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International Immunology Advance Access originally published online on December 16, 2005
International Immunology 2006 18(1):211-220; doi:10.1093/intimm/dxh364
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© The Japanese Society for Immunology. 2005. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

The amino acid at position 97 is involved in folding and surface expression of HLA-B27

M. A. Blanco-Gelaz1, B. Suárez-Alvarez1, Segundo González2, A. López-Vázquez1, J. Martínez-Borra1 and Carlos López-Larrea1

1 Department of Immunology, Hospital Universitario Central de Asturias, 33006 Oviedo, Spain
2 Functional Biology Department, University of Oviedo, Asturias, Spain

Correspondence to: C. López-Larrea; E-mail: inmuno{at}hca.es


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
HLA-B27 confers susceptibility to ankylosing spondylitis (AS) but the mechanism linking this association remains unknown. Other properties unrelated to its natural role of antigen presenting function may be important in disease pathogenesis. We determined here the impact of N97D substitution on the folding and expression of HLA-B*2704 transfected in the 721.221 cell line. The mutation at position 97 abolishes the surface expression of non-conformational (HC10) and conformational (ME1) forms. The expression of ME1 forms was found to be absent in B*2704 N97D by immunoprecipitation and flow cytometry of fixed and permeabilized cell experiments with the conformation-sensitive ME1 antibody. However, immunoblotting cell lysates with HC10 revealed the presence of unfolded heavy chain (HC) and HC-dimer forms. The impact of the N97D mutation in the exit from the endoplasmic reticulum (ER) was analysed by western blot after endoglycosidase-H treatment, and it was found that B*2704 N97D was retained and accumulated as unfolded molecules. We tested for mutant association with transporter associated with antigen processing (TAP), calnexin (CNX), calreticulin (CLR) and ß2 microglobulin (ß2m). The wild-type B*2704 and N97D mutants were associated with TAP, CNX and CLR, although HC10 forms of mutant N97D interact more weakly with TAP. Only folded molecules of HLA-B*2704 were associated with ß2m. Surprisingly, the peptide-binding assay demonstrated the ability of unfolded N97D molecules to bind high-affinity peptides. It has been suggested that AS may arise because of aberrant folding of HLA-B27 molecules within the ER. Future work must therefore aim to clarify the functional connection between the unfolded protein response pathway in response to the accumulation of HLA-B27 in the ER. This mutant could be useful as a model for the misfolding of HLA-B27.

Keywords: antigen processing, MHC, peptide binding, spondyloarthropathies, TAP


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
The human MHC class I molecule HLA-B27 is strongly associated with spondyloarthropathies (SpA) (1, 2), a group of closely related inflammatory arthritic diseases, the most common of which is ankylosing spondylitis (AS). Although this association suggests direct involvement of HLA-B27 in disease pathogenesis, the mechanism remains unclear (3–6). HLA-B27 represents a family of 24 subtypes that only differ in a few amino acid substitutions in the region of the peptide-binding groove, that are mostly located in the C/F pocket. Among the 31 different HLA-B27 subtypes that have been identified to date, there are two subtypes (B*2706 and B*2709) for which only a very limited association with SpA has been proven (7–9). A number of theories explaining the pathogenic role of HLA-B27 have been proposed and the ‘arthritogenic peptide’ theory is the most popular (10). The identification of different molecular subtypes of HLA-B27, which differ in the amino acid composition of their peptide-binding grooves, has led to studies of the disease association and peptide-binding specificity of different subtypes (11–14). The failure to identify arthritic peptide ligands of HLA-B27 has resulted in more recent studies suggesting that this molecule may have disease-related properties other than specific peptide binding.

Recent studies have focused on the stability and expression of the HLA-B27 molecule and suggest that abnormal processing, transport and/or folding of HLA-B27 could be relevant to understanding its pathogenic role. It has been described that the HLA-B*2705 molecule achieves high levels of surface expression and presents specific viral peptides in the absence of tapasin (15). Significant differences in the peptide repertoire selected by B*2705 molecules in the absence or presence of this chaperone have been reported. Particularly efficient presentation of cytosolic peptides with low concentration may also play a role in the implication of HLA-B27 in AS (16).

Allen et al. (17) reported that HLA-B27 heavy chains (HCs) can be expressed as novel ß2 microglobulin (ß2m)-free HC homodimers as a consequence of an unusual cysteine-reactive residue (C67) located in the B pocket. HLA-B27 (B*2705) has an increased tendency, compared with some other HLA-B proteins, to misfolding after biosynthesis, leading to increased retrotranslocation and degradation in the cytosol (18). Accumulation of misfolded protein could result in a potentially pro-inflammatory intracellular stress response. This property appears to be related to the B pocket. However, the specificity and pathogenetic significance of these features are unclear. In addition, a direct pathogenic role of class I HCs and their expression levels has been suggested by HLA-B27 transgenic models of SpA where ß2m-deficient mice develop arthritis (19).

