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(Received for publication, February 2,
1995; and in revised form, January 26, 1996) From the
The mutation at codons 670/671 of
In Alzheimer's disease a characteristic pathological
finding in the brains of affected individuals is the deposition of
amyloid
Figure 1:
A Evidence that A In this
report, biosynthetic analyses confirmed the increase in A
Figure 2:
Secretion of
a shortened
The
onset of secretion of total
Figure 3:
Incremental release of
Figure 4:
Profiles of
Regarding A
Figure 5:
Generation of intracellular
These results suggested that A
Figure 6:
Release of A
Two additional observations are noteworthy from
these experiments. First,
Figure 7:
Effects of various treatments on A
A double mutation in the Our results showed that, as anticipated, A number of observations suggest
that A Regarding the
endocytic processing of Our studies have defined a number of
similarities in In summary, our data
suggest that there is a similar mechanism for A
Note Added in Proof-Similar findings of
intracellular A
Volume 271,
Number 15,
Issue of April 12, 1996 pp. 9100-9107
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
-Protein from Codon 670/671 
Swedish
Mutant -Amyloid Precursor Protein Occurs in Both Secretory and
Endocytic Pathways (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-amyloid precursor
protein (PP) dramatically elevates amyloid -protein (A)
production. Since increased A may be responsible for the disease
phenotype identified from a Swedish kindred with familial
Alzheimer's disease, evaluation of the cellular mechanism(s)
responsible for the enhanced A release may suggest potential
therapies for Alzheimer's disease. In this study, we analyzed
Chinese hamster ovary cells stably transfected with either wild type
PP (PP-wt) or ``Swedish'' mutant PP
(PP-sw) for potential differences in PP processing. We
confirmed that increased amounts of A and a
-secretase-cleaved COOH-terminally truncated soluble PP
(PP
) were secreted from PP-sw cells. As shown
previously for PP-wt cells, A was released more slowly than
the secretion of PP from surface-labeled PP-sw
cells, indicating that endocytosis of cell surface PP is one
source of A production. In contrast, by
[
S]methionine metabolic labeling, the rates of
A and PP release were virtually identical for
both cell lines. In addition, the identification of intracellular
PP and A shortly after pulse labeling suggests
that A is produced in the secretory pathway. Interestingly, more
A was present in medium from PP-sw cells than PP-wt
cells after either cell surface iodination or
[S]methionine labeling, indicating that
PP-sw cells have enhanced A release in both the endocytic and
secretory pathways. Furthermore, a variety of drug treatments known to
affect protein processing similarly reduced A release from both
PP-wt and PP-sw cells. Taken together, the data suggest that
the processing pathway for PP is similar for both PP-wt and
PP-sw cells and that increased A production by PP-sw
cells arises from enhanced cleavage of mutant PP by
-secretase, the as-yet unidentified enzyme(s) that cleaves at the
NH
terminus of A.
-protein (A) (
)in senile
plaques(1) . A is the 39-43-amino acid proteolytic
cleavage product of the type I integral membrane protein -amyloid
precursor protein (PP). The PP gene is encoded on chromosome
21, and alternative exon splicing produces three major isoforms of 695,
751, or 770 amino acids(2) . During constitutive secretion some
full-length PP molecules are proteolytically cleaved between
lysine and leucine residues at positions 16 and 17 of A (Fig. 1) by an enzyme termed
-secretase(3, 4) . Cleavage of PP at this
position creates a soluble 
100-120-kDa
NH-terminal fragment (PP) (5) and
a COOH-terminal membrane-retained fragment of 10 kDa(6) .
Generation of these fragments by -secretase precludes formation of
an intact A sequence from full-length PP.
PP structure, enzymatic cleavage
sites, COOH-terminal fragments, and antibody epitopes. Schematic
diagram of PP
. The vertical cross-hatched box represents the plasma membrane. The white box labeled A represents the A peptide (also shown enlarged with
the amino acid sequence listed). The horizontally striped box labeled KPI represents the Kunitz protease inhibitor
domain alongside the adjacent exon indicated by the small open
box; the NH-terminal black box represents the
signal sequence. , , and 
mark the sites of the
enzymatic cleavages by -, -, and -secretases,
respectively. Also indicated are the 10-kDa fragment (including
the p3 region, transmembrane region, and COOH terminus) and the
12-kDa fragment (including the A region, transmembrane
region, and COOH terminus). -NPTY- indicates the putative
clathrin internalization signal. Horizontal black bars indicate the approximate epitopes of antibodies B5, C7, 6E10,
MMAb, R1280 and R1282, and R1736.
