Characterization of presenilin complexes from mouse and human brain using Blue Native gel electrophoresis reveals high expression in embryonic brain and minimal change in complex mobilit
Trang 1Characterization of presenilin complexes from mouse and human brain using Blue Native gel electrophoresis reveals high expression
in embryonic brain and minimal change in complex mobility with pathogenic presenilin mutations
Janetta G Culvenor1,2,3, Nancy T Ilaya1,2,3, Michael T Ryan4, Louise Canterford1,3, David E Hoke1,3, Nicholas A Williamson1,3, Catriona A McLean5, Colin L Masters1,3and Genevie`ve Evin1,3
1 Department of Pathology and 2 Centre for Neuroscience, The University of Melbourne, Australia; 3 Mental Health Research Institute
of Victoria, Australia; 4 Department of Biochemistry, La Trobe University, Bundoora, Australia; 5 National Neuroscience Facility, University of Melbourne, Australia
The presenilin proteins are required for intramembrane
cleavage of a subset of type 1 membrane proteins including
the Alzheimer’s disease amyloid precursor protein Previous
studies indicate presenilin proteins form enzymatically
act-ive high molecular mass complexes consisting of
hetero-dimers of N- and C-terminal fragments in association with
nicastrin, presenilin enhancer-2 and anterior pharynx
defective-1 proteins Using Blue Native gel electrophoresis
(BN/PAGE) we have studied endogenous presenilin 1
complex mass, stability and association with nicastrin,
presenilin enhancer-2 and anterior pharynx defective-1
Solubilization of mouse or human brain membranes with
dodecyl-D-maltoside produced a 360-kDa species reactive
with antibodies to presenilin 1 Presenilin 1 complex levels
were high in embryonic brain Complex integrity was
sen-sitive to Triton X-100 and SDS, but stable to reducing
agent Addition of 5Murea caused complex dissolution and
nicastrin to migrate as a subcomplex Nicastrin and pre-senilin enhancer-2 were detected in the prepre-senilin 1 complex following BN/PAGE, electroelution and second-dimension analysis Anterior pharynx defective-1 was detected as an 18-kDa form and 9-kDa C-terminal fragment by standard SDS/PAGE of mouse tissues, and as a predominant 36-kDa band after presenilin 1 complex second-dimension analysis Membranes from brain cortex of Alzheimer’s disease patients, or from cases with presenilin 1 missense mutations, indicated no change in presenilin 1 complex mobility Higher molecular mass presenilin 1-reactive species were detected in brain containing presenilin 1 exon 9 deletion mutation This abnormality was confirmed using cells transfected with the same presenilin deletion mutation Keywords: Alzheimer’s disease; brain; Blue Native PAGE; presenilin complex
The presenilins are multispanning membrane proteins
(presenilin 1, PS1 and presenilin 2, PS2) first identified for
their genetic association with Alzheimer’s disease (AD)
They are essential components of a multiprotein protease
complex implicated in regulated intramembrane proteolysis
of several type 1 membrane proteins including the amyloid
precursor protein (APP) and developmentally important
Notch receptors [1] The variety of identified substrates
indicates a critical role for PS in cell metabolism involving controlled cleavage of protein transmembrane domains and signal transduction The role of PS in c-secretase activity
is compelling and includes lack of Ab amyloid peptide generation and Notch signalling in PS double knockout cells, cofractionation of activity with PS, abolition of activity with mutation of conserved aspartates in trans-membrane domains six and seven, binding of c-secretase aspartyl protease inhibitors to PS, as well as identification of homologues with protease-associated domains [2–6] Fol-lowing a primary cleavage event that causes ectodomain shedding, PS-dependent Ôc-secretaseÕ proteolysis results in generation of a membrane spanning stub (such as Ab peptide) and a cytoplasmic fragment such as the APP intracellular domain or the Notch intracellular domain which translocates to the nucleus for regulation of gene expression [1]
The PS proteins undergo endoproteolysis between trans-membrane domains six and seven to generate heterodimers
of N-terminal fragments (NTF) and C-terminal fragments (CTF) [7] Many binding partners for the PS proteins have been identified including substrates and the catenins (b, d and p0071) [8] The interacting components that are required for c-secretase activity besides PS include the
Correspondence to J G Culvenor, Department of Pathology,
The University of Melbourne, Parkville, Victoria, 3010 Australia.
