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The Journal of Immunology, 1998, 161: 2307-2316.
Copyright © 1998 by The American Association of Immunologists

The Assembly and Stability of MHC Class II-({alpha}ß)2 Superdimers1

Peter H. Schafer2, Sunil Malapati, Kimberly K. Hanfelt and Susan K. Pierce3

Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
X-ray crystallography of several MHC class II molecules revealed a structure described as a dimer of heterodimers, or a superdimer. This discovery led to the hypothesis that MHC class II molecules may interact with the TCR and CD4 as an ({alpha}ß)2 superdimer, potentially providing more stable and stimulatory interactions than can be provided by the simple {alpha}ß heterodimer alone. In this study, using chemical cross-linking, we provide evidence for the existence of the superdimers on the surface of B cells. We further characterize the superdimers and demonstrate that in lysates of B cells, I-Ek dimers and superdimers are derived from the same population of I-Ek molecules. Purified, I-Ek molecules in solution also exist as a mixture of 60-kDa dimers and 120-kDa superdimers, indicating that I-Ek has an intrinsic ability to form 120-kDa complexes in the absence of other cellular components. Peptide mapping showed that the {alpha}ß and ({alpha}ß)2 complexes are closely related and that the superdimers do not contain additional polypeptides not present in the dimers. The ({alpha}ß)2 complex displays thermal and pH stability similar to that of the {alpha}ß complex, both being denatured by SDS at temperatures above 50°C and at a pH below 5. These data support the model that MHC class II has an intrinsic ability to assume the ({alpha}ß)2 superdimeric conformation, which may be important for interactions with the TCR and CD4 coreceptor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD4+ Th cells recognize peptide fragments of Ags bound to class II molecules presented on the surface of APCs (reviewed in 1 . MHC class II molecules consist of a heterodimer of noncovalently associated polymorphic {alpha} and ß subunits, each type I integral membrane glycoproteins, with molecular mass of approximately 34 and 29 kDa, respectively (2, 3, 4). Upon synthesis in the endoplasmic reticulum, the {alpha}- and ß-chains immediately associate and assemble noncovalently with a third protein, the invariant chain (Ii),4 forming a nine-chain ({alpha}ßIi)3 complex (5, 6). The Ii serves both to prevent peptides from binding to the class II molecules en route to the appropriate subcellular site for peptide binding (7, 8, 9), and to direct the class II molecules to that site (10, 11, 12). Ii is proteolytically removed from the class II molecules in the peptide-loading compartments (13, 14, 15), allowing the class II molecules to bind antigenic peptides (16, 17, 18). Peptide binding completes the protein-folding process and maturation of class II molecules (19, 20). Upon antigenic peptide binding, the association between the {alpha}- and ß-chains becomes remarkably strong, withstanding the denaturing effects of SDS-PAGE at room temperature in the absence of reducing agents (21, 22, 23).

Studies over the last several years have yielded a detailed picture of the structure of class II molecules (reviewed in 24 . The three-dimensional structures of the human class II molecules, HLA-DR1 and HLA-DR3 (25, 26, 27, 28, 29, 30), and the mouse class II molecule I-Ek (31) have been determined by x-ray crystallography. The binding site for CD4, the Th cell coreceptor for class II molecules, has been mapped to a loop in the ß2 domain (32, 33, 34) and possibly the {alpha}2 domain (25, 35). One striking feature of the class II crystal structures is that the {alpha}ß heterodimer dimerized with itself to form an ({alpha}ß)2 complex. The fact that these dimers of {alpha}ß heterodimers, or superdimers, appeared in several different types of DR crystals, as well as in I-Ek crystals, suggests that the tendency to superdimerize may represent an intrinsic property of MHC class II molecules (reviewed in 36 . It has been postulated that the superdimerization of class II molecules may serve to both facilitate the activation of T cells by cross-linking TCRs and signaling the APC to express essential costimulatory molecules for T cell activation (25). These authors envisioned a synergistic process in which a pair of TCRs interacts with a pair of class II molecules, resulting in stable, dimerized class II-TCR complexes. Indeed, the x-ray crystal structures of the ß-chain (37) and the V{alpha} domain (38) of the TCR have been solved, and the V{alpha} domain shows a tendency to form pairs of homodimers potentially able to interact with two class II molecules (38); however, homodimers were absent in the heterodimeric TCR-{alpha}ß structures (39, 40). Recently, Reich et al. (41) showed that TCR/peptide-MHC class II complexes oligomerized in solution in a ligand-dependent fashion to form supramolecular structures containing two to six TCR/peptide/MHC class II complexes. These results provided direct evidence for models of T cell signaling based on specific multimerization of TCR/peptide/MHC complexes. Others have hypothesized that CD4 oligomerization may promote MHC class II and TCR oligomerization (42). The model of CD4-mediated oligomerization is attractive because it does not depend upon two MHC class II molecules containing identical or closely related antigenic peptides required for TCR dimerization.

Our previous studies provided evidence for the existence of both a 60-kDa {alpha}ß heterodimer and a 120-kDa ({alpha}ß)2 superdimer of the mouse I-Ek molecules, in detergent lysates of B cells (43). A mAb with demonstrated specificity for the ({alpha}ß)2 superdimer inhibited a low affinity, but not a high affinity T cell response, suggesting that the superdimers exist on the B cell surface and that they may play a role in presenting low affinity Ags (43). Subsequently, Roucard et al. (44) provided biochemical evidence that HLA-DR existed as a superdimer in detergent solutions. Very recently, Cherry et al. (45), using single-particle fluorescence imaging, showed that HLA-DR exists as both dimers and superdimers on the surfaces of human fibroblasts transfected with the genes encoding HLA-DR {alpha} and ß and Ii, and estimated that 25% of class II molecules were in the superdimer form. In this study, using chemical cross-linking, we demonstrate that the mouse I-Ek superdimer exists on the surface of B cells in the absence of detergent. We also provide evidence that both {alpha}ß and ({alpha}ß)2 are derived from a single population of I-Ek molecules in B cell lysates and that purified I-Ek in detergent solution assumes both {alpha}ß dimers and ({alpha}ß)2 superdimers. The class II superdimers are present in significant amounts, are free of components other than {alpha}- and ß-chains, and have the same general properties of SDS stability as {alpha}ß complexes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Abs and cell lines

The mouse mAbs 17-3-3S (46) and Y-17 (47), which recognize different but overlapping determinants on the I-Ekß-chain (48, 49, 50, 51), were obtained from hybridomas provided by American Type Culture Collection (ATCC, Manassas, VA). 17-3-3S is of the IgG2a,{kappa} isotype, and Y-17 is of the IgG2b,{kappa} isotype. The mAbs were purified by protein A-Sepharose chromatography. The Ii-specific rat mAb IN-1 (isotype IgG2b,{kappa}) was kindly provided by Dr. N. Koch (University of Bonn, Bonn, Germany). The isotype control mAb IgG2a,{kappa} was purchased from PharMingen (San Diego, CA), and rat IgG was purchased from Jackson ImmunoResearch (West Grove, PA). The horseradish peroxidase (HRP)-conjugated secondary Abs, rabbit Abs specific for mouse IgG2a, and goat Abs specific for rat IgG heavy and light chains were obtained from Zymed Laboratories (South San Francisco, CA).

The B cell lymphoma CH27 (52) was characterized and kindly provided by Dr. G. Haughton (University of North Carolina, Chapel Hill, NC). The mouse T cell hybridoma TPc9.1 generated in this laboratory is specific for cytochrome c presented by I-Ek-expressing APC, and secretes IL-2 upon activation (53, 54). The CTLL-2 cell line (55), obtained from ATCC, is an IL-2-dependent cell line. All cells were grown in complete medium (CM) (56) containing 15% FCS (15% CM). The CTLL-2 cells were maintained in 15% CM containing 10% T-Stim (Collaborative Biomedical Products, Bedford, MA) as an IL-2 source.

Immunoprecipitation

CH27 cells (2.5 x 106 cells/ml) were incubated in Met- 5% CM for 30 min, labeled with [35S]Met,Cys (NEN, Wilmington, DE) at 150 µCi/ml for 30 min, and washed in 15% CM. The cells were cultured in 15% CM at 3 x 105 cells/ml for 4 h. At the conclusion of the chase, cells were washed twice in cold PBS and lysed at 5 x 106 cells/ml in ice-cold Nonidet P-40 lysis buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 5 mM EDTA, and 0.5% Nonidet P-40) containing 200 µg/ml PMSF, 25 µg/ml aprotinin, and 10 mM iodoacetamide. The lysate was divided into two equal portions of 5 x 106 cell equivalents each, centrifuged at 16,000 x g for 30 min at 4°C, and precleared twice with 200 µl protein A-Sepharose (Pharmacia, Piscataway, NJ) slurry. I-Ek was immunoprecipitated with mAbs 17-3-3S or Y-17 and 60 µl protein A-Sepharose slurry at 4°C. Supernatants were precipitated a second and third time with 30 µg mAb and 60 µl protein A-Sepharose slurry. Beads were washed three times with ice-cold Nonidet P-40 wash buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 5 mM EDTA, and 0.2% Nonidet P-40). Bound proteins were eluted with 80 µl of nonreducing SDS-loading mixture (250 mM Tris-HCl, pH 6.8, 50% (v/v) glycerol, 5% (w/v)). Eluates were subjected to 10% SDS-PAGE and fluorography using Fluoro-Hance (Research Products International, Mt. Prospect, IL).

