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INTRODUCTION |
Cellular immune system defense against the invasion of
viruses and tumors depends on the presentation of peptides at the cell surface for recognition by cytotoxic T lymphocytes (1). These peptides
must be bound by cell surface class I major histocompatibility complex
(MHC)1 heavy chains. Assembly
of the MHC class I heavy chain, a light chain called
2-microglobulin (
2m), and peptide into
the heterotrimeric, complete MHC class I molecule occurs in the
endoplasmic reticulum (ER) (2, 3). ER resident proteins such as
calreticulin, the transporter associated with antigen processing (TAP),
tapasin, and ERp57 interact with peptide-free class I heavy
chain/
2m to assist with peptide binding (4-11).
Although many aspects of chaperone assistance with the folding of class
I molecules have been elucidated, the rules governing chaperone
selection have not been fully defined. It has been shown that the
products of certain class I alleles may interact disparately with
different chaperones. Natural HLA molecules with limited amino acid
changes in the
2 domain differ in respect to whether they associate
with TAP (12). Certain HLA-A and -B subtypes strongly
associate with TAP, tapasin, and calreticulin, while others differing
only at a single amino acid residue show little to no association with
these same proteins (13-15). Furthermore, the HLA subtypes that lack
association with these ER proteins are more poorly retained and are
present in greater quantity at the cell surface, indicating that
differences in the ability of natural MHC heavy chains to interact with
these ER proteins are functionally relevant (13-15).
Amyloid precursor-like protein 2 (APLP2) is a type I transmembrane
protein and a member of the amyloid precursor protein family that is
implicated in the etiology of Alzheimer's disease; however, APLP2
lacks the
-amyloid peptide domain (A
) that is deposited as
extracellular amyloid in the brain parenchyma of Alzheimer's disease
patients (16, 17). Although APLP2 is ubiquitously expressed, its
function is unknown. In the ER, APLP2 associates with the murine class
I MHC molecule Kd and with some HLA molecules (18).
APLP2/Kd interaction has been demonstrated in
FO-1Kd
(human melanoma cell line), B7 (H-2d
murine fibroblast cell line), and 293-Kd (human embryonic
kidney cell line) (18). Experimental retention of Kd with
brefeldin A, which induces retrograde Golgi
ER transport, increases
the number of Kd molecules associated with APLP2 (18).
APLP2 can be dissociated from Kd by the addition of a
Kd-specific peptide ligand (19); therefore, APLP2 may play
a role in the assembly of Kd and possibly other MHC class I
molecules as well.
In this study, we examined the role of APLP2 as a possible chaperone of
the Kd molecule, utilizing an epitope-tagged Kd
(etKd) heavy chain that can be specifically detected in the
open as well as the folded form (20). Our findings indicate that APLP2 binds only to the folded form of MHC class I, which is also
preferentially bound by the adenovirus protein E3/19K. This specificity
for the folded form differentiates APLP2 and E3/19K from other proteins known to bind to MHC class I heavy chains. When intracellular expression of APLP2 was experimentally down-regulated, the surface expression of Kd rose, indicating that APLP2 not only
associates with Kd but also regulates its expression at the
cell surface.
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EXPERIMENTAL PROCEDURES |
Cell Lines--
HeLa cells and DAP-3 L cells were grown in RPMI
1640 medium (Invitrogen) supplemented with 15% fetal bovine serum,
glutamine, pyruvate, and penicillin/streptomycin. The HeLa cell line
was a gift from Dr. Wendy Maury (University of Iowa, Iowa City, IA). Human embryonic kidney 293 cells (ATCC CRL 1573) were transfected with
the Ad2 EcoRI-D fragment, and positive expressors were
selected with Geneticin (Invitrogen) to give rise to the 293.12 cell
line, which constitutively expresses E3/19K (21). (The 293 cell line expresses Ad5 E1 but not E3 (21-25).) The 293.12 E3/19K+ cell line was transfected with the
Kd gene to create the cell line
293.12Kd8 (26). These cell lines were maintained in
Dulbecco's modified Eagle's medium (Invitrogen) supplemented as
described above with the addition of 0.2 mg/ml Geneticin (Invitrogen)
or 0.2 mg/ml Geneticin and 0.1 mg/ml hygromycin B (Calbiochem),
respectively. All cells were maintained at 37 °C in 5%
CO2.