Fax: +61 38344 4004, Tel.: +61 38344 3990,
E-mail: janetta@unimelb.edu.au
Abbreviations: AD, Alzheimer’s disease; APH, anterior pharynx
defective; APP, amyloid precursor protein; BACE, b-site
APP-clea-ving enzyme; BN/PAGE, Blue Native polyacrylamide gel
electro-phoresis; CTF, carboxyl-terminal fragment; DDM, n-dodecyl
b- D -maltoside; ECL, enhanced chemiluminescence; FAD, familial
Alzheimer’s disease; FTD, frontotemporal dementia; NCT, nicastrin;
NTF, amino-terminal fragment; PEN, presenilin enhancer;
PS1, presenilin 1; PS2, presenilin 2.
(Received 2 October 2003, revised 9 November 2003,
accepted 20 November 2003)
Trang 2glycoprotein, nicastrin (NCT) identified by affinity
purifi-cation of PS1 complex using digitonin-solubilization of
membranes from human embryonic kidney cells [9], and
two further membrane proteins PEN-2 and APH-1,
iden-tified by genetic screening in Caenorhabditis elegans [10,11]
NCT is a heavily glycosylated type 1 membrane protein
Maturation of NCT glycosylation is impaired in
PS1-deficient cells, and down-regulation of NCT inhibits
c-secretase cleavage of APP and Notch [11–13] PEN-2 is
a small double membrane-spanning protein with N- and
C-termini facing the lumen [14] APH-1 contains seven
putative transmembrane domains and has a variety of
isoforms with differing C-terminal domains; in humans,
APH-1a long and short forms are encoded by a gene on
chromosome 1 and APH-1b by chromosome 15 [15,16]
Glycerol velocity gradient centrifugation and gel filtration
of detergent-solubilized membrane extracts have
character-ized PS complexes of sizes ranging between 150 kDa and
two million kDa [5,17,18] There is limited knowledge about
the assembly and role of the PS complex components other
than PS We have applied Blue Native gel electrophoresis
(BN/PAGE) [19] to analyse further and characterize
endo-genous native complexes from mouse and human brain
tissue and from the neuroblastoma line SH-SY5Y (SY5Y)
Experimental procedures
Antibodies
Synthetic peptides derived from human sequences were
conjugated to diphtheria toxoid and rabbit antibodies
generated as follows: Ab 98/1, PS1 NT (1–20) [20]; Ab 00/
1 and Ab 00/2 PS1 loop (301–317) [21]; Ab 00/12, PS2 loop
(307–336); Ab 00/19 NCT CT (691–709]) [22]; Ab 00/22
NCT ectodomain (331–346); Ab 02/45 PEN-2 NT (1–15)
and Ab 02/41 APH-1b CT (244–257) Ab 00/6 was raised to
human BACE CT (485–501) [23] NCT, PEN-2 and APH-1
antibodies were affinity-purified using immunizing peptide
coupled to SulfoLink Coupling Gel (Pierce) Mouse
mAb C1/6.1 raised to APP (676–695) was a gift from
P Mathews, Nathan Kline Institute for Psychiatric
Research, NY, USA [24]
Cell lines
Human neuroblastoma SY5Y wild-type cells were cultured
as described previously and stably transfected with cDNA
for PS1, PS1 with exon 9 deletion mutation, or PS1 with
D257A artificial mutation following published protocols
[20] Mouse embryonic neural stem cell lines NS51 (PS1–/–)
and NS52 (PS1 wt) were derived and cultured from
PS1-deficient mice as described [25]
Brain bank tissue
Samples of frontal cortex from human brain were obtained
from the NHMRC Tissue Resource Centre (Melbourne,
Australia) and were well characterized pathologically and
clinically [23] Details of pathology or mutation analysis of
tissue used in this study is published as follows: FTD1 and
FTD2 [26], FAD PS1 L271V [27], FAD PS1 exon 9 deletion
[28], FAD PS1 S169L [29] and FAD PS1 L219P [30]
Membrane preparations Cells were washed in NaCl/Piand pellets stored at)70 C Mouse brain tissue was obtained from C57/Bl6 mice, APPC100 transgenic mice (over-expressing last 99 residues