Chemical cross-linking

CH27 cells (1.6 x 106 cells/ml) were labeled with [35S]Met,Cys (150 µCi/ml) for 14 h at 37°C. The cells were washed with ice-cold PBS at pH 8.5 (PBS 8.5). The cells were resuspended in PBS 8.5 at a concentration of 25 x 106 cells/ml, and aliquots (200 µl) were incubated with 2 µl of a 200 mM solution of a cross-linking reagent in DMSO or 2 µl of DMSO alone for 2 h at 4°C. The cross-linking reagents tested included: dithiobis[succinimidylpropionate] (DSP); 1,5-difluoro-2,4-dinitrobenzene (DFDNB); 3,3'-dithiobis[sulfosuccinimidylpropionate] (DTSSP); ethyleneglycolbis[succinimidylsuccinate] (EGS); bis[sulfosuccinimidyl]suberate (BS3); dimethyladipimidate.2HCl (DMA); dimethylpimelimidate.2HCl (DMP); disulfosuccinimidyltartarate (sulfo-DST); and dimethyl 3.3'-dithiobispropionimidate.2HCl (DTBP) (Pierce, Rockford IL). The cross-linking was quenched by adding 20 µl of 1 M glycine and incubating at 4°C for 5 min. The cells were pelleted and lysed in 1 ml Triton X-100 lysis buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 5 mM EDTA, 0.02% sodium azide, and 1% Triton X-100) containing 200 µg/ml PMSF. I-Ek was immunoprecipitated with mAbs Y-17 or 17-3-3S, as described above. The bound I-Ek was eluted from the protein A-Sepharose with 40 µl of SDS-loading mixture alone or in the presence of either ß-mercaptoethanol (2% v/v) or iodoacetamide (10 mM). The samples were centrifuged to pellet the protein A-Sepharose, the eluates were removed and either boiled for 5 min or not, and subjected to 10% SDS-PAGE. Where indicated, bands were excised from the gel, rehydrated as described below, and subjected to 8% SDS-PAGE under reducing conditions.

Purification of I-Ek

CH27 cells were grown, lysed, and I-Ek purified, as described (54), with the following modifications. The mAbs 17-3-3S and Y-17 were coupled to CNBr-activated Sepharose 4B (Pharmacia Biotech, Uppsala, Sweden) and used for affinity chromatography of I-Ek. I-Ek was eluted using ß-octylglucoside (ß-OG) elution buffer (15 mM triethanolamine, pH 11.5, 140 mM NaCl, and 30 mM ß-OG), neutralized with a measured amount of 1 M 2-(N-morpholino)ethanesulfonic acid, pH 5, dialyzed against PBS 30 mM ß-OG, concentrated in a centrifuge microconcentrator (Centricon 10; Amicon, Beverly, MA), and centrifuged through a 0.2-µm filter (Schleicher & Schuell, Keene, NH). Protein concentration was determined by bicinchoninic assay (Micro BCA Assay; Pierce, Rockford, IL).

Immunoblotting

I-Ek purified by immunoaffinity chromatography on a Y-17 mAb column was divided into aliquots of 8 µg each, diluted 1/1 in nonreducing SDS-loading mixture, and subjected to 10% SDS-PAGE without boiling. Proteins were transferred to 0.2-µm-pore nitrocellulose membrane (Schleicher & Schuell) at 0.5 amps for 2 h. The membrane was Ponceau S stained and cut into strips. The strips were soaked in 150 mM NaCl, 10 mM sodium phosphate, pH 7.4 (PBS), containing 5% (w/v) nonfat dry milk and 0.01% NaN3, washed in PBS containing 0.1% (v/v) Tween-20 (PBS Tween), and soaked 2 h in a 3 µg/ml solution of one of the following primary mAbs: 17-3-3S, mouse IgG2a,{kappa}, IN-1, or rat IgG at 3 µg/ml. These primary mAbs were diluted in PBS Tween containing 1% (w/v) BSA. The strips were washed four times in PBS Tween and soaked in a 1/10,000 dilution of either HRP-rabbit anti-mouse IgG2a or HRP-goat anti-rat IgG for 30 min. The strips were washed four times in PBS Tween, and developed using enhanced chemoluminescence (ECL; Amersham, Arlington Heights, IL).

Silver staining

Gels were fixed in 30% ethanol, 10% acetic acid, then soaked in 1 mg/ml sodium thiosulfate, 30% ethanol, and 10 mM sodium acetate, pH 6, for 30 min. The gels were then washed thoroughly in distilled water, and soaked in 0.1% silver nitrate, 0.01% formaldehyde for 30 min. The gel was developed in 2.5% sodium carbonate, 0.02% formaldehyde, and the development was arrested by glacial acetic acid to yield a final concentration of 5% acetic acid. Gels were soaked in glycerin and stored in plastic.

Gel electroelution

I-Ek complexes were analyzed after electrophoresis by excising bands and electroeluting the proteins from the polyacrylamide for 100 V-h into 300 µl of 4x SDS-PAGE electrode buffer using a SixPac GE200 Gel Eluter (Hoefer Scientific Instruments, San Francisco, CA). This volume was concentrated in a Centricon 10 to a final volume of 50 µl, diluted 1/1 with SDS-loading mixture containing 4% (v/v) ß-mercaptoethanol, boiled 10 min, subjected to SDS 10%-PAGE, and silver stained.

Peptide mapping

Peptide mapping of I-Ek complexes was performed by the method of Cleveland et al. (57), as modified by Lamb and Choppin (58). [35S]Met, Cys-labeled I-Ek was synthesized by growing CH27 cells for 21 h at 1 x 106 cells/ml in Met-Cys- 5% CM containing 45 µCi/ml [35S]Met,Cys. The cells were washed and lysed, and the I-Ek was purified, as described above. A 10-µg aliquot of I-Ek was electrophoresed without reducing or boiling. The gel was dried and exposed to film, and the 60- and 120-kDa I-Ek bands were excised. The gel pieces were rehydrated by soaking in sample buffer (SB, 0.125 M Tris-HCl, pH 6.8, 0.1% SDS, and 1 mM EDTA) containing 0.5% DTT for 45 min, and inserted into the wells of a 12% SDS polyacrylamide gel and overlaid with 20 µl SB containing 20% glycerol/bromphenol blue and 0.5% DTT. The gel pieces were further overlaid with 20 µl of 200 µg/ml Staphylococcus aureus V8 protease (Promega, Madison, WI) in SB containing 10% glycerol, and the gel was electrophoresed. When the dye front passed the stacking gel/separating gel interface, the current was turned off for 30 min to allow the protease to digest the I-Ekin situ. The gel was then run for the remainder of the electrophoresis as usual and subjected to fluorography.

Peptide presentation assay

For presentation of purified I-Ek, I-Ek, purified by immunoaffinity chromatography on either 17-3-3S or Y-17 mAb columns, was serially diluted in PBS across a 96-well tissue culture plate starting at 1 µg/well, in a final volume of 0.1 ml. The plate was incubated for 4 h at 25°C, then washed three times with 0.15 ml citrate buffer (100 mM sodium citrate, pH 4.5). A peptide corresponding to the C terminus of tobacco hornworm moth cytochrome c residues 82–103 (THMc 82–103) was synthesized, as detailed previously (59). THMc (82–103) was added to each well at a concentration of 1 µM in citrate buffer, and incubated at 25°C for 5 h. The wells were then washed three times with 0.15 ml 5% CM. For presentation by APCs, CH27 cells were cultured at 1.3 x 106 cells/ml with 2 µM THMc (82–103) for 2 h at 37°C, in a 5% CO2 atmosphere. The CH27 cells were fixed by incubation in 0.1% glutaraldehyde at 1 to 5 x 106 cells/ml in DMEM at 4°C for 30 s. The cell suspension was diluted twofold with 0.2 M lysine in DMEM and washed in 5% CM. The fixed CH27 cells were serially diluted across a 96-well tissue culture plate starting at 2 x 105 cells/well. TPc9.1 cells were added to the wells containing either the purified I-Ek or the CH27 cells at 5 x 104 cells/well in 200 µl and incubated at 37°C for 24 h. Supernatants (75 µl) were harvested and tested for IL-2 content by their ability to support the growth of the IL-2-dependent T cell line CTLL-2, as previously described (60).

