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*
Department of Cellular Biochemistry, The Netherlands Cancer Institute, Amsterdam, The Netherlands; and
Department of Immunohematology and Blood Bank, University Hospital Leiden, Leiden, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Correct assembly and peptide loading of MHC class I molecules in the ER are required for proper cell surface expression. This involves the interaction of class I molecules with other proteins such as calnexin (8), tapasin (9), and, for a subset of class I alleles, calreticulin (9), and TAP (9, 10, 11, 12, 13). Upon peptide binding, the MHC class I molecules dissociate from these molecules (10, 11), which allows their transport from the ER to the cell surface.
The biochemical basis for the reduced surface expression of HLA-C molecules is still under debate. McCutcheon et al. (3) have suggested that the low HLA-C surface expression is caused by the instability of HLA-C mRNA. It also has been reported that HLA-C alleles were poorly expressed at the cell surface, although expressed intracellularly in amounts similar to HLA-A and HLA-B heavy chains (14). These studies suggested that HLA-C molecules were assembled inefficiently and that the heavy chains were degraded in the ER. Finally, HLA-C locus products often prefer peptides with a Pro at position 2 or 3 as an anchor residue (15, 16). However, peptides with a Pro at these positions are poor substrates for TAP (17, 18), which may contribute to the reduced assembly and surface expression of HLA-C locus products.
In this study, we have investigated HLA-C alleles, and the HLA-C4 molecules in particular. HLA-C4 is expressed intracellularly in amounts comparable with HLA-A and HLA-B alleles, but is poorly expressed at the cell surface, as previously reported (14). In contrast to most HLA-A and HLA-B alleles, the majority of the HLA-C4 molecules is not transported, but is retained in the ER in complex with TAP. The pool of TAP-associated HLA-C4 molecules does not appear to be an intermediate in the assembly of HLA-C4/peptide complexes, but is ultimately degraded in the ER. HLA-C4 molecules dissociate from TAP equally efficiently as HLA-A2 when supplied with allele-specific peptides. However, HLA-C4, as well as HLA-C2, require about a 10-fold higher concentration of a completely degenerated 9-mer peptide for dissociation from TAP as compared with the HLA-A and HLA-B alleles. This suggests that these HLA-C alleles are more selective in peptide binding than the HLA-A and HLA-B molecules. As a result of this limited peptide binding, the majority of the HLA-C molecules remains associated with TAP in the ER and never reaches the cell surface. The restricted peptide binding of certain HLA-C alleles provides an additional explanation to their low cell surface expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human B lymphoblastoid cell line LCL721 (HLA-A1, HLA-A2, HLA-B8, HLA-B51, and HLA-C1) (19) and the following human EBV-transformed B cell lines were used: OSH cells (HLA-A2, HLA-A11, HLA-B27, HLA-B35, HLA-C2, and HLA-C4) and Fivo cells (HLA-A2, HLA-A3, HLA-B35, HLA-C4, and HLA-C7) were obtained from Dr. F. Claas, Bloodbank (Leiden, The Netherlands), and BM28.7 cells (HLA-A1, HLA-B35, and HLA-C4) were obtained from Dr. A. Ziegler (Freie Universität, Berlin, Germany) (20). Cells were cultured in RPMI 1640 supplemented with 10% FCS.
Antibodies
The following Abs were used: mAb W6/32 (recognizing assembled
HLA-A, HLA-B, and HLA-C molecules) (21), the rabbit polyclonal
antiserum 
HC (recognizing free HLA-A, HLA-B, and HLA-C heavy chains
and free ß2m) (14), and the polyclonal rabbit
anti-human TAP2 serum (TAP2), which recognizes the ATP-binding
domain of TAP2 as well as TAP1/TAP2 complexes (12). The rabbit
anti-TAP2 serum was made by constructing a glutathion S-transferase
fusion protein containing the ATP-binding domain of TAP2 (amino acids
434–703). Templates for PCR were a generous gift from Dr. J. Trowsdale
(Imperial Cancer Research Fund, London, U.K.), and the fragment was
cloned in the pGEX vector. The glutathion S-transferase fusion protein
was overexpressed in Escherichia coli, isolated as inclusion
bodies by centrifugation, and injected into rabbits.
