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Misfolding of HLA-B27 as a Result of Its B
Pocket Suggests a Novel Mechanism for Its
Role in Susceptibility to
Spondyloarthropathies
John P. Mear, Kathy L. Schreiber, Christian Münz,
Xiaoming Zhu, Stefan Stevanovic, Hans-Georg Rammensee,
Sarah L. Rowland-Jones and Robert A. Colbert
J Immunol 1999; 163:6665-6670; ;
http://www.jimmunol.org/content/163/12/6665
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Copyright © 1999 by The American Association of
Immunologists All rights reserved.
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References
Misfolding of HLA-B27 as a Result of Its B Pocket Suggests a
Novel Mechanism for Its Role in Susceptibility to
Spondyloarthropathies
John P. Mear,* Kathy L. Schreiber,* Christian Münz,† Xiaoming Zhu,* Stefan Stevanović,†
Hans-Georg Rammensee,† Sarah L. Rowland-Jones,‡ and Robert A. Colbert2*
H
LA-B27 designates a group of MHC class I molecules or
subtypes strongly associated with susceptibility to the
spondyloarthropathies (1). Although evidence supports a
direct role in pathogenesis (2–5), the mechanism remains unknown. One hypothesis predicts that HLA-B27, as a result of its
particular peptide binding specificity, presents self peptides mimicking pathogen-derived epitopes that then become the target of
autoreactive CTL (6). However, no arthritogenic peptides have
been identified, and extensive efforts to isolate HLA-B27-restricted autoreactive CD81 T cells from affected individuals have,
with few exceptions (7), been unsuccessful. Of interest, some studies imply an important role for CD41 T cells in humans (8, 9) and
transgenic rodents (4, 10). Consequently, the arthritogenic peptide
hypothesis remains unproven, suggesting a need to consider alternative mechanisms (11).
Peptide binding differences between alleles result from extensive polymorphisms, particularly in amino acids within the peptide
binding groove. One region, the B pocket, is especially significant
for HLA-B27, as it is conserved among the subtypes unique to this
group of alleles (12) and is probably responsible for the large overlap in their peptide repertoires (13–18). Since many of the subtypes
sufficiently prevalent to be assessed are associated with disease
*William S. Rowe Division of Rheumatology, Children’s Hospital Medical Center,
Cincinnati, OH 45229; †Department of Immunology, Institute of Cell Biology, Eberhard-Karls-Universität Tubingen, Tubingen, Germany; and ‡Nuffield Department of
Clinical Medicine and Institute of Molecular Medicine, University of Oxford, Oxford,
United Kingdom
(19), the B pocket is thought to be critical for the arthritogenic
phenotype (18). Considering the lack of evidence for arthritogenic
peptides, we hypothesized that this pocket might confer other
unique characteristics to HLA-B27. To address this question we
compared peptide binding and assembly characteristics of HLAB27 with B27.A2B, an HLA-B27 molecule substituted with an
HLA-A2-like B pocket (20). Our results indicate that in addition to
its role in peptide selection, the B pocket also causes heavy chains
to misfold, which suggests a novel mechanism to explain the role
of HLA-B27 in disease susceptibility.
Materials and Methods
DNA and cell lines
B27.A2B was generated by site-directed mutagenesis of B*2705 (20) and
has the following amino acid substitutions: H9F (F for H at position 9),
T24A, E45M, I66K, C67V, and H70K. Minigenes encoding the HIV-1 gag
p24 epitope (sequence KRWIIMGLNK), or position 2 (P2)3-substituted
variants, were constructed using overlapping oligonucleotides and ligated
into pCEP4 (Invitrogen, Carlsbad, CA) (21, 22). HMy2.C1R (C1R) are
HLA-A negative and -B35 low (23), and 0.174 X CEMR (T2) (24) are TAP
deficient. C1R.B*2705, C1R.B27.A2B, T2.B*2705, and T2.B27.A2B were
produced as described previously (20). Cells were grown in R-10 (RPMI
1640 supplemented with 10% FCS, 2 mM L-glutamine, and 10 U/ml penicillin/streptomycin) with geneticin (Life Technologies, Gaithersburg, MD)
at 0.5 mg of activity/ml. T5-1 is a B cell line expressing HLA-A1, -A2, B8,
-B27, and -Cw4 (25). C1R.B*2705 and C1R.B27.A2B were transfected
with minigenes, selected with hygromycin (Calbiochem, La Jolla, CA; 1.5
mg/ml) (21, 22), and maintained in R-10 with hygromycin (0.5 mg/ml) and
G418 (0.4 mg activity/ml).
