From the Graduate School of Biotechnology, Korea University, Seoul 136-701, Korea
Received for publication, December 18, 2002
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ABSTRACT |
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Tapasin plays an important role in the quality
control of major histocompatibility complex (MHC) class I assembly, but
its precise function in this process remains controversial. Whether tapasin participates in the assembly of HLA-G has not been studied. HLA-G, an MHC class Ib molecule that binds a more restricted set of
peptides than class Ia molecules, is a particularly interesting molecule, because during assembly, it recycles between the endoplasmic reticulum (ER) and the cis-Golgi until it is loaded with a
high affinity peptide. We have taken advantage of this unusual
trafficking property of HLA-G and its requirement for high affinity
peptides to demonstrate that a critical function of tapasin is to
transform class I molecules into a high affinity, peptide-receptive
form. In the absence of tapasin, HLA-G molecules cannot bind high
affinity peptides, and an abundant supply of peptides cannot overcome
the tapasin requirement for high affinity peptide loading. The addition of tapasin renders HLA-G molecules capable of loading high affinity peptides and of transporting to the surface, suggesting that tapasin is
a prerequisite for the binding of high-affinity ligands.
Interestingly, the "tapasin-dependent" HLA-G
molecules are not empty in the absence of tapasin but are in fact
associated with suboptimal peptides and continue to recycle between the
ER and the cis-Golgi. Together with the finding that empty
HLA-G heterodimers are strictly retained in the ER and degraded, our
data suggest that MHC class I molecules bind any available peptides to
avoid ER-mediated degradation and that the peptides are in turn
replaced by higher affinity peptides with the aid of tapasin.
After being targeted to the endoplasmic reticulum
(ER),1 nascent MHC class I
heavy chains associate with a multiprotein complex that assists in
their assembly with peptides and Recent studies have implied that tapasin serves several
functions in the assembly of class I molecule-peptide complexes.
It functions to bridge the heavy chain complexes to TAP (8). Tapasin expression also enhances the stability of TAP heterodimers, increasing overall peptide transport into the ER (9, 10). In insect cells, tapasin
retains empty Kb molecules in the ER (11). Likewise, in
tapasin-deficient human 721.220 cells, tapasin prevents premature
release of Kb molecules from the ER, suggesting that
tapasin might participate in retaining class I molecules in the ER
until an optimal peptide is loaded (12). Finally, other recent studies
have suggested that tapasin might be involved in the peptide editing of
class I molecules (13-15). Although there is little doubt that tapasin is a class I-dedicated chaperone, the actual mechanism for each proposed function of tapasin during its interactions with class I
alleles remains to be elucidated. Tapasin dependence could reflect a
direct role for tapasin in ER retention of heavy chains, TAP stabilization, peptide editing, or any combination of these functions. Determining which of these interrelated functions are primary rather
than secondary manifestations of tapasin's interactions with class I
molecules has proven to be difficult. In addition to the uncertainty
regarding the precise functions of tapasin, various class Ia molecules
differ in their dependence on tapasin for both efficient surface
expression and presentation of antigenic determinants to CTL, as
observed in studies of 721.220 transfectants (13, 16-18). In 721.220 cells, tapasin is not required for high levels of surface expression of
the HLA-B2705 allele or for presentation of viral determinants
to cytotoxic T lymphocytes (16). In contrast, for the HLA-B4402
allele, functional antigen presentation and surface expression are
highly dependent on tapasin; HLA-B0801 falls between the B2705 and
B4402 alleles in the spectrum of tapasin dependence (16). These
relationships reflect the complexity of tapasin function. In fact, the
aforementioned studies regarding the function of tapasin took place
before the concept was established that the tapasin dependence of class
I molecules is allele-specific. Thus, the conclusions of these studies
might be biased, depending on which class I alleles were analyzed.
HLA-G, an MHC class Ib molecule, is expressed primarily in trophoblast
cells and has limited polymorphism (2). Due to a scarcity of natural
endogenous peptide ligands in most cells, the supply of peptides is the
rate-limiting factor for the intracellular transport kinetics of HLA-G
(19). Recent evidence shows that HLA-G protects trophoblast cells from
recognition by NK cells (20, 21); expression of HLA-G on melanoma cells
protects them from lysis by NK cells (22). HLA-G is a particularly
interesting molecule in protein trafficking, because it recycles
between the ER and the cis-Golgi until it is loaded with a
high affinity peptide (19). This feature makes it possible to estimate
whether HLA-G molecules are loaded with high affinity or low affinity
peptides. Whether the process involved in the assembly of MHC class Ia
molecule-peptide complexes applies to the assembly of
HLA-G-peptide complexes has not been studied. Because HLA-G binds a
restricted set of peptides (23, 24) and recycles between the ER and
cis-Golgi, it is an attractive model for investigating the
roles of individual components of the ER peptide-loading complex in the
assembly of functional MHC class I molecules.
