(Received for publication, June 11, 1997)
From Pharma Division, Preclinical Central Nervous System
Research-GeneTechnology, F. Hoffmann-La Roche Ltd., Ch-4070 Basel,
Switzerland and the Department of Molecular Biology,
Central Institue of Mental Health, Mannheim D-68159, Germany
Presenilin 1 (PS1) and presenilin 2 (PS2) are endoproteolytically processed in vivo and in cell transfectants to yield 27-35-kDa N-terminal and 15-24-kDa C-terminal fragments. We have studied the cleavage of PS1 and PS2 in transiently and stably transfected hamster kidney and mouse and human neuroblastoma cells by immunoblot and pulse-chase experiments. C-terminal fragments were isolated by affinity chromatography and SDS-polyacrylamide gel electrophoresis and sequenced. The processing sites identified in PS1 and PS2 (Asp345/Ser346 and Asp329/Ser330, respectively) are typical for caspase-type proteases. Specific caspase inhibitors and cleavage site mutations confirmed the involvement of caspase(s) in PS1 and PS2 processing in cell transfectants. Fluorescent peptide substrates carrying the PS-identified cleavage sites were hydrolyzed by proteolytic activity from mouse brain. The PS2-derived peptide substrate was also cleaved by recombinant human caspase-3. Additional processing of PS2 by non-caspase-type proteases was also observed.
Mutant presenilin 1 and 2 (PS1,
PS2)1 genes are
linked to early-onset familial Alzheimer's disease (1-5). The PS gene
products are integral membrane proteins localized in Golgi and ER
compartments (6-8). They share ~65% sequence identity and similar
structural features, i.e. 6-8 transmembrane domains,
hydrophilic regions at their N and C termini, and a large hydrophilic
loop C-terminal to transmembrane domain 6 (6, 9, 10). The loop and the N terminus are the regions of significant sequence divergence between
PS1 and PS2. More than 30 mutations in PS1 and 2 mutations in PS2 have
been genetically linked to early-onset FAD (3, 4). The biological
functions of PS currently remain unknown. Studies on the structural
homologue in Caenorhabditis elegans, SEL-12,
suggest a role in protein trafficking and in the Notch signaling pathway (11). Human carriers of PS FAD mutations show a small
but significant increase (approximately 1.5-3-fold) in plasma levels
of A1-42 compared with normal controls, whereas levels
of A
1-40 are unchanged (12-14). A specific effect on
-secretase processing of
-amyloid precursor protein has been proposed, and this enhanced misprocessing of
-amyloid precursor protein is thought to be crucial for FAD pathogenesis. Recent experiments have indicated a regulatory role for PS2 in cell apoptosis. C-terminal fragment of mouse PS2 reportedly protected T-cells or PC12
cells from different apoptosis-inducing challenges, whereas the
susceptibility to the same challenges was enhanced when full-length PS2
was overexpressed (15, 16).
Extensive proteolytic cleavage of PS1 resulting in a 27-28-kDa N-terminal fragment and a 16-17-kDa C-terminal fragment was reported for PS1-transfected COS-1 cells, mice transgenic for human PS1, and endogenous PS1 in human and murine tissues (12, 17-19). Proteolytic processing has also been reported for human PS2 resulting in a 35-kDa N-terminal fragment and a 20-kDa C-terminal peptide (3, 10, 13). In the present study we have identified proteolytic processing sites in PS1 and PS2 and demonstrate that processing occurs through caspase-type protease(s). We also observed additional, caspase-independent cleavage products suggesting alternative pathways of presenilin processing.
Amino acids 1-80 of human PS2 (N terminus) and 270-387 of murine PS2 (large hydrophilic loop between transmembrane domains 6 and 7) were expressed in Escherichia coli as (NANP)19-fusion proteins and used for immunization of mice. PST20 is a monoclonal antibody specific for the hydrophilic N-terminal region of human and mouse PS2. PST18, PST8, and PST10 react with the PS2 hydrophilic loop region, PST18 having preference for murine PS2. The rabbit antiserum 53217 is directed against the hydrophilic loop of PS2. The M2 is a commercially available anti-FLAG antibody (Kodak Scientific Imaging Systems Products).