Received for publication July 8, 1999. Accepted for publication October 7, 1999.
Peptides
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
The HPLC-purified peptides were obtained from the University of North
Carolina MicroProtein Chemistry Facility (Chapel Hill, NC). Purity and
expected molecular mass were confirmed by mass spectroscopy.
1
This work was supported in part by the Children’s Hospital Research Foundation of
Cincinnati, the Schmidlapp Foundation, and National Institutes of Health Grant
P60AR44059-01. R.A.C. received support from a Pfizer Scholar Award.
2
Address correspondence and reprint requests to Dr. Robert A. Colbert, William S.
Rowe Division of Rheumatology, Children’s Hospital Medical Center, 3333 Burnet
Avenue, Cincinnati, OH 45229-3039. E-mail address: [email protected].
chmcc.org
Copyright © 1999 by The American Association of Immunologists
3
Abbreviations used in this paper: P2, second peptidic amino acid; T2, 0.174 X
CEMR; b2m, b2-microglobulin; C1R, HMy2.C1R; EC50, effective concentration
yielding 50% maximum binding; ER, endoplasmic reticulum; LLnL, N-acetyl-Lleucyl-L-leucyl-L-norleucinal; TS, 10 mM Tris (pH 7.4) and 150 mM NaCl; IEF,
isoelectric focusing.
0022-1767/99/$02.00
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
The MHC class I protein HLA-B27 is strongly associated with susceptibility to spondyloarthropathies and can cause arthritis when
expressed in rats and mice, implying a direct role in disease pathogenesis. A prominent hypothesis to explain this role suggests that
the unique peptide binding specificity of HLA-B27 confers an ability to present arthritogenic peptides. The B pocket, a region of
the peptide binding groove that is an important determinant of allele-specific peptide binding, is thought to be critical for
arthritogenicity. However, this hypothesis remains unproven. We show that in addition to its role in peptide selection, the B pocket
causes a portion of the pool of assembling HLA-B27 heavy chains in the endoplasmic reticulum to misfold, resulting in their
degradation in the cytosol. The misfolding phenotype is corrected by replacing the HLA-B27 B pocket with one from HLA-A2.
Our results suggest an alternative to the arthritogenic peptide hypothesis. Misfolding and its consequences, rather than allelespecific peptide presentation, may underlie the strong link between the HLA-B27 B pocket and susceptibility to
spondyloarthropathies. The Journal of Immunology, 1999, 163: 6665– 6670.
6666
The CTL and cytotoxicity assays
HLA-B27 MISFOLDING
protease inhibitors and 1% Triton X-100 for 30 min on ice before
immunoprecipitation.
The CTL lines recognizing the influenza A nucleoprotein peptide
(sequence SRYWAIRTR; HF CTL), and the HIV-1 gag epitope (sequence
KRWIIMGLNK; 868 CTL) bound to B*2705 have been described previously (21, 22). Standard 4-h 51Cr release assays were performed using C1R
transfectants pulsed with synthetic peptides or expressing minigenes as
targets. Specific lysis was calculated using the formula (E 2 M/T 2 M) 3
100, where E is experimental release, M is release in the presence of medium alone, and T is total release in the presence of 5% Triton X-100
(Sigma, St. Louis, MO).
Immunoprecipitations were performed after preclearing samples with
washed formalin-fixed Staphylococcus aureus (Sigma), using purified
mAbs at a final Ab concentration of 30 mg/ml for 1 h at 4°C. Protein
A-Sepharose (Sigma) was then added (100 ml of 50 mg/ml suspension in
lysis buffer) for 1 h at 4°C. Protein A-Sepharose pellets were washed and
stored at 220°C until electrophoresis.
Cell surface binding assay
Gel electrophoresis and phosphorimaging
Peptide binding was measured with a stabilization assay using TAP-deficient T2 cells as described previously (22, 26). Cell surface B*2705 or
B27.A2B complexes were measured by staining with ME.1, which recognizes HLA-B7, B27, and Bw22 (27) and affinity-purified FITC-F(ab9)2
goat anti-mouse IgG (Fc-specific; Organon Technika, West Chester, PA),
followed by FACS analysis (FACScan, Becton Dickinson, Palo Alto, CA).
