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The Journal of Immunology, 2002, 169: 4379-4387.
Copyright © 2002 by The American Association of Immunologists

Uniquely Conformed Peptide-Containing {beta}2-Microglobulin- Free Heavy Chains of HLA-B2705 on the Cell Surface1

Pratap Malik, Polina Klimovitsky, Lih-Wen Deng, Jonathan E. Boyson and Jack L. Strominger2

Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The human class I MHC molecules are known to generally exist on the cell surface either as peptide-containing complexes of H chain ({alpha}-chain) and {beta}2-microglobulin ({beta}2m) or as {beta}2m-free H chains incapable of binding peptides. In this study, a uniquely conformed peptide-containing {beta}2m-free HLA-B2705 H chain has been isolated using the recently described highly efficient perfusion-affinity chromatography system for purification of class I MHC protein molecules. This form recognized by the mAb MARB4 is very closely associated with the remainder of the peptide containing HLA-B2705/{beta}2m complex reactive with mAb ME1 and is present to ~1–10% of mAb ME1 reactive forms on the cell surface. Also, HLA-B2705 purified using the mAb ME1 affinity column includes this unique mAb MARB4-reactive, unusually stable peptide-containing {beta}2m-free form. A peptide nonamer GRWRGWYTY was isolated and identified from this {beta}2m-free HLA-B2705 H chain and was used to assemble the mAb MARB4 reactive form efficiently on the surface of cells expressing HLA-B2705. The discovery of this form opens new avenues for further investigation of the role of HLA-B27 in spondyloarthropathies.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Class I MHC protein molecules are highly polymorphic surface glycoproteins that are involved in immune recognition. Most of the class I MHC protein molecules on the cell surface are present as dimers of two noncovalently associated chains, a 44,000-Da transmembrane H chain ({alpha}-chain) and {beta}2-microglobulin ({beta}2m),3 a 14,000-Da soluble protein (1). In this form, these MHC protein molecules are strongly associated with a broad spectrum of peptides which are presented to T cells via interaction with the TCR (2, 3). A smaller fraction of the MHC molecules on the cell surface exist as functionally inactive {beta}2m-free H chains believed to be formed by the dissociation of {beta}2m and peptide from the previously cell surface-expressed heterodimers (4). This form does not bind peptides and is recognized by mAbs that recognize denatured H chains. The biological role of these denatured {beta}2m-free class I H chains is not clear. It has been shown that {beta}2m-free class I H chains are spontaneously released from the surface of activated cells and that cross-linking of {beta}2m-free class I H chains with specific mAbs results in the rapid down-regulation and internalization of these molecules (5).

The MHC class I allele HLA-B27 is strongly associated with human spondyloarthropathies including ankylosing spondylitis, Reiter’s syndrome, reactive arthritis, and uveitis. Over 90% of patients with ankylosing spondylitis have HLA-B27 (reviewed in Ref. 6 and references therein). Prior enterobacterial infections have been linked with the pathogenesis of several spondyloarthropathies. Several models explaining the role of HLA-B27 in the disease pathogenesis have been proposed, e.g., molecular mimicry to enterobacterial peptides, presentation of arthritogenic peptide, and more recently the role of {beta}2m-free H chains from transgenic mice (7, 8).

A mouse mAb, MARB4, has previously been shown to recognize a subset (5–10%) of the HLA-B2705 present on the surface of an EBV-transformed human B cell line, LG2 (9). Furthermore, mAb MARB4-reactive HLA-B2705 molecules bound peptides and the pooled sequencing of these peptides suggested the presence of peptides much longer than nonamers which are conventionally bound to class I MHC proteins, although they were not able to isolate an individual long peptide due to a low total yield of peptides. To further investigate and explore this mAb MARB4-reactive subset of HLA-B2705 molecules, a recently described advance in technology for purification of MHC molecules using the highly efficient method of perfusion-affinity chromatography (10) was employed. A complete characterization of this mAb MARB4-reactive form as {beta}2m-free, but with peptide-containing conformed HLA-B2705 H chain, is presented in this study. The discovery of this unique form of HLA-B2705 could be a significant first step in elucidating the exact role of HLA-B27 with spondyloarthropathies, especially because strong evidence directly links HLA-B27 to their pathogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and mAbs

The EBV-transformed human lymphoblastoid cell line (LCL) LG2 is homozygous for HLA-B2705 and HLA-DR1 as previously described (11). The 721.221 cells are EBV-transformed, MHC class I-negative human B cells (12). The HLA-B2705 transfectant of 721.221 cells has been described previously (13). The mutant human cell line T2 is derived from a hybrid of the B-LCL.174 and the T-LCL.CEM (14) which produces HLA-A0201 and HLA-B5101 encoded by its parent B-LCL.174, but fails to express normal amounts of class I MHC at the cell surface due to the lack of a functional TAP heterodimer. T2 cells transfected with the HLA-B2705 gene and the recombinant form HLA-B27A2B (in which the B pocket residues of HLA-B2705 have been mutated to an HLA-A2-like B pocket viz H9F, T24A, E45 M, I66K C67V, and K70H; Ref. 15) were a kind gift from Dr. R. A. Colbert (Cincinnati Children’s Hospital, Cincinnati, OH). HMy2.C1R (C1R) are HLA-A-negative and HLA-B35-low (16). The parent C1R cells and the transfectants HLA-B2705, HLA-B2703, HLA-B27A2B were also kind gifts from Dr. R. A. Colbert. Cells were grown in RPMI 1640 supplemented with 10% heat inactivated FBS (HyClone Laboratories, Logan UT), 2 mM glutamine (Life Technologies, Grand Island, NY), 50 U/ml penicillin (Life Technologies), and 50 µg/ml streptomycin (Life Technologies). For the transfectants, the above medium was made in 1 mg/ml geneticin (G418 sulfate; Life Technologies). Splenocytes from HLA-B2705 transgenic mice (17) were a kind gift from Dr. C. S. David (Mayo Clinic, Rochester, MN).

