From § Nervous System, Novartis Pharma AG,
CH-4002 Basel, Switzerland, ¶ Functional Genomics, Novartis
Pharmaceuticals Corp., Summit, New Jersey 07901, and
Center for Molecular Biology, University of Heidelberg,
D-69120 Heidelberg, Germany
Received for publication, September 28, 2000, and in revised form, January 3, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The Recently, BACE was identified as an enzyme having all the
characteristics expected for BACE overexpression in cultured cells increases the generation of
sAPP In situ hybridization studies demonstrated homogenous
expression of the BACE mRNA in neurons of all brain regions,
whereas glial cells appeared to express little or no BACE (12).
Accordingly, BACE protein is detected in neurons but not glial cells
(15). BACE enzymatic activity is extracted from human brain (13) in line with the observation that Antibodies--
The rabbit polyclonal antisera GM190 (20), 815, and 818 were raised against synthetic antigens corresponding to
peptides 22-45, 46-61 (with an additional carboxyl-terminal
cysteine), and 484-501 of BACE501. Antisera 815 and 818 were affinity
purified using commercially available reagents with the corresponding
covalently coupled peptide. All antisera reacted equally well against
BACE501 and BACE457. The monoclonal antibody Polymerase Chain Reaction and Plasmid Construction--
The
BACE501 and the BACE457 cDNAs were amplified by seminested
polymerase chain reaction from a human brain cDNA library. For the
first amplification, the primers BetaBHS1 (GATGTAGCGGGCTCCGGATC) and
BetaR33 (GGGGAATTCACTTCAGCAGGGAGATGTCATCAG) were used. For the second
reaction, the primer pair BetaBHS2 (GCGGATCCACCATGGCCCAAGCCC) and
BetaR33 was chosen. Subsequently the cDNA fragments were subcloned into the BamHI/EcoRI sites of an appropriate
vector for cytomegalovirus-regulated mammalian expression. For specific
detection of BACE501 or BACE457, primers overlapping the respective
exon/exon boundaries were chosen. Primers 501s (TGCCAGGCCTGACG) or 457s
(GGCACCGACCTGCCT) were used in combination with primer BetaR33 in a
seminested polymerase chain reaction on human pancreas and brain
cDNA libraries (CLONTECH), as well as on
cDNA synthesized from total human pancreas poly(A+)
RNA (CLONTECH). Pancreatic cDNA and
poly(A+) RNA were obtained from different pools of human
tissues. cDNA synthesis was performed with Moloney murine leukemia
virus reverse transcriptase (Life Technologies, Inc.) using the random
priming method according to the manufacturer's protocol. The BACE501
and BACE457 plasmids were used as positive or negative template controls.
Cell Extracts and Pancreas Homogenates for Western Blot
Analysis--
Cultured cells were extracted 48 h
post-transfection in RIPA buffer (10 mM Tris, pH 7.5, 150 mM sodium chloride, 1 mM EDTA, 1%
Nonidet P-40, 0.5% sodium-deoxycholate, 1% SDS) containing protease
inhibitors (Roche Molecular Biochemicals). Tissue samples from human
cortex or pancreas were homogenized in homogenization buffer (10 mM Tris, pH 7.5, 150 mM sodium chloride, 1 mM EDTA) and centrifuged at 4 °C for 10 min at
10,000 × g. Subsequently the supernatants were
centrifuged at 4 °C for 90 min at 100,000 × g. The
new supernatants were discarded, and the pellets were resuspended in
RIPA buffer by ultrasonic treatment. For PNGase F treatment
pancreas homogenate or cell extracts were incubated with PNGase F
overnight at 30 °C in 100 mM Tris/HCl, pH 7.5, 1% Cell Culturing, Transient Transfection, Metabolic Labeling, and
Immunoprecipitation--
HEK-293 and COS-7 cells were cultured in DMEM
supplemented with 10% FCS and 1% penicillin/streptomycin. Transient
transfections were performed using the SuperFect transfection reagent
(Qiagen) according to the manufacturer's protocol. For metabolic
labeling, cells were starved for 30 min in Met/Cys-free DMEM (Sigma)
supplemented with 5% dialyzed FCS and 1% penicillin/streptomycin.
