From the
Deposition of
Amyloid precursor protein (APP)
In previous efforts, we and
others
(7, 8) have examined the maturation and sorting
of human APP in Madin-Darby canine kidney (MDCK) cells, a prototypic
polarized epithelial cell line, and demonstrated that full-length APP
is sorted to the basolateral compartment. Furthermore, C-terminally
truncated APP derivatives are secreted into the basolateral medium.
Because APP and APLP2 mature through a similar secretory/cleavage
pathway in non-polarized cells, we were prompted to investigate the
sorting of APLP2-751, a CS proteoglycan (PG), and APLP2-763
in MDCK cells. We demonstrate that both APLP2-751 and
APLP2-763 are directly sorted to the basolateral surface of MDCK
cells, releasing
To generate stable cell lines that express either mouse
APLP2-751 or mouse APLP2-763, MDCK cells were transfected
with CsCl-purified plasmid DNA using a high efficiency calcium
phosphate method
(10) . After selection in medium containing 500
µg/ml G418 (Life Technologies, Inc.), stable transfectants were
assayed for steady-state expression of the transgene by Western blot
analysis of cell lysates using D2-I antibody
(5) .
For pulse-chase polarity assays, cells growing on Transwell units
were starved for 30 min and labeled with 200 µCi of
[
For immunoprecipitation
analysis, aliquots of the media and detergent-soluble lysates were
incubated with D2-I or Myc-I antiserum, respectively, in
immunoprecipitation buffer. Immune complexes were collected with
immobilized protein A-agarose beads (Pierce) and analyzed by
SDS-polyacrylamide gel electrophoresis and autoradiography.
Quantitation was performed using a Molecular Dynamics (Sunnyvale, CA)
PhosphorImager.
Expression and Post-translational Modification of Mouse
APLP2-751 and Mouse APLP2-763 in MDCK Cells-To
examine the metabolism of APLP2-751 and APLP2-763, we
generated MDCK cells stably transfected with cDNAs encoding
APLP2-751 and APLP2-763. The transgenes were epitope-tagged
at the extreme C terminus in order to distinguish the transgene-encoded
polypeptides from endogenously synthesized APLP2-751 and/or
APLP2-763. Three independent clones expressing each APLP2 isoform
were examined by polarity assays. Cells were plated onto Transwell
units, induced with butyrate, and labeled for 2 h with
[
APP, the precursor protein of A
Our studies also demonstrate that
APLP2-763, an isoform that is not modified by CS GAG chains, is
trafficked to the basolateral compartment in a manner similar to
APLP2-751. Hence, we conclude that intracellular trafficking and
appearance of APLP2 on the basolateral cell surface are mediated by
signals contained within the core protein, independent of the CS GAG
chain. The specific sequences that mediate APLP2 sorting in MDCK cells
have not yet been determined. However, recent studies indicate that
specific tyrosine-containing signals contained within the APP
cytoplasmic domain are essential for basolateral targeting of the
parent molecule
(15) . Considering the high degree of homology
throughout the cytoplasmic domains of APP and APLP2, we speculate that
APLP2 trafficking and targeting to the basolateral surface are mediated
by signals contained within the cytoplasmic domain.
Our observation
that the CS GAG chain has little, if any, influence on the targeting of
the APLP2 core protein in polarized cells is unique. On the other hand,
studies of the invariant chain (Ii), a nonpolymorphic glycoprotein that
associates with the major histocompatibility complex class II molecules
during antigen presentation, reveal that CS GAG chains are an important
requirement for intracellular trafficking and cell surface expression
of the core protein in non-polarized cells. The predominant
localization of Ii is in endosomal vesicles, a localization specified
by sequences within the cytoplasmic N terminus
(16, 17) .
However, a fraction (2-5%) of Ii is expressed at the cell
surface
(18) , and these cell surface Ii molecules are modified
by the addition of CS GAG at a single serine
residue
(19, 20) . The presence of CS GAG-modified Ii on
the cell surface indicates that the glycosaminoglycan moiety is a
dominant signal that overrides the endosomal sorting signal.
The
basolateral trafficking of APLP2 may also be related to one of the
typical pathological features in Alzheimer's disease (i.e. the deposition of amyloid fibrils in cerebral microvessels).
