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TAP Association Influences the Conformation of Nascent MHC Class I Molecules

Barbara A. L. Owen^* and Larry R. Pease^1,

Departments of ^* Biochemistry and Molecular Biology, and Immunology, Biochemistry, and Molecular Biology, Mayo Clinic/Foundation, Rochester, MN 55905

	Abstract

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The influence of TAP-MHC class I interactions on peptide binding to the class I heavy chain is assessed during TAP-dependent assembly using K^b-specific Abs that recognize conformational changes induced by assembly with ß₂-microglobulin (ß₂m) and by peptide binding. A significant portion (45%) of K^b molecules in TAP⁺, RMA-derived microsomes are associated with the TAP complex as measured by coimmunoisolation of K^b using anti-TAP1 Abs, while only 20% of the K^b heavy chain molecules are isolated as K^bß₂m complexes with the -K^b-specific Abs, Y-3 or K-10-56. The amount of K^b isolated with Y-3 and K-10-56 increases in proportion to transport and binding of peptide to the K^b molecules within the RMA microsomes. In contrast, less than 5% of the K^b within TAP2-RMA-S microsomes associated with the remaining TAP1 subunit. However, greater than 60% of K^b heavy chain is isolated as K-10-56- and Y-3-reactive K^bß₂m complexes. We propose that a TAP-MHC class I interaction serves to stabilize the MHC class I:ß₂m complex in an immature conformation (Y-3 and K-10-56 nonreactive) prior to high affinity peptide binding, preventing the export of class I molecules complexed with low affinity peptide ligands from the ER.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Assembly of the MHC class I complex occurs very shortly after biosynthesis of the MHC class I heavy chain. The nascent MHC class I protein is cotranslationally inserted into the endoplasmic reticulum (ER),² associates with the ß₂-microglobulin (ß₂m) subunit within minutes of its synthesis, and shortly thereafter is competent to bind peptide (1). Cofactors in MHC class I assembly have been identified, but the exact pathway is not completely understood. One aspect of MHC class I complex assembly that remains to be fully characterized is how the TAP/tapasin complex mediates peptide binding to the MHC class I heavy chain.

Cell surface expression of MHC class I is barely detectable in ß₂m-deficient cell lines (2, 3) or ß₂m knockout mice (4). Likewise, a requirement for the ER membrane protein, TAP, for normal cell surface expression of MHC class I complex has been demonstrated through the use of a variety of TAP-mutant cell lines (5, 6, 7, 8) and TAP-knockout mice. (9). Deficiencies in TAP or ß₂m are associated with aberrantly glycosylated (10) and partially assembled MHC class I molecules, which are retained in the ER/cis-Golgi (11, 12). Therefore, lack of either ß₂m or peptide results in the inability of the MHC class I heavy chain to traffic efficiently through the secretory pathway, resulting in the severely reduced expression of MHC class I on the cell surface.

MHC class I proteins associate with calnexin (13) and calreticulin (14, 15), two chaperones which reside in the ER and are sensitive to the glycosylation state of their targets. The contributions of these chaperones is not fully understood, but may be overlapping since MHC class I expression is normal in a calnexin-deficient cell line (16). The recently discovered tapasin protein (14, 15, 17, 18), which has been shown to associate with TAP and calreticulin, also modulates the level of MHC class I cell surface expression. The basis of the tapasin requirement has not been determined, but a function similar to that of HLA-DM in MHC class II assembly has been proposed (19). In a tapasin-deficient cell line, 721.220, the levels of MHC class I expression are variable and allele dependent (17).

Although MHC class I heavy chains have been coimmunoprecipitated with anti-TAP Abs, a requirement for a direct TAP-MHC class I interaction for efficient peptide loading remains controversial. Allele-specific differences have been noted with regard to TAP-MHC class I association; for example, some HLA-B heavy chains do not demonstrably associate with TAP, but are able nonetheless to present antigenic peptides (20). Additionally, both membrane-bound HLA-G and soluble HLA-G class I proteins bind a similar peptide repertoire, while only the membrane-bound form has been coimmunoprecipitated with TAP (21). These findings must be interpreted in the context of the fact that failure to demonstrate associations experimentally is not equivalent to the absence of biological associations. Evidence in support of the importance of a TAP-MHC class I interaction is based on the findings that a mutant HLA-A2.1 molecule, T134K, does not associate demonstrably with TAP and is expressed at 20% of the level of the parent molecule (22). However, HLA-A2.1-T134K also apparently does not interact demonstrably with calreticulin, and therefore, the cause of impaired peptide binding may not be solely due to inefficient TAP association with the mutant molecule (23).