The ability of each peptide to stabilize cell surface class I molecules is used
as an indirect measure of peptide binding, and was determined as follows:
% maximum change in fluorescence 5 [(MFIsample peptide 2 MFIdiluent)/
(MFISXYWAIRTR 2 MFIdiluent)] 3 100.
SRYWAIRTR (for B*2705) or SQYWAIRTR (for B27.A2B; 100 mM)
was used in each assay to establish maximum binding. The concentration
of peptide required to achieve 50% maximum change in fluorescence is
referred to as the EC50 and is used for all comparisons.
Isoelectric focusing (IEF) and SDS-10.5% PAGE were performed according to established methods (29). For phosphorimage analysis, dried gels
were exposed to plates for 48 h, then 35S-labeled proteins were visualized
and quantitated with ImageQuant software (Molecular Dynamics,
Sunnyvale, CA).
Cells (107/time point) were preincubated for 1 h at 37°C in medium lacking
Met and Cys, then pulse-labeled with 2 mCi of [35S]Met/Cys (Easytag,
New England Nuclear, Boston, MA) and chased with a 10-fold excess of
medium supplemented with 2 mM Met/Cys. Protein synthesis was stopped
by the addition of 5 vol of ice-cold PBS and cells were harvested by
centrifugation. Cells were washed, then lysed in 500 ml of lysis buffer (20
mM Tris (pH 7.8), 100 mM NaCl, 10 mM EDTA, and 1% Triton X-100)
supplemented with protease inhibitors (5 mM iodoacetamide, 0.5 mM
PMSF, and 0.1 mM Na-p-tosyl-L-lysine chloromethyl ketone; Sigma). After 20 min on ice, nuclei were removed with a 10,000 3 g spin for 5 min
at 4°C, and supernatants were used for immunoprecipitations.
Cell fractionation
Membrane and soluble fractions were prepared as described by Hughes et
al. (28). Briefly, pelleted cells (1.5 3 107/condition) were washed and
lysed by two cycles of freezing and thawing in dry ice/ethanol, and then
lysates were suspended in 1 ml of TS (10 mM Tris (pH 7.4) and 150 mM
NaCl) supplemented with protease inhibitors. Nuclei were removed by
centrifugation, and postnuclear supernatants were further centrifuged at
100,000 3 g for 1 h at 4°C to produce a pellet (membrane fraction) and
supernatant (soluble fraction). One-tenth volume of 10% Triton X-100 in
TS was added to supernatants, and pellets were dissolved in TS containing
Immunoblotting
Proteins were transferred onto a polyvinylidene difluoride membrane
(Westran, Schleicher & Schuell, Keene, NH) using methods adapted for
IEF gels (29). Following transfer, the polyvinylidene difluoride membrane
was blocked for 1 h with 1% blocking powder in PBS (Schleicher &
Schuell), then HC10 (1 mg/ml) was added for an additional 30 min at room
temperature. After washing, blots were incubated with a 1/1000 dilution of
alkaline phosphatase-conjugated goat anti-mouse IgG heavy and light
chain (Southern Biotechnology Associates, Birmingham, AL) for 30 min,
then protein bands were visualized using 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium for color development.
Results
Peptide binding specificity and loading efficiency
Previously we showed that B27.A2B, which is identical with
HLA-B*2705 except for six amino acid substitutions in the B
pocket (see Materials and Methods), can be recognized by HLAB27-restricted flu-specific CTL, provided the flu peptide has Leu
instead of Arg at P2 (20). To determine the characteristics of peptides naturally presented by B27.A2B, it was immunopurified from
C1R transfectants, and peptides were eluted and sequenced. Gln,
Met, and Leu were found to predominate at P2 (data not shown)
instead of Arg seen in the majority of peptides presented by various HLA-B27 subtypes (13–17). This P2 specificity for B27.A2B
is similar to HLA-A2 (A*0201 subtype), but not identical, since
Gln has not been reported (30). HLA-A*0205, a subtype with Tyr
instead of Phe at position 9 in its B pocket, has a similar Leu/Met/
Gln P2 preference (31), suggesting that a relatively conservative
change in this pocket can produce an environment suitable for Gln.
