From the
Alzheimer's disease is characterized by the presence of
parenchymal and cerebrovascular deposits of
Alzheimer's disease is pathologically characterized by the
deposition of
APP is a member of a larger family of proteins that includes amyloid
precursor-like proteins, termed APLP1 (12) and
APLP2(13, 14, 15) . Like APP pre-mRNA, the
primary APLP2 transcript undergoes alternative splicing, and resultant
transcripts encode APLP2 isoforms containing a Kunitz protease
inhibitory domain (16, 17) (APLP2-751 and APLP2-763) or
a 12-amino acid peptide of unknown function (APLP2-706 and APLP2-763)
(13-15, 18). In earlier efforts, we documented that in cultured
cells, APLP2-751 matures through a secretory/cleavage pathway similar
to that utilized by APP(15, 19) . Moreover, a
chondroitin sulfate glycosaminoglycan (CS GAG) chain is added to
Ser-614 of APLP2-751(19) . The sequence flanking Ser-614
(ENEGSGMA) is remarkably similar to a previously described recognition
signal for CS GAG attachment(20) , i.e. S-G-X-G/A flanked by acidic amino acids. In the APLP2-706
and APLP2-763 isoforms, however, a 12-amino acid peptide, presumably
encoded by an alternatively spliced exon, is inserted between Glu-612
and Gly-613. We have examined the metabolism of the APLP2-763 isoform,
and now report that the 12 amino acid peptide abolishes CS GAG
modification of the core APLP2. Thus, our data are consistent with the
idea that the level of CS GAG modification of the APLP2 core protein
can be modulated by insertion of sequences encoded by an alternatively
spliced exon.
To assess the generality of the novel mechanism of
regulation of CS GAG modification in members of the APP/APLP family, we
examined the levels of CS GAG modification of APP isoforms encoded by
alternatively spliced APP mRNAs. Indeed, earlier reports indicated that
APP is a substrate for modification by CS GAG in C6 glioma
cells(21, 22) , despite our failure to demonstrate CS
GAG modification of transiently expressed APP-695 or -770 isoforms in
Chinese hamster ovary and COS-1 cells(19) . L-APP isoforms,
which lack 18 amino acids encoded by exon 15, contain the sequence
ENEGSGL(7) ; this sequence is generated by a fusion of sequences
encoded by exons 14 and 16 and is surprisingly similar to sequences
flanking the CS GAG attachment site of APLP2-751 (see above). In the
present report, we demonstrate that like APLP2-751, L-APP isoforms are
also substrates for CS GAG addition. The molecular mechanisms involved
in regulating the levels of alternatively spliced transcripts which
encode APP/APLP2 isoforms is unknown. Furthermore, the biochemical
mechanism(s) by which exon 15 of APP-770/751/695 and the 12-amino acid
insert in APLP2-763/706 disrupt CS GAG modification of respective core
proteins have not been fully clarified. Nevertheless, we suggest that
CS GAG addition represents a cell or developmental-specific mechanism
to generate additional functional diversity for each polypeptide.
The primer pair SAPP and AsAPPmyc was used in PCR to amplify cDNA
fragments encoding the C terminus of APP and L-APP polypeptides using
the indicated DNA as template: plasmid pEFmo695 (provided by Dr. David
Borchelt, The Johns Hopkins University School of Medicine, Baltimore,
MD) for the C-terminal 184 amino acids of mouse APP and
reverse-transcribed mouse lung RNA for the C-terminal 166 amino acids
of mouse L-APP. The PCR products were digested with XhoI and XbaI and ligated to a vector fragment generated by digesting
plasmid p770SP (23) with XhoI and XbaI. The
resulting plasmids encode polypeptides in which the N-terminal 380
amino acids of human APP-770 are fused to the C terminus of mouse APP
(770
APLP2-751 appears as an immature
A
Our recent studies of the
expression of APLP2 in the mouse nervous system support the view that
alternatively spliced APLP2 mRNAs might encode proteins which are
functionally distinct.
We thank Dr. Philip Wong (Neuropathology Laboratory,
The Johns Hopkins University School of Medicine) and Dr. Steve Wagner
(SIBIA, La Jolla, CA) for providing Myc-I and P2-1 antibodies,
respectively; Frances Davenport for assistance with cell culture; Dr.
Jeffrey D. Esko (University of Alabama at Birmingham, Birmingham, AL)
for helpful suggestions; and Drs. David Borchelt and Donald Price
(Neuropathology Laboratory, The Johns Hopkins University School of
Medicine) for advice on the manuscript.