The mAbs used were ME1, an anti-HLA-B27 mouse IgG1 mAb which recognizes a conformational epitope that maps to amino acid residues A 69 and A 71 in the {alpha}1 helix in the H chain (18, 19); MARB4, an IgG2a mouse mAb that recognizes a subset of HLA-B2705 expressed on the cell surface (9) and was generously provided by B. Uchanska-Ziegler and A. Ziegler (Institut für Immungenetik, Universitätsklinikum Charite, Berlin, Germany); W6/32, a mouse IgG2a anti-human MHC class I mAb that recognizes a conformational epitope on the intact MHC molecule containing both {beta}2m and the H chain (20, 21, 22); HC10, a mouse IgG2a mAb that recognizes denatured human MHC class I H chains with arginine at position 62 (23, 24); BBM.1, a mouse IgG2b anti-human {beta}2m (25); and LB3.1, an anti-HLA-DR mouse IgG2b mAb (26). The hybridoma cells were grown in hybridoma serum-free medium (Life Technologies) or RPMI 1640 supplemented with 0–1% low IgG FBS (HyClone Laboratories), 2 mM glutamine (Life Technologies), 50 U/ml penicillin (Life Technologies), and 50 µg/ml streptomycin (Life Technologies). The mAbs were purified by running the cell culture supernatant on either POROS 20 A (Applied Biosystems, Foster City, CA) (protein A-coupled POROS 20 medium) or POROS 20 G (protein G-coupled to POROS 20 medium used for IgG1 Abs) columns using a BioCAD workstation for perfusion chromatography (Applied Biosystems). The mAbs were labeled with FITC (Molecular Probes, Eugene, OR) or with Alexa Fluor 488 (Molecular Probes) per the manufacturer’s instructions.

Iso-osmotic low pH treatment of cells

The low pH treatment of cells was done essentially as described previously (27). Approximately 5–10 x 106 cells were washed twice with PBS and resuspended in 250 µl of 1% BSA, 300 mM glycine, pH 2.4, for 2 min at room temperature. The cells were neutralized by the addition of 50 ml of RPMI 1640 medium to the cells. The cells were washed three times with staining buffer for FACScan analysis.

FACScan analysis

Typically, 2 x 105 cells were dispensed into a well of 96-well U- or V-bottom 96-well plates (Corning, Corning, NY), washed three times with 200 µl of ice-cold staining buffer (PBS containing 2% FBS, 0.05% sodium azide). For staining, the washed cells were incubated with 100 µl of primary mAb solution (2 µg/ml in staining buffer) for 30 min on ice and washed three times with 200 µl of staining buffer. Cells were then further incubated with a 1/100 dilution in staining buffer of F(ab')2 FITC-conjugated goat anti-mouse whole IgG (ICN Pharmaceuticals, Costa Mesa, CA) for 30 min, washed three times with 200 µl of staining solution, resuspended in 133 µl of staining buffer, and fixed by adding 66 µl of 3x fixing solution (3% paraformaldehyde in PBS, pH 7.2). Analysis was conducted on a FACSCalibur (BD Biosciences, Mountain View, CA).

Peptide loading on T2 cells

The protocol was based on as described in Ref. 28 . The cells were made to 1,000,000/ml in serum-free medium (Life Technologies). Cells (2 ml) were cultured in presence of 10 or 100 µM peptide for 12–18 h in a 24-well plate (Corning). The peptides were purchased from ResGen (Carlsbad, CA) and were >85% pure. The peptides were resuspended in DMSO.

Perfusion-immunoaffinity purification of MHC protein molecules

The affinity columns on POROS 20 beads were prepared using the mAbs MARB4, ME1, and LB3.1. Cell culture, lysate preparation, and purification were conducted essentially as described previously (9). Purification from the cell lysate was done using a series of columns in the following order: POROS 20 AL-normal mouse serum, POROS 20 A, POROS 20 AL-LB3.1, and POROS 20 AL-MARB4 or POROS 20 AL-ME1. The columns were washed extensively in each case with 0.1% deoxycholic acid (DOC), 10 mM Tris, pH 8.0, eluted with 0.1% DOC, 50 mM glycine, pH 11.0. Each eluant was neutralized with 2 M glycine, pH 2.0, and dialysed against 0.1% DOC, 10 mM Tris, pH 8.0, in a cold room at 4°C, concentrated in Centricon 10 (Amicon, Beverly, MA).

Peptide isolation

The peptides were eluted and isolated from the mAb MARB4 column essentially as described in Ref. 9 . For eluting peptides, the column was washed extensively with 1% (3–3[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate), 150 mM NaCl, 20 mM Tris-HCl, pH 8.0, then by 20 mM Tris-HCl, pH 8.0, PBS containing 0.05% sodium azide and water, and eluted with 2% acetic acid at a flow rate of 2–5 ml/min. The eluant was made in 10% acetic acid concentrated in a speed vacuum and passed through Microcon 3 (Amicon) to obtain peptides <3000 Da.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The mAb MARB4 recognizes a conformational epitope of HLA-B2705 on the cell surface

Although the mAb MARB4 recognizes a subset of HLA-B2705 on the cell surface as seen by FACS analysis, it did not work on Western blots (data not shown). This was unlike the mAb HC10 that recognizes denatured human class I MHC H chains with arginine at position 62 (23, 24) and is used extensively for visualizing the class I H chains. This indicated that mAb MARB4 recognized a conformational epitope on the H chain. LG2 cells exposed briefly to pH 2.4 in 300 mM glycine buffer completely lost the signal with mAb MARB4 as well as lost at least 99% of the signal with mAb ME1 on FACScan (Fig. 1Go). Moreover, the signal with mAb HC10, which recognizes denatured {beta}2m-free HLA B27 H chain, increased ~10 times (Fig. 1Go). Thus the mAb MARB4-reactive HLA-B2705 subset on the cell surface retains a unique conformation and is not a denatured class I H chain.



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  		FIGURE 1. FACScan analysis using the mAbs W6/32, HC10, MARB4, ME1, and BBM.1 of the 721.221 transfectants expressing MHC class I molecule HLA-B2705 with and without pretreatment with glycine buffer, pH 2.4.

 
A mAb MARB4 immunoaffinity column binds {beta}2m-free HLA B2705 H chain

Two separate sets of perfusion-affinity purifications of the LG2 lysate each from 100 g of cells were performed using the mAb MARB4-coupled POROS AL matrix and the mAb ME1-coupled POROS AL matrix. These were done as described in Materials and Methods and run on 12.5% polyacrylamide gel. Surprisingly, perfusion-affinity purification of the LG2 lysate using the POROS 20 AL-MARB4 yielded a protein that contained an HLA H chain but showed only a very faint band for the {beta}2m as visualized by reducing SDS-PAGE (Fig. 2Go, lane 2).



