupage gels in MOPS running
buffer and processed for western blotting using pAb B59.2.
Pulse-chase experiments
MEFs at 80% confluency were metabolically labeled for 15 minutes with
methionine-free DMEM medium supplemented with 100 µCi/ml
[35S]-methionine. After labeling, cells were washed once and
incubated for the indicated time periods in DMEM/F12 with 1% FCS. Next, cells
were extracted in 250 mM sucrose, 5 mM Tris-HCl (pH 7.4), 1 mM EGTA and 1%
Triton X-100 in the presence of protease inhibitors, and cleared by
centrifugation (20,800 g, 15 minutes). Labeled protein was
immunoprecipitated using 30 µl protein G sepharose and specific antibodies
against PS1, APP and nicastrin. Immunoprecipitates were finally eluted in 25
µl Nupage sample buffer containing 1% ß-mercapto-ethanol and
electrophoresed on 7% Tris-Acetate Nupage gels in Tris-Acetate running buffer
(Invitrogen). Radiolabeled bands were visualized by phosphorimaging and
ImageQuant 4.1 software (Molecular Dynamics).
Co-immunoprecipitation experiments
Cell pellets were resuspended and homogenized in 250 mM sucrose, 5 mM
Tris-HCl (pH 7.4) and 1 mM EGTA supplemented with protease inhibitors using a
ball-bearing cell cracker (10 passages, clearance 10 µm). After low-speed
centrifugation (800 g, 10 minutes), the postnuclear
supernatant was ultracentrifuged (100,000 g, 1 hour) and the
membrane pellet resuspended in 10 mM Tris and 1mM EDTA containing 0.5% CHAPS,
and incubated at 4°C for 1 hour. For immunoprecipitation, the cleared
extracts (100,000 g, 1 hour) were incubated overnight
(4°C) with protein G sepharose and specific antibodies as indicated.
Immunoprecipitates were solubilized in 25 µl Nupage sample buffer
(Invitrogen), electrophoresed on 4-12% Nupage Bis-Tris Nupage gels in MOPS
running buffer and processed for western blotting.
Immunofluorescence experiments
PS1- and 2-deficient MEFs were rescued with human PS1 using the retroviral
system for stable transduction (Clontech). By western blot analysis, expressed
human PS1 is fully endoproteolytically processed and restores both APP
processing and nicastrin maturation (G.V.G. and W.A., unpublished). This cell
line allowed us to apply triple immunofluorescent staining using mouse mAb 5.2
against human PS1, rabbit pAb against calnexin, and guinea-pig pAb against
nicastrin (Chemicon). Briefly, cells were grown to 30-50% confluency on glass
coverslips in DMEM/F12 containing 10% FCS, washed twice in Dulbecco's PBS and
fixed for 10 minutes in ice-cold methanol and for 2 minutes in acetone. Cells
were subsequently washed three times with Dulbecco's PBS and blocked for 2
hours in blocking buffer (Dulbecco's PBS containing 2% BSA, 2% FCS, 0.2 % fish
gelatine and 5% of normal rabbit, goat and guinea-pig whole serum). Fixed
cells were incubated with the three primary antibodies diluted in blocking
buffer for 1 hour. After washing in PBS, immune complexes were visualized
using alexa-488, -555 and -647 conjugated antibodies (Molecular Probes).
Finally, cover slips were mounted with moviol and analyzed on a Biorad MRC1024
confocal microscope equipped with a Nikon Axiophot inverted microscope and a
60x plan apochromat (Nikon) oil-immersion objective. The different
fluorochromes were sequentially captured using LaserSharp 3.0 and finally
processed in Adobe Photoshop 5.0.2 (Adobe, CA).
Cell free -secretase assay
Microsomal membranes, prepared as described above, were washed twice in
0.02% saponin and finally resuspended in Tris-EDTA containing 0.5% CHAPS, and
incubated at 4°C for 1 hour. Cleared extracts (100,000 g,
1 hour) were incubated overnight (37°C) with recombinant APP
C100-flag (Li et al.,
2000a). Finally, de novo formed Aß was analyzed by SDS-PAGE
on 10% Bis-Tris Nupage gels in MES running buffer and followed by western
blotting using mAb WO2.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
The specific binding of lectins to carbohydrate moieties was used to analyze the glycosylation pattern of nicastrin in wild-type MEFs in further detail (Fig. 1B). The immature glycosylated nicastrin reacted strongly with Galanthus nivalis agglutinin (GNA), indicating the presence of terminally linked mannose sugar residues, in agreement with its endoH sensitivity. The mature nicastrin also reacted weakly with this lectin, confirming the presence of at least one high-mannose oligosaccharide on mature nicastrin as well. The mature nicastrin protein also reacted with the lectin Datura stramonium agglutinin (DSA), which indicates the presence of complex and hybrid N-glycan structures. Finally, the reactivity of the lectin Maackia amurensis agglutinin (MAA) indicated that sialic acid residues are added to the oligosaccharide side chains of mature nicastrin. No reaction was observed with the lectin peanut agglutinin (PNA), suggesting the absence of O-glycan modifications. This was further confirmed by the insensitivity of the nicastrin proteins to O-glycosidase treatment (not shown). However, the higher Mr form of nicastrin was sensitive to neuraminidase, a glycosidase that removes sialic acid residues from oligosaccharides, further confirming the presence of sialic acid on its oligosaccharide side chains (not shown). The addition of sialic acid occurs in the trans-Golgi network, and we conclude therefore that nicastrin reaches the trans-cisternae of the Golgi in wild-type MEFs.