Heat and pH treatment of I-Ek

For thermal stability experiments, the aliquots were diluted 1/1 with nonreducing SDS-loading mixture and incubated at 25°, 37°, 50°, or 65°C for 30 min. For pH stability experiments, the aliquots of Y-17 affinity-purified I-Ek were diluted in Tris/acetate buffers to give the final conditions of 25 mM Tris-HCl, 25 mM acetate, 1% (w/v) SDS, and 15% (v/v) glycerin, pH 3, 4, 5, 6, 7, 8, or 9. One aliquot was diluted in nonreducing SDS-loading mixture, pH 7.4, and one was diluted in ß-OG elution buffer, pH 11.5. The final volume of each sample was 50 µl. The samples were incubated at these pH levels for 30 min at 37°C and neutralized with 50 µl nonreducing SDS-loading mixture. Following treatment, all samples were electrophoresed on 10% SDS polyacrylamide gels and silver stained.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Chemical cross-linking of the 120-kDa class II superdimers on the surfaces of B cells

We previously reported the existence of both 60-kDa dimers and 120-kDa superdimers of the mouse class II I-Ek molecules in detergent lysates of B lymphocytes by immunoprecipitation using the I-Ek-specific mAb Y-17, which differentially recognized the 120-kDa superdimeric form of I-Ek (43). We further showed by Western blotting of the B cell lysate that the existence of the 120-kDa superdimer was not dependent on the presence of the Y-17 mAb (43). To determine whether the superdimer exists on the surfaces of B cells in the absence of detergents, B cells were treated with chemical cross-linking reagents to covalently cross-link the components of the superdimer. Because it is difficult to predict the ability of a chemical cross-linking reagent to covalently link the components of an oligomeric structure, several chemical cross-linkers were tried, including: DTSSP, DSP, DFDNB, EGS, BS3, DMA, DMP, sulfo-DST, and DTPB. Treatment of B cells with three cross-linkers (DSP, DTSSP, and DFDNB) resulted in some degree of covalent cross-linking of the {alpha}- and ß-chains within the 60-kDa dimer. However, only DFDNB showed covalent cross-linking of the ({alpha}ß)2 superdimer. The results for DFDNB and DSP are shown (Fig. 1Go). DSP is a long (12A°) homobifunctional NHS-ester NH2-linker that cross-links through -NH2 amines. The presence of a disulfide bond within DSP allows for reversal of cross-linking with a reducing reagent (61). DFDNB is a small (3A°) bifunctional aryl halide that reacts with -NH2 groups to form a nonreducible bond (62).



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  		FIGURE 1. The chemical cross-linker DFDNB cross-links 120-kDa superdimers of I-Ek on B cell surfaces. Metabolically labeled CH27 cells were either untreated (-) or treated with the cross-linkers DSP or DFDNB, as described in Materials and Methods. Lysates were immunoprecipitated using the superdimer-specific mAb Y-17 (A) or the dimer-specific mAb 17-3-3S (B), or as a control, isotype-matched IgG (data not shown). Shown is a representative experiment in which the number of samples and the differences in SB within the same experiment required analysis on three separate gels for nonreduced (Nonboiled NonReduced, Boiled NonReduced), reduced (Boiled Reduced), and iodoacetamide-containing samples (Boiled and Iod NonReduced). Molecular mass markers for each gel allowed the assignment of the 60-kDa ({alpha}ß), 120-kDa ({alpha}ß)2, and 240-kDa ({alpha}ß)4 bands. In a separate experiment, the 60-kDa band in the unboiled Y-17 mAb immunoprecipitates of uncross-linked B cells (labeled 1), and the 240-kDa band in the boiled Y-17 mAb immunoprecipitates of DFDNB-treated cells (labeled 2) were excised, rehydrated, and subjected to SDS-PAGE under reducing conditions (C). The representative bands 1 and 2 are indicated by arrows in A.

 
B cells were treated with the cross-linking reagents and subjected to immunoprecipitation using either the Y-17 mAb (Fig. 1GoA) or the 17-3-3S mAb (Fig. 1GoB), which differentially immunoprecipitate the 120-kDa superdimer or the 60-kDa dimer, respectively. The immunoprecipitates were subjected to SDS-PAGE under conditions in which the 60-kDa dimer and 120-kDa superdimer are stable (without reducing or boiling) or under conditions in which both the dimer and superdimer are unstable and migrate as {alpha}- and ß-chains (boiling with or without reduction). Immunoprecipitation of either untreated cells or cells cross-linked with DSP or DFDNB using the Y-17 mAb yields the predicted 120-kDa ({alpha}ß)2 superdimers when the samples are not boiled (Fig. 1GoA). In contrast, the 17-3-3S immunoprecipitates contain only the 60-kDa dimers and no 120-kDa superdimers (Fig. 1GoB). When the samples are boiled, but not reduced, the Y-17-immunoprecipitated class II molecules from untreated cells migrate as free {alpha}- and ß-chains, and the class II molecules from DSP- and DFDNB-treated cells migrate as 60-kDa {alpha}ß dimers (Fig. 1GoA), indicating that both DSP and DFDNB cross-linked the {alpha}- and ß-chains within the heterodimer. DFDNB appears to be a more efficient cross-linker as compared with DSP, as the immunoprecipitates from DFDNB-treated cells have less free {alpha}ß-chains compared with the immunoprecipitates from the DSP-treated cells. The DSP- and DFDNB-cross-linked {alpha}ß dimers migrate slightly differently, suggesting that the cross-links introduced into the {alpha}ß dimers are not the same for the two different chemical cross-linkers. In addition to the cross-linked 60-kDa {alpha}ß dimer, a 240-kDa band is present in the immunoprecipitates from the DFDNB-treated cells. This 240-kDa band is the correct size to be as dimer of the superdimer or ({alpha}ß)4. The 240-kDa band is only present in the Y-17 superdimer-specific immunoprecipitates, and not in the 17-3-3S immunoprecipitates (Fig. 1GoB), indicating that the 240-kDa band is related to the superdimer. When the Y-17 mAb immunoprecipitates from DFDNB-treated cells are run under reducing conditions with boiling, the 240-kDa band disappears and the 120-kDa superdimer appears (Fig. 1GoA), suggesting that the 240-kDa band is a disulfide-linked dimer of DFDNB-cross-linked ({alpha}ß)2 superdimers. Reduction of the Y-17 immunoprecipitate also reduces the DSP chemical cross-links in the 60-kDa dimers, resulting in free {alpha}- and ß-chains. By densitometry, the cross-linked ({alpha}ß)2 superdimers in the reduced and boiled Y-17 immunoprecipitates represent approximately 18% of the total population of class II molecules.

To further characterize the 240-kDa band, the band was excised, rehydrated, and subjected to SDS-PAGE under reducing conditions. The 240-kDa band resolves into 240-, 120-, and 60-kDa bands (Fig. 1GoC), indicating that the 240-kDa band is a disulfide-linked oligomer of DFDNB-cross-linked 60-kDa ({alpha}ß) dimers and 120-kDa ({alpha}ß)2 superdimers. When the Y-17 immunoprecipitates from the DFDNB-treated cells are reduced, all of the 240-kDa resolves as 120-kDa superdimers and 60-kDa dimers (Fig. 1GoA). The failure to completely reduce the excised, rehydrated 240-kDa band (Fig. 1GoC) may be due to some degree of irreversible denaturation of the material in the gel. A disulfide-linked oligomer of class II molecules was unexpected because the predominant chemical cross-linking reaction of DFDNB is through amines at the pH used in this study (pH 8.5), although it is possible for DFDNB to cross-link through sulfhydryl groups. However, the only free cysteines in the I-Ek molecules are in the transmembrane region or buried in the peptide binding site, making it unlikely that the 240-kDa ({alpha}ß)4 was the result of DFDNB sulfhydryl cross-linking. To determine whether disulfide bonds formed between DFDNB-cross-linked {alpha}ß and ({alpha}ß)2 in the immunoprecipitates, iodoacetamide was added to the SDS SB to block free sulfhydryls. The 120-kDa, but not the 240-kDa band is present in iodoacetamide-treated Y-17 immunoprecipitates of DFDNB-treated cells (Fig. 1GoA), indicating that the disulfide bonds were formed between DFDNB-cross-linked superdimers in the immunoprecipitate. In the Y-17 immunoprecipitates, which are neither boiled nor reduced, the 240-kDa band does not appear, suggesting that either the disulfide bonds are formed during boiling or that in the absence of boiling the ({alpha}ß)4 may aggregate and not enter the gel. The fact that the 240-kDa ({alpha}ß)4 was not present in immunoprecipitates from untreated cells or DSP-treated cells suggests that the formation of the disulfide bond was favored between DFDNB-cross-linked {alpha}ß and ({alpha}ß)2.

Taken together, the results presented in this study indicate that treating cells with DFDNB results in covalently cross-linked 120-kDa ({alpha}ß)2 superdimers, providing direct biochemical evidence for the existence of the 120-kDa superdimers on the B cell surface. Y-17 immunoprecipitation of DFDNB-treated cells also results in the formation of a disulfide-linked 240-kDa ({alpha}ß)4 oligomer composed of DFDNB-cross-linked ({alpha}ß)2 and {alpha}ß.