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  		FIGURE 2. SDS-PAGE analysis of perfusion affinity-purified HLA-B2705 protein molecules from LG2 cells. Lane M contains the molecular mass markers, lane 1 is the material eluted from the POROS 20 AL-ME1, and lane 2 is the material eluted from the POROS 20 AL-MARB4 column. The gel was stained with Coomassie blue.

 
mAb ME1 column-purified HLA-B2705 molecules include the mAb MARB4-reactive protein

LG2 cells (50 g) were lysed and the mAb ME1-reactive HLA-B2705 molecules were purified using a POROS 20 AL-ME1 column as described in Materials and Methods and dialysed against 0.1% DOC, 10 mM Tris, pH 8.0, in a cold room at 4°C. After 2 days at that temperature, the HLA-B2705 protein molecules were loaded on a POROS 20 AL-MARB4 column. The column was washed extensively and the eluted protein was immediately neutralized and concentrated using Microcon 3 (Amicon). The material eluted from the mAb MARB4 column was ~10–15% of the original mAb ME1 column eluted material as estimated from the approximate size of the elution peaks. Thus, the mAb MARB4-reactive form was included in the mAb ME1 column-eluted protein. This mAb MARB4-bound material, when run on SDS-PAGE, again showed an extremely faint band for the {beta}2m (Fig. 3Go, lane 2).



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  		FIGURE 3. SDS-PAGE analysis of perfusion affinity-purified HLA-B2705 protein molecules. Lane 1 has material purified from the POROS 20 AL-ME1column. The column (with precolumns) was loaded with cell lysate from 50 g of LG2 cells. Lane 2 has material purified from the POROS 20 AL-MARB4 column. The column (with precolumns) was loaded with the material eluted from the POROS 20 AL-ME1 column run in lane 1. Lane 3 has material purified from the POROS 20 AL-MARB4 column. This column was loaded with the material that flowed through the POROS 20 AL-ME1 column in lane 1. The gel was stained with Coomassie blue.

 
In another experiment, the HLA-B2705 molecules affinity-purified using POROS 20 AL-ME1 column from the lysate of 50 g of LG2 cells (prepared as above) and dialysed against 0.1% DOC, 10 mM Tris, pH 8.0, in a cold room at 4°C for 2 days were reloaded on a POROS 20 AL-ME1 column. About 5–10% of the material ran through without binding to this column. This flow-through was collected and loaded on a POROS 20 AL-MARB4 column. The column was washed extensively and the eluted protein was immediately neutralized and concentrated using Microcon 3 (Amicon). This eluant, when run on SDS-PAGE, was completely devoid of the {beta}2m and had only the HLA-B2705 H chain (Fig. 3Go, lane 3). The presence of the HLA-B2705 H chains in each of the three samples was confirmed by Western blotting using the mAb HC10 and the complete absence of the {beta}2m in the sample in lane 3 (Fig. 3Go) was confirmed by Western blotting using mAb BBM.1 (data not shown).

The mAb MARB4-reactive uniquely conformed {beta}2m-free HLA-B2705 H chains retain peptides

To investigate whether the conformed mAb MARB4-reactive material contained peptides, HLA-B2705 protein molecules were first purified using an mAb ME1 column and the eluted material was then loaded on the mAb MARB4 column essentially as described in the previous section. This was chosen over directly purifying the mAb MARB4-reactive material with the mAb MARB4 affinity column because purifying from the mAb MARB4 material from the mAb ME1 column-eluted pool consistently gave a much purer preparation with much less cocontaminating proteins (typically actin). The peptide elution step was conducted as described in Materials and Methods. The material was checked for the presence of peptides by amino acid analysis, followed by pool sequencing. In a single experiment, the pool sequencing clearly showed enrichment of amino acids glycine, serine, and arginine at P1 and of arginine at P2 (Table IGo). Matrix-assisted laser desorption ionization time of flight mass spectrometric analysis of the pool showed a predominance of material between 900 and 1300 molecular mass (Fig. 4Go). A predominant peptide sequence present in this pool was identified to be GRWRGWYTY by HPLC separation and N-terminal Edman sequencing. A prominent peak of molecular mass 1244.43 was evident in the mass spectrum analysis of the mixture (Fig. 4Go), exactly corresponding to the molecular mass of the identified peptide.


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  		Table I. Pooled sequencing of peptides from mAb MARB4-reactive HLA-B2705a

 


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  		FIGURE 4. Matrix-assisted laser desorption ionization time of flight mass spectrometry analysis of the peptide mixture eluted from the POROS 20 AL-MARB4 column. The POROS 20 AL-MARB4 column was loaded with HLA-B2705 protein purified from 50 g of LG2 cells using the POROS 20 AL-ME1 column.

 
Incubation of cells with peptides generates mAb MARB4 reactivity on the cell surface

To examine whether the peptide GRWRGWYTY could indeed generate mAb MARB4 reactivity in vitro, cells were cultured in the presence of this peptide as described in Materials and Methods. Incubating the HLA-B2705-transfected 721.221, C1R, and T2 cells with this peptide caused a dramatic increase in the mAb MARB4 signal as well as the expected increase (15) in the ME1 signal (Fig. 5Go). However, no increase in mAb MARB4 signal was seen when the cells were incubated with SRYWAIRTR which, as expected, caused a severalfold increase in the mAb ME1 signal. Substitution of glycine at P1 by alanine, methionine, isoleucine, and serine also resulted in an increase, although a smaller one in the mAb MARB4 mean fluorescence intensity (MFI) in T2/HLA-B2705 cells but other amino acid residues at this position had no effect whatsoever (Table IIGo). Also, the peptide GQWRGWYTY in which the arginine in position P2 is substituted with glutamine caused an increase in the mAb MARB4. However, SQWRGWYTY failed to show an increase in the mAb MARB4 signal. T2/HLA-B2705 cells were incubated with several other peptides (GRIDKPILK, GRAFVTIGK, KRWIIMGLNK, RRIKEIVKK, RRYQKSTEL) previously reported to bind HLA-B2705 (29). All these peptides did not give an enhanced mAb MARB4 signal despite the usual severalfold increase in the mAb ME1 signal (data not shown).



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  		FIGURE 5. FACScan analysis using the mAbs MARB4 and ME1 of the (A) T2, (B) 721.221, and (C) C1R transfectants expressing MHC class I molecule HLA-B2705 with incubation with synthetic peptides. Two milliliters of 1 x 106 cells in serum-free medium were incubated with 100 µM peptide (20 µl of 10 mM solution in DMSO) or DMSO (20 µl). The staining was conducted as described in Materials and Methods.

 

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  		Table II. Effect of substitution of amino acid residues on surface expression of HLA-B2705a

 
Next, the binding of peptides to HLA-B27A2B (the HLA-B2705 molecule in which the B pocket has been substituted by that of HLA-A2) was examined. The peptide SQYWAIRTR that had been reported to bind to this molecule (15) also caused an increase in both mAb MARB4 and mAb ME1 signals when T2 cells expressing HLA-B27A2B were incubated with it (Fig. 6Go). None of the other amino acids had a comparable effect when present as P1 although several had small effects (Table IIIGo). No significant increase in the mAb MARB4 signal was produced in these cells on incubation with GRWRGWYTY or GQWRGWYTY (Fig. 6Go). These results clearly indicated the strong role of the peptide in stabilizing this unusual conformation.