In primary cultures of cortical neurons, a different picture was observed:
only one single protein band reacted with nicastrin antibodies
(Fig. 1C). Removing all
N-glycans by N-glycosidase F digestion yielded a protein core with
Mr 70 kDa. EndoH treatment resulted in a loss of
Mr of
20 kDa, indicating that part of the
oligosaccharide side chains of nicastrin in neurons is of the high-mannose
type as in fibroblasts. Further experiments
(Fig. 1C) demonstrated that
nicastrin in neurons was neuraminidase but not O-glycosidase sensitive.
Overall, neurons apparently contain mainly mature nicastrin, whereas a
considerable pool of immature nicastrin is observed in fibroblasts.
Importantly, glycosylation was severely impaired when nicastrin was
exogenously overexpressed as, under these conditions, nicastrin, pulse-labeled
for 15 minutes, remained entirely endoH sensitive even after 24 hours chase
(results not shown).
PS1 interacts with mature nicastrin and is required for its complex
maturation
PS1 was immune precipitated from (metabolically labeled) MEFs using PS1
NTF- and CTF-specific antibodies. Mainly the mature form of nicastrin and only
a very small amount of immature nicastrin co-precipitated with the PS complex
(Fig. 2A). To analyze to what
extent the oligosaccharide modifications of nicastrin were involved in the
interaction with PS1, we treated MEFs with MNJ, a mannosidase type I
inhibitor. This treatment inhibits the trimming of the high-mannose
oligosaccharides upon arrival in the cis-Golgi, and therefore prevents their
further maturation to complex oligosaccharides in the distal Golgi
compartments. As expected, this treatment resulted in fully endoH-sensitive
nicastrin (Fig. 2B).
Surprisingly, under these conditions, immature nicastrin still co-precipitated
well with PS1, indicating that the carbohydrate modifications themselves are
not required for the binding to PS (Fig.
2C).
|
We next investigated whether the absence of PS or APP [which both bind to
nicastrin (Esler et al., 2002;
Yu et al., 2000
)] affected the
expression of nicastrin (Fig.
1A,D). In the complete absence of PS
(PS1/PS2/ MEFs), only the
lower immature endoH-sensitive nicastrin band is observed
(Fig. 1A, four right-hand
lanes). Interestingly, it appears that PS1 contributes significantly to the
maturation of nicastrin. Indeed, in single PS2/ MEFs,
we found similar amounts of mature nicastrin as in wild-type MEFs whereas, in
PS1/ MEFs, only some mature glycosylated nicastrin
was visible after prolonged exposure (Fig.
1A, inset showing overexposed parts of the blots). When analyzed
in APP-deficient cells (Fig.
1D), no effect on the expression or glycosylation pattern of
nicastrin was observed. Also, PS1 expression was unaffected in APP-knockout
cells (not shown).
Finally, in PS1-deficient neurons, one single nicastrin band is observed,
as in wild-type neurons (Fig.
1C). However, after endoH treatment, most of the nicastrin protein
migrates at 70 kDa (Fig. 1C,
right panel, lane 2), indicating that it contains only glycosyl residues of
the high-mannose, immature type. This confirms that PS1 is also needed in
neurons for an efficient maturation of nicastrin. Since we are not able to
generate PS1 and 2 double-deficient neurons owing to the early lethality of
the embryos (Herreman et al.,
1999), it is not possible to confirm that the residual maturation
of nicastrin in the PS1-deficient neurons was, as in fibroblasts, indeed
dependent on PS2.
Subcellular localization of endogenous nicastrin
We next investigated whether the major defects in glycosylation caused by
PS deficiency also affected the subcellular localization of nicastrin. We
first confirmed that nicastrin could reach the cell surface by performing
surface biotinylation experiments (Fig.