, however,
is known to be released during normal cellular metabolism both in
vivo(7, 8) and in a number of cell culture
systems(9, 10) . Cleavage of PP at the NH terminus of the A sequence by an enzyme designated
-secretase creates a shortened form of PP and the
12-kDa COOH-terminal fragment(11, 12) . An
additional enzymatic cleavage at the COOH terminus of the A
sequence by the as yet unidentified enzyme designated -secretase
generates the 4-kDa A peptide. The -secretase enzyme is also
hypothesized to generate p3, the 3-kDa NH-terminal piece of
the membrane-retained 10-kDa COOH-terminal fragment of PP
produced by -secretase cleavage (7, 8, 9, 13) . In addition to the
secretory cleavage, PP can also be processed in an
endosomal/lysosomal
pathway(14, 15, 16, 17) . Although
A-containing COOH-terminal fragments are generated in lysosomes,
evidence suggests that these are not an important source of
A(18) . Recently, it was shown that cell surface PP
molecules can be processed in the endocytic pathway and may be the
direct precursors of A, presumably by recycling internalized
molecules from the cell surface(19) .
and PP contribute to the pathogenesis of Alzheimer's disease
comes from the findings of missense mutations within and adjacent to
the A region of the PP gene in families with autosomal
dominant forms of Alzheimer's disease(20) . The
concurrence of the mutations with the disease phenotype suggests that
altered PP function or processing may be pathogenic. A double
mutation at amino acids 670 and 671 (PP numbering)
changing Lys
to Asn and Met
to Leu (K670N/M671L) was identified in a Swedish
pedigree with familial Alzheimer's disease(21) . In
vitro analyses of transfected cells expressing the Swedish form of
PP (12, 22) and primary cell cultures of
fibroblasts obtained from affected individuals (23) reveal a
dramatic increase in A production. However, the mechanism by which
A generation is increased has not been elucidated. Furthermore, a
detailed analysis of cellular processing of PP with this mutation
has not been reported. Because recent studies have implicated the
endocytic pathway in A production(19) , we speculated that
A production may be similarly enhanced in this pathway in cells
expressing the K670N/M671L PP
mutation.
production and the abundant secretion of a shorter PP species by Chinese hamster ovary (CHO) cells stably transfected
with the PP K670N/M671L mutation. Furthermore,
A generation was increased in both the secretory and endocytic
pathways. We postulate that this increase in A production is the
result of enhanced proteolytic cleavage of the mutant PP by the
-secretase enzyme.
Cell Culture
Stably transfected CHO cell lines
were generated with wild type PP(19) or
with 670/671 PP ``Swedish'' mutation
cloned into pcDNA3 (Invitrogen, San Diego) by CaPO
transfection and selection by G418 resistance. The mutant PP
construct was obtained by subcloning the 500-base pair BamHI-EcoRI fragment containing the mutation from
PP
K670N/M671L (22, generously provided by Dr.
Martin Citron) into the wild type PP expression
vector. Cells were grown in Dulbecco's modified Eagle's
medium (DMEM, BioWhitaker, Walkersville, MD) with 10% Fetal Clone II
(HyClone Laboratories, Logan, UT) at 37 °C, with 5% CO. Antibodies
Several anti-PP antibodies were
used (see Fig. 1). The monoclonal antibodies 5A3 and 1G7
(referred to as MMAb when used together), recognize a midregion
extracellular PP domain (19) . PP monoclonal antibody
6E10 (from K. S. Kim and H. Wisniewski) recognizes
A(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17) and
-secretase-cleaved PP(24) . Five
previously described polyclonal antibodies were also used: R1280 and
R1282 raised against synthetic
A(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40) precipitate
A and p3 fragments(25, 26) . R1282 (1:100) gave
consistent recovery of both A and the p3 fragment by
immunoprecipitation. C7 was raised against PP(751-770) and
recognizes full-length PP and COOH-terminal fragments of
PP(27) . B5 raised against the midregion of
PP(519-667) precipitates both -secretase- and
-secretase-cleaved PP(28) , and R1736
raised against PP(670-686) recognizes
-secretase-cleaved PP(15) .Metabolic Labeling
Confluent cultures of
PP-transfected CHO cells were incubated in methionine-free DMEM
for 15 min followed by incubation with methionine-free DMEM
supplemented with 200 µCi/ml [S]methionine
for 10 min (pulse labeling) or for 2-4 h with 50-100
µCi/ml [S]methionine (long labeling). Cells
were lysed immediately or incubated with 2-fold unlabeled methionine
(chase) in DMEM from 10 min to 4 h. For some experiments, single dishes
of confluent cells were pulse labeled and chased at multiple time
points with repeated collection of medium to evaluate the incremental
secretion of PP products at each time point of the chase period.
PPs were immunoprecipitated using PP-specific antibodies and
separated by SDS-polyacrylamide gel electrophoresis (using 6-10%
Tris-glycine gels for high molecular weight proteins and 16.5%
Tris-Tricine gels for low molecular weight proteins). Gels were either
fluorographically enhanced and exposed to x-ray film or dried and
exposed on a Phosphor screen (Molecular Dynamics). All experiments
reported herein were performed two to six times, and a representative
example of each is shown. Where applicable, average values ±
S.E. are given.Assessment of Total
PP and
PPPP expression and PP quantity were determined using parallel triplicate cultures of
stably transfected CHO cells expressing wild type PP or
``Swedish'' PP. One set of cultures was lysed
immediately after a 10-min pulse labeling and immunoprecipitated with
C7 to determine total PP. The other set was chased for 4 h, and
media were collected and immunoprecipitated with MMAb to determine
PP secretion. Samples were separated by
SDS-polyacrylamide gel electrophoresis and analyzed by Phosphorimaging.