Peptides
The HLA-A*0201-binding HPV16 E7-derived peptide TLGIVCPI
(peptA2) (22) and the HLA-C4-binding peptide QFDDAVYKL (peptC4) were
synthesized with a free carboxyl terminus on a multiple peptide
synthesizer (Abimed AMS 422). The design of peptC4 was based on
published peptide-binding motifs of HLA-C4 (15, 16). The degenerated
peptide peptDEG was a 9-mer peptide with a free carboxyl terminus and
synthesized such that all 20 natural amino acids could be incorporated
at all nine positions, theoretically resulting in a peptide mixture
containing all possible 9-mer peptides. The amino acid composition of
the degenerated peptide mixture that dissolved in PBS was determined by
amino acid analysis using the Pico Tag method (23), and the result is
shown in Table I
. The 20 amino acids are
present in the peptide mixture in roughly equal amounts. All peptides
were dissolved in PBS and stored at -20°C.
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One-dimensional isoelectric focusing (1D-IEF) was performed as described (24). Gels were fluorographed using DMSO/2,5-diphenyloxazol (PPO; Merck Co., Rahway, NJ) and exposed to Kodak XAR-5 films. Quantitation was performed using a FUJIX BAS 2000 phosphor imager with TINA software.
Biochemical experiments
Analysis of cell surface MHC class I molecules. A quantity amounting to 15 x 106 LCL721 or OSH cells was surface labeled using lactoperoxidase-catalyzed iodination with 500 µCi of Na125I. Cells were washed four times with cold PBS and lysed in 1% Nonidet P-40-containing lysis mixture (1% (w/v) Nonidet P-40, 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, and 150 mM NaCl). Nuclei were removed (5 min, 12,000 x g) and the lysate was precleared three times with normal rabbit serum (nrs) at 4°C. The lysate was split into two portions and immunoprecipitated with W6/32. After recovering the immunoprecipitates with protein A-Sepharose and washing the beads in 1% Nonidet P-40-containing lysis mixture, one-half was digested with 0.2 U of neuraminidase type V (N.A.) (Sigma Chemical Co., St. Louis, MO) in 25 µl of 1% Nonidet P-40 lysis mixture for 1.5 h at 37°C, whereas the other half was left untreated. All immunoprecipitates were subsequently analyzed by 1D-IEF.
Analysis of intracellular MHC class I molecules.
A quantity amounting to 15 x 106 LCL721 or OSH
cells was starved for 30 min in 1 ml of methionine/cysteine-free RPMI
1640 medium supplemented with 10% FCS. The cells were pelleted,
resuspended in 200 µl fresh methionine/cysteine-free RPMI 1640 medium
supplemented with 10% FCS, and metabolically labeled with 125
µCi [35S]methionine/cysteine for 15 min at 37°C.
Cells were lysed in a digitonin-containing lysis mixture (1% (w/v)
digitonin, 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, and 150 mM
NaCl), the nuclei were removed, and the lysate was precleared three
times with nrs at 4°C. The precleared lysate was split into three
equal portions, followed by isolation of MHC class I molecules (W6/32),
TAP-associated MHC class I molecules (TAP2), or free MHC class I
heavy chains (HC). The immunoprecipitates were recovered with
protein A-Sepharose and subsequently analyzed by 1D-IEF.
Pulse-chase analysis of free and TAP-associated MHC class I
molecules to follow the fate of the intracellular HLA-C molecules.
A quantity amounting to 90 x 106 OSH cells was
starved for 1 h in methionine/cysteine-free RPMI 1640 medium
supplemented with 10% FCS. The cells were pelleted, resuspended in 500
µl fresh methionine/cysteine-free RPMI 1640 medium supplemented with
10% FCS, and metabolically labeled with 500 µCi
[35S]methionine/cysteine for 15 min at 37°C. Cells were
chased for different time periods (indicated in figures) until 10
h in RPMI 1640 medium supplemented with 1 mM cold methionine/cysteine
and 10% FCS. Equal amounts of cells were pelleted and lysed in 1 ml
1% digitonin-containing lysis mixture, and the lysates were precleared
with nrs, as described above. Each lysate was divided into two portions
and immunoprecipitated with either W6/32 or TAP2.
Cycloheximide treatment. Where indicated, labeled cells were treated with the protein synthesis inhibitor cycloheximide (CHX) (200 µM; Sigma Chemical Co.), which was added after the pulse and present throughout the chase period. All immunoprecipitates were analyzed by 1D-IEF.
Analysis of peptide binding to TAP-associated MHC class I
molecules.