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  		FIGURE 6. FACScan analysis using the mAbs MARB4 and ME1 of the T2 transfectant expressing MHC class I molecule HLA-B27A2B with incubation with synthetic peptides. Two milliliters of 1 x 106 cells in serum-free medium were incubated with 100 µM peptide (20 µl of 10 mM solution in DMSO) or DMSO (20 µl). The staining was conducted as described in Materials and Methods.

 

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  		Table III. Effect of substitution of amino acid residues on surface expression of HLA-B27A2Ba

 
Cells expressing the allele HLA-B2703 (which differs from HLA-B2705 in having a histidine residue at position 59 in place of tyrosine) are not stained by mAb MARB4 (Ref. 9 and Fig. 7Go). The HLA-B2703 C1R transfectants showed minimal up-regulation (if any) in the mAb MARB4 signal on incubation with GRWRGWYTY and none with SRYWAIRTR (Fig. 7Go) or with any of the other peptides (GRIDKPILK, GRAFVTIGK, KRWIIMGLNK, RRIKEIVKK, RRYQKSTEL) (data not shown).



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  		FIGURE 7. FACScan analysis using the mAbs MARB4 and ME1 of the C1R transfectant expressing MHC class I molecule HLA-B2703 with incubation with synthetic peptides. Two milliliters of 1 x 106 cells in serum-free medium were incubated with 100 µM peptide (20 µl of 10 mM solution in DMSO) or DMSO (20 µl). The staining was conducted as described in Materials and Methods.

 
Also, human PBLs positive for HLA-B27 and splenocytes from mice transgenic for HLA-B2705 and human {beta}2m were both mAb MARB4-reactive (Fig. 8Go). The FACScan mAb MARB4 signal was significantly up-regulated when these cells were preincubated with the peptide GRWRGWYTY (Fig. 8Go).



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  		FIGURE 8. FACScan analysis using the mAbs MARB4 and ME1 of the (A) human HLA-B27-positive PBLs (HLA-A2, B18, B27) and (B) splenocytes from HLA-B2705/human {beta}2m+/mouse {beta}2m- with incubation with synthetic peptides. Two milliliters of 1 x 106 cells in serum-free medium were incubated with 100 µM peptide (20 µl of 10 mM solution in DMSO) or DMSO (20 µl). The staining for human PBLs was conducted as described in Materials and Methods. The mouse splenocytes were first stained with goat-anti-mouse IgG PE-labeled Ab and washed before carrying out the staining with the mAbs. The cell population that was negative for the mouse IgG is shown in B.

 
The mAb MARB4 is blocked by the mAb ME1 on the cell surface and not vice versa

To examine cross-blocking, 721.221/HLA-B2705 cells pretreated with one mAb were stained by a labeled second mAb. The mAb ME1 blocked the binding by mAb MARB4. However, the reverse, i.e., the blocking of mAb ME1 by mAb MARB4 could not be observed by this method as the signal with mAb ME1 is severalfold higher than with mAb MARB4 and a small change in the mAb ME1 signal, if any, will not be observed. To overcome this, cross-blocking was examined using T2/HLA-B2705 precultured in the presence of the GRWRGWYTY peptide. Cells pretreated with one Ab were stained by Alexa Fluor 488 (Molecular Probes) -labeled second mAb (Fig. 9Go). mAb ME1 clearly blocks mAb MARB4 binding; however, mAb ME1 binding is unaffected by mAb MARB4.



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  		FIGURE 9. FACScan analysis to show the cross-blocking of mAbs ME1 and MARB4 using the T2 transfectant expressing MHC class I molecule HLA-B2705, preincubated with synthetic peptide GRWRGWYTY. Cells in serum-free medium were incubated with 100 µM GRWRGWYTY peptide for 18 h. The cells were washed and first incubated with the unlabeled Ab (20 µg/ml) and then with the Alexa Fluor 488-labeled Ab (20 µg/ml) in the presence of the unlabeled Ab. Staining and washings were conducted essentially as described in Materials and Methods.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, the HLA-B2705 form recognized by the mAb MARB4 has been characterized. Surprisingly, the material eluted from the mAb MARB4 column was devoid of {beta}2m. It had previously been reported that mAb MARB4 immunoprecipitated an HLA-B2705 H chain that appeared identical to that precipitated by other HLA-B27-specific mAbs and that {beta}2m was coprecipitated with it (9). Immunoprecipitation with mAb MARB4 of surface biotinylated HLA-B2705-expressing LG2 (or transfected 721.221/HLA-B2705) cell lysate did show the presence of {beta}2m although the H chain band was much brighter than the {beta}2m band (data not shown). The mAb MARB4-reactive form of HLA-B2705 obtained in this study was conformed and free of {beta}2m, although different from the mAb HC10 reactive form that recognizes the denatured peptide-free and {beta}2m-free form of HLA-B2705 (Fig. 1Go). Furthermore, HLA-B2705 molecules eluted from a mAb ME1 column included the uniquely conformed mAb MARB4-reactive form because a fraction of the material eluted from the mAb ME1 column bound to the mAb MARB4 column. Although some of the mAb MARB4-reactive material could possibly have been generated on treatment with high pH in the mAb ME1 column elution step, its inclusion in the eluate with the mAb ME1-reactive form could have been due to the intrinsic ability that major histocompatibility molecules have to form homotypic associations on the cell surface (30). The slight amount of {beta}2m in the SDS-PAGE (Fig. 2Go, lane 2) may be due to this intrinsic ability to form homotypic associations. A very small fraction of the mAb ME1-reactive heterodimer and the conformed mAb MARB4-reactive monomer could have been copurified as a result of homotypic association. The combination of efficient multiple washing steps (that can be easily done in the perfusion-affinity chromatography system) and the high pH elution step may help in breaking up this homotypic association and therefore result in an effective separation of the mAb ME1 {beta}2m-containing and mAb MARB4 {beta}2m-free forms of the HLA-B2705 protein molecule.

Although part of the mAb MARB4-reactive form is copurified with the mAb ME1 form (Fig. 3Go, lanes 1 and 2), the fact that material in the flow-through from the mAb ME1 column binds to the mAb MARB4 column (Fig. 3Go, lane 3) also indicates that mAb ME1 did not recognize another part of the mAb MARB4-reactive form. These results may be due to differing affinities of the two mAbs for the two forms of HLA-B2705 or alternatively homotypic association could result in the two forms being copurified. The cell surface blocking experiments in which the mAb MARB4 did not block the binding with mAb ME1, although mAb ME1 completely blocked the binding by mAb MARB4 (Fig. 9Go), could also be based on the relative affinities of the two mAbs for the two forms. It could also be envisaged that prebinding of mAb ME1 to its epitope occludes the mAb MARB4 epitope while prebinding of mAb MARB4 to its epitope does not interfere with access to mAb ME1 epitope. This could imply some special spatial organization of the two forms.