Subsequently, cells were pulse-labeled for 10 min with 100 µCi/ml
[35S]Cys/Met (Expre35S35S protein
labeling mix; PerkinElmer Life Sciences) in Met/Cys-free DMEM,
5% dialyzed FCS and chased for 1 h or as indicated in DMEM, 10%
FCS, 1% penicillin/streptomycin. Cell extracts were obtained in RIPA
buffer after a short centrifugation. Equal amounts of labeled proteins
were immunoprecipitated using 1.5 µl of 818 or GM190 antibody and
protein A-Sepharose (Amersham Pharmacia Biotech). For Endo H
(Roche Molecular Biochemicals) treatment, immunoprecipitated BACE501 or
BACE457 was eluted from protein A-Sepharose with a 10-min, 100 °C
treatment in 0.5% SDS, 1% Immunocytochemistry--
Transfected cells were plated on
poly-D-lysine-coated chamber slides (Costar). For
immunostaining, cells were fixed in 3% paraformaldehyde or 5%
methanol/acetic acid (95/5) at Bioinformatic Approach--
To assemble the genomic sequence of
BACE, component genomic fragments representing the BACE exons were
identified by searching GenBankTM with the cDNA
sequence. high-throughput genomic sequencing (HTGS) draft
sequences AC020997 and AP001822 were found to include sequences
representing the full-length cDNA. These entries were split into
their component contigs. An iterative method alternating sequence
search and assembly steps was then applied to these contigs to build
the intervening introns.
To address the discrepancy observed in pancreas,
i.e. efficient transcription of the BACE mRNA
in the absence of recovered enzymatic activity, we searched for
differential mRNA splicing. Initially, we amplified the BACE
cDNA from a human brain cDNA library by nested polymerase chain
reaction. Restriction mapping was performed with restriction enzymes
expected to digest the full-length BACE cDNA. Using this strategy,
we identified a DNA fragment that was not digested by NcoI
and StuI (not shown). This suggested the existence of a BACE
transcript lacking a fragment of at least 120 base pairs. We then
designed several polymerase chain reaction primers specific for the
region around the NcoI and StuI sites.
Accordingly, we confirmed the existence of a differentially spliced
BACE mRNA in human pancreas that was not detectable in human brain
(Fig. 1). The amplification of the splice
variant was confirmed using a cDNA pancreatic library, as well as
using a distinct poly(A+) RNA pool. In contrast to this, the
full-length form of BACE was expressed in brain but was not detected in
pancreas (Fig. 1). Sequencing analysis indicated that 132 base pairs
were missing in this cDNA compared with that of full-length BACE.
To validate these findings, we screened the GenBankTM data
base to gain information on the structure of the human BACE gene. We
found that the BACE open reading frame was distributed over nine exons
covering about 26 kilobases of the human genome (Fig.
2). The first intron accounted for about
18.6 kilobases. All other introns ranged in length between 1526 bases
(intron 2) and 240 bases (intron 7). The 132 base pairs missing in the proposed differentially spliced mRNA appeared to match the terminal two-thirds of exon 3 (Fig. 2). Alignment of the exon/exon boundaries between several members of the human aspartyl protease family at the
protein level showed no conserved pattern (Fig. 2). Interestingly, the
only exon/exon boundary that was located in a conserved position corresponded to the 5' start of the differentially spliced intron. Lack
of the 132 base pairs would cause an in-frame deletion of 44 amino
acids in the full-length BACE (hereafter referred to as BACE457; see
Fig. 2). Moreover, the deletion in BACE457 would eliminate in the
full-length BACE (BACE501) two of the four consensus sites for
N-glycosylation (Fig. 2). Based on this, BACE457 should migrate faster than BACE501 when separated by polyacrylamide gel electrophoresis. In fact, recombinant BACE457 expressed in HEK-293 cells was revealed as an immunoreactive band with an apparent molecular
mass of 50 kDa using the specific antibody 815 raised against
the amino terminus of BACE (Fig.