Note Added in
Proof-While our manuscript was in review, we learned of a
report by Pangelos et al. (Pangelos, M. N., Efthimiopoulos,
S., Shioi, J., and Robakis, N. K.(1995) J. Biol. Chem.270, 10388-10391) which demonstrated that APP lacking exon 15 is
modified by CS GAGs.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-amyloid (A
).
A
is derived from larger amyloid precursor proteins (APP), a
member of a family of related polypeptides that includes amyloid
precursor-like proteins, APLP1 and APLP2. APP and APLP2 isoforms are
encoded by several alternatively spliced APP and APLP2 transcripts, respectively. We previously reported that the
APLP2-751 isoform is modified by the addition of chondroitin sulfate
glycosminoglycan (CS GAG) at Ser-614. In this report, we demonstrate
that the APLP2-763 isoform, which contains an insertion of 12 amino
acids immediately N-terminal to Ser-614, is not modified by CS GAG.
Finally, we demonstrate that like APLP2-751, APP isoforms that lack
sequences encoded by exon 15 (L-APP) are also modified by CS GAG,
whereas APP forms containing exon 15 are not. We suggest that CS GAG
modification of a subset of APP and APLP2 isoforms represents a means
of generating functional diversity for these polypeptides.
-amyloid (A
)
(
)in senile
plaques and cerebrovascular vessels(1, 2) . A
, an
4-kDa peptide, is derived from larger integral membrane
glycoproteins, termed amyloid precursor proteins (APP) (3) encoded by several alternatively spliced
transcripts(4, 5, 6) . APP lacking sequences
encoded by exon 15, termed L-APP, are expressed in leukocytes,
microglial cells, and several nonneuronal
tissues(7, 8, 9, 10, 11) .
Oligonucleotide Primers
The following
oligonucleotide primers were used in this study: SD2,
5`-CCGCTCGAGGAACAGCGAGCG-3` (encodes residues 567-570 of mouse
APLP2); AsD2myc, 5`-CCGTCTAGATTAATTCAAGTCCTCTTCAGAAATGAGCTTTT
GCTCCATAATCTGCATCTGCTCCAGG-3` (complementary to sequences encoding the
C-terminal 6 amino acids of APLP2 and 12 amino acids (MEQKLISEEDLN) of
c-Myc); SAPP, 5`-CCGCTCGAGATCAGTTACGGA-3` (encodes amino acids
586-589 of human APP-770); AsAPPmyc,
5`-CC-GTCTAGATTAATTCAAGTCCTCTTCAGAAATG
AGCTTTTGCTCCA-TGTTCTGCATCTGCTC-3` (complementary to sequences encoding
the C-terminal 5 amino acids of APP and 12 amino acids (MEQKLISEEDLN)
of c-Myc).
Plasmid Construction
Sequences encoding the
C-terminal 196 amino acids of mouse APLP2-763 were amplified by
polymerase chain reaction (PCR) from reverse-transcribed E16 embryonic
mouse head RNA using the primer pairs SD2 and AsD2myc. The resulting
PCR product was digested with XhoII and XbaI and
ligated to pSVAPLP2myc(19) , previously digested with XhoII and XbaI, to generate plasmid pSVAPLP2-763myc.
mAPP) or mouse L-APP (770
mL-APP).
Transfection and Immunoprecipitation
Analysis
COS-1 cells were transfected using a high efficiency
calcium phosphate method(24) . Cells were labeled 36 h after
transfection with 250 µCi/ml [S]methionine
for 3 h in Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) supplemented with 1% dialyzed fetal bovine serum.
Detergent lysates were prepared by lysing the cells in
immunoprecipitation buffer(23) . Exogenous Myc epitope-tagged
APLP2 and APP polypeptides were immunoprecipitated from cell lysates
using polyclonal Myc-I antibody(25) . Soluble APLP2 derivatives
were immunoprecipitated from culture medium using polyclonal antibody
D2-I raised against baculovirus-synthesized mouse
APLP2-751(19) . Soluble chimeric APP-APLP2 derivatives were
immunoprecipitated from culture medium with monoclonal antibody P2-1
which recognizes an epitope in the APP ectodomain(26) . Immune
complexes were collected with protein A-agarose (Pierce).
Chondroitinase enzyme digestions of immobilized immunoprecipitates were
carried out as described(19) .