3). The specificity of this finding is clear from the fact that
immature nicastrin was not biotinylated in these experiments, either in
wild-type or in PS-deficient cells (Fig.
3). Also, at the immunocytochemical level, the absence of PS
expression causes changes in the distribution of nicastrin. In wild-type
cells, only a limited overlap of nicastrin with the ER marker protein BIP is
observed (Fig. 4, top), with
important amounts of nicastrin distributed in vesicular structures in the
cytoplasm. In PS1/PS2/ MEFs,
nicastrin distribution was far more restricted and the overlap with BIP was
much more prominent (Fig. 4,
bottom and compare insets in merged pictures).
|
|
To define more precisely to what extent nicastrin co-distributed with PS1,
we triple labeled PS1/PS2/
MEFs stably transduced with human PS1 using antibodies against endogenous
calnexin, human PS1 and endogenous nicastrin. The calnexin antibodies showed
the typical reticular structure of the ER
(Fig. 5, insets in bottom
panel). As described before (Annaert et
al., 1999), the subcellular distribution of PS1 largely overlaps
with calnexin, but also the non-ER structures (such as the intermediate
compartment) are immunostained. The overall pattern of nicastrin is in general
different from both PS1 and calnexin staining, displaying a more perinuclear
dot-like pattern. However, some discrete structures show co-localization of
nicastrin with PS1 and/or with calnexin
(Fig. 5, insets in merged
pictures).
|
Turnover of nicastrin is slow in wild-type and PS-deficient
cells
The turnover and maturation of nicastrin was further analyzed in wild-type
and PS1- and 2-deficient MEFs by metabolic pulse labeling (15 minutes)
followed by different chase periods (Fig.
6). At the end of the pulse, only immature nicastrin was clearly
visible in the wild-type fibroblasts (Fig.
6A, upper panel). After 6 hours, most of the protein was chased
into the mature nicastrin and little precursor remained visible. The
generation of mature nicastrin is relatively slow, reaching a maximum after 3
hours of chase. The mature nicastrin was remarkable stable during the next 24
hours, indicating an extremely slow degradation of the mature protein. This is
not an artefact of the cell culture system, since APP for instance becomes
fully glycosylated and is already degraded after 3 hours of chase
(Fig. 6B, upper panel).
In PS1/PS2/ MEFs, the precursor was synthesized to a similar extent as in the wild-type cells (Fig. 6A, lower panel). However, even after prolonged chase periods, no mature nicastrin was observed, confirming that PS is absolutely required for the maturation of nicastrin. The pool of immature nicastrin was remarkably slowly degraded in the PS-deficient cells (Fig. 6A, lower panel) indicating that PSs are not needed for the stability of nicastrin. Again, the global pattern of maturation and degradation of APP was fast in the PS1- and 2-deficient MEFs (Fig. 6B, lower panel).
As mentioned above, similar pulse-chase experiments performed in wild-type
neurons indicated that overexpressed nicastrin remains endoH sensitive even
after prolonged chase periods, indicating that, as is the case for PS
(Thinakaran et al., 1997),
limiting factor(s) are responsible for the maturation of this protein.
The glycosylation of nicastrin is not required for -secretase
activity
Given the close correlation between mature nicastrin and its association
with PS, we investigated whether glycosylation of nicastrin is required for
-secretase cleavage of APP and Notch. We prepared membranes from
fibroblasts treated with MNJ, an inhibitor of mannosidase I, and used them in
the cell-free
-secretase assay using recombinant synthetic APP-C99
peptides (Li et al., 2000a
).
Similar amounts of Aß were produced from solubilized membranes generated
from control cells (MNJ in Fig.
7A) and from treated cells (+MNJ in
Fig. 7A). Western blot
confirmed that almost all nicastrin in the treated cell membranes was of the
`high-mannose' type (Fig. 7B).
In a second experiment, we treated HEK cells stably transfected with APP695/Sw
with the mannosidase I inhibitor kifunensine and measured the effect on
Aß production. Kifunensine treatment effectively inhibited nicastrin
maturation beyond the level of the high-mannose type of glycosylation
(Fig. 7C). This treatment did
not inhibit Aß production (Fig.
7C). In a similar experiment, we transfected these cells
transiently with mNotchDE and analyzed NICD generation
(De Strooper et al., 1999
). No
differences between untreated or kifunensine-treated cells on NICD production
could be observed (Fig. 7C).
