From the
Appicans are secreted and cell-associated chondroitin sulfate
proteoglycans containing Alzheimer amyloid precursor (APP) as their
core protein. Appicans are found in brain tissue, and in cell cultures
their expression depends on both cell type and growth conditions. Here
we report that the core protein of appicans derives from an APP mRNA
lacking exon 15. Splicing out of this exon creates a new consensus
sequence for the attachment of a chondroitin sulfate chain in the
resulting APP product. Transfection of C6 glioma or 293 kidney
fibroblast cells with APP cDNAs containing exon 15 produced no appican,
while transfection with an APP cDNA lacking this exon induced high
levels of appican production. Polymerase chain reactions indicated that
appican-producing cells contained an APP mRNA species without exon 15,
whereas cells without this mRNA produced no appican. Site-directed
mutagenesis combined with immunoreactivity experiments showed that the
chondroitin sulfate chain is attached to a serine residue 16 amino
acids upstream of the amino terminus of the A
The amyloid precursor proteins (APPs)
Chondroitin sulfate proteoglycans are a class
of molecules consisting of a core protein to which one or more
chondroitin sulfate (CS) glycosaminoglycan (GAG) chains are covalently
attached. The CS GAG chains are bound via a xylose sugar to a serine
residue of the core protein. Although the structural characteristics
that determine the attachment site of a CS GAG chain are poorly
understood, the serine residue part of the consensus sequence Ser-Gly,
usually preceded by acidic amino acids, is the most commonly found site
for CS attachment
(11, 12) . Due to the large size of
the GAG chains, proteoglycans can be considerably larger than their
core proteins. These macromolecules are postulated to be involved in a
number of key cellular events (for review see Ref. 12), including
neuroprotection from excitotoxic agents
(13) , and in brain they
have been suggested to modulate cell adhesion, axonal growth, and
neural patterning
(14, 15) . Proteoglycans, including
heparan, dermatan, and chondroitin sulfates, have been found in and
around senile plaques and neurofibrillary tangles and have been
suggested to play a role in the pathogenesis of AD
(16, 17, 18) .
Protein sequencing and
immunoreactivity studies showed that a novel CSPG secreted by C6 glioma
cells and migrating on SDS-PAGE between 140 and 250 kDa had a
KPI-containing 120-kDa secreted APP as its core protein
(19) .
This secreted CSPG is derived from a membrane-bound cellular
full-length precursor, after the core APP is cleaved by secretase
(20) . In cell cultures, expression of the APP proteoglycans
(appicans) is cell-specific and depends on the growth conditions
(21) . Appicans are found in the brain and in rat brain primary
cultures are produced by glia, but not neurons
(26) . Recent
evidence suggested that the CS chain of APP is attached near to or
within the A
Metabolic labeling and immunoprecipitation
of media from semi-confluent C6 cells was performed as described
previously
(20) . Chondroitinase ABC or AC digestions and
treatment of the resulting digests were performed as described
(21) . SDS-electrophoresis, immunoblotting, and quantitation of
the obtained signals by densitometry using a computerized Magiscan
Joyce Lobel image analysis system were performed as described
(19, 21) .
A construct, KL-APP
C6 and 293 cells were either stably
transfected with CsCl-purified APP
Close inspection of the
SDS-PAGE mobility of the appican core APP produced after chondroitinase
digestion indicated that this protein migrated slightly faster than the
secreted unmodified K-APP (Fig. 1 B), suggesting that
these two proteins may differ in structure.
Treatment of media samples with
chondroitinase ABC eliminated the appican staining, with a concomitant
increase in the human KL-APP
Appicans are CSPGs found in brain,
and in vitro they are produced endogenously by neural cells
(21, 26) . CSPGs have been suggested to have important
functions in the central nervous system, including cell adhesion,
guidance of growing axons
(14, 15, 27) , and
participation in the healing process following brain injury
(28) . Interestingly, CSPGs have been found in both senile
plaques and neurofibrillary tangles, suggesting that these molecules
are involved in the pathology of AD
(18) . The detection of
appicans suggests that APP has additional biological functions deriving
from its proteoglycan nature. Some of these functions may be involved
in the development of AD. Recent evidence indicates that brain appican
is produced by astrocytes
(26) . Because these cells are found
associated with neuritic plaques, and participate in the formation of
brain scars following neuronal injury, astrocytic appican may well be
involved in the development of these pathological structures.