PeptidesThe tetrapeptide derivatives used in this study were obtained from the following sources: Ac-DSYD-AMC and Ac-AQRD-AMC from E. Kitas, Hoffmann-La Roche, Ac-DEVD-AMC from Alexis Corp.; b-DEVD-CHO and Ac-YVAD-dbmk from Bachem AG, b-DSYD-CHO and b-AQRD-CHO from SynPep Corp.
Cloning and Expression of Mouse and Human PresenilinsMouse and human PS1 and PS2 cDNAs were amplified by PCR from a mouse brain cDNA library in pcDNA1 and a human brain cDNA library in pACT2 (CLONTECH) and inserted into pcDNA3 as BamHI/XbaI fragments. FLAG epitopes (DYKDDDDK) were added at the C terminus using appropriate primers and PCR amplification. The respective PS1 and PS2 fragments were cloned into pcDNA3 (Invitrogen) and pSFV1 (Life Technologies, Inc.) for stable expression in mouse Neuro2a and transient expression in BHK21, respectively. BHK cells were transfected by electroporation (20 µg of RNA per 107 cells) and harvested 20-24 h later. For stable tetracycline-controlled expression in SK-N-MC cells, mouse PS2-FLAG and mouse PS2-N141I-FLAG were placed under control of the tTA-dependent promoter and cloned into ptetREP, a pREP9 (Invitrogen) derivative containing a modified tTA under control of the tTA dependent promoter2 (20). Clones were selected in medium containing 1.5 mg/ml G418. For expression experiments, cells were grown with or without 1 µg/ml tetracycline for 3 days.
ImmunoblottingBHK cells expressing the various PS2 or PS1 constructs were collected, washed in phosphate-buffered saline, and dissolved without heating in SDS sample buffer containing 8 M urea and 0.2 M dithiothreitol. The proteins were separated by SDS-PAGE and electrophoretically transferred to a nitrocellulose membrane. Blocking, incubation with the various antibodies, and ECL detection were performed according to the recommendation of the manufacturer (Amersham).
Isolation of the C-terminal Fragment and Sequencing~5 × 108 BHK cells transfected with PS2-FLAG or PS1-FLAG constructs were washed in 5 mM Hepes, pH 7.2, 250 mM sucrose, 1 mM EDTA, 10 mM benzamidine, 100 units/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, 1 mM phenylmethylsulfonyl fluoride (breaking buffer) and mechanically disrupted in a mini-bead-beater using 0.1-mm glass beads. After removing nuclei and large cell debris by low speed centrifugation a crude membrane fraction was collected at 100,000 × g for 45 min and resuspended in breaking buffer. The suspension was adjusted to 0.5% SDS and incubated for 10 min at 37 °C. The solution was then diluted 1:5 with 1.25 × RIPA buffer, not containing SDS (25 mM Tris, pH 7.4, 171 mM NaCl, 12.5% glycerol, 0.625% sodium deoxycholate, 1.25% Triton X-100, 2.5 mM EDTA), and clarified by centrifugation at 100,000 × g for 30 min. The clear supernatant was applied to an M2 anti-FLAG immunoaffinity column (0.5-ml bed volume) equilibrated in 1 × RIPA buffer. After extensive washing with 1 × RIPA buffer and phosphate-buffered saline containing 0.1% octyl glucoside the column was eluted with 100 mM glycine, pH 2.6, 0.1% octyl glucoside. Fractions containing the C-terminal fragment were concentrated, subjected to SDS-PAGE, and electrophoretically transferred to an Immobilon-PSQ membrane (Millipore). The membrane was stained with 0.1% Amido Black in 45% methanol, 7% acetic acid for 10 min, and the band corresponding to the PS2 fragment was cut out and N-terminally sequenced by Edman degradation in an ABI 490cLC sequencer. The same procedure was applied to isolate human PS2 from ~109 Neuro-2a cells except that a mixture of immobilized antibodies PST8, PST10, and PST18 was used to affinity purify the C-terminal fragment.
Expression of Recombinant Caspase-3The active form of
human caspase-3 was amplified by PCR from Ser29 to the end
of the coding region and inserted as a BamHI fragment into
pET15b (Novagen). Transformants in E. coli BL21(DE3)pLysS (Novagen) were grown in 5 ml of LB medium, induced at
A600 = 0.6 with 0.2 mM
isopropyl-1-thio--D-galactopyranoside for 2 h
before extracts were prepared by sonification in 0.5% Nonidet P-40, 20 mM Hepes (pH 7.4), 100 mM NaCl (21).