Comparison of PP levels was made after normalization
for total PP expression.Intracellular
Separate dishes
of stably transfected CHO cells, one dish for each chase time, were
pulse labeled for 10 min and chased for either 10, 20, 30, or 60 min.
Chase media were collected, and cells were washed with ice-cold DPBS
and rapidly chilled to 4 °C. After washing, DPBS was replaced with
DPBS containing 0.1% saponin plus protease inhibitors (leupeptin and
Pefabloc, Boehringer Mannheim). Cells were treated with saponin buffer
for 40 min at 4 °C as described previously(29) , to allow
the release of intracellular PPPP. Saponin buffer was
then collected, and both the chase media and saponin buffers were
immunoprecipitated using antibody B5 for total PP and
antibody C7 for full-length PP.Cell Surface Iodination
Derivatized Bolton-Hunter
reagent, sulfosuccinmidyl-3-(4-hydroxyphenyl)propionate (sulfo-SHPP,
Pierce) was labeled with Na
I in the presence of IODO-GEN
(Pierce) at room temperature essentially as described(30) .
After 5 min, the iodination reaction was quenched with p-hydroxyphenylacetic acid (Sigma), diluted with DPBS, and
immediately added to chilled washed cells for 40 min at 4 °C. After
iodination, the cells were extensively washed in DPBS containing 1
mg/ml lysine followed by incubation in prewarmed CHO medium at 37
°C. Three independent experiments were performed.Surface Antibody Binding
To determine the amount
of cell surface and total PP in stably transfected CHO cells, 5A3
monoclonal antibody Fab fragments were radioiodinated with IODO-GEN to
2-4 µCi/µg(19) . Confluent cell cultures were
chilled and washed. One set of cultures was treated with 0.1% saponin
in DPBS for 30 min at 4 °C to permeabilize cells gently and permit
the labeling of both cell surface and intracellular PP. Parallel
cultures for each cell line were treated for 30 min at 4 °C with
DPBS for evaluation of cell surface PP. Both sets were incubated
with radioiodinated antibody at 10 nM in binding medium (RPMI
1640 medium supplemented with 0.2% bovine serum albumin) at 4 °C
for 1 h, followed by two washes with binding medium and two washes with
DPBS. The cells were then lysed with 0.2 M NaOH, and
radioactivity was determined by -counting. To calculate specific
binding, background levels of radioactivity were determined from
parallel cultures of untransfected CHO cells to subtract from the
counts obtained from PP-transfected CHO cells. Four separate
experiments were performed using triplicate cultures.Drug Studies
Confluent CHO cells were
metabolically labeled with 50-100 µCi/ml
[S]methionine for 2 h and then chased for 2 h in
media containing either chloroquine (100 µM), bafilomycin
A1 (0.25 µM, Wako BioProducts, Richmond, VA), or brefeldin
A (35 µM, Epicentre Technologies, Madison, WI). These
drugs are known to affect protein processing as described below. Some
bafilomycin experiments were performed using a 10-min pulse with
[S]methionine followed by a 20-min chase in 0.25
µM bafilomycin A1. PP products were
immunoprecipitated and visualized as described previously. Six separate
experiments were performed.
Multiple stably transfected PP Processing by -Secretase Is Enhanced in
CHO Cells Stably Expressing ``Swedish'' Mutant
PPPP-sw CHO
cell lines were selected based on their equivalent PP expression
levels to PP-wt cell lines. Consistent with earlier reports (12, 22, 31, 32, 33) all of
the PP-sw cell lines released severalfold more of the 4-kDa A
peptide than wild type cells, which had comparable levels of PP
expression (not shown). Pulse-chase experiments also showed that the
timing of appearance and disappearance of labeled PP was
essentially identical in PP-wt and PP-sw cells (Fig. 2A). Both PP-wt and PP-sw cells
produced abundant N-glycosylated PP during the 10-min
labeling reaction (time 0, Fig. 2A). At 1 h of the
chase, higher molecular weight N+O-glycosylated
PP molecules were seen in addition to the N-glycosylated
forms. Full-length PP decreased by 2 h; and by 4 h, little
full-length PP remained (Fig. 2A).