Although the phenotype of cell surface MHC class I complexes derived from TAP-deficient cells has been studied extensively, there is less known about the conformation of intracellular MHC class I/ß₂m complexes in TAP-deficient cells. To better define aspects of the MHC class I assembly process in the endoplasmic reticulum, the role that TAP plays in the formation of early MHC class I-ß₂m complexes was investigated. We find that the conformation of K^bß₂m complexes formed in TAP2-deficient microsomes differs substantially from those formed in TAP-positive microsomes. Our findings demonstrate that in the presence of a functional TAP (TAP/tapasin) complex, a significant portion of the nascent MHC class I molecules in sealed microsomes does not assemble into structures recognized by K^b-specific Abs until appropriate peptides are added. In contrast, in the absence of functional TAP molecules, irrespective of whether K^b-binding peptides are added to microsomes, the majority of the class I molecules assemble as if they are bound to low affinity ligands within the endoplasmic reticulum. We interpret these findings as evidence that the TAP complex performs regulatory functions beyond peptide transport. We raise the possibility that TAP may be an integral part of an editing process, functioning to assure that most class I molecules exported to the cell surface are bound to high affinity peptides.

	Materials and Methods

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Cell lines, Abs, and peptides

The murine adenoma cell lines RMA and RMA-S were obtained from P. Cresswell (Yale University, New Haven, CT). The cells were maintained at 37°C, 5% CO₂ in RPMI 1640 (Life Technologies, Grand Island, NY) containing 10% bovine calf serum (Hyclone Laboratories, Logan, UT), 2 mM L-glutamine, and 100 U penicillin/streptomycin/ml. The mAbs Y-3 (12-K^b-specific, ß₂m-dependent, peptide insensitive) and 28-14-8 (3-L^d-specific) were purchased from American Type Culture Collection (Rockville, MD) and the mAb K-10-56 (12-K^b-specific, ß₂m-dependent, peptide sensitive) was originally supplied by G. Hammerling (German Institute for Immunology and Genetics, Heidelberg, Germany). The 100.3 Ab is an 12-K^b-specific, ß₂m-dependent, peptide-sensitive mAb and was supplied by K. Hogquist (University of Minnesota, Minneapolis, MN). T. Hansen (Washington University, St. Louis, MO) generously supplied the anti-TAP antiserum, raised against the 19-carboxyl amino acids of the murine TAP1 protein. The Mayo Peptide Synthesis Core Facility synthesized peptides using standard f-moc synthesis and verified the sequences by both HPLC and mass spectrometric analysis. All peptides were synthesized with a free carboxyl terminus, except OVA8 (SIINFEKL), which was synthesized with either a free carboxyl or amide terminus.

Cloning (cDNA), RNA transcription, and in vitro translation

A cDNA for the murine class I MHC heavy chain, K^b, obtained from D. Margulies (National Institutes of Health, Bethesda, MD) was excised with EcoRI and subcloned into the PGEM7zf (Promega, Madison, WI) vector behind the T7 promoter. Sequence was confirmed by dideoxy sequencing; this particular splice variant of K^b is missing exon-8, which encodes the last 9 amino acids of the cytoplasmic tail of most K^b proteins. K^b cDNA was linearized using HindIII and RNA was transcribed from 4 µg of linearized insert with T7 polymerase (Boehringer Mannheim, Indianapolis, IN). RNA was capped during synthesis with p'-5'-(7-methyl)-guanosine-p3,5-guanosine triphosphate, dilithium salt (Calbiochem, San Diego, CA). The RNA was treated with DNase I (Boehringer Mannheim) to remove template DNA, twice extracted with TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA)-saturated phenol:CHCl₃ (1:1), and extracted once with isoamyl alcohol:CHCl₃ (1:24). The RNA was precipitated overnight at -20°C with 2.5 vol 100% ethanol and 0.5 vol 7.5 M ammonium acetate. Precipitated RNA was washed once with 70% ethanol, dried in a vacuum centrifuge, and then reconstituted to 1 mg/ml in sterile, HPLC grade water (Burdick and Jackson, Dade International, Muskegon, MI). The K^b RNA was translated using rabbit reticulocyte lysate prepared in our laboratory by the method of Jackson and Hunt (24). The lysate was stored in liquid N₂ and refrozen no more than once. [³⁵S]Methionine (SJ 204, Amersham Life Sciences, Arlington Heights, IL) was added at 16.0 µl/250 µl lysate resulting in an additional 0.58 mM ß-mercaptoethanol; no additional reducing agent was added to the translation mixture. No oxidized glutahione was added during translation; we estimate the final concentration of free SH in the final translation mixture to be about 1.5 mM. This was determined by summing the concentrations of reduced glutathione within the lysate, the added ß-mercaptoethanol contained within the [³⁵S]methionine, and the DTT added to the microsomes for storage at -70°C. Translations were performed at 30°C for 90 min, in the absence or presence of potassium/EDTA-extracted microsomes prepared from cultured RMA or RMA-S cells by the method of Walter and Blobel (25).