The uniquely conformed mAb MARB4-reactive form retains bound peptides despite the absence of {beta}2m. A nonamer peptide, GRWRGWYTY, was isolated from this {beta}2m-free HLA-B2705 form. A basic local alignment search tool search for this peptide yielded a hypothetical protein of human origin (31). The mAb MARB4-reactive form could be generated in high yield on the surface of HLA-B2705-expressing cells by incubating them with this peptide. mAbs showing peptide-dependent binding to HLA-B27 have been generated in the past (32, 33). The peptide-dependent recognition is due either to conformational changes of class I molecules induced by peptide-binding or due to epitopes jointly generated by a peptide and the MHC molecule. For example, two mAbs, MARB3 and MARB7, that recognize only peptide filled HLA-B27 molecules (as opposed to empty) have been characterized (33). mAb MARB7 binding is influenced by the C terminus of peptide loaded on the HLA-B2705; acidic residues at the C terminus abolish recognition. The fact that the peptide SQYWAIRTR gives rise to a mAb MARB4 form on HLA-B27A2B-expressing cells and that the same peptide, or SRYWAIRTR, does not on HLA-B2705-expressing cells indicates that mAb MARB4 is not recognizing merely a subset of peptides in the context of the MHC heterodimer but a different conformation of the H chain. This form could arise by a certain subset of peptides giving unusual conformational stability to the generally very unstable {beta}2m-free class I MHC H chain. In the case of HLA-B27A2B, the peptide is SQYWAIRTR and in the case of HLA-B2705, it is GRWRGWYTY. The sensitivity to the amino acid residue at P1 (Tables IIGo and IIIGo) shows that this anchor is important for this stability. It remains a puzzle that only the peptide with glycine (but not serine) at P1 confers mAb MARB4 reactivity to HLA-B2705, while that with serine (but not glycine) at P1 confers mAb MARB4 reactivity to HLA-B27A2B.

Cells expressing the HLA-B2703 protein, even after incubation with the GRWRGWYTY peptide, did not react with mAb MARB4. In fact, this is the only subtype of HLA-B27 that is not recognized by mAb MARB4 (9). HLA-B2703 is found predominantly in a Black African population and has been found not to be associated with spondyloarthropathy (34). Previous studies (35) have shown the HLA-B2703 subtype of HLA-B27, which differs from HLA-B2705 and other MHC class I molecules by having a histidine substituted for tyrosine at position 59 in the A pocket (a residue that forms part of a pentagonal hydrogen bonding network that stabilizes the NH3+ end of the peptide in the A pocket), inefficiently presents certain HLA-B2705-restricted peptides. Also, the P1 residue had a marked effect on relative affinities for both of these subtypes; the EC50 of P1-substituted peptides spanned four orders of magnitude. Self peptides eluted from HLA-B2703 have a motif similar to HLA-B2705 peptides, except for a stronger preference for lysine or arginine at P1, consistent with peptide binding data. Computer modeling (35) of HLA-B2703 revealed movement of a water molecule and the {alpha}1 {alpha}-helix to allow histidine at position 59 to maintain important hydrogen bonds with the peptide N terminus in the A pocket. However, these bonds are weaker, and the water molecule movement results in the loss of a hydrogen bond with glutamic acid 45 in the B pocket. They concluded that HLA-B2703, as a consequence of its unique A pocket polymorphism, appears to have a greater dependency on an accessory anchor residue at P1 to maintain tight binding of peptides. The A pocket contains the amino acid residue at position 59 and this negative reactivity of HLA-B2703 to mAb MARB4 indicates that the A pocket may be involved in the adoption of this conformation. The crystal structure of the HLA-B2705 protein molecule shows a deep B pocket with glutamic acid at its base (36, 37) and has a strong specificity for an arginine side chain at this pocket (29, 38). However, glutamine at P2 is accepted as well and the mAb MARB4-reactive form is generated with the peptide GQWRGWYTY although to a lesser extent (Table IIGo).

The strong association of HLA-B27 with spondyloarthropathies has been known for almost three decades (39, 40). More than 95% of patients with this disease carry this class I gene. It has been proposed that the role of HLA-B27 in spondyloarthropathy involves binding and presentation of "arthritogenic" peptides. Studies in transgenic rodents suggest a direct role of HLA-B27 H chains in disease pathogenesis. The disease has been suggested to be associated with infection by one or more enterobacteria, possibly through a mechanism involving molecular mimicry. This idea is supported by the finding that mice or rats transgenic for the HLA-B27 genes do not develop any disease under specific pathogen-free conditions, but develop arthritis when removed from the barrier facility (41). In rats, the arthritis is accompanied by inflammatory bowel disease and/or uveitis, both of which can be present in the human disease. More recently, a role for misfolded HLA-B27 H chains, due to a relatively unstable B pocket, has also been hypothesized (15). The unusual ability of HLA-B2705 to form peptide complexes without associating with TAP or tapasin confers flexibility in the repertoire of peptides presented by this molecule (42). It is also speculated that these properties might contribute to the role of HLA-B27 in conferring susceptibility to inflammatory spondyloarthropathies (42).

The unusual presence of cysteine 67 in the B pocket of HLA-B27 protein has led to speculation about its role in disease, which may have been refuted by the finding that the C67S mutant was able to induce arthritis in the rat transgenic model (41). It has been recently demonstrated that the cysteine 67 residue influences cell surface stability, peptide specificity, and T cell Ag presentation (43). These authors suggest that the cysteine to serine mutation weakens B pocket interactions, leading to decreased stability of the mutant-peptide complexes. It has been suggested that cysteine 67 plays a crucial role in controlling the thermodynamic stability of the HLA-B2705-peptide complexes as they found a faster unfolding for the cysteine to serine mutant (44). It could be speculated that the unusual cysteine residue at position 67 may be somehow responsible for the mAb MARB4-reactive form. However, our current study indicates that the cysteine 67 residue of HLA-B2705 H chain has no role in its adoption of the mAb MARB4 conformation. TAP-deficient T2 cells transfected with a mutated HLA-B2705 molecule in which the residues that constitute the B pocket (that includes the amino acid at position 67) are replaced by those that constitute the B pocket of the HLA-A2, when incubated with another nonamer peptide SQYWAIRTR also show a mAb MARB4-reactive form on the cell surface (Fig. 6Go). Homodimerization of the HLA-B27 H chains through the unusual cysteine 67 has been suggested to generate a potential immunogenic agent and is believed to occur on intact cells (7). However, repetition of these experiments by us with untransfected T2 cells and T2 cells transfected with HLA-B2705 failed to reveal the presence of such a homodimer when lysis of cells was conducted in the presence of iodoacetamide to prevent homodimerization during the isolation procedure (data not shown and compare Ref. 45). Also, T2 cells have considerable cell surface expression of HLA-A2 molecules (as seen by FACScan) (46) which can easily be immunoprecipitated using mAb BBM.1 or, less efficiently (13), with mAb W6/32 from surface-biotinylated cells. This is also in contradiction to the result, reported in Ref. 7 , that protein could not be immunoprecipitated from surface-biotinylated untransfected T2 cells. Although Allen et al. (7) state in Methods that lysis was conducted in the presence of iodoacetamide, it is difficult to reconcile with the fact that tetramerization occurred through the cysteine residues in the cytoplasmic tails (see Fig. 4Go in Ref. 7 and compare Ref. 45).