APLP2 Isoforms Encoded by Alternatively Spliced
Transcripts Are Differentially Modified by CS GAG
In previous
studies, we reported that APLP2-751 is modified by the addition of CS
GAG at a single serine residue (Ser-614)(19) . Interestingly,
the APLP2-763 isoform contains an additional 12-amino acid peptide
inserted two amino acids N-terminal to the CS GAG addition site. In
order to assess the effect of the 12-amino acid peptide on CS GAG
attachment, we transiently transfected cDNA encoding a Myc
epitope-tagged mouse APLP2-763 (pSVAPLP2-763myc) into COS-1 cells in
parallel with cDNA encoding Myc-tagged mouse APLP2-751 (pSVAPLP2-751).
Transfected cells were biosynthetically labeled with
[S]methionine, and radiolabeled mouse
APLP2-related molecules were immunoprecipitated from cell lysates with
Myc epitope-specific polyclonal antiserum Myc-I. The levels of CS GAG
modification was assessed by digestion of the Myc-I immunoprecipitates
with chondroitinase ABC prior to electrophoresis on SDS-polyacrylamide
gels.
120-kDa polypeptide and
a heterogeneous population of molecules with a mobility of
120-200 kDa which result from the addition of variable
length CS GAG to the core protein (Ref. 19; Fig. 1B, lane
3). Chondroitinase ABC digestion of APLP2-751 resulted in the
conversion of the heterogeneous set of polypeptides to a species of
130 kDa (Fig. 1B, lane 4). In contrast, APLP2-763
appears as an
118-kDa molecule and a mature, chondroitinase
ABC-insensitive polypeptide of
130 kDa (Fig. 1B, compare lanes 5 and 6). The latter result was
indistinguishable from the behavior of APLP2-751S/A, a molecule that
harbors a serine-alanine substitution at Ser-614 and which fails to be
modified by CS GAG(19) . Notably, the immature form of the
APLP2-763 molecule exhibits somewhat accelerated migration relative to
the immature form of the shorter APLP2-751 molecule. This deviation
from the expected electrophoretic mobility was also observed when newly
synthesized APLP2-763 molecules were immunopreciptated from lysates of
cells which were pulse-labeled for 5 min (not shown). The reason for
this discrepancy is unclear but appears to be dependent of the presence
of the APLP2 transmembrane and cytoplasmic segments, since the cognate
secreted derivatives migrated as expected (see below, Fig. 1B, compare lanes 8 and 9). As we
have demonstrated previously, cells expressing APLP2-751 secreted APLP2
derivatives of
100-150 kDa, generated following
endoproteolytic cleavage of the
120-200-kDa cell-associated
species (Fig. 1B, lane 8). On the other hand, cells
expressing APLP2-763 secreted a fairly homogeneous species of
110
kDa (Fig. 1B, lane 9) which is likely generated
following cleavage of the
130-kDa cell-associated form. The
cellular site and sequence requirements for the endoproteolytic
cleavage of APLP2-751 and APLP2-763 remain to be determined. In any
event, our studies clearly document that the 12-amino acid peptide
inhibits CS GAG modification of the APLP2-763 isoform.
Figure 1:
Insertion of alternatively spliced
exon-encoded 12-amino acid peptide regulates CS GAG modification of
APLP2. A, schematic representation of APLP2 polypeptides.
Isoforms APLP2-751 and APLP2-763 differ by the presence of a 12-amino
acid sequence (stippled box) encoded by an alternatively
spliced exon. TMD, transmembrane domain; KPI, Kunitz
protease inhibitor domain (hatched box); solid box,
C-terminal Myc epitope. B, analysis of APLP2 maturation in
transiently transfected COS-I cells. Myc epitope-tagged APLP2
polypeptides were immunoprecipitated with Myc-I antibody from lysates
of [S]methionine-labeled cells transfected with
plasmid vector (lanes 1 and 2) or plasmids encoding
APLP2-751 (lanes 3 and 4) or APLP2-763 (lanes 5 and 6). The immune complexes were incubated at 37 °C
without (-, lanes 1, 3, 5) or with (+, lanes 2,
4, 6) chondroitinase ABC prior to electrophoresis. Soluble
APLP2-related polypeptides were immunoprecipitated with D2-I antibody
from conditioned medium of cells transfected with plasmid vector (lane 7) or plasmids encoding APLP2-751 (lane 8) or APLP2-763 (lane 9). Heterogeneous CS GAG-modified APLP2-751
immunoprecipitated from cell lysate is marked by a dotted
line; APLP2-751 product resulting from chondroitinase enzyme
digestion is indicated by an arrowhead. Molecular mass of
standards are in kilodaltons.