This analysis was performed essentially as described (10). Briefly, 30
to 50 x 106 LCL721 or EBV-transformed B cells
were labeled metabolically with 250 µCi
[35S]methionine/cysteine for 15 min at 37°C. Cells were
lysed in 1% digitonin-containing lysis mixture, and lysate was
precleared with nrs, as described above. Precleared lysate was split
into equal portions and TAP-associated MHC class I molecules were
co-isolated with TAP2. After recovering the immunoprecipitates with
protein A-Sepharose and washing the beads in digitonin-containing lysis
mixture, the precipitates were incubated in 1% digitonin-containing
lysis mixture with different concentrations of the peptides peptA2,
peptC4, or peptDEG (total volume 100 µl), as indicated in the
figures. After incubation overnight at 4°C, to allow peptide binding
to TAP-associated MHC class I molecules, the precipitates were washed
in digitonin-containing buffer, followed by incubation in 500 µl 1%
Triton X-100 buffer (1% (w/v) Triton X-100, 50 mM Tris-HCl (pH 7.5), 5
mM MgCl2, and 150 mM NaCl) for 2 h in a 37°C water
bath to release the MHC class I molecules from TAP. The supernatants
were then precleared with nrs before specific immunoprecipitation of
the pool of released MHC class I molecules with W6/32. These samples
were analyzed by 1D-IEF.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We studied HLA-C alleles expressed in LCL721 cells or the
EBV-transformed B cell line OSH to determine their intracellular as
well as their surface expression. Cells were labeled metabolically for
15 min and lysed under mild detergent conditions using 1% digitonin,
followed by immunoprecipitation with mAb against MHC class I heavy
chain/ß2m complexes (W6/32), antiserum against TAP2
(TAP2), or antiserum recognizing free class I heavy chains (HC)
(Fig. 1, A and
C). These Abs precipitate or coprecipitate different
nonoverlapping pools of MHC class I complexes (12). In accordance with
previous studies (14), only trace amounts of HLA-C4 were recovered with
the class I mAb W6/32, whereas the intracellular protein level of
HLA-C4 heavy chains was comparable with that of HLA-A and HLA-B
heavy chains (Fig. 1A, lane HC). However, a relatively
large pool of HLA-C4 molecules could be found in association with TAP
(Fig. 1A, lane TAP2), an interaction that requires the
presence of ß2m (10, 11). Similar results were obtained
in other HLA-C4-expressing cell lines (12) (data not shown) and
indicate that HLA-C4 heavy chain/ß2m complexes associate
very efficiently with TAP. Both the HLA-C2 molecules, expressed
intracellularly at lower levels than HLA-C4 molecules (Fig. 1A), and the HLA-C1 alleles (Fig. 1C) were also efficiently associating with TAP,
whereas they are poorly recovered by W6/32. It should be noted that
the TAP-associated class I molecules are not recognized by W6/32 (12),
probably because the epitope is shielded from the Ab in these
complexes.
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, B and
D). One-half of the immunoprecipitate was treated
with N.A., which removes sialic acids from the surface class I
molecules, resulting in a shift to a more basic (higher) position in
the 1D-IEF gel, thereby facilitating the identification of the
different (nonsialylated) MHC class I alleles. The detection of the
desialylated form of surface HLA-C4 molecules was hampered by the
presence of a background band migrating at the same position (Fig. 1B, lane +). This band, as well as a band migrating slightly
below the HLA-B35 allele (Fig. 1B), was also present
in precipitates from LCL721 cells (Fig. 1D), and may
represent oxidized forms of ß2m. However, the relative
HLA-C4 surface expression can be deduced from the samples not treated
with N.A. and showed that it was strongly reduced compared with the
HLA-A and HLA-B alleles (Fig. 1B, lane -). Surface
expression of HLA-C2 and HLA-C1 molecules was below detection level
(Fig. 1, B and D, respectively). These results confirm previous observations that HLA-C molecules are relatively poorly expressed at the cell surface (2, 3). Especially the low surface expression of the HLA-C4 allele is of interest, since our data show that HLA-C4 heavy chains are expressed intracellularly in amounts similar to HLA-A and HLA-B heavy chains, and a large pool of assembled HLA-C4 molecules is found associated efficiently with TAP in the ER.