Antibodies--
The mAb 64-3-7 is an IgG2 Ab that recognizes a
short stretch of amino acids in the
1 domain of open, peptide-free
Ld (27, 28). The mAb 34-1-2 is directed against the
1/
2 domain of folded, peptide-occupied Kd (29).
SF1.1.1 mAb recognizes both open and folded forms of Kd as
it is directed toward the
3 region of the molecule (30). Ascites
containing the 64-3-7, 34-1-2, and SF1.1.1 mAbs were kind gifts from
Dr. Ted Hansen (Washington University, St. Louis, MO). W6/32 recognizes
2m-associated HLA-A, -B, and -C (31) and can recognize
Kd if Kd is associated with bovine
2m (obtained from fetal calf serum in tissue culture
medium) (32). HC10 is an IgG2a mAb that reacts preferentially with open
HLA-B and -C heavy chains (33, 34). Generation of the polyclonal rabbit
antisera AP 206 and AP tail has been described previously (18).
Briefly, rabbits were immunized with peptides representing amino acids
206-219 of APLP2 (for AP 206) or corresponding to the C terminus of
APLP2 (for AP tail). AP 206 recognizes human APLP2, while AP tail
cross-reacts with both human and murine APLP2. Although the AP 206 serum is specific for APLP2, the AP tail serum also binds to amyloid
precursor protein at a very low level (18). Since the AP tail serum
reacted much better with APLP2 than did AP 206 and because
co-precipitation of endogenous amyloid precursor protein using the AP
tail serum was at a negligible level, the AP tail serum was used for
some experiments.
Mutagenesis and Transfection--
The etKd cDNA
was generated by site-directed mutagenesis at position 48 (arginine
glutamine) using the QuikChange mutagenesis kit from Stratagene Cloning
Systems (La Jolla, CA) as described previously (20). This substitution
confers the epitope for the 64-3-7 mAb. The etKd has been
thoroughly tested for proper assembly, folding, and trafficking and
shown to function equivalently to wild type Kd (20). The
etKd was cloned into the mammalian expression vector
RSV.5-neo and stably transfected into HeLa and DAP-3 L cells with
Lipofectin (Invitrogen). Cells were selected in culture with 0.4 mg/ml
Geneticin. Levels of surface expression of the etKd
proteins were determined by indirect immunofluorescence with mAb as
described below. For transient transfections, 293 and 293.12 cells
(1 × 106 each) were plated into 60-mm plates and
transfected with etKd using a calcium phosphate
transfection system (Invitrogen) or Effectene (Qiagen, Valencia, CA).
Infection with Recombinant Adenovirus--
The adenoviral
vectors used had deletions of the E1 and E3 regions of the adenovirus,
and a dual expression cassette was inserted in place of the E1 region
(35). The expression cassette carries E3/19K driven by the
cytomegalovirus promoter with or without the reporter gene
-glucuronidase driven from the Rous sarcoma virus promoter.
-Glucuronidase expression allows the assessment of infection
efficiency by immunohistochemistry. The null control vector has the
E3/19K sequence deleted. For infections, untransfected HeLa cells and
HeLa-etKd cells (1 × 107 of each) were
plated and infected 24 h later in 3 ml of serum-free medium
at a multiplicity of infection of 20 for 2 h at 37 °C. The
virus was then washed off the cells with phosphate-buffered saline
(PBS), and medium with serum was added. Twenty-four hours later,
the cells were washed two times with PBS. Radiolabeling and
immunoprecipitations were then carried out as described below.