Microsome isolation, proteinase K protection, and the MHC class I assembly assay

Microsomes were added at a final concentration of 0.1 A₂₈₀ units (absorbance determined in 0.1% SDS) before the addition of the RNA to the translation mixture to allow cotranslational insertion of the K^b protein into the microsomal membrane. Translation was stopped by dilution into 50 mM triethanolammonium acetate, pH 7.5, 150 mM K acetate, and 2.5 mM Mg acetate (microsome isolation buffer) at room temperature. Microsomes were isolated immediately by centrifugation through a sucrose cushion at 90,000 rpm for 20 min in a Beckman Airfuge at room temperature. Microsomes were washed in 100 µl of microsome isolation buffer and either were treated with 50 µg/ml proteinase K (PK) (Boehringer Mannheim) for 45 min in 50 µl 10 mM Tris-HCl, pH 7.5, at 4°C or left untreated. PK treatment has been used extensively to determine both the topology of membrane proteins and the portion of in vitro translated proteins that reside within the interior of the microsome (26). Additionally, PK treatment results in the removal of hydrophobic peptides on the exterior of the microsome (see Results). We have found that inclusion of hydrophobic peptides during translation (cotranslationally (CO-T)) results in an additional 20% loss of the PK-protected microsome fraction. The specificity of PK is determined by bulky aromatics and large hydrophobic side chains of amino acids, residues that are contained frequently in K^b restricted peptides. After PK treatment, the microsomes were made 1 mM (final concentration) in PMSF (Sigma, St. Louis, MO) for 10 min at 4°C in order to inactivate the protease prior to solubilization.

Isolation of in vitro-translated K^b protein from protease-protected microsomes was used to determine the impact of peptide acquired during K^b synthesis (CO-T) or posttranslationally, concomitant with solubilization (SOL) had on K^bß₂m-peptide association. K^b RNA was translated, as described above, either in the presence of RMA- or RMA-S-derived microsomes. Peptide was added CO-T and/or SOL at 25 µM. In experiments in which microsomes and peptide were both added CO-T, the microsomes were added first, followed by the peptide and then the RNA. We have determined that in TAP-positive, RMA-derived microsomes the yield of conformed K^b, as determined by immunoisolation (see below), is directly proportional to the amount of high affinity K^b-binding peptide added.

Immunoisolation of K^bß₂m complexes and SDS-PAGE analysis

Immunoisolation was performed on K^bß₂m complexes solubilized away from microsomes recovered from in vitro translation reactions as described above. Preadsorption consisted of a 30-min incubation at 4°C with protein A-sepharose (Sigma). For immunoisolation using the mAbs Y-3, K-10-56, or 100.3, microsomes were solubilized with 50 µl Nonidet P-40 (NP-40) (Sigma) lysis buffer (1% NP-40, 10 mM Tris-HCl, pH 7.5, and 1 mM PMSF) for 30 min on ice. Ab (5 µg/ml, final concentration) was added and incubated overnight at 4°C. Ab-K^b complexes were recovered on 60 µl of protein A-sepharose (1:1 slurry in NP-40 lysis buffer) after an additional 2-h incubation at 4°C. The entire supernatant fluid (135 µl) was retained for analysis by SDS-PAGE as the "unbound" fraction of K^b and the protein A-Sepharose pellet was collected by centrifugation (14,000 rpm, 10 min, 4°C) for extensive washing. The first wash consisted of 100 µl NP-40 lysis buffer, followed by 100 µl Tris-saline (0.05 M Tris-HCl, pH 7.5, 0.1 M NaCl), followed by 100 µl 0.05 Tris, 1.0 M NaCl, and then a final wash with 100 µl Tris-saline. The washes were collected and assayed for [³⁵S]methionine-labeled K^b by scintillation counting. It was determined, on multiple samples, that the amount of [³⁵S]methionine-labeled K^b contained within the washes represented less than 5% of the total [³⁵S]methionine-labeled K^b contained within the total microsome fraction. Nonspecific binding by K^b to either an Ab that recognizes the 3-domain of L^d (28-14-8) or to protein A-Sepharose alone was not detectable.