Furthermore, since proteoglycans have also been shown to bind A
We thank Dr. Claudia Schmauss and Kai Liu for help
with RNA preparation and PCR techniques, Dr. Kevin Felsenstein for
providing plasmid KL-APP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
sequence of APP. The
attachment of a glycosaminoglycan chain close to the A
sequence of
APP may affect the proteolytic processing of APP and production of
A
. The proteoglycan nature of APP suggests that addition of the
chondroitin sulfate glycosaminoglycan is important for the
implementation of the biological function of these proteins.
(
)
are a family of type I transmembrane proteins involved in
the development of Alzheimer disease (AD). They are the precursors of
A
peptide, which aggregates to form the amyloid depositions
characteristic of AD pathology
(1, 2, 3, 4) . The A
sequence
includes the last 28 extracytoplasmic residues and 11-15 amino
acids of the adjacent transmembrane domain of APP. In addition, certain
mutations of the APP gene seem sufficient for the induction of the AD
phenotype
(5, 6, 7) . The APP gene locus
contains at least 18 exons, and several distinct APP mRNA species have
been detected as a result of alternative exon splicing. Certain APP
isoforms contain a 56-amino acid insert with high sequence homology to
the Kunitz-type serine protease inhibitors (KPI), encoded by a single
exon
(8) . Additional APP mRNA species have also been identified
where exon 14 is fused to exon 16 by splicing out the 54-bp exon 15 of
the APP gene (L-APP
(9) ). APPs are extensively modified
post-translationally, a process that may affect both the production of
A
and the biological function of these proteins. Secreted APP,
containing almost all of the extracytoplasmic sequence but without the
transmembrane and cytoplasmic regions, is produced when membrane
full-length APP is cleaved by the unidentified enzyme(s) secretase (for
review see Ref. 10).
sequence of APP, but the resolution of the
methodology used did not allow a distinction between these two
possibilities
(21) . In this study we present evidence that the
core protein of appicans is derived from the translation of an APP mRNA
missing exon 15 and that the CS GAG chain is attached to a serine
residue located 16 amino acids upstream of the A
sequence of APP.
Cell Cultures and Treatments
Rat C6 glioma (C6),
mouse neuro-2a neuroblastoma (N2a), human SY5Y neuroblastoma (SY5Y),
and human 293 embryonic kidney fibroblast
(293) cells were
obtained from the ATCC cell bank. Cultures were grown in
Dulbecco's modified Eagle's medium (Life Technologies,
Inc.) supplemented with 10% (v/v) fetal bovine serum (JRH Biosciences).
Medium was collected for analysis after 2-3 days of confluent
cell growth. Cultures were maintained at 37 °C, as described
(19, 21) .
Antibodies
For APP detection, the following
antisera and monoclonal antibodies were used (APP amino acid (aa)
positions are based on APP): GID specific for aa
179-185
(22) , R7 specific for aa 296-315 of the KPI
insert
(23) ,
5 specific for aa 444-592 of human APP
(Athena Neurosciences), R3 and monoclonal antibody 1G6 specific for aa
628-652
(21) , R47 specific for aa 652-667
(19) (see also Fig. 1 C).
Figure 1:
Immunoprecipitation of
appican by different antibodies. A, C6 cultures were labeled
with [S]sulfate for 22 h (see
``Experimental Procedures''). Conditioned medium was
collected and heat-treated in the presence of 0.1% SDS, and 2.0
10
trichloroacetic acid-precipitable counts were
immunoprecipitated with anti-APP antibodies GID ( lane1), R7 ( lane2), R47 ( lane3), R3 ( lane4) and 1G6 ( lane5). Samples were separated by SDS-PAGE and fluorography.
Exposure times to x-ray film were adjusted to give similar APP signals
with the different antibodies. Arrowhead represents unmodified
APP. B, chondroitinase digestion was performed before
immunoprecipitation to show disappearance of the appican smear.
Lane1, minus chondroitinase; lane2, plus chondroitinase. Arrowhead and arrow represent unmodified APP and unmodified APP plus appican core
protein, respectively. Samples were immunoprecipitated with R47
antiserum. Numbers at the right of A represent mobilities of molecular mass markers in kDa. C,
diagrammatic representation of the APP regions recognized by the
antibodies used in the present study.
Plasmid Construction and
Transfections
APP, APP
, and a
KPI-containing L-APP (KL-APP
) cDNA were subcloned into
pRc-CMV between restriction sites HindIII and XbaI.