In this study, we examined the tapasin dependence of HLA-G in terms of
tapasin's critical role in the assembly and intracellular transport of
HLA-G. Our findings demonstrate that in the absence of tapasin, HLA-G
molecules are not able to bind high affinity peptides despite an
abundant supply of peptides, suggesting that tapasin is a prerequisite
for the binding of high affinity peptides. Interestingly, immediately
after synthesis, these tapasin-dependent HLA-G molecules
associate constitutively with low affinity endogenous peptides and exit
the ER, thereby avoiding ER-mediated degradation. We propose that a
critical function of tapasin is to transform the peptide-binding groove
of HLA-G into a high affinity, peptide-receptive form, which could
promote the replacement of low affinity peptides with high affinity peptides.
DNA Constructs--
All HLA cDNAs and their mutagenized
derivatives were inserted into the mammalian expression vector
pcDNA3.1 (Invitrogen). The cDNA encoding human tapasin was
kindly provided by Dr. Cresswell (Yale University, New Haven, CT) and
was subcloned into the pcDNA3.1/Hygro vector (Invitrogen). The
cDNA encoding human Stable Cell Lines and Antibodies--
NIH3T3 cells were cultured
in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with
10% fetal bovine serum (Hyclone, Logan, Utah), penicillin (50 units/ml), and streptomycin (50 µg/ml). NIH3T3 cells were transfected
with either human Pulse-Chase Labeling and Immunoprecipitation--
Cells (5 × 106) were transfected by electroporation, starved for 40 min in medium lacking methionine, labeled for 20 min with 0.1 mCi/ml
[35S]methionine (TranS-label; PerkinElmer Life
Sciences), and chased for the indicated times in normal medium.
Cells were lysed by using 1% Nonidet P-40 (Sigma) in
phosphate-buffered saline (PBS) with protease inhibitor mixture (Sigma)
for 30 min at 4 °C. After preclearing lysates with protein
G-Sepharose (Amersham Biosciences), primary antibodies and protein
G-Sepharose were added to the supernatant and incubated at 4 °C with
rotation for 2 h. The beads were washed three times with 0.1%
Nonidet P-40 in PBS. Proteins were eluted from the beads by boiling in
SDS sample buffer and separated by 12% SDS-PAGE. The gels were dried,
exposed to BAS film for 14 h, and analyzed with the
phosphorimaging system BAS-2500 (Fuji Film, Tokyo, Japan). For
endoglycosidase-H (endo-H) treatment, immunoprecipitates were digested
with 3 milliunits of endo-H (Roche Molecular Biochemicals) at 37 °C
overnight in 50 mM sodium acetate (pH 5.6), 0.3% SDS, and
150 mM Flow Cytometry and Immunofluorescence Microscopy--
The
surface expression of HLA-G molecules was determined by flow cytometry
(FACScalibur, Becton Dickinson Biosciences, Mountain View, CA). Cells
(1 × 106) were washed twice with cold PBS containing
1% bovine serum albumin and incubated for 1 h at 4 °C with a
saturating concentration of mAb G233. Normal mouse IgG was used as a
negative control for each test. The cells were washed twice with cold
PBS containing 1% bovine serum albumin and then stained with
fluorescein isothiocyanate-conjugated goat anti-mouse IgG for 30 min. A
total of 10,000 gated events were collected by the FACScalibur
cytometer and analyzed with CellQuest software (Becton Dickinson
Biosciences). For immunofluorescence staining of permeabilized cells,
NIH3T3 cells were fixed in 3.7% formaldehyde, made permeable with
0.1% Triton X-100, and incubated with the appropriate primary antibody
for 1 h. MRC-1024 confocal microscopy was used for confocal
imaging (Bio-Rad).