The I-Ek 60-kDa dimer and 120-kDa superdimer are derived from the same cellular pool of I-Ek molecules

To determine whether class II molecules that form the 60-kDa dimer versus the 120-kDa superdimers exist as one single or two separate pools in cell lysates, exhaustive immunoprecipitations were performed. CH27 cells were metabolically labeled with [35S]Met for 30 min, chased for 4 h, and lysed. The cell lysate was divided in two, lysate 1 and lysate 2 (Fig. 2Go). Lysate 1 was subjected to two successive immunoprecipitations using the {alpha}ß dimer-specific mAb, 17-3-3S, followed by a single immunoprecipitation using the ({alpha}ß)2 superdimer-specific mAb, Y-17. Conversely, lysate 2 was immunoprecipitated twice using the Y-17 mAb, then once using the 17-3-3S mAb. The immunoprecipitates were subjected to SDS-PAGE without reducing or boiling, conditions under which peptide-loaded {alpha}ß dimers are stable. The 17-3-3S mAb immunoprecipitates I-Ek, which migrates as a 60-kDa {alpha}ß band. The 17-3-3S mAb depleted nearly all of the I-Ek from the lysate, leaving only a small amount to be immunoprecipitated by Y-17, which migrates as a 120-kDa ({alpha}ß)2 band. The I-Ek immunoprecipitated by Y-17 from lysate 2 also migrates as a 120-kDa band. After two successive Y-17 immunoprecipitations, no detectable I-Ek remained to be immunoprecipitated by 17-3-3S (Fig. 2Go). This result indicates that the 60-kDa {alpha}ß heterodimers and 120-kDa ({alpha}ß)2 superdimers are derived from the same pool of I-Ek molecules in the cell lysate, and that all class II {alpha}ß dimers can dimerize to form 120-kDa superdimers.



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  		FIGURE 2. Successive immunoprecipitations show that the 60- and 120-kDa I-Ek complexes are derived from the same pool of I-Ek in cell lysates. CH27 cells (107) were [35S]Met labeled for 30 min and chased with unlabeled Met for 4 h. Cells were lysed, and the lysate was divided into two, lysate 1 and lysate 2. Lysate 1 was immunoprecipitated twice in succession with 30 µg of the mAb 17-3-3S, followed by a third immunoprecipitation with 30 µg of the mAb Y-17. Lysate 2 was immunoprecipitated twice with Y-17, followed by 17-3-3S. All immunoprecipitates were subjected to 10% SDS-PAGE without reducing or boiling.

 
Purified I-Ek molecules form both 60-kDa dimers and 120-kDa superdimers in vitro

Our previous studies demonstrated the presence of 120-kDa I-Ek complexes in cell lysates both by immunoprecipitation and by immunoblotting (43). Cell lysates are complicated mixtures that potentially contain cellular components that could affect I-Ek superdimerization. To determine whether purified I-Ek has the intrinsic ability to form 120-kDa complexes in the absence of other cellular components, I-Ek was purified by Y-17 immunoaffinity chromatography and analyzed by SDS-PAGE without reducing or boiling. A portion of the gel was silver stained (Fig. 3Go, right panel), and the remainder was used to transfer the proteins to nitrocellulose for immunoblotting. Immunoblots were probed with the mAb 17-3-3S and its isotype control mAb mouse IgG2a,{kappa}, or the Ii-specific mAb IN-1 and its isotype control mAb rat IgG2b,{kappa}. The 17-3-3S immunoblots were stained with a IgG2a-specific Ab, and the IN-1 immunoblots were stained with rat IgG heavy and light chain-specific Ab and developed by ECL (Fig. 3Go, left and center panels). Two major bands are detected in the silver-stained gel having molecular mass of 60 and 120 kDa, and these bands stain positively for I-Ek in immunoblotting. Thus, the Y-17 immunoaffinity-purified I-Ek appears to be a mixture of both 60-kDa dimers and 120-kDa superdimers. It should be noted that in contrast to the preparations of Y-17 immunoaffinity-purified I-Ek, the Y-17-immunoprecipitated I-Ek is almost entirely in the superdimer form (see Fig. 3Go). This difference may be explained by the fact that during immunoaffinity purification, the I-Ek is eluted from the Y-17 column at high pH and, as will be shown below (Fig. 8Go), the superdimers dissociate at extreme pHs. Thus, it is likely that the mixture of dimers and superdimers in the Y-17 immunoaffinity-purified preparation represents the reassociation of I-Ek molecules at neutral pH.



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  		FIGURE 3. Affinity-purified I-Ek forms both 60- and 120-kDa complexes. I-Ek was purified from CH27 cells on a Y-17-Sepharose column. Aliquots (8 µg) were subjected to 10% SDS-PAGE without reducing or boiling, and when indicated, samples were transferred to nitrocellulose for immunoblotting. Left panel, Immunoblot of I-Ek preparation, using 17-3-3S or the isotype control mouse IgG2a,{kappa}, followed by HRP-conjugated secondary Abs specific for mouse IgG2a,{kappa}. Center panel, Immunoblot of I-Ek preparation using the Ii-specific mAb, IN-1, or the isotype control rat IgG2b,{kappa}, followed by HRP-conjugated secondary Abs specific for rat IgG heavy and light chains. Immunoblots were developed using ECL. Right panel, Silver-stained gel of I-Ek preparation and m.w. markers (Rainbow markers; Amersham Life Science).

 


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  		FIGURE 8. The pH stability of 60- and 120-kDa I-Ek complexes is similar. Affinity-purified I-Ek was aliquoted into 8-µg portions and diluted in SDS-containing Tris-acetate buffers (25 mM Tris-HCl, 25 mM acetate, pH 4–9, 1% w/v) 1% SDS, and 15% glycerin) (left panel), or into nonreducing SDS-loading mixture, pH 7.4, or ß-OG elution buffer, pH 11.5 (right panel), and incubated at 37°C for 30 min. The samples were diluted 1/1 with nonreducing SDS-loading mixture, electrophoresed on a 10% SDS polyacrylamide gel, fixed, and silver stained.

 
In addition to the 60- and 120-kDa bands, a faint third band of 90 kDa is visible in the silver-stained gels and in the 17-3-3S immunoblot (Fig. 3Go). This band does not stain with the IgG2a-specific secondary Ab alone, but does stain with the heavy and light chain-specific secondary Ab. This 90-kDa band most likely represents small amounts of contaminating Y-17 mAb from the affinity column, which is of the IgG2b isotype. A faint 35-kDa band is also detected in immunoblots using the heavy and light chain-specific secondary Ab, and this is most likely the contaminating Y-17 light chain. Significantly, the 120-kDa I-Ek band does not stain with either of the secondary Abs alone, indicating that there is no Ab present in this band.

The IN-1 immunoblot of the affinity-purified I-Ek preparations shows bands at 35 to 40 kDa and 65 kDa, indicating that Ii monomers and disulfide-linked dimers (5) are present in this I-Ek preparation. The 65-kDa Ii dimer migrates slightly higher than the bulk of the 60-kDa I-Ek. Significantly, the 120-kDa band does not stain with the IN-1 mAb, indicating that it does not contain Ii. Taken together, these results provide evidence that affinity-purified I-Ek forms both 60- and 120-kDa complexes in vitro, and that these complexes do not contain Ii or Ab.

Analysis of I-Ek molecules purified using the dimer-specific 17-3-3S mAb also showed these to be a mixture of dimers and superdimers (see Fig. 7Go below). Moreover, the ratio of {alpha}ß dimers to ({alpha}ß)2 superdimers in Y-17 and 17-3-3S affinity-purified preparations was the same. In addition, the I-Ek purified using the Y-17 or the 17-3-3S Ab were functionally equivalent in their ability to present peptide in vitro. To demonstrate this, the purified I-Ek preparations were adsorbed to tissue culture plates, incubated with the cytochrome c peptide THMc (82–103) at pH 4.5, and cultured with the I-Ek-restricted cytochrome c-specific T cell hybrid TPc9.1. The two sources of I-Ek were equivalent in their ability to stimulate the TPc9.1 cells (Fig. 4Go). The magnitude of stimulation is the same as that attained by whole CH27 cells and peptides.



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  		FIGURE 7. The thermostability of 60- and 120-kDa I-Ek complexes is similar. I-Ek was affinity purified using either Y-17-Sepharose (leftmost four lanes) or 17-3-3S-Sepharose (rightmost lane) aliquoted into 8-µg portions and diluted with an equal volume of 2x nonreducing SDS-PAGE mixture (pH 6.8). The aliquots were incubated at 25, 37, 50, or 65°C for 30 min and electrophoresed on a 10% SDS polyacrylamide gel, fixed, and silver stained.

 


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  		FIGURE 4. The mAbs 17-3-3S and Y-17 purify functionally similar I-Ek. I-Ek was purified on either a 17-3-3S or a Y-17-Sepharose column and adsorbed to tissue culture plates in graded amounts. The I-Ek was loaded with the antigenic peptide THMc (82–103) at pH 4.5, neutralized, and cultured with the T cell hybrid TPc 9.1. T cell stimulation was measured by quantifying the amount of IL-2 secretion into the supernatant by its ability to support the growth of the IL-2-dependent CTLL-2. 17-3-3S-purified I-Ek ({circ}) and Y-17-purified I-Ek (•) are compared with peptide-loaded fixed CH27 cells ({square}).