FIGURE 1. Specificity of exogenous and intracellular peptide loading by HLA-B*2705 and B27.A2B. A, C1R.B*2705 and B, C1R.B27.A2B were
incubated with peptides, then tested for recognition by HF CTL at an E:T cell ratio of 5. The natural epitope SRYWAIRTR (SRY) or variants containing
L (SLY) or M (SMY) at P2 were used. C, The same target cells expressing minigenes encoding the HIV gag peptide KRWIIMGLNK (KRW) or variants
with Q (KQW), A (KAW), or K (KKW) at P2 were tested for recognition by 868 CTL at an E:T cell ratio of 15.
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Metabolic labeling
Immunoprecipitations
The Journal of Immunology
6667
pared, and in each case binding to HLA-B*2705 was less efficient
(data not shown). It is important to note that EC50 measured by this
assay does not represent an affinity constant, and as we have shown
previously, this measure better represents peptide loading efficiency than complex stability (26). Thus, these results suggest
HLA-B*2705 is relatively inefficient at loading peptide as a result
of its B pocket. This may reflect poor formation of peptide-receptive HLA-B*2705:b2m complexes, and/or a greater dependence on
peptide to undergo the conformational change required for Ab recognition measured as binding in the cell surface stabilization assay.
B pocket affects heavy chain folding
FIGURE 2. Peptide loading efficiency of HLA-B*2705 and B27.A2B.
Peptide binding was compared using TAP-deficient T2 cells expressing
B*2705 or B27.A2B as described in Materials and Methods. Peptides
are SM/RYWAIRTR (SMY, SRY), NQ/RHGIILKY (NQH, NRH),
GQ/RIDKPILK (GQI, GRI), and FL/RANVSTVL (FLA, FRA). NQHGI
ILKY and FLANVSTVL are two natural ligands of B27.A2B determined
from peptide elutions.
FIGURE 3. Inefficient HLA-B*2705 folding. Cells
were pulsed for 5 min with [35S]Met/Cys, then chased
for up to 2 h in the presence of excess nonradioactive
Met/Cys. At each time point sequential immunoprecipitations were performed with W6/32 followed by
HC10. Phosphorimages of IEF gel regions containing
immunoprecipitated B*2705 (A) and B27.A2B (B) are
shown. Nonsialylated (form 0) and sialylated (forms 1
and 2) heavy chains are indicated. C and D, Quantitative results expressed as the amount of [35S]Met/Cys
in W6/32 or HC10-reactive heavy chain (sum of 0, 1,
and 2 forms for each Ab) as a percentage of the total
(W6/32 1 HC10) are shown. Resolution of bands into
doublets represents a focusing artifact.
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Recognition of B27.A2B presenting two different HLA-B27-restricted epitopes with Leu, Met, or Gln at P2 is shown in Fig. 1. In
addition to exogenous peptide loading (Fig. 1, A and B), B27.A2B
loads an endogenously synthesized HIV gag peptide with Gln, but
not Ala, Arg, or Lys at P2 (Fig. 1C). This indicates that the antigenic surface of B27.A2B is similar to HLA-B*2705 and suggests
that the B pocket substitution does not have a major effect on
amino acid preference at peptide positions outside the B pocket.
To compare peptide binding characteristics of HLA-B*2705
with two different B pockets, known ligands containing either an
HLA-B*2705 or B27.A2B-specific anchor residue at P2 were
tested using a cell surface class I stabilization assay (22). Peptides
with Gln, Met, or Leu at P2 did not bind appreciably to HLAB*2705, and Arg-P2 peptides only showed limited binding to
B27.A2B at the highest concentrations (50 –100 mM; data not
shown), indicating that binding was specific. The most striking
finding was that in each case equivalent binding to HLA-B*2705
required much higher peptide concentrations than B27.A2B (Fig.
2), with EC50 ratios ranging from approximately 30 for NRH/NQH
to 60 for GRI/GQI. Several other cognate peptides have been com-
To assess formation of peptide-receptive complexes in vivo, pulsechase experiments were performed. C1R transfectants were labeled for 5 min with [35S]Met/Cys, and chased in the presence of
excess nonradioactive Met/Cys. Class I molecules were immunoprecipitated from cell lysates first using W6/32, which recognizes
folded heavy chain-b2m complexes (32), then HC10, which recognizes unfolded free (non-b2m-associated) heavy chains (33).