A
We thank Dr. Philip Wong for providing Myc-I antiserum
and Dr. Mike Stinski for providing the pCB6 vector. We also thank Drs.
David Borchelt and Ora Weisz for helpful comments and discussions.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-amyloid peptide in senile plaques is a
principal neuropathological hallmark of Alzheimer's disease.
-Amyloid peptide is derived from larger amyloid precursor
proteins. Amyloid precursor protein is a member of a family of integral
membrane glycoproteins that includes amyloid precursor-like protein
(APLP) 1 and 2. Alternatively spliced pre-mRNAs encode several APLP2
isoforms; the APLP2-751 isoform is a substrate for modifications
by a chondroitin sulfate glycosaminoglycan (CS GAG) chain, whereas the
APLP2-763 isoform does not undergo CS GAG modification. In this
report, we have examined the sorting and metabolism of APLP2-751
and APLP2-763 in polarized epithelial Madin-Darby canine kidney
(MDCK) cells. We demonstrate that, despite differences in
post-translational modifications, both the APLP2-751 proteoglycan
and APLP2-763 isoform were targeted and secreted to the
basolateral compartment of MDCK cells. We document that the kinetics of
intracellular maturation of full-length forms and secretion of soluble
derivatives generated from each isoform were indistinguishable. Our
results are consistent with the view that, in MDCK cells, the CS GAG
chain of APLP2 has little influence on intracellular trafficking and
that the principal basolateral targeting determinants are likely to
reside in the APLP2 core protein.
(
)
is a
member of a family of integral membrane glycoproteins that includes
amyloid precursor-like proteins (APLP) 1
(1) and
2
(2, 3, 4) . We have demonstrated that, in
cultured cells of non-neuronal or neuronal origin, mouse APLP2, like
APP, matures through the constitutive secretory
pathway
(4, 5) . We and others
(2, 4, 6) have also shown that APLP2 is encoded by several
alternatively spliced transcripts. One isoform, APLP2-751, is
modified by the addition of a chondroitin sulfate (CS)
glycosaminoglycan (GAG) chain at a single serine residue (Ser-614) in
the ectodomain
(5) . Interestingly, the APLP2-763 isoform
contains an additional 12-amino acid sequence, presumably encoded by an
alternatively spliced exon, inserted two amino acids N-terminal to
Ser-614 of APLP2-751. Insertion of this 12-amino acid peptide
abolishes CS GAG modification of the APLP2-763
isoform.
(
)
95% of the total soluble derivatives into the
basolateral compartment. Furthermore, the kinetics of secretion of
soluble derivatives generated from either isoform is indistinguishable.
Thus, the CS GAG chain has little influence on trafficking of the core
protein in MDCK cells, and we argue that the principal basolateral
targeting information resides within the core protein itself.
Antibodies
The following antibodies were used in
the experiments. D2-I antiserum was raised against a mouse
APLP2-751 polypeptide synthesized in Sf9 cells using a
baculovirus expression system
(5) . Myc-I antiserum was raised
against a synthetic peptide, MEQKLISEEDLN, corresponding to a region of
the c-Myc oncoprotein
(9) .
Plasmid Construction
To distinguish
transgene-encoded APLP2 from endogenous APLP2, sequences encoding a
region of the human c-Myc oncoprotein (MEQKLISEEDLN) were engineered
into the extreme C terminus of either mouse APLP2-751 or
APLP2-763 cDNA. Sequences encoding Myc-tagged mouse
APLP2-751 were excised from pSVAPLP2-751myc
(5) and
subcloned downstream of the cytomegalovirus promotor in the pCB6 vector
containing a gene that encodes resistance to the neomycin analog G418
(a kind gift of Dr. Mike Stinski, University of Iowa, Iowa City, IA).
To generate expression plasmid pCB6APLP2-763myc, sequences
encoding a Myc-tagged mouse APLP2-763 were excised from
pSVAPLP2-763myc with EcoRI and
XbaI. The gel-purified insert was subcloned into pBSII
KS+ (Stratagene, La Jolla, CA) previously digested with
EcoRI and XbaI to generate pBSAPLP2-763myc.