Immunoisolation using the anti-TAP Ab was performed as described above, with the following modifications. Microsomes were not treated with PK, as the epitope that the anti-TAP antiserum recognizes is present on a cytoplasmic domain of the TAP1 protein. The lysis buffer was digitonin based (Calbiochem, San Diego, CA) (0.5% digitonin, 150 mM NaCl, 20 mM Tris-HCl, pH 7.5) instead of NP-40. The incubation time with the anti-TAP antiserum was 2 h (4°C) instead of overnight and the K^b-immune complexes were washed three times with only the digitonin lysis buffer. In the absence of PK treatment, several species of K^b were resolved on the gels that are not derived from the interior of microsomes. We have determined that only the glycosylated-K^b species resides within PK-protected microsomes and only this species was used in determining Ab "bound" and unbound fractions (see below). Control translations with either RMA or RMA-S microsomes using the yeast pre-pro- protein confirmed our results with K^b, as unprocessed, or partially glycosylated pre-pro- proteins were degraded after PK treatment.

After the immunoisolation procedure, the washed beads were solubilized into 30 µl 5% SDS containing 0.1% 2-mercaptoethanol, heated at 100°C for 5 min, and the entire sample, beads included, was analyzed by SDS-PAGE. The total microsomal K^b is comprised of the fraction of K^b in the supernatant fluid, the unbound fraction and the fraction of K^b bound to the Ab and therefore the protein A-Sepharose. SDS-PAGE (12%, reduced) was performed on the bound and unbound fractions, and the gels were fixed, enhanced with (Fluoro-en³Hance Research Products International, Boston, MA) for 45 min at room temperature, and dried prior to autoradiography with Bio-MAX (Kodak, Rochester, NY) film. K^b bound and unbound SDS-PAGE bands were measured by densitometry with an Ambis Systems (San Diego, CA) imaging and data analysis system. Identical, size-matched scans of lane backgrounds were subtracted from each protein band to account for film or individual lane variations. Multiple film exposures were used in order to ensure that K^b protein bands were within the linear range of the analytical system.

Flow cytometric analysis

Surface expression of K^bß₂m complexes was induced on RMA-S cells by incubating with 10 µM of the indicated peptide at 26°C for 12 h. A total of 5 x 10⁵ cells were incubated with either 50 µl of K-10-56 cultured supernatant or 50 µl 100.3 ascites (diluted 1:100) for 25 min at 4°C in FACS buffer (HBSS, Life Technologies) containing 10% BSA, 0.2% sodium azide, and 5 mM sodium bicarbonate, pH 7.4). After incubation, cells were washed three times with FACS buffer and then incubated with fluorescein-conjugated goat anti-mouse IgG and IgM (Biosource International, Camarillo, CA) in FACS buffer for 20 min at 4°C. Cells were then washed three times with FACS buffer at 4°C. Flow cytometric analysis was performed using a FACScan (Becton Dickinson, Mountain View, CA) instrument and mean channel fluorescence (MCF) values were determined.

	Results

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Conformational analysis of K^bß₂m complexes within microsomes

Conformational differences of K^b synthesized into microsomes derived from the TAP-positive, murine lymphoma cell line RMA and the variant TAP-negative cell line RMA-S (27) are shown by their differential reactivity with the conformationally sensitive mAb Y-3 (Fig. 1). In peptide-untreated microsomes, about 20% of the intramicrosomal K^b is Y-3 positive, while in RMA-S microsomes, approximately 80% of the K^b molecules can be isolated with the Ab. The specificity of the immunoisolation procedure was confirmed by the absence of K^b recovered with an Ab that does not bind specifically to K^b (L^d-specific, 28-14-8, data not shown, see Materials and Methods). The fraction of K^b in any particular conformation can be assessed accurately by analyzing both the Ab bound and unbound fractions after immunoisolation with K^b-specific Abs by SDS-PAGE of [³⁵S]met-labeled, in vitro-translated K^b. The percentage of K^b that is reactive with a particular Ab is calculated by dividing the amount of K^b in the bound fraction by the total amount of K^b in the microsome fraction (bound/(bound + unbound) x 100 = % Y-3 reactive, for example). For the analysis of peptide-dependent conformational changes within microsomal K^b, the microsomes were treated with PK. This well-established technique has been used extensively to define the fraction of in vitro-translated protein that actually resides within the microsome and is therefore resistant to digestion by the protease (26). Additionally, PK treatment removes any cytoplasmic domains of the translated protein, such as the cytoplasmic tail of K^b, and also eliminates hydrophobic peptides, which do not get transported across but may associate with the membrane. The details of this assay are described in Materials and Methods.

View larger version (75K): [in this window] [in a new window]   FIGURE 1. Y-3 complexes formed in TAP-positive and TAP-deficient microsomes. Kbß2m complexes formed in either RMA or RMA-S microsomes, as indicated, were subjected to immunoisolation with the ß2m-dependent mAb Y-3. All microsomes were treated with PK after isolation and washing and prior to SOL with NP-40 lysis buffer. This treatment is necessary to determine the intramicrosomal fraction of in vitro-translated Kb, which has been radiolabeled during its synthesis with [35S]methionine. Both Y-3-bound Kbß2m complexes and unbound Kb were analyzed by SDS-PAGE. The percentage of Y-3-reactive Kb was quantified as the ratio of bound Kb to unbound Kb measured by densitometric image analysis of [35S]methionine-exposed film. The absence or presence of the OVA8amide (SIINFEKL) peptide (25 µM) is as indicated. Peptide was added either during in vitro translation (CO-T) or concomitant with SOL. See Material and Methods for additional details.