3A, upper panel,
lane 2). In contrast, BACE501 migrated mainly as a diffuse
band of about 65 kDa (Fig. 3A, upper panel,
lane 1). Analysis of membrane protein isolated from three
distinct human pancreatic samples revealed a specific immunoreactive
protein comigrating with the recombinant BACE457 (Fig. 3A,
upper panel, lanes 4-6). Practically identical
results were obtained when the carboxyl-terminal-specific 818 antibody
was used, indicating that BACE457 extends between the same amino and
carboxyl termini as full-length BACE501 (Fig. 3A,
lower panel). BACE457, but not BACE501, was also detected in
total pancreatic homogenates (not shown). When brain membranes or total
brain homogenates were used, we were not able to detect either BACE457
or BACE501 (Fig. 3A, lane 7 or data not shown). Full-length BACE has been reported to be expressed in the brain in a
barely detectable amount (12). Both recombinant and pancreatic BACE457
were glycosylated, most likely at both remaining sites, as suggested by
the 4-kDa shift to 46 kDa after PNGase digestion (Fig. 3B).
In overexpressing HEK-293 cells, in addition to mature BACE501
migrating as a diffuse protein band of about 65 kDa, the deglycosylated
and Endo H-sensitive forms of BACE501 migrated at 48 and 55 kDa,
respectively (Fig. 3C, lanes 1 and 2),
as reported by others (19, 20, 22). Notably, we observed that the
50-kDa BACE457, in contrast to BACE501, did not acquire Endo H
resistance (Fig. 3C, lanes 3 and 4),
suggesting that the transport along the secretory pathway of the
pancreatic variant of BACE was blocked at the level of the endoplasmic
reticulum. To further explore this hypothesis, we analyzed whether the
propeptide of BACE457 was cleaved off, a process shown to occur after
exit from the endoplasmic reticulum for BACE501. As expected (19,
22), propeptide removal and full glycosylation of BACE501 were
completed in about 3.5 h when analyzed in a pulse-chase experiment
(Fig. 4, upper panel). On the
other hand, we found that virtually all of BACE457 remained in the
proenzymatic form over a period of 16 h (Fig. 4, lower
panel). The presence of the propeptide would reduce the length of
BACE457 to 20 and not 44 amino acids when compared with BACE501. This
may explain the small difference observed in the apparent molecular
masses (48 and 46 kDa, respectively) between the two deglycosylated
forms. Immunofluorescence studies revealed that BACE501 is present on
the cell surface (Fig. 5, panel
1), as well as along the secretory pathway,
i.e. in the endoplasmic reticulum and Golgi
apparatus (Fig. 5, panels 2 and 3) when expressed in cells. Strengthening the evidence that BACE457 is retained in the
early secretory pathway, the splice variant was not detected on the
cell surface (Fig. 5, panel 4) but found
exclusively in the endoplasmic reticulum where it colocalized with the
specific marker BiP (Fig. 5, panels 5 and 6).
Overexpression of BACE501 was shown previously to increase
We have identified a differentially spliced form of BACE. The
novel variant of BACE was found in the pancreas but was not detected in
the brain. In contrast, we observed an opposite expression pattern of
full-length BACE. In the human pancreas, we demonstrate BACE457
expression to exist at the mRNA and at the protein level. Previous
studies (12-15) have demonstrated transcription of BACE in pancreas in
the absence of recovered enzymatic activity. The novel splice form
suggests an explanation for this apparent discrepancy. BACE457 lacks 44 amino acids located between the two catalytic aspartyl residues and
containing two of four putative glycosylation sites. Such a
deletion can be expected to cause misfolding of the protein, explaining
the observed retention within the endoplasmic reticulum,
e.g. by a quality control mechanism. With the
strategy of analysis chosen, we cannot exclude at this point that
additional splice variants of BACE may exist.