Coimmunoprecipitation and Western Blot Analysis--
Cells were
lysed in 1% digitonin in digitonin buffer containing 25 mM
HEPES, 100 mM NaCl, 10 mM CaCl2,
and 5 mM MgCl2 (pH 7.6) supplemented with
protease inhibitors. Lysates were precleared with protein G-Sepharose
(Amersham Biosciences) for 1 h at 4 °C. For
immunoprecipitation, samples were incubated with the appropriate antibodies for 2 h at 4 °C before protein G-Sepharose beads
were added. Beads were washed four times with 0.1% digitonin, and
bound proteins were eluted by boiling in SDS sample buffer. Proteins were separated by 12% SDS-PAGE, transferred onto a nitrocellulose membrane, blocked with 5% skim milk in PBS with 0.1% Tween 20 for
2 h, and probed with the appropriate antibodies for 4 h.
Membranes were washed three times in PBS with 0.1% Tween 20 and
incubated with horseradish peroxidase-conjugated streptavidin (Pierce)
for 1 h. The immunoblots were visualized with ECL detection
reagent (Pierce).
Microsomes and Peptide Loading Assays--
Microsomes from
721.220 and 721.220.Tpn cells expressing HLA-G or HLA-G/E114H heavy
chains were prepared and purified as previously described (25).
Biotinylated peptide KIPAQFYIL was conjugated to the photoreactive
cross-linker N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOS; Pierce) as described (9). For the peptide-loading assay,
reporter peptides, with or without various concentrations of the
unlabeled peptide (KIPAQFYIL), were mixed with 15 µl of microsomes
(concentration of 60 A280/ml) in a total volume
of 50 µl of RM buffer (250 mM sucrose, 50 mM
triethanolamine-HCl, 50 mM potassium acetate, 2 mM magnesium acetate, 1 mM dithiothreitol, and
10 mM ATP). The mixture was incubated for 30 min at
26 °C in a flat-bottomed 96-well tissue culture plate. The samples
were maintained on ice during a 3-min exposure to shortwave (365-nm) ultraviolet irradiation. After centrifugation, the membranes were washed once with cold RM buffer and lysed with 1% digitonin, and the
cross-linked proteins were immunoprecipitated with mAb G233. The
precipitates were separated by 12% SDS-PAGE and transferred to an
Immobilon-P membrane (Millipore Corp., Bedford, MA). The membrane was
incubated with horseradish peroxidase-conjugated streptavidin for
1 h, and biotinylated proteins were visualized by using ECL
Western blotting reagent (Pierce). Peptide translocation was determined
after incubating microsomes with biotin-conjugated reporter peptides in
the absence of competitor for 30 min at 26 °C with or without 1 mM ATP. Microsomal membranes were recovered by
centrifugation at 75,000 × g for 10 min through a 0.5 M sucrose cushion in cold RM buffer. After washing with
cold RM buffer twice, the membrane pellet was directly dissolved in
sample buffer. The samples were analyzed on Tricine/SDS-PAGE,
appropriate for resolution of low mass polypeptides as described (26),
and probed with horseradish peroxidase-conjugated streptavidin. The
relative densities of the peptide bands were determined by use of an
imaging densitometer (GS-700; Bio-Rad) and MultiAnalyst densitometer
software (Bio-Rad).
HLA-G Is Highly Dependent on Tapasin for Cell Surface Expression
and Intracellular Maturation--
To determine the function of tapasin
for the assembly of HLA-G-peptide complexes, we first examined the
tapasin dependence of HLA-G for cell surface expression. The genes
encoding HLA-G heavy chains were independently transfected into both
721.220 and 721.220.Tpn cell lines. In the absence of tapasin, low
levels of surface expression were observed (Fig.
1A). Significantly, upon
expression of tapasin, surface expression increased more than 5-fold,
indicating the dependence of HLA-G on tapasin for its surface
expression. In recent efforts to identify the factors that determine
the relative tapasin dependence of various class Ia alleles, we have
found that the nature of the amino acid residues present at the
naturally polymorphic position 114 determines tapasin dependence.2 To test whether
this is also the case for the HLA-G molecules, we constructed
substitution mutants and examined their dependence on tapasin for
surface expression (Fig. 1A). Surprisingly, a glutamic acid
to histidine substitution at position 114 (HLA-G/E114H) allows the
otherwise tapasin-dependent HLA-G to have levels of surface expression comparable with the levels seen in the presence of tapasin.