Metabolic Labeling, Immunoprecipitation, and Western
Blotting--
Cells were preincubated for 30 min at 37 °C in
methionine-free culture medium. For each immunoprecipitation,
1 × 107 cells were used. The cells that were compared
within each experiment were of matched, high viability and of matched
confluence. Next [35S]methionine (100 µCi/ml) was
added, and the cells were radiolabeled for 30 min. The cells were then
washed three times in PBS containing iodoacetamide (Sigma) and lysed in
1% CHAPS (Roche Molecular Biochemicals) or 1% digitonin in
Tris-buffered saline (pH 7.4) with freshly added 0.2 mM
phenylmethylsulfonyl fluoride (Sigma) and 20 mM
iodoacetamide. L-1-Chloro-3-(4-tosylamido)-7-amino-2-heptanone
hydrochloride (Roche Molecular Biochemicals) was also added to the
digitonin lysis buffer. The lysis buffer was supplemented with a
saturating volume of mAb before its addition to pelleted cells. After
incubation for 1 h on ice, nuclei were removed by centrifugation,
and lysates were incubated with protein A-Sepharose beads (Amersham
Biosciences). The beads were washed three times with ice-cold
0.1% CHAPS or 0.1% digitonin in Tris-buffered saline (pH 7.4), and
protein was eluted from the beads by boiling in 0.125 M
Tris (pH 6.8), 0.2% SDS, 12% glycerol, 2% bromphenol blue.
All immunoprecipitates were electrophoresed on 4-20% Tris-glycine
acrylamide gels (Invitrogen). In instances where comparisons were made,
all samples were electrophoresed on the same gel. For autoradiographs,
gels were soaked in Amplify (Amersham Biosciences) plus 2% glycerol,
dried, and exposed to Biomax MR film (Eastman Kodak Co.).
For Western blotting, immunoprecipitates were electrophoresed by
SDS-PAGE as described above and were transferred to Immobilon-P membranes (Millipore, Bedford, MA). After blocking overnight at 4 °C
in 10% (w/v) dry milk, 0.05% Tween 20 in PBS, membranes were incubated in a dilution of Ab for 2 h, washed three times with 0.05% Tween 20 in PBS, and incubated for 1 h with
biotin-conjugated goat anti-rabbit IgG (Caltag Laboratories, San
Francisco, CA). Following three washes with 0.05% Tween 20 in PBS,
membranes were incubated for 1 h with streptavidin-conjugated
horseradish peroxidase (Zymed Laboratories Inc., San
Francisco, CA), washed three times with 0.3% Tween 20 in PBS, and
incubated with enhanced chemiluminescence Western blot developing
reagents (Amersham Biosciences). Membranes were exposed to Biomax MR
film for various periods of time.
Reverse Transcription-PCR--
RNA was prepared from 4 × 106 DAP-3 L cell fibroblasts by the Qiagen RNeasy minikit.
The reverse transcription-PCR was performed with the Gene-Amp kit
(Applied Biosystems, Branchburg, NJ) using primers designed with
APLP2-specific sequences (5'-ACCAATGATGTTGATGTGTATTTT-3' and
5'-TAAGGAACTTTGTACAGAAGAGA-3').
Flow Cytometry--
Cells were centrifuged and resuspended at
5 × 106/ml in PBS containing FACS medium (0.2%
bovine serum albumin, 0.1% sodium azide). Cell suspension aliquots
(0.1 ml) were added to a 96-well plate. The cells were incubated with
saturating concentrations of antibody or FACS medium alone for 30 min
at 4 °C, washed two times, and incubated with
phycoerythrin-conjugated, Fc-specific, affinity-purified
F(ab')2 fragment of donkey anti-rabbit IgG (if AP 206 had
been used as the primary Ab) or goat anti-mouse IgG (if 34-1-2 had been
used as the primary Ab) for 30 min at 4 °C. The cells were then
washed two times, resuspended, and analyzed on a FACS-Calibur flow
cytometer (BD Immunocytometry Systems) at the University of Nebraska
Medical Center Cell Analysis Facility.