Full glycosylation and activation by propeptide removal are
post-translational modifications acquired during maturation and transport of BACE to a subcellular compartment where APP processing occurs. Failure to detect said modifications for the pancreatic variant
is consistent with the observation that BACE457 was not found to
contribute to the amyloidogenic processing of APP,
i.e. increased formation of the carboxyl-terminal
amyloidogenic intermediate C99. Lack of increased production of C99
also appears to exclude the notion that BACE457 may represent an
endoplasmic reticulum form of active BACE.
We also report the structure of the BACE gene. Both a novel pattern of
exon/exon boundaries when compared with prorenin and pepsinogen A, as
well as the first description of differential splicing for a gene
encoding an aspartyl protease, might indicate that the BACE gene is
derived by convergent evolution rather than by a gene duplication
mechanism. The homology between BACE and the aspartyl protease family
members (13) appears to span the open reading frame encoded by exon 2 to exon 8 and excluding exon 1 and exon 9. Exon 1 encodes the signal
peptide, the propeptide, and the poorly conserved amino terminus of
mature BACE. Exon 9 encodes the characteristic carboxyl terminus of
BACE that includes the transmembrane spanning domain and the cytosolic tail.
The expression of an inactive form of BACE in pancreas, as opposed to
having the gene repressed transcriptionally, suggests an unknown
function of this form of BACE that may be independent of proteolytic
activity. Alternatively, because propeptide removal may not be required
for activity, a difference in substrate specificity between the two
differentially spliced forms of BACE cannot yet be excluded.
-Secretase (BACE) initiates the
amyloidogenic processing of the amyloid precursor protein leading to
the generation of the
-amyloid, the main component of Alzheimer's
disease senile plaques. BACE is a type I transmembrane aspartyl
protease of 501 amino acids. Here we describe a novel BACE mRNA
lacking 132 base pairs that is expressed in the pancreas but not in the
brain. Sequence alignment indicates that the deleted fragment matches
the terminal two-thirds of exon 3. The new BACE variant is short of a
44-amino acid region located between the two catalytic aspartyl
residues. Accordingly, a 50-kDa form of BACE (BACE457) is detected in
the human pancreas. When expressed in cells, BACE457 colocalizes with the marker for the endoplasmic reticulum BiP. Moreover, BACE457 remains
in a proenzymatic and endoglycosidase H-sensitive state, suggesting
that its transport along the secretory pathway is blocked at the level
of the endoplasmic reticulum. Notably, this novel form of BACE does not
contribute to the processing of the amyloid precursor protein.
Our findings suggest that tissue-specific splicing of the BACE mRNA
may explain the observation that in the human pancreas robust
transcription of the BACE gene does not translate into recovered
enzymatic activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amyloid peptide
(A
)1 is the main component
of the senile plaques found in the brain of Alzheimer's disease
patients (1, 2). Amyloidogenic processing of the amyloid precursor protein (APP) is initiated by
-secretase cleavage generating a
100-kDa soluble form of the ectodomain (sAPP
) and a 12-kDa membrane-associated intermediate of 99 amino acids (C99). The latter is
then processed by
-secretase to A
(3).
-Secretase cleaves
typically after Met596 of the APP695 isoform
generating the amino terminus of A
at Asp1 (4).
