From the Laboratory of Neurobiophysics, School of
Pharmaceutical Sciences, the University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033 Japan, the ¶ Division of Biochemistry,
Chiba Cancer Center Research Institute, 666-2, Nitona, Chuou-ku,
Chiba 260-0801, Japan, and
Bio-oriented Technology Research
Advancement Institution, 3-18-19 Toranomon, Minato-ku, Tokyo 105-0001, Japan
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ABSTRACT |
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A novel protein, human X11-like (human X11L),
contains a phosphotyrosine interaction (PI) domain and two PDZ domains
and displays 55.2% amino acid homology with the human X11 (human X11).
The PI domain of human X11L interacts with a sequence containing the NPXY motif found in the cytoplasmic domain of Alzheimer's
amyloid precursor protein. A construct lacking the carboxyl-terminal
domain, which comprises two PDZ domains (N + PI), enhances PI binding to APP, whereas another construct lacking an amino-terminal domain relative to PI domain (PI + C) suppresses PI binding to APP.
Overexpression of full-length human X11L (N + PI + C) in cells that
express APP695 stably decreased the secretion of A Alzheimer's amyloid precursor protein
(APP)1 is an integral
membrane protein with a receptor-like structure (1). A principal component of parenchymal amyloid deposits in Alzheimer's disease (AD)
is The X11 gene, which is located on chromosome 9, was
originally isolated as a gene candidate for Friedreich ataxia (21) and its partial cDNA was identified as a clone encoding a protein that
associates with APPCOOH (22). The PI domain of X11
interacts with the YENPTY motif of APPCOOH (20). In the
present study, we isolated a complete cDNA encoding an X11-like
protein (X11L) from a human adult brain cDNA library, utilizing the
yeast two-hybrid system and APPCOOH as a bait. A partial
short cDNA encoding approximately 190 amino acids in the PI domain
of X11L has already been isolated using a similar procedure (22).
However, detailed characterization of X11L binding to APP and
identification of the role X11L plays in the physiological function of
APP have not been performed. We found that human X11L requires a
sequence containing the NPXY motif of APPCOOH
for APP binding and that association of the PI domain with APP was
suppressed by a deletion of a amino-terminal domain fused to the PI
domain (PI + C construct) but enhanced by a deletion of a
carboxyl-terminal domain fused to the PI domain (N + PI construct).
Co-transfection of full-length human X11L into cells that express
stably transfected human APP695 cDNA resulted in decreased
secretion of A Yeast Two-hybrid System, cDNA Cloning of Human and Mouse
X11-like Protein, and Plasmid Construction--
The yeast two-hybrid
system, MATCH MAKER Two-Hybrid System, was purchased from
CLONTECH Laboratories, Inc. (Palo Alto, CA). The
cDNA encoding APPCOOH, located between amino acids 649 and 695 (numbering as for the APP695 isoform), was amplified by PCR using the APP695 cDNA present in pcDNA3 (23). The PCR product, which was produced by additional EcoRI sequence in 5'
upstream and BamHI sequence in 3' downstream, was digested
with EcoRI plus BamHI, and the restriction
fragment was inserted into pGBT9 at identical restriction sites
in-frame with the GAL4 encoding sequence present on this vector. The
resulting pGBT9APPCOOH "bait" plasmid was used to
isolate the cDNA of the APP-binding protein from a human adult
brain cDNA library cloned into the vector pGAD424, which contains
the GAL4 transactivation domain. Both plasmids were co-transfected into
the yeast HF7c strain (MATa, ura3-52, his3-200, ade2-101,
lys2-801, trp1-901, leu2-3, 112, gal4-542, gal80-538,
LYS::GAL1-HIS3, URA:: (GAL4
17-mer)3-CYC1-LacZ) (24) using the lithium
acetate/heat shock procedure according to the manufacturer's product
protocol (catalog no. PT1265-1, CLONTECH Laboratories). Because the HF7c strain harbors the reporter genes lacZ and HIS3, co-transformants were streaked first on
selection medium lacking tryptophan, leucine, and histidine to assay
for activation of the HIS3 reporter gene. Positive colonies
were then picked and assayed for activation of the lacZ
reporter gene as evidenced by their blue color. Plasmids from colonies
positive for both reporter genes were rescued into Escherichia
coli HB101. The rescued plasmids were reassayed for their binding
activity to the cytoplasmic domain of APP by transfection into HF7c
harboring the "bait" plasmid. Finally, the nucleotide sequences of
the positive clones were determined by the dideoxy terminator cycle
sequencing using a Prism 377 DNA Sequencer (Perkin-Elmer), and the
homology of the deduced amino acid sequence to existing sequences in
the data bases was investigated using the BLAST program.
The 5' nucleotide sequence of one cDNA, pGAD424#104, isolated by
the yeast two-hybrid system encoded the 624-amino acid sequence of
human X11L protein. The deleted amino-terminal region consisting of 125 amino acids was determined by the RACE procedure (Life Technologies,
Inc.; 5'-RACE system version 2) using human whole brain
poly(A)+ RNA (CLONTECH Laboratories;
catalog no. 6516-1) and primers corresponding to the amino acid
sequence indicated by the underline in Fig. 1a.
The 5'-RACE product was inserted into the pCRII TA cloning vector
(Invitrogen), pCRII5'hX11L. Mouse X11L (previously referred to as mouse X11 in Refs. 22 and 25) cDNA was isolated by
reverse transcription-PCR using two sets of primers. The primer
sequence was designed from the known partial nucleotide sequence of
mouse X11 (DDBJ/EMBL/GenBankTM accession no.
L34676). A combination of primers consisting of primer 1 corresponding
to nucleotide positions 5-22 (forward, numbering for L34676) and
primer 2 corresponding to nucleotide positions 839-856 (reverse,
numbering for L34676) and another combination of primers consisting of
primer 3 corresponding to nucleotide positions 839-856 (forward,
numbering for L34676) and primer 4 corresponding to nucleotide
positions 2041-2059 (reverse, numbering as for L34676) were used to
amplify whole mouse brain poly(A)+ RNA using Pwo
DNA polymerase (Boehringer Mannheim). The resulting 0.8-kb (primers 1 and 2) and 1.2-kb PCR (primers 3 and 4) products were cloned separately
into pCRII to give, respectively, pCRIImX11L-1, which contains the
nucleotide sequence encoding the amino acid sequence between positions
70 and 353 (numbering as for Fig. 1a), and pCRIImX11L-2,
which contains the nucleotide sequence encoding the amino acid sequence
between positions 348 and 748 (numbering as for Fig. 1a).
The 5' unknown nucleotide sequence was also determined by the RACE
procedure using the mouse brain poly(A)+ RNA and primers
corresponding to the amino acid sequences indicated by
underlines in Fig. 1. The 5'-RACE product, corresponding to amino acid positions 1-69 (numbering for Fig. 1a), was
inserted into pCRII to give pCRIITA5'mX11L.
The insert from pGAD424#104 and the cDNA encoding the 5' region of
human X11L, pcRIITA5'hX11L, were ligated. The resulting entire cDNA
encoding the entire human X11L protein was inserted into pcDNA3
(Invitogen, Carlsbad, CA) with (pcDNA3-FLAG-hX11L) or without
(pcDNA3-hX11L) the addition of a synthetic linker encoding the FLAG
epitope on the carboxyl-terminal side of the inserts. The amino acid
sequence of human X11L, consisting of 749 amino acids, and that of
mouse X11L, consisting of 748 amino acids, were deduced from the
nucleotide sequence of each cDNA and are indicated in Fig. 1.
The cDNA clones encoding the partial amino-terminal domain
(pGAD424#6; amino acids 121-367), PI domain (pGAD424#PI; 368-555), carboxyl-terminal domain (pGAD424#7; 556-749), amino-terminal domain
attached to the PI domain (pGAD424#1; 121-555), PI domain attached
with the carboxyl-terminal domain (pGAD424#4; 368-749), and almost the
entire sequence of human X11L (pGAD424#104; 121-749) were
subcloned into pGAD424 harboring the GAL4 transactivation domain. These
plasmids were used for the APP binding assay in co-transfections with
pGBT9APPCOOH using the yeast two-hybrid system. The
cDNA encoding APPCOOH was truncated internally at regions 681-690 (pGBT9APP695
Antibodies--
The polyclonal anti-X11L amino-terminal domain
antibody UT-29 was raised against a peptide, "Cys" plus containing
the amino acid sequence between positions 207 and 223 of human X11L.