SA, was engineered with serine 619,
16 amino acids upstream of A
, mutated to an alanine. Briefly, the
KL-APP
/pRc-CMV plasmid was partially digested with
HindIII and BglII, and a 6089-bp fragment (A)
containing 3` KL-APP
sequences and the intact pRc-CMV
sequence was isolated. KL-APP
/pRc-CMV was also digested
with HindIII and BsmI to isolate a 1930-bp fragment
product (B) containing 5` KL-APP
sequences. Two synthetic
APP oligonucleotides were made, APP-S2 (nucleotides 1931-2007)
and APP-AS1 (antisense nucleotides 1929-2011), in which
nucleotide 1978 was changed from T to G (nucleotide numbering based on
KL-APP
). These were then phosphorylated and annealed to
form a double-stranded sticky end product (C). Fragments A, B, and C
were ligated to give construct KL-APP
SA/pRc-CMV and
subsequently cloned and sequenced (Sequenase 2.0 kit, U. S. Biochemical
Corp.) to confirm the mutation.
, APP
, or
KL-APP
DNA (pRc-CMV) or transiently transfected with
KL-APP
SA/pRc-CMV, using the DOTAP protocol according to
manufacturer's instructions (Boehringer Mannheim). Transfectants
were selected and maintained in 400 µg/ml Geneticin (Life
Technologies, Inc.).
Isolation of RNA, Reverse Transcription and Polymerase
Chain Reaction (RT-PCR)
Total RNA was isolated from C6, 293,
N2a, and SY5Y as described
(24) . Ten µg of total RNA from
each cell line was reverse transcribed, using Moloney murine leukemia
virus reverse transcriptase (250 units, U. S. Biochemical Corp.) and
oligo(dT) as a primer (100-µl reaction volume) to make
first strand cDNA. Three µl of each cDNA reaction mixture was used
for amplification by PCR with Taq polymerase (2.5 units,
Stratagene) using APP-specific primers 5`-TGTGATTTATGAGCGCA-3`
(nucleotides 1672-1688) and 5`-GCCGTTCTGCTGCATCTTGGA-3`
(nucleotides 2315-2335). The reaction volume of 100 µl
contained 50 pmol of primers and 2.5 mM of each dNTP
(Pharmacia Biotech Inc.) and was overlaid with 100 µl of mineral
oil. Reactions were for 30 cycles: 0.5 min at 92 °C, 1 min at 48
°C, and 3 min at 72 °C. PCR products were separated on a 4%
non-denaturing polyacrylamide gel and visualized by ethidium bromide
staining.
Immunoprecipitation of Appican with Various
Antibodies
Recent studies using Western blotting
(21) suggested that the CS GAG chain(s) of appican was attached
proximal to or within the A sequence of APP, either at Ser-637
located 16 residues upstream from the N terminus of A
or at
Ser-660, which corresponds to A
residue 8 (numbering according to
APP
). Each of these serines is part of a Ser-Gly
sequence. To further characterize the appican APP core protein and to
determine the exact site of attachment of the CS GAG chain, conditioned
media from
SO
-labeled C6 cell cultures were
immunoprecipitated using various antibodies. GID antibodies that
recognize all APP isoforms and R7 antibodies specific to the
KPI-containing APP (K-APP) immunoprecipitated approximately equal
amounts of appican, in agreement with earlier results suggesting that
most of the C6 appican derives from K-APP
(19, 20, 21) . In addition, both of these
antibodies immunoprecipitated the secreted unmodified APP
(Fig. 1 A, lanes1 and 2).
Both GID and R7 antibodies recognize the appican core protein after
chondroitinase digestion
(19, 21) . R47 antisera
specific to K-APP
sequence 653-668, which includes
Ser-660, also immunoprecipitated appican, although less efficiently
than GID (Fig. 1 A, lane3). R47
recognized the core APP produced after chondroitinase treatment of
appican (Fig. 1 B, lane2). In
contrast, neither monoclonal antibody 1G6 nor polyclonal R3, both
specific to K-APP
sequence 628-657, which includes
serine 637, was able to immunoprecipitate appican although they
recognized the unmodified secreted APP (Fig. 1 A,
lanes4 and 5). In addition, these
antibodies failed to recognize the appican core protein produced after
chondroitinase treatment (data not shown). This failure may be due to
the interference from the disaccharide stub that remains attached to
the core protein after chondroitinase digestion of appican
(19) . Taken together, these data suggested, although did not
prove, that Ser-637 rather than Ser-660 was the major site for
attachment of the CS GAG chain of appican.