The proper folding and assembly of proteins in the endoplasmic reticulum (ER) is dependent on a number of chaperones that are subject to stringent quality control measures to ensure that the improperly folded are destroyed. It has been described that the polymorphism of HLA class I influences their binding interaction with peptide loading complex (20–22). MHC class I assembly is a highly regulated process with coordinated ER chaperones and accessory molecule interactions. Peptide binding occurs when class I molecules fold and associate with a loading complex consisting of interaction with the transporter associated with antigen processing (TAP), tapasin, ERp57 and calreticulin (CLR) (23). Different sites in the HLA-B27 HC might be implicated in the ER chaperone association (24, 25). These combined findings indicate the importance of defining the precise mechanism of the HLA-B27 assembly and folding.

The residue 97 located in the C/F pocket is polymorphic in HLA-B27 alleles and may influence the strength of the loading complex interaction. We generated a mutant in the allele B*2704 at position 97 (N97D) which abolishes the surface expression of MHC and that is involved in transport and accumulation in the ER of unfolded protein. Previous findings obtained with H-2Ld mutant have shown that modifications at residue 97 alter the peptide-binding cleft, suggesting that this position is involved in antigen presentation (26). Our mutant could be useful as a misfolding model that could be used to study the molecular mechanism that regulates the unfolded protein response (UPR).


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Cell lines and culture conditions
The LCL 721.221 cells (American Type Tissue Collection CRL1855, USA) are EBV-transformed human lymphoblastoid cell line, MHC class I negative (HLA-A–, -B–, -C–) (27). Wewak 1 (ECACC 94022553) is an EBV-transformed B cell line HLA-B*2704 positive. Cells were grown in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 10 U ml–1 streptomycin/penicillin (all from Invitrogen, Carlsbad, CA, USA) and 2 mM glutamine (Sigma, St Louis, MO, USA) at 37°C in a humidified atmosphere containing 5% CO2. Antibiotic-resistant transfectants were maintained in the above medium with hygromycin B at 800 µg ml–1 (Invitrogen).

Cloning of HLA-B*2704 subtype, mutagenesis and generation of stable transfectant cell lines
To create the B*2704 and B*2704 N97D transfectants, full-length cDNA clone of HLA-B*2704 subtype was obtained from an EBV-transformed cell line wewak 1. Briefly, RNA was extracted using TRI reagent (Sigma) and first-strand cDNA prepared according to standard procedures with an oligo-p(dT)18 primer. HLA class I cDNA was then amplified using primers HLA5UT and HLA3UTB (28), which introduced SalI and HindIII restriction sites, respectively. The PCR product was then subcloned into pBS II SK+ (Stratagene, San Diego, CA, USA) using these sites, and was sequenced in both directions. This construct was then subjected to site-directed mutagenesis using the QuickChange site-directed mutagenesis kit (Stratagene), and the sequence was confirmed after mutagenesis. The following oligonucleotides were used for mutagenesis (lower-case letters denote mutated bases): N97D F, 5'-TCTCACACCCTCCAGgATATGTATGGCTGCGAC-3', and N97D R, 5'-GTCGCAGCCATACATATcCTGGAGGGTGTGAGA-3'.

Subcloned HLA-B*2704 and HLA-B*2704 N97D were then transferred into the pRep4 vector (Invitrogen) using the XhoI and BamHI sites, and stable 221.B*2704 and 221.B*2704 N97D clones were generated by electroporation from a BIO-RAD Gene Pulser with a capacitance extender unit at 220 V and 975 µF (BIO-RAD, Hercules, CA, USA). Stable transfectants were selected with hygromycin B at 800 µg ml–1.

Antibodies
The mouse mAb HC10 (IgG2a), generously provided by J. A. López de Castro (Centro de Biología Molecular ‘Severo Ochoa’, Madrid, Spain), reacts with a determinant on ß2m-free HCs of HLA-B, -C and some HLA-A alleles (29, 30) and was used in western blot, immunoprecipitation and FACScan analysis. The mAb ME1 (IgG1), generously provided by K. Granfors (National Public Health Institute, Turku, Finland), recognizes HLA-B27, -B7, -B42, -B67, -B73 and -Bw22 (31) and is conformation dependent; this was used in immunoprecipitation and FACScan analysis. The mAb BBM.1, which recognizes human ß2m, was generously provided by J. R. Parra Cuadrado (Universidad Complutense de Madrid, Spain) and was used in western blot analysis. These antibodies were purified from hybridoma supernatants by AFFI-T gel affinity column (KE-MEN-TEC, Copenhagen, Denmark) according to the manufacturer's instructions. Rabbit anti-TAP1 (CSA-620), rabbit anti-CLR (SPA-600) and rabbit anti-calnexin (CNX) (SPA-860) were purchased from Stressgen Biotechnologies (Victoria, BC, Canada). Rabbit anti-tapasin anti-serum was generously provided by T. Hansen (Washington University School of Medicine, St Louis, MO, USA).