5 µg of E. coli extract containing recombinant caspase-3 or 0.5 mg of mouse brain homogenate were incubated with 20 µM aminomethylcoumarin (AMC) tetrapeptide substrates in 100 mM Hepes, pH 7.0, 5 mM dithiothreitol, 0.5 mM EDTA, 0.1% CHAPS, 20% glycerol at 25 °C. Total volume was 1 ml. The release of AMC as a function of time was measured in a fluorescence spectrophotometer using 380 nm and 460 nm as excitation and emission wavelengths, respectively. Background reagent fluorescence was determined and subtracted. The relative fluorescence intensities were correlated with AMC concentrations by standard curve titrations. In the inhibition experiments the release of AMC from Ac-DSYD-AMC was measured in the presence of different concentrations of the tetrapeptide aldehydes after incubation for 3 h (E. coli extract) or 4.5 h (brain homogenate) at 25 °C.
We have studied the
expression and processing of murine and human PS2 and PS1 constructs
tagged with a C-terminal FLAG epitope in transient and stable
transfectants. Immunoblot experiments with the M2 anti-FLAG antibody
confirmed expression of full-length murine PS2 in Semliki Forest virus
(SFV) transfected BHK cells migrating at around 55 kDa and the presence
of a major C-terminal fragment at around 18 kDa (Fig.
1A). This fragment was
recognized by the monoclonal antibody PST18 directed against the C
terminus of PS2. Using monoclonal antibody PST20 directed against the N terminus of PS2, a major N-terminal fragment migrating at ~34 kDa and
a weaker band at ~31 kDa were detected by immunoblotting. The 18-kDa
C-terminal fragment together with a double band at 22 and 24 kDa were
detected using the rabbit antiserum 53217. The poor but occasionally
clearly visible immunoreactivity of the 22- and 24-kDa double band with
the anti-FLAG and PST18 antibodies probably reflects a rather low
abundance of these fragments. The processing pattern of murine PS2
carrying the FAD mutation N141I was similar to that of wild-type PS2 as
revealed by the antiserum 53217. Likewise, processing of human PS2
lacking the C-terminal FLAG epitope was identical to that of murine
PS2-FLAG, indicating that the introduction of the FLAG sequence into
the C terminus of PS2 did not affect its processing pattern (Fig.
1A).
Transient expression of the murine PS2-FLAG construct in CHO cells under the control of a cytomegalovirus promoter (data not shown) or in stably transfected human neuroblastoma SK-N-MC cells under a tetracycline-regulated promoter yielded, similar to the SFV/BHK expression system, full-length murine PS2 migrating at 55 kDa and a major C-terminal fragment at 18 kDa when probed with the anti-FLAG antibody (Fig. 1B). A faint band migrating at 22 kDa could also be seen which most likely corresponds to the 22-kDa fragment easily detected by the antiserum 53217 in BHK cells. Introduction of the N141I FAD mutation did not result in a different processing pattern of murine PS2 stably expressed in SK-N-MC cells.
The precursor-product relation for full-length PS2 and its fragments was established by a pulse-chase experiment using BHK cells transfected with murine PS2. Over a 6-h chase period, an increase in the 34-kDa N-terminal fragment was clearly observed in immunoprecipitations with the PST20 antibody (data not shown). Together, these experiments proved the processing of PS2 into N- and C-terminal fragments in transient and stable expression systems and suggested a slow proteolytic processing of the full-length protein to yield the processing products.
Determination of the Proteolytic Cleavage Site(s)To determine the sites of proteolytic cleavage, the 18-kDa C-terminal fragment of murine PS2-FLAG was isolated from a BHK cell lysate by M2 anti-FLAG affinity chromatography and SDS-PAGE. N-terminal sequencing of the fragment indicated cleavage between Asp329/Ser330 (Table I). In a second independent experiment an additional minor sequence was determined starting at Ser327 and representing about 5% of the material that started at Ser330. Cleavage of PS2 between Asp329 and Ser330 results in two processing products with predicted molecular masses of 37 kDa and 14 kDa (including the FLAG epitope) which is in reasonable agreement with the observed mobilities on SDS-PAGE/immunoblots. An 18-kDa C-terminal fragment was also isolated from mouse Neuro-2a cells stably expressing the human PS2 cDNA under the control of the cytomegalovirus promoter by immunoaffinity chromatography using a mixture of PST8, PST10, and PST18 antibodies. The fragment was shown to start at the same N terminus as its mouse homologue (Table I).
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A similar approach was chosen to map the cleavage site in PS1. We isolated and expressed the murine PS1 cDNA, purified its 15-kDa C-terminal fragment from BHK cell lysates, and determined the proteolytic processing site (Table I). The N-terminal residue of this fragment corresponded to cleavage between Asp345/Ser346 of the full-length protein.
Both the PS2 and PS1 cleavage sites yielding the 18-kDa and 15-kDa
fragments, respectively, are located within the predicted hydrophilic
loop C-terminal of transmembrane domain 6 but in a region where the PS1
and PS2 primary structures are highly divergent. However, the mapped
cleavage sites shared the same residues in the P1 position (Asp) and in
the P1 position (Ser). Interleukin-1
-converting enzyme-type
proteases (or caspases, following a recently introduced nomenclature)
cleave their substrates at positions following Asp residues (22, 23).
In particular the PS2 cleavage site (Asp-Ser-Tyr-Asp/Ser) corresponds
closely to sites which are recognized and cleaved by caspase-3 (CPP32
or apopain). Such sites are found in poly(A)DP-ribose polymerase and
the Huntington protein and follow the general structure Asp-X-X-Asp for the P1 to P4 positions (24,
25).
Podlisny et al. (26) reported the isolation and radiosequencing of a C-terminal fragment from human PS1 transfected into HEK-293 cells. They mapped a major fragment starting at Ala299 and two minor fragments starting at positions Met292 and Val293. These sites are clearly distinct from the position Asp345/Ser346 mapped by us and, moreover, bear no similarities to caspase-type cleavage sites. This indicates that PS1 can be processed at multiple sites and by presumably different proteases. For PS2 we also have identified larger C-terminal fragments of about 22 and 24 kDa (see Fig. 1A). While the exact identity of these fragments remains to be determined, they clearly contain the C-terminal domain of PS2 and correspond in size to the larger C-terminal fragment of PS1. Thus, there may be alternate processing pathways also for PS2 with possibly different functional properties.
Cleavage Site InhibitorsSubstrate mimetics of naturally
occurring caspase cleavage sites have been described which act as
specific caspase inhibitors in vitro and in vivo
(25, 27). We tested the activity of a caspase-3-type inhibitor
(Ac-DEVD-CHO) and of a caspase-1-type inhibitor (Ac-YVAD-dbmk) on PS2
and PS1 processing in BHK cells transfected with SFV constructs. The
caspase-3-selective inhibitor reduced the accumulation of the 18-kDa
C-terminal fragment of PS2 almost completely in a
concentration-dependent manner, whereas the
caspase-1-selective inhibitor had only a minor effect (Fig. 2A). The processing of PS1 was
also affected by both types of inhibitors although the extent of
inhibition was less pronounced (Fig. 2B). The clear
inhibition of PS2 and PS1 processing by the general serine and cysteine
protease inhibitor TLCK suggests a common pathway of PS1 and PS2
fragmentation. TLCK may interfere with serine-type protease(s) acting
upstream in a cascade of proteolytic events which finally leads to the
activation of caspases selective for PS2 and PS1.
Cleavage Site Mutations
The cleavage site in murine PS2 was
further characterized by analyzing the effects of mutations around the
cleavage site upon expression in the SFV/BHK cell system. Deletion of
residues 326-331 completely abolished processing as did the mutation
of the P1 residue Asp329 Arg (Fig. 2C).
Mutant Asp326
Asn showed some residual activity whereas
mutations of Ser327
Phe or Tyr328
Ala
had little effect on PS2 processing (not shown). The mutants thus
confirmed the importance of an aspartic acid residue in the P1 and, to
a lesser extent, in the P4 position for PS2 processing. Interestingly,
the caspase cleavage site mutations did not induce a significant
increase of the 22-24-kDa PS2 fragments strongly suggesting
independent processing pathways. The slightly smaller size of the
caspase-independent processing product of the
326-331 PS2 mutant
might reflect a subtle structural change or a secondary modification
induced by the deletion.
The proteolytic activity of
caspase-type proteases can be assayed in vitro by
fluorogenic peptide substrates. We have tested homogenates of mouse
brains for proteolytic activity on fluorogenic substrates based on the
PS2 and PS1 cleavage sites. The strongest endopeptidase activity was
measured with the caspase-3-specific substrate DEVD-AMC followed by the
PS2-peptide DSYD-AMC. We also observed a weak but significant activity
on the PS1 substrate peptide AQRD-AMC (Fig.
3A). Thus, caspase-type
activities are present in mouse brain which also accept, in addition to
the caspase-3 substrate, peptide substrates derived from both PS2 and
PS1. Caspase substrate peptides, if derivatized with an aldehydic
or dimethylbenzoyloxymethyl ketone function at the C-terminal Asp, act
as potent caspase inhibitors. Indeed, the aldehyde derivatives of all
three tetrapeptides used here were found to inhibit DSYD-AMC cleavage
by mouse brain homogenates in a concentration-dependent
fashion, the most potent inhibitor being the caspase-3 inhibitor
followed by the PS2- and PS1-derived peptides (Fig. 3C).
Several caspases, including caspase-3, show activity when expressed in E. coli as an N-terminally truncated protein (21). To further substantiate the role of caspases in PS processing and to characterize the caspase-type activity found in mouse brain we inserted the human caspase-3 cDNA (from Ser29 to the end of its open reading frame) into a pET15 vector and prepared crude cell lysates from selected transformants. The lysates were assayed for activity on the fluorogenic peptides based on the PS1 or PS2 cleavage sites and on the prototype caspase-3 substrate peptide to confirm expression of a functional enzyme. We observed a time-dependent cleavage activity with the PS2-derived substrates whereas, in contrast to the mouse brain homogenate, no activity was measured with the PS1 peptide (Fig. 3B). Inhibition of recombinant caspase-3 activity was most pronounced with the caspase-3-type inhibitor DEVD-aldehyde with an IC50 of about 2 nM (Fig. 3D). The same inhibitor was 50-100 times less efficient in inhibiting the endopeptidase(s) from mouse brain indicating that the caspase involved in the hydrolysis of the PS2-derived substrate might be different from caspase-3. Although the PS1-derived tetrapeptide AQRD-AMC was not hydrolyzed by recombinant caspase-3, the corresponding aldehyde derivative was capable of inhibiting DYSD-AMC cleavage by caspase-3 similar to the DSYD-aldehyde analogue (Fig. 3D). This apparent discrepancy might reflect improper binding and processing of the AQRD-AMC substrate peptide at the active site of caspase-3, whereas the inhibitory function of the aldehyde derivative is maintained by forming a thiohemiacetal with the catalytic cysteine.
In conclusion, our results demonstrate that PS2 and PS1 are substrates for caspase-type proteases. Alternate processing by non-caspase-type proteases yields fragments of different sizes. Hartmann et al. (28) recently reported constitutive expression of a 20-kDa C-terminal fragment of rat PS1 in all tissues and stages of development. A second C-terminal fragment of 14 kDa was observed in the brain of adult and aged animals which corresponds in size to the caspase-generated PS1 fragment described here. At least two C-terminal PS2 fragments of about 21 kDa and 18 kDa were detected in mouse tissues indicating alternative processing pathways for PS2 also in vivo.3 A C-terminal PS2 fragment of about 10 kDa protected T-cells against apoptotic challenge whereas overexpression of PS2 restored their sensitivity (15, 16). Cleavage of PS2 by a caspase-type protease generating a similar fragment suggests a role of PS2 processing in the cellular response to apoptotic signals. Elucidating the significance of presenilin processing will help to understand apoptotic or neurodegenerative phenomena in Alzheimer's disease.4
We thank D. Jermann and U. Roethlisberger for protein sequencing, H. Doebeli for purification of the (NANP)19 constructs, E. Kitas for peptide synthesis, M. Haenggi, A. Hayes, N. Holzwarth, V. Krumb, and H. P. Kurt for skilled technical assistance, and T. Bartfai for helpful comments and suggestions.