Transfection of Cells with APP Isoforms
To further
define the APP isoform containing the CS chain(s), C6 cells that
produce appican endogenously
(19) or 293 cells that produce no
endogenous appican
(21) were stably transfected with the human
cDNAs encoding APP, K-APP
, or K-APP
without exon 15 (KL-APP
). Possible involvement of
the latter isoform as an appican core protein was suggested by the
observation that fusion of exon 14 to exon 16 results in the formation
of the consensus sequence Glu-Xaa-Ser-Gly (Fig. 2 C),
where Ser-619, corresponding to Ser-637 of K-APP
, becomes
an excellent candidate for the attachment of the CS chain
(11, 12) . Absence of the 18 amino acids encoding exon
15 from the appican core protein was also consistent with the slightly
faster mobility exhibited by this protein compared with the secreted
unmodified K-APP (Fig. 1 B).
Figure 2:
APP and appican overexpression in
transfected C6 and 293 cells. Media from untransfected and transfected
( A) rat C6 or ( B) human 293 cells were collected
after 3 days of confluent culture and separated by SDS-PAGE prior to
immunoblotting with antibody 5. Volumes loaded were adjusted to
account for differences in total cellular protein present in each
culture, as determined by BCA assay of cell homogenates. Lane1, untransfected cells; lanes2 and
3, cells transfected with KL-APP
; lane4, cells transfected with K-APP
; and
lane5, cells transfected with APP
.
Medium collected from cells transfected with KL-APP
was
digested with chondroitinase ABC ( lane3).
Numbers on the right represent mobilities of
molecular mass markers in kDa. C, splicing out of exon 15
results in fusion of exon 14 to exon 16 and formation of the consensus
sequence EXSG for the attachment of a CS chain to the serine.
This site is 16 residues upstream of the A
sequence of APP.
Openarrow indicates amino acid position of possible
CS chain attachment
Colonies overexpressing
each APP isoform were cultured in the presence of 10% fetal bovine
serum for 2-3 days, and conditioned media were collected and
analyzed by SDS-PAGE and immunoblotting. Appican or APP was detected
with 5 antisera, specific to human APP
(25) . Following
stable transfection of C6 or 293 cells with either APP
or
APP
cDNA, increased APP expression was clearly seen in
both transfectants with no increase in appican secretion
(Fig. 2 A, lanes4 and 5). At
least 10 independent clones of each transfection were analyzed with
identical results. In contrast, C6 cells transfected with the
KL-APP
cDNA showed a marked increase in the secretion of
the 140-250-kDa diffuse staining characteristic of appican
(Fig. 2 A, lane2) (see also Refs.
19-21). Similarly, 293 cells, which do not produce endogenous
appican, secreted high amounts of appican into the media upon
transfection with KL-APP
(Fig. 2 B,
lane2).
core protein (Fig. 2,
A and B, lane3). Treatment with
chondroitinase AC yielded similar results, showing that the GAG chains
attached to the KL-APP
core protein consist of
chondroitin sulfate and not dermatan sulfate (data not shown; see also
Ref. 19). Densitometric quantitation of the relative amounts of the
transfected KL-APP
core protein before and after
chondroitinase treatment indicated that in all C6 transfected cell
cultures more than 90% of the total secreted KL-APP
was
in the CSPG form. In contrast, in 293 cells only about 40-50% of
the expressed KL-APP
was in the PG form.
APP mRNA Expression in Different Cell Lines as Determined
by RT-PCR
To examine whether there is any relationship between
appican production and expression of APP mRNA lacking exon 15, we
performed polymerase chain reactions using reverse-transcribed RNA from
different cell lines. Of these cell lines, C6 and N2a synthesize
appican endogenously, whereas SY5Y and 293 do not
(21) . First
strand cDNA was prepared from total RNA of these cultures, and PCR was
then performed on these cDNA preparations using APP-specific primers,
flanking exon 15 (see ``Experimental Procedures''). Under
these conditions, the predicted PCR product derived from APP cDNA
containing the 54-bp exon 15 is a transcript of 664 bp, while the
predicted PCR product derived from APP cDNA without exon 15 is a
transcript of 610 bp. Plasmids K-APP/pRc-CMV and
KL-APP
/pRc-CMV were used as controls (Fig. 3). The
cDNA samples from the SY5Y neuroblastoma and 293 fibroblast cultures
yielded a single PCR transcript of 660 bp, equal in size to the
transcript derived from the K-APP
plasmid DNA. In
contrast, cDNA samples from C6 and N2a cells produced two PCR
transcripts, one equal in size to that produced from the K-APP
plasmid DNA and an additional smaller transcript of about 610 bp,
identical in size to that obtained from the KL-APP
plasmid DNA (Fig. 3). To ensure that exon 15 was absent
from the lower molecular weight PCR product, we cloned and sequenced
this transcript from C6 cells. The obtained sequence confirmed both the
APP nature of the transcripts and the absence of exon 15 from the
smaller transcript (data not shown). These data show that cells
producing L-APP mRNA also produce appican, while cells that do not
produce L-APP mRNA produce no appican. Combined with the
immunoprecipitation and transfection experiments, these observations
suggested that 1) the core protein of appican is the translation
product of an APP mRNA lacking exon 15 and 2) Ser-619 of KL-APP
(corresponding to Ser-637 of K-APP
) is the
attachment site of the CS GAG chain.