Much data suggest that a conformed peptide-binding {beta}2m-free form of HLA-B27 may be involved in the spondyloarthropathies and their rodent models (reviewed in Refs. 6 and 47). The unusually important observation was made that introduction of the HLA-B27 transgene into {beta}2m-/- mice was able to induce arthritis in the same manner as its introduction into {beta}2m+/+ mice (17). The idea that a peptide derived from the HLA-B27 H chain itself was presented by a class II MHC protein to T cells to initiate disease was negated by the finding that the HLA-B27 transgene in mice with a class II-/-, {beta}2m-/- background was still able to initiate arthritis (48). These studies of {beta}2m-/- mice have intersected with our examination of the reactivity of the mAb MARB4 with a subset of HLA-B27 molecules on the surface of human B LCLs. An earlier examination of the nature of this material had revealed that it bound unusually long peptides, i.e., longer than the 9–12 residues found bound to all other class I MHC proteins (9). In the current study using improved methods, the mAb MARB4-reactive protein was found to normally exist on the surface of human cells without {beta}2m and that the {beta}2m-free form retains bound peptides. Although it is very unusual for the MHC class I protein to exist in this form, it has been shown that in the mouse, the H-2Db protein exists in a functionally conformed state on the surface of {beta}2m-negative cells (49). Not only could the {beta}2m- cells be stained by a Db {alpha}1 domain-specific mAb (which detects a conformation-dependent epitope), but also {beta}2m-negative Con A blast target cells could be lysed by alloreactive CTL, even in serum-free conditions. It was also shown that a subset of properly conformed H chains, free of {beta}2m, may have almost equal representation on {beta}2m-positive and {beta}2m-negative cells (49). However, to our knowledge this is the first report in which a peptide has been isolated and identified from conformed class I MHC H chains free of {beta}2m.

More recently the development of spontaneous arthritis in {beta}2m-negative mice without expression of HLA-B27 has also been seen (50). Under certain conditions, {beta}2m deficiency alone was sufficient to cause spontaneous inflammatory arthritis. Although in the above study (50), it was concluded that class I deficiency was sufficient to cause spontaneous inflammatory arthritis in mice, it is quite conceivable given the present discovery that a combination of a lack of {beta}2m and a certain genetic background could cause stable expression of conformed class I MHC H chain molecules in significant concentration to generate an autoreactive immune response.

Additional studies are needed to determine the exact role, if any, of the mAb MARB4-reactive {beta}2m-free conformed peptide-containing HLA-B2705 in spondyloarthropathies. In a preliminary experiment, antisera from five ankylosing spondylitis patients had no detectable effect on the binding of mAb MARB4 to 721.221/HLA-B2705. This experiment was designed to examine the possibility that ankylosing spondylitis patients might have generated Abs against the mAb MARB4 epitope. The negative result does not preclude that these patients may have generated Abs with a significantly lower affinity than mAb MARB4 or that they were present at a very low concentration. Another question to be explored is the origin and fate of this form on the cell surface. Is it formed by dissociation of {beta}2m from the preformed peptide containing heterodimer at the cell surface or is it transported to the cell surface independently of the heterodimeric form? Also, whether this form can exist in significant concentration on HLA-B2705-transfected cells or transgenic animals that do not express {beta}2m remains to be seen. It has been shown using an HLA-A2 mutant (with lysine at position 242) with a reduced affinity for binding {beta}2m that both conformed and nonconformed {beta}2m-free H chains can exist on the surface of C1R cells and that these are cleaved by metalloproteinase (51). It has also been shown that nonmutant soluble conformed {beta}2m-free H chains can be detected in supernatants of activated cells (52). The results with HLA-B2705/{beta}2m- mouse splenocytes suggest that in the absence of {beta}2m, low surface expression of the HLA-B2705 H chain occurs and the mAb MARB4-reactive species cannot be detected by FACScan even in the presence of the GRWRGWYTY peptide. Also, in vitro attempts to fold bacterially produced mutants of HLA-B2705 H chain with the cysteine 67 to serine substitution with this peptide in the absence of {beta}2m were unsuccessful, although a good yield of the refolded HLA-B2705 (serine 67) heterodimer was obtained when the refolding was done with {beta}2m. These data suggest that the FACScan-detectable mAb MARB4-reactive form originates from already existing peptide-containing HLA-B2705/{beta}2m complexes at the cell surface and that the presence of a subset of special peptides confers unusual stability to this form.


   Acknowledgments
 
We thank Dr. W. S. Lane and J. M. Neveu for mass spectrometry and sequencing of peptides; Drs. A. Zeigler and B. Uchanska-Ziegler for providing the cell line-producing mAb MARB4; Dr. R. A. Colbert for kindly providing the C1R cells and the transfectants (HLA-B2705, HLA-B2703, HLA-B27A2B) and the T2 cells and the transfectants (HLA-B2705, HLA-B27A2B); Dr. C. S. David for the splenocytes of the transgenic mice; and Dr. R. Sorrentino for providing the antisera from ankylosing spondylitis patients.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants CA47554 and AI49524. Back

2 Address correspondence and reprint requests to Dr. Jack L. Strominger, Department of Molecular and Cellular Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138. E-mail address: jlstrom{at}fas.harvard.edu Back

3 Abbreviations used in this paper: {beta}2m, {beta}2-microglobulin; LCL, lymphoblastoid cell line; DOC, deoxycholic acid; MFI, mean fluorescence intensity. Back