These data demonstrate that the maturation of the glycosyl residues on
nicastrin is not required for
-secretase activity.
|
Treatment with kifunensine also did not affect the transport of nicastrin
to the cell surface as measured by cell-surface biotinylation
(Fig. 7D). This indicates that
the maturation of the glycosyl residues of nicastrin reflects in the first
place its trafficking through the compartments of the biosynthetic pathway,
and has little or no importance for its incorporation into the
-secretase complex or its cell-surface localization.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Since, under normal conditions, mainly mature nicastrin co-precipitates
with PS1 (Fig. 2A), we
investigated the possibility that glycosylation of nicastrin has direct
functional implications for the activity of -secretase. However, we
found normal
-secretase activity towards Aß and NICD generation
when glycosylation of nicastrin was inhibited
(Fig. 7). Therefore, the
complex glycosylation of nicastrin is not directly needed for the
-secretase processing of APP or Notch substrates. In previous work, we
noticed that brefeldin A treatment of neurons, which fuses several components
of the Golgi apparatus with the ER, is able to activate
-secretase
cleavage of an APP substrate that is specifically retained in the ER
(Cupers et al., 2001a
). The
good correlation between complex glycosylation of nicastrin, PS binding and
-secretase activity documented in the current work reinforces the
hypothesis that passage of nicastrin and probably other components of the
-secretase complex through the Golgi apparatus is a prerequisite for
the maturation and activation of the protease. Thus, whereas the glycosylation
of nicastrin is not a direct requisite for activity, it reflects faithfully
the acquisition of one or more additional components/factors associated with
the trafficking through the Golgi apparatus that makes the protease fully
operational.
Previous work (Chung and Struhl,
2001; Goutte et al.,
2002
; Hu et al.,
2002
) showed that PS becomes destabilized in the absence of
nicastrin. In the current work, we find an inverse relationship: PSs are
needed for the trafficking and maturation of nicastrin. The effects caused by
PS1 or PS2 deficiency on nicastrin maturation reflects very closely the
effects on
-secretase inhibition by the different PS-deficient
genotypes. Thus, in PS1- and 2-deficient cells, which do not display
-secretase activity, no glycosyl maturation of nicastrin was observed
at all. In PS1-deficient cells that display a quantitatively important but not
complete
-secretase deficiency, a tiny fraction of nicastrin became
complex glycosylated. Finally, in PS2-deficient cells that display an almost
normal
-secretase activity
(Herreman et al., 1999
;
Herreman et al., 2000
;
Zhang et al., 2000
), no
changes in the glycosylation pattern of nicastrin compared with wild-type
cells were observed. These observations, together with the profound effects of
PS deficiency on nicastrin subcellular localization, underscore the
possibility that nicastrin and PS travel, maturate and become stabilized
together to constitute a functional unit displaying
-secretase
activity. It should be noted that a fraction of PS and nicastrin do not
co-distribute in the cell (Fig.
5), perhaps reflecting dynamic changes in the stoichiometry of the
proteins or indicating that both proteins could be involved in functions where
they are not operating in one functional unit. Pulse-chase experiments and
immunocytochemical data demonstrate that, in the absence of PS, nicastrin
remains largely caught in the ER. This suggests that nicastrin needs to
associate with PS to leave the ER, or at least that PSs are involved in the
transport of nicastrin out of the ER. Since only little or no immature
nicastrin is associated with PS in wild-type cells, we suggest that nicastrin
and PS rapidly leave the ER once they become associated and travel together
towards the Golgi apparatus and perhaps to the cell surface. In the Golgi
compartments, nicastrin undergoes post-translational modifications, but this
maturation is not required for
-secretase function nor for further
trafficking to the cell surface, as convincingly demonstrated using the
mannosidase I inhibitors kifunensine or MNJ
(Fig. 7). Our biotinylation
data unequivocally demonstrate the presence of mature nicastrin at the cell
surface in wild-type cells. This could also indicate the presence of PSs at
the cell surface (Ray et al.,
1999a
; Kaether et al.,
2002
). By contrast, several groups have demonstrated that the bulk
of PS immunoreactivity is distributed in the ER, intermediate compartment and
to a certain extent into the early Golgi
(Annaert et al., 1999
;
Culvenor et al., 1997
;
Lah et al., 1997
). Since
-secretase activity is believed to occur mainly in endosomes, Golgi and
at the cell surface, some discrepancy still exists between the subcellular
distribution of PS, which is the putative catalytic subunit, and the
-secretase activity, agreeing with the `spatial paradox'
(Annaert and De Strooper,
1999
). The current data indicate that this spatial paradox is not
absolute since small amounts of PS are apparently leaving the ER/intermediate
compartment/cis-Golgi, as deduced from the appearance of nicastrin at the cell
surface. Nevertheless, it should be stressed that this does not answer several
questions. For example, what is the function of the bulk of PS in the ER or
why are the PSs, containing the putative catalytic site of the complex, not
functionally active in the ER (Cupers et
al., 2001a
; Maltese et al.,
2001
)?