PP turnover, PP species, and precursor product relationship of 12-kDa
fragments and A in CHO cells stably transfected with
PP. Panel A, turnover of full-length
PP immunoprecipitated with antibody C7 from PP-wt and
PP-sw cells pulse-labeled for 10 min with
[S]methionine and chased for 0, 1, 2, or 4 h. Panel B, immunoprecipitation of PP from
conditioned medium with antibodies B5, R1736, and 6E10 from PP-wt
and PP-sw cells labeled for 4 h with
[S]methionine. R1736 and 6E10 are specific for
-secretase-cleaved PP. Panel C,
COOH-terminal fragments and A release from PP-sw cells
following a 10-min pulse with [S]methionine and
10- or 20-min chase. The COOH-terminal fragments were
immunoprecipitated with antibody C7 from cell lysates after the 10- or
20-min chase. The 12-kDa fragments (at arrowhead),
clearly apparent by 10 min, increased by 20 min. Media from parallel
cultures immunoprecipitated with antibody R1282 show an A (at arrowhead) signal by 20 min. No A signal is observed at
10 min even when the signal is intentionally amplified as in the lanes on the right. For comparison, the unamplified
A image is presented to the left of the darkened
image. Molecular weights determined from prestained standards are
indicated. wt = PP-wt cells; sw =
PP-sw cells.
PP species has been reported from a
PP chimeric molecule expressing the ``Swedish'' mutation (31) . To confirm this finding with authentic PP
molecules, PP was immunoprecipitated from conditioned
media using B5 antibody, which recognizes both - and
-secretase species of PP, and two antibodies that
recognize only -secretase-cleaved PP (R1736 and
6E10). As observed for untransfected CHO cells (not shown),
PP from transfected CHO cells migrates as a doublet of
bands on low percentage polyacrylamide gels. As a result, the higher
molecular weight PP cleaved by -secretase and the
slightly lower molecular weight PP cut by
-secretase can best be compared by observing the lower of the two
bands of each doublet (Fig. 2B). The PP from PP-sw cells migrated at an M
consistently lower than that of PP-wt cells, indicating the
secretion of a shorter PP species. Although both cell
lines secreted comparable levels of total PP by B5
antibody immunoprecipitation (Fig. 2B), PP-sw
cells had dramatically reduced levels of -secretase-cleaved
PP (6 ± 1.3-fold less) than PP-wt cells
using antibodies R1736 and 6E10 (Fig. 2B). Consistent
with this finding, and as reported by
others(12, 31, 32, 33) , PP-sw
cells also had correspondingly higher levels of 12-kDa
COOH-terminal PP fragments (see below).Timing of Secretion of
Since the
turnover rate of full-length PP, A, and
p3 Is Identical in PP-wt and PP-sw CellsPP was essentially the same in the
PP-wt and PP-sw cells, we next examined the biosynthetic rate
for the generation of PP-secreted products (PP,
A, and p3). Following a 10-min pulse labeling, media were
collected from a single dish each from PP-wt and PP-sw cells
and reapplied at 10-min intervals to define the incremental release of
PP secretion products during the 1st h of the chase period.PP was first detectable at
10 min as determined with B5 immunoprecipitation (Fig. 3B). However, at this first time interval only
minute amounts of PP were secreted from both
PP-wt and PP-sw cells because the signal could be seen in the
10-min lane only after prolonged autoradiographic exposures (Fig. 3B). PP became pronounced at 20
min for both cell lines with peak secretion at approximately 30 min (Fig. 3A and Fig. 4). The profile of
PP secretion as a function of time was essentially
identical for the two cell lines (Fig. 4). In the experiment
shown, although PP secretion by PP-sw cells was
lower because of diminished expression of full-length PP, the
profile of secretion is essentially identical to that of PP-wt
cells. This profile of PP secretion did not depend on
the level of PP expression because other wild type and Swedish
cell lines exhibited the same patterns of release (not shown).
Furthermore, comparison of PP-wt and PP-sw cells that
expressed equivalent levels of PP confirmed that PP secretion by both cell lines was essentially the same (within
10% of each other as determined by Phosphorimage analysis of media
from triplicate cultures from each cell line, Student's t test, p = 0.49).
PP, A, and p3 from CHO cells transfected with
wild type or K670N/M671L mutant PP.
Immunoprecipitations of PP, A, and p3 from
conditioned chase media from single cultures of PP-wt and
PP-sw cells following a 10-min
[S]methionine pulse label were collected at
10-min intervals. The level of PP holoprotein expression was
somewhat lower in PP-sw cells in this experiment. On low
percentage polyacrylamide gels, PP from CHO cells
migrates as a doublet. Panel A, total PP was
immunoprecipitated with antibody B5. Note the lower molecular weight
species of PP, indicated by the arrowhead,
from PP-sw cell media. Panel B, the presence of
PP at 10 min is confirmed by this long exposure of the
gel shown in panel A. The shortened PP form,
at the arrowhead, is apparent at the earliest time point. Panel C, A and p3 immunoprecipitated by R1282 from the
same media as panels A and B. Positions of A and
p3 are indicated. Molecular weights determined from prestained
standards are indicated on the right. wt =
PP-wt cells; sw = PP-sw
cells.