Another polyclonal anti-X11L carboxyl-terminal domain antibody, UT-30, was raised against the peptide "Cys" plus containing the amino acid
sequence between positions 735 and 749 of human X11L. These peptides
were synthesized at Quality Controlled Biochemicals Inc. (Hopkinton,
MA). UT-29 and UT-30 are specific to X11L (see "Results") and
appear not to react with X11. These antibodies were affinity-purified using antigen coupled to SulfoLink gel (Pierce). Anti-APP polyclonal antibody UT-18 was prepared against APP695-(676-695). The specificity was identical to that of another anti-APP polyclonal antibody, UT-421,
which has been fully characterized (15, 23). Anti-A Preparation of Proteins and in Vitro Interaction of Human X11L
with APP--
The cDNA clones encoding human X11L, the domain
structures of human X11L, and the cytoplasmic domain of human APP695
were subcloned into pGEX-4T-1 (Amersham Pharmacia Biotech) to produce GST fusion protein. The recombinant plasmids were introduced into E. coli JM109. Production of the fusion protein was induced
by culturing the recombinant E. coli strains in the presence
of 1 mM isopropyl-
Whole cell extracts of 293 cells that expressed stably transfected
APP695 cDNA (pcDNA3APP695) (23), human X11L cDNA
(pcDNA3-hX11L), and cDNA encoding the domain structures of
human X11L were prepared by lysing cells in lysis buffer (PBS
containing 10 mM CHAPS, 5 µg/ml chymostatin, 5 µg/ml
leupeptin, 5 µg/ml pepstatin A, 1 mM Na3VO4, and 1 mM NaF) for 1 h
on ice, followed by centrifugation at 12,000 × g for
10 min at 4 °C. The resulting supernatant containing APP695 and
protein constructs from human X11L were used for the binding assay.
The protein samples, which were adjusted to contain approximately 2-4
nmol of protein, were applied to a column (100-µl bed volume) of
glutathione-Sepharose beads bearing GST fusion protein or GST alone
(control). The beads were washed with binding buffer three times, and
the protein complex was eluted with elution buffer. Following SDS-PAGE,
the proteins were analyzed by immunoblotting with the indicated
antibodies and 125I-Protein A (Amersham Pharmacia Biotech).
Protein concentration was determined by the micro-Lowry method with
bovine serum albumin as a standard (26).
Co-immunoprecipitation--
COS7 cells (~1 × 107 cells) were doubly transfected with 2 µg of
pcDNA3APP695 and 2 µg of pcDNA3-hX11L in Lipofectamine (Life Technologies) for 18 h. After changing the medium, the cells were cultured for 48 h in Dulbecco's modified Eagle's medium
containing 10% (v/v) heat-inactivated fetal bovine serum. The cells
were harvested and lysed in 1 ml of lysis buffer (PBS containing 10 mM CHAPS, 5 µg/ml chymostatin, 5 µg/ml leupeptin, 5 µg/ml pepstatin A, 1 mM Na3VO4,
and 1 mM NaF) for 1 h on ice and centrifuged at 12,000 × g for 10 min at 4 °C. The affinity-purified polyclonal antibodies (50 µg) UT-18 and UT-30 and monoclonal antibodies (50 µg) 2D1 and 25C were added to the supernatant and incubated on ice
for 1 h. Protein G-Sepharose beads were then added and incubated for 1 h at 4 °C, and the samples were centrifuged. The pellets were washed with the lysis buffer three times, and proteins were eluted
by boiling the pellet in SDS-sample buffer (23). Proteins were then
analyzed by SDS-PAGE (6% (w/v) polyacrylamide) and immunoblotted with
the indicated antibodies, and immunocomplexes were revealed by ECL
(Amersham Pharmacia Biotech).
Immunocytochemistry--
293 cells that express stably
transfected pcDNA3-hX11L were fixed for 20 min with 4% (w/v)
paraformaldehyde in PBS (pH 7.4) containing 4% (w/v) sucrose,
permeabilized with 0.2% (v/v) Triton X-100 for 5 min and blocked for
1 h in PBS containing 0.2% (v/v) goat serum. The same cells were
double-stained with rhodamine-conjugated Con A (Vector Laboratories,
Inc., Burlingame, CA), which binds with high affinity to glycoproteins
in the ER and cis-Golgi, or with rhodamine-conjugated WGA
(Vector Laboratories), which binds with high affinity to glycoproteins
in medial and trans-Golgi (27, 28). In a separate
study, 293 cells that express stably transfected pcDNA3-APP695 were
further transfected with pcDNA3-hX11L transiently. The cells were
incubated with anti-human X11L antibody UT-30 and anti-APP monoclonal
antibody LN27 and then with fluorescein isothiocyanate- or rhodamine
isothiocyanate-conjugated secondary antibody (Zymed
Laboratories Inc., San Francisco, CA). The coverslips were
mounted in immersion oil type B (R. P. Cargille Laboratories Inc.,
Cedar Grove, NJ), and the cells were viewed using a confocal laser
scanning microscope, Bio-Rad MRC 600.
Western Blot Analysis of Mouse X11L from Adult
Tissues--
Tissues from adult ddY mice (4-5 weeks) were homogenized
and sonicated in a solution containing 50 mM Tris-HCl (pH
7.4), 1% (w/v) SDS, 2.7 M urea, 25 µg/ml pepstatin A, 25 µg/ml leupeptin, and 25 µg/ml chymostatin. Samples were centrifuged
(10,000 × g, 10 min), the resulting supernatant (150 µg of protein) was subjected to SDS-PAGE (7.5% (w/v)
polyacrylamide), and proteins were electrophoretically transferred to a
nitrocellulose membrane. The membrane was probed with UT-30 and
125I-Protein A (Amersham Pharmacia Biotech). Radioactivity
was analyzed by autoradiography.
Comprehensive Gene Mapping--
The somatic cell hybrid Southern
DNA membranes, BIOSMAP, were purchased from BIOS Laboratories (New
Haven, CT). Southern hybridization was performed with a probe prepared
from a template containing the insert from pcDNA3-hX11L with random
oligonucleotides (High Prime DNA labeling kit; Boehringer Mannheim) and
[ Northern Analysis of Human X11L--
Human multiple tissue
Northern blots (catalog no. 7760-1) were purchased from
CLONTECH Laboratories. Hybridizations were carried out using the standard procedure (29). Radioactive probes with a
specific activity equal to, or higher than, 1 × 108
cpm/µg were prepared from a template of human X11L cDNA coding for the amino acid sequence between positions 126 and 749 using [ ELISA Analysis--
The 293 cell line that expresses stably
transfected pcDNA3APP695 (15, 23) was co-transfected with 10 µg
of pcDNA3-hX11L, which contained the cDNA sequence encoding the
full-length form of human X11L (amino acid sequence 1-749),
pcDNA3-N + PI, which contained the cDNA encoding the
amino-terminal region attached to the PI domains (amino acid sequence
1-555), pcDNA3-PI, which contained the cDNA encoding the PI
domain (amino acid sequence 368-555), or the control plasmid
pcDNA3, which lacked a cDNA sequence (Invitrogen, Carlsbad,
CA). The cells were supplied with fresh growth media 12 h after
the start of transfection, and conditioned medium from cells (2 × 10 6 cells) was collected 72 h after the medium
change. A Molecular Cloning of a cDNA Encoding Human X11L Protein:
Primary Structure and Chromosome Location--
The short cytoplasmic
domain of APP is the sole region exposed to the cytoplasm and contains
several functional amino acid motifs that regulate the metabolism of
APP (13-15, 30, 31). Therefore, the isolation and characterization of
proteins which interact with APPCOOH are important for our
understanding the physiological function and metabolism of APP.