Immunoprecipitated heavy chains were then resolved on IEF gels
(Fig. 3, A and B). The 0 form contains unsialylated N-linked glucosyl residues, indicating they remain in the ER, while forms 1 and
2 contain increasing amounts of sialic acid, reflecting ER to Golgi
transport (33) and thus focus to more acidic positions. There is a
striking difference in the rate of conversion of unfolded HC10reactive heavy chain into folded W6/32-reactive material (Fig. 3, C
and D), with HLA-B*2705 requiring ;30 min for 50% conversion, while for B27.A2B this occurs within the first 5 min of the
pulse. Inefficient folding is not due to overexpression, since only
slightly higher levels of HLA-B27 mRNA (per microgram of 18S
RNA) were found on Northern blots (data not shown), and if anything, there appears to be greater synthesis of B27.A2B on a per
cell basis. We also considered the possibility that HC10 might not
react as well with B27.A2B, and thus free heavy chains might
remain unprecipitated. To test this, a third immunoprecipitation
was performed (after W6/32 and HC10) using 5H7, which recognizes a determinant in the a3 domain of the heavy chain (34) and
is not dependent on the conformation of the a1 and a2 domains.
Only trace amounts of B27.A2B could be recovered (data not
shown), confirming that the majority of this molecule folds and
becomes recognizable by W6/32 shortly after synthesis.
6668
HLA-B27 MISFOLDING
FIGURE 6. Newly synthesized heavy chains misfold and accumulate in
the soluble fraction. T5-1 cells were incubated in the absence (2) or the
presence (1) of LLnL. After 1 h, they were pulsed for 15 min with
[35S]Met/Cys and chased for up to 4 h with excess nonradioactive Met/Cys.
At each time point aliquots were harvested, and soluble fractions were
prepared. Heavy chains were immunoprecipitated with HC10 and separated by 10.5% SDS-polyacrylamide gels, then visualized by
phosphorimaging.
Misfolding and cytosolic degradation of HLA-B27
Typically, heavy chains fold and associate with b2m shortly after
synthesis and then load peptides that have been transported into the
ER from the cytosol before being released to traffic to the cell
surface (35). However, when synthesized in mutant cell lines deficient in b2m or peptide transporters (TAP) or even when TAP
function is inhibited, heavy chains can misfold and are dislocated
into the cytosol and degraded by proteasomes (28). Due to the
rapidity of this process, detection of cytosolic heavy chains requires proteasome inhibition. In light of the inefficient folding of
HLA-B*2705, we were interested in whether some of the heavy
chains might misfold and enter the dislocation pathway. To test
this, cells were incubated with the proteasome inhibitor N-acetylL-leucyl-L-leucyl-L-norleucinal (LLnL) and lysed in the absence of
detergents by freezing and thawing, and soluble (cytosolic) and
membrane fractions were prepared (28). Heavy chains were then
immunoprecipitated from these fractions using HC10, separated on
IEF gels, and visualized by immunoblotting. HLA-B*2705 heavy
chains can be found accumulating in the cytosolic fraction in the
presence of LLnL, suggesting that they are normally rapidly degraded (Fig. 4). This is even more readily apparent in a B cell line
(T5-1) expressing HLA-B*2705 (as part of its full complement of
class I molecules) than in C1R transfectants. In contrast, no accu-
FIGURE 5. Molecular weights of soluble heavy chains. Cytosolic (S)
and membrane (M) fractions were prepared from cells treated as described
in Fig. 4. Following immunoprecipitation with HC10, heavy chains were
separated on 10.5% SDS-polyacrylamide gels and visualized by immunoblotting. Prestained m.w. standards (not shown) were used to determine the
apparent m.w. of heavy chain bands.
mulation of B27.A2B can be detected. The predominant form of
cytosolic heavy chain is more acidic than the ER (0) form (Fig. 4),
consistent with conversion of Asn to Asp during N-glycanase-mediated carbohydrate removal during heavy chain dislocation (36).
Removal of carbohydrate is also expected to reduce the m.w. of the
heavy chain. To confirm this, the experiment was repeated, and the
size of cytosolic heavy chains was found to be ;3 kDa less (Fig.