Treatment with APLP2-specific Short Interfering RNA
(siRNA)--
RNA interference treatments were performed using an
established technique (36). The duplex siRNA and inverse siRNA used were obtained from Dharmacon Research, Inc. (Lafayette, CO) in the
annealed and purified form. The APLP2 siRNA sense sequence used
was 5'-GTTCTTCAGTACTGTCAGGdTdT-3'. This sequence corresponds to an
APLP2 sequence that follows an AA plus it has two dT residues added to the 3'-end of the RNA. The APLP2 inverse siRNA sense sequence
used was 5'-GGACTGTCATGACTTCTTGdTdT-3'. In the design of the
siRNA, a National Center for Biotechnology Information data base BLAST
search was performed to ensure that the siRNA would target only
APLP2.
For each duplex, 20 nmol of purified, annealed duplex in annealing
buffer was received from Dharmacon Research, Inc. to which we added 1 ml of RNase-free water (also supplied by Dharmacon Research, Inc.) to
make a 20 µM stock solution. The day prior to
transfection, HeLa-etKd cells were plated in six-well
plates at 1 × 105 cells/well in 3 ml of
antibiotic-free medium per well. After overnight incubation at 37 °C
in 5% CO2, the cells were transfected as follows. For each
well, 12 µl of Oligofectamine (Invitrogen) and 3 µl of Opti-MEM
(Invitrogen) were combined in a polystyrene tube, mixed gently without
vortexing, and incubated for exactly 5 min at room temperature. In
separate tubes, the siRNA or inverse siRNA was diluted as follows. For
each well of a six-well plate, 175 µl of Opti-MEM was mixed with 10 µl of duplexed 20 µM siRNA, inverse duplexed 20 µM siRNA, or water. For each well, diluted Oligofectamine
solution (15-µl total volume) was combined with diluted siRNA
solution (185-µl total volume) in a polystyrene tube, mixed gently by
pipetting, and incubated 15 min at room temperature. The plates were
then incubated at 37 °C in 5% CO2.
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RESULTS |
APLP2 Associates with etKd in HeLa-Kd
Cells--
We examined whether APLP2 associates with etKd
in two cell types: HeLa-etKd (human cervical carcinoma
cells) and DAP-3-etKd (murine L cell fibroblasts).
etKd was immunoprecipitated from radiolabeled cell lysates
with SF1.1.1 mAb, which recognizes total Kd. Fig.
1 shows etKd and APLP2
association in HeLa-etKd was easily detectable by
immunoprecipitation and Western blotting. In contrast, interaction
between APLP2 and etKd in DAP-3-etKd was
relatively weak (Fig. 1), although it could be detected in prolonged
film exposures. The level of expression of APLP2 is not low in DAP-3 as
we could readily detect it by reverse transcription-PCR (data not
shown). These observations suggest that the strength of APLP2 and
Kd association may be cell-dependent.

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Fig. 1.
APLP2 interacts with etKd more
strongly in HeLa-etKd cells than in DAP-3-etKd
L cell fibroblasts. etKd was immunoprecipitated from
HeLa-etKd with SF1.1.1 and from DAP-3-etKd with
mAbs SF1.1.1, 64-3-7, and 34-1-2. The precipitated proteins were
electrophoresed, autoradiographed (top panel), and probed on
a Western blot (bottom panel) with AP tail anti-serum. From
left to right: lane 1,
HeLa-etKd/SF1.1.1; lane 2,
DAP-3-etKd/64-3-7; lane 3,
DAP-3-etKd/34-1-2; lane 4,
DAP-3-etKd/SF1.1.1. This experiment was repeated five times
with similar results. On shorter exposures of the Western blots to
film, no APLP2 was apparent in association with etKd, but
with very prolonged exposure APLP2 could be detected.