Plasmid pBSAPLP2-763myc was subsequently digested with
KpnI and XbaI, and the
2.2-kilobase fragment was
subcloned into pCB6 vector containing KpnI and XbaI
ends to generate pCB6APLP2-763myc.
Cell Culture and Transfection
MDCK cells (type II,
ATCC CCL 34) (Rockville, MD) were maintained in Dulbecco's
modified Eagle's medium (Life Technologies, Inc.) supplemented
with 10% heat-inactivated fetal bovine serum (Life Technologies, Inc.),
penicillin (100 units/ml), and streptomycin (100 µg/ml) (complete
medium).
Cell Labeling
For polarity assays, MDCK cells (1.5
10
cells) were plated onto 24-mm clear Transwell
filters (Costar Corp., Cambridge, MA) and grown for 5 days. Transgene
expression was induced with 10 mM butyrate at least 18 h
before each experiment. Prior to labeling, the integrity of the tight
junctions in the monolayer was assessed using a
[
H]inulin test as described
previously
(8) . Filter-grown cells were labeled with 500 µCi
of [
S]methionine (DuPont NEN) for 2 h in
methionine-free Dulbecco's modified Eagle's medium
supplemented with 10% dialyzed fetal bovine serum. At the end of the
labeling period, apical and basolateral media were collected and
centrifuged at 14,000 rpm for 1 min to remove particulate materials. To
prepare detergent-soluble lysates, the filters were excised from the
Transwell units and washed with phosphate-buffered saline. Cells were
lysed in immunoprecipitation buffer containing freshly added protease
inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 10 µg/ml
aprotinin, 5 µg/ml leupeptin, 5 µg/ml pepstatin A)
(11) .
S]methionine for 10 min. At the end of the
labeling period, cells were rinsed once with medium containing 1
mML-methionine. One Transwell of cells was
immediately harvested and lysed in immunoprecipitation buffer; and the
remaining Transwell units were incubated for various time periods in
methionine-free Dulbecco's modified Eagle's medium
supplemented with 10% heat-inactivated fetal bovine serum, 10
mM butyrate, and 1 mML-methionine. After
each chase period, apical and basolateral media were collected; cells
were harvested and lysed as described above.
Chondroitinase Digestions
Chondroitinase
digestions were performed as described previously
(5) . Briefly,
biosynthetically labeled APLP2-751 or APLP2-763 was
immunoprecipitated in duplicate with Myc-I antiserum.
Immunoprecipitates were collected with protein A-agarose beads. One set
of the resulting immunoprecipitates was then subjected to digestion
with 0.1 unit of chondroitinase AC (Sigma) in 100 µl of 100
mM Tris-HCl (pH 7.0), 30 mM sodium acetate, and a
protease inhibitor mixture (10 µg/ml aprotinin, 5 µg/ml
leupeptin, 5 µg/ml pepstatin A, and 0.5 mM
phenylmethylsulfonyl fluoride) at 37 °C for 1 h. The other set of
immunoprecipitates was mock treated by incubation at 37 °C in
digestion buffer lacking chondroitinase AC.
Biotinylation
MDCK cells plated on Transwell
filters were metabolically labeled with 500 µCi of
[S]methionine for 1 h. At the end of the
labeling period, the filters were washed with ice-cold
phosphate-buffered saline containing CaCl
and
MgCl
. Cell surface molecules on duplicate filters were
biotinylated on ice with 0.5 mg/ml sulfosuccinimidyl
2-(biotinamido)ethyl-1,3-dithiopropionate/biotin (Pierce) in 10
mM borate buffer (pH 9) as described
(12) from either
the apical or basolateral side. After the biotinylation, filters were
rinsed three times with ice-cold complete medium, incubated in ice-cold
complete medium for 5 min, and then excised. Cells were lysed and
processed as described above. Cellular APLP2 molecules were collected
by immunoprecipitation with Myc-I antiserum. One-twentieth of the
resulting immunoprecipitates was analyzed directly, whereas the
remaining fraction was reimmunoprecipitated with immobilized
streptavidin-agarose beads (Pierce) to isolate cell surface forms of
APLP2 as described previously
(8) . Myc-I-immunoprecipitated
APLP2 and streptavidin-bound APLP2 were fractionated by
SDS-polyacrylamide gel electrophoresis. The levels of surface-bound
APLP2 molecules were quantitated using a PhosphorImager.