Western blot analysis and endoglycosidase-H digestion
The cells were washed three times in PBS and lysis was carried out in an NP40 lysis buffer [1% NP40, 20 mM Tris–HCl, pH 8, 130 mM NaCl, 10 mM iodoacetamide (Calbiochem, Schwalbach, Germany) and 1 mM phenylmethylsulphonyl fluoride (PMSF) (Sigma)] on ice for 30 min. Nuclei and cell debris were removed by centrifugation (13 000 r.p.m. for 15 min at 4°C). Protein was quantified by using RC DCTM Protein Assay (BIO-RAD) and an aliquot of lysate was boiled (5 min) in SDS sample buffer [50 mM Tris–HCl, pH 6.8, 2% SDS, 10% (v/v) glycerol and bromophenol blue] with or without reducing agent 2-mercaptoethanol (6%) and run on a 10% SDS-PAGE gel (32) using the Mini-PROTEAN 3 system (BIO-RAD). Proteins were transferred onto nitrocellulose membranes using Trans-Blot SD semi-dry transfer cell (BIO-RAD). The membranes were blocked for 1 h at room temperature in blocking buffer [PBS containing 1% (w/v) skimmed milk powder and 0.1% Tween 20]. Antigens on membranes were detected using antibodies diluted in blocking buffer and incubated for 2 h at room temperature. Following washes with washing buffer (PBS and 0.1% Tween 20), binding was detected by incubating membranes with peroxidase-conjugated goat anti-mouse or goat anti-rabbit immunoglobulins (Dako, Copenhagen, Denmark) and was visualized using ECL (Amersham Biosciences, Uppsala, Sweden) and exposure to film. The films were analysed on a GS-800 calibrated densitometer using Quantity One software (BIO-RAD) to quantify the volume of pixels contained in each band analysed.

For endoglycosidase-H (endo-H) digestion, lysates were diluted on 50 mM NaH2PO4, pH 5.5, 0.1% SDS and 50 mM 2-mercaptoethanol and digested overnight at 37°C with 5 mU of endo-ß-N-acetylglucosaminidase H (Calbiochem).

Immunoprecipitations
The cells were washed three times in PBS and lysis was carried out in digitonin lysis buffer [50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1mM MgCl2, 0.1 mM EDTA, 10 mM iodoacetamide, 1 mMPMSF and 1% digitonin (Calbiochem)] on ice for 30 min. Nuclei and cell debris were removed by centrifugation (13 000 r.p.m. for 15 min at 4°C). Lysates were pre-cleared overnight at 4°C with protein G Sepharose beads (Amersham Biosciences) and later with protein G Sepharose beads and normal mouse serum. For immunoprecipitation, pre-cleared lysates were incubated with HC10 or ME1 mAbs bound to protein G Sepharose beads for 2 h at 4°C. Beads were washed twice with digitonin lysis buffer, another two times with PBS containing 0.1% digitonin and 1 mM PMSF, and a further two times with PBS. Bound proteins were eluted in SDS sample buffer without reducing agent 2-mercaptoethanol and run on a 10% SDS-PAGE gel.

Peptide-binding assay
A total of 10 x 106 cells were washed three times in PBS and lysis was carried out in digitonin lysis buffer on ice for 30 min. Nuclei and cell debris were removed by centrifugation (13 000 r.p.m. for 15 min at 4°C). Lysates were pre-cleared overnight at 4°C with Sepharose CL-4B beads (Amersham Biosciences) and were later incubated for 4 h at 4°C with 20 µM biotin-RRIYDLIEL (33) (EUROGENTEC, Seraing, Belgium). HLA–peptide complexes were purified with streptavidin-agarose (Sigma) for 2 h at 4°C. Beads were washed twice with digitonin lysis buffer, another two times with PBS containing 0.1% digitonin and 1 mM PMSF and a further two times with PBS. Bound proteins were eluted in an SDS sample buffer without reducing agent 2-mercaptoethanol and run on a 10% SDS-PAGE gel.