5), consistent with loss of carbohydrate seen following in vitro
digestion with N-glycanase (28) or endoglycosidase H (data not
shown). Since LLnL may inhibit other proteases (37), including
those that may affect peptide trimming in the ER (38), we have
performed the same experiment with lactacystin, a more specific
proteasome inhibitor (39) and obtained similar results (data not
shown). Thus, it is unlikely that HLA-B*2705 heavy chain misfolding is due to a nonspecific action of LLnL.
To determine whether newly synthesized molecules are misfolding, T5-1 cells were pulse-chased with [35S]Met/Cys in the absence or the presence of LLnL. A soluble 40-kDa heavy chain
band accumulates and is most notable at 1–2 h of chase (Fig. 6). A
portion of the samples run on an IEF gel confirmed the identity of
the heavy chain band as HLA-B*2705 (data not shown). These
results indicate that HLA-B*2705 heavy chains are dislocated into
the cytosol within 1–2 h of synthesis, as evidenced by their accumulation when proteasomes are inhibited.
Discussion
Formation of stable heavy chain:b2m:peptide complexes in the ER
is necessary before trafficking to the cell surface (35). Following
synthesis and glycosylation, free heavy chains are initially stabilized by chaperones such as calnexin (40, 41) until a conformation
suitable to bind b2m and peptide is achieved. In the absence of
b2m, heavy chains can be retained by calnexin (42), but ultimately
misfold and are degraded (28). They have a similar fate in the
absence of peptide, either in TAP-deficient cells or when TAP is
inhibited (28). Our studies demonstrate that a1-domain amino acids in HLA-B27 that ultimately form the B pocket have a dramatic
effect on folding efficiency and cause misfolding, even in the presence of an intact Ag presentation pathway. Although allelic differences in rates of folding and assembly have been noted previously (33, 43), and in one case attributed to a2-domain
polymorphisms (43), misfolding in the presence of a normal assembly pathway has not been reported. Furthermore, we have not
detected misfolding of HLA-B7, -B53, or even -B8, despite its
relatively slow folding (data not shown). Thus, although we cannot
rule out misfolding of other class I molecules, to date this phenotype appears to be limited to HLA-B27 and is related to its unique
B pocket.
The B pocket could affect the formation of heavy chain:b2m:
peptide complexes by directly influencing heavy chain folding or
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FIGURE 4. Accumulation of soluble HLA-B27 heavy chains. C1R.
B*2705, T5-1, and C1R.B27.A2B were incubated in the absence (2) or the
presence (1) of LLnL (250 mM) for 3 h, lysed by freezing and thawing,
and separated into cytosolic (S) and membrane (M) fractions. Heavy chains
were immunoprecipitated with HC10, separated on IEF gels, then visualized by immunoblotting.
The Journal of Immunology
tion of misfolded forms that, in turn may be responsible for the
development of high frequency spontaneous arthritis.
Another consequence of misfolding with potential relevance to
autoimmune disease is the inadvertent entry of HLA-B27 heavy
chains into the cytosol. This can result in cytosolic degradation by
proteasomes, which may lead to enhanced presentation of heavy
chain-derived peptides by class I. Indeed, a peptide matching part
of the a2 domain of HLA-B27 has been eluted from HLA-B27
expressed in C1R (15), and it is likely that different HLA-B27
breakdown products could be presented by alleles with other specificity. If the degree of misfolding varies among different cell types
or is affected by physiological changes such as infection with intracellular bacteria known to be triggers of spondyloarthropathies,
then tolerance to HLA-B27-derived self-peptides may be lost.
Since attempts to find autoreactive CTL have largely focused on
recognition of HLA-B27 (7), other class I molecules presenting
bits of HLA-B27 could have been missed. Alternatively, misfolded
cytosolic heavy chains could interact with Hsc73 and enter the
endosomal-lysosomal pathway (55) in a manner different from
their usual recycling from the cell surface. It has been argued that
increased heavy chain turnover and presentation of HLA-B27
breakdown products by class II may be important in disease (11),
in part to account for the apparent role of CD41 T cells in pathogenesis (4, 10). In support of this, one study has shown that T cells
from patients have greater reactivity against HLA-B27-derived
peptides than T cells from healthy controls (9). However, development of arthritis in HLA-B27 transgenic, b2m-deficient mice
also lacking class II molecules argues against this (56).