The HLA-G/E114Q mutant, in which residue 114 was replaced by the
neutrally charged glutamine, fell between HLA-G wild type and
HLA-G/E114H in the spectrum of tapasin dependence. These results indicate that, like HLA class Ia molecules, the tapasin dependence of
HLA-G is also influenced by the nature of the amino acid at position
114. Since it is unlikely that the point mutation causes a gross
conformational change in class I molecules, we used the HLA-G/E114H
mutant in parallel with wild-type HLA-G as a control for the
tapasin-independent phenotype.
To investigate the mechanism by which tapasin affects surface
expression of HLA-G, we compared the intracellular maturation and
transport of HLA-G in the 721.220 and 721.220.Tpn cells. In the absence
of tapasin, 50% of HLA-G heavy chains remained sensitive to endo-H
digestion, even after 8 h (Fig. 1B), reflecting
retention of these molecules in the ER. Conversely, in the presence of
tapasin, most HLA-G molecules had become endo-H-resistant by this time (Fig. 1B). These findings suggest that the impaired
intracellular transport of HLA-G molecules in the absence of tapasin
accounts for their low levels of surface expression. Pulse-chase
experiments from tapasin-negative and -positive cells revealed
comparable acquisition of endo-H resistance by HLA-G/E114H (Fig.
1C), indicating its tapasin independence for normal
intracellular transport.
We have shown that the availability of high affinity peptides dictates
the transport kinetics of HLA-G (19). To determine whether the
inefficient transport of HLA-G in the absence of tapasin can be
overcome by supplying high affinity peptides, we examined the
intracellular trafficking of HLA-G and HLA-G/E114H upon expression of
high affinity peptides by the minigene expression system. The minigene
encoding MIPAQFYIL, a high affinity peptide ligand for HLA-G (23), was
co-expressed with the gene encoding either HLA-G or HLA-G/E114H in
721.220 and 721.220.Tpn cells. In the presence of tapasin, no
discernible difference in the transport rate between HLA-G and
HLA-G/E114H was seen upon expression of high affinity peptides (Fig.
1D), but both HLA-G and HLA-G/E114H molecules were transported with much faster kinetics when compared with the kinetics of molecules without the supply of high affinity peptides (Fig. 1,
B and C, right). In the absence of
tapasin, the supply of high affinity peptides increased the transport
kinetics of HLA-G/E114H (Fig. 1, compare C (left)
and E (right)), whereas this supply did not
influence the transport kinetics of HLA-G (Fig. 1, compare B
(right) and E (left)). These results indicate
that for efficient intracellular transport of HLA-G molecules, an
abundance of high affinity peptides cannot compensate for the lack of tapasin.
Tapasin Is a Prerequisite for Loading High Affinity
Peptides--
In the absence of tapasin, the impaired intracellular
transport and the low surface expression of HLA-G might be due to its inability to load high affinity peptides. To test this possibility, we
examined peptide loading into HLA-G or HLA-G/E114H molecules as a
function of tapasin by using the reporter peptide, KIPAQFYIL, which is
known to be a high affinity ligand for HLA-G (23), in the presence of
the competitors at different concentrations. We were surprised at the
marked differences in the ability of HLA-G and HLA-G/E114H to load
peptides in the absence of tapasin. In the case of no competitor added,
the level of binding of the reporter peptide by HLA-G was only 25% of
the binding observed in the HLA-G/E114H substitution mutant (Fig.
2A). Furthermore, the reporter
peptides loaded into HLA-G were completely outcompeted by a lower
concentration (1.6 µM) of unlabeled peptide, whereas as
much as 6.4 µM of unlabeled peptide was not sufficient
for completely outcompeting reporter peptide binding to HLA-G/E114H. However, in the presence of tapasin, no discernible difference in the
peptide binding ability of HLA-G and HLA-G/E114H was seen (Fig.
2B). These results indicate that HLA-G molecules are highly dependent on tapasin for efficient loading with high affinity peptides,
but the substitution of glutamic acid to histidine at position 114 renders the molecules independent of tapasin for high affinity peptide
loading. Accordingly, the impaired intracellular transport and reduced
surface expression of HLA-G in the absence of tapasin are probably the
result of the inadequacy of HLA-G for high affinity peptide
loading.
To test whether the differential peptide loading observed in the
absence of tapasin might be due to differences in the luminal availability of the peptide, we quantitated the amount of reporter peptides that were translocated into the ER lumen. Comparable amounts
of peptides were recovered between HLA-G and HLA-G/E114H (Fig.