View larger version (42K): [in this window] [in a new window]   FIGURE 2. Y-3 complexes formed in microsomes. A, Kbß2m complexes formed in RMA microsomes were analyzed by SDS-PAGE and quantified as shown in Fig. 1. Kb was translated into RMA microsomes either in the absence of peptide (none) or peptide was added CO-T or with SOL, as described in Fig. 1. Either the OVA8amide peptide (SIINFEKL) or the SEV9 peptide (FAPGYNPAL) was added as indicated. B, Kbß2m complexes formed in RMA-S microsomes were analyzed exactly as described for A. The results are representative of six independent experiments, with error bars depicting SD.

Due to the unexpected finding that the majority of K^bß₂m complexes formed within RMA-S microsomes in the absence of high affinity peptide are Y-3-reactive, we looked for further detectable differences in the association of RMA-S-derived-K^b molecules with other ER components. It has been previously shown that a significant portion of MHC class I molecules in peptide-untreated TAP-positive cells are associated with TAP. To determine whether a measurable K^b-TAP association occurs within purified microsomes, we compared the amount of K^b cotranslated into either peptide-untreated RMA or RMA-S microsomes by coimmunoisolation with an anti-TAP1 Ab. This Ab, which was raised against the 19 carboxyl-terminal residues of murine TAP1, has been shown to coprecipitate "open" forms of the murine MHC class I protein, L^d (28).

We find that over 40% of the glycosylated-K^b molecules synthesized into peptide-untreated RMA microsomes can be coimmunoisolated with an anti-TAP1 Ab (Fig. 3). Forty percent is a minimal estimate of the TAP-associated molecules, as weak complexes could dissociate during isolation. Our results are comparable with previously described amounts of endogenous K^b coprecipitated from detergent-lysed, K^b-expressing cells using an Ab raised against the terminal one-third of the TAP1 protein (29). The epitope recognized by the anti-TAP1 Ab is contained within the cytoplasmic tail of TAP. PK-treatment most likely destroys this epitope as evidenced by the failure of PK-treated microsomes to yield any K^b in the TAP-bound fraction, as shown in Fig. 3. Therefore, anti-TAP immunoisolation must be performed on microsomes that have not been treated with protease. It is important to only quantify the glycosylated form of K^b, K^b_gly, which is the highest m.w. species visible in SDS-PAGE of class I molecules coprecipitated with the anti-TAP1 Ab. We have determined that, when microsomes have been treated with PK, only the glycosylated fraction of the total synthesized K^b is protected from digestion. Therefore, it is this species that represents the fraction of K^b molecules that resides within sealed microsomes. When peptide is added SOL, the amount of K^b associated with TAP drops to 15%. This result is consistent with previously published reports (29, 30), showing that MHC class I molecules dissociate from TAP after binding peptide. In contrast, we find that barely detectable levels of the glycosylated form of K^b cotranslated into RMA-S microsomes is associated with the intact TAP1 subunit, in the presence or absence of high affinity peptide as shown in Fig. 3. A 6x exposure of the bound SDS-PAGE bands to film is required to determine that less than 5% of the K^b_gly fraction can be coimmunoisolated from RMA-S microsomes. Therefore, the association of K^b with TAP1 is much weaker in RMA-S microsomes than with intact TAP1/TAP2 complexes in RMA microsomes. The apparent association of unglycosylated forms of K^b with TAP1 Ab is likely artifactual, as unglycosylated forms of K^b do not reside within either intact RMA-S or RMA microsomes. This is shown clearly by the presence of only a single m.w. species of K^b, which migrates at the expected size for the glycosylated protein after PK treatment (Figs. 1 and 5).