Peptide-binding assay on plates was performed on digitonin-lysed 721.221 Rep4, 721.221 B*2704 and 721.221 B*2704 N97D cells as follows: Flat-bottom plates (Nunc) were coated with mAb HC10, 2 µg per well in 100 µl of 50 mM sodium carbonate buffer, pH 9.6, for 2 h at 37°C. Subsequently, the wells were incubated with PBS/2% gelatine (w/v) overnight at 4°C and washed before use. Lysates of PBS-washed cells (10 x 106 cells ml–1) were prepared in digitonin lysis buffer as above and 100 µl of the lysates was applied to the coated wells, followed by incubation overnight at 4°C. The plates were washed and biotin-labelled peptide was applied in a concentration range (0–100 µM) in binding buffer [5% dimethyl sulphoxide/0.05% NP40/0.05% Tween 20/16.8 mM citric acid/36 mM Na2HPO4 and protease inhibitors (Complete Mini tablets, Roche)] and incubated for 48 h at 37°C. Plates were washed and incubated with 100 µl of ExtrAvidin®–HRP diluted 800x in assay buffer (Sigma) for 2 h at room temperature. After extensive washing, o-phenylenediamine dihydrochloride (Sigma) was added at 0.4 mg ml–1 in 50 mM phosphate/citrate buffer, pH 5.5, until colour development. Optical densities (ODs) were measured at 492 nm. All results refer to the net OD value after subtraction of the background OD. The results represent the mean ± SD of three independent assays; each assay was done in triplicate.

Flow cytometry
Cell-surface staining for HLA-B27 expression was performed by indirect immunofluorescence on stable 221.B*2704 and 221.B*2704 N97D clones. Cells were washed three times with PBS (5 x 105 cells per sample) and stained with a saturating concentration of antibody for 30 min at 4°C. Normal mouse IgG antibody was used as a negative control for each test. HC10 and ME1 were used as primary antibodies followed by goat anti-mouse IgG conjugated to FITC (Dako). Dead cells were excluded by staining with 7-amino-actinomycin D (34) (BD PharMingen, San Jose, CA, USA). Samples were analysed in a Becton Dickinson FACScan with CellQuest software (Becton Dickinson, San Jose, CA, USA). Detection of intracellular HLA-B*2704 N97D expression was performed by immunofluorescence staining of fixed and permeabilized cells. Cells were washed three times with PBS (1 x 106 cells per sample), fixed on PBS containing 2% paraformaldehyde and incubated 1 h at 4°C. After washing on PBS, cells were permeabilized on PBS containing 0.2% Tween 20 and incubated for 15 min at 37°C. mAbs HC10 and ME1 were used as primary antibodies followed by FITC-conjugated goat anti-mouse IgG, and samples were analysed in a Becton Dickinson FACScan with CellQuest software.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Effect of N97D substitution on surface expression of HLA-B*2704
mAbs ME1 and HC10 recognizes distinct populations of HLA class I molecules. ME1 is conformation dependent, recognizing HLA-B27 folding complex containing peptides. In contrast, HC10 recognizes free HC not associated with ß2m. Here we refer to ME1- and HC10-reactive HC as folded and unfolded, respectively.

Using flow cytometry we examined the surface expression of the B*2704 N97D mutation in 721.221 transfectants. The levels of cell-surface expression were compared in relation with B*2704 wild type using the conformation-sensitive mAb ME1 and non-conformational mAb HC10. As shown in Fig. 1, the mutation at position 97 abolished the expression of non-conformational (HC10) and conformational (ME1) forms. In contrast, the B*2704 wild type exhibits surface-level expression comparable to other HLA-B27 alleles analysed (data not shown). It might be questioned whether the absence of surface expression of B*2704 N97D mutant correlates with the intracellular forms. Detection of intracellular B*2704 N97D expression was made by immunofluorescence staining of fixed and permeabilized cells. The expression of ME1 was also found to be absent in N97D mutant and the whole intracellular form correlates with HC10 non-conformational molecules.



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  		Fig. 1. Expression of HLA-B*2704 and HLA-B*2704 N97D mutants in 721.221 cells. Flow cytometry analysis on cells stained with ME1 (solid line) and HC10 (dotted line). Filled histogram shows mouse IgG control (negative control). (A) Surface expression of 721.221 cells transfected with pRep4, (B) surface expression of 721.221 cells transfected with HLA-B*2704, (C) surface expression of 721.221 cells transfected with HLA-B*2704 N97D, (D) intracellular expression of 721.221 cells transfected with pRep4, (E) intracellular expression of 721.221 cells transfected with HLA-B*2704 and (F) intracellular expression of 721.221 cells transfected with HLA-B*2704 N97D (note that this transfectant does not show ME1 expression). Histograms are representative of repeated experiments.