The mechanism
by which the 12-amino acid insert inhibits CS GAG modification of
APLP2-763 is not fully clear. In order to assess the requirement for
the presence of acidic residues upstream of the CS GAG attachment site
on CS GAG addition, we examined the maturation of APLP2-751 in which
glutamate residues Glu-610 and Glu-612 were substituted to glutamine.
We have demonstrated that the latter molecules fail to be modified by
CS GAG (data not shown), a finding that suggests that one, or both, of
the acidic residues N-terminal to the Ser-Gly dipeptide is (are)
essential for efficient CS GAG modification of APLP2-751. Hence, we
propose that one potential mechanism by which the 12-amino acid insert
inhibits CS GAG addition is by displacing the acidic residues from the
CS GAG attachment site, thus altering the optimal xylosyltransferase
recognition sequence(20) . However, several caveats to the
latter interpretation remain; it is presently uncertain whether
presence of the two upstream acidic residues are the sole requirement
for efficient CS GAG addition of APLP2-763; confirmation awaits a
systematic analysis of a spectrum of APLP2 substrates harboring
additional site-directed mutations surrounding the CS GAG attachment
site or by analysis of APLP2-763 with dominant mutations that restore
CS GAG addition. Moreover, it is not presently clear whether the
12-amino acid peptide inhibits CS GAG addition at the level of CS GAG
chain initiation or elongation.
APP Lacking Exon 15 (L-APP) Is a Substrate for CS GAG
Modification
Earlier studies demonstrated that APP expressed in
C6 glioma cells were modified by CS GAG(21, 22) .
However, transiently expressed APP-695, -751, or -770 do not appear to
be modified by CS GAG in human 293(27, 28) , COS-1, or
Chinese hamster ovary cells(19, 23) , although
heterogeneous [S]sulfate-labeled APP-related
species have been described in PC12 cells(29) . However, little
information is available about the metabolism of the L-APP isoforms
that lack 18 amino acids encoded by exon 15(7) . Notably, the
L-APP isoforms contain a sequence ENEGSGL, generated by fusing exons 14
and 16, which is strikingly similar to the region surrounding the CS
GAG attachment site of APLP2-751; remarkably, comparison of a 111-amino
acid region immediately N-terminal to the transmembrane domains of APP
and APLP2 reveals that the only stretch of identity resides in the
ENEGSG sequence (Fig. 2). In order to assess whether L-APP is
modified by CS GAG, we transfected COS-1 cells with APP minigenes that
encode polypeptides in which the N-terminal 380 amino acids of human
APP-770 are fused to the C-terminal 184 amino acids of mouse APP
(770
mAPP) or 166 amino acids of mouse L-APP (770
mL-APP) (Fig. 3A). 770
mAPP is synthesized as a polypeptide
of
80 kDa (Fig. 3B, lane 1) and matures to discrete
species of
100 and
110 kDa (Fig. 3B, lane 3).
However, 770
mL-APP appears as an immature species of
85 kDa (Fig. 3B, lane 2) and matures to a heterogeneous set of
polypeptides of
150-200 kDa (Fig. 3B, lane
5). As expected, mature forms of 770
mAPP were insensitive to
chondroitinase AC digestion (Fig. 3B, lane 4). However,
chondroitinase AC converted the heterogeneous
150-200-kDa
770
mL-APP-related species into a homogeneous polypeptide of
105 kDa, confirming that the chimeric L-APP forms were modified by
CS GAG (Fig. 3B, lane 6). Moreover, the vast majority of
soluble 770
mL-APP derivatives were modified by CS GAG (Fig. 3B, compare lanes 9 and 10;
phosphorimaging analysis revealed that >70% of the soluble forms of
770
mL-APP were modified), whereas the soluble derivatives of
770
mAPP were insensitive to digestion by chondroitinase AC (Fig. 3B, compare lanes 7 and 8).
These results demonstrate that mouse L-APP isoforms are substrates for
CS GAG attachment and that insertion of sequences encoded by exon 15
into the CS GAG recognition site significantly inhibits CS GAG
modification of the precursor.
Figure 2:
Sequence comparison of the C-terminal 210
amino acids of mouse APLP2 and mouse L-APP. Amino acid alignment was
generated using the Genetics Computer Group software (7.0) PILEUP (30).
Transmembrane domain is boxed. The boxed region marked by arrows identifies the amino acid identity
between L-APP and CS GAG attachment site of APLP2. Note that the
stretch of 111 amino acids of L-APP immediately N-terminal to the
transmembrane domain has little homology with APLP2 outside the boxed region marked by arrows.