PP, A,
and p3 release from CHO cells transfected with wild type or K670N/M671L
mutant PP. Data from Phosphorimage analysis of gels
in Fig. 3represent the percent secretion for each time point
relative to the cumulative (100%) secretion during the entire 60-min
chase. The top panel shows the PP release
from PP-wt (designated by the solid line and circles in all graphs) and PP-sw cells (designated by the dotted
line and triangles in all graphs) from antibody B5
immunoprecipitation. A (middle panel) and p3 (bottom
panel) release from PP-wt and PP-sw cells,
immunoprecipitated with R1282 antibody, are also shown. wt = PP-wt cells; sw = PP-sw
cells.
release, the
timing of A secretion during the 1st h from PP-wt and
PP-sw cells was also identical (Fig. 3C and Fig. 4). The A signal was first apparent at the 20-min
collection time by autoradiography (Fig. 3C) and
reached a peak at 30-40 min. Although no discernible A
signal was ever seen on either autoradiograms or Phosphorimages at the
10-min chase time, after long exposures a few Phosphorimage counts
higher than background were detected in the 10-min lane (Fig. 4). At each chase time, PP-sw cells consistently
released more A than PP-wt cells. The timing of p3 secretion
mirrored that of A in both PP-wt and PP-sw cells
throughout the chase period (Fig. 3C), although
PP-wt cells consistently released more p3 relative to A than
did PP-sw cells. Authentication of A (beginning at
Asp) and p3 (beginning at Lys
) was obtained by
radiosequencing (not shown), as reported
previously(15, 19) . Thus, a difference in the ratios
of -secretase- and -secretase-generated molecules was also
reflected by the levels of p3 and A released by these cell lines.
Finally, the formation of the -secretase-generated 12-kDa
PP COOH-terminal fragments preceded the release of A from
pulse-labeled PP-sw cells (Fig. 2C). After a
10-min labeling with [S]methionine, the
12-kDa fragment was apparent by the 10-min chase time in
PP-sw cells and increased at 20 min (Fig. 2C).
Consistent with the above results, A was not apparent in the
corresponding media until 20 min of the chase period (Fig. 2C). This earlier generation of the 12-kDa
fragment prior to A release, consistently seen in three
experiments, indicates a precursor-product relationship between the two
molecules.
The release
of PP and A Appear to Be Present
Intracellularly in PP-wt and PP-sw CellsPP at very early chase times suggested that PP
may be cleaved by -secretase in the secretory pathway. To
determine if soluble PP was present intracellularly,
metabolically labeled cells were treated with 0.1% saponin in buffer.
Saponin is a mild detergent that permeabilizes cells but does not
solubilize the lipid bilayer (29) and therefore allowed the
intracellular PP to diffuse into the buffer.
Essentially no full-length PP was detected in the saponin buffer
of treated cells. However, soluble intracellular PP was recovered from the saponin buffer from both PP-wt and
PP-sw cells (Fig. 5, A and B).
Intracellular PP species from PP-wt cells (a
finding previously reported by others; see (34, 35, 36) ) migrated with an M consistent with -secretase-cleaved
molecules (Fig. 5A). A shorter PP species with an M identical to secreted
PP and consistent with -secretase-cleaved
molecules was observed from PP-sw cells (Fig. 5A; (32) ). Furthermore, in pulse-chase experiments a
precursor-product relationship could be demonstrated between
intracellular PP from the saponin-treated cells and
PP secreted into the medium (Fig. 5B).
PP and A from CHO cells transfected with wild type or
K670N/M671L mutant PP. Panel A, B5 antibody
immunoprecipitation of PP from chase media and saponin
buffers of PP-wt and PP-sw cells pulse-labeled for 10 min
with [S]methionine and chased for 20 min. Since
PP from CHO cells migrates as a doublet, the higher
molecular weight PP cleaved by -secretase (
at arrow) and the slightly lower molecular weight
PP cut by -secretase ( at arrow)
can best be appreciated by observing the lower of the two
bands. The shorter PP species is observed both
intracellularly (intra) and secreted into the medium (sec) of PP-sw cells. The faint bands that run
below 97 kDa in the saponin lanes are degradation products.
Molecular weights determined from prestained standards are indicated on
the right. Panel B, antibody B5 immunoprecipitations
of intracellular and secreted PP from PP-wt and
PP-sw cells following a 10-min pulse with
[S]methionine and 10-60-min chase.
Intracellular PP was immunoprecipitated from saponin
buffers; secreted PP was obtained from chase media. Panel C, immunoprecipitation of PP-wt and PP-sw
control (cont) cell lysates after 4 h
[S]methionine labeling with antibody R1280 or
R1280 that had been preabsorbed (abs) with the A
1-40 peptide. The positions of the 12-kDa COOH-terminal
fragments and A are indicated at arrows on the left. wt = PP-wt cells; sw = PP-sw cells.