S]methionine. Detergent-soluble cell lysates
were immunoprecipitated with Myc-I antiserum (Fig. 1a).
In all clones expressing APLP2-751, Myc-I antiserum
immunoprecipitated a full-length APLP2-related
120-kDa protein and
a heterogeneous set of polypeptides migrating between 130 and 160 kDa.
This pattern is similar to that observed in Chinese hamster ovary cells
expressing Myc-tagged mouse APLP2-751, wherein an
120-kDa
immature form matured to a set of heterogeneous polypeptides migrating
between 120 and 200 kDa
(5) . Thus, it is likely that the
polypeptide of
120 kDa in MDCK cells represents the immature form
of APLP2-751 that is subject to post-translational modifications.
We recently showed that APLP2-751 is modified by a CS GAG
chain
(5) . To address whether MDCK-synthesized APLP2-751
is also a substrate for CS GAG addition, we immunoprecipitated
full-length APLP2-751 molecules with Myc-I antiserum in
duplicate. One set of the immunoprecipitates was treated with
chondroitinase AC, an enzyme that cleaves CS GAG chains from core
proteins. As shown in Fig. 1b, chondroitinase AC
treatment of the Myc-I immunoprecipitates from cell line B17 expressing
APLP2-751 converted the heterogeneous population of
130-160-kDa molecules seen in the undigested sample
(Fig. 1b, lane 1) to a single band migrating at
130 kDa (Fig. 1b, lane 2), reflecting the
removal of variable-length CS GAG chains from the APLP2-751 core
protein. PhosphorImager analysis revealed that the radioactivity
contained within the
130-kDa band (Fig. 1b,
lane 2) was essentially identical to that contained within the
heterogeneous polypeptides of 130-160 kDa in Fig. 1b (lane 1). Therefore, in MDCK cells, CS GAG modification
of APLP2-751 occurs in a fashion similar to that shown in
non-polarized Chinese hamster ovary and COS-1 cells.
Figure 1:
Expression and CS GAG modification of
APLP2 in MDCK cells. a, filter-grown MDCK cells stably
transfected with cDNA encoding either mouse APLP2-751 or
APLP2-763 were labeled for 2 h with
[S]methionine. Myc-tagged exogenous APLP2
molecules were immunoprecipitated from detergent-soluble cell lysates
with Myc-I antiserum. Lanes 1-3 represent
immunoprecipitates from three independent clones expressing
APLP2-751 (751). Note the heterogeneity in the migration
pattern of the mature forms marked by the bracket; lanes
4-6 represent Myc-I immunoprecipitates from three
independent clones expressing APLP2-763 (763).
b, MDCK cells expressing APLP2-751 and APLP2-763
were labeled for 2 h with [
S]methionine.
Exogenous APLP2 molecules were immunoprecipitated with Myc-I antiserum
and collected with immobilized protein A-agarose beads. Immobilized
immunoprecipitates were incubated without (-) or with (+)
chondroitinase AC (ChAC) at 37 °C for 1 h. Lanes 1 and 2 represent immunoprecipitates from B17 cells
expressing APLP2-751 (751); lanes 3 and 4 represent immunoprecipitates from A15 cells expressing
APLP2-763 (763).