Stable interaction of HLA-C4 and HLA-C2 with TAP is prolonged by inhibition of ER degradation
Normal intracellular expression, but poor cell surface expression,
of HLA-C4 molecules may be the result of intracellular retention of
HLA-C4 by TAP and/or other molecules. Hence, the intracellular fate of
HLA-C4 molecules was followed in OSH cells that were labeled
metabolically for 15 min, chased for the times indicated, and lysed in
digitonin-containing lysis mixture (Fig. 2). Each lysate was split into two equal
portions and immunoprecipitated with either W6/32 or TAP2 (Fig. 2, A and B (left panels),
respectively). Only trace amounts of HLA-C4 molecules were recovered
with W6/32, whereas the majority of the HLA-C4 complexes was associated
with TAP. The association of HLA-C4 with TAP remained relatively stable
for a long period of time compared with HLA-A and HLA-B locus products
(Fig. 2B, left panel), and started to
decrease after 2 to 4 h. In principle, free class I complexes
(recognized by W6/32) should be the result of either ß2m
association with free class I heavy chain or dissociation of class I
complexes from TAP. However, no increase in the recovery of free HLA-C4
molecules with W6/32 was observed as a result of release from TAP (Fig. 2A, left panel). Furthermore, the amount
of HLA-C4 released from TAP after 4 to 8 h did not result in an
increase of the intracellular pool of free HLA-C4 heavy chains, as
assessed by sequential immunoprecipitation with HC (Fig. 2C, left panel). Note that a relatively
large pool of free HLA-C4 heavy chains does not participate in the
formation of assembled HLA-C4 complexes (Fig. 2C).
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HC pool, they may instead be degraded. Hence, the same pulse-chase
experiment was performed, but now the labeled cells were treated with
CHX throughout the chase period (Fig. 2, right panels,
+CHX). CHX inhibits protein synthesis, but also has been shown to
inhibit protein degradation in the ER
(25).4 As anticipated, CHX
treatment of the cells did not influence the recovery of properly
assembled MHC class I molecules (Fig. 2A), or the
rate of intracellular transport of class I molecules (visualized by
disappearance of nonsialylated and concomitant appearance of sialylated
class I molecules). Furthermore, CHX treatment had only a minor effect
on the TAP interaction of the HLA-A and HLA-B alleles (Fig. 2B). Quantitations of the TAP-associated MHC class I
molecules depicted in Figure 2B by phosphor imaging show
that release of HLA-A2, HLA-A11, and HLA-B27 from TAP follows similar
kinetics in the absence or presence of CHX (Fig. 2D).
The majority of these TAP-associated class I molecules are released
after 2 to 4 h. However, the disappearance of TAP-associated
HLA-C4 molecules was markedly delayed in CHX-treated cells, in which
the HLA-C4 allele remained associated with TAP for at least 12 h,
compared with 4 to 8 h in the absence of CHX (Fig. 2, B
and D). Quantitations of the TAP-associated HLA-C2
allele show that, like HLA-C4, the HLA-C2 allele also associated stably
with TAP and that the interaction was prolonged in the presence of CHX
(Fig. 2, B and D). Note that the free
class I heavy chains become more stable upon CHX treatment (Fig. 2C). Taken together, these data show that the HLA-C4 and HLA-C2 alleles are stably associated with TAP in living cells for a prolonged period of time compared with HLA-A and HLA-B alleles. Release of the HLA-C molecules from TAP can be delayed by CHX treatment, suggesting that the majority of HLA-C molecules associated with TAP is removed by the ER-degradation machinery and not by conversion into the pool of stable HLA-C/peptide complexes.
TAP-associated HLA-C4 molecules can be released by allele-specific peptide
In vitro studies have shown that binding of appropriate
peptide to the class I molecules induces their release from TAP (10, 11). The poor release of HLA-C molecules from TAP in living cells could
therefore result from impeded generation and/or transport of peptides
that bind to HLA-C alleles. Alternatively, TAP-associated HLA-C
molecules may bind peptides poorly. We tested the ability of
TAP-associated HLA-C4 molecules to bind peptides in vitro by following
the peptide-dependent release of HLA-C4 from TAP, using a previously
described protocol (10). OSH cells were labeled metabolically, lysed in
1% digitonin lysis mixture, and split into equal portions. TAP/class I
complexes were isolated with TAP2 antiserum, and the
immunoprecipitates were incubated with different concentrations of an
HLA-C4-specific peptide (peptC4) or an HLA-A2-specific peptide
(peptA2). The peptide-bound MHC class I molecules were released from
TAP after incubation in 1% Triton X-100 at 37°C and isolated with
the mAb W6/32 recognizing free class I complexes. The released HLA-C4
molecules were recovered specifically by W6/32 only when the
HLA-C4-specific peptide had been present, whereas HLA-A2 molecules were
recovered only after incubation with the HLA-A2-binding peptide (Fig. 3A). The HLA-C4
molecules bound peptide and were released from TAP in a
concentration-dependent fashion similar to HLA-A2. Both alleles were
released at a peptide concentration of at least 0.01 µM peptide.