 
The 120-kDa I-Ek complex is a dimer composed of only {alpha}- and ß-chains

To determine the composition of the 120-kDa band observed in preparations of purified I-Ek, both the 60- and the 120-kDa bands were excised from gels. The proteins were electroeluted, reduced, boiled, reelectrophoresed on a second gel, and silver stained (Fig. 5Go). Under these conditions, the constituent proteins in both of the 60- and 120-kDa bands migrate as two bands, {alpha} and ß, of 34 and 29 kDa, respectively. The excised 120-kDa band may have also contained peptide that was not resolved in the system.



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  		FIGURE 5. Reelectrophoresis shows that both 60- and 120-kDa I-Ek complexes are composed of {alpha}- and ß-chains. Left panel, Affinity-purified I-Ek was electrophoresed on a 10% SDS polyacrylamide gel without reducing or boiling, and the areas of the gel in which the 60- and 120-kDa I-Ek complexes are known to migrate were excised. Right panel, The polyacrylamide gel pieces were subjected to electroelution, and the eluate was concentrated, reduced, boiled, and electrophoresed on a second 10% SDS polyacrylamide gel. Both gels were silver stained.

 
To further demonstrate the identity of the 60- and 120-kDa I-Ek complexes, peptide mapping was performed. CH27 cells were metabolically labeled, washed, and lysed, and the I-Ek was purified, as described above. Approximately 10 µg of 35S-labeled I-Ek was loaded onto a 10% SDS polyacrylamide gel without reducing or boiling. The gel was electrophoresed and dried and exposed to film. The autoradiogram revealed the location of the 60- and 120-kDa I-Ek complexes, allowing the precise excision of a portion of each band. Care was taken to avoid the upper region of the 60-kDa band, as this is likely to contain some contaminating Ii dimers (see Fig. 3Go). The gel pieces were rehydrated in buffer containing DTT and inserted into the wells of a 12% SDS polyacrylamide gel. The proteins were partially digested with protease from S. aureus V8, separated by electrophoresis, and imaged by fluorography (Fig. 6Go). Two different exposures are shown, to allow for optimal analysis of the digestion products from each gel piece. The products of proteolytic digestion derived from the 60- and 120-kDa I-Ek complexes appear to be highly related and within the resolution of the method that they can be considered the same protein. Peptide mapping of these complexes using the protease chymotrypsin also produced closely related patterns of digestion products (data not shown). This result, together with the silver-stained gels in Figures 3Go and 5Go, confirms that the 60- and 120-kDa I-Ek complexes are composed of the same constituent proteins, and that the 120-kDa complex does not result from the addition of extraneous protein to the 60-kDa complex. The proteolytic digestion products of the 60- and 120-kDa complexes shown in Figure 6Go, as well as the electroeluted {alpha}- and ß-chains shown in Figure 5Go, are present in the same relative amounts. Therefore, the stoichiometry of the constituent proteins in each of the two complexes is the same, and the 120-kDa complex observed in this study represents a dimer of the {alpha}ß heterodimer, the superdimer ({alpha}ß)2 complex.



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  		FIGURE 6. Peptide mapping shows that the 60- and 120-kDa I-Ek complexes are identical in composition. CH27 cells were metabolically labeled with [35S]Met,Cys, and I-Ek was purified on a Y-17-Sepharose column. Approximately 10 µg of I-Ek was electrophoresed in one lane of a 10% SDS polyacrylamide gel without reducing or boiling. The gel was dried, and an autoradiogram was obtained to facilitate excision of the 60- and 120-kDa I-Ek complex bands. The gel pieces were soaked in SB containing DTT, inserted into the lanes of a 12% SDS polyacrylamide gel, and overlaid with 23 U of S. aureus V8 protease. Voltage was applied until the proteins reached the stacking gel/separating gel interface, interrupted for 30 min to permit partial proteolytic digestion, then reapplied to separate peptide fragments. The gel was fixed, soaked in fluorographic reagent, dried, and exposed to film.

 
Thermal and pH stability of the 60-kDa {alpha}ß and 120-kDa ({alpha}ß)2 I-Ek complexes to SDS denaturation

Having established that purified I-Ek has the tendency to form {alpha}ß and ({alpha}ß)2 complexes in detergent solution, we examined the stability of these complexes to SDS denaturation. The observation that these complexes exist during SDS-PAGE indicates that they are held together either by very stable noncovalent associations, or by covalent bonds, perhaps an interchain disulfide bond, which stabilizes the complex against SDS-mediated denaturation. To address this question, Y-17-purified I-Ek was incubated in the absence of reducing agents at various temperatures for 30 min, in the presence of 2.5% SDS at pH 6.8 before electrophoresis on a 10% SDS polyacrylamide gel (Fig. 7Go). At 25° or 37°C, the I-Ek migrates as {alpha}ß and ({alpha}ß)2 complexes. However, at 50°C, there is partial denaturation of both the {alpha}ß and ({alpha}ß)2 complexes into individual {alpha}- and ß-chains. At 65°C, all of the I-Ek is denatured into free {alpha}- and ß-chains. The ability of these complexes to become denatured solely by the addition of heat indicates that they are noncovalent complexes. Both the {alpha}ß and ({alpha}ß)2 appear to be partially decomposed at 50°C, suggesting that the two complexes have similar thermal stabilities. Since thermal denaturation does not result in an increase in the amount of ({alpha}ß)2, it is unlikely that this complex forms as a result of nonspecific aggregation of I-Ek.

The superdimer ({alpha}ß)2 complex has previously been observed in immunoprecipitates of newly synthesized I-Ek that has acquired peptide (43). The intravesicular pH of the subcellular compartment in which peptide loading onto class II molecules occurs has been measured to be approximately 4.6 (13). To examine the pH stability of {alpha}ß and ({alpha}ß)2 complexes, aliquots of affinity-purified I-Ek were incubated at various pH levels for 30 min at 37°C in the presence of 2.5% SDS. These aliquots were then neutralized, electrophoresed on a 10% SDS polyacrylamide gel, and silver stained (Fig. 8Go). Both {alpha}ß and ({alpha}ß)2 complexes are stable at pH levels between 6 and 9. At pH 5, these complexes partially denature and run as individual {alpha}- and ß-chains. At pH 4 and 3, the complexes completely dissociate into the free chains. The dissociation is accompanied by a conversion of a portion of the {alpha}ß heterodimers into a complex having a m.w. slightly higher than that of the major {alpha}ß band, the compact heterodimer. This may be the floppy I-Ek, a conformational intermediate observed by Dornmair et al. (22) during the denaturation of compact {alpha}ß heterodimers to individual {alpha}- and ß-chains. At pH 11.5, the ({alpha}ß)2 and compact {alpha}ß complexes are unstable (Fig. 8Go). Taken together, these studies demonstrate that {alpha}ß and ({alpha}ß)2 complexes display similar pH and thermal stability to SDS denaturation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we further characterize the dimers and superdimers of I-Ek in B lymphocytes. We initially observed the 120-kDa superdimer of class II molecules using the mAbs 17-3-3S and Y-17, which immunoprecipitated I-Ek {alpha}ß dimers and ({alpha}ß)2 superdimers, respectively (43). The molecular basis of the apparent specificity of these two mAbs is not known. Our previous flow-cytometric analysis showed that these mAbs bind simultaneously to I-Ek molecules expressed on cell surfaces and show no competition for binding to I-Ek (43). Mutational studies of I-Ek have shown that the epitopes recognized by the 17-3-3S and Y-17 mAb are largely distinct (48, 51). However, the epitopes for these two mAbs must be somewhat overlapping, because immobilized purified 17-3-3S/I-Ek complexes were shown not to bind to Y-17 by plasmon resonance (49). The best assessment of the epitopes that these two mAbs recognize is that 17-3-3S reacts with the ß1 domain, while Y-17 reacts either with an epitope on ß1, which is dependent on the proximal {alpha}1 domain for conformation, or on an epitope including both the ß1 and {alpha}1 domains (50).

The exhaustive immunoprecipitation analysis presented in this work confirms the specificity of the 17-3-3S and Y-17 mAbs for the {alpha}ß heterodimers and ({alpha}ß)2 superdimers, respectively, and demonstrates that the {alpha}ß and ({alpha}ß)2 complexes are derived from the same pool of I-Ek in cell lysates. Thus, all of the I-Ek molecules in the cell are capable of forming superdimers, under the conditions used in this study. Exhaustive immunoprecipitation also illustrates that the mAbs themselves can influence the conformation of the I-Ek. The form of the I-Ek complex observed following SDS-PAGE ({alpha}ß or ({alpha}ß)2) depends solely on which mAb was used for immunoprecipitation (17-3-3S or Y-17). Thus, it is likely that the mAbs actually drive the I-Ek conformation toward one form or the other during immunoprecipitation. The 17-3-3S epitope may block superdimer formation by interfering with the ß1-ß1 interactions (25) at the most membrane-distal part of the ({alpha}ß)2 superdimer interface. Y-17 may avoid such interference, and may actually bind to a ß1-{alpha}1 epitope of an {alpha}ß heterodimer with one F(ab')2 arm and to another ß1-{alpha}1 epitope with another Fab arm, thus bringing two {alpha}ß heterodimers together to form the ({alpha}ß)2 superdimer.