Figure 3:
RT-PCR analysis of APP mRNA isoform
expression in transformed cell lines. RT-PCR products were separated by
4% non-denaturing PAGE and analyzed by ethidium bromide staining. APP
specific primers used flanked exon 15 APP DNA sequences. Products are
shown from C6 glioma, SY5Y neuroblastoma, N2a neuroblastoma, and 293
fibroblasts. RT-PCR was also performed on control samples of
K-APP/pRc-CMV (APP) and KL-APP
/pRc-CMV
(L-APP). Numbers on the right represent mobilities of
molecular mass markers from
phage digested with BstE II
in bp.
Transfection of C6 and 293 Cells with Mutated
KL-APP
To test
the hypothesis that Ser-619 of KL-APP to Identify CS Attachment Site
is the CS
attachment site (Fig. 2 C), C6 and 293 cells were
transfected with a mutated KL-APP
cDNA, in which the T
residue at coding position 1977 was substituted with a G (see
``Experimental Procedures''). The translation product of this
mutated mRNA, designated KL-APP
SA, will contain an
alanine residue, instead of a serine, at position 619. Since this is
the postulated GAG attachment site, the effect of such a mutation
should be the inhibition of appican production. Indeed,
Fig. 4
shows that although C6 and 293 cells transfected with
KL-APP
cDNA secreted appican, which appeared as the
characteristic heterogenous staining between 140 and 250 kDa,
transfection of these cells with KL-APP
SA resulted in the
secretion of only a homogenous 120-kDa molecule that was insensitive to
chondroitinase ABC treatment. These results further confirm that
Ser-619 of KL-APP
, located 16 residues N-terminal to
A
sequence, is the primary attachment site of the CS GAG chain of
appicans.
Figure 4:
Site-directed mutagenesis of Ser-619 to
alanine in KL-APP prevents CS chain attachment. C6
( lanes1-4) and 293 cells ( lanes5-8) were transfected with either KL-APP
( lanes1, 2 and 5, 6) or
KL-APP
SA ( lanes 3, 4 and 7,
8). Medium was collected after 3 days of confluent culture,
incubated in the presence (+) or absence (-) of
chondroitinase ABC, and separated by SDS-PAGE prior to immunoblotting
with human APP specific antibody
5. Numbers on the
right represent mobilities of molecular mass markers in
kDa.
Taken together, our results indicate the core proteins of
appicans lack exon 15 of the APP gene. Splicing out of this exon
creates a sequence -Glu-Gly-Ser-Gly-, which constitutes an excellent
consensus sequence for the attachment of the CS chain
(Fig. 2 C). The absence of appican production in cells
transfected with KL-APPSA suggests that appicans contain
only one CS chain. These data, as well as earlier data
(19, 20, 21, 26) , suggest that most of
the appican core proteins are derived from the K-APP isoforms, although
isoforms missing both the KPI insert and exon 15 may also occur as
appicans. However, the percent of APP that receives a CS chain appears
to be dependent on the cell type. Thus, in C6 cells more than 90% of
the produced transfected KL-APP
was in appican form
compared with only 40-50% in 293 cells. In addition, Chinese
hamster ovary cells transfected with KL-APP
contained
50-60% of the produced protein as appican.
(
)
Interestingly, leucocytes, where L-APPs without exon 15
were first described
(9) , produced little or no
appican.
(
)
(29, 30) , appicans may interact with A
in ways
that result in either inhibition or stimulation of A
aggregation.
In addition, the attachment of the GAG chains close to the A
sequence of APP could alter its proteolytic processing and thus affect
production of A
.
/pRc-CMV, and Dr. Ivan Lieberburg
for providing antibody
5.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.