Our data suggest that the PSs have, in addition to their role in
-secretase activity, an important function in the trafficking and
maturation of nicastrin. One could argue that the disturbances in nicastrin
trafficking and maturation observed in PS-deficient cells does not necessarily
imply such an intrinsic function of PS since it is not unusual that an
oligomeric complex needs to be fully assembled before all subunits can travel
through the biosynthetic pathway. By contrast, deficiency of PSs also affects
the subcellular distribution of other proteins, such as APP and the Trk
receptor (Naruse et al., 1998
)
and telencephalin (Annaert et al.,
2001
). Therefore, it becomes likely that PS has, in addition to
its role as a
-secretase catalytic subunit, an important function in
subcellular trafficking of a selected group of proteins. This dual function
could probably explain the large amounts of PSs in the early biosynthetic
compartments having no role in proteolysis
(Cupers et al., 2001a
).
In conclusion, the current manuscript provides basic information regarding the cellular distribution of nicastrin, its post-translational modifications and their role in the generation of the PS/nicastrin complex, and the effect of PS deficiency on nicastrin biosynthesis and trafficking.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Annaert, W. and de Strooper, B. (1999). Presenilins: molecular switches between proteolysis and signal transduction. Trends Neurosci. 22,439 -443.[CrossRef][Medline]
Annaert, W. and de Strooper, B. (2002). A cell biological perspective on Alzheimer's disease. Annu. Rev. Cell Dev. Biol. 18,25 -51.[CrossRef][Medline]
Annaert, W. G., Levesque, L., Craessaerts, K., Dierinck, I.,
Snellings, G., Westaway, D., George-Hyslop, P. S., Cordell, B., Fraser, P. and
de Strooper, B. (1999). Presenilin 1 controls gamma-secretase
processing of amyloid precursor protein in pre-golgi compartments of
hippocampal neurons. J. Cell Biol.
147,277
-294.
Annaert, W. G., Esselens, C., Baert, V., Boeve, C., Snellings, G., Cupers, P., Craessaerts, K. and de Strooper, B. (2001). Interaction with telencephalin and the amyloid precursor protein predicts a ring structure for presenilins. Neuron 32,579 -589.[Medline]
Baek, S. H., Ohgi, K. A., Rose, D. W., Koo, E. H., Glass, C. K. and Rosenfeld, M. G. (2002). Exchange of N-CoR corepressor and Tip60 coactivator complexes links gene expression by NF-kappaB and beta-amyloid precursor protein. Cell 110, 55-67.[Medline]
Cao, X. and Sudhof, T. C. (2001). A
transcriptionally [correction of transcriptively] active complex of APP with
Fe65 and histone acetyltransferase Tip60. Science
293,115
-120.
Chen, F., Yu, G., Arawaka, S., Nishimura, M., Kawarai, T., Yu, H., Tandon, A., Supala, A., Song, Y. Q., Rogaeva, E. et al. (2001). Nicastrin binds to membrane-tethered Notch. Nat. Cell Biol. 3,751 -754.[CrossRef][Medline]
Chung, H. M. and Struhl, G. (2001). Nicastrin is required for Presenilinmediated transmembrane cleavage in Drosophila.Nat. Cell Biol. 3,1129 -1132.[CrossRef][Medline]
Culvenor, J. G., Maher, F., Evin, G., Malchiodi-Albedi, F., Cappai, R., Underwood, J. R., Davis, J. B., Karran, E. H., Roberts, G. W., Beyreuther, K. et al. (1997). Alzheimer's disease-associated presenilin 1 in neuronal cells: evidence for localization to the endoplasmic reticulumGolgi intermediate compartment. J. Neurosci. Res. 49,719 -731.[CrossRef][Medline]
Cupers, P., Bentahir, M., Craessaerts, K., Orlans, I.,
Vanderstichele, H., Saftig, P., de Strooper, B. and Annaert, W.
(2001a). The discrepancy between presenilin subcellular
localization and gamma-secretase processing of amyloid precursor protein.
J. Cell Biol. 154,731
-740.