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APLP2 Associates with Only the Folded Form of
etKd--
To determine at what point in the process of
etKd assembly the association between etKd and
APLP2 first occurs, we used the 64-3-7 mAb, which allows immunoprecipitation specifically of the open form of etKd.
etKd was immunoprecipitated from HeLa-etKd
lysates with mAb SF1.1.1 (for total etKd), 34-1-2 (for
folded etKd), or 64-3-7 (for open etKd). The
immunoprecipitates were probed on a Western blot with anti-APLP2 serum
for co-precipitated APLP2. Both open and folded forms of etKd were present in the cell lysates (Fig.
2); however, no association was found
between APLP2 and open Kd. Thus APLP2 recognizes a
conformational binding site in the
1/
2 region.

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Fig. 2.
APLP2 interacts exclusively with folded
etKd. Radiolabeled HeLa or HeLa-etKd cell
lysates were treated with mAb SF1.1.1 (for total Kd),
34-1-2 (for folded Kd), or 64-3-7 (for open
etKd). The precipitated proteins were electrophoresed, and
the gel was autoradiographed (top panel). These samples were
also electrophoresed on a separate gel, transferred to a membrane, and
probed with anti-APLP2 antiserum AP 206 (bottom panel). From
left to right: lane 1, HeLa/SF1.1.1; lane
2, HeLa-etKd/SF1.1.1; lane 3, HeLa/34-1-2;
lane 4, HeLa-etKd/34-1-2; lane 5,
HeLa/64-3-7; lane 6, HeLa-etKd/64-3-7. This
experiment was repeated three times with similar results obtained each
time.
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Two bands, migrating closely to each other, are visible in the 64-3-7 lane in Fig. 2. The identity of the upper band in the doublet is not
known for certain. It may be a differentially glycosylated form of
etKd, or it may be tapasin, which has a molecular weight
slightly larger than that of the MHC class I heavy chain and which
associates with open MHC class I heavy chains, including
64-3-7+ etKd (8, 10, 37, 38). Two bands have
previously been distinguished in autoradiographs of 64-3-7 immunoprecipitates of Ld and etKd (7, 38).
Interestingly the higher molecular weight band is not visible in the
SF1.1.1 lane in Fig. 2, although SF1.1.1 recognizes unfolded as well as
folded etKd. It has been previously observed that TAP (and
therefore also possibly tapasin) does not co-precipitate with
28-14-8+ Ld (7). The 28-14-8 antibody, like
SF1.1.1, is an anti-
3 domain antibody that recognizes both open and
folded forms. Anti-
3 domain antibodies may be unable to
co-precipitate TAP and tapasin with open forms of MHC class I due to
steric hindrance, consistent with the finding that the
3 domain of
class I is involved in TAP and tapasin association (7,
39-42).
APLP2 Molecules Are Present at the Cell Surface--
By flow
cytometry, we investigated whether any complete APLP2 molecules could
be detected at the surface of HeLa and HeLa-etKd cells. The
AP 206 anti-serum can immunoprecipitate APLP2 even after
glycosaminoglycan modification (18, 43) and therefore should be able to
detect mature, complete APLP2 if it is present at the cell surface.
HeLa-etKd cells and HeLa cells were incubated with the
anti-APLP2 antiserum AP 206 or with FACS medium alone. Following
incubation with fluorescently labeled secondary antibody, the cells
were analyzed by flow cytometry. APLP2 was detected at equivalent
levels on HeLa and HeLa-etKd (Fig.
3). Thus, despite the strong
intracellular association between APLP2 and etKd, the
expression of etKd does not affect the level of APLP2 at
the cell surface.

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Fig. 3.
Expression of etKd is not
associated with an altered level of APLP2 at the cell surface.