 
B*2704 N97D only exist as unfolded molecules retained within ER
We determined the expression of intracellular HLA-B*2704 and HLA-B*2704 N97D HC. In HC10 western blot analysis of 221.B*2704 lysates, bands around 45 and 90 kDa were detected (Fig. 2). The last band corresponds to homodimers that are dependent on the presence of an unpaired cysteine residue at position 67 (C67). The complete spectrum of bands is similar in 221.B*2704 N97D. However, immunoblotting cell lysates with HC10 revealed that N97D mutant significantly increased the number of HC-dimer molecules. The 90-kDa molecule, which represents 50% of the total HC in the mutant as determined by densitometry, resolves to a 45 kDa after reduction. Dimers account only for 5–10% of the total HC10-reactive form in B*2704 wild type. We determined expression levels of B*2704 by immunoprecipitation with conformation-dependent (ME1) and -independent (HC10) antibodies. Figure 3 shows that the B*2704 wild type was immunoprecipitated by HC10 and ME1 antibodies. However, the experiment indicated that the mutant N97D failed to provide results of conformational forms as seen by immunoprecipitation with ME1 antibody. This may reflect that the N97D substitution induces and/or increases an accumulation of non-conformational HC and HC-dimer forms.



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  		Fig. 2. Lysates of 721.221 cells transfected with the construct indicated at the top of the figure were tested by western blot analysis. Iodoacetamide-containing lysates were run on 10% SDS-PAGE in non-reducing (NR) and reducing (R) conditions followed by HC10 immunoblotting. Arrows indicate the position of dimer and monomer HLA-B27. The upper band in the B*2704 N97D lysate (*) is under investigation and is seen in all immunoblottings which have been made with HC10 antibody.

 


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  		Fig. 3. 721.221 B*2704 and 721.221 B*2704 N97D cells were lysed in digitonin and immunoprecipitated with ME1 (folded B27) and HC10 (unfolded B27). Samples were analysed in non-reducing conditions followed by HC10 immunoblotting. Note that in 721.221 B*2704 N97D the expression of ME1 forms was not detected. Similar findings were seen in three separate experiments.

 
To investigate the mechanism by which the amino acid at position 97 influences surface expression, we analysed the extent of intracellular transport of HLA-B27 molecules by examining the acquisition of endo-H resistance and oligosaccharide modification (Fig. 4). The total of B*2704 N97D remained sensitive to endo-H digestion, reflecting the complete retention of these molecules within the ER. In contrast, the HC10-reactive material from B*2704 lysates remained endo-H resistant. The B*2704 mutant forms intracellular HC but these do not egress to the cell surface. These findings suggest that the impaired intracellular transport of HLA-B*2704 N97D accounts for their absence of surface expression and the nature of the amino acid residue at position 97 is involved in the maturity, transport and surface expression of MHC class I.



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  		Fig. 4. Lysates of 721.221 cells transfected with the construct indicated at the bottom of the figure were tested by western blot analysis after endo-H digestion. Respective lysates were digested overnight with 5 mU endo-H at 37°C and run on 10% SDS-PAGE followed by HC10 immunoblotting. Tapasin immunoblotting (TPN) was performed to ensure total digestion of samples.

 
Effect of N97D substitution on interaction with chaperones
Given that the N97D substitution abolishes the surface expression of B*2704, we undertook the molecular association with peptide loading complex. To further understand the molecular basis for the critical role of residue 97 in defining chaperone dependence, we examined, by co-immunoprecipitation, the interaction of HC from B*2704 and its mutant derivates with proteins associated with antigen processing (Fig. 5). HC10 was used to immunoprecipitate B*2704 and B*2704 N97D and to check for association with chaperone proteins (CNX, CLR and TAP) by western blotting. We reacted the same blots with HC10 and verified that each lane contained a comparable amount of MHC class I molecules. We found that TAP was weakly associated with B*2704 N97D in comparison with B*2704, secondary to tapasin association, but the mutagenesis may not totally ablate HC interaction with tapasin–TAP. Co-immunoprecipitation of the B*2704 mutant with TAP is reduced to about 50% of the level of association of the wild-type B*2704. Both the B*2704 wild type and the mutant retained the ability to associate with CNX and CLR, but the level of B*2704 N97D association seems to be higher with these chaperones, especially with CLR. In conclusion, the N97D substitution debilitates the association to tapasin–TAP but remains capable of binding CNX and CLR as well as the B*2704 wild type. The presence of aspartic acid at position 97 in the B*2704 mutant, compared with asparagine in the B*2704 natural subtype correlates with the absence of surface expression, with accumulation as unfolded molecule and with poorer assembly to TAP. This finding is surprising taking into account that residue 97 has not been shown to be involved in this association. In fact, it has been described that residues around 134 and 227 might play a role in MHC class I association with tapasin that forms a bridge with TAP (24). It could be possible that N97 is involved in ß2m and peptide association, and poorer tapasin–TAP association could be secondary.