Cupers, P., Orlans, I., Craessaerts, K., Annaert, W. and de Strooper, B. (2001b). The amyloid precursor protein (APP)-cytoplasmic fragment generated by gamma-secretase is rapidly degraded but distributes partially in a nuclear fraction of neurones in culture. J. Neurochem. 78,1168 -1178.[CrossRef][Medline]
De Strooper, B., Simons, M., Multhaup, G., van Leuven, F., Beyreuther, K. and Dotti, C. G. (1995). Production of intracellular amyloid-containing fragments in hippocampal neurons expressing human amyloid precursor protein and protection against amyloidogenesis by subtle amino acid substitutions in the rodent sequence. EMBO J. 14,4932 -4938.[Abstract]
De Strooper, B., Beullens, M., Contreras, B., Levesque, L.,
Craessaerts, K., Cordell, B., Moechars, D., Bollen, M., Fraser, P.,
George-Hyslop, P. S. et al. (1997). Phosphorylation,
subcellular localization, and membrane orientation of the Alzheimer's
disease-associated presenilins. J. Biol. Chem.
272,3590
-3598.
De Strooper, B., Saftig, P., Craessaerts, K., Vanderstichele, H., Guhde, G., Annaert, W., von Figura, K. and van Leuven, F. (1998). Deficiency of presenilin-1 inhibits the normal cleavage of amyloid precursor protein. Nature 391,387 -390.[CrossRef][Medline]
De Strooper, B., Annaert, W., Cupers, P., Saftig, P., Craessaerts, K., Mumm, J. S., Schroeter, E. H., Schrijvers, V., Wolfe, M. S., Ray, W. J. et al. (1999). A presenilin-1-dependent gamma-secretase-like protease mediates release of Notch intracellular domain. Nature 398,518 -522.[CrossRef][Medline]
Edbauer, D., Winkler, E., Haass, C. and Steiner, H.
(2002). Presenilin and nicastrin regulate each other and
determine amyloid beta-peptide production via complex formation.
Proc. Natl. Acad. Sci. USA
99,8666
-8671.
Esler, W. P., Kimberly, W. T., Ostaszewski, B. L., Diehl, T. S., Moore, C. L., Tsai, J. Y., Rahmati, T., Xia, W., Selkoe, D. J. and Wolfe, M. S. (2000). Transition-state analogue inhibitors of gamma-secretase bind directly to presenilin-1. Nat. Cell Biol. 2,428 -434.[CrossRef][Medline]
Esler, W. P., Kimberly, W. T., Ostaszewski, B. L., Ye, W.,
Diehl, T. S., Selkoe, D. J. and Wolfe, M. S. (2002).
Activity-dependent isolation of the presenilin-gamma-secretase complex reveals
nicastrin and a gamma substrate. Proc. Natl. Acad. Sci.
USA 99,2720
-2725.
Francis, R., McGrath, G., Zhang, J., Ruddy, D. A., Sym, M., Apfeld, J., Nicoll, M., Maxwell, M., Hai, B., Ellis, M. C. et al. (2002). aph-1 and pen-2 are required for Notch pathway signaling, gamma-secretase cleavage of betaAPP, and presenilin protein accumulation. Dev. Cell. 3,85 -97.[Medline]
Gao, Y. and Pimplikar, S. W. (2001). The
gamma-secretase-cleaved C-terminal fragment of amyloid precursor protein
mediates signaling to the nucleus. Proc. Natl. Acad. Sci.
USA 98,14979
-14984.
Goutte, C., Tsunozaki, M., Hale, V. A. and Priess, J. R.
(2002). APH-1 is a multipass membrane protein essential for the
Notch signaling pathway in Caenorhabditis elegans embryos.
Proc. Natl. Acad. Sci. USA
99,775
-779.
Herreman, A., Hartmann, D., Annaert, W., Saftig, P.,
Craessaerts, K., Serneels, L., Umans, L., Schrijvers, V., Checler, F.,
Vanderstichele, H. et al. (1999). Presenilin 2 deficiency
causes a mild pulmonary phenotype and no changes in amyloid precursor protein
processing but enhances the embryonic lethal phenotype of presenilin 1
deficiency. Proc. Natl. Acad. Sci. USA
96,11872
-11877.
Herreman, A., Serneels, L., Annaert, W., Collen, D., Schoonjans, L. and de Strooper, B. (2000). Total inactivation of gamma-secretase activity in presenilin-deficient embryonic stem cells. Nat. Cell Biol. 2,461 -462.[CrossRef][Medline]
Hu, Y., Ye, Y. and Fortini, M. E. (2002). Nicastrin is required for gamma-secretase cleavage of the Drosophila notch receptor. Dev. Cell 2, 69-78.[Medline]
Kaether, C., Lammich, S., Edbauer, D., Ertl, M., Rietdorf, J.,
Capell, A., Steiner, H. and Haass, C. (2002). Presenilin-1
affects trafficking and processing of betaAPP and is targeted in a complex
with nicastrin to the plasma membrane. J. Cell Biol.
158,551
-561.
Kimberly, W. T., Zheng, J. B., Guenette, S. Y. and Selkoe, D.