Approximately 106 independent clones from an adult human
brain cDNA library were screened by the yeast two-hybrid system
with a "bait" vector containing the cDNA encoding the 47-amino
acid APPCOOH region, pGBT9APPCOOH. Ultimately, 125 clones were isolated as positive for nutrient (His)
selection and
The human X11L cDNA encoded a protein consisting of 749 amino acids
and presented 55.2% amino acid homology to the partially known amino
acid sequence of human X11 and 90.2% homology to the complete sequence
of mouse X11, which consists of 748 amino acids. Thus, we designated
this protein as human X11-like protein (human X11L), whose partial
sequence consisting of 188 amino acids comprising the PI domain has
already been reported (22). Furthermore, mouse X11 (25) is thought to
be a homologue of human X11L because it possesses higher homology to
human X11L than to human X11. Thus, we surmise that the mouse
counterpart of the human X11 gene has not yet been
identified. Therefore, we have referred to mouse X11 as mouse X11L in
the present paper (Fig. 1a).
The human X11L protein contains a PI domain (Fig. 1a,
yellow box) and two PDZ domains (Fig.
1a, blue boxes) (the repeated sequences of the brain-specific protein PSD-95,
Drosophila septate junction protein disks-large,
and the epithelial tight-junction protein ZO1) (32) in its
carboxyl-terminal domain, as does human X11 (Fig. 1b). The
PI domain (amino acid positions 368-555, 82.4% homology) as well as
the region that lies on the carboxyl-terminal side (amino acid
positions 556-749, 85.6% homology) of the PI domain, but not the
amino-terminal domain (amino acid positions 1-367, 20.6% homology),
are highly conserved between the human X11L and X11 proteins (Fig.
1).
The chromosomal location of the human X11 gene has been
fully characterized (21). The gene is located on 9q13-q21 within 0.7 centimorgans of the D9S5 and D9S15 loci. We
identified the chromosome on which human X11L gene is
located using a comprehensive gene mapping procedure with the somatic
cell hybrid Southern DNA membrane as described under "Experimental
Procedures". Southern hybridization with a probe of human X11L whole
cDNA, pcDNA3-hX11L, indicated that the human X11L
gene is located on human chromosome 15 (data not shown), a location
that is distinct from that of the X11 gene located on
chromosome 9.
Binding of Human X11L Protein to APP--
X11 and Fe65 have
been shown to interact with the YENPTY (APP-(682-687) of APP695
isoform) motif of APPCOOH through their PI domains. In
contrast to the interaction involving the Shc PI domain, the
interaction is independent of APP tyrosine phosphorylation (20, 33). To
characterize the binding of X11L to APP, we performed a yeast
two-hybrid study using a combination of the cDNA encoding the
domain sequence derived from human X11L in pGAD424 and the cDNA of APPCOOH, pGBT9APPCOOH (Fig.
2), and another combination of the
cDNA coding for almost the whole length of human X11L, pGAD424#104,
and the cDNA of APPCOOH truncated internally in pGBT9 (Fig. 3a). The
When the binding site of the cytoplasmic domain of APP was examined
(Fig. 3a), we found that a cDNA,
pGBT9APP695
APP is a member of a gene family that includes
APLP1 (34) and APLP2 (35). The amino acid
sequences of these proteins are highly conserved, especially within
their cytoplasmic domains. The binding ability of human X11L to the
cytoplasmic domain of APLPs was examined using the yeast two-hybrid
system (Fig. 3b). When pGAD424#104 was co-transfected with
pGBT9APLP2COOH consisting of the cDNA encoding the
47-amino acid human APLP2 cytoplasmic domain (Fig. 3b,
APLP2), or pGBT9APLP1COOH consisting of the
cDNA encoding the 46-amino acid human APLP1 cytoplasmic domain
(Fig. 3b, APLP1) in yeast, equal or greater
Interaction of APP with Human X11L Using GST Fusion Proteins in
Vitro--
The protein-protein interaction of human X11L with APP was
examined in vitro using GST-human X11-like fusion protein
(GST-hX11L) and human APP695 (Fig. 4,
a and b). Glutathione-Sepharose beads bearing GST
fusion proteins, whose constructs are shown in Fig. 4d, were
incubated with the lysate of 293 cells that were stably transfected
with pcDNA3APP695 containing human APP695 cDNA. The beads were
washed with binding buffer three times, and protein complexes were
eluted with PBS containing 5 mM reduced glutathione. Following SDS-PAGE, the proteins were analyzed by immunoblotting with
anti-APP antibody UT-18 plus 125I-Protein A
(arrows in Fig. 4, a and b (longer
exposure of a), indicate mature (m,
upper) and immature (im, lower) APP695
isoforms). APP was found to associate with the PI domain
(PI, amino acid positions 368-555) as well as with
full-length human X11L (human X11L). The PI domain attached
to the amino-terminal domain (N + PI; amino acid positions
1-555) enhanced binding to APP, and the PI domain attached to the
carboxyl-terminal domain (PI + C, amino acid positions
368-749) suppressed binding to APP. The amino-terminal (N,
amino acid positions 1-367) and carboxyl-terminal (C, amino acid positions 556-749) domains alone did not bind to APP. The results
of these assays corroborate the results obtained using the yeast
two-hybrid assay (Fig. 2). In addition, the results suggested that the
amino- and/or carboxyl-terminal domains may be regulatory element(s) of
PI domain binding to APP.
In order to confirm the protein-protein interaction of human X11L with
APPCOOH, the studies described in Fig. 4, a and
b, were performed again but with the difference that
glutathione-Sepharose beads bearing GST- APPCOOH fusion
protein (APPCOOH) were incubated with the
cell lysates from 293 cells that expressed stably transfected pcDNA3-hX11L (human X11L), pcDNA3-N + PI (N + PI), or pcDNA3-N (N) (Fig. 4c). These
cell lysates contain the respective protein constructs as shown in
"crude" of Fig. 4c. The beads were washed with binding
buffer three times, and protein complexes were eluted with PBS
containing 5 mM reduced glutathione. Following SDS-PAGE, the proteins were analyzed by immunoblotting with anti-X11L antibody UT-29 plus 125I-Protein A. Full-length human X11L was found
to associate with GST-APPCOOH fusion protein on the beads
(human X11L). The PI domain fused to the amino-terminal
domain (amino acid positions 1-555) associated strongly with the
GST-APPCOOH fusion protein on the beads (N + PI). However, the amino-terminal domain (amino acid positions
1-367) alone did not associate with GSTAPPCOOH fusion protein (N). The beads carrying GST peptide alone
(GST) did not present any appreciable ability to associate
with human X11L or its domain structure proteins. We have not studied
binding of the PI domain (PI) alone, nor the PI domain fused
to the carboxyl-terminal domain (PI + C), nor the
carboxyl-terminal domain alone (C), because we had no
PI-specific antibody and could not establish 293 cell lines stably
transfected with pcDNA3-PI + C or pcDNA3-C. However, the
results broadly confirmed the data presented in Fig. 4, a and b, and, especially, proved that deletion of the human
X11L carboxyl-terminal domain enhances the binding of human X11L to APPCOOH.
Specificity of the Anti-X11L Antibody and Brain-specific Expression
of X11L--
Antibodies raised against sequences in the amino-terminal
(UT-29) and the carboxyl-terminal (UT-30) regions of the PI domain were
examined for their specificity. Human X11L tagged with the FLAG peptide
on its carboxyl-terminal side (FLAG-hX11L) was prepared. From the
lysates of 293 cells, which were stably transfected with FLAG-hX11L
cDNA (pcDNA3-FLAG-hX11L), proteins were immunoprecipitated (IP) with anti-FLAG (M2), UT-29, or UT-30
antibodies, separated by SDS-PAGE, transferred to nitrocellulose
membrane, and probed (WB) with the indicated antibodies
(Fig. 5a). Because a
~135-kDa protein (arrow) was detected with all three types
of antibodies, we designated the ~135-kDa protein as FLAG-hX11L. When
crude cell lysates were probed with these three antibodies, the
~135-kDa protein was also specifically detected (Fig. 5a,
crude). Preincubation of antibodies with the corresponding
antigen peptides (10 µM) resulted in the failure of these
antibodies to recognize the ~135-kDa protein (data not shown). The
results demonstrate that both UT-29 and UT-30 antibodies are highly
specific for human X11L and that they are potentially useful for
detecting endogenous X11L. Endogenous X11L was also analyzed (Fig.