Received for publication June 7, 2002. Accepted for publication August 12, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Jones, E. Y.. 1997. MHC class I and class II structures. Curr. Opin. Immunol. 9:75.[Medline]
  2. Madden, D. R.. 1995. The three-dimensional structure of peptide-MHC complexes. Annu. Rev. Immunol. 13:587.[Medline]
  3. Engelhard, V. H.. 1994. Structure of peptides associated with class I and class II MHC molecules. Annu. Rev. Immunol. 12:181.[Medline]
  4. Rock, K. L., S. Gamble, L. Rothstein, C. Gramm, B. Benacerraf. 1991. Dissociation of {beta}2-microglobulin leads to the accumulation of a substantial pool of inactive class I MHC heavy chains on the cell surface. Cell 65:611.[Medline]
  5. Demaria, S., R. Schwab, Y. Bushkin. 1992. The origin and fate of {beta}2m-free MHC class I molecules induced on activated T cells. Cell. Immunol. 142:103.[Medline]
  6. Allen, R. L., P. Bowness, A. J. McMichael. 1999. The role of HLA-B27 in spondyloarthritis. Immunogenetics 50:220.[Medline]
  7. Allen, R. L., C. A. O’Callaghan, A. J. McMichael, P. Bowness. 1999. HLA-B27 can form a novel {beta}2-microglobulin-free heavy chain homodimer structure. J. Immunol. 162:5045.[Abstract/Free Full Text]
  8. Lopez-Larrea, C., S. Gonzalez, J. Martnez-Borra. 1998. The role of HLA-B27 polymorphism and molecular mimicry in spondylarthropathy. Mol. Med. Today 4:540.[Medline]
  9. Urban, R. G., R. M. Chicz, W. S. Lane, J. L. Strominger, A. Rehm, M. J. Kenter, F. G. UytdeHaag, H. Ploegh, B. Uchanska-Ziegler, A. Ziegler. 1994. A subset of HLA-B27 molecules contains peptides much longer than nonamers. Proc. Natl. Acad. Sci. USA 91:1534.[Abstract/Free Full Text]
  10. Malik, P., J. L. Strominger. 2000. Perfusion chromatography for very rapid purification of class I and II MHC proteins. J. Immunol. Methods 234:83.[Medline]
  11. Gorga, J. C., V. Horejsi, D. R. Johnson, R. Raghupathy, J. L. Strominger. 1987. Purification and characterization of class II histocompatibility antigens from a homozygous human B cell line. J. Biol. Chem. 262:16087.[Abstract/Free Full Text]
  12. Shimizu, Y., R. DeMars. 1989. Production of human cells expressing individual transferred HLA-A, -B, -C genes using an HLA-A, -B, -C null human cell line. J. Immunol. 142:3320.[Abstract]
  13. Malik, P., E. Baba, J. L. Strominger. 1999. Biotinylation of class I MHC molecules abrogates recognition by W6/32 antibody. Tissue Antigens 53:576.[Medline]
  14. Salter, R. D., P. Cresswell. 1986. Impaired assembly and transport of HLA-A and -B antigens in a mutant TxB cell hybrid. EMBO J. 5:943.[Medline]
  15. Mear, J. P., K. L. Schreiber, C. Munz, X. Zhu, S. Stevanovic, H. G. Rammensee, S. L. Rowland-Jones, R. A. Colbert. 1999. Misfolding of HLA-B27 as a result of its B pocket suggests a novel mechanism for its role in susceptibility to spondyloarthropathies. J. Immunol. 163:6665.[Abstract/Free Full Text]
  16. Zemmour, J., A.-M. Little, D. J. Schendel, P. Parham. 1992. The HLA-A, B "negative" cell line C1R expresses a novel HLA-B35 allele, which also has a point mutation in the translation initiation codon. J. Immunol. 148:1941.[Abstract]
  17. Khare, S. D., H. S. Luthra, C. S. David. 1995. Spontaneous inflammatory arthritis in HLA-B27 transgenic mice lacking {beta}2-microglobulin: a model of human spondyloarthropathies. J. Exp. Med. 182:1153.[Abstract/Free Full Text]
  18. Ellis, S. A., C. Taylor, A. McMichael. 1982. Recognition of HLA-B27 and related antigen by a monoclonal antibody. Hum. Immunol. 5:49.[Medline]
  19. Benjamin, R., P. Parham. 1990. Guilt by association: HLA-B27 and ankylosing spondylitis. Immunol. Today 11:137.[Medline]
  20. Barnstable, C. J., W. F. Bodmer, G. Brown, G. Galfre, C. Milstein, A. F. Williams, A. Ziegler. 1978. Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens-new tools for genetic analysis. Cell 14:9.[Medline]
  21. Parham, P., C. J. Barnstable, W. F. Bodmer. 1979. Use of a monoclonal antibody (W6/32) in structural studies of HLA-A, B, C, antigens. J. Immunol. 123:342.[Abstract/Free Full Text]
  22. Brodsky, F. M., P. J. Parham. 1982. Monomorphic anti-HLA-A, B, C monoclonal antibodies detecting molecular subunits and combinatorial determinants. J. Immunol. 128:129.[Abstract]
  23. Stam, N. J., H. Spits, H. L. Ploegh. 1986. Monoclonal antibodies raised against denatured HLA-B locus heavy chains permit biochemical characterization of certain HLA-C locus products. J. Immunol. 137:2299.[Abstract]
  24. Stam, N. J., T. M. Vroom, P. J. Peters, E. B. Pastoors, H. L. Ploegh. 1990. HLA-A- and HLA-B-specific monoclonal antibodies reactive with free heavy chains in Western blots, in formalin-fixed, paraffin-embedded tissue sections and in cryo-immuno-electron microscopy. Int. Immunol. 2:113.[Abstract/Free Full Text]
  25. Brodsky, F. M., W. F. Bodmer, P. Parham. 1979. Characterization of a monoclonal anti-{beta}2-microglobulin antibody and its use in the genetic and biochemical analysis of major histocompatibility antigens. Eur. J. Immunol. 9:536.[Medline]
  26. Gorga, J. C., P. J. Knudsen, J. A. Foran, J. L. Strominger, S. J. Burakoff. 1986. Immunochemically purified DR antigens in liposomes stimulate xenogeneic cytolytic T cells in secondary in vitro cultures. Cell. Immunol. 103:160.[Medline]
  27. Smith, K. D., B. E. Mace, A. Valenzuela, J. L. Vigna, J. A. McCutcheon, J. A. Barbosa, E. Huczko, V. H. Engelhard, C. T. Lutz. 1996. Probing HLA-B7 conformational shifts induced by peptide-binding groove mutations and bound peptide with anti-HLA monoclonal antibodies. J. Immunol. 157:2470.[Abstract]