In transiently
transfected COS-1 cells, APLP2-763 isoform is not modified by the
addition of CS GAG. A similar result is obtained in MDCK
cells expressing APLP2-763 (Fig. 1a, lanes
4-6); although an immature form of
120 kDa was
detected, these forms appeared not to be modified to heterogeneous
species (Fig. 1a, lanes 4-6). Instead, a
single band of
130 kDa is now apparent. As expected,
chondroitinase AC failed to alter the electrophoretic mobility of the
130-kDa species (Fig. 1b, lane 4); hence,
we argue that insertion of the 12-amino-acid peptide inhibits CS GAG
modification of the APLP2-763 core protein. Sorting of APLP2-751 and APLP2-763 to the Basolateral
Compartment-Stimulated by the observation that APLP2-751
and APLP2-763 are differentially modified, we asked whether the
CS GAG chain has any effect on the intracellular trafficking of the
core proteins. To examine the delivery of each precursor to either the
apical or basolateral surface, we selected cell lines B17 and A15,
which express APLP2-751 and APLP2-763, respectively. Cells
were grown on Transwell filters, labeled for 1 h, and then placed on
ice. Surface molecules were biotinylated from either the apical or the
basolateral compartment. Full-length APLP2 forms were first
immunoprecipitated with Myc-I antibody. A fraction of the Myc-I
immunoprecipitates was saved, and the remainder was subjected to a
second round of binding with streptavidin. As expected, the total
levels of labeled APLP2 forms immunoprecipitated with Myc-I were
identical irrespective of whether biotinylation was performed from the
apical or basolateral surface (Fig. 2a, lanes
1, 2, 5, and 6). On the other hand,
streptavidin only detected modified, full-length forms of
APLP2-751 and APLP2-763 when biotinylation was performed
from the basolateral side (Fig. 2a, lanes 4 and
8, respectively). Thus, full-length forms of both
APLP2-751 and APLP2-763 were preferentially delivered to
the basolateral surface. As quantitated using a PhosphorImager, 95% of
the surface-bound APLP2-751 and APLP2-763 molecules were
found to reside on the basolateral side.
Figure 2:
Polarized delivery and secretion of APLP2.
A, apical; B, basolateral. a, filter-grown
B17 and A15 cells were labeled with
[S]methionine, and the cell surfaces were
biotinylated as described under ``Materials and Methods.''
APLP2 molecules immunoprecipitated from cell lysates using Myc-I were
subjected to a second round of binding with streptavidin. Lanes 1 and 2 and lanes 5 and 6 represent total
Myc-I-immunoprecipitable (Myc) full-length forms from B17 and
A15 cells, respectively. Lanes 3, 4, 7, and
8 represent cell surface biotinylated APLP2 molecules isolated
with streptavidin (SAV). Note the preferential basolateral
surface localization of both APLP2-751 and APLP2-763
molecules. b, B17 and A15 cells were grown on Transwell units
and labeled for 2 h with [
S]methionine. Equal
aliquots of apical and basolateral media were immunoprecipitated with
D2-I antiserum and analyzed by SDS-polyacrylamide gel electrophoresis.
c, PhosphorImager analysis of APLP2 secretion from cells
expressing APLP2-751 (751) and APLP2-763
(763) in 2-h labeling experiments. The values indicated
represent average values obtained from three independent clones in two
separate experiments. Solid columns, apical; shaded
columns, basolateral.
To examine the secretion of
soluble forms of APLP2-751 or APLP2-763, cells grown on
Transwell filters were labeled for 2 h, after which both apical and
basolateral media were collected and immunoprecipitated with D2-I
antibody. As shown in Fig. 2b, cell line B17 expressing
APLP2-751 secreted 94% of total soluble
APLP2-751-related derivatives to the basolateral compartment
(Fig. 2b, compare lanes 1 and 2, and
Fig. 2c). These soluble derivatives migrated as a
heterogeneous species on SDS-polyacrylamide gel electrophoresis
reflecting the CS GAG modification of the secreted ectodomain. In
addition, >95% of total soluble APLP2-763-related molecules
were secreted into the basolateral medium in cells expressing
APLP2-763 (Fig. 2b, lanes 3 and
4, and Fig. 2c). Thus, we conclude that,
despite differences in post-translational modification of
APLP2-751 and APLP2-763, both molecules appear to be
targeted preferentially to the basolateral cell surface in polarized
MDCK cells, and soluble APLP2-751 and APLP2-763 derivatives
are secreted predominantly into the basolateral media. Kinetics of Secretion of APLP2-751 and APLP2-763 Are
Indistinguishable-To examine the maturation and kinetics of
secretion of APLP2-751 and APLP2-763 in MDCK cells, we
performed pulse-chase experiments. Polarized MDCK cells grown on
Transwell filters were pulse-labeled for 10 min with
[
S]methionine and chased for various periods of
time in medium containing excess unlabeled methionine.
Detergent-soluble lysates and conditioned media were subjected to
immunoprecipitation with Myc-I and D2-I antisera, respectively.