5b); mouse X11L was immunoprecipitated (IP) from
mouse brain lysate with UT-29 or UT-30. The immunoprecipitate was
analyzed by Western blot (WB) with UT-29 or UT-30. When
crude brain lysate (Fig. 5b, crude) was probed
directly, the ~135-kDa protein (arrow) was also detected
by both UT-29 and UT-30. The efficiency of immunoprecipitation of mouse
X11L by UT-30 is much higher than that of UT-29 (compare
left part with middle part of Fig. 5b), although UT-29 and UT-30 immunoprecipitate
human X11L equally well as shown in Fig. 5a. The amino acid
sequence of the region used to raise the UT-29 antibody was not
identical between human and mouse (15/17 amino acid identity) X11L,
although that used to raise UT-30 was identical (15/15 amino acid
identity). Since UT-29 was raised against the human sequence, the
antibody may not be as effective in recognizing the mouse protein,
especially in immunoprecipitation. The molecular size of X11L deduced
from the cDNA is 82.5 kDa, which is much smaller than 135 kDa, the value determined by SDS-PAGE. One explanation for this discrepancy could be that X11L is subject to modifications such as glycosylation. However, this does not seem plausible, because the GST-hX11L fusion synthesized in E. coli also migrated as a 160-kDa protein
during SDS-PAGE, and a ~135-kDa protein after the removal of the GST peptide by digestion with thrombin (data not shown). The most plausible
explanation is that X11L adopts a secondary structure that results in
anomalous migration during SDS-PAGE.
It has been reported that human X11 is expressed dominantly (21) but
not specifically2 in brain
tissues. However, the expression patterns of human and mouse X11L have
not been examined, although both X11 and X11L proteins are similar with
respect to structure and APP binding. The expression of mouse X11L in
various tissues was examined (Fig. 5c). Protein extracts
(150 µg of protein) from various adult mouse tissues were analyzed by
Western blot with UT-30 antibody. A ~135-kDa mouse X11L protein (Fig.
5c, arrow) was detected specifically in brain but
not in other nonneuronal tissues such as heart, lung, liver, testis,
spleen, thymus, or kidney. The results indicated brain-specific
expression of X11L protein.
We further performed Northern blot analysis using a probe derived from
human X11L cDNA as described under "Experimental Procedures." This Northern blot analysis showed a strong 4.4-kb and a comparatively weak 4.0-kb message in brain tissue but not in the other nonneuronal tissues examined (Fig. 6a).
The actin gene was found to exist in all tissues (Fig. 6b).
The size of the mRNA is in good agreement with the size of human
X11L cDNA and clearly differs from the mRNA size of human X11
(21). Thus, we concluded that the major 4.4-kb product is the mRNA
that encodes human X11L. The minor 4.0-kb product may be a truncated
mRNA or a minor product derived from alternative splicing of human
X11L gene transcripts. These results demonstrate that
X11L gene is specifically expressed in neuronal tissue and
suggests that the protein product may play a role in neuron-specific
function(s) through association with APP.
In Vivo Interaction of Human X11L with APP--
To examine whether
human X11L and APP interacted in cells, co-immunoprecipitation
experiments were performed. COS7 cells that transiently express human
X11L and APP695 were lysed in the presence of 10 mM CHAPS.
The detergent-soluble fraction was subjected to immunoprecipitation
(IP) using affinity-purified UT-30 or anti-APP extracellular
domain monoclonal antibodies 2D1 and 25C (Fig.
7). The same quantity of normal rabbit or
mouse IgG was used as a control. The immunoprecipitates were analyzed
by Western blot (WB) using UT-18 (Fig. 7a) or
UT-30 (Fig. 7b) antibody. APP was detected in the sample
immunoprecipitated with UT-30 (Fig. 7a). Human X11L was
detected in the sample immunoprecipitated with 2D1 and 25C (Fig.
7b). APP and human X11L could not be detected in the sample
immunoprecipitated using normal rabbit or mouse IgG, indicating that
the co-immunoprecipitation of APP and human X11L was specific. The
result suggests that APP and X11L interact in mammalian cells.
Intracellular Distribution of Human X11L and APP--
The
intracellular distribution of human X11L was examined by an
immunocytochemical study using 293 cells stably expressing human X11L
protein. The 293 cells that express stably transfected pcDNA3-human
X11L were double-stained with UT-30 and WGA (medial- plus
trans-Golgi marker) or ConA (ER plus cis-Golgi
marker) (23, 27, 28) to analyze the intracellular distribution of human X11L (Fig. 8, a and
b). In a separate study, the 293 cells stably transfected
with pcDNA3-APP695 and transiently transfected with pcDNA3-hX11L were double-stained with UT-30 and LN-27 antibodies to
analyze for co-distribution of human X11L and APP (Fig. 8c). The cells were then observed under a confocal laser scanning
microscope. We found that the staining of human X11L with UT-30
(green in Fig. 8, a and b) partially
co-localized with the staining obtained using WGA (red in
Fig. 8a) and ConA (red in Fig. 8b). It
is also clear that UT-30 did not stain the plasma membrane. These
results show that X11L protein is present in the cytoplasm and to a
lesser extent in the ER and Golgi apparatus as shown by the
yellow color (hX11L/WGA in Fig.
8a and hX11L/ConA in Fig. 8b). LN27
stained the Golgi apparatus strongly (red in Fig. 8c), which
agrees with a previous report on the cellular distribution of APP (23). Taken together, these observations indicate that APP (red)
may be colocalized with human X11L (green) mostly in the
Golgi apparatus (hX11L/APP in Fig. 8c), although
the majority of human XIIL was localized in the cytoplasm. The antibody
that had been preadsorbed to antigen did not show any staining (data
not shown).
The distribution of X11L was confirmed by a biochemical study. Mouse
brain homogenate was fractionated into nuclear, membrane, and cytosolic
fractions and then analyzed by immunoblot analysis with the UT-30
antibody. Most of the mouse X11L protein was detected in the cytosolic
fraction with a smaller quantity of mouse X11L being also detected in
crude membrane fractions consisting of the plasma membrane, ER, and
Golgi apparatus membranes. This indicates that X11L may be attached to
these membranes through association with APP (data not shown). The
results of the immunocytochemical (Fig. 8) and biochemical studies
indicate that X11L is most likely a cytosolic protein with a tendency
to co-distribute with APP.
Role of X11L Protein in APP Processing: Generation of APP was identified as a causative factor in the pathogenesis of AD
(1, 5-11). Several mutations of APP and other AD-related genes have been found in familial AD, and it has been reported that
these mutations contribute to the pathogenesis of familial AD (reviewed
in Refs. 36 and 37). However, familial AD represents only a small
minority of AD cases. The majority of AD cases are of the sporadic
type, whose patients are not thought to carry the mutation. Therefore,
elucidation of the physiological function(s) and metabolism of APP is
critical for our understanding of the pathogenesis of sporadic type AD.
The cytoplasmic domain of APP, or APPCOOH, which is highly
conserved phylogenetically in vertebrates (38), contains signals for
APP metabolism (13-15) and is thought to contain the amino acid
sequence essential for the transduction of signals from an
extracellular ligand that remains to be identified (reviewed in Ref.
16). Consequently, research based on the identification and isolation
of factors that interact with APPCOOH has been pursued (17-20, 39, 40). In the present paper, we isolated a cDNA encoding a novel protein, human X11L, as a protein that binds to
APPCOOH. This human X11L possesses 55.2%
homology to human X11, which has been identified as a gene
in the region of the Friedreich ataxia locus (21), a degenerative
disorder involving both the central and peripheral nervous system (41).