  28. Brooks, J. M., R. A. Colbert, J. P. Mear, A. M. Leese, A. B. Rickinson. 1998. HLA-B27 subtype polymorphism and CTL epitope choice: studies with EBV peptides link immunogenicity with stability of the B27:peptide complex. J. Immunol. 161:5252.[Abstract/Free Full Text]
  29. Jardetsky, T. S., W. S. Lane, R. A. Robinson, D. R. Madden, D. C. Wiley. 1991. Identification of self peptides bound to purified HLA-B27. Nature 353:326.[Medline]
  30. Triantafilou, K., M. Triantafilou, K. M. Wilson, N. Fernandez. 2000. Human major histocompatibility molecules have the intrinsic ability to form homotypic associations. Hum. Immunol. 61:585.[Medline]
  31. Yu, W., B. Andersson, K. C. Worley, D. M. Muzny, Y. Ding, W. Liu, J. Y. Ricafrente, M. A. Wentland, G. Lennon, R. A. Gibbs. 1997. Large-scale concatenation cDNA sequencing. Genome Res. 4:353.
  32. Wang, J., D. T. Y. Yu, T. Fukazawa, H. Kellner, J. Wen, X.-K. Cheng, G. Roth, K. M. Williams, R. B. Raybourne. 1994. A monoclonal antibody that recognizes HLA-B27 in the context of peptides. J. Immunol. 152:1197.[Abstract]
  33. Rehm, A., A. Rohr, C. Seitz, K. Wonigeit, A. Ziegler, B. Uchanska-Ziegler. 2000. Structurally diverse forms of HLA-B27 molecules are displayed in vivo in a cell type-dependent manner. Hum. Immunol. 61:408.[Medline]
  34. Hill, A. V., C. E. Allsopp, D. Kwiatkowski, N. M. Anstey, B. M. Greenwood, A. J. McMichael. 1991. HLA class I typing by PCR: HLA-B27 and an African B27 subtype. Lancet 337:640.[Medline]
  35. Griffin, T. A., J. Yuan, T. Friede, S. Stevanovic, K. Ariyoshi, S. L. Rowland-Jones, H. G. Rammensee, R. A. Colbert. 1997. Naturally occurring A pocket polymorphism in HLA-B*2703 increases the dependence on an accessory anchor residue at P1 for optimal binding of nonamer peptides. J. Immunol. 159:4887.[Abstract]
  36. Madden, D. R., J. C. Gorga, J. L. Strominger, D. C. Wiley. 1991. The structure of HLA-B27 reveals nonamer self-peptides bound in an extended conformation. Nature 353:321.[Medline]
  37. Madden, D. R., J. C. Gorga, J. L. Strominger, D. C. Wiley. 1992. The three-dimensional structure of HLA-B27 at 2.1: a resolution suggests a general mechanism for tight peptide binding to MHC. Cell 70:1035.[Medline]
  38. Rotzschke, O., K. Falk, S. Stevanovic, V. Gnau, G. Jung, H. G. Rammensee. 1994. Dominant aromatic/aliphatic C-terminal anchor in HLA-B*2702 and B*2705 peptide motifs. Immunogenetics 39:74.[Medline]
  39. Schlosstein, L., P. I. Terasaki, R. Bluestone, C. M. Pearson. 1973. High association of an HL-A antigen, W27, with ankylosing spondylitis. N. Engl. J. Med. 288:704.
  40. Brewerton, D. A., F. D. Hart, A. Nicholls, M. Caffrey, D. C. James, R. D. Sturrock. 1973. Ankylosing spondylitis and HL-A 27. Lancet 1:904.[Medline]
  41. Taurog, J. D., S. D. Maika, N. Satumtira, M. L. Dorris, I. L. McLean, H. Yanagisawa, A. Sayad, A. J. Stagg, G. M. Fox, A. Le O’Brien, et al 1999. Inflammatory disease in HLA-B27 transgenic rats. Immunol. Rev. 169:209.[Medline]
  42. Peh, C. A., S. R. Burrows, M. Barnden, R. Khanna, P. Cresswell, D. J. Moss, J. McCluskey. 1998. HLA-B27-restricted antigen presentation in the absence of tapasin reveals polymorphism in mechanisms of HLA class I peptide loading. Immunity 8:531.[Medline]
  43. Alvarez, I., M. Marti, J. Vazquez, E. Camafeita, S. Ogueta, J. A. Lopez De Castro. 2001. The Cys-67 residue of HLA-B27 influences cell surface stability, peptide specificity, and T-cell antigen presentation. J. Biol. Chem. 276:48740.[Abstract/Free Full Text]
  44. Reinelt, S., S. Dedier, G. Asuni, G. Folkers, D. Rognan. 2001. Mutation of Cys-67 alters the thermodynamic stability of the human leukocyte antigen HLA0-B*2705. J. Biol. Chem. 276:18472.[Abstract/Free Full Text]
  45. Springer, T. A., R. J. Robb, C. Terhorst, J. L. Strominger. 1977. Submit and disulfide structure of monomeric and dimeric forms of detergent-soluble HLA antigens. J. Biol. Chem. 252:4694.[Abstract/Free Full Text]
  46. Wei, M. L., P. Cresswell. 1992. HLA-A2 molecules in an antigen-processing mutant cell contain signal sequence-derived peptides. Nature 356:443.[Medline]
  47. Khare, S. D., H. S. Luthra, C. S. David. 1998. HLA-B27 and other predisposing factors in spondyloarthropathies. Curr. Opin. Rheumatol. 10:282.[Medline]
  48. Khare, S. D., M. J. Bull, J. Hanson, H. S. Luthra, C. S. David. 1998. Spontaneous inflammatory disease in HLA-B27 transgenic mice is independent of MHC class II molecules: a direct role for B27 heavy chains and not B27-derived peptides. J. Immunol. 160:101.[Abstract/Free Full Text]
  49. Bix, M., D. Raulet. 1992. Functionally conformed free class I heavy chains exist on the surface of {beta}2 microglobulin negative cells. J. Exp. Med. 176:829.[Abstract/Free Full Text]
  50. Kingsbury, D. J., J. P. Mear, D. P. Witte, J. D. Taurog, D. C. Roopenian, R. A. Colbert. 2000. Development of spontaneous arthritis in {beta}2-microglobulin-deficient mice without expression of HLA-B27: association with deficiency of endogenous major histocompatibility complex class I expression. Arthritis Rheum. 43:2290.[Medline]
  51. Demaria, S., L. D. DeVito-Haynes, R. D. Salter, W. J. Burlingham, Y. Bushkin. 1999. Peptide-conformed {beta}2m-free class I heavy chains are intermediates in generation of soluble HLA by the membrane-bound metalloproteinase. Hum. Immunol. 60:1216.[Medline]
  52. Demaria, S., Y. Bushkin. 2000. Soluble HLA proteins with bound peptides are released from the cell surface by the membrane metalloproteinase. Hum. Immunol. 61:1332.[Medline]



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