J. (2001). The intracellular domain of the beta-amyloid
precursor protein is stabilized by Fe65 and translocates to the nucleus in a
notch-like manner. J. Biol. Chem.
276,40288
-40292.
Kopan, R. and Goate, A. (2000). A common enzyme
connects notch signaling and Alzheimer's disease. Genes
Dev. 14,2799
-2806.
Lah, J. J., Heilman, C. J., Nash, N. R., Rees, H. D., Yi, H.,
Counts, S. E. and Levey, A. I. (1997). Light and electron
microscopic localization of presenilin-1 in primate brain. J.
Neurosci. 17,1971
-1980.
Leem, J. Y., Vijayan, S., Han, P., Cai, D., Machura, M., Lopes, K. O., Veselits, M. L., Xu, H. and Thinakaran, G. (2002). Presenilin 1 is required for maturation and cell surface accumulation of nicastrin. J. Biol. Chem. 24,19236 -19240.[CrossRef]
Leissring, M. A., Murphy, M. P., Mead, T. R., Akbari, Y.,
Sugarman, M. C., Jannatipour, M., Anliker, B., Muller, U., Saftig, P., de
Strooper, B. et al. (2002). A physiologic signaling role for
the gamma-secretase-derived intracellular fragment of APP. Proc.
Natl. Acad. Sci. USA 99,4697
-4702.
Li, Y. M., Lai, M. T., Xu, M., Huang, Q., DiMuzio-Mower, J.,
Sardana, M. K., Shi, X. P., Yin, K. C., Shafer, J. A. and Gardell, S. J.
(2000a). Presenilin 1 is linked with gamma-secretase activity in
the detergent solubilized state. Proc. Natl. Acad. Sci.
USA 97,6138
-6143.
Li, Y. M., Xu, M., Lai, M. T., Huang, Q., Castro, J. L., DiMuzio-Mower, J., Harrison, T., Lellis, C., Nadin, A., Neduvelil, J. G. et al. (2000b). Photoactivated gamma-secretase inhibitors directed to the active site covalently label presenilin 1. Nature 405,689 -694.[CrossRef][Medline]
Lopez-Schier, H. and St Johnston, D. (2002). Drosophila nicastrin is essential for the intramembranous cleavage of notch. Dev. Cell 2,79 -89.[Medline]
Maltese, W. A., Wilson, S., Tan, Y., Suomensaari, S., Sinha, S.,
Barbour, R. and McConlogue, L. (2001). Retention of the
Alzheimer's amyloid precursor fragment C99 in the endoplasmic reticulum
prevents formation of amyloid beta-peptide. J. Biol.
Chem. 276,20267
-20279.
Marambaud, P., Shioi, J., Serban, G., Georgakopoulos, A.,
Sarner, S., Nagy, V., Baki, L., Wen, P., Efthimiopoulos, S., Shao, Z. et
al. (2002). A presenilin-1/gamma-secretase cleavage releases
the E-cadherin intracellular domain and regulates disassembly of adherens
junctions. EMBO J. 21,1948
-1956.
May, P., Reddy, Y. K. and Herz, J. (2002). Proteolytic processing of low density lipoprotein receptor-related protein mediates regulated release of its intracellular domain. J. Biol. Chem. 21,18736 -18743.[CrossRef]
Naruse, S., Thinakaran, G., Luo, J. J., Kusiak, J. W., Tomita, T., Iwatsubo, T., Qian, X., Ginty, D. D., Price, D. L., Borchelt, D. R. et al. (1998). Effects of PS1 deficiency on membrane protein trafficking in neurons. Neuron 21,1213 -1221.[Medline]
Ni, C. Y., Murphy, M. P., Golde, T. E. and Carpenter, G.
(2001). Gamma-secretase cleavage and nuclear localization of
erbb-4 receptor tyrosine kinase. Science
294,2179
-2181.
Nyabi, O., Pype, S., Mercken, M., Herreman, A., Saftig, P., Craessaerts, K., Serneels, L., Annaert, W. and de Strooper, B. (2002). No endogenous A beta production in presenilin-deficient fibroblasts. Nat. Cell Biol. 4, E164
Ponting, C. P., Hutton, M., Nyborg, A., Baker, M., Jansen, K.
and Golde, T. E. (2002). Identification of a novel family of
presenilin homologues. Hum. Mol. Genet.
11,1037
-1044.
Ray, W. J., Yao, M., Mumm, J., Schroeter, E. H., Saftig, P.,
Wolfe, M., Selkoe, D. J., Kopan, R. and Goate, A. M. (1999a).
Cell surface presenilin-1 participates in the gamma-secretase-like proteolysis
of notch. J. Biol. Chem.