Substitution of Met596 of APP with a Leu increases whereas
a Val decreases cleavage rate by the
-secretase (5). Indeed, the
Swedish variant of APP Lys595-Met596 to Asn-Leu
(APPSWE) linked to Alzheimer's disease is a better substrate for
-secretase (6, 7). Sensitivity to alkalizing agents
indicates an acidic pH value optimum for
-secretase as found in
Golgi-derived vesicles or in the endosomal/lysosomal compartment (8,
9). This is consistent with cellular studies demonstrating the
involvement of the endocytic pathway in A
generation (10, 11).
-secretase (12-16). The BACE gene is encoded on chromosome 11 (11q23.2) (17, 18) and was shown to generate
transcripts whose expression is robust in pancreas, moderate in brain,
and low in several peripheral tissues (12-14, 16). BACE is a type I
transmembrane aspartyl protease of 501 amino acids (15). All six
ectodomain cysteine residues appear to be involved in disulfide bonds
tethering the catalytic domain in an atypical pattern for an aspartyl
protease (19). Maturation of BACE occurs by propeptide removal and
complex N-glycosylation at four Asn residues (12, 13, 19),
both of which take place almost simultaneously after exit from the
endoplasmic reticulum. Immunohistochemistry studies localized BACE at
the same intracellular sites as APP, consistent with Golgi and
endosomal distribution (12, 15, 16, 20). When expressed in cells,
mature BACE has a half-life of more than 9 h (19).
, C99, and A
(12-16). Notably, the amount of
full-length APP remains unchanged under these conditions,
whereas processing of APP by the nonamyloidogenic pathway is reduced,
i.e. cleavage after Lys16 of A
by
-secretase. In contrast,
-secretase antisense application leads
to opposite effects (12, 14). Purified recombinant BACE cleaves more
efficiently a substrate carrying the Swedish mutation when compared
with the wild-type peptide (12, 14). The aspartyl protease inhibitor
pepstatin, as well as several inhibitors for the other protease
classes, were found inactive (12-14, 16). The pH value optimum in the
slight acidic range (4.5-5.5) is consistent with the involvement of an
intracellular acidic compartment in
-secretase processing (12-14,
16).
-secretase activity is enriched in
cells of central nervous system origin (21). In contrast, little or no
BACE activity is detected in peripheral tissues including the human
pancreas, although high expression of its mRNA is found in this
tissue (13). Here we show that an endoplasmic reticulum-retained pancreatic form of BACE resulting from differential mRNA splicing is deficient in APP processing.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 was raised as
described previously (9). The mouse monoclonal antibodies 6E10 and
-GRP 78 are commercially available (Senetek; StressGen
Biotechnologies Corp.).
-octyl glycoside. For immunoprecipitation of C99 from extracts of
HEK-293 cells expressing wild-type APP (9), antibody
1 and protein
A-Sepharose were used. Subsequently, the precipitates or cell extracts
were resolved by SDS polyacrylamide gel electrophoresis transferred to
Immobilon-P membrane (Millipore) and detected using primary antibodies
as indicated.
-mercaptoethanol. Each sample was split
into two aliquots. Sodium citrate (to a final concentration of 50 mM, pH 5.5) and, if indicated, 5 milliunits of Endo H were
added to the samples and incubated for 1.5 h at 37 °C.
20 °C. After blocking with 3%
bovine serum albumin in phosphate-buffered saline, cells were incubated
with the antibodies
-GRP 78 (anti-BiP) at 1:200 dilution, 815 at 1:500 dilution, and 818 at 1:2000 dilution. Alexa-488 and Alexa-594
secondary antibodies (Molecular Probes) were used at 1:500 dilution.
Stained cells were embedded in Mowiol (Calbiochem).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-secretase processing of APP (12-14). This can be monitored by an
increase in cell-associated C99, the membrane-bound precursor of A
generated by
-secretase cleavage. Indeed, when BACE501 was expressed
in cells, C99 production was increased as expected (Fig.