Figure 3:
L-APP is modified by the addition of CS
GAG. A, structure of chimeric APP polypeptides. Human APP-770
residues 1-380 and mouse APP-770 residues 586-770 are in open box; TMD, transmembrane domain: KPI,
Kunitz protease inhibitor domain (hatched box); solid
box, C-terminal Myc epitope tag. B, analysis of chimeric
APP and L-APP polypeptides in transfected COS-1 cells. Chimeric APP
polypeptides were immunoprecipitated from lysates of cells labeled with
[S]methionine for 5 min (lanes 1 and 2) or 3 h (lanes 3-6); immunoprecipitates are
from lysates of cells transfected with plasmids encoding 770
mAPP (lanes 1, 3, and 4) or 770
mLAPP (lanes 2,
5, and 6). Soluble derivatives of chimeric APP
polypeptides were immunoprecipitated with monoclonal antibody P2-I from
conditioned medium of cells transfected with plasmids encoding
770
mAPP (lanes 7 and 8) or 770
mLAPP (lanes 9 and 10). The immune complexes were incubated
at 37 °C without (-, lanes 3, 5, 7, 9) or with
(+, lanes 4, 6, 8, 10) chondroitinase AC prior to
electrophoresis. The CS GAG-modified L-APP immunoprecipitated from the
cell lysate is marked by a bracket.
, the principal component of senile plaques of
Alzheimer's disease, Down's syndrome, and to a lesser
extent, normal aging is derived from APP. APP is a member of a larger
family of integral membrane glycoproteins that includes APLP1 and
APLP2(12, 13, 14, 15) . Several
alternatively spliced APP and APLP2 mRNA have been described that
encode a family of APP and APLP2 isoforms. We recently demonstrated
that the APLP2-751 isoform is post-translationally modified by the
addition of CS GAG at a single serine residue contained within the
sequence ENEGSGMA(19) . To that initial observation, the present
report provides two novel insights. First, we document that the
APLP2-763 isoform, in which a 12-amino acid peptide is inserted two
amino acids N-terminal to the CS GAG attachement site, is not modified
by CS GAG. Second, we demonstrate that like APLP2-751, APP isoforms
that lack sequences encoded by exon 15, termed L-APP, are also
post-translationally modified by the addition of CS GAG. We suggest
that L-APP are modified at Ser-638 (of L-APP 752) contained within a
junctional sequence, ENEGSGLT, that is remarkably similar to sequences
surrounding the CS GAG attachment site of APLP2-751. The latter result
resolves the apparent discrepancy between studies which documented that
APP is modified by CS GAG in C6 glioma cells (21, 22) and others which failed to demonstrate that
human APP-695, -751, and -770 isoforms, when transiently overexpressed
in mammalian cells, are substrates for CS GAG
addition(19, 23, 27, 28, 29) .
Notably, our recent RT-PCR analysis of C6 glioma cell mRNA reveal that
approximately 35% of APP mRNA encodes L-APP.
(
)These latter results are consistent with earlier
studies showing that L-APP is highly expressed in microglial
cells(7, 8) . In any event, the sequence ENEGSG,
contained within the most highly divergent domain between APP and
APLP2, appears to be essential for efficient CS GAG modification of
both APLP2 and APP. Moreover, CS GAG addition is effectively disrupted
by insertion of sequences encoded by an alternatively spliced exon, two
amino acids N-terminal to the CS GAG attachment site. To the best of
our knowledge, this is the first demonstration that addition of CS GAG
to core proteins can be modulated by sequences encoded by alternatively
spliced exons. The generality of this novel mechanism of regulation of
GAG addition to other proteoglycans awaits a systematic biochemical and
molecular analysis of variants of each core protein encoded by
alternatively spliced transcripts.
(
)For example, APLP2
isoforms containing the 12-amino acid insert are expressed in mature
neuronal populations in cortex, hippocampus, and cerebellum (18) which have established stable connections with respective
targets. On the other hand, APLP2 isoforms lacking the insert are
highly and selectively expressed in olfactory sensory neurons, the only
neuronal population in the adult mammalian central nervous system which
are continuously replaced
; axonal outgrowth from newly born
sensory neurons to the olfactory bulb requires elaborate mechanisms to
regulate guidance, adhesion and synaptogenesis. Thus, we argue that
APLP2 isoforms which are differentially modified by CS GAG are utilized
for functionally distinct aspects of neuronal biology.
,
-amyloid; APLP, amyloid precursor-like protein; APP, amyloid
precursor protein; CS, chondroitin sulfate; GAG, glycosaminoglycan;
L-APP, APP lacking exon 15; PCR, polymerase chain reaction.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.