Although a partial amino acid sequence consisting of 165 amino acids in
the PI domain of X11L has been identified as the APP binding motif (22)
and the interaction between the PI domain of X11 and a sequence
containing the NPTY motif of APPCOOH has been fully
analyzed (20, 33), the biological function of these proteins has not
been determined. Because the amino acid homology between the PI domains
from human X11 and human X11L is very high (82.9%), the architecture
and APPCOOH recognition mechanism of the PI domains from
both proteins may be similar. In the present study, we demonstrate that
the interaction of human X11L with APPCOOH is enhanced by
deletion of the carboxyl-terminal domain and suppressed by deletion of the amino-terminal domain (Figs. 2 and 4). One possible explanation for
these results is that both domains function as regulatory elements of
PI domain binding to APPCOOH, possibly indicating that the
amino-terminal domain contains an "enhancer" sequence and the
carboxyl-terminal domain contains a "suppressor" sequence for PI
domain binding to APPCOOH. The deletion of the amino- or carboxyl-terminal domain may alter the conformation of the PI domain,
and this may affect the binding affinity to APPCOOH.
Although these deleted constructs of human X11L are not physiological
products, in vivo, proteins that can interact with the
amino- and/or the carboxyl-terminal domains of X11L may regulate APP
binding to the PI domain by inducing conformational changes in human
X11L. We propose a possible hypothesis for the regulation of X11L
binding to APPCOOH (Fig.
10). In cells, X11L exists in a free
form and shows a weak affinity for APPCOOH (Fig.
10a). When an unidentified "factor X" binds to the
amino-terminal domain of X11L, the PI domain adopts an altered
conformation and acquires strong binding affinity for
APPCOOH (Fig. 10b). The construct deleted for
the carboxyl-terminal domain (N + PI construct) may mimic this strong binding state of X11L (Fig. 10d). Then the PDZ domain(s) of
the stable X11L·APP complex, associate with another factor, "factor Y," resulting in the suppression of A40 but not that of
A
42. However, overexpression of the PI domain alone and the N + PI
construct in cells did not affect the secretion of A
despite their
ability to bind to the cytoplasmic domain of Alzheimer's amyloid
precursor protein. These observations suggest that the amino-terminal
domain regulates PI binding to APP and that the carboxyl-terminal
domain containing PDZ motifs is essential to modulate APP processing. Because expression of the human X11L gene is specific to
brain, the present observations should contribute to shedding light on the molecular mechanism of APP processing in Alzheimer's disease.
INTRODUCTION
Top
Abstract
Introduction
References
-amyloid (A
) (2-4), which is derived from APP by proteolytic cleavage (1, 5-11). A
is thought to be generated through an intracellular protein secretory pathway of APP (for a review, see Ref.
12). The short APP cytoplasmic domain consisting of 47 amino acid
residues is thought to be responsible for determination of APP
metabolism (13-15) and possible signal transduction from a putative
extracellular ligand that has yet to be identified (for a review, see
Ref. 16). Because APP is a candidate pathogenic factor of AD,
elucidation of the physiological function of APP as well as the
determination of the metabolic mechanism of A
production should
increase our understanding of the pathogenesis of AD. Using a yeast
two-hybrid system, we isolated cDNA of proteins that interact with
the cytoplasmic domain of APP (APPCOOH) in order to
elucidate the molecular mechanisms of APP metabolism and function of
APP. Previous efforts to identify and isolate proteins associating with
APPCOOH, utilizing the yeast two-hybrid system, have
resulted in the isolation of proteins carrying phosphotyrosine binding/phosphotyrosine interaction (PI) domains such as Fe65 (17),
Fe65-like (18, 19), and X11 (20).
40 but not A
42. However, co-transfection into cells
of the cDNA lacking the carboxyl-terminal domain (N + PI construct)
or a cDNA encoding only the PI domain (PI construct), whose protein
products preserve the ability to bind to APPCOOH, did not
present the ability to modulate A
production. The present results
suggest that the amino-terminal region of the PI domain is needed to
regulate binding affinity to APPCOOH and that the carboxyl-terminal region of the PI domain is essential for modulation of APP metabolism. Furthermore, Northern blot and Western blot analyses
demonstrated strong specific expression of the X11L gene in
brain tissues. Together with the present results, this suggests that
X11L may play an important role in the physiological function and
metabolism of APP in neuronal tissues.
EXPERIMENTAL PROCEDURES
681-690) and 669-680
(pGBT9APP695
669-680). The cDNA encoding the cytoplasmic
domain of amyloid precursor-like protein 2 (APLP2) was cloned from an
adult human brain cDNA library by the reverse yeast two-hybrid
system with the "bait vector," pGBT9#104, and the cDNA
encoding the cytoplasmic domain of human amyloid precursor-like protein
1 (APLP1) was cloned by reverse transcription-PCR using human brain
poly(A)+ RNA and primers with nucleotide sequences
corresponding to positions 605-611 (forward) and 644-650 (reverse)
(numbering as for the human APLP1 genome sequence,
DDBJ/EMBL/GenBankTM accession no. AD000864). These
cDNAs encoding 53 and 47 amino acid residues of the cytoplasmic
domain of APLP1 and APLP2, respectively, were inserted into pGBT9
to produce pGBT9hAPLP2COOH and pGBT9hAPLP1COOH. These constructs in pGBT9 vectors were used to examine the binding ability for human X11L (pGAD424#104) in the yeast two-hybrid system.
-Galactosidase activity in the yeast two-hybrid system was measured
in a liquid assay using o-nitrophenylgalactopyranoside according to the manufacturer's protocol (CLONTECH
Laboratories; catalog no. PT1265-1) and was expressed in Miller units.
monoclonal
antibody, 2D1, recognizes a human-specific epitope FRH-(600-602),
which lies between the
- and
-secretase sites (23); 25C was
prepared against human APP695-(621-631) (A
-(25-35)). Anti-FLAG
monoclonal antibody M2 (Eastman Kodak Co.) and anti-APP extracellular
domain monoclonal antibody LN27 (Zymed Laboratories Inc., San Francisco, CA) were purchased.
-D-thiogalactopyranoside.
Cells were lysed in phosphate-buffered saline (PBS; 140 mM
NaCl and 10 mM sodium phosphate (pH 7.4)) by sonication,
and the lysate was centrifuged at 10,000 × g for 20 min at 4 °C. The resulting supernatant was applied to a
glutathione-Sepharose 4B (Amersham Pharmacia Biotech) column (1-ml bed
volume), and proteins bound to the beads were recovered with an elution
buffer consisting of 5 mM glutathione and 50 mM
Tris-HCl (pH 7.4). Affinity-purified GST fusion proteins were dialyzed
against a buffer consisting of 20 mM Tris-HCl (pH 7.5)/2
mM EDTA/1 mM dithiothreitol, and the purity of
the protein was examined by staining with Coomassie Brilliant Blue
R-250 following SDS-PAGE. To prepare the affinity beads coupled to the
GST fusion protein, 2-4 nmol of GST fusion proteins were incubated
with 100 µl of glutathione-Sepharose 4B for 1 h at 4 °C with
rotation. The efficiency of coupling, almost greater than 95% under
these conditions, was determined by examining the protein content of
the uncoupled protein.
-32P]dCTP (3000 Ci/mmol; NEN Life Science Products)
according to the user guidelines.
-32P]dCTP (3000 Ci/mmol, NEN Life Science Products)
and random oligonucleotides (High Prime DNA labeling kit; Boehringer Mannheim).