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  		Fig. 5. Immunoprecipitations with mAb HC10 were performed on digitonin-lysed 721.221 Rep4, 721.221 B*2704 and 721.221 B*2704 N97D cells. (A) The immunoprecipitates were electrophoresed on 8% Tris–glycine polyacrylamide gels. Following transfer of the protein to blots, the same membrane was probed with mAb HC10 and anti-serum against CNX, CLR and TAP1. The blots were developed with ECL reagents and exposed to film. (B) CNX, CLR and TAP1 band densities normalized for the background and the ratio of the HC10-reactive form immunoprecipitated. Quantity One software and GS-800 calibrated densitometer were used to quantify the volume of pixels contained in each band analysed.

 
B*2704 N97D molecule is able to bind high-affinity peptide in the absence of ß2m association
Examination of the B27 X-ray crystal structure raises an intriguing question concerning how N97 impacts the association of B*2704 with ß2m (35). N97 points up from the ß-pleated sheet that forms the floor of the peptide-binding cleft. A detailed analysis of the C/F pocket reveals a different effect of the N97D replacement. In fact, N97, H114 and D116 residues form a bridge of hydrogen bonds in the peptide-binding cleft that accommodates the peptide at P7 position (Fig. 6). When N97 is substituted for D97 in the HLA-B27 structure, computer modelling indicates that the hydrogen bonds formed to N97 with H114 and D116 are not retained in the B*2704 mutant. The disruption of these interactions could alter the conformation of the ß-strand and influence the assembly of tapasin–TAP, ß2m and/or peptide binding. Although it has not been previously reported that unfolded HLA class I HCs (HC10 detected) bind peptides (29, 34), N97D substitution in HLA-B27 could alter the three-dimensional structure of mutant N97D and influence its binding to ß2m and peptides. To investigate this possibility we examined, by co-immunoprecipitation, the interaction of HC from B*2704 and its mutant with ß2m (Fig. 7). HC10 was used to immunoprecipitate B*2704 and B*2704 N97D and to check for association with ß2m by western blotting. We reacted the same blot with HC10 and verified that each lane contained MHC class I molecules except in the control lane. We found, as previously described, that ß2m was only associated with folded HLA-B*2704 molecules (indicated by ß2m co-immunoprecipitation with ME1 antibody) but was not associated either with unfolded HLA-B*2704 molecules or with N97D mutant (HC10 immunoprecipitation). Thus, it might be questioned whether the HLA-B*2704 and N97D mutant could bind to high-affinity peptide. Biotin-RRIYDLIEL (EBNA-3C, 258–266) was chosen as a representative high-affinity peptide because it possesses a canonical sequence to bind to HLA-B*2704 (33). Peptide binding to unfolded and/or folded HLA-B*2704 and N97D mutant was detected by streptavidin-agarose binding of peptide–HLA complexes and western blotting (Fig. 8A). We transferred to the membrane the material isolated with streptavidin-agarose beads and probed with mAb HC10. As shown in Fig. 8(A), there was recovery (undistinguishing folded and unfolded molecules) of HLA-B*2704 HC in the wewak 1 lymphoblastoid cell line and in HLA-B*2704 transfectant after incubation with EBNA-3C peptide. Surprisingly, we also detected that the biotin-peptide was associated with the N97D HC, but only unfolded molecules as demonstrated in Figs 1 and 3. Thus, the loss of surface expression of HLA-B*2704 N97D is not due to lack of association with the peptide of unfolded molecules. HLA-B*2704 N97D is able to bind high-affinity peptides and this finding has not been previously described for the unfolded HLA class I HC. Considering the induction of conformational changes in HLA class I HC when it binds to antigenic peptides, we attempted to study whether the phenotype observed in transfectant 221.R04 N97D was reverted when the cellular extracts were incubated with the peptide EBNA-3C and the conformational and non-conformational forms were selectively immunoprecipitated. In order to do this, we incubated the peptide with lysates of transfectants 221.R04, 221.R04 N97D and 221.Rep4 as control, in the presence and absence of the peptide. The result of the immunodetection of HLA class I HC after immunoprecipitation with mAbs HC10 and ME1 is shown in Fig. 8(B). Thus, and as can be seen in the figure, the incubation of the peptide with the lysates of transfectant 221.R04 N97D does not induce conformational changes in the HC that favour the folding, since the ME1-negative phenotype remains. Taking into account that the peptide binds to non-conformational molecules in transfectant 221.R04 N97D, we set out to study whether the substitution introduced exerted some effect at the level of binding affinities in comparison with present HC10 molecules in transfectant 221.R04. In order to analyse such cellular lysates of both transfectants and transfectant 221.Rep4 as control were incubated on plates coated with mAb HC10 and were later incubated for 48 h in the presence of several peptide concentrations (0–100 µM). The analysis of the collected data (Fig. 9) shows that substitution N97D in HLA-B*2704 HC does not exert an effect on the affinity of the peptide biotin-RRIYDLIEL. Consequently, it is possible that this peculiar phenotype is not due to peptide binding but to primary misfolding of the N97D molecule and the inability to acquire a folded structure probably due to not binding to ß2m.