of human APP) [31], APPsw transgenic mice Tg 2576 (over-expressing APP with Swedish mutation) [32], or transgenic mice homozygous for human PS1M146 L [33] Samples were homogenized in 250 mM sucrose, 20 mM Hepes
pH 7.4 with protease inhibitors (Sigma P-2714) at 4C with a glass Dounce homogenizer Centrifugation at 1000 g for 10 min, 4C, yielded a post-nuclear supernatant that was further centrifuged at 100 000 g for 1 h, 4C, and the resultant membrane pellets were resuspended in sucrose homogenization buffer with protease inhibitors and stored
at )70 C Protein concentration was determined with bicinchoninic acid reagent (Pierce)
BN/PAGE Membrane protein (30–100 lg protein per gel lane) was resuspended in 0.2–0.5% n-dodecyl b-D-maltoside (DDM) (or alternate treatment as indicated) in buffer composed of
50 mM NaCl, 5 mM 6-aminohexanoic acid and 50 mM imidazole pH 7.0 and then clarified after 15 min at room temperature by microfuge centrifugation for 5 min before addition of 0.5% Coomassie G250 (Sigma) and 50 mM 6-aminohexanoic acid Samples were resolved on 4–16.5% gradient acrylamide gels with cooling; anode buffer con-tained 25 mMimidazole pH 7.0; cathode buffer contained
50 mM tricine, 7.5 mM imidazole pH 7.0 with 0.02% Coomassie G 250 [34] High molecular mass standards for native electrophoresis were from Amersham and were stained with Coomassie R250 Following electrophoretic transfer to poly(vinylidene difluoride) membrane (Immobi-lon-P, Millipore), proteins were identified by immunoblot-ting and detection with enhanced chemiluminescence (ECL; Amersham) For second-dimension analysis, gel bands corresponding to the PS complex region derived from 2-cm wide lanes, 16-cm length, 1.5-mm thickness slab gels were excised at 1 cm below the ferritin marker as indicated, proteins were electroeluted with BN/PAGE buffers, and samples were solubilized with 1.5% SDS, 2.3Murea, 0.1M dithiothreitol, 15 mM Tris/HCl pH 6.8 with heating at
50C for 15 min before electrophoresis on 10% Tris/tricine gels
Immunoblotting For analysis of PS1, NCT, PEN-2 and APH-1, membrane proteins were separated with reducing agent on Tris/glycine/ SDS or Tris/tricine/SDS polyacrylamide gels and analysed
by immunoblotting as described previously [20,35]
Results
PS1 is detected in a 360 kDa complex with nicastrin
in embryonic mouse brain and SY5Y cells Preliminary investigation of optimal conditions for PS1 complex analysis from mouse brain by BN/PAGE indicated that solubilization of membranes with 0.5% DDM
Trang 3generated a complex of mobility 360 kDa, which
migra-ted ahead of the ferritin marker (440 kDa) as detecmigra-ted by
Western analysis (Fig 1A) The complex was reactive with
both PS1 N-terminal (98/1) and loop antibodies (00/1)
Digitonin was also compatible with maintaining the
com-plex stability and yielded a comcom-plex of higher apparent
molecular weight of about 460 kDa (Fig 1A) Chapso
solubilization was also tested as it has proved useful for
maintaining c-secretase activity [5,36] but did not produce a
distinct band of PS1 complex immunoreactivity under these
electrophoresis conditions (data not shown) Embryonic
mouse brain produced stronger PS1 complex
immunoreac-tivity (Fig 1A) compared to adult tissue Results for brain
tissue from adult PS1 [M146L] transgenic mice is shown but was the same for mouse wild-type brain NCT was readily detected in association with PS1 complex in embryonic tissue as indicated by reactivity of NCT ectodomain antibody NCT was barely detectable in the PS complex from adult mouse brain as shown in the last lane of Fig 1A PS1 and NCT were also strongly detected as an apparent complex in membranes from the human neuroblastoma cell line SY5Y using PS1 Ab 98/1 and C-terminal NCT Ab 00/
19 (Fig 2A)