Because of the influences these mAbs may have on I-Ek conformation, it was important to demonstrate the presence of the I-Ek superdimer in the absence of the Y-17 mAbs. This was accomplished in three ways. First, as previously shown, both 60-kDa {alpha}ß and 120-kDa ({alpha}ß)2 complexes were detected by immunoblotting of untreated whole cell lysates (43). Second, both {alpha}ß and ({alpha}ß)2 complexes were observed in preparations of affinity-purified I-Ek. Third, the 120-kDa superdimer can be chemically cross-linked on the cell surface in the absence of the Y-17 mAb. Therefore, while ({alpha}ß)2 superdimer formation can be facilitated by the Y-17 mAb, I-Ek has the intrinsic ability to superdimerize and does not require Y-17 to do so.

It was also important to demonstrate that the superdimers exist in the absence of detergent, given the potential for detergent-induced artifactual associations. The fact that the superdimer did not appear in all class II preparations, in particular in the 17-3-3S immunoprecipitates, argues against detergent-induced class II dimerization. If exposure to SDS induces dimerization, then the 17-3-3S-immunoprecipitated class II molecules, which as discussed above have the inherent ability to dimerize, should dimerize. In addition, our previous studies, showing a differential effect of the Y-17 and 17-3-3S mAbs on the presentation of peptide by B cells, suggested that the superdimers did indeed exist on B cell surfaces (43). In this study, we provide direct biochemical evidence for the presence of superdimers in the absence of detergent by showing that the ({alpha}ß)2 superdimer can be chemically cross-linked on B cell surfaces using the cross-linker DFDNB. DFDNB chemically cross-linked the {alpha}- and ß-chains in {alpha}ß dimers as well as portions of ({alpha}ß)2. In addition, we observed a phenomenon seen only with chemically cross-linked {alpha}ß and ({alpha}ß)2, which was further oligomerized in vitro to form ({alpha}ß)4 stabilized by disulfide bonds. Because the ({alpha}ß)4 did not form in the 17-3-3S immunoprecipitates, we assume that the Y-17 mAb held the ({alpha}ß)2 in a position that facilitated the disulfide bond formation.

The presence of ({alpha}ß)2 class II superdimers was confirmed very recently by Cherry et al. (45), who used single-particle fluorescence imaging to demonstrate the presence of HLA-DR superdimers on the surface of a fibroblast cell line transfected with the genes encoding HLA-DR {alpha}- and ß- and Ii chains. Their results also indicated the existence of class II oligomers larger than ({alpha}ß)2. They report that approximately 25% of class II molecules on the cell surface are present as superdimers. By densitometry of the DFDNB-cross-linked ({alpha}ß) and ({alpha}ß)2, we estimated that approximately 18% of class II molecules on the cell surface are present as superdimers in good agreement with the estimate of Cherry et al.

The formal proof that the 120-kDa band seen in this study in preparations of I-Ek purified by immunoaffinity chromatography is an ({alpha}ß)2 complex was achieved by a comparison of the 60- and 120-kDa complexes following fully denaturing reelectrophoresis and peptide mapping. The similarity of the polypeptide patterns derived from each complex by these two methods verifies that they are composed of {alpha}- and ß-chains in the same stoichiometric amounts. Therefore, the 120-kDa complex can only be a dimer of the 60-kDa complex.

The nature of the 120-kDa ({alpha}ß)2 I-Ek complex remains to be determined. The structure of this complex may be similar to that which has been determined by x-ray crystallography of I-Ek (31). Alternatively, the 120-kDa complex may be stabilized by hydrophobic interactions between the transmembrane regions of the {alpha}- and ß-chains. These two scenarios are not mutually exclusive, as it has been established that specific residues in the transmembrane domain of I-Ak facilitate assembly of the {alpha}ß heterodimer (63). The 120-kDa complex could also be the result of two I-Ek molecules binding to a single long peptide. The ability of the Y-17 mAb to drive total 120-kDa complex formation would argue against this possibility, because of the improbability of having enough peptides present that are capable of binding all I-Ek molecules in pairs, without having any monomers, trimers, tetramers, etc.

The ({alpha}ß)2 superdimers observed in affinity-purified I-Ek preparations seem to have a stability to SDS denaturation similar to that of the {alpha}ß heterodimer. Both complexes partially decomposed at 50°C, with total dissociation into {alpha}- and ß-chains at 65°C. This heat-sensitive nature of I-Ek under nonreducing conditions is evidence that the interactions involved are noncovalent, and the data in this study are similar to those in previous studies that showed that compact {alpha}ß heterodimers in ß-OG micelles are stable at 50°C, but begin to dissociate at 65°C (22). Thus, it would appear that the stability of the ({alpha}ß)2 superdimer to SDS denaturation depends on the stability of the constituent {alpha}ß heterodimers themselves.

The pH stability exhibited by the ({alpha}ß)2 complexes derived from affinity-purified I-Ek appears to correlate with previously measured pH stabilities of I-Ek-peptide complexes (22). Dissociation into free {alpha}- and ß-chains most likely reflects the loss of the heterodimer-stabilizing peptide. Recent studies have confirmed the longstanding model of a pH-dependent conformational change in MHC class II to facilitate peptide binding (64, 65). Furthermore, the intravesicular pH of the subcellular compartment, where peptide loading onto MHC class II molecules occurs, has been measured to be approximately 4.6 (13). While these conditions used to test the stability of {alpha}ß and ({alpha}ß)2 complex described in this study are certainly different from those found in the peptide-loading compartment in vivo, these findings suggest that ({alpha}ß)2 complexes may indeed be able to form intracellularly upon peptide binding to I-Ek.

A class II superdimer would have potentially important implications for the mechanism of T cell stimulation. One possibility is that a pair of class II molecules might interact with a pair of TCRs to help strengthen the rather weak affinity of MHC class II for the TCR (25). This model is supported by the crystal structure of the TCR V{alpha} domain, which suggests an ({alpha}ß)2 TCR conformation complementary to the HLA-DR1 structure (38). However, it is statistically unlikely that both MHC class II molecules in a superdimer would be filled with identical or closely related peptides, given the large number of peptides available in an APC. Even if the superdimer did contain two of the same peptides, simple dimerization of the TCR alone may provide only weak activation. Reich et al. (41) recently provided evidence for ligand-specific oligomerization of TCR/peptide/MHC complexes to form complexes containing two to six ternary complexes. The superdimers we observed may not be required for TCR dimerization, but may indicate the intrinsic ability to oligomerize when containing ligands for peptide-specific TCR.

Others have proposed that MHC class II superdimers may play a role in T cell signaling through CD4, a mechanism that would be independent of the peptides bound to MHC class II. These models depend largely on the ability of the superdimer to cross-link or oligomerize CD4 (reviewed in Refs. 42 and 66). The models are supported by evidence for multiple sites on MHC class II that are important for CD4 binding. Originally, the CD4 binding site was shown to lie only on the ß2 domain of HLA-DR (32, 33, 67). More recently, however, studies have shown that there is a second site of interaction with CD4 on the {alpha}2 domain, oriented such that it cannot interact simultaneously with the same CD4 molecule as the ß2 site (35). To accommodate these data, either monomeric CD4 would cross-link MHC class II monomers, or MHC class II superdimers would interact with CD4 oligomers. It has recently been demonstrated that oligomers of CD4 are required for stable interaction with MHC class II (68), and an interaction between CD4 tetramers and MHC class II superdimers has been modeled (42). Thus, the weight of the data is consistent with a role for MHC class II superdimers in the binding of CD4. Such interactions of CD4 with the superdimer could contribute either to the overall adherence of the T cell and the APC and/or stabilize the TCR/class II dimer interaction. In this regard, it is interesting to note that the superdimer-specific mAb Y-17 (43) had the same effect as a CD4-specific mAb (53) on an Ag-specific T cell response, blocking the low affinity, but not high affinity, T cell response. Finally, a mutational analysis of residues on the putative dimer-dimer interface of HLA-DR3, identified on the basis of the residues that participate in the HLA-DR1 superdimer (25), has not supported a critical role for these residues in maintaining DR3 conformation peptide binding, or SDS-stable heterodimer formation (69). However, this study did not directly measure the ability of these mutant DR3 molecules to form superdimers. In addition, these two residues represent only a small part of the overall dimer-dimer interface, which encompasses more than 1300 Å2 of surface area and includes seven salt bridges (69).

The data presented in this study have verified that the full-length mouse MHC class II molecule I-Ek has the intrinsic ability to superdimerize in detergent micelles and remain stable during SDS-PAGE under nonreducing, nonboiled conditions. Superdimerization may only be one step in the formation of larger order MHC class II/TCR/CD4 multimers, but its effects should nevertheless be significant for both T cell and APC signal transduction.