View larger version (45K): [in this window] [in a new window]   FIGURE 3. Kb-TAP complexes isolated from RMA or RMA-S microsomes. Kb was in vitro translated into either RMA or RMA-S microsomes as indicated, and TAP-associated Kb was immunoisolated with an anti-TAP1 antiserum. Microsomes were not treated with PK, as had been done previously for immunoisolation of Y-3 complexes, as the epitope recognized by the anti-TAP1 Ab resides within the cytoplasmic tail of TAP1 and is proteolytically removed. This is shown in the third lane of the RMA microsome data; PK treatment results in one m.w. species of Kb, which is not coimmunoisolated (ND) by the anti-TAP1 antiserum. In contrast, in peptide-untreated RMA microsomes, 45% of the glycosylated Kb fraction, designated Kbgly in the figure, was coimmunoisolated with the anti-TAP Ab. The Kbgly fraction, which is the highest m.w. species isolated from protease-untreated microsomes, is the only species that resides within microsomes after treatment with protease: see Materials and Methods for details. When peptide is added with SOL, only 15% of the Kbgly fraction can be coimmunoisolated with the anti-TAP Ab. Trace quantities (essentially none detected) amounts of the Kbgly fraction within RMA-S microsomes were coimmunoisolated with the anti-TAP antiserum. The absence (none) or presence of peptide added with SOL had no impact upon the amount of the Kbgly fraction detected. An increased exposure time (288 h vs 48 h) of only the bound lanes of the SDS-PAGE revealed that less than 5% of the Kbgly fraction isolated from RMA-S microsomes could be coimmunoisolated with the anti-TAP antiserum. Only the OVA8amide derivative of the OVA8 peptide was used for this particular experiment.

View larger version (79K): [in this window] [in a new window]   FIGURE 5. K-10-56-positive Kbß2m-ligand complexes isolated from RMA and RMA-S microsomes. Kbß2m complexes formed in either RMA or RMA-S microsomes, as indicated, were subjected to immunoisolation with the peptide-sensitive, ß2m-dependent mAb K-10-56. Microsome isolation, PK-treatment and SOL, peptide addition, SDS-PAGE, and data analysis were exactly as described in Fig. 1. Only the OVA8amide derivative of the OVA8 peptide was used for this particular experiment.

Abs sensitive to conformation changes induced by bound peptides were used to establish that peptide added CO-T is transported across the microsome membrane and binds to K^b within RMA-derived microsomes. The mAbs K-10-56 and 100.3 bind different epitopes on K^b that are sensitive to peptide occupancy. Both K-10-56 and 100.3 can distinguish the difference between the binding of OVA8 with an amidated or free carboxyl leucine at position 8 (31, 32). As shown in Fig. 4, K-10-56 stains K^b molecules on RMA-S cells cultured at 26°C 150 channels more intensely than cells preincubated with the amidated peptide, OVA8. Binding of K^b to either SEV9 or OVA8 with a free carboxyl terminus results in a 20- to 30-channel increase in the K-10-56 signal. However, the binding of the amidated version of OVA8 to K^b causes a 60-channel decrease in 100.3 binding relative to no peptide, whereas the binding of the OVA8-free COOH results in a drop of over 150 channels of relative staining intensity. The SEV9 peptide produces a comparable increase in 100.3 binding as it does for K-10-56, approximately 30 channels. These data are summarized in Table I.

View larger version (26K): [in this window] [in a new window]   FIGURE 4. The K-10-56 Ab distinguishes peptide occupancy by Kb. RMA-S cells were cultured overnight at 26°C in RPMI 1640 medium containing 8% bovine calf serum containing 10 µM peptide as indicated. Cells were stained with the mAb K-10-56 and fluorescein-conjugated goat anti-mouse IgG and then analyzed by flow cytometry. The control was a nonspecific isotype-matched mAb.

The fact that K-10-56 recognizes molecules bearing a mature folding pattern is demonstrated using a second peptide, SEV9, which does not mask the epitopes bound by the Abs K-10-56 or 100.3. When K^b is translated in the presence of the SEV9 peptide, the amount of K-10-56-positive molecules was nearly doubled to 39%, and increased to 54% when SEV9 was added SOL (see Fig. 6A). Comparable results were obtained with the Ab 100.3, as summarized in Table II.

View larger version (37K): [in this window] [in a new window]   FIGURE 6. K-10-56-positive Kbß2m complexes formed in RMA and RMA-S microsomes. A, Kbß2m complexes formed in RMA microsomes were analyzed by SDS-PAGE and quantified as described in Figs. 1 and 2. B, Kbß2m complexes formed in RMA-S microsomes were analyzed exactly as described for A. Microsome isolation, PK treatment and SOL, peptide addition, SDS-PAGE, and data analysis were exactly as described in Figs. 1 and 2. The results are representative of six independent experiments, with error bars depicting SD. Only the OVA8amide derivative of the OVA8 peptide was used for this particular experiment.

The fact that the K-10-56-reactive K^b fraction does not decrease when the OVA8_amide peptide is added CO-T to RMA-S microsomes demonstrates the effectiveness of the PK treatment in removing hydrophobic peptides from the surface of microsomes. Had any OVA8_amide peptide remained associated with the RMA-S microsomes after the PK treatment, it would be expected to bind to preexisting K^bß₂m complexes during SOL and result in the loss of the K-10-56 signal. Addition of OVA8_amide concomitant with SOL does result in the loss of the K-10-56 epitope, demonstrating that the peptide-binding site of microsomal K^b, which has an antigenic phenotype of a folded molecule, is accessible to peptide.