We investigated further these apparent changes in PS1 and NCT detection with developmental stages NCT levels
in neural stem cell lines and mouse brain are compared in Fig 1B after analysis using 12% SDS/PAGE and immu-nodetection with NCT CT Ab 00/19 (left panel) Antibody reactivity was removed by preabsorption with immunizing peptide (middle panel) Consistent with previous reports [12,37–39] we found that cells derived from PS1 knockout mice express high levels of less mature NCT ( 110 kDa) Embryonic mouse brain contained a more intermediate glycosylated form of NCT ( 120 kDa) than adult brain where the predominant NCT species was a 140-kDa form NCT ectodomain Ab 00/22 reactivity indicated detection of only the less mature or fully deglycosylated forms of NCT (data not shown), this finding may explain the poor detection of NCT in adult mouse brain complex as high glycosylation may impair antibody access to the epitope As expected, the PS1 N-terminal antibody detected a 29-kDa immunoreactive band from these tissues corresponding to PS1 NTF which was not detectable in PS1-deficient cells
Amyloid precursor protein, b-catenin, and b-secretase are not tightly associated with the PS1 complex
To investigate possible association of the c-secretase substrate APP or APP C-terminal fragments with the complex, BN/PAGE blots of membranes from SY5Y, APPC100 transgenic mouse brain and APP over-expressing transgenic mouse brain (Tg2576) were probed with APP mAb C1/6.1 to the APP C terminus Fig 2A shows that APP was detected as a smear of reactivity that spanned the PS1 complex mobility Some increase in APP reactivity corresponding to PS1 complex mobility was observed for APP Tg2576 tissue when contrast of the blot was modified, indicated as Tg2576* on Fig 2A Antibody for the PS1 binding partner, b-catenin, produced a smear after BN/ PAGE of molecular mass greater than the 440 marker consistent with interaction with many alternate binding proteins
Fig 2B compares immunoreactivity for b-site APP-cleaving enzyme (BACE), PS2 and PS1 in wild-type mouse brain membrane extracts after BN/PAGE Immunoreac-tivity in a broad band of molecular mass greater than
550 kDa did not suggest association of BACE with the PS1 complex PS2 immunoreactivity indicated that the PS2 complex has a slightly higher complex mass of 420 kDa compared with 360 kDa for PS1
Complex stability to detergents and denaturing agents Samples of adult mouse brain membranes were solubilized with alternative detergents and subjected to BN/PAGE to
Fig 1 BN/PAGE of PS1 complex reveals a 360-kDa species reactive
with antibodies to PS1 and NCT (A) Protein (100 lg) from membranes
of embryonic day 14 (E14)
4 mouse brain (E14MB), or from adult
mouse brain with PS1 [M146L] (AdMB), were treated with DDM or
digitonin before BN/PAGE and Western blotting for PS1 or NCT
detection Sharp bands of PS1 reactivity indicated that the 360-kDa
PS1 complex contained both PS1 N-terminal reactivity and PS1
C-terminal reactivity in DDM A broader band of 460 kDa PS1
immunoreactivity was found after digitonin solubilization Reactivity
for PS1 and NCT was stronger in E14 mouse brain than for adult
mouse brain NCT was detected in a 360-kDa complex with DDM and
low levels were detected as apparent monomer of 125 kDa; NCT
was detected only weakly in adult brain (B) Reactivity of NCT
C-terminal antibody after SDS/PAGE for PS1-deficient mouse neural
stem cells, PS1 wild-type neural cells, E16 mouse brain and adult
mouse brain showed three major bands of 110, 120 and 140 kDa which
were absent after preabsorption with immunizing peptide Right panel
shows PS1 antibody detection of PS1 29-kDa NTF after SDS/PAGE
(20 lg membrane protein per lane).