HeLa or HeLa-etKd cells (5 × 105 each)
were placed in a 96-well plate with anti-APLP2 antiserum AP 206 or FACS
medium alone (0.2% bovine serum albumin, 0.1% sodium azide) on ice
for 30 min. After washing, cells were incubated with
phycoerythrin-conjugated donkey anti-rabbit secondary Ab (for AP 206)
and analyzed by flow cytometry for surface expression of APLP2. The
mean fluorescence obtained for APLP2 on HeLa was 28.79 ± 0.76, and the mean fluorescence obtained for APLP2 on HeLa-etKd
was 28.70 ± 0.58. Thus there was no significant difference in the
expression of APLP2 on HeLa versus HeLa-etKd
(p = 0.07).
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E3/19K Increases the Association of APLP2 with
etKd--
The adenoviral protein E3/19K is known to bind
to class I molecules and retain them in the ER through an ER retrieval
sequence in its cytoplasmic tail (21, 44, 45). E3/19K also binds independently to TAP and thereby prevents tapasin from bridging TAP to
class I (46). We found that the association of APLP2 with
etKd increased when HeLa-etKd cells expressed
E3/19K (Fig. 4), consistent with a
similar observation by Sester et al. (18).

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Fig. 4.
The association of APLP2 with
etKd is increased in the presence of E3/19K. HeLa and
HeLa-etKd cells were infected with adenoviral vectors
expressing E3/19K and -glucuronidase (Ad19K/G), E3/19K alone
(Ad19K), or a null vector with the E3/19K sequence deleted (AdNull/G).
The cells were infected at a multiplicity of infection of 20 for 2 h at 37 °C in serum-free medium. After 24 h, the cells
were washed and radiolabeled, and etKd was
immunoprecipitated with SF1.1.1. The immunoprecipitates were
electrophoresed on a Tris-glycine acrylamide gel and autoradiographed
(top panel). The immunoprecipitates were also
electrophoresed, transferred to a membrane, and probed with AP 206 (bottom panel). From left to right: lane
1, HeLa-etKd; lane 2, HeLa-Ad19K/G;
lane 3, HeLa-etKd-Ad19K/G; lane 4,
HeLa-Ad19K; lane 5, HeLa-etKd/Ad19K; lane
6, HeLa-AdNull/G; lane 7,
HeLa-etKd/AdNull/G. This experiment was performed three
times with similar results each time.
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Since the binding of APLP2 to etKd was limited to the
folded form of etKd (Fig. 2), we tested whether binding of
E3/19K was similarly restricted. For this experiment, 293-E3/19K
(293.12) cells were transiently transfected with etKd,
radiolabeled, and lysed. The cell lysates were incubated with 34-1-2 to
immunoprecipitate folded Kd and 64-3-7 to immunoprecipitate
open Kd. E3/19K associated preferentially with the folded
(34-1-2+) form of etKd and not the open
(64-3-7+) form (Fig. 5). Thus
E3/19K seems to interact preferentially with the folded form of class I
as does APLP2.

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Fig. 5.
E3/19K associates preferentially with the
folded form of etKd. 293-E3/19K (293.12) cells were
transiently transfected with etKd or were mock-transfected,
and immunoprecipitations were done on radiolabeled cell lysates with
mAb 34-1-2 (recognizes folded etKd) or 64-3-7 (recognizes
open, peptide-free etKd). The immune complexes were eluted
from protein A-Sepharose beads and electrophoresed on a Tris-glycine
acrylamide gel, which was dried and autoradiographed. From left
to right: lane 1, 293-E3/19K; lane 2,
293-E3/19K-etKd; lane 3, 293-E3/19K; lane
4, 293-E3/19K-etKd. The lower band in this
figure was identified as E3/19K on the basis of appropriate molecular
weight and co-precipitation with etKd. A previous
publication from one of our laboratories had shown that a protein of
the correct molecular weight to be E3/19K can be co-precipitated with
Kd from lysates of 293 cells that express E3/19K and
Kd but not from 293 cells that express Kd alone
(19). This experiment was performed three times with similar results
each time.