6, lane 1). In sharp contrast to this, overexpression of BACE457 did not enhance the amyloidogenic processing of APP (Fig. 6, lane 2) when compared with the
control (Fig. 6, lane 3). This was consistent with the
observation that exit from the endoplasmic reticulum,
i.e. full glycosylation and propeptide removal,
is required to produce a proteolytic competent
-secretase/BACE.
View larger version (53K):
[in a new window]
Fig. 1.
Expression of a differentially spliced BACE
mRNA in the human pancreas. Shown are DNA fragments
separated on an agarose gel and obtained by seminested polymerase chain
reaction using BACE457-specific primers (left panel) and
BACE501-specific primers (right panel) with total human
brain or two different pancreas cDNAs as templates. The BACE501
plasmid, the BACE457 plasmid, and water (control) served as
template in specificity control reactions. The migration of DNA size
markers is given on the right.
View larger version (23K):
[in a new window]
Fig. 2.
Schematic representation of the 44-amino acid
deletion generating BACE457. Shown is the alignment of prorenin,
pepsinogen A, and BACE with the location of the exon/exon boundaries
for these aspartyl proteases. The BACE501 open reading frame is
distributed over nine exons of the indicated size in base pairs.
Vertical dotted lines frame the 132 base pairs missing in
BACE457 and leading to an in-frame deletion of 44 amino acids. The
deletion is located between the two catalytic aspartyl residues
(D) and also eliminates two of the four
N-glycosylation sites (asterisks) present in
BACE501.
View larger version (45K):
[in a new window]
Fig. 3.
Immunological detection, glycosylation, and
Endo H sensitivity of BACE457. A, immunoblots of recombinant
BACE501 and BACE457 expressed in human HEK-293 cells (lanes
1 and 2). The specificity of both the carboxyl- and
amino-terminal antibodies (Ab) is demonstrated by using
mock-transfected cell lysates (lane 3) or by using preimmune
serum (not shown). BACE457, but not BACE501, is detected in human
pancreas homogenates (lanes 4-6). The signal at 68 kDa
obtained only with the 818 antibody is due to an unspecific reaction to
a protein present in cell and brain homogenates. The migration of
molecular mass markers is given on the right. B,
PNGase F treatment of recombinant (lanes 1 and
2), as well as pancreatic (lanes 3-6), BACE457
showed the same shift in molecular mass indicative of glycosylation in
both cases. pancr., pancreas. C, Endo H treatment
of recombinant 35S-labeled BACE501 (lanes 1 and
2) and BACE457 (lanes 3 and 4).
Extracts of cells expressing BACE501 or BACE457 were immunoprecipitated
with the carboxyl-terminal 818 antiserum and treated in the presence
(+) or absence ( ) of Endo H as indicated. unglycosyl.,
unglycosylated.
View larger version (56K):
[in a new window]
Fig. 4.
Lack of propeptide cleavage in BACE457.
Complex glycosylation and propeptide removal of BACE501 is completed
shortly after the 3.5-h time point in a [35S] pulse-chase
experiment (upper panel). In contrast, BACE457 remained in
the Endo H-sensitive, proenzymatic form over a period of 16 h
(lower panel). Protein was immunoprecipitated
(IP) with the 818 carboxyl-terminal antiserum to visualize
all forms of BACE or with the GM190 propeptide-specific antiserum to
detect the immature and unglycosylated (unglycosyl.) forms
of BACE, as indicated. Ab, antibodies.
View larger version (53K):
[in a new window]
Fig. 5.
BACE457 is retained in the endoplasmic
reticulum. Immunofluorescence micrographs of COS-7 cells
expressing BACE501 (panels 1-3) or BACE457 (panels
4-6) are shown. In intact cells, BACE501 (panel 1),
but not BACE457 (panel 4), is detected on the cell
surface and along most cellular protrusions using the amino-terminal
815 antibody against the BACE ectodomain. In permeabilized cells,
BACE501 and BACE457 colocalized with BiP, a marker of the endoplasmic
reticulum (panels 3 and 6). To immunostain
permeabilized cells the carboxyl-terminal 818 antibody was used
(panels 2 and 5).