Trang 4determine PS1 complex stability Fig 3A shows that sample
preparation with 1% SDS, or a combination of 1% Triton
X-100/1% Nonidet P-40 abolished the 360-kDa PS1
complex signal that was observed with 0.5% DDM
solubilization Treatment with 1% Triton X-100 caused
incomplete complex dissociation as a weak 360-kDa signal
was observed together with smearing in the 100–200-kDa
range after long exposure (Fig 3A; ECL 60 min compared
with ECL 5 min) Thus, we confirm that detergent type is
critical for solubilization of the complex without disruption,
as shown by others [5,17] The complex was stable in
0.5% DDM even after addition of reducing agent (1%
b-mercaptoethanol), indicating strong interaction,
inde-pendent of maintenance of disulfide bonds Stability to
2Murea further supports this strong interaction However,
the complex breaks down with increased urea concentration
of 5M in presence of reducing agent and 0.2% DDM
(Fig 3B, left panel) At the low SDS concentration of 0.1%
with 0.2% DDM, PS1 subcomplexes of 90 and 60 kDa
were detected (Fig 3B)
We also investigated the association of NCT with PS
complex using embryonic mouse brain where we have
shown optimal NCT detection Paralleling the results
observed for PS1, we found that the low SDS concentration
of 0.1% caused a loss of 360-kDa immunoreactivity for
NCT, using either ectodomain or C-terminal directed
antibodies (Fig 3B, right panels) A 140-kDa signal was
observed that is consistent with the apparent molecular
mass of NCT The intermediate complex of 280 kDa has
a size consistent with that expected for a NCT dimer, and
was sensitive to the ionic detergent SDS but stable to
b-mercaptoethanol and 5M urea (Fig 3B) It was hardly
detectable with antibody 00/22, suggesting that the
mole-cular interaction prevented antibody access to its epitope
that is located within the median region of the ectodomain
Detection with the C-terminal antibody 00/19 was robust, excluding interaction through the C-terminal domain This intermediate NCT complex was similar to the subcomplex described previously on BN/PAGE for transfected cells solubilized with digitonin [40,41]
Second dimension denaturing electrophoretic analysis
of semipurified PS complex Prior to investigation of PEN-2 and APH-1 association with the PS complex, mouse- and human-derived membrane preparations were examined for PEN-2 immunoreactivity and revealed strong detection of a 10-kDa band in P S1+/ +neural stem cells, embryonic brain, and SY5Y cells; lower levels were detected in PS1–/– cells, and in adult brain from mouse and human (Fig 4A) Down-regulation of PEN-2 with PS1-deficiency is in agreement with other reports [42] APH-1 analysis using APH-1b CT [244–257] antibody in the same samples detected APH-1b immunoreactivity at
18 kDa and 9 kDa (Fig 4B) Levels of APH-1b were similar for PS1-deficient and wild-type neural stem cells indicating that levels are not coordinated with PS1 expres-sion Detection was low in adult human brain as compared with SY5Y cells Detection of the APH-1b products was reduced by peptide absorption (data not shown) APH-1a expression was also investigated but was detected more weakly with the reagents currently available
To analyse further the composition of the PS1 complex, samples with high expression of PS1 complex were selected for further study BN/PAGE gel bands corresponding to the region of PS1 complex were excised as indicated in Fig 4C Fig 4C shows that treatment of membranes with 0.1M sodium carbonate, pH 11.3, for removal of nonintegral membrane proteins, enriched for PS1 immunoreactivity This treatment was previously shown to be compatible with
Fig 2 PS complex does not contain b-catenin or BACE (A) Membrane protein samples (50 lg) from SY5Y, C100 adult brain, or Tg2576 adult brain in 0.4% DDM were resolved by BN/PAGE and immunoblotted with the indicated antibody PS1 detection was enriched in SY5Y as a 360-kDa band APP was detected as a broad band above 140 360-kDa; contrast adjustment for Tg2576* indicated increased APP immunoreactivity with the PS1 complex region NCT was strongly detected in SY5Y membrane complex and weakly in adult mouse C100 brain complex b-catenin was detected as a broad smear above 440 kDa as indicated (B) Membrane protein (50 lg) from adult mouse brain were compared for immuno-reactivity with antibody as indicated BACE was detected as a broad smear of apparent molecular mass greater than 440 kDa PS2 was detected as
a band of 420 kDa.
Trang 5Fig 3 Stability of PS1 complex to detergents
and urea analysed by BN/PAGE (A) Adult
mouse brain samples (50 lg) were treated with
0.5% DDM or as indicated before loading.
Triton X-100 1%/1% NP40, or 1% SDS
eliminated the PS1 complex at 360 kDa ECL
exposure of 1 h revealed low detection of PS1
complex treated with 1% Triton compared
with 0.5% DDM Addition of 2 M urea or 1%
b-mercaptoethanol to 0.5% DDM did not
affect complex detection by PS1 antibody.
(B) Treatment of 50 lg E14 mouse brain with
0.2% DDM and 0.1% SDS or 5 M urea
indicated disruption of PS1 complex With
0.1% SDS, PS1 was detected as bands of 90
and 60 kDa With 0.1% SDS, NCT was
detected largely as monomer with minimal
reactivity corresponding to possible dimer
or subcomplex After treatment with 1%
b-mercaptoethanol/5 M urea, NCT was
detected primarily at 280 kDa by NCT CT
antibody and only weakly as a monomer
with NCT ectodomain antibody.