These results were confirmed in the EBV-transformed B cell line Fivo
(Fig. 3B). The data show that TAP-associated HLA-C4
molecules are able to bind peptide in vitro, and thereby dissociate
from TAP, with similar kinetics to HLA-A2. Thus, poor dissociation of
HLA-C4 from TAP in vivo cannot be explained by an inability of HLA-C4
molecules to bind peptide.
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The observed peptide-induced release of HLA-C4 and HLA-A2 from TAP
was based on two defined peptides, and the result does not explain the
stable interaction of HLA-C4 molecules with TAP in living cells.
However, it is possible that HLA-C4 remains associated with TAP because
it binds a more restricted set of peptides than HLA-A and HLA-B locus
products. If so, one would predict that higher concentrations of a
random mixture of peptides are required for release of HLA-C4 molecules
from TAP than for HLA-A and HLA-B locus products. We used a completely
degenerated 9-mer peptide mixture (peptDEG), in which all possible
combinations of the 20 natural amino acids were present at every
position, thereby mimicking the pool of peptides that theoretically can
be generated in vivo. The amino acid composition of the degenerated
peptide mixture was calculated by hydrolysis and amino acid
determination, which showed that all amino acids were present in
similar amounts (Table I).
Immunoprecipitated TAP/class I complexes were isolated from OSH cells
and incubated with increasing concentrations of the degenerated 9-mer
peptide mixture. Released class I molecules were immunoprecipitated
with W6/32, as described above. Peptide-induced release of all of the
class I alleles from TAP was observed and reached a maximum at a
concentration of 100 µM of degenerated peptide, as determined by
quantitation (Fig. 4A and data
not shown). A time course experiment with TAP/class I complexes
incubated with a defined concentration of peptDEG showed that the
release of the different class I alleles from TAP occurred at the same
rate (data not shown). The HLA-B35 allele is absent in Figure 4A, since this allele is known to associate inefficiently
with TAP (12) (Fig. 1A). Peptide titrations showed
that HLA-A2, HLA-A11, and HLA-B27 could be released specifically from
TAP at peptide concentrations as low as 1 µM. However, about a
10-fold higher concentration of the degenerated peptide was needed for
TAP dissociation of HLA-C4. This was confirmed by quantitation of the
bands by phosphor imaging, which showed that the concentration of
peptDEG required to release 50% of the TAP-associated HLA-C4 molecules
was 7- to 14-fold higher than the concentration required for 50%
release of HLA-A2/HLA-A11 and HLA-B27, respectively (data not shown).
The relative amount of the individual class I alleles released from
TAP and recovered with W6/32 is comparable with that of HLA class I
molecules directly coprecipitated with TAP (Fig. 4B,
TAP2). Note that W6/32 efficiently recognizes the HLA-C alleles
released from TAP (Fig. 4A), showing that the trace
amounts of HLA-C molecules recovered with W6/32 in Figures 1 and 2 are
not due to inefficient recognition of HLA-C alleles by W6/32, but
reflect the small pool of free HLA-C molecules. The HLA-C2 allele also
required about a 10-fold higher concentration of degenerated peptide
for TAP release than for HLA-A and HLA-B alleles (Fig. 4A). The more restricted peptide binding of HLA-C4
molecules was confirmed by isolating TAP/class I complexes from the B
cell line BM28.7 (Fig. 4C). In this study,
HLA-C4 also requires 10-fold higher concentrations of the degenerated
peptide mixture than HLA-A1 to bind peptide and become released from
TAP. Similar data were obtained with the HLA-C1 allele, which has also
been shown to associate stably with TAP (12) (data not shown). These
results suggest that in a completely degenerated peptide mixture,
the HLA-C molecules tested bind a more restricted set of peptides than
HLA-A and HLA-B alleles.
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D). TAP/class I
complexes isolated from OSH cell lysates were incubated in the absence
(lanes -peptide) or presence of peptide peptA2, peptC4, or peptDEG.