View larger version (22K):
[in a new window]
Fig. 6.
BACE457 is deficient in the processing of the
amyloid precursor protein. HEK-293 cells overexpressing the human
APP were transiently transfected with the BACE501 and BACE457 plasmids
or mock-transfected with an empty vector. BACE501 (lane 1),
but not BACE457 (lane 2), caused an increase in the
steady-state levels of C99, the processing product of APP generated by
-secretase, when compared with mock-transfected cells. C99 was
visualized specifically after immunoprecipitation with
1 and
immunoblotting with 6E10.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Muriel Stefani for excellent technical assistance, Karl-Heinz Wiederhold and Alphonse Probst for supplying pancreatic tissue samples, Mauro Zurini for the affinity purification of the antisera, Christian Ostermeier for the gift of purified PNGase, Robert Crowl for comments on an early version of the manuscript, and Matthias Staufenbiel and Bernd Sommer for support throughout this study.
![]() |
FOOTNOTES |
---|
* 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.
Contributed equally to this work.
** To whom correspondence should be addressed: WSJ.386.8.28, Novartis Pharma AG, CH-4002 Basel, Switzerland. Tel.: 41 61 324 3481; Fax: 41 61 324 5524; E-mail: paolo.paganetti@pharma.novartis.com.
Published, JBC Papers in Press, January 10, 2001, DOI 10.1074/jbc.M008861200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
A,
-amyloid
peptide;
APP,
-amyloid precursor protein;
BACE,
-site
APP-cleaving enzyme;
C99, the carboxyl-terminal 99-amino acid
intermediate generated by BACE;
HEK, human embryonic kidney;
DMEM, Dulbecco's modified Eagle's medium;
FCS, fetal calf serum;
Endo H, endoglycosidase H;
PNGase F, peptide N-glycosidase F.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Glenner, G. G., and Wong, C. W. (1984) Biochem. Biophys. Res. Commun. 120, 885-890[Medline] [Order article via Infotrieve] |
2. | Masters, C. L., Simms, G., Weinman, N. A., Multhaup, G., McDonald, B. L., and Beyreuther, K. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 4245-4249[Abstract] |
3. | Selkoe, D. J. (1998) Trends Cell Biol. 8, 447-453[CrossRef][Medline] [Order article via Infotrieve] |
4. | Golde, T. E., Estus, S., Younkin, L. H., Selkoe, D. J., and Younkin, S. G. (1992) Science 255, 728-730[Medline] [Order article via Infotrieve] |
5. | Citron, M., Teplow, D. B., and Selkoe, D. J. (1995) Neuron 14, 661-670[Medline] [Order article via Infotrieve] |
6. | Citron, M., Oltersdorf, T., Haass, C., McConlogue, L., Hung, A. Y., Seubert, P., Vigo-Pelfrey, C., Lieberburg, I., and Selkoe, D. J. (1992) Nature 360, 672-674[CrossRef][Medline] [Order article via Infotrieve] |
7. | Cai, X. D., Golde, T. E., and Younkin, S. G. (1993) Science 259, 514-516[Medline] [Order article via Infotrieve] |
8. |
Haass, C.,
Hung, A. Y.,
Schlossmacher, M. G.,
Teplow, D. B.,
and Selkoe, D. J.
(1993)
J. Biol. Chem.
268,
3021-3024 |
9. | Schrader-Fischer, G., and Paganetti, P. A. (1996) Brain Res. 716, 91-100[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Perez, R. G.,
Soriano, S.,
Hayes, J. D.,
Ostaszewski, B.,
Xia, W.,
Selkoe, D. J.,
Chen, X.,
Stokin, G. B.,
and Koo, E. H.