can be formed within the
secretory pathway. Indeed, intracellular A appeared to be present
in both PP-wt and PP-sw cell lysates labeled for 4 h (Fig. 5C). Preabsorption of R1280 antibody with the
A 1-40 peptide totally eliminated immunoprecipitation of
A from the cell lysates by R1280 antibody (Fig. 5C), and no 4-kDa band was observed from the same
lysate using antibody C7. Treatment with trypsin prior to
immunoprecipitation did not diminish the A signal (not shown),
indicating that A was present inside the cells. In addition, cells
pulse labeled with [S]methionine followed by a
20-min or 30-min chase had both A and p3 isolated from cell
lysates (not shown). Thus, the immunoprecipitated A had not been
derived from secreted molecules present on the extracellular plasma
membrane at the time of cell lysis. Furthermore, the appearance of
these intracellular A and p3 molecules after short pulse-chase
intervals provides indirect evidence of their production in the
secretory and not the endosomal/lysosomal pathway. Nevertheless, A
and p3 bands were visualized only after 8-10 weeks of
autoradiographic exposure, suggesting that only very low levels of
A were ever present intracellularly. The minute amounts of
intracellular A precluded definitive identification by amino acid
radiosequencing.
To determine
if the endocytic pathway contributed to APP from the Cell Surface Contributes to A
Production in Both PP-wt and PP-sw Cells production in PP-sw
cells, release of A was analyzed after selective cell surface
radioiodination. Consistent with an earlier report(19) , little
radiolabeled A was secreted within the first 10 min by either cell
line, but considerable A was released by 2 h from both PP-wt
and PP-sw cells (Fig. 6A), with PP-sw cells
releasing greater than 2-fold more A than PP-wt cells at the
2-h collection time (2.4 ± 0.4). The timing of A release
following labeling of cell surface PP was essentially the same in
the two cell lines (Fig. 6A). However, in sharp
contrast to the timing of A secretion, the majority of the
iodinated PP was released within the first 5 min of
incubation at 37 °C from both PP-wt and PP-sw cells (Fig. 6B). In addition, these profiles of both A
and PP release are distinctly different from the
timing observed for A and PP release observed
using [S]methionine labeling ( Fig. 3and Fig. 4).
and PP from cell surface-iodinated PP molecules. Panel A,
immunoprecipitation of A with antibody R1280 from chase media of
PP-wt and PP-sw cells following iodination of cell surface
PP. The timing of release of A was the same from both cell
lines. Molecular weights determined from prestained standards are
indicated. Panel B, rapid release of PP was
observed from both PP-wt and PP-sw cells after surface
iodination and immunoprecipitation by antibody B5. Panel C,
immunoprecipitation of cell lysates with antibody C7 after iodination
revealed more full-length PP on the surface of PP-wt cells
than PP-sw cells. PP-sw cells, however, had more iodinated
12-kDa COOH-terminal fragments and fewer 10-kDa fragments
than PP-wt cells. wt = PP-wt cells; sw = PP-sw cells.
PP derived from cell surface
PP by PP-sw cells had an M compatible
with -secretase-cleaved PP (Fig. 6B). A lower M -secretase-cleaved PP species was not
readily apparent after surface labeling. However, resolution of the
labeled bands is significantly less distinct from an iodine signal
because of radiographic intensification, and minor differences may be
undetectable. Second, we consistently observed more full-length PP
on the surface of PP-wt cells than PP-sw cells (Fig. 6C) expressing the same amount of PP. To
confirm and quantitate this difference, the levels of cell surface and
total PP were measured by an antibody binding assay using
radioiodinated antibody 5A3 Fab fragments, which bind to an
extracellular PP epitope(19) . Treatment with 0.1% saponin
permitted labeling of both cell surface and intracellular PP.
Multiple repetitions of this experiment showed that PP-sw cells
had approximately 50% less cell surface PP than PP-wt cells
(49.8% ± 0.7, p < 0.0001). Interestingly, PP-sw
cells showed more of the COOH-terminal 12-kDa fragment and less of
the 10-kDa fragment than PP-wt cells (Fig. 6C) present on the cell surface.Drug Treatments Affect
To assess further whether the processing of PP-wt and PP-sw Cells
SimilarlyPP
molecules is similar in PP-wt and PP-sw cells, both cell
lines were treated with a variety of compounds known to affect protein
processing. Control lanes reveal the higher production of both A
and 12-kDa COOH-terminal PP fragments from PP-sw cells (Fig. 7A). Treatment with brefeldin A, a drug that
blocks the maturation of proteins by collapsing the Golgi into the
endoplasmic reticulum, inhibited the production of both A and the
12-kDa fragment in both PP-wt and PP-sw cells (Fig. 7A). Treatments that alkalinize intracellular
vesicles were also used because A generation in cultured cells
appears to require an acidic compartment(8, 13) . To
determine if -secretase cleavage of both PP-wt and PP-sw
cells occurs in an acidic intracellular compartment, cells were exposed
to chloroquine or bafilomycin A1(37) . In response to
chloroquine presented during the 2-h chase, both PP-wt and
PP-sw cells released 30%-60% less A than untreated controls (Fig. 7A). As anticipated(10, 13) ,
chloroquine also dramatically elevated the level of COOH-terminal
fragments in both cell lines (Fig. 7A). Exposure of
cells to bafilomycin A1, a drug that specifically inhibits vacuolar
H
-ATPases and thus prevents vesicular
acidification(37) , produced a 60% decrease in A
release from both PP-wt and PP-sw cells (as measured by
Phosphorimaging, Fig. 7A). A corresponding increase in
10-kDa COOH-terminal fragments was observed following exposure to
bafilomycin A1 (Fig. 7A), suggesting that less PP
was cleaved by -secretase in the presence of the drug. Consistent
with this interpretation is the finding of a dramatic decrease in the
amount of 12-kDa fragments in PP-sw cells when bafilomycin
was presented using a short pulse-chase paradigm (Fig. 7B). This reduction of the 12-kDa fragment (Fig. 7B) was seen at a time when abundant 12-kDa
COOH-terminal fragments were normally observed from PP-sw cell
lysates following a 10-min pulse-labeling (Fig. 2C and Fig. 7B). In sum, the processing of PP and release
of A by CHO cells expressing either wild type or mutant PP
were similarly affected by these drug treatments.