After incubation at 37°C, the peptide-bound MHC class I molecules
were released from TAP and recovered by W6/32 in the presence of both
Triton X-100 and the milder detergent digitonin (Fig. 4D). Thus, the observed release of MHC class I
molecules from TAP is caused by peptide and not by the type of
detergent used in the assay. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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More recently, it was suggested by McCutcheon et al. (3) that the reduced HLA-C surface expression resulted from instability of HLA-C mRNA. They found that EBV-transformed B cell lines expressed lower levels of HLA-C mRNA than of HLA-B mRNA, and suggested that this was due to rapid degradation of HLA-C heavy chain mRNA. This would result in a lower rate of protein synthesis and limited cell surface expression of HLA-C alleles. We show that the HLA-C4 allele is poorly expressed at the cell surface, although translated in amounts similar to HLA-A and HLA-B alleles. Thus, the instability of HLA-C mRNA is not reflected at the protein level of the HLA-C4 allele investigated in this study. However, inefficient translation of other HLA-C alleles may explain their low surface expression.
An additional explanation for the poor HLA-C cell surface expression could be a restriction in the peptide binding of HLA-C molecules. HLA-C alleles bind peptides of eight to nine amino acids in length and utilize a C-terminal anchor similar to HLA-A and HLA-B alleles (15, 16). Based on these similarities, Falk et al. (15) suggested that HLA-C molecules should be able to bind about the same numbers of different peptides as other classical class I molecules, although no evidence for this assumption was provided. In contrast to this, a molecular analysis of HLA class I polymorphism indicated that HLA-C alleles have less variation in the amino acid sequence of the peptide-binding groove than HLA-A and HLA-B heavy chains (28). The limited polymorphism of HLA-C molecules was suggested to result in binding of a more restricted set of peptides to HLA-C alleles than to HLA-A and HLA-B alleles (28). Although this proposal would be correct for peptide differences within the HLA-C locus, it is unclear how this would result in binding of a limited set of peptides to a defined HLA-C allele.
In this study, we provide biochemical evidence that the TAP-associated
HLA-C molecules that we tested do bind a more restricted set of peptide
than the HLA-A and HLA-B locus products. We made use of the observation
that TAP-associated class I molecules require binding of specific
peptides for release from TAP (10, 11). In an in vitro peptide-binding
assay, the TAP-associated HLA-C alleles needed a 10-fold higher
concentration of a completely degenerated 9-mer peptide mixture for
peptide binding, and thus release from TAP than the HLA-A and HLA-B
alleles. Reduced peptide binding of TAP-associated HLA-C molecules in
vivo may explain the stable and prolonged interaction observed between
HLA-C molecules and TAP in the ER. The TAP-associated HLA-C molecules
did not appear to be an intermediate in the generation of HLA-C/peptide
complexes, nor did they rapidly dissociate into free heavy chains.
Instead, they were retained in the ER in association with TAP and were
only slowly released compared with HLA-A and HLA-B alleles. Treatment
of cells with CHX inhibits translation and has been reported to inhibit
protein degradation in the ER by an as yet unknown mechanism
(25).4 The addition of CHX results in a prolonged
association of HLA-C4 and HLA-C2 with TAP, whereas the HLA-A and HLA-B
alleles continue to dissociate from TAP for at least 8 h (Fig. 2D, 480 min). During this time period, processes involved in
MHC class I peptide loading and release from TAP are apparently not
affected by CHX. We observe slow release from TAP of HLA-C4 and HLA-C2
and no recovery of these molecules in the W6/32 pool (Fig. 2, A and B). This slow release is most likely
due to degradation in the ER, and not to peptide-induced release from
TAP, since HLA-C4 and HLA-C2 remain stably associated with TAP under
conditions in which ER degradation is inhibited. Note that a fraction
of the other TAP-associated class I alleles, and especially HLA-A2, is
also rescued by CHX treatment, suggesting that degradation of
TAP-associated class I molecules is a general phenomenon, albeit more
extreme for HLA-C alleles.
The peptide-binding motif of several HLA-C molecules has been determined and shows that HLA-C alleles often prefer peptides with a Pro at position 2 or 3 and a hydrophobic C-terminal anchor residue (15, 16). We have shown previously that peptides containing a Pro at these positions are transported very inefficiently into the ER by TAP (17, 18). It is unclear to what extent this might influence peptide loading, since HLA class I alleles, such as HLA-B7, HLA-B35, and HLA-B53, also seem to bind peptides with a Pro at position 2 (29, 30), whereas they are expressed at the cell surface at normal levels.