40 and A
42 were quantified with sandwich ELISA using
three types of A
-specific monoclonal antibodies as described
previously (15, 23). Briefly, wells were coated with the A
40 (4D1)
or A
42 (4D8) end-specific monoclonal antibodies (0.3 µg of IgG in
PBS), washed with PBS containing 0.05% (v/v) Tween 20 (washing
buffer), blocked with bovine serum albumin (3% (w/v) in PBS), and
washed with washing buffer, and then a sample (100 µl) diluted
suitably with washing buffer containing 1% (w/v) bovine serum albumin
(dilution buffer) was incubated together with a standard dose of
synthetic A
-(1-40) and A
-(1-42) peptides (synthesized using
solid phase t-BOC
(N-tert-butyloxycarbonyl)-chemistry by Dr. James I. Elliott
at the W. M. Keck Foundation Biotechnology Resource Laboratory,
Yale University). After washing, wells were treated with biotinized 2D1
(12. 5 ng in dilution buffer), washed, and incubated with 100 µl of a
streptavidin-horseradish peroxidase complex (1:2000 dilution; Amersham
Pharmacia Biotech catalog no. RPN1051). The plates (96 wells) were
washed further, 100 µl of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid solution (KPL
5062-01, Kirkegaard & Perry Laboratories, Gaithersburg MD) was added
to the wells, and incubation was continued at room temperature. The
reaction was stopped by adding 100 µl of 1% (w/v) SDS, and the
absorbance at 405 nm was measured.
RESULTS
-galactosidase activity. The nucleotide sequences of
all the positive candidates were determined. Out of the 125 clones, 88 encoded the amino acid sequence of Fe65 (17) or Fe65-like (18)
proteins, which have previously been reported to be APP-binding proteins. Nucleotide sequences of the other four clones revealed a
novel protein sequence with higher homology to mouse X11 than to human
X11 (25). Other sequences were nonsense clones, which were not thought
to encode significant proteins. One of the four cDNA clones,
pGAD424#104, contained an open reading frame coding for a potentially
novel protein of 624 amino acids, as shown in amino acid positions
126-749 of human X11L (Fig.
1a), and contained a
3'-untranslated and poly(A)+ sequence. To identify the 5'
nucleotide sequence of this clone, we performed the RACE procedure
using poly(A)+ RNA from human adult brain and the indicated
primer (underlined in Fig. 1a), which corresponds
to amino acid positions 130-135. An approximately 620-nucleotide
sequence including the missing 260 base pairs of the 5'-untranslated
region was determined, and the whole amino acid sequence of this clone
was deduced (Fig. 1a). This protein showed high homology to
the partial amino acid sequence of mouse X11 rather than to human X11.
Because the amino-terminal sequence of mouse X11 has not been reported
(25), we also determined the 5' nucleotide sequence of mouse
X11 by the RACE procedure using poly(A)+ RNA
from mouse adult brain and the indicated primer (underlined in Fig. 1a), which corresponds to amino acids 80-85. The
entire amino acid sequence of the amino-terminal part of mouse X11 was deduced, and the sequence was used to look for homology between the
three related proteins (Fig. 1).
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Fig. 1.
Comparison of amino acid sequences and
homology between the human X11-like, mouse X11-like, and human X11
proteins. a, alignment of human X11L
(hX11L), mouse X11L (mX11L), and human X11
(hX11) protein amino acid sequences. Gaps produced by the
alignment are indicated by a hyphen in the sequence.
Predicted phosphotyrosine interaction (PI) domain (yellow
box) and PDZ domains (blue boxes) are
indicated. The first methionine residue was chosen as the methionine
after the termination codon. The nucleotide sequence surrounding the
methionine seems to match the Kozak consensus sequence (55). Although
mouse X11-like was previously reported as a partial amino acid sequence
of mouse X11 (25), we designated this mouse X11 as mouse X11L (see
"Experimental Procedures"). The positions of amino acids are
relative to those of human X11L. b, general characteristics
and homology between the three proteins. Amino acid homologies between
human X11L and other proteins are indicated. PI,
phosphotyrosine interaction domain (yellow); PDZ,
PDZ domain (blue) predicted from the consensus sequence of
other PDZ domains (50). Numbers indicate amino acid
positions of human X11L. Amino acid homology (percentage of human X11L)
of mouse X11L and human X11 to human X11L is indicated.
-galactosidase activities (Miller units) of both combinations were
measured and compared (Figs. 2 and 3a). The PI domain
(pGAD424#PI, containing the cDNA coding for amino acids 368-555)
was essential for binding to APP and gave almost the same activity as
that of the plasmid coding for the full-length human X11L,
pGAD424#104. We found that when the PI domain was attached to the
amino-terminal domain (pGAD424#1, containing the cDNA coding for
amino acids 126-555) the activity was enhanced (2-fold). However, when
the PI domain was attached to the carboxyl-terminal domain (pGAD424#4,
containing the cDNA coding for amino acids 368-749) APP binding
ability was markedly suppressed (Fig. 2). The amino-terminal (pGAD424#6
containing cDNA coding for amino acids 126-367) and
carboxyl-terminal (pGAD424#7 containing cDNA coding for amino acids
556-749) domains alone were negative for both
-galactosidase
activity and His nutrient selection and had no ability by themselves to
bind to APPCOOH. A cDNA construct coding for amino
acids 1-125 gave a nonspecific and constitutive positive signal in the
nutrient and
-galactosidase assays of the yeast two-hybrid system,
which indicates that this region contains an artificial trans-activator
sequence of Gal4-mediated transcription. Therefore, we deleted this
region from the cDNA constructs for the two-hybrid assays.
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Fig. 2.
Binding of the human X11-like protein
constructs to the cytoplasmic domain of APP in yeast.
a, binding between constructs derived from human X11L and
APPCOOH was quantified by the liquid -galactosidase
assay. Results are an average of six independent studies
(n = 6), and the error bar
indicates S.D. Control indicates a study with the plasmid
alone.
-Galactosidase activity is indicated in Miller units.
b, schematic structures of human X11L and the constructs
derived from human X11L used in this study. PI,
phosphotyrosine interaction domain; PDZ, PDZ domain.
Numbers indicate amino acid position.
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Fig. 3.
Binding of human X11-like protein to protein
constructs derived from the cytoplasmic domain of APP and to the
cytoplasmic domain of APP family proteins in yeast. a,
binding between human X11L (hX11L) and the protein
constructs derived from APPCOOH was quantified by the
liquid -galactosidase assay. Results are the average of six
independent studies (n = 6), and the error
bar indicates S.D. wt, the cytoplasmic domain of
APP695;
669-680, the cytoplasmic domain of APP695
lacking the amino acid sequence of APP695-(669-680);
681-690, the cytoplasmic domain of APP695 lacking the
amino acid sequence of APP695-(681-690). The amino acid sequences of
the protein constructs are also indicated. b, binding
activity of human X11L to the cytoplasmic domain of APP gene family,
APP695, APLP1650, and APLP2763. Results are the average of six
independent studies (n = 6), and the error
bar indicates S.D. The amino acid sequence of the
cytoplasmic domain of the APP gene family proteins is also indicated.
Control indicates a study with the pGBT9 plasmid alone.
-Galactosidase activity is indicated in Miller units.
681-690, encoding the cytoplasmic domain with a
deletion of amino acids 681-690 containing the NPTY sequence had lost
binding ability for human X11L. Another construct,
pGBT9APP695
669-680, with a deletion of the amino-terminal 15 amino
acid residues to the NPTY motif retained binding ability, but the
binding was slightly weaker compared with that of the whole sequence
(wild type). The results indicate that the sequence containing the NPTY
motif of APP is essential for association with human X11L as well as
with X11 and Fe65/Fe65-like (17-20).
-galactosidase activities were observed than for APPCOOH
(Fig. 3b, APP). These results indicate that human
X11L associates with APP family proteins, APP, APLP1, and APLP2, all of
which contain NPXY motifs. The control vector, pGBT9, which
did not contain cDNA gave no
-galactosidase activity (Fig.
3b, control).
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Fig. 4.