production and COOH-terminal fragments from CHO cells transfected with
wild type or K670N/M671L mutant PP. Panel
A, immunoprecipitation of PP-wt and PP-sw conditioned
media with antibody R1282, and cell lysates with antibody C7 from a 2-h
[S]methionine label followed by a 2-h chase
containing either no drug (Cont), brefeldin A (Bref),
chloroquine (Cq), or bafilomycin A1 (Balfilo). Less
A was released in the presence of drugs compared with the control
condition for both cell lines. Panel B, antibody C7
immunoprecipitation of PP-sw cell lysates after a 10-min
[S]methionine pulse followed by a 20-min chase
in the absence (0) or presence (.25) of bafilomycin
A1. Note the presence of the 12-kDa COOH-terminal fragment
generated by -secretase cleavage at 20 min in the control lane (0) and its near absence after bafilomycin A1 treatment. wt = PP-wt cells; sw =
PP-sw.
PP gene from a Swedish kindred
with familial Alzheimer's disease is invariably linked with
Alzheimer's disease(21) . All cells reported to date
which express the PP mutation produce dramatically more A
peptide than do cells expressing wild type
PP(12, 22, 23, 31, 32, 33) .
Since excess A production may be causally related to the
Alzheimer's phenotype in individuals affected with the
``Swedish'' mutation(21) , it is important to
evaluate the mechanism by which A is produced from PP with
this alteration. In this study we performed a detailed analysis of the
biosynthetic processing of PP in PP-wt and PP-sw CHO
cells.PP-sw cells
released substantially more A than PP-wt cells.
Interestingly, the timing of onset and the duration of A secretion
during the 1st h following a short pulse labeling were coincident with
p3 release for both cell lines. Only the amounts of A and p3
varied between PP-wt and PP-sw cells. Furthermore, treatments
known to decrease A in PP-wt cells (8, 13, 38) also affected PP-sw cells.
We interpret our data to suggest that the pathway of A production
is similar for PP-wt and PP-sw cells. In contrast, however,
the timing of A release differed substantially depending on
whether cells were [S]methionine-labeled or
surface-iodinated. In both cell lines A was released with a
shorter time course from [S]methionine-labeled
cells than from cells that were surface-iodinated. This difference in
the timing of A secretion leads us to propose that A is
generated in both the secretory and endocytic pathways from both
PP-wt and PP-sw cells. is generated in the secretory
pathway(10, 39) . First, the timing of secretion of
PP, A, and p3 was essentially identical at early
chase times in both cell lines. Specifically, within the first 30 min
in a short pulse-chase experiment, the profiles of PP,
A, and p3 secretion were remarkably similar. This chase paradigm
was chosen specifically to reveal the incremental release of these
early secretory products. Second, permeabilization of
[S]methionine pulse-labeled cells followed by
immunoprecipitation with a PP midregion antibody (B5) showed that
intracellular soluble PP was present in both
PP-wt (34, 35, 36) and PP-sw cells
as reported previously(32) . The major intracellular species of
soluble PP from PP-sw cells had a lower M than PP from PP-wt cells,
consistent with production by -secretase cleavage. Significantly,
intracellular PP was present before abundant
PP was secreted into the culture medium, thus
demonstrating a precursor-product relationship. Third, the 12-kDa
COOH-terminal fragment of PP and A showed a precursor-product
relationship, with the 12-kDa molecules apparent 10 min prior to
the appearance of A. Moreover, consistent with a recent
report(12) , this COOH-terminal 12-kDa fragment was
specifically increased in PP-sw cells compared with PP-wt
cells. Fourth, our data suggest that intracellular A is present in
both PP-wt and PP-sw cells. Based on results from trypsin
digestion using a short pulse-chase paradigm, A in cell lysates
did not appear to represent extracellular A attached to the cell
surface or to be derived from the lysosomal pathway. However, the
exceedingly small amount of intracellular A suggests that A
turnover and secretion are rapid. This is consistent with the earlier
postulation that A is released from cells soon after it is formed
and suggests that -secretase cleavage occurs at or near the cell
surface(19) . Previously, intracellular A has only been
detected in neurons(40) . Thus our preliminary findings suggest
that the pathways of A production in neurons and non-neuronal
cells may be more similar than was previously thought.PP, cells labeled by selective cell
surface iodination confirmed that PP-sw cells produced more A
from cell surface precursors than did PP-wt cells. However, the
timing of A release after surface labeling was essentially
identical for both PP-wt and PP-sw cells. As shown previously
for cells expressing wild type PP(19) , A generated