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APLP2 Regulates the Surface Expression of Kd--
To
determine whether interaction between APLP2 and etKd has
functional consequences, we down-regulated the expression of APLP2 in
HeLa-etKd with siRNA corresponding to an APLP2 sequence. As
controls, mock treatment or treatment with an inverse of the correct
APLP2 siRNA sequence was performed. APLP2-specific siRNA, but not mock
or inverse siRNA, significantly down-regulated APLP2 intracellular expression by 3 and 4 days post-treatment (Fig.
6). The down-regulation in APLP2
expression was accompanied by a relative increase in cell surface
etKd expression (Fig. 7 and Table I). Similar
results were obtained with 34-1-2 and with SF1.1.1 in three separate
experiments. APLP2 seems to retain
etKd and depress its surface expression, supporting a
possible role for APLP2 in regulating the trafficking of Kd
out of the ER. Thus APLP2, a secreted protein, may regulate ER/Golgi export of Kd.

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Fig. 6.
The expression of APLP2 was down-regulated by
siRNA. Mock transfection, transfection with siRNA, and
transfection with inverse siRNA (negative control) were performed on
HeLa-etKd cells as described under "Experimental
Procedures." At day 3 and day 4 post-transfection, etKd
molecules were immunoprecipitated from lysates of the mock,
siRNA-transfected, or inverse siRNA-transfected HeLa-etKd
cells with mAb 34-1-2. The etKd immunoprecipitates were
electrophoresed, transferred to blotting membranes, and probed with
64-3-7 for etKd (top panel) or with the AP 206 antiserum that recognizes APLP2 (bottom panel). From
left to right: lane 1, day 3 mock transfection;
lane 2, day 3 siRNA transfection; lane 3, day 3 inverse siRNA transfection; lane 4, day 4 mock transfection;
lane 5, day 4 siRNA transfection; lane 6, day 4 inverse siRNA transfection.
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Fig. 7.
Down-regulation of APLP2 expression results
in an up-regulation of MHC class I surface expression. A histogram
is shown from flow cytometric analysis of HeLa-etKd cells
with anti-Kd mAb 34-1-2 at 4 days after transfection of
cells with APLP2-specific siRNA or inverse siRNA. As the secondary
antibody, phycoerythrin (PE)-conjugated, Fc-specific,
purified F(ab')2 fragment of goat anti-mouse IgG was used.
The y axis indicates relative cell counts, and
the x axis indicates the relative mean
fluorescence obtained with 34-1-2 and phycoerythrin. The dark
line corresponds to cells treated with siRNA to reduce APLP2
expression (mean fluorescence value, 208.8). The light line
corresponds to cells treated with inverse siRNA (mean fluorescence
value, 148.4). The background mean fluorescence values obtained with
secondary antibody only were 5.3 for the siRNA-treated cells and 2.96 for the inverse siRNA-treated cells.
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Considering our results with APLP2-specific siRNA (Fig. 7 and Table
I), since APLP2 interacts with
etKd more strongly in HeLa-etKd than in
DAP-3-etKd (Fig. 1) it can be questioned whether there are
indications of possible functional effects of this difference. We
compared the level of etKd at the surface of the two cell
types by flow cytometry and found that HeLa-etKd
consistently expressed a lower level of etKd at the surface
than did DAP-3-etKd (data not shown) despite the matched
level of intracellular etKd (shown by the
immunoprecipitations in Fig. 1). HeLa-etKd and
DAP-3-etKd could presumably have other cell- or
species-specific differences that might influence etKd
surface expression in addition to the disparity in
APLP2/etKd interaction. However, our finding that
etKd expression is lower on HeLa-etKd than on
DAP-3-etKd is certainly consistent with our findings using
siRNA to down-regulate APLP2 expression in HeLa-etKd (Fig.
7 and Table I). In both cases, reduction of APLP2 association with
etKd correlated with increase in the quantity of cell
surface etKd.