2C). Therefore, we exclude the notion that luminal
availability of the peptide plays an important role in dictating class
I loading in tapasin-deficient cells. In control experiments in which
ATP was omitted, little peptide was recovered. Since peptide
translocation via TAP requires ATP (27), we conclude that the modified
reporter peptides entered the ER lumen in a TAP-dependent manner.
Without Tapasin, HLA-G Recycles between the ER and Golgi Despite an
Abundant Supply of High Affinity Peptides--
HLA-G molecules that
are loaded with peptides of suboptimal affinity are retrieved back to
the ER. Loading of HLA-G with high affinity peptides abrogates this
retrieval and allows HLA-G-peptide complexes to transport forward to
the cell surface (19). To examine whether tapasin affects the recycling
behavior of HLA-G as a function of the availability of high affinity
peptides, we stably expressed minigenes encoding MIPAQFYIL in
NIH3T3.h
To further dissect the fate of HLA-G on the secretory pathway in the
absence of tapasin, we examined the intracellular localization of HLA-G
and HLA-G/E114H molecules by immunofluorescence staining and confocal
microscopy. In the absence of tapasin and high affinity peptides, both
HLA-G and HLA-G/E114H molecules colocalize with GM130 (Fig.
4A), which is a
cis-Golgi marker (29). Upon supply of high affinity
peptides, most HLA-G molecules are still found in the
cis-Golgi, whereas the predominant distribution of
HLA-G/E114H is shown by the pattern of ER and surface staining (Fig.
4B). These results indicate that in the absence of tapasin,
HLA-G molecules are not able to transport beyond medial Golgi despite
the availability of high affinity peptides. In the presence of tapasin
but absence of high affinity peptides, the surface staining of HLA-G
molecules is clearly observed (Fig. 4C, top
panel). Upon expression of high affinity peptides, the
surface staining patterns of HLA-G and HLA-G/E114H are much stronger
than the patterns without high affinity peptides (Fig. 4D),
suggesting that in the presence of tapasin, the availability of high
affinity peptides can be a limiting factor in determining the surface
level of HLA-G molecules. Taken together, these results suggest that in
the absence of tapasin, HLA-G molecules are incapable of loading high
affinity peptides, and, subsequently, they are retrieved from the
cis-Golgi to the ER. In contrast, HLA-G/E114H molecules can
load high affinity peptides in the absence of tapasin and are directly
transported to the cell surface without recycling.
To understand the biochemical basis for the retrieval phenomenon of
HLA-G in the absence of tapasin, we analyzed by coimmunoprecipitation experiments the interaction of HLA-G or HLA-G/E114H with Tapasin-dependent HLA-G Molecules Are Not Empty in the
Absence of Tapasin--
In wild-type cells, only peptide-filled and
fully conformed classical MHC class I heterodimers transit from the ER
to the cell surface (31-33). Given the well established concept that
tapasin plays a critical role in the proper assembly of class I
molecules in the ER, we were surprised at the finding that the
so-called tapasin-dependent HLA-G molecules were able to
exit the ER in the absence of tapasin. It appeared important,
therefore, to determine whether the HLA-G molecules that were in
transit to the cis-Golgi in the absence of tapasin were actually filled
with peptides. Empty MHC class I molecules are unstable and undergo an
irreversible conformational change at 37 °C due to dissociation of
the heavy chains from Constitutively Associated Peptides Confer Resistance to ER-mediated
Degradation of HLA-G Molecules--
The findings that HLA-G molecules
can assemble with low affinity self-peptides in the absence of tapasin
and that the resulting complexes are not able to transport beyond
medial-Golgi raise a question as to the physiological function of such
assembly. In the absence of peptides, empty class I molecules are
retained in the ER and degraded by a quality control mechanism
(35-37). To test whether this is also the case for HLA-G, an MHC class Ib molecule, we measured the turnover rate of HLA-G molecules in
ICP47-expressing cells. In these ICP47-expressing cells, the resulting
empty HLA-G and HLA-G/E114H molecules were being degraded rather
rapidly during the longer chases so that we could not detect HLA-G
molecules at the 2-h time point (Fig.