Fig. 3
(a and b, top panels) depicts
the pattern of intracellular Myc-I-immunoprecipitated forms of
APLP2-751 and APLP2-763, respectively, during the
pulse-chase experiment. Maturation of each precursor to their
respective modified forms occurred with similar kinetics and peaked at
between 30 min and 1 h into the chase period. In addition, the levels
of accumulated secreted APLP2-related derivatives in either the apical
or the basolateral medium of both cell lines followed indistinguishable
kinetics (Fig. 3, a and b, middle and
bottompanels).
Figure 3:
Kinetics of maturation and secretion of
APLP2-751 (a) and APLP2-763 (b).
Filter-grown cells were pulse-labeled with
[S]methionine for 10 min and chased for the
times indicated. At the end of each time point, cells and media were
collected. Exogenous full-length APLP2 molecules were
immunoprecipitated with Myc-I antiserum, whereas secreted APLP2
derivatives were immunoprecipitated with D2-I antiserum. Values
indicated in the graphs (bottom panels) represent the average
values obtained from two independent experiments. A, apical;
B, basolateral.
, is a member of a family
of integral membrane glycoproteins that includes APLP1 and APLP2. We
previously documented that APLP2, like APP, matures through the
secretory pathway and that truncated soluble APLP2 derivatives are
secreted
(4) . We and others have also reported that APLP2
pre-mRNA is alternatively spliced to encode several APLP2
isoforms
(2, 4, 6) . Here, we demonstrate that in
stably transfected MDCK cells, a prototypic polarized epithelial cell
line, the APLP2-751 isoform is modified by the addition of CS GAG
chains, consistent with findings in non-polarized cells
(5) .
Furthermore, CS GAG modification is not apparent in the APLP2-763
isoform, which contains an additional 12-amino acid sequence,
presumably encoded by an alternatively spliced exon that is inserted
two amino acids upstream of the CS GAG addition site. In order to
address whether the CS GAG chain plays a role in intracellular
trafficking of the APLP2 core protein, we examined the polarized
delivery and secretion of the precursor and its soluble derivatives,
respectively, in stably transfected MDCK cells. In this report, we
demonstrate that APLP2-751 is targeted to the basolateral plasma
membrane and that truncated derivatives are released into the
basolateral medium. The observation that a CS GAG modified form of
APLP2 is targeted to the basolateral domain is consistent with earlier
reports that demonstrate that several proteoglycans expressed
endogenously in polarized epithelial cells are also delivered
vectorially to the basolateral surface. One of these molecules,
syndecan-1, a CS PG, is localized at the basolateral cell surface in
mammary epithelial cells
(13) . Moreover, a heparan sulfate PG of
unknown core protein is released predominantly into the basolateral
medium of MDCK cells
(14) . Because both syndecan-1 and heparan
sulfate PG are components of the extracellular matrix and are
implicated to be involved in cell-matrix adhesion, the basolateral
targeting and secretion of APLP2-751 may have functional
implications for this molecule.
, a major constituent of amyloid fibrils, is localized to the
basement membrane of capillaries, including vessels in the meningeal
space
(21) . This preferential localization might be explained by
the observation that A
is delivered to the basolateral compartment
of MDCK cells
(7, 8) . Although APLP2 cannot generate
A
, the similarity in trafficking of A
and APLP2 to the
basolateral compartment may have some functional implications related
to A
deposition and amyloid fibril formation in cerebral
vasculature. Indeed, the close association of proteoglycans with
amyloid fibrils is well documented (for review, see Ref. 22). Moreover,
both heparan sulfate and chondroitin sulfate proteoglycans have been
localized within senile
plaques
(23, 24, 25, 26) . In addition,
A
binds with high affinity to glycosaminoglycans
(27) , an
interaction that results in the formation of fibrillar
A
-proteoglycan complexes that are resistant to both removal and/or
degradation by cultured microglial cells
(28, 29) . These
latter findings are consistent with a postulated role for proteoglycans
in amyloid accumulation and persistence. The role of CS PG in A
aggregation and fibril formation and the persistence of amyloid
deposits in vivo remain to be elucidated.
,
-amyloid peptide; APLP, amyloid
precursor-like protein; CS, chondroitin sulfate; GAG,
glycosaminoglycan; PG, proteoglycan; MDCK, Madin-Darby canine kidney;
Ii, invariant chain.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.