The SEV9 peptide also is believed to replace endogenous ligands when added with SOL. As expected, however, no significant change in the K-10-56 signal was discernible (Fig. 6B). By manipulating the amount and nature of peptides added during translation and SOL, we were able to demonstrate that high affinity peptides in the class I-binding site are not readily displaced, even by other high affinity peptides. When K^bß₂m complexes formed with the high affinity peptide, SEV9, were solubilized away from TAP⁺ RMA microsomes and subsequently incubated with 25 µm OVA8_amide peptide added with SOL, replacement of SEV9 was minimal (30% binding to K-10-56 following SOL incubation with OVA8_amide vs 33% without OVA8_amide addition) (data not shown). This provides the basis of our conclusion that K^bß₂m complexes formed in the absence of added high affinity peptide are occupied by what are functionally defined as low affinity ligands. Very few (7%) of K^b molecules synthesized in the absence of high affinity peptide are peptide unreceptive (Fig. 6A).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The possibility that a direct TAP-MHC class I interaction is important for peptide acquisition is intriguing, but strong evidence supporting this hypothesis has been elusive (13, 33, 34). Although TAP has been shown to associate only transiently with MHC class I molecules, this association may serve to increase interactions with other accessory proteins within the ER that are required for the folding or peptide binding of MHC class I molecules, such as tapasin and/or calreticulin, calnexin, or as yet unidentified ER-resident proteins. For example, the HLA-A 0201 mutant (T134K), defined previously by the absence of demonstrable association with TAP, improper folding, and an inability to present Ag at the cell surface, also does not interact demonstrably with calreticulin (22). Additionally, in cells lacking TAP, a redistribution of MHC class I associations with other ER resident proteins has been demonstrated. Specifically, MHC class I molecules within TAP-mutant cells lose associations with calreticulin and tapasin and concomitantly increase their associations with calnexin and BiP (35). Finally, changes in trafficking and maturation of carbohydrate moieties on class I molecules have been shown in the TAP-deficient RMA-S cells and tapasin-deficient 721.220 cells (17, 33).

In order to determine whether a measurable difference in an MHC class I-TAP interaction might influence early MHC class I complex assembly, we assayed specific conformational changes in ER-resident K^bß₂m complexes within microsomes derived from TAP-positive or TAP-negative cells. High affinity peptide added CO-T to K^b synthesized in the presence of TAP-positive microsomes induced folding with endogenous ß₂m as measured by the gain of the ß₂m-dependent and peptide-inducible Y-3 epitope within the K^b heavy chain. The K-10-56 and 100.3 antigenic profiles of K^b synthesized in the presence of OVA8_amide peptide indicate specific peptide transport and binding within TAP-positive, protease-resistant microsomes. When peptide is added concomitant with SOL of the microsomes, preexisting-K^bß₂m complexes generated within intact microsomes also bind peptide. TAP, a protein that should span the membrane multiple times, should contain several extracytoplasmic loops that most likely are destroyed by the PK treatment step that precedes SOL. In support of this hypothesis, Fig. 3 shows the loss of the coimmunoisolated K^b fraction after PK treatment, suggesting the loss of the epitope recognized by the anti-TAP antiserum. K^bß₂m-peptide complexes that bind peptide added with SOL therefore are most likely doing so in a TAP-independent manner.

In contrast, K^bß₂m complexes synthesized within TAP-deficient microsomes can only bind high affinity peptide when added concomitant with SOL. As predicted from previous findings, peptide added CO-T to TAP-deficient microsomes is not transported as determined by the retention of the K-10-56-defined conformation of K^bß₂m molecules within sealed RMA-S microsomes. Interestingly, in peptide-untreated RMA-S microsomes, the majority of the microsomal K^b is present as Y-3- or K-10-56-reactive K^bß₂m complexes. This differs from the K^b molecules within RMA microsomes in which only 20% of the total can be isolated as Y-3- or K-10-56-reactive K^bß₂m complexes. Since the RMA-derived mirosomes have a functional TAP (TAP/tapasin) complex one would predict that the concentration of available peptide ligands would be substantially higher compared with RMA-S. However, the majority of K^b molecules in RMA microsomes are not only unoccupied by peptide, but do not maintain association with ß₂m after SOL as determined by low Y-3 and K-10-56 binding (Figs. 1 and 5). This finding implies that the presence of intact TAP molecules plays an important function in preventing the premature assembly of class I molecules before high affinity peptides are available for binding to the peptide-binding site.