7A). In sharp contrast, in the
absence of ICP47, both HLA-G and HLA-G/E114H molecules were relatively
stable, even after a 4-h chase. In repeated experiments, we have
consistently observed that HLA-G molecules in the presence of tapasin
become more stable over time than do HLA-G molecules in its absence
(Fig. 7B, compare lanes 11 and
12 and lanes 3 and 4,
respectively). Since the stability of the class I molecules depends on
high affinity association with peptides, this observation suggests that
HLA-G molecules are loaded with higher affinity peptides in the
presence of functional tapasin. Overall, our data indicate that even in
the absence of tapasin, HLA-G molecules are constitutively associated
with peptides, albeit low affinity peptides, and that the binding of
such peptides might stabilize the intracellular HLA-G pool by
preventing their degradation. This strategy could confer a longer
half-life on HLA-G and provide more chances for HLA-G to exchange the
suboptimal peptide ligands for ones with higher affinity.
Recent studies have implicated that in the assembly of class I
molecule-peptide complexes, tapasin serves several functions, including ER retention of heavy chains, TAP stabilization, and peptide
editing. In this study, we addressed the question of whether tapasin is
involved in the assembly and trafficking of HLA-G. More importantly, by
taking advantage of the unusual trafficking property of HLA-G and the
unique feature of peptide ligands of HLA-G, we were able to delineate
the precise function of tapasin in the quality control of class I molecules.
Our results demonstrate that tapasin is not required for suboptimal
peptide binding but is critical for loading of high affinity peptides
onto HLA-G. Given the tapasin requirement of HLA-E (5) and H2-M3 (6,
7), class Ib molecules, for their proper assembly, this suggests that
class Ib molecules resemble "tapasin-dependent" class
Ia alleles in their dependence for tapasin. Our data rule out retention
of class I molecules in the ER as the primary function of tapasin. The
rate at which HLA-G/E114H exits the ER is identical with and without
tapasin (Fig. 1C). The assembly of HLA-G in 721.220.Tpn cells showed even enhanced transport of HLA-G heavy chains to the cell
surface, as compared with the transport of tapasin-deficient 721.220 cells (Fig. 1B). These results indicate that ER retention of
HLA-G is relatively unaffected by tapasin. In line with this interpretation, soluble tapasin also fails to retain class I molecules in the ER but still enhances class I surface expression to levels similar to the levels observed for the full-length tapasin construct (10). McCluskey and colleagues (12) have shown that, based on the
endo-H assay, tapasin prevents premature release of Kb from
the ER, which led them to conclude that tapasin plays a role in the
retention of suboptimally loaded class I molecules in the ER. Taking
into account the observation that HLA-G associated with suboptimal
peptides is not statically retained in the ER but recycles between the
ER and the cis-Golgi, together with the fact that the
sensitivity of proteins to endo-H digestion cannot distinguish between
true retention and retrieval from the cis-Golgi, the
apparent steady-state distribution of Kb in the ER might be
reminiscent of Kb retrieval from the cis-Golgi
to the ER rather than Kb retention in the ER. In support of
this view, in the murine mutant cell line (CMT), class I molecules seem
to recycle through the cis-Golgi (37), suggesting that similar quality
control by recycling can occur in class Ia molecules. It appears that
tapasin does not affect the function of TAP in translocating
peptides into the ER lumen, because peptide transport in microsomes
derived from 721.220 cells was similar to peptide transport in
microsomes derived from 721.220.Tpn cells (data not shown).
Furthermore, since loading of peptides onto HLA-G/114H proceeds
normally in the absence of tapasin (Fig. 2A), bridging of
class I molecules to TAP, which is one of the proposed functions of
tapasin, appears to be unnecessary for enhanced peptide loading. Thus,
the most important function of tapasin is exerted by a process that is independent of tapasin's role in TAP stabilization and in bridging MHC
class I molecules with TAP.
In the final analysis, our data favor the hypothesis that a critical
function of tapasin is to transform class I molecules into the high
affinity peptide-receptive form. In the absence of tapasin, HLA-G
molecules cannot bind their natural ligands with high affinity
peptides, despite an abundant supply. Increasing the high affinity
peptide pool cannot overcome the requirement of tapasin for fast
transport kinetics and surface expression of HLA-G. These results
support the notion that a conformational change of HLA-G by tapasin is
a prerequisite for the binding of high affinity peptides. In contrast
to tapasin-dependent HLA-G, HLA-G/E114H, the
tapasin-independent phenotype, has a peptide-binding groove that
seemingly forms the open state to fit the appropriate peptides,
regardless of tapasin. Accordingly, HLA-G/E114H molecules are capable
of binding a broad spectrum of the peptide repertoire without the
assistance of tapasin and are subsequently transported to the cell
surface. The functional consequence of the failure of HLA class I heavy
chains to associate with tapasin is substantial impairment in the
loading of endogenously generated peptides. Interestingly, our results
show that the tapasin-dependent HLA-G molecules are not
empty in tapasin-negative cells but are associated with endogenous
self-peptides, ones with low affinity. These HLA-G complexes recycle
between the ER and the cis-Golgi until they bind high
affinity peptides, underscoring the role of not only the ER but also
the post-ER compartments in the quality control of HLA-G molecules.