(1999)
J. Biol. Chem.
274,
18851-18856 |
11. | Cescato, R., Dumermuth, E., Spiess, M., and Paganetti, P. A. (2000) J. Neurochem. 74, 1131-1139[Medline] [Order article via Infotrieve] |
12. |
Vassar, R.,
Bennett, B. D.,
Babu-Khan, S.,
Kahn, S.,
Mendiaz, E. A.,
Denis, P.,
Teplow, D. B.,
Ross, S.,
Amarante, P.,
Loeloff, R.,
Luo, Y.,
Fisher, S.,
Fuller, J.,
Edenson, S.,
Lile, J.,
Jarosinski, M. A.,
Biere, A. L.,
Curran, E.,
Burgess, T.,
Louis, J. C.,
Collins, F.,
Treanor, J.,
Rogers, G.,
and Citron, M.
(1999)
Science
286,
735-741 |
13. | Sinha, S., Anderson, J. P., Barbour, R., Basi, G. S., Caccavello, R., Davis, D., Doan, M., Dovey, H. F., Frigon, N., Hong, J., Jacobson-Croak, K., Jewett, N., Keim, P., Knops, J., Lieberburg, I., Power, M., Tan, H., Tatsuno, G., Tung, J., Schenk, D., Seubert, P., Suomensaari, S. M., Wang, S., Walker, D., Zhao, J., McConlogue, L., and John, V. (1999) Nature 402, 537-540[CrossRef][Medline] [Order article via Infotrieve] |
14. | Yan, R., Bienkowski, M. J., Shuck, M. E., Miao, H., Tory, M. C., Pauley, A. M., Brashier, J. R., Stratman, N. C., Mathews, W. R., Buhl, A. E., Carter, D. B., Tomasselli, A. G., Parodi, L. A., Heinrikson, R. L., and Gurney, M. E. (1999) Nature 402, 533-537[CrossRef][Medline] [Order article via Infotrieve] |
15. | Hussain, I., Powell, D., Howlett, D. R., Tew, D. G., Meek, T. D., Chapman, C., Gloger, I. S., Murphy, K. E., Southan, C. D., Ryan, D. M., Smith, T. S., Simmons, D. L., Walsh, F. S., Dingwall, C., and Christie, G. (1999) Mol. Cell. Neurosci. 14, 419-427[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Lin, X.,
Koelsch, G.,
Wu, S.,
Downs, D.,
Dashti, A.,
and Tang, J.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
1456-1460 |
17. | Saunders, A. J., Kim, T. W., and Tanzi, R. E. (1999) Science 286, 1255[CrossRef] |
18. | Fan, W., Bennett, B. D., Babu-Kahn, S., Luo, Y., Louis, J. C., McCaleb, M. M., Citron, M., Vassar, R., and Richards, W. G. (1999) Science 286, 1255[CrossRef] |
19. |
Haniu, M.,
Denis, P.,
Young, Y.,
Mendiaz, E. A.,
Fuller, J.,
Hui, J. O.,
Bennett, B. D.,
Kahn, S.,
Ross, S.,
Burgess, T.,
Katta, V.,
Rogers, G.,
Vassar, R.,
and Citron, M.
(2000)
J. Biol. Chem.
275,
21099-21106 |
20. |
Capell, A.,
Steiner, H.,
Willem, M.,
Kaiser, H.,
Meyer, C.,
Walter, J.,
Lammich, S.,
Multhaup, G.,
and Haass, C.
(2000)
J. Biol. Chem.
275,
30849-30854 |
21. | Seubert, P., Oltersdorf, T., Lee, M. G., Barbour, R., Blomquist, C., Davis, D. L., Bryant, K., Fritz, L. C., Galasko, D., Thal, L. J., Lieberburg, I., and Schenk, D. (1993) Nature 361, 260-263[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Huse, J. T.,
Pijak, D. S.,
Leslie, G. J.,
Lee, V. M. Y.,
and Doms, R. W.
(2000)
J. Biol. Chem.
275,
33729-33737 |