   Footnotes
 
1 This work is supported by Grants AI 40309, AI 27957, and AI 18939 from the National Institutes of Health. Back

2 Current address: R.W. Johnson Pharmaceutical Research Institute, Route 202, P.O. Box 300, Raritan, NJ 08869. Back

3 Address correspondence and reprint requests to Dr. Susan K. Pierce, Department Biochemistry, Molecular Biology and Cell Biology, 2153 N. Campus Drive, Northwestern University, Evanston, IL 60208. E-mail address: Back

4 Abbreviations used in this paper: Ii, invariant chain; ß-OG, ß-octylglucoside; BS3, bis[sulfosuccinimidyl]suberate; CM, complete medium; DFDNB, 1,5-difluoro-2,4-dinitrobenzene; DMA, dimethyladipimidate.2HCl; DMP, dimethylpimelimidate.2HCl; DSP, dithiobis[succinimidylpropionate]; DTSSP, 3,3'-dithiobis[sulfosuccinimidylpropionate]; ECL, enhanced chemoluminescence; EGS, ethyleneglycolbis[succinimidylsuccinate]; HRP, horseradish peroxidase; SB, sample buffer; sulfo-DST, disulfosuccinimidyltartarate; THMc, tobacco hornworm moth cytochrome c. Back

Received for publication January 21, 1998. Accepted for publication May 4, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Germain, R. N.. 1994. MHC-dependent antigen processing and peptide presentation: providing ligands for T lymphocyte activation. Cell 76:287.[Medline]
  2. Owen, M. J., A.-M. Kissonerghis, H. F. Lodish, M. J. Crumpton. 1981. Biosynthesis and maturation of HLA-DR antigens in vivo. J. Biol. Chem. 256:8987.[Abstract/Free Full Text]
  3. Mathis, D. J., C. O. Benoist, II V. E. William, M. R. Kanter, H. O. McDevitt. 1983. The murine E{alpha} immune response gene. Cell 32:745.[Medline]
  4. Mengle-Gaw, L., H. O. McDevitt. 1983. Isolation and characterization of a cDNA clone for the murine I-Eß polypeptide chain. Proc. Natl. Acad. Sci. USA 80:7621.[Abstract/Free Full Text]
  5. Roche, P. A., M. S. Marks, P. Cresswell. 1991. Formation of a nine-subunit complex by HLA class II glycoproteins and the invariant chain. Nature 354:392.[Medline]
  6. Lamb, C. A., P. Cresswell. 1992. Assembly and transport properties of invariant chain trimers and HLA-DR-invariant chain complexes. J. Immunol. 148:3478.[Abstract]
  7. Roche, P. A., P. Cresswell. 1990. Invariant chain association with HLA-DR molecules inhibits immunogenic peptide binding. Nature 345:615.[Medline]
  8. Teyton, L., D. O’Sullivan, P. W. Dickson, V. Lotteau, A. Sette, P. Fink, P. A. Peterson. 1990. Invariant chain distinguishes between the exogenous and endogenous antigen presentation pathways. Nature 348:39.[Medline]
  9. Busch, R., I. Cloutier, R. P. Sekaly, G. J. Hammerling. 1996. Invariant chain protects class II histocompatibility antigens from binding intact polypeptides in the endoplasmic reticulum. EMBO J. 15:418.[Medline]
  10. Anderson, M., J. Miller. 1992. Invariant chain can function as a chaperone protein for class II major histocompatibility complex molecules. Proc. Natl. Acad. Sci. USA 89:2282.[Abstract/Free Full Text]
  11. Lotteau, V., L. Teyton, A. Peleraux, T. Nilsson, L. Karlsson, S. L. Schmid, V. Quaranta, P. A. Peterson. 1990. Intracellular transport of class II MHC molecules directed by invariant chain. Nature 348:600.[Medline]
  12. Bakke, O., B. Dobberstein. 1990. MHC class II-associated invariant chain contains a sorting signal for endosomal compartments. Cell 63:707.[Medline]
  13. Xu, X., W. Song, H. Cho, Y. Qiu, S. K. Pierce. 1995. Intracellular transport of invariant chain-MHC class II complexes to the peptide-loading compartment. J. Immunol. 155:2984.[Abstract]
  14. Amigorena, S., P. Webster, J. Drake, J. Newcomb, P. Cresswell, I. Mellman. 1995. Invariant chain cleavage and peptide loading in major histocompatibility complex class II vesicles. J. Exp. Med. 181:1729.[Abstract/Free Full Text]
  15. Morton, P. A., M. L. Zachais, K. S. Giacoletto, J. A. Manning, B. D. Schwartz. 1995. Delivery of nascent MHC class II-invariant chain complexes to lysosomal compartments and proteolysis of invariant chain by cysteine proteases precedes peptide binding in B-lymphoblastoid cells. J. Immunol. 154:137.[Abstract]
  16. Roche, P. A., P. Cresswell. 1991. Proteolysis of the class II-associated invariant chain generates a peptide binding site in intracellular HLA-DR molecules. Proc. Natl. Acad. Sci. USA 88:3150.[Abstract/Free Full Text]
  17. Neefjes, J. J., H. L. Ploegh. 1992. Inhibition of endosomal proteolytic activity by leupeptin blocks surface expression of MHC class II molecules and their conversion to SDS resistant {alpha}ß heterodimers in endosomes. EMBO J. 11:411.[Medline]
  18. Maric, M. A., M. D. Taylor, J. S. Blum. 1994. Endosomal aspartic proteinases are required for invariant-chain processing. Proc. Natl. Acad. Sci. USA 91:2171.[Abstract/Free Full Text]
  19. Germain, R. N., L. R. Hendrix. 1991. MHC class II structure, occupancy and surface expression determined by post-endoplasmic reticulum antigen binding. Nature 353:134.[Medline]
  20. Sadegh-Nasseri, S., R. N. Germain. 1992. How MHC class II molecules work: peptide-dependent completion of protein folding. Immunol. Today 13:43.[Medline]
  21. Springer, T. A., J. F. Kaufman, L. A. Siddoway, D. L. Mann, J. L. Strominger. 1977. Purification of HLA-linked B lymphocyte alloantigens in immunologically active form by preparative sodium dodecyl sulfate-gel electrophoresis and studies on their subunit association. J. Biol. Chem. 252:6201.[Abstract/Free Full Text]
  22. Dornmair, K., B. Rothenhausler, H. M. McConnell. 1989. Structural intermediates in the reactions of antigenic peptides with MHC molecules. Cold Spring Harbor Symp. Quant. Biol. 54:409.
  23. Sadegh-Nasseri, S., R. N. Germain. 1991. A role for peptide in determining MHC-class II structure. Nature 353:167.[Medline]
  24. Madden, D. R.. 1995. The three-dimensional structure of peptide-MHC complexes. Annu. Rev. Immunol. 13:587.[Medline]
  25. Brown, J. H., T. S. Jardetzky, J. C. Gorga, L. J. Stern, R. G. Urban, J. L. Strominger, D. C. Wiley. 1993. Three-dimensional structure of the human class II histocompatibility antigen HLA-DR1. Nature 364:33.[Medline]
  26. Jardetzky, T. S., J. H. Brown, J. C. Gorga, L. J. Stern, R. G. Urban, Y. Chi, C. Stauffacher, J. L. Strominger, D. C. Wiley. 1994. Three-dimensional structure of a human class II histocompatibility molecule complexed with superantigen. Nature 368:711.[Medline]
  27. Stern, L. J., J. H. Brown, T. S. Jardetzky, J. C. Gorga, R. G. Urban, J. L. Strominger, D. C. Wiley. 1994. Crystal structure of the human class II MHC protein HLA-DR1 complexed with an influenza virus peptide. Nature 368:215.[Medline]
  28. Kim, J. R., R. J. Urban, J. L. Strominger, D. C. Wiley. 1994. Toxic shock syndrome toxin-1 complexed with a class II major histocompatibility molecule, HLA-DR1. Science 266:1870.[Abstract/Free Full Text]
  29. Ghosh, P., M. Amaya, E. Mellins, D. C. Wiley. 1995. The structure of an intermediate in class II MHC maturation: CLIP bound to HLA-DR3. Nature 378:457.[Medline]
  30. Stern, L. J., J. H. Brown, R. S. Jardetzky, J. C. Gorga, R. G. Urban, J. L. Strominger, D. C. Wiley. 1994. Crystal structure of the human class II MHC protein HLA-DR1 complexed with an influenza virus peptide. Nature 368:215.
  31. Fremont, D. H., W. A. Hendrickson, P. Marrack, J. Kappler. 1996. Structures of an MHC class II molecule with covalently bound single peptides. Science 272:1001.[Abstract]
  32. Konig, R., L.-Y. Huang, R. N. Germain. 1992. MHC class II interaction with CD4 mediated by a region analogous to the MHC class I binding site for CD8. Nature 356:796.[Medline]
  33. Cammarota, G., A. Scheirle, B. Takacs, D. M. Doran, R. Knorr, W. Bannwarth, J. Guardiola, F. Sinigaglia. 1992. Identification of a CD4 binding site on the ß2 domain of HLA-DR molecules. Nature 356:799.[Medline]