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Table I
Surface MHC class I is increased by inhibition of APLP2 biosynthesis
Flow cytometric analysis was performed on HeLa-etKd cells 3 and
4 days after mock transfection or transfection of cells with siRNA or
inverse siRNA. Mean fluorescence obtained with no primary antibody or
anti-Kd antibodies 34-1-2 and SF1.1.1 is shown.
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DISCUSSION |
Our results showed that APLP2 interacted with etKd in
HeLa-etKd cells more strongly than in the L cell line
DAP-3-etKd (Fig. 1). Our findings suggest that a
cell-specific factor might be involved in the interaction between
Kd and APLP2. Another possibility is that the difference is
related to the presence of human versus murine
2m in these two cell types since
2m is a
requirement for APLP2/Kd interaction (18).
Since APLP2 binds to folded and not to open etKd, it
interacts with etKd relatively late in maturation.
Interestingly previous studies showed that addition of peptide caused
dissociation of APLP2 from Kd (19). Therefore APLP2
presumably binds to a form of Kd that is serologically
recognized as folded but still is peptide-free. The specificity of
APLP2 can be compared with that of other proteins that bind to MHC
class I heavy chains in the ER. Calnexin binds to open as well as
folded forms of Ld and does not release from Ld
upon peptide addition to cell lysates (7). TAP, tapasin, calreticulin, and ERp57 interact specifically with the open, peptide-free form of
class I (5-11). Thus the specificity of APLP2 is distinct from that of
other proteins recognized as MHC class I chaperones.
A new model for Kd assembly that incorporates APLP2 would
include interaction of Kd with calnexin, association of the
peptide-free heavy chain with tapasin, calreticulin, ERp57, and TAP,
and then release of these proteins and interaction with APLP2 prior to
peptide binding and migration to the cell surface. Since it has been
shown that Kd interacts with glycosaminoglycan-modified
APLP2, which is created in the Golgi (18), then
Kd·APLP2 complexes presumably exist beyond the ER.
Interestingly the exit of MHC class I molecules from the ER has
recently been shown to be regulated at a point subsequent to TAP
binding (47). Our results demonstrate that some APLP2 molecules are
present even at the surface of HeLa and HeLa-etKd cells
(Fig. 3).
Our results show that the presence of the adenoviral protein E3/19K
results in increased association of APLP2 with etKd. The
E3/19K protein, like APLP2, seems to bind preferentially to the folded
form of Kd (Fig. 5), indicating that it also has
specificity for a conformational determinant. Similarly, when we
immunoprecipitated folded forms of the endogenous HLA class I molecules
present in 293-E3/19K cells with mAb W6/32, E3/19K was co-precipitated;
in contrast, HC10 immunoprecipitates of open HLA class I molecules did
not contain associated E3/19K (data not shown).
Viruses are known to use numerous mechanisms to evade recognition by
the immune system, and it is possible that the adenovirus recruits
APLP2 for its benefit. Our finding that APLP2 reduces the level of cell
surface etKd is consistent with this notion. Adenovirus
E3/19K down-regulates class I expression by direct retention of class I
(21, 44, 45) and by binding to TAP (46). By retaining APLP2 in the ER,
E3/19K may be acting to reduce Kd expression further. This
would be a third mechanism whereby this single adenoviral protein
down-regulates the surface expression of the MHC class I molecule.
Although APLP2 binds well to Kd, it does not bind to all
MHC class I molecules (18, 19). This attribute is not unique to APLP2
since MHC class I allele specificity is also exhibited by TAP, tapasin,
and calreticulin (12-15). Disparity among class I molecules in regard
to chaperone dependence may provide protection against attack from
viruses against any particular chaperone. In this case, if adenovirus
is indeed using APLP2 to increase the down-regulation of
Kd, the inability of APLP2 to interact with all class I
molecules limits the extent to which APLP2 can be exploited by the virus.