The Amyloid Precursor-like Protein 2 and the Adenoviral E3/19K Protein Both Bind to a Conformational Site on H-2Kd and Regulate H-2Kd Expression*

Chantey R. MorrisDagger , Jason L. PetersenDagger , Shanna E. VargasDagger §, Heth R. TurnquistDagger §, Mary M. McIlhaneyDagger , Sam D. Sanderson, Joseph T. Bruder||, Yik Y. L. Yu**, Hans-Gerhard BurgertDagger Dagger , and Joyce C. SolheimDagger §§§¶¶

From the Dagger  Eppley Institute for Research in Cancer and Allied Diseases, the Departments of § Pathology and Microbiology and §§ Biochemistry and Molecular Biology, and the  School of Allied Health Professions, University of Nebraska Medical Center, Omaha, Nebraska 68198, || GenVec, Gaithersburg, Maryland 20878, ** NCI, National Institutes of Health, Bethesda, Maryland 20892, and the Dagger Dagger  Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom

Received for publication, August 11, 2002, and in revised form, December 10, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A protein of unknown physiological function, called amyloid precursor-like protein 2 (APLP2), forms an association with the murine class I molecule Kd that is up-regulated by the presence of the adenoviral protein E3/19K. We have extended these findings to show that APLP2 and E3/19K associate preferentially with folded Kd and not with the open form. APLP2 was detectable at the cell surface, but its surface expression was not up-regulated by the concurrent expression of Kd. Experimental down-regulation of APLP2 expression caused a consistent increase in the surface expression of Kd, indicating that APLP2 normally reduces Kd surface expression. These data suggest a role for APLP2 in controlling the maturation of major histocompatibility complex class I molecules.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta 2-microglobulin (beta 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/beta 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 alpha 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 beta -amyloid peptide domain (Abeta ) 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-1Kdbeta (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 right-arrow 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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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 alpha 1 domain of open, peptide-free Ld (27, 28). The mAb 34-1-2 is directed against the alpha 1/alpha 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 alpha 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 beta 2m-associated HLA-A, -B, and -C (31) and can recognize Kd if Kd is associated with bovine beta 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 right-arrow 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 beta -glucuronidase driven from the Rous sarcoma virus promoter. beta -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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

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 alpha 1/alpha 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.

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-alpha 3 domain antibody that recognizes both open and folded forms. Anti-alpha 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 alpha 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).

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 beta -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.

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.

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.

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.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta 2m in these two cell types since beta 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.

    ACKNOWLEDGEMENTS

We thank Dr. Charles Kuszynski and Linda Wilkie of the University of Nebraska Medical Center Cell Analysis Core Facility for assistance with flow cytometry; Dr. Ted Hansen for the generous gifts of ascites containing 34-1-2, SF1.1.1, and 64-3-7 mAbs and the DAP-3 L cell line; and Dr. Wendy Maury for the HeLa cell line.

    FOOTNOTES

* This work was supported by Deutsche Forschungsgemeinschaft Grant Bu 642/1 (to H.-G. B.), National Institutes of Health Grant GM57428 (to J. C. S.), and National Institutes of Health Training Grant T32 CA09476 and University of Nebraska Medical Center Graduate Studies Fellowships (to J. L. P., S. E. V., and H. R. T.).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.

¶¶ To whom correspondence should be addressed: Eppley Inst. for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198-6805. Tel.: 402-559-4539; Fax: 402-559-4651; E-mail: jsolheim@unmc.edu.

Published, JBC Papers in Press, December 27, 2002, DOI 10.1074/jbc.M208203200

    ABBREVIATIONS

The abbreviations used are: MHC, major histocompatibility complex; APLP2, amyloid precursor-like protein 2; beta 2m, beta 2-microglobulin; CHAPS, 3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; ER, endoplasmic reticulum; et, epitope-tagged; FACS, fluorescence-activated cell sorting; Ab, antibody; mAb, monoclonal antibody; TAP, transporter associated with antigen processing; Ad, adenovirus; PBS, phosphate-buffered saline; siRNA, short interfering RNA.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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