Interaction of APP695 and the cytoplasmic
domain of APP695 with protein constructs derived from human X11-like
protein in vitro. a, binding of APP695 to
protein constructs derived from human X11L. Whole cell extracts
(approximately 1 mg of protein) of 293 cells stably transfected with
APP695 cDNA were applied to columns of glutathione beads bearing
the different GST fusion protein constructs as indicated in
d. The bound APP695 protein was eluted together with the
protein constructs of human X11L, separated by SDS-PAGE (10% (w/v)
polyacrylamide), transferred to a nitrocellulose membrane, and detected
by anti-APP antibody UT-18. Arrows indicate APP695
(m, mature APP; im, immature APP).
hX11L, full-length human X11L; N + PI,
amino-terminal domain attached to the PI domain; N,
amino-terminal domain alone; PI + C, PI domain attached to
the carboxyl-terminal domain; PI, PI domain alone;
C, carboxyl-terminal domain alone. b, longer
exposure of the autoradiogram of a. c, whole cell
extract (approximately 1 mg of protein) of 293 cells stably transfected
with cDNAs encoding constructs derived from human X11L was applied
to a column of glutathione beads bearing the GST-APPCOOH
fusion protein (APPCOOH) and GST protein alone
(GST). The protein constructs from the human X11L-bound
fraction of the GST-APPCOOH beads were recovered, separated
by SDS-PAGE (7.5% (w/v) polyacrylamide), transferred to a
nitrocellulose membrane, and detected by immunoblotting with UT-29
antibody. Crude indicates the result of Western blot
analysis with cell lysates (10 µg of protein) containing the
full-length human X11L protein (hX11L), the amino-terminal
domain attached with the PI domain (N + PI) or the
amino-terminal domain alone (N). d, schematic
structure of the human X11L construct used in this study. N,
amino-terminal region of the PI domain; PI, phosphotyrosine
interaction domain; C, carboxyl-terminal region of the PI
domain; PDZ, PDZ domain. Amino acid numbers of human X11L
are indicated. 170, 116, and 76 refer
to the molecular masses (kDa) of the protein standards.
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Fig. 5.
Specificity of antibodies and identification
of the X11-like protein. a, crude lysate
(crude, 100 µg of protein) from 293 cells stably
transfected with pcDNA3-FLAG-hX11L was subjected to
immunoprecipitation (IP) using anti-FLAG (M2), anti-X11L
amino-terminal domain (UT-29), or anti-X11L carboxyl-terminal domain
(UT-30) antibodies. Immunoprecipitates from the lysate (approximately 1 mg of protein) were subjected to SDS-PAGE (6% (w/v) polyacrylamide),
proteins were transferred to a nitrocellulose membrane, and the
membrane was probed (WB) with M2, UT-29, or UT-30
antibodies. Immunocomplexes were detected by incubation with anti-mouse
IgG peroxidase or anti-rabbit IgG peroxidase followed by ECL. The
~135-kDa protein indicated with an arrow is FLAG-hX11L.
b, mouse brain lysate (crude, 100 µg of
protein) was subject to immunoprecipitation (IP) using UT-29
and UT-30 antibodies. Immunoprecipitates from the lysate (approximately
1 mg of protein) were probed (WB) with UT-29 and UT-30
antibodies, and immunocomplexes were detected by ECL with anti-rabbit
IgG following SDS-PAGE (6% (w/v) polyacrylamide) and transfer of
proteins to a nitrocellulose membrane. PC, sample was
subjected to preclearing with Protein A-Sepharose prior to the addition
of the antibody; w/o PC, sample was immunoprecipitated without
preclearing; Comp, sample was immunoprecipitated with
indicated antibodies in the presence of antigen peptide (10 µM). The ~135-kDa protein indicated with an
arrow is mouse X11L. UT-29 does not react with mouse X11L in
immunoprecipitation (see text). Numbers are the molecular masses (kDa)
of the protein standards. c, mouse tissues lysate (150 µg
of protein) was subject to SDS-PAGE (6% (w/v) polyacrylamide), and
proteins were transferred to a nitrocellulose membrane. The membrane
was probed with UT-30 and 125I-Protein A and then analyzed
by autoradiography. An arrow indicates the position of mouse
X11L.
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Fig. 6.
Northern blot analysis with probes for human
X11-like (hX11L) and -actin genes. Human multiple
tissue Northern blots containing 2 µg of poly(A)+ RNA
were hybridized with the probe, which was radiolabeled by random
priming of human X11L cDNA coding for amino acids 126-749
(a) and with a radioactive probe derived from the cDNA
of human
-actin (b). The 4.4- and 4.0-kb bands in brain
hybridized with the human X11L probe (a). The 2-kb band was
hybridized ubiquitously, and an additional 1.8-kb band was hybridized
in heart and skeletal muscle with
-actin probe (b).
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Fig. 7.
Co-immunoprecipitation of the human X11-like
protein and APP. COS7 cells transiently expressing human X11L and
APP were lysed in PBS containing 10 mM CHAPS, and the
lysate (crude) was used for immunoprecipitation
(IP) with the indicated antibodies. The immunoprecipitates
were analyzed by Western blot (WB) with antibodies UT-18
(a) and UT-30 (b) following SDS-PAGE (6% (w/v)
polyacrylamide) and transfer to a nitrocellulose membrane. Mature
(m) and immature (im) APP695 and immature
endogenous APP (imAPP endogenous) were co-immunoprecipitated
with human X11L using anti-X11L antibody UT-30 (a). The
human X11L protein was also co-immunoprecipitated with APP using
anti-APP extracellular domain antibodies 2D1 and 25C (b).
Rabbit IgG, normal rabbit IgG; mouse
IgG, normal mouse IgG; 2D1 and 25C,
anti-A monoclonal antibodies; UT-30, anti-human X11L
polyclonal antibody.
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Fig. 8.
Localization of the human X11-like protein in
293 cells. Intracellular localization of human X11L is shown as
follows (left parts of a-c):
medial- and trans-Golgi (a), ER and
cis-Golgi (b), and APP (c). 293 cells
that express stably transfected pcDNA3-hX11L were stained with
UT-30 antibody (a and b, green) and
observed under a confocal laser scanning microscope. The cells were
also double-stained with rhodamine-conjugated WGA (a,
red) to identify the location of medial- and
trans-Golgi and rhodamine-conjugated ConA (b,
red) to identify the location of ER and
cis-Golgi. c, 293 cells stably transfected with
pcDNA3-APP695 were further transiently transfected with
pcDNA3-hX11L. The cells were double-stained with UT-30
(green) and LN27 (red) antibodies.
Co-localization of human X11L and WGA (hX11L/WGA), human
X11L and ConA (hX11L/ConA), and human X11L and APP
(hX11L/APP) are indicated by the yellow.
Scale bar, 20 µm.
-Amyloid
Peptides--
To examine whether X11L regulates APP processing through
its interaction with APPCOOH, the effect of X11L on the
generation of both A
40 and A
42 peptides was examined (Fig.
9). The 293 cells that stably expressed
human APP695 (15, 23) were co-transfected transiently with the cDNA
encoding full-length human X11L, N + PI (amino acid positions 1-555),
PI (amino acid positions 369-555), or vector alone (control) (see Fig.
4 for protein construction). We have already demonstrated that these
three constructs, full-length human X11L, N + PI, and PI, can associate
with APPCOOH (Figs. 2 and 4). At 72 h after
supplementation of the cells with fresh medium following transfection,
the conditioned media were collected, and the levels of A
40 and
A
42 were quantified by sandwich ELISA (15, 23). The relative ratios
of the levels of A
to the control level (reference value set to 1.0)
were determined. The full-length human X11L construct decreased the
level of A
40 secretion but not that of A
42. The levels of A
40
produced from cells expressing N + PI or PI domains did not change
compared with the levels produced by control cells, despite the fact
that the N + PI and PI domain have binding ability for
APPCOOH (Fig. 4). The secretion level of A
42 was not
altered remarkably by co-transfection with human X11L or its
domain structures (Fig. 9b). These results suggest that
human X11L functions to modulate APP processing. The constructs N + PI
and PI did not have this modulator function, indicating that the
carboxyl-terminal region is important for APP processing. The study
indicates that the PDZ domains in the carboxyl-terminal domain of human
X11L play an important role in APP processing. Furthermore, the results
suggest that regulation of A
42 production may differ from that of
A
40.
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Fig. 9.