from surface-labeled molecules was released more slowly than
PP by PP-sw cells. These profiles of A and
PP release from surface-labeled molecules are
dramatically different from the [S]methionine
labeling experiments in which A and PP were
released simultaneously. Interestingly, PP and A
release from [S]methionine pulse-chase
experiments showed that PP secretion peaked at 30
min followed by a sharp decrease, whereas A release continued at
the same level until later chase times. We interpret the sustained
A release into the medium at a time when PP secretion decreased (40-50 min) to represent the addition
of newly generated A, derived from the endocytic pool, after the
contribution of the secretory pool of A has peaked. Therefore, our
data indicate that A can be derived from both the secretory and
endocytic pathways and that more A is formed within each pathway
by PP-sw cells.PP processing between PP-wt and PP-sw
cells. First, the timing of secretion of PP, A,
and p3 is essentially the same for both cell lines within the 1st hour
following a 10-min pulse label. Second, various drug treatments
decrease A in both PP-wt and PP-sw CHO cells. Third,
intracellular PP species and A appear to be
present in both cell lines. Fourth, both secretory and endocytic
pathways appear to contribute to A generation and release. Fifth,
both PP-wt and PP-sw cells secrete primarily
-secretase-cleaved PP from surface-labeled
PP. Thus, within the limits and sensitivity of our experimental
system, the timing and the pathway of A secretion appear to be
identical in PP-wt and PP-sw cells. Only the amounts of
A and -secretase-cleaved precursors differed in PP-wt
and PP-sw cells. Our data and interpretation are therefore
consistent with the results of previous investigators who have
suggested that the ``Swedish'' mutation at the NH terminus of A enhances -secretase
cleavage(12, 22, 23) . This altered
-secretase cleavage produces abundant -secretase-cleaved
PP in the secretory pathway in PP-sw cells,
leading to excess A production. However, it remains unclear at
present which pathway, secretory or endocytic, plays the greater role
in A production.PP-sw cells did show some differences in
PP processing from PP-wt cells. In addition to the increase
in -secretase-cleaved products described above, there was a 50%
reduction in the amount of cell surface PP in PP-sw cells.
Concomitantly, there was an increase in the 12-kDa
membrane-retained PP fragments present on the cell surface of
PP-sw cells. Whether this increase in 12-kDa fragments is
sufficient to account for the decrease in full-length PP molecules
at the cell surface of PP-sw cells is unclear. Because secreted
PP levels are similar between PP-wt and
PP-sw cells, the reduction in full-length PP at the surface
of PP-sw cells suggests that the amount of PP targeted to the
cell surface may represent a minor fraction of the total PP
processed in the secretory pathway. Otherwise, one would expect to see
a substantial increase in PP released into the medium
from PP-sw cells, which was not detected. Furthermore, this
interpretation is also consistent with reports of other cell types that
express little or no PP on the cell
surface(34, 35, 36) . generation in both
PP-wt and PP-sw cells. The increased A production from
PP-sw cells appears to result from enhanced -secretase
cleavage of the mutant PP in both the secretory and endocytic
pathways. A recent report has demonstrated altered PP processing
in mutant PP molecules with natural or designed mutations in codon
692(41) , whereas another report demonstrated an increased
percentage of longer A peptides from PP with codon 717
mutations (42) . Taken together, it appears that FAD PP
mutations lead to pleiotropic effects on PP and A metabolism.
The Alzheimer phenotype associated with these dominant mutations may
therefore result from different cellular perturbations that
specifically modify PP processing.
), amyloid -protein; PP, -amyloid precursor
protein; PP, soluble PP; PP-sw,
``Swedish'' mutant PP; PP-wt, wild type PP;
CHO, Chinese hamster ovary; DMEM, Dulbecco's modified
Eagle's medium; MMAb, monoclonal antibodies 5A3 and 1G7 used
together; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
DPBS, Dulbecco's phosphate-buffered saline.
We thank Dr. Martin Citron for the PP K670N/M671L cDNA and for helpful discussions, Dr. Margaret Kruse
and Deborah Watson for critical reading of the manuscript, and Dr.
Dennis Selkoe for the generous contribution of various PP
antibodies. recently have been reported by Martin et al. (Martin, B. L., Schrader-Fischer, G., Busciglio, J., Duke, M.,
Paganetti, P., and Yankner, B. A.(1995) J. Biol. Chem.270, 26727-26730) in cells transfected with Swedish mutant
[Abstract/Full Text]
PP.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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