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  		Fig. 6. Structural model of the C/F pocket of HLA-B*2704. This model was generated from HLA-B*2705 structure (1HSA in www.rcsb.org/pdb) using the graphic program DeepView v3.7 SP5. The structure of HLA-B*2704 C/F pocket is shown in (A) and the structure generated for HLA-B*2704 N97D C/F pocket is shown in (B).

 


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  		Fig. 7. Immunoprecipitations with mAb HC10 were performed on digitonin-lysed 721.221 Rep4, 721.221 B*2704 and 721.221 B*2704 N97D cells. A 721.221 B*2704 immunoprecipitation with mAb ME1 and pre-immunoprecipitation lysate serves as ß2m control. The immunoprecipitates and lysate were electrophoresed on 10% Tris–glycine polyacrylamide gels, and following transfer of the protein to blots, the same membrane was probed with mAbs HC10 and BBM.1. The blots were developed with ECL reagents and exposed to film.

 


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  		Fig. 8. (A) Peptide-binding assay with biotin-RRIYDLIEL peptide was performed on digitonin-lysed 721.221 Rep4, 721.221 B*2704 and 721.221 B*2704 N97D cells. An assay with and without peptide of digitonin-lysed wewak 1 lymphoblastoid cell line serves as control. Upper panel: western blotting using HC10 antibody was performed to ensure the presence of HC in all samples before peptide was added; note that 721.221 Rep4 serves as control. Lower panel: streptavidin-agarose-eluted material was electrophoresed on 10% Tris–glycine polyacrylamide gels, and following transfer of the protein to blots, the membrane was used for immunodetection of HLA-B27 HC. (B) Immunoprecipitations with mAbs HC10 and ME1 were performed on digitonin-lysed 721.221 Rep4, 721.221 B*2704 and 721.221 B*2704 N97D cells without (upper panel) and with (lower panel) biotin-RRIYDLIEL. Note that the presence of high-affinity peptide does not induce conformational changes of B*2704 N97D HC (immunoprecipitation ME1 negative with biotin-RRIYDLIEL).

 


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  		Fig. 9. Peptide-binding assay on plates coated with mAb HC10 was performed on digitonin-lysed 721.221 Rep4, 721.221 B*2704 and 721.221 B*2704 N97D cells. Six concentrations of biotin-RRIYDLIEL were tested in the range from 5 to 100 µM. These results are representative of three independent experiments in triplicate using 100 µl of lysate equivalent to 2 x 105 cells.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
HLA-B27 polymorphism determines its peptide preferences but may also affect its association with other chaperones. Previous findings obtained with H-2Ld point mutant have shown that single amino acid change at position 97 (W97R) alters the peptide-binding cleft and its affinity for endogenous peptides and ß2m (26). On the basis of this finding, it has been suggested that position 97 is involved in antigen presentation. In fact, we found that a mutant of the allele HLA-B*2704 in which N97 was substituted by D97 (negatively charged) dramatically abolishes the surface expression. B*2704 N97D only exist as unfolded molecules within ER and possess less tapasin–TAP association than the B*2704 wild type. In addition to these findings, N97D molecules are able to bind high-affinity peptides. We demonstrated that unfolded HLA class I HC bind peptides, thus indicating the capacity to load unfolded molecules without the ß2m association. N97D substitution does not abolish the possibility to bind peptides but these N97D molecules are unable to gain a stable conformation and leave the ER. This substitution could affect the three-dimensional HLA-B27 structure and could prevent ß2m association.