Quantification of A in the medium of cells
expressing human X11-like protein and derived constructs. The 293 cells stably transfected with pcDNA3APP695 (15, 23) were
co-transfected transiently with human X11L and derived
constructs. Fresh medium was supplied 12 h after transfection,
conditioned media were collected 72 h after the medium change, and
the concentrations of A
40 (a) and A
42 (b)
in the medium were measured using sandwich ELISA as described under
"Experimental Procedures." The quantity of A
is indicated as the
relative ratio of the levels of A
observed using cells transfected
with "control" or the pcDNA3 vector alone (a reference value of
1.0). hX11L, full-length hX11L; N + PI,
amino-terminal domain attached to the PI domain; PI, PI
domain. Results are the average of 12-16 independent assays. The
error bar indicates S.D. (**, p = 0.005).
DISCUSSION
40 production (Fig.
10c). We have already isolated several novel proteins that
bind to the amino-terminal
domain.3 One of these
proteins may be "factor X."
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Fig. 10.
Possible role of X11L in APP
processing. a, the X11L protein binds to the
APPCOOH domain weakly via its PI domain ("weak associated
state"). b, when factor X binds to the amino-terminal
domain, the conformation of X11L changes and the X11L·factor X
complex binds to APPCOOH with high affinity ("strong
associated state"). c, when the PDZ domain(s) in the
carboxyl-terminal domain of the X11L are in a strong associated state,
another "factor Y" binds. This binding may be essential for
suppression of A 40 secretion. d, A construct with a
deletion of the carboxyl-terminal domain (N + PI) can bind
to APPCOOH with high affinity. In this case, the
conformation of N + PI is thought to mimic the strong associated state.
This construct does not suppress the secretion of A
40, because it
lacks the carboxyl-terminal domain. e, the construct
containing the PI domain alone can bind to APPCOOH perhaps
without any regulation. f, the construct with a deletion of
the amino-terminal domain (PI + C) cannot bind to
APPCOOH because of the suppressor effect of the
carboxyl-terminal domain and the missing amino-terminal domain.
M, membrane; A
,
-amyloid; N,
amino-terminal domain of X11L; PI, PI domain of X11L;
C, carboxyl-terminal domain of X11L; X, a
putative factor X, which binds to the amino-terminal domain of X11L;
Y, a putative factor Y, which binds to the carboxyl-terminal
domain of X11L.
When the ability of full-length human X11L, N + PI, or PI constructs on
A production was examined in cells, only full-length human X11L
reduced the secretion of A
40. This result agrees well with our
hypothesis. The constructs deleted for the carboxyl-terminal domain (N
+PI construct), which preserves human X11L binding ability for
APPCOOH, did not have this modulator activity to regulate A
40 secretion. This observation would also appear to support our
hypothesis. Neither the PI domain alone nor the N + PI domain is
sufficient to regulate APP metabolism if it binds to
APPCOOH with higher affinity (Fig. 10, d and
e). The human X11L construct lacking the amino-terminal
domain (PI + C) loses its binding affinity for APPCOOH
(Fig. 10f). The carboxyl-terminal domain containing the PDZ
domains may be essential for the regulation of A
40 production. We
have not examined how the PDZ domains function to regulate A
40
production in detail. However, it has been well established that the
PDZ domain interacts with other proteins (32, 42, 43). Fig. 10 proposes
that an association of an unidentified "factor Y" to the PDZ
domain(s) may be necessary to regulate APP processing.
APPCOOH is not subject to tyrosine phosphorylation,4 and it is believed that the mode of binding of X11 and X11L to a sequence containing the NPXY motif in APPCOOH is a phosphotyrosine-independent interaction such as in the case of Shc phosphotyrosine binding/PI (44). However, APPCOOH contains other phosphorylation sites (45-47). One of these, Thr668, is located at position 16 in the amino-terminal region of the NPTY motif and is phosphorylated in brain tissue (48). Furthermore, we have found that Thr668 phosphorylation of mature APP (mAPP, N- and O-glycosylated form) is neuron-specific,5 although Thr668 phosphorylation of immature APP (N-glycosylated form) is observed in nonneuronal cells during the mitotic phase of cell division (47-49). Therefore, we examined the effect of phosphorylation of APP at Thr668 on the interaction between X11L and APPCOOH. A single amino acid mutation that changed this threonine to alanine or glutamic acid did not alter the binding of APP to the PI domain of human X11L.2 This result indicates that the association of X11L with APPCOOH is not dependent on the phosphorylation of APP.
Because proteins containing the PDZ domain are known to play a role in the clustering of membrane receptors and channels (50) and because the expression of X11L is brain-specific, X11L may also play an important role in neuronal function(s) through its association with APP. While we were preparing this manuscript, we found that a cDNA similar to human X11L could be isolated from rat as a protein interacting with Munc 18, Mint (51). Munc 18 is thought to be a protein required for synaptic vesicle exocytosis. Mint-1 and Mint-2 seem to be rat homologues of human X11 and X11L, respectively. The Munc 18 binding domain is located in the amino-terminal region of the PI domain of X11L/Mint-2, and a complex consisting of Mint and Munc18 is thought to play a role in the docking of synaptic vesicles to the active zone (51). This observation suggests that APP may play an important role in neurotransmitter release at nerve terminals or that Mint-2 may regulate the binding ability of X11L to APP. Since we have demonstrated that mAPP localizes in the nerve terminals of cholinergic neurons (38), one may hypothesize that APP interacts with the exocytotic machinery proteins of synaptic vesicles via X11L/Mint-2. Since cholinergic neurotransmission is thought to be involved in learning and memory processes (52) and loss of function in cholinergic neurotransmission has been observed in senile dementia of AD (53), further characterization of X11L should increase our understanding of the physiological function of APP and the pathogenesis of sporadic type AD.
Furthermore, while this manuscript was being reviewed, a report
appeared showing that X11 has an inhibitory effect on both A40 and
A
42 secretion using a double transient expression system with APP
and X11 cDNAs (54). The X11 and X11L proteins may regulate APP
metabolism in a different fashion due to differences in their amino-terminal domain structures.
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ACKNOWLEDGEMENTS |
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We thank Drs. M. Oishi (Montifore Medical Center, New York), T. Seki (Juntendo University School of Medicine, Tokyo, Japan), and S. Itohara (RIKEN: The institute of Physical and Chemical Research, Wako, Japan) for critical reading and comments on the manuscript and Y. Morimoto and N. Kobayashi for technical assistance
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FOOTNOTES |
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* This work was supported in part by a Grant from Program for Promotion of Basic Research Activities for Innovative Biosciences.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB014719 (human X11-like) and AB014720 (mouse X11-like).
§ Recipient of Japan Society for the Promotion of Science Research Fellowships for Young Scientists.
** To whom correspondence should be addressed. Tel./Fax: 81-3-3814-6937; E-mail: tsuzuki{at}mayqueen.f.u-tokyo.ac.jp.
The abbreviations used are:
APP, -amyloid
precursor protein; A
,
-amyloid; AD, Alzheimer's disease; APPCOOH, the cytoplasmic domain of APP; CHAPS, 3-[(3-cholamidpropyl)dimethylammonio]-1-propanesulfonic acid; ConA, concanavalin A; COS7 cells, SV40-transformed kidney cells from African
green monkey; ELISA, enzyme-linked immunosorbent assay; ER, endoplasmic
reticulum; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PDZ, repeated sequences in the
brain-specific protein PSD-95, the Drosophila
septate junction protein disks-large, and the epithelial tight junction protein ZO-1; PI, phosphotyrosine
interaction; RACE, rapid amplification of cDNA ends; WGA, wheat
germ agglutinin; kb, kilobase pair(s); X11, a gene in the
region of the Friedreich ataxia locus; X11L, X11-like.
2 S. Tomita and T. Suzuki, unpublished observation.
3 S. Tomita, D.-S. Lee, and T. Suzuki, unpublished observations.
4 T. Suzuki, unpublished observation.
5 Iijima, K., Satoh, Y., Ando, K., Seki, T., Arai, Y., Greengard, P., Nairn, A. C., Kirino, Y., and Suzuki, T., manuscript in preparation.
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