274,36801
-36807.
Ray, W. J., Yao, M., Nowotny, P., Mumm, J., Zhang, W., Wu, J.
Y., Kopan, R. and Goate, A. M. (1999b). Evidence for a
physical interaction between presenilin and Notch. Proc. Natl.
Acad. Sci. USA 96,3263
-3268.
Roth, J., Taatjes, D. J., Lucocq, J. M., Weinstein, J. and Paulson, J. C. (1985). Demonstration of an extensive trans-tubular network continuous with the Golgi apparatus stack that may function in glycosylation. Cell 43,287 -295.[Medline]
Seiffert, D., Bradley, J. D., Rominger, C. M., Rominger, D. H.,
Yang, F., Meredith, J. E., Jr, Wang, Q., Roach, A. H., Thompson, L. A., Spitz,
S. M. et al. (2000). Presenilin-1 and -2 are molecular
targets for gamma-secretase inhibitors. J. Biol. Chem.
275,34086
-34091.
Selkoe, D. J. (1999). Translating cell biology into therapeutic advances in Alzheimer's disease. Nature 399,A23 -31.[CrossRef][Medline]
Sisodia, S. S. and St George-Hyslop, P. H. (2002). Gamma-secretase, Notch, Abeta and Alzheimer's Disease: where do the presenilins fit in? Nat. Rev. Neurosci. 3, 281-290.[CrossRef][Medline]
Steiner, H. and Haass, C. (2000). Intramembrane proteolysis by presenilins. Nat. Rev. Mol. Cell Biol. 1, 217-224.[CrossRef][Medline]
Steiner, H., Winkler, E., Edbauer, D., Prokop, S., Basset, G., Yamasaki, A., Kostka, M. and Haass, C. (2002). PEN-2 is an integral component of the gamma-secretase complex required for coordinated expression of presenilin and nicastrin. J. Biol. Chem. 42,39062 -39065.[CrossRef]
Struhl, G. and Greenwald, I. (1999). Presenilin is required for activity and nuclear access of Notch in Drosophila.Nature 398,522 -525.[CrossRef][Medline]
Thinakaran, G., Borchelt, D. R., Lee, M. K., Slunt, H. H., Spitzer, L., Kim, G., Ratovitsky, T., Davenport, F., Nordstedt, C., Seeger, M. et al. (1996). Endoproteolysis of presenilin 1 and accumulation of processed derivatives in vivo. Neuron 17,181 -190.[Medline]
Thinakaran, G., Harris, C. L., Ratovitski, T., Davenport, F.,
Slunt, H. H., Price, D. L., Borchelt, D. R. and Sisodia, S. S.
(1997). Evidence that levels of presenilins (PS1 and PS2) are
coordinately regulated by competition for limiting cellular factors.
J. Biol. Chem. 272,28415
-28422.
Weihofen, A., Binns, K., Lemberg, M. K., Ashman, K. and
Martoglio, B. (2002). Identification of signal peptide
peptidase, a presenilin-type aspartic protease.
Science 296,2215
-2218.
Wolfe, M. S., Xia, W., Ostaszewski, B. L., Diehl, T. S., Kimberly, W. T. and Selkoe, D. J. (1999). Two transmembrane aspartates in presenilin-1 required for presenelin endoproteolysis and gamma-secretase activity. Nature 398,513 -517.[CrossRef][Medline]
Ye, Y., Lukinova, N. and Fortini, M. E. (1999). Neurogenic phenotypes and altered Notch processing in Drosophila Presenilin mutants Nature 398,525 -529.[CrossRef][Medline]
Yu, G., Chen, F., Levesque, G., Nishimura, M., Zhang, D. M.,
Levesque, L., Rogaeva, E., Xu, D., Liang, Y., Duthie, M. et al.
(1998). The presenilin 1 protein is a component of a high
molecular weight intracellular complex that contains beta-catenin.
J. Biol. Chem. 273,16470
-16475.
Yu, G., Nishimura, M., Arawaka, S., Levitan, D., Zhang, L., Tandon, A., Song, Y. Q., Rogaeva, E., Chen, F., Kawarai, T. et al. (2000). Nicastrin modulates presenilin-mediated notch/glp-1 signal transduction and betaAPP processing. Nature 407, 48-54.[CrossRef][Medline]
Zhang, Z., Nadeau, P., Song, W., Donoviel, D., Yuan, M., Bernstein, A. and Yankner, B. A. (2000). Presenilins are required for gamma-secretase cleavage of beta-APP and transmembrane cleavage of Notch-1. Nat. Cell Biol. 2, 463-465.[CrossRef][Medline]
Related articles in JCS: