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J Biol Chem, Vol. 274, Issue 12, 7615-7618, March 19, 1999
Exon 9 Mutation Is Independent of Its Defect to Undergo Proteolytic
Processing*
,,
,**
From the Central Institute of Mental Health,
Department of Molecular Biology, J5, 68159 Mannheim, Germany,
§ Boehringer Ingelheim KG, CNS Research, 55216 Ingelheim,
Germany, ¶ Genzentrum, Feodor-Lynen-Str.25, 81377 Munich, Germany,
and Amgen Inc., Thousand Oaks, California 91320-1789
 |
ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
REFERENCES
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The two homologous presenilins are
key factors for the generation of amyloid Early onset Alzheimer's disease
(AD)1 can occur due to
mutations within the genes encoding the Cell Culture and Cell Lines--
K293 cells were cultured in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum, 1% penicillin/streptomycin, 200 µg/ml G418 (to select
for Construction of the cDNA Encoding PS1
After gel purification the PCR products were mixed and subjected to a
final PCR with primers PS1-187-F and PS1-STOP-R. The resulting PCR
product was digested with EcoRI/XhoI and cloned into the pcDNA3.1/Zeo(+) expression vector (Invitrogen). The
cDNA was sequenced to verify successful mutagenesis.
Antibodies--
The polyclonal antibodies against amino acids
263-407 of PS1 (3027) and amino acids 297-356 of PS2 (3711) were
described previously (19, 20). The monoclonal antibodies to the PS1 and
PS2 loop were raised against fusion proteins containing amino acids
263-407 (BI.3D7) or 297-356 (BI.HF5C).
Metabolic Labeling and Immunoprecipitation of PS--
Stably
transfected K293 cell lines were grown to confluence in 10-cm dishes.
After starvation for 1 h in 4 ml of methionine- and serum-free MEM
(MEM supplemented with 1% L-glutamine and 1% penicillin/streptomycin) cells were metabolically labeled with 700 µCi of [35S]methionine (Promix, Amersham Pharmacia
Biotech) in 4 ml of methionine- and serum-free MEM for 1 h. Cell
extracts were prepared and subjected to immunoprecipitation of PS as
described (12). PS immunoprecipitates were solubilized in sample buffer
containing 4 M urea for 10 min at 65 °C, separated on
SDS-urea gels, and analyzed by fluorography (19).
Combined Immunoprecipitation/Western Blotting--
Stably
transfected K293 cell lines were grown to confluence. Cell extracts
were prepared and subjected to immunoprecipitation as described (12).
Following gel electrophoresis, immunoprecipitated proteins were
identified by immunoblotting (12). Bound antibodies were detected by
enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech).
Analysis of A Transgenic Lines of Caenorhabditis elegans and Rescue
Assays--
Expression constructs were generated as described
previously (22). Transgenic lines were established by microinjection of plasmid DNA mixtures into the C. elegans germ line to create
extrachromosomal arrays as described previously (22). All plasmids used
in this study were injected at a concentration of 20 ng/µl into
sel-12(ar171) or sel-12(ar171)
unc-1(e538) hermaphrodites along with ttx-3:GFP as a cotransformation marker. Successful transformation was monitored by the expression of GFP in the AIY interneurons of F1 and F2 generation animals. Four independent lines from the progeny of F2
generation animals were established.
As the sel-12(ar171) animals never lay eggs (23), rescue of
the sel-12 defect can be quantified by scoring egg laying
behavior in transgenic animals (22, 24). Consequently, for each
transgenic line, we examined 50 transgenic animals for their ability to
lay eggs and determined their brood size. The number of eggs laid by
individual transgenic animals was counted and placed into four categories: "Egl+++," robust egg laying, more than 30 eggs laid (wt
phenotype); "Egl++," 15-30 eggs laid; "Egl+," 5-15 eggs laid; "Egl The PS1
-peptide (A), since
Alzheimer's disease (AD)-associated mutations enhance the production
of the pathologically relevant 42-amino acid A (A42), and a gene
knockout of presenilin-1 (PS1) significantly inhibits total A
production. Presenilins undergo proteolytic processing within the
domain encoded by exon 9, a process that may be closely related to
their biological and pathological activity. An AD-associated mutation
within the PS1 gene deletes exon 9 (PS1
exon9) due to a splicing
error and results in the accumulation of the uncleaved full-length
protein. We now demonstrate the unexpected finding that the
pathological activity of PS1exon9 is independent of its lack to
undergo proteolytic processing, but is rather due to a point mutation
(S290C) occurring at the aberrant exon 8/10 splice junction.
Mutagenizing the cysteine residue at position 290 to the original
serine residue completely inhibits the pathological activity in regard
to the elevated production of A42. Like PS1exon9, the resulting
presenilin variant (PS1exon9 C290S) accumulates as an uncleaved
protein and fully replaces endogenous presenilin fragments. Moreover,
PS1exon9 C290S exhibits a significantly increased biological
activity in a highly sensitive in vivo assay as compared
with the AD-associated mutation. Therefore not only the increased
A42 production but also the decreased biological function of
PS1exon9 is due to a point mutation and independent of the lack of
proteolytic processing.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
-amyloid precursor protein (APP) and the two presenilins, PS1 and PS2 (Refs. 1-3; summarized in Refs. 4 and 5). The mutations increase the generation of the
42-amino acid version of A (A42), which aggregates more readily
(6) and is therefore preferentially deposited in senile plaques (5). PS
proteins are also involved in the physiological production of A,
since the lack of PS1 expression in PS1
/ mice results
in a dramatically reduced A production (7). All but one PS mutations
are point mutations affecting conserved amino acids (4, 5). However,
due to a splicing error, the PS1exon9 mutation results in the
deletion of the domain encoded by exon 9 (8). This domain contains the
cleavage site for proteolytic processing (9, 10), and therefore
PS1exon9 accumulates as an uncleaved protein (9). Since proteolytic
processing is highly regulated (9, 11, 12) and appears to be altered by
PS mutations (Refs. 13-15; summarized in Ref. 16), the lack of
proteolytic processing caused by the exon 9 deletion was expected to be
responsible for its pathological activity. We now demonstrate the
unexpected finding that the pathological function of PS1exon9 as
well as its reduced biological activity is independent of its lack to undergo proteolytic processing, but is rather due to a point mutation (S290C) that is the result of the aberrant exon 8/10 splice junction.
EXPERIMENTAL PROCEDURES
APP expression) and 200 µg/ml zeocin (to select for presenilin
expression). K293 cells stably expressing PS1exon9 C290S were
generated by transfection of K293 cells stably expressing APP
containing the Swedish mutation (17). K293 cells stably transfected
with Swedish APP695, wt PS1, and PS1exon9 were described
previously (18).
Exon9
C290S--
The cDNA encoding PS1exon9 C290S was constructed by
mutagenizing the cysteine at codon 290 of the PS1exon9 cDNA (8)
to serine in a two-step PCR procedure. The following primers were designed: first PCR, PS1-187-F (5'-CCGAATTCAAGAAAGAACCTCAA-3') and
E9-C290S-R (5'-GTGACTCCCTTTCTGTGGAGGAGTAAATGAGAGC-3'); second PCR,
E9-C290S-F (5'-GCTCTCATTTACTCCTCCACAGAAAGGGAGTCAC-3') and PS1-STOP-R (5'-CGCCTCGAGGCAAATATGCTAGATATA-3').
40 and A42--
Culture medium (2 ml) was
collected from confluent K293 cells grown in six-well dishes for
24 h. The medium was assayed for A40 and A42 using a
previously described ELISA assay (12). A peptides were
immunoprecipitated using antibody 6E10 (Senetek) according to
established protocols (21).
," no eggs laid.
RESULTS
exon9 mutation changes a G to a T at the splice site for
exon 9. It therefore destroys the minimal required consensus sequence
for the splice acceptor site (8), which then results in an aberrant
deletion of the domain encoded by exon 9 (Ref. 8; Fig.
1A). However, the mutation
also changes codon 290 at the exon 8/10 splice junction from a serine
to a cysteine (Ref. 8; Fig. 1A). We therefore have
investigated if the single amino acid exchange or the deletion of the
domain required for proteolytic processing is responsible for the
pathological activity of the PS1exon9 mutation in regard to A42
generation. For this purpose we mutagenized amino acid 290 of
PS1exon9 to a serine (PS1exon9 C290S), thus correcting the point
mutation (Fig. 1A). cDNAs encoding PS1exon9 and
PS1exon9 C290S were stably transfected into kidney 293 cells
overexpressing APP containing the Swedish mutation. This cell line
was used previously to determine the pathological effect of PS
mutations on A production (12, 18). Untransfected K293 cells
expressing endogenous PS, as well as cell lines overexpressing PS1exon9 and PS1exon9 C290S were pulse-labeled with
[35S]methionine for 1 h, and cell lysates were
immunoprecipitated with antibody 3027 to the large loop of PS1. Both
PS1exon9 and PS1exon9 C290S accumulated as full-length proteins
(Fig. 1B). As reported previously (9, 10) very little
endogenous full-length PS1 could be detected in the untransfected cell
line.
View larger version (21K):
[in a new window]
Fig. 1.
Generation and expression of
PS1 exon9 and PS1exon9
C290S. A, schematic representation of the genomic
structure (exon 8 to exon 10) of wt PS1, PS1exon9 and the amino acid
sequence at the splice junctions of exons 8, 9, and 10. The nucleotide
exchange (G to T) in the PS1 gene identified in the DNA of patients
with the PS1exon9-linked mutation results in an amino acid exchange
at the exon 8/10 splice junction, which changes codon 290 from a serine
to a cysteine (bold letters). B, K293 cells
expressing endogenous presenilins (control) as well as K293 cells
stably overexpressing PS1exon9, or PS1exon9 C290S were pulse
labeled with [35S]methionine for 1 h, and cell
lysates were immunoprecipitated with antibody 3027 (19) to the large
loop of PS1. Full-length PS (fl-PS) proteins accumulate in
the cell lines expressing PS1exon9 and PS1exon9 C290S but not in
cells expressing endogenous presenilins. The asterisk
indicates dimeric forms of PS1 (19). C and
D, stable expression of PS1exon9 and PS1exon9
C290S inhibits formation of endogenous PS1 (C) and PS2 C-terminal
fragments (CTF) (D). Cell lysates from K293 cells
and cell lines overexpressing PS1exon9 and PS1exon9 C290S were
immunoprecipitated with the previously described antibodies (see
"Experimental Procedures") specific to the large loop of PS1 (3027;
Ref. 19) and PS2 (3711; Ref. 20) and detected by immunoblotting using
the monoclonal antibodies BI.3D7 and BI.HF5C.
It has been shown before that overexpression of PS proteins results in
the replacement of endogenous PS fragments and the accumulation of
PS1exon9 prevents the formation of stable PS fragments (9, 11, 12).
To prove that overexpression of PS1exon9 C290S still allows the
reduction of endogenous PS fragments, cell lysates from unlabeled cells
were immunoprecipitated with antibodies specific to the PS1 or PS2 loop
domain (see "Experimental Procedures"), and PS fragments were
identified with the PS1/PS2-specific monoclonal antibodies BI.3D7 and
BI.HF5C (see "Experimental Procedures"). Whereas cell lines
expressing endogenous presenilins produced PS1 as well as PS2
C-terminal fragments (Fig. 1, C and D),
overexpression of PS1exon9 and PS1exon9 C290S inhibited the
formation of endogenous PS1 and PS2 fragments (Fig. 1, C and
D). This demonstrates that PS1exon9 C290S, like
PS1exon9, fully replaced presenilins derived from the endogenous
genes and also proves that PS1exon9 C290S does not undergo
proteolytic processing but rather accumulates as an uncleaved
full-length protein.
In order to investigate the pathological activity of the mutant PS
derivatives on A production, control cells as well as cells
overexpressing PS1exon9 and PS1exon9 C290S were metabolically labeled with [35S]methionine, and A40 and A42
peptides were immunoprecipitated from the conditioned medium using
antibody 6E10. This antibody is raised to A1-17 and therefore
recognizes A40 as well as A42 (see "Experimental
Procedures"). Isolated A peptides were then separated on a
previously described gel system, which allows the specific resolution
of A40 and A42 (25). As shown in Fig. 2A, cells overexpressing PS1exon9
secreted elevated levels of A42 as compared with control cells. In
contrast, the cell line stably transfected with PS1exon9 C290S
produced significantly lower amounts of A42 as cells expressing the
Alzheimer's disease-associated PS1exon9 mutation. This suggests
that the pathological activity of the PS1exon9 mutation is due to
the point mutation generated at the aberrant splice junction. In order
to quantitate A42 and A40 production, we used a previously
described specific ELISA (12). In this assay, two highly specific
monoclonal antibodies are used for the discrimination of both peptide
species (12). As reported before (12, 18, 26), the PS1exon9 mutation results in an approximately 3-fold increase of the
A42/Atotal ratio (Fig. 2B). In contrast,
the cell line stably overexpressing PS1exon9 C290S showed no
increased A42/Atotal ratio (Fig. 2B). Therefore using two different approaches we can show that the pathological activity of PS1exon9 is independent of its lack of
proteolytic processing but is rather caused by the single amino acid
change at the aberrant splice junction.
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Having demonstrated that PS1exon9 C290S is pathologically inactive,
we also wanted to test if this protein is sufficient to rescue a
lin-12-mediated signaling defect that is a result of a
mutant PS homologue (sel-12) in Caenorhabditis
elegans (23). As reported previously, transgenic expression of
human presenilins rescues the phenotype caused by mutations of
sel-12 (22, 24). In contrast, transgenic expression of the
PS1exon9 variant in C. elegans resulted in an incomplete
rescue of the sel-12 mutant phenotype, as indicated by a
significantly reduced brood size and reduced egg laying (Refs. 22 and
24; see also Table I). In order to assess
the in vivo function of PS1exon9 C290S, we tested its
ability to rescue the putative sel-12 null allele
ar171 (23). Four independent transgenic strains expressing
PS1exon9 C290S displayed robust egg laying (Table I). Based on both
numbers of laid progeny and numbers of eggs in utero, the
phenotype of these strains is almost indistinguishable of that of wild
type worms (Table I). These results demonstrate that PS1exon9 C290S rescues the egg laying phenotype of the sel-12(ar171) mutant
animals significantly better than PS1exon9 (Table I). Therefore, the reduced biological activity of PS1exon9 is also due to a single point mutation and completely independent of the lack of proteolytic processing.
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DISCUSSION |
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Our results demonstrate that the pathological effect of the
PS1exon9 mutation is independent of its lack of proteolytic cleavage and the deletion of the exon 9-encoded domain. Reverting the cysteine residue generated by the exon 8/10 splice junction at codon 290 back to
its wt residue (serine) still inhibits its proteolytic processing,
causes an accumulation of the uncleaved protein, and prevents formation
of endogenous PS fragments. Although the artificially generated
PS1exon9 C290S variant behaves like PS1exon9 in regard to the
characteristic biochemical features described above, it does not allow
pathological overproduction of A42. Moreover, PS1exon9 C290S
regains full biological activity in a very sensitive in vivo
assay system. This demonstrates that not only the pathological overproduction of A42 but also the reduced biological function is
due to the single amino acid exchange. Therefore, similar to all other
known PS mutations, the pathological effect of the PS1exon9 mutation
is due to a rather subtle amino acid exchange at a single highly
conserved codon whereas the large deletion of the complete domain
encoded by exon 9 does not affect the biological and pathological function. However, consistent with our previous results (27), we
suggest that PS molecules lacking the domain encoded by exon 9 mimic a
proteolytically processed and biologically active PS complex and
therefore rescue the phenotype of the mutant nematode. Consequently,
the rescuing activity of PS1exon9 is significantly enhanced when the
pathologically relevant point mutation at codon 290 is reverted to the
wt residue.
It remains to be shown if the mutation at codon 290 is also
pathologically active within the full-length protein like all other
AD-associated point mutations. It may however be possible that the
mutation only exhibits a pathological activity if it is aberrantly
flanked by the domains encoded by exons 8 and 10. Structural changes
that may specifically occur in the PS1exon9 molecule (12, 19) might
be responsible for the pathological activity of this very unusual mutation.
Based on our results it would be interesting to investigate if the
point mutations in the PS genes require the endoproteolytic cleavage
for their pathological activity. Therefore artificial PS molecules
should be generated containing an AD associated mutation as well as the
smallest possible alteration of the sequence at the cleavage site which
would inhibit PS processing. However, such mutations might be very
difficult to generate since PS can be cleaved at multiple sites (10,
12) and PS molecules containing larger deletions at the cleavage sites
might mimic a proteolytically processed PS molecule (27).
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ACKNOWLEDGEMENTS |
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We thank Roland Donhauser for injecting PS constructs into C. elegans and Drs. Bernard Lakowski and Helmut Jacobsen for critical discussion.
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Note Added in Proof |
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While this manuscript was in press, we
found that expression of PS1exon9 C290S containing a FAD-associated
mutation (M146L) results in elevated A42 production, in further
support of the findings presented here.
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FOOTNOTES |
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*
This work was supported by grants from the Verum Foundation
(A42 generation) and the Deutsche Forschungsgemeinschaft (DFG) (to
C. H. and R. B.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
** To whom correspondence should be addressed: Central Institute of Mental Health, Dept. of Molecular Biology, J5, 68159 Mannheim, Germany. Tel.: 49-621-1703-884; Fax: 49-621-23429; E-mail: Haass{at}as200.zi-mannheim.de (for C. H.) or Genzentrum, Feodor-Lynen-Str.25, 81377 Munich, Germany. Tel.: 49-89-7401-7347; Fax: 49-89-7401-7314; E-mail: bmeister{at}LMB.uni-muenchen.de (for R. B., for correspondence regarding C. elegans).
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ABBREVIATIONS |
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The abbreviations used are:
AD, Alzheimer's
disease;
A, amyloid -peptide;
APP, amyloid precursor
protein;
PS, presenilin;
wt, wild type;
PCR, polymerase chain reaction;
MEM, minimal essential medium;
ELISA, enzyme-linked immunosorbent
assay.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
REFERENCES
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H. Steiner, T. Revesz, M. Neumann, H. Romig, M. G. Grim, B. Pesold, H. A. Kretzschmar, J. Hardy, J. L. Holton, R. Baumeister, et al. A Pathogenic Presenilin-1 Deletion Causes Abberrant Abeta 42 Production in the Absence of Congophilic Amyloid Plaques J. Biol. Chem., March 2, 2001; 276(10): 7233 - 7239. [Abstract] [Full Text] [PDF]
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C. A. Saura, T. Tomita, S. Soriano, M. Takahashi, J.-Y. Leem, T. Honda, E. H. Koo, T. Iwatsubo, and G. Thinakaran The Nonconserved Hydrophilic Loop Domain of Presenilin (PS) Is Not Required for PS Endoproteolysis or Enhanced Abeta 42 Production Mediated by Familial Early Onset Alzheimer's Disease-linked PS Variants J. Biol. Chem., May 26, 2000; 275(22): 17136 - 17142. [Abstract] [Full Text] [PDF]
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M. S. Wolfe and C. Haass The Role of Presenilins in gamma -Secretase Activity J. Biol. Chem., February 16, 2001; 276(8): 5413 - 5416. [Full Text] [PDF]
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L. Kulic, J. Walter, G. Multhaup, D. B. Teplow, R. Baumeister, H. Romig, A. Capell, H. Steiner, and C. Haass Separation of presenilin function in amyloid beta -peptide generation and endoproteolysis of Notch PNAS, May 23, 2000; 97(11): 5913 - 5918. [Abstract] [Full Text] [PDF]
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