Trang 6preservation of c-secretase activity [36] Samples were
processed by second-dimension analysis to investigate
composition and presence of complex components
Second-dimensional analysis of complex from carbonate-washed membranes of SY5Y and 5-day-old mouse brain were examined using 10% Tris/tricine gels with reducing
Trang 7conditions and SDS (Fig 4D and E) PS1 NTF and CTF
and PS2 CTF were detected robustly in these samples Low
levels of full-length PS1 were also detected with N-terminal
antibody NCT and PEN-2 were also detected in this
semi-purified complex APH-1 was not detectable with the
reagents tested which included affinity-purified antibodies to
the C-terminal regions of 1a (short form) and
APH-1b, and to the loop four region of APH-1b (data not
shown)
Investigation of noncarbonate washed embryonic mouse
brain PS complex revealed similar detection of PS1 NTF,
PS1 CTF, low level of full-length PS1, mature NCT, and
PEN-2 (Fig 4F) PS1 CTF was detected with PS1 loop
antibody as well as low amounts of full-length PS1 The
epitope for this antibody is largely buried in full-length PS1
as shown previously by us and others [26,43] APH-1b was
detected for this complex as immunoreactivity at about
36 kDa with low level detection also at about 18 kDa and
54 kDa suggestive of self-association as dimer and trimer;
mobility corresponding to putative association with mature
NCT (140 kDa) was also detected Evidence for
self-association of APH-1a with itself and with APH-1b was
also reported previously [16] Morais et al demonstrated
that NCT and APH-1 were able to interact in vitro in the
absence of PS [44] Our results suggest that all four putative complex components could be detected in the PS complex for embryonic mouse brain after BN/PAGE, and that APH-1 association may be diminished by carbonate wash Analysis of complex components by second-dimension analysis for control and AD human brain samples without carbonate wash revealed detection of PS1 NTF, PS1 CTF, full-length PS1, NCT and PEN-2 (data not shown) but not APH-1 which may be below detection levels
Analysis of PS1 complex from sporadic AD and tissue with early onset familial AD mutations
Membrane fractions were prepared from frontal cortex of cases with sporadic AD and compared with age-matched controls BN/PAGE analysis indicated no major differences
in PS1 complex mobility with AD (Fig 5A) Human brain cortex had lower detectable levels of PS1 complex compared with preparations from SY5Y No major differences in expression of PS1 and NCT levels were found in these tissues as shown in Fig 5C using Western analysis after standard SDS gel electrophoresis
We next examined cases of early onset familial AD with pathogenic PS1 missense mutations and found no alteration
in complex mobility by BN/PAGE analysis for tissue containing PS1 [L271V], PS1 [S169L] or PS1 [L219P] (Fig 5B) No change in PS1 complex was found for two fronto-temporal dementia cases associated with altered PS1 transcript expression [26] However tissue from a case with pathogenic PS1 exon 9 deletion showed an additional oligomeric species of about 600 kDa (demonstrated twice) PS1 expression and NCT levels for some of these early onset cases is shown in Fig 5D
SY5Y cells stably transfected with PS1 cDNA constructs were used to examine effects of over-expression of PS1 mutations on complex formation Abnormal accumulation
of PS1 at about 600 kDa was detected in SY5Y cells over-expressing the PS1 delta 9 construct Increased PS1 expression or expression of the artificial loss of function D257A mutation did not alter complex mobility Interest-ingly for all cell lines over-expression of PS1 did cause increased complex detection by PS1 antibody (Fig 5E) but significant amounts of PS1 were not incorporated in the complex, as indicated by a smear of reactivity between 50 and 100 kDa
Discussion
The PS complex is critical for normal biological processes such as Notch signalling in development in addition to the generation of Ab peptides in pathological processes Study
of the characteristics of the PS complex will facilitate elaboration of the molecular processes involved in the production of Ab Analysis of these complexes requires detergent solubilization in conditions that do not disrupt protein interactions This can lead to loss of weakly interacting proteins or generation of artefact from nonspe-cific associations BN/PAGE utilizes Coomassie Blue G250 rather than SDS in the cathode buffer and sample buffer to assist with protein solubilization and addition of charge for electrophoretic field migration of proteins according to mass [19] It is ideal for analysis and partial isolation of membrane
Fig 4 PEN-2 and APH-1 expression in mouse and human tissues and
detection in PS complex after 2D-complex analyis (A) Membranes
from tissues and cells were analysed for PEN-2 (1–15) reactivity
Sig-nificant expression was detected as a 10-kDa band in PS1 wild-type
neural stem cells, E15 mouse brain and SY5Y cells Low-level
expression was present in PS1 deficient cells, adult mouse brain and in
control and AD human brain cortex (20 lg protein per lane, 15%
Tris-tricine gel) (B) Tissues were also examined for APH-1b CT reactivity.