We also found an increased accumulation of HLA-B27 dimers in the N97D mutant. HC dimerization was previously observed after ß2m had dissociated from previously assembled complexes. In fact, C67 appears to allow HLA-B27 HC to dimerize when they are refolded in vitro in the absence of ß2m, a phenomenon that was also observed in TAP-deficient T2 cells expressing HLA-B27 (17) and in ß2m-deficient HCT cells transfected with HLA-B*2705 (36). Similar to our findings, it has been reported that B27 forms intracellular homodimers in the HCT cell line but that these do not egress to the cell surface as seen by flow cytometry and western blot experiments. Lévy et al. (37) showed that the assembly of HLA-B27 HC with ß2m precedes binding of the peptide, although these authors do not exclude interactions between HC and peptide before assembly with ß2m. These authors were able to show an association between HLA-B27 HC and peptide, as seen in long exposure times of their streptavidin-agarose precipitations. All our experiments are in agreement with the findings of these investigators, demonstrating that HLA-B27 HC is able to bind peptides independently of ß2m association. Therefore, when certain mice class I alleles are expressed in ß2m-deficient cells, misfolding is associated with HC-dimers in the ER via an unpaired cysteine in the cytoplasmic tail. This has led to the suggestion that HC dimerization might be a mechanism by which dysfunctional molecules are removed (38). The impaired interaction with the class I assembly pathway may contribute to the formation of dimers.

Previous studies have demonstrated that the N-linked oligosaccharide is involved in class I association with CLR/tapasin/TAP (39, 40). Furthermore, the location of an oligosaccharide at N97 residue has not been demonstrated but this is not critical since natural polymorphism of HLA-B27 alleles at this position does not affect its ability to associate with the peptide loading complex. Our findings suggest that position 97 influences the association with tapasin–TAP probably throughout the interaction between this position with H114 and D116 and the effect that these positions have on the global structure of the protein. Amino acids 114 and 116 have been previously reported to influence the association of HLA class I HC with tapasin–TAP complex (21, 25). The positions 97 (N), 114 (H) and 116 (D) of B*2704 are reaching up from the floor of the cleft and are able to accommodate P7 at different bound orientations. The side chain of D97 located on the floor of the groove points downwards towards D116. Based on the available class I crystal structures (33, 41), some considerations on the peculiar behaviour of position 97, an integral part of the C/F pocket of the peptide-binding groove, may be discussed. The introduction of N97D into the C/F pocket is accompanied by the destruction of hydrogen bonds with H114 and D116 and displacement of the {alpha}2 complex altering the conformation of the ß-strand of the C/F pocket. The mutation of N97 to negatively charged D97 residue possibly causes perturbation in neighbouring residues that make contact as H114 and D116. This change prevents ß2m association and abrogates the exit from ER by misfolding and being retained with an unfolded conformation. This could be the reason why we observed an absence of surface expression. It remains in a peptide-receptive open form but the substitution modifies the C/F binding pocket and does not interfere with the acquisition of peptide binding. This modified pocket retains an affinity similar to that of B*2704 wild type.

When taken together, these studies argue that the residue at position 97 in HLA-B27 has a critical effect on the folding and structure but not on peptide specificity or affinity, which subsequently induces misfolding of the molecule and ER retention. HLA-B27 displays several unusual characteristics which may contribute to its strong association with human SpA. One such characteristic is the formation of HC-dimers (42). It has also been suggested that HLA-B27 misfolds within the ER and the accumulation of misfolded molecules within the environment of the ER results in a potentially pro-inflammatory intracellular stress response (43). Support for the idea that HLA-B27 misfolding can contribute to inflammatory arthritis has come from transgenic mice expressing HLA-B27 which are unable to assemble due to the lack of ß2m, and consequently develop a form of spontaneous inflammatory arthritis (19, 44). Homodimer formation is a symptom of HLA-B27 ‘misfolding’ within the ER, and these aberrant structures have been postulated to be involved in SpA disease (36).

Empty MHC class I molecules are strictly retained in the ER and degraded by quality control mechanisms. Although our data suggest that B*2704 N97D binds high-affinity peptides, the intracellular form of HLA-B*2704 N97D does not reach a mature phenotype, and it does not bind to ß2m and could be targeted to degradation by proteasome. Our mutant could be used as a model for peptide binding to open forms of HLA class I molecules and as a misfolding model that could be useful for studying the UPR pathway associated with HLA-B27 expression.


   Acknowledgements
 
We would like to thank J. A. Lopez de Castro, K. Granfors and J. R. Parra Cuadrado for their very generous gift of the HC10, ME1 and BBM.1 antibodies, respectively. We also wish to thank D. H. Wallace for his critical reading of the manuscript. This work was supported by Spanish grant SAF 2004/02669 from Ministerio de Educación y Ciencia.


   Abbreviations
 
AS   ankylosing spondylitis
CLR   calreticulin
CNX   calnexin
endo-H   endoglycosidase-H
ER   endoplasmic reticulum
HC   heavy chain
ß2m   ß2 microglobulin
OD   optical density
SpA   spondyloarthropathies
TAP   transporter associated with antigen processing
UPR   unfolded protein response

   Notes
 
Transmitting editor: G. Hammerling

Received 26 April 2004, accepted 1 November 2005.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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