Since HLA-C alleles are expressed at such low levels at the cell surface compared with HLA-A and HLA-B locus products, the function and, therefore, physiologic relevance of HLA-C alleles have been questioned. However, it is now evident that HLA-C molecules (like some HLA-A and HLA-B molecules) can protect target cells from lysis by NK cells via a direct interaction between the NK receptor and HLA-C molecules on the target cell (6, 7).
It appears unlikely, however, that the polymorphic C locus has evolved to function exclusively as a ligand for NK receptors. Indeed, HLA-C-restricted CTL recognizing HIV-derived peptides have been isolated, indicating that HLA-C molecules can function as restriction elements in CTL responses (4, 5, 31). Similarly, HLA-C alleles are correlated with susceptibility to certain diseases such as leukemia (32) and psoriasis (33), and have been shown to be involved in graft rejections (34, 35). It may be that HLA-C alleles have a specialized function in the immune system, perhaps by binding and presenting a restricted set of peptides that are not presented efficiently by the HLA-A and HLA-B locus products.
In conclusion, we have shown that a number of HLA-C alleles that are poorly expressed at the cell surface efficiently and stably interact with TAP, which retains them in the ER. TAP-associated HLA-C alleles are able to bind peptide, and thus become released from TAP in vitro, but the HLA-C alleles tested bind a more restricted set of peptides than HLA-A and HLA-B alleles. As a consequence of the limited peptide binding, the HLA-C molecules are only poorly released from TAP, and thus retained in the ER, where the majority of the HLA-C molecules is ultimately degraded. The restricted peptide binding of certain HLA-C alleles in the ER provides an additional explanation to the reduced cell surface expression of HLA-C molecules.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jacques Neefjes, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. E-mail address:
3 Abbreviations used in this paper: ß2m, ß2-microglobulin; 1D-IEF, one-dimensional isoelectric focusing; HC, rabbit anti-human class I heavy chain; TAP2, rabbit anti-human TAP2 serum; CHX, cycloheximide; ER, endoplasmic reticulum; N.A., neuraminidase; nrs, normal rabbit serum.
4 S. Dusseljee, R. Wubbolts, D. Verwoerd, A. Tulp, H. Janssen, J. Calafat, and J. Neefjes. Removal and degradation of the free MHC class II ß-chain in the endoplasmic reticulum requires proteasomes and is accelerated by BFA. Submitted for publication.
Received for publication March 11, 1997. Accepted for publication September 3, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 domain of HLA-A2.1 define a functionally relevant interaction with TAP. Curr. Biol. 7:873.
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 W. D. Wick, P. B. Gilbert, and O. O. Yang Predicting the Impact of Blocking Human Immunodeficiency Virus Type 1 Nef In Vivo J. Virol., March 1, 2009; 83(5): 2349 - 2356. [Abstract] [Full Text] [PDF]
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M. R. Schaefer, M. Williams, D. A. Kulpa, P. K. Blakely, A. Q. Yaffee, and K. L. Collins A Novel Trafficking Signal within the HLA-C Cytoplasmic Tail Allows Regulated Expression upon Differentiation of Macrophages J. Immunol., June 15, 2008; 180(12): 7804 - 7817. [Abstract] [Full Text] [PDF]
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 L. Sibilio, A. Martayan, A. Setini, E. L. Monaco, E. Tremante, R. H. Butler, and P. Giacomini A Single Bottleneck in HLA-C Assembly J. Biol. Chem., January 18, 2008; 283(3): 1267 - 1274. [Abstract] [Full Text] [PDF]
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 S. Adnan, A. Balamurugan, A. Trocha, M. S. Bennett, H. L. Ng, A. Ali, C. Brander, and O. O. Yang Nef interference with HIV-1-specific CTL antiviral activity is epitope specific Blood, November 15, 2006; 108(10): 3414 - 3419. [Abstract] [Full Text] [PDF]
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L. H. Boyle, A. K. Gillingham, S. Munro, and J. Trowsdale Selective Export of HLA-F by Its Cytoplasmic Tail. J. Immunol., June 1, 2006; 176(11): 6464 - 6472. [Abstract] [Full Text] [PDF]