APH-1b was detected at 18 kDa and as an apparent CTF of 9 kDa
except in adult human brain Expression was unchanged with PS1
deficiency (C) Sodium carbonate wash of membranes before BN/
PAGE analysis increased intensity of PS1 complex reactivity (50 lg
membrane protein per lane in 0.4% DDM) A region of the unstained
BN/polyacrylamide gel was excised as indicated by alignment with
standards (D) Carbonate washed SY5Y membranes (500 lg) were
run on BN/PAGE, proteins electroeluted from the PS complex region,
treated with reducing sample buffer and analysed on 10% Tris/tricine
gels using mini Biorad apparatus PS1 NTF and full-length PS1 were
detected with PS1 [1–20] antibody, PS1 loop antibody detected PS1
CTF, NCT was detected at 140 kDa, PEN-2 at 10 kDa and PS2
CTF detected with PS2 CTF antibody Parallel poly(vinylidene
diflu-oride) strips were also probed with APH 1a and 1b antibodies but
APH-1 was not detected (E) Carbonate washed membranes (500 lg)
from mouse brain post-natal day 5, were similarly probed after PS
complex isolation from BN/PAGE and second-dimension Tris/tricine
gel analysis using a large gel (16 · 18 cm) format PS1 and PEN-2
were easily detected in these samples NCT was clearly detected as
the mature form with very low detection of an immature form Again
APH-1 was not detected with the available reagents (F) E16
membranes were analysed in the absence of carbonate treatment.
Second-dimension analysis after BN/PAGE of the PS complex
detected full-length PS1, NTF, CTF and PEN-2 NCT was detected as
the mature form APH-1b was detected primarily as a major band of
36 kDa (possible dimer) and as minor bands of 18 kDa and possible
trimer at 54 kDa (after long exposure); immunoreactivity with
mobility corresponding to NCT was also indicated.
Trang 8Fig 5 BN/PAGE analysis of PS1 complex in membranes from human brain control and AD brains (A) After BN/PAGE, PS1 complex from control (CT) or AD cortex was detected in 50 lg membrane protein at 360 kDa (B) Analysis of samples from AD cases with PS1 missense mutations and fronto-temporal dementia (FTD) indicated no change in PS1 complex mobility by BN/PAGE In familial AD (FAD) with exon 9 deletion, a higher band of 600 kDa was detected as well as the major band for PS1 at 360 kDa (shown twice for confirmation) (C) Detection of PS1 and NCT after 12% Tris-glycine SDS/PAGE of 20 lg membrane samples of human brain analysed in (A) indicated no major difference in PS1 or NCT expression
in AD (D) Analysis of samples used in (B) for PS1 and NCT expression with 12% Tris-glycine SDS/PAGE, indicated additional full-length PS1 detection with exon 9 deletion and no alteration in NCT expression (E) Detection of PS1 in SY5Y stably transfected with PS1, PS1 exon 9 deletion (delta E9), and PS1-D257A following BN/PAGE, indicated increased levels of PS1 complex with PS1 over-expression Increased immunoreactivity was detected at 600 kDa with P S1 delta E9 (30 lg protein per lane from 18 000 g · 20-min post-nuclear pellets).