Under the otherwise identical experimental conditions, the addition of
tapasin renders HLA-G molecules capable of loading peptide ligands with
high affinity and allows their transport beyond the medial Golgi to the
cell surface. The phenotype of HLA-G in the absence of tapasin clearly
differs from the phenotype of class I molecules expressed in cells
either lacking functional TAP or In summary, the role of tapasin in peptide loading can be viewed as a
process with several distinct steps. To avoid ER-mediated degradation,
newly synthesized HLA-G initially binds a variety of peptides with
different affinities, whichever peptides are available in the vicinity.
Upon association with tapasin, the binding cleft of heavy chains
undergoes the conformational change into a high affinity,
peptide-receptive form, probably by breaking conserved hydrogen bonds
between the peptide backbone and MHC class I side chains. In turn, the
substitution of suboptimal to optimal peptides occurs by the
competition of high affinity peptides with preloaded low affinity
peptides in the ER. Finally, high affinity peptides close the
peptide-binding groove efficiently and thereby initiate dissociation of
peptide-loaded MHC class I molecules from tapasin and subsequent
transport to the cell surface. In support of this model, it has been
reported that tapasin-associated and peptide-loaded H-2Ld
class I molecules display different conformations (41) and that tapasin
interacts with regions of the class I heavy chains that are sensitive
to the presence of peptide in the antigen binding cleft (42). These
events reflect the potential of tapasin to discriminate between the
high affinity and low affinity peptide-loaded conformation. Independent
evidence supporting an intracellular editing mechanism for class I
molecules has been provided by the observation that a specific
peptide-Kd complex dissociates more rapidly when retained
in the ER than when expressed at the cell surface and that sequential
peptide binding can occur in the ER (43). In this regard, tapasin
function might be comparable with the function proposed for HLA-DM,
which influences peptide loading and peptide selection in the MHC class II pathway. HLA-DM is suggested to keep "empty" MHC class II
molecules in a peptide-receptive, and probably more open, conformation
(44, 45).
The findings presented in this study not only reveal a critical
function of tapasin in class I assembly in the ER but also reveal
cooperation of post-ER subcellular compartments for maximizing the
quality control of proteins. Similar quality control mechanisms might
be imposed upon other class Ib molecules, such as HLA-E or HLA-F, for
effective presentation of a restricted set of peptide antigens.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-microglobulin (
2m). This complex includes calnexin, calreticulin,
ERp57, transporter associated with antigen processing (TAP), and
tapasin (1). MHC class I molecules are divided into two types based on
their polymorphism and levels of expression. The highly expressed
polymorphic class Ia molecules bind a diverse set of peptides derived
from the cytosol, whereas the less abundant, tissue-specific,
nonpolymorphic class Ib molecules bind a more restricted set of
peptides (2). Tapasin is indispensable for the proper function of the
class Ia antigen presentation pathway. In tapasin mutant mice, the
expression and stability of surface class I molecules are strongly
reduced. In tapasin double-negative cells, the presentation of
cytosolic antigens is markedly impaired (3). Defects in the development of CD8+ T cells and immune responses against some viruses were also
noted in tapasin double-negative mice (4). Like class Ia molecules,
tapasin plays an important role in the assembly and surface expression
of certain class Ib molecules such as HLA-E (5) and H2-M3 (6, 7).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2m was contained in the
pcDNA3.1/Neomycin vector (Invitrogen). Point mutations of HLA-G
were made by changing the codons by PCR with Pfu DNA polymerase (Stratagene). The sequences for all mutations were confirmed
by sequencing.
2m cDNA or human
2m
and human tapasin cDNAs. Stable transfectants expressing human
2m (NIH3T3.h
2m) were selected with 1 mg/ml G418 (Sigma). Stable transfectants expressing both human
2m and human tapasin (NIH3T3.h
2m.hTpn) were selected with 1 mg/ml G418 and 0.35 mg/ml hygromycin (Invitrogen). Tapasin expression was restored in 721.220 cells by transfection with
the cDNA encoding human tapasin, and transfectants were selected with 0.35 mg/ml hygromycin, giving rise to the 721.220.Tpn cell line.