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 R. J. Boyton, J. Smith, R. Ward, M. Jones, L. Ozerovitch, R. Wilson, M. Rose, J. Trowsdale, and D. M. Altmann HLA-C and Killer Cell Immunoglobulin-like Receptor Genes in Idiopathic Bronchiectasis Am. J. Respir. Crit. Care Med., February 1, 2006; 173(3): 327 - 333. [Abstract] [Full Text] [PDF]
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 M. T. Barel, N. Pizzato, P. Le Bouteiller, E. J. H. J. Wiertz, and F. Lenfant Subtle sequence variation among MHC class I locus products greatly influences sensitivity to HCMV US2- and US11-mediated degradation Int. Immunol., January 1, 2006; 18(1): 173 - 182. [Abstract] [Full Text] [PDF]
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L. Sibilio, A. Martayan, A. Setini, R. Fraioli, D. Fruci, J. Shabanowitz, D. F. Hunt, and P. Giacomini Impaired Assembly Results in the Accumulation of Multiple HLA-C Heavy Chain Folding Intermediates J. Immunol., November 15, 2005; 175(10): 6651 - 6658. [Abstract] [Full Text] [PDF]
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 D. Zernich, A. W. Purcell, W. A. Macdonald, L. Kjer-Nielsen, L. K. Ely, N. Laham, T. Crockford, N. A. Mifsud, M. Bharadwaj, L. Chang, et al. Natural HLA Class I Polymorphism Controls the Pathway of Antigen Presentation and Susceptibility to Viral Evasion J. Exp. Med., November 8, 2004; (2004) jem.20031680. [Abstract] [Full Text] [PDF]
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M. T. Barel, M. Ressing, N. Pizzato, D. van Leeuwen, P. Le Bouteiller, F. Lenfant, and E. J. H. J. Wiertz Human Cytomegalovirus-Encoded US2 Differentially Affects Surface Expression of MHC Class I Locus Products and Targets Membrane-Bound, but Not Soluble HLA-G1 for Degradation J. Immunol., December 15, 2003; 171(12): 6757 - 6765. [Abstract] [Full Text] [PDF]
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S. Tourdot and K. G. Gould Competition Between MHC Class I Alleles for Cell Surface Expression Alters CTL Responses to Influenza A Virus J. Immunol., November 15, 2002; 169(10): 5615 - 5621. [Abstract] [Full Text] [PDF]
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P. Tan, H. Kropshofer, O. Mandelboim, N. Bulbuc, G. J. Hammerling, and F. Momburg Recruitment of MHC Class I Molecules by Tapasin into the Transporter Associated with Antigen Processing-Associated Complex Is Essential for Optimal Peptide Loading J. Immunol., February 15, 2002; 168(4): 1950 - 1960. [Abstract] [Full Text] [PDF]
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 E. W. Petersdorf, J. A. Hansen, P. J. Martin, A. Woolfrey, M. Malkki, T. Gooley, B. Storer, E. Mickelson, A. Smith, and C. Anasetti Major-Histocompatibility-Complex Class I Alleles and Antigens in Hematopoietic-Cell Transplantation N. Engl. J. Med., December 20, 2001; 345(25): 1794 - 1800. [Abstract] [Full Text] [PDF]
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L. Lybarger, Y. Y. L. Yu, T. Chun, C.-R. Wang, A. G. Grandea III, L. Van Kaer, and T. H. Hansen Tapasin Enhances Peptide-Induced Expression of H2-M3 Molecules, but Is Not Required for the Retention of Open Conformers J. Immunol., August 15, 2001; 167(4): 2097 - 2105. [Abstract] [Full Text] [PDF]
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S. D. Wainwright, P. A. Biro, and C. H. Holmes HLA-F Is a Predominantly Empty, Intracellular, TAP-Associated MHC Class Ib Protein with a Restricted Expression Pattern J. Immunol., January 1, 2000; 164(1): 319 - 328. [Abstract] [Full Text] [PDF]
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Q. R. Fan and D. C. Wiley Structure of Human Histocompatibility Leukocyte Antigen (Hla)-Cw4, a Ligand for the Kir2d Natural Killer Cell Inhibitory Receptor J. Exp. Med., July 1, 1999; 190(1): 113 - 124. [Abstract] [Full Text] [PDF]
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M. R. Knittler, K. Gulow, A. Seelig, and J. C. Howard MHC Class I Molecules Compete in the Endoplasmic Reticulum for Access to Transporter Associated with Antigen Processing J. Immunol., December 1, 1998; 161(11): 5967 - 5977. [Abstract] [Full Text] [PDF]
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