Trang 9protein complexes under nondenaturing conditions This
technique complements immunoprecipitation and velocity
gradient analyses that have been applied more extensively to
examine PS interactions with binding partners and complex
size The molecular mass of PS1 complex determined here
with BN/PAGE corresponds to the size of 400 kDa
recently determined by velocity gradient centrifugation of
digitonin-solubilized membranes from HEK293 cells [45] It
is also consistent with the size of Chapso-solubilized PS
complex obtained after over-expression of Drosophila P S in
Drosophilacells or N2a neuroblastoma cells (between 232
and 443 kDa) [46] The detection of some full-length PS1
after second-dimension analysis of complex components
suggests assembly with full-length PS before
endoproteo-lysis to generate heterodimers of NTF and CTF
compo-nents consistent with previous reports [47]
Sensitivity of the PS complex to SDS and Triton X-100
and stability to reducing agent is consistent with previous
studies [17,48] Conditions causing partial complex
disrup-tion can contribute to knowledge of complex assembly
With 0.1% SDS, PS1 high molecular mass complex was
disrupted, and some PS1 with mobility of about 90 kDa
was detected, which may correspond to a subcomplex or
PS1 dimer A recent report by He´bert et al showed evidence
for PS1 dimerization using 0.1% SDS treatment and
alternative native electrophoresis conditions as well as by
yeast two-hybrid analysis [49] We also document that NCT
migrates in BN/PAGE as a subcomplex or possible dimer
stable to reducing agent and high urea concentration This
finding supports previous evidence, that NCT and APH-1
may associate independently of PS1 complexes [40,41,44]
and that NCT may form a subcomplex in the absence of
PS1 [13] Low or no detection of APH-1 in the partially
purified endogenous PS complex by second-dimension
analysis, and minimal effect on APH-1 expression level
with PS1 deficiency supports the proposal that APH-1 may
be important for early assembly and stabilization of the
complex [50–52]
High levels of PS complex were detected in mouse
embryonic brain consistent with high expression of PS1
in embryogenesis and supporting an important role for
PS in development, presumably due to its involvement in
the Notch signalling system [53] Indeed studies with PS1
knockout mouse embryos demonstrated severe neural
and skeletal defects and PS1/PS2 double knockouts
display a severe phenotype resembling Notch gene
knockout [3]
Alternative approaches have provided evidence that PS
and cofactors interact directly: affinity isolation of the
complexes [9], coimmunoprecipitation studies [12,37–39],
and complex isolation with c-secretase inhibitor [4,22,54]
Other recent studies have also utilized BN/PAGE for
analysis of PS complexes DDM at 0.5% has been
recently reported independently to preserve PS complex
integrity in BN/PAGE with reports of complex size from
440 to 600 kDa [12,13,42,55,56] Digitonin (1%) and BN/
PAGE produced PS1 complex mobility of 250–270 kDa
in cells over-expressing the four main constituents of the
c-secretase complex [40,41] Estimates of complex mobility
may vary depending on sample preparation, detergent
type and concentration, as well as buffer and gel
composition
Detection of APH-1b as an 18-kDa and CTF 9-kDa form indicates that native APH-1 undergoes endoproteo-lysis Lack of detection of a free 9-kDa form after second-dimension complex analysis indicates that this fragment is unlikely to be associated with the mature complex This is in agreement with the report by Kimberley et al that AP H-1a also generated a small CTF which did not associate with other PS complex components after glycerol velocity gradient fractionation for tagged APH-1 in transfected cells [40]
This is the first report of native PS1 complex analysis from human brain of late onset sporadic AD cases and from cases with early onset PS1 mutations We found that PS1 complex from cortex of sporadic late-onset AD cases
or from pathogenic PS1 point mutations did not have altered apparent mobility in BN/PAGE This was consis-tent with study of PS1 complex from transfected SY5Y cells reported here for the artificial PS1 Asp257Ala mutation, and for Asp257 or Asp385 mutations in transfected mouse embryonic fibroblasts [56] Additional high molecular weight PS1 species of 600 kDa were detected for brain carrying the severe PS1 exon 9 deletion and for SY5Y cells over-expressing this mutation, indica-ting impairment of normal complex formation in the presence of the mutation
In vitroco-expression studies in transfected cell systems indicates PS1 or PS2 and NCT, APH-1, and PEN-2 are all required for generation of active c-secretase activity [40,50,52,57] The current study of endogenous tissue levels and detection within native endogenous complex for these membrane proteins from mouse and human brain contri-butes to further understanding of the nature of the native mature presenilin/c-secretase complex complementing
in vitrocell-based studies
Acknowledgements
We thank P M Mathews and R.M.D Holsinger for antibodies, F B Reinhard for SY5Y-PS1 mutant cell lines, and Q.-X Li for transgenic mouse tissue The work was supported by the Australian NHMRC (Grants 114132 and 208978) and ANZ Charitable Trusts D H was supported by an NIH post doctoral fellowship.
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