When peptide is added CO-T to RMA-derived microsomes, peptide translocation and peptide binding by K^bß₂m occurs readily as measured both by changes in the Y-3 and K-10-56 conformations. Since K-10-56 is able to distinguish whether the OVA8_amide or SEV9 peptide is bound to K^b, it is possible to speculate about the nature of the ligands bound to K^bß₂m in peptide-untreated microsomes. Nearly half of the K^bß₂m complexes that are K-10-56 reactive in RMA microsomes prior to addition of high affinity peptide exchange endogenous ligands for the OVA8_amide peptide. Additionally, when OVA8_amide is added with SOL, nearly 70% of the endogenous ligands are replaced by the high affinity K^b-binding peptide. However, OVA8_amide added upon SOL replaces very little of the high affinity SEV9 peptide added CO-T. Therefore, the low amount of K^bß₂m complexes formed in TAP-positive microsomes are occupied by what functionally resembles low affinity ligands.

In TAP-deficient, RMA-S microsomes, most of the K^b molecules are associated with ß₂m, but appear to be bound to low affinity ligands. A majority of these molecules readily bind the OVA8_amide peptide-added SOL. The nature of the low affinity ligands has, as yet, not been determined, but several potential candidates exist. The most obvious candidate is low affinity peptide which, in the case of TAP-deficient microsomes, should be predominantly signal peptides or, perhaps, incompletely synthesized and, therefore, misfolded secretory proteins that normally traffic through the ER. However, one would assume that both RMA and RMA-S microsomes would have equivalent amounts of such peptides. Given this assumption, the importance of the finding that RMA microsomes untreated with high affinity peptide maintain such low levels of MHC class I-ß₂m complexes is accentuated. We could not detect a significant portion of the K^bß₂m complexes formed in RMA-S microsomes associated with the remaining TAP1 subunit. In TAP⁺ RMA microsomes, more K^b heavy chain was found associated with TAP by coimmunoisolation than could be recovered by direct immunoisolation with K^b-specific Abs. It is possible that this association is important for the maintenance of peptide-receptive class I molecules prior to peptide loading and could serve a peptide editing function, permitting only higher affinity peptides to induce the mature folding phenotype required for class I export from the ER. Such a proposed role for the TAP in this pathway does not minimize the possibility that other ER-resident proteins could also contribute by binding to and stabilizing K^bß₂m complexes or precursors.

To date, no other deficiencies in the cofactors involved in MHC class I assembly have been detected within RMA-S cells. Therefore, we conclude from our findings that a functional TAP1/TAP2 complex, either directly or indirectly through its association with tapasin (tapasin/calreticulin), prevents the premature folding of nascent MHC class I proteins in the endoplasmic reticulum. Recent reports indicated that some class I molecules, such as HLA-B8, are able to assemble efficiently without apparent TAP association (36). A similar finding was reported for the HLA-A2 mutant T134K, a molecule that is not detected in complexes with either TAP or calreticulin, but nonetheless assembles and is exported as a peptide-receptive class I molecule to the cell surface (22, 23). While it has long been clear that TAP is not absolutely required for surface expression of class I, none of these early studies addresses the regulatory role TAP expression may have on selection of peptides that bind maturing class I molecules, or on the influence of TAP on the range of peptide concentrations that are required to efficiently package Ag-presenting molecules with appropriate peptides. Our finding that TAP molecules associate with nascent class I heavy chains and prevent them from folding into a mature antigenic configuration is consistent with the view expressed earlier that retention of class I molecules in the ER plays an important role in the process by which class I molecules acquire high affinity peptides for presentation as Ags (23).

	Acknowledgments

 
We thank Scott Kuhns for his critical review of the manuscript, Dr. Peter Cresswell for providing the cell lines RMA and RMA-S, Dr. Ted Hansen for generously supplying the anti-TAP1 antiserum and the 28-14-8 mAb, and Dr. Kristin Hogquist for supplying the 100.3 Ab. Mike Hansen prepared K-10-56 and Y3 ascites, Rudy Hanson assisted with the cDNA construction, Kathleen Allen performed the flow cytometric analysis, and Becky Sanford provided secretarial assistance in preparation of this manuscript. Ben Madden and Jane Liebenow of the Mayo Peptide Core synthesized and characterized all of the peptides used in this study.

	Footnotes

 
¹ Address correspondence and reprint requests to Dr. Larry R. Pease, Department of Immunology, Mayo Clinic, Guggenheim 3, 200 First Street SW, Rochester, MN 55905.

² Abbreviations used in this paper: ER, endoplasmic reticulum; ß₂m, ß₂-microglobulin; PK, proteinase K; NP-40, Nonidet P-40; MCF, mean channel fluorescence, CO-T, cotranslationally; met, methionine.

Received for publication June 8, 1998. Accepted for publication January 14, 1999.

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