  34. Sant, A. J.. 1993. Isotypic residues in the membrane proximal domain of MHC class II ß chains control activation of CD4+ T cells. J. Immunol. 150:5299.[Abstract]
  35. Konig, R., X. Shen, R. N. Germain. 1995. Involvement of both major histocompatibility class II {alpha} and ß chains in CD4 function indicates a role for ordered oligomerization in T cell activation. J. Exp. Med. 182:779.[Abstract/Free Full Text]
  36. Schafer, P. H., S. K. Pierce, T. S. Jardetzky. 1995. The structure of MHC class II: role for the dimer of dimers. Semin. Immunol. 7:389.[Medline]
  37. Bentley, G. A., G. Boulot, K. Karjalainen, R. A. Mariuzza. 1995. Crystal structure of the ß chain of a T cell antigen receptor. Science 267:1984.[Abstract/Free Full Text]
  38. Fields, B. A., B. Ober, E. L. Malchiodi, M. I. Lebedeva, B. C. Braden, X. Ysern, J.-K. Kim, X. Shao, E. S. Ward, R. A. Mariuzza. 1995. Crystal structure of the V{alpha} domain of a T cell antigen receptor. Science 270:1821.[Abstract/Free Full Text]
  39. Garcia, K. C., M. Degano, R. L. Stanfield, A. Brunmark, M. R. Jackson, P. A. Peterson, L. Teyton, I. A. Wilson. 1996. An {alpha}ß T cell receptor structure at 2.5 A and its orientation in the TCR-MHC complex. Science 274:209.[Abstract/Free Full Text]
  40. Garboczi, D. N., P. Ghosh, U. Utz, Q. R. Fan, W. E. Biddison, D. C. Wiley. 1996. Structure of the complex between human T-cell receptor, viral peptide and HLA-A2. Nature 384:134.[Medline]
  41. Reich, Z., J. J. Boniface, D. S. Lyons, N. Borochov, E. J. Wachtel, M. M. Davis. 1997. Ligand-specific oligomerization of T-cell receptor molecules. Nature 387:617.[Medline]
  42. Sakihama, T., A. Smolyar, E. L. Reinherz. 1995. Molecular recognition of antigen involves lattice formation between CD4, MHC class II, and TCR molecules. Immunol. Today 16:581.[Medline]
  43. Schafer, P. H., S. K. Pierce. 1994. Evidence for dimers of MHC class II molecules in B lymphocytes and their role in low affinity T cell responses. Immunity 1:699.[Medline]
  44. Roucard, C., F. Garban, N. A. Mooney, D. J. Charron, M. L. Ericson. 1996. Conformation of human leukocyte antigen class II molecules. J. Biol. Chem. 271:13993.[Abstract/Free Full Text]
  45. Cherry, R. J., K. M. Wilson, K. Triantafilou, P. O’Toole, I. E. G. Morrison, P. R. Smith, N. Fernandez. 1998. Detection of dimers of dimers of human leukocyte antigen (HLA)-DR on the surface of living cells by single-particle fluorescence imaging. J. Cell Biol. 140:71.[Abstract/Free Full Text]
  46. Ozato, K., N. Mayer, D. H. Sachs. 1980. Hybridoma cell lines secreting monoclonal antibodies to mouse H-2 and Ia-antigens. J. Immunol. 124:533.[Abstract]
  47. Lerner, E. A., L. A. Matis, C. A. Janeway, P. P. Jones, R. H. Schwartz, D. B. Murphy. 1980. Monoclonal antibody against an Ir gene product?. J. Exp. Med. 152:1085.[Abstract/Free Full Text]
  48. Quill, H., R. H. Schwartz, L. H. Gilmcher. 1986. Ekß mutant antigen-presenting cell lines expressing altered Ak{alpha} molecules. J. Immunol. 136:3351.[Abstract]
  49. Kozono, H., D. Parker, J. White, P. Marrack, J. Kappler. 1995. Multiple binding sites for bacterial superantigens on soluble class II MHC molecules. Immunity 3:187.[Medline]
  50. Barber, L. D., R. I. Lechler. 1991. Interactions between the amino-terminal domains of MHC class II molecules have a profound effect on serologic and T cell recognition: an analysis using recombinant HLA-DR/H-2E molecules. J. Immunol. 147:2346.[Abstract]
  51. Griffith, I. J., F. M. Carland, L. H. Glimcher. 1987. Alteration of a non-polymorphic residue in a class II Eb gene eliminates an antibody-defined epitope without affecting T cell recognition. J. Immunol. 138:4480.[Abstract]
  52. Pennell, C. A., L. W. Arnold, P. M. Lutz, N. J. LoCasio, P. B. Willoughby, G. Haughton. 1985. Cross-reactive idiotypes and common antigen binding specificities expressed by a series of murine B cell lymphomas: etiological implications. Proc. Natl. Acad. Sci. USA 82:3799.[Abstract/Free Full Text]
  53. Lakey, E. K., E. Margoliash, F. W. Fitch, S. K. Pierce. 1986. Role of L3T4 and Ia in the heteroclitic response of T cells to cytochrome c. J. Immunol. 136:3933.[Abstract]
  54. Srinivasan, M., S. K. Pierce. 1990. Isolation of a functional antigen-Ia complex. Proc. Natl. Acad. Sci. USA 87:919.[Abstract/Free Full Text]
  55. Gillis, S., M. M. Ferm, W. Ou, K. A. Smith. 1978. T cell growth factor: parameters of production and a quantitative microassay for activity. J. Immunol. 120:2027.[Abstract/Free Full Text]
  56. Schafer, P. H., J. M. Green, S. Malapati, L. Gu, S. K. Pierce. 1996. HLA-DM is present in one-fifth the amount of HLA-DR in the class II peptide-loading compartment where it associates with leupeptin-induced peptide (LIP)-HLA-DR. J. Immunol. 157:5487.[Abstract]
  57. Cleveland, D. W., S. G. Fischer, M. W. Kirschner, U. K. Laemmli. 1977. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J. Biol. Chem. 252:1102.[Abstract/Free Full Text]
  58. Lamb, R. A., P. W. Choppin. 1977. The synthesis of Sendai virus polypeptides in infected cells. III. Phosphorylation of peptides. Virology 81:382.[Medline]
  59. Srinivasan, M., S. Z. Domanico, P. T. P. Kaumaya, S. K. Pierce. 1993. Peptides of 23 residues or greater are required to stimulate a high affinity class II-restricted T cell response. Eur. J. Immunol. 23:1011.[Medline]
  60. Cristau, B., P. H. Schafer, S. K. Pierce. 1994. Heat shock enhances antigen processing and accelerates the formation of compact class II {alpha}ß dimers. J. Immunol. 152:1546.[Abstract]
  61. Lomant, A. J., G. Fairbanks. 1976. Chemical probes of extended biological structures: synthesis and properties of the cleavable protein cross-linking reagent [35S]dithiobis(succinimidyl propionate). J. Mol. Biol. 104:243.[Medline]
  62. Kornblatt, J. A., D. F. Lake. 1980. Cross-linking of cytochrome oxidase subunits with difluorodinitrobenzene. Can. J. Biochem. 58:219.[Medline]
  63. Cosson, P., J. S. Bonifacino. 1992. Role of transmembrane domain interactions in the assembly of class II MHC molecules. Science 258:659.[Abstract/Free Full Text]
  64. Runnels, H. A., J. C. Moore, P. E. Jensen. 1996. A structural transition in class II major histocompatibility complex proteins at mildly acidic pH. J. Exp. Med. 183:127.[Abstract/Free Full Text]
  65. Boniface, J. J., D. S. Lyons, D. A. Wettstein, N. L. Allbritton, M. M. Davis. 1996. Evidence for a conformational change in a class II major histocompatibility complex molecule occurring in the same pH range where antigen binding is enhanced. J. Exp. Med. 183:119.[Abstract/Free Full Text]
  66. Konig, R., S. Fleury, R. N. Germain. 1996. The structural basis of CD4-MHC class II interactions: coreceptor contributions to T cell receptor antigen recognition and oligomerization-dependent signal transduction. Curr. Top. Microbiol. Immunol. 205:19.[Medline]
  67. Nag, B., D. Passmore, T. Kendrick, H. Bhayani, S. D. Sharma. 1992. N-linked oligosaccharides of murine major histocompatibility complex class II molecule: role in antigenic peptide binding, T cell recognition, and clonal nonresponsiveness. J. Biol. Chem. 267:22624.[Abstract/Free Full Text]
  68. Sakihama, T., A. Smolyar, E. L. Reinherz. 1995. Oligomerization of CD4 is required for stable binding to class II major histocompatibility complex proteins but not for interaction with human immunodeficiency virus gp120. Proc. Natl. Acad. Sci. USA 92:6444.[Abstract/Free Full Text]
  69. Goodman, S., T. Sawada, J. A. Barbosa, B. Cole, R. Pergolizzi, J. Silver, E. Mellins, M. Y. Chang. 1995. Mutational analysis of two DR{alpha} residues involved in dimers of HLA-DR molecules. J. Immunol. 155:1210.[Abstract]



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