In summary, our studies were undertaken to determine whether
B pocket amino acids confer more than just peptide binding specificity to HLA-B27, in particular characteristics that might ultimately underlie the ability of this allele to cause disease. As we
have demonstrated, this pocket affects folding efficiency and confers a misfolding phenotype on HLA-B27. Although the mechanism of HLA-B27-associated disease remains to be determined,
these observations emphasize the need to consider misfolding and
its sequelae in pathogenesis, rather than a unique ability to select
and present arthritogenic peptides.
Acknowledgments
We thank J. A. Frelinger, H. Ploegh, P. Cresswell, and E. Mellins for their
gifts of reagents or cells, and D. N. Glass for critically reading the
manuscript.
References
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by altering its affinity for b2m or peptide. HLA-A2 assembles normally in calnexin-deficient cells (44), and when calnexin binding
is blocked with glycosylation inhibitors, HLA-B27 is less stable
than HLA-A2 (33). Although this could indicate that alleles such
as HLA-A2 are inherently better at folding and do not require
calnexin, the involvement of other chaperones has not been ruled
out. Since b2m and peptide binding to heavy chains can facilitate
release from calnexin (40, 45), increased affinity and/or availability
of these components could enhance folding efficiency. In this regard, since the B pocket in HLA-B27 is highly selective for Arg,
while in HLA-A2 it accepts Met and Leu (and Gln for B27.A2B),
HLA-A2 and B27.A2B may be more promiscuous in peptide interactions. Furthermore, our peptide binding data suggest that even
for individual peptides, HLA-B27 stabilization requires higher
concentrations. Since HLA-B27 heavy chains tend to misfold even
in the presence of TAP, we considered the possibility that this
result might be due to a large population of misfolded HLA-B27
heavy chains on the surface of T2 cells. However, while HC10reactive material is abundant, there is actually less on T2.B*2705
than on T2.B27.A2B (data not shown), and therefore, this does not
explain the peptide binding differences we observe. Thus, while
the molecular mechanism by which the B pocket affects folding
efficiency remains to be determined, it seems likely that peptide
interaction is important.
The strong association between several HLA-B27 subtypes containing the same B pocket and disease susceptibility (19) and the
lack of evidence implicating arthritogenic peptides suggest that
alternative hypotheses incorporating misfolding should be considered. Typically, misfolded ER proteins are degraded as part of a
quality control process (46). When a large percentage of the pool
misfolds, protein deficiency diseases may occur (47). However,
misfolded proteins can also accumulate in the ER and trigger a
stress response (48) or escape the quality control process and traffic
to other cellular compartments (49) or to the cell surface (50).
HLA-B27 expression in mice has been reported to cause arthritis,
but only in the absence of mouse b2m, a condition that eliminates
cell surface HLA-B27 complexes but enhances expression of unfolded (HC10-reactive), or perhaps misfolded, free heavy chain
(4). Although implicated in pathogenesis (4, 51), the precise structure or role of free heavy chains is not known. Recently, it has been
shown that HLA-B27 heavy chains can form homodimers when
synthesized in cells deficient in TAP (52). Whether this also occurs
in b2m-deficient mice or perhaps at low frequency in the presence
of peptide is not known, but, interestingly, it appears to be dependent on Cys67, a B pocket amino acid (52). In HLA-B27 transgenic
rats, b2m deficiency is not required for disease; however, there is
a dependence on high copy number and high levels of HLA-B27
expression (3, 53). Although it has been difficult to reconcile results from rats and mice mechanistically, both could be explained
by a requirement for misfolded HLA-B27 molecules. It should be
noted that coexpression of an HLA-B27-binding peptide in the ER
of transgenic rats reduces the incidence of arthritis (54). These
findings were interpreted as support for a class I-restricted mechanism, since the spectrum of peptides presented by HLA-B27 was
affected. However, an alternative explanation is that overexpression of an HLA-B27-binding peptide may reduce misfolding. In
summary, a plausible hypothesis is that as a result of its unique B
pocket, HLA-B27 has a tendency to misfold even when all components of the Ag presentation pathway are present, as would be
expected in most individuals with HLA-B27. This may result in
the formation of neoantigens such as the homodimers found in
TAP-deficient cells (52). In the animal models misfolding is exacerbated: in mice due to the absence of endogenous b2m, and in
rats by overexpression. This would lead to the enhanced produc-
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HLA-B27 MISFOLDING