The 721.220 and 721.220.Tpn cells were cultured in RPMI 1640 medium
(Invitrogen) containing 10% fetal bovine serum, 2 mM
glutamine, penicillin (50 units/ml), and streptomycin (50 µg/ml). The
HLA-G-specific monoclonal antibody (mAb) G233 was a gift from Dr. Loke
(University of Cambridge). The mAb W6/32 recognizes only MHC class I
heavy chains associated with
2m. Polyclonal rabbit K455
antibody reacts with MHC class I heavy chains and
2m in both assembled and nonassembled forms. The rabbit polyclonal antibody to human tapasin (R.gp48N) was a gift from Dr. Cresswell. The rabbit
polyclonal antibody to PDI (SPA-890) and the anti-
-COP mAb M3A5 were
purchased from Stressgen (Victoria, Canada) and Sigma, respectively.
The mAb to the cis-Golgi marker GM130 was purchased from BD
Transduction Laboratories (Franklin Lakes, NJ.). Fluorescein
isothiocyanate-conjugated goat anti-mouse IgG and Texas Red-conjugated
goat anti-mouse IgG were purchased from Jackson ImmunoResearch
Laboratories, Inc. (West Grove, PA). Horseradish peroxidase-conjugated
streptavidin was purchased from Pierce.
-mercaptoethanol (Sigma).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (41K):
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Fig. 1.
Tapasin dependence of HLA-G for cell surface
expression and intracellular maturation. 721.220 and 721.220.Tpn
cells were transfected with DNA encoding the indicated heavy chains.
A, surface expression level of HLA-G and its mutant alleles
was measured by fluorescence-activated cell sorting analysis.
Fluorescence-activated cell sorting histograms are shown for cells
without tapasin (thin lines) and with tapasin
(thick lines). Staining of mock-transfected cells
is shown by dotted lines (control). To examine
the effect of tapasin on intracellular transport of HLA-G
(B) and HLA-G/E114H (C), cells were labeled with
[35S]methionine for 25 min and chased for the indicated
times. To test whether a supply of high affinity peptide ligands can
overcome the lack of tapasin for efficient transport of HLA-G, we
cotransfected 721.220.Tpn (D) and 721.220 cells
(E) with minigenes encoding high affinity peptides for HLA-G
and DNAs encoding HLA-G or HLA-G/E114H. Cell lysates were
immunoprecipitated with mAb G233 and then digested for 16 h with
(+) or without ( ) endo-H. Proteins were separated by 12% SDS-PAGE.
Endo-H-resistant (r) and endo-H-sensitive (s)
protein bands are indicated.
View larger version (33K):
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Fig. 2.
Tapasin dependence of HLA-G for high affinity
peptide loading. Peptide loading of HLA-G and HLA-G/E114H was
measured by using microsomal membranes derived from 721.220 cells
(A) and 721.220.Tpn (B). The peptide transport
assay was performed using the microsomal membrane derived from
tapasin-negative 721.220 cells. The amount of translocated peptides
into the microsomal membrane derived from expressing HLA-G/E114H was
compared with the value obtained from cells expressing wild-type HLA-G
(C).
2m cells and NIH3T3.h
2m.hTpn
cells and then treated cells with nocodazole. Nocodazole disrupts
microtubules, leading to disintegration of the Golgi and interruption
of traffic between the Golgi and the ER (28). On the basis of cell size
and ease of manipulation, we used NIH3T3 cells for the
immunofluorescence and confocal microscopy experiments throughout the
study. In the absence of high affinity peptides, in nocodazole-treated
cells, both HLA-G and HLA-G/E114H exhibited the punctate staining
pattern around the perinuclear region regardless of tapasin (Fig.
3, A and B), an
indication of recycling. In the presence of high affinity peptides and
tapasin, the staining pattern for HLA-G and HLA-G/E114H remained
unchanged after nocodazole treatment (Fig. 3D), indicating
that these molecules were not recycled back to the ER. In the presence
of high affinity peptides but in the absence of tapasin, the
distribution of wild-type HLA-G changed to a punctate perinuclear
pattern after nocodazole treatment, but the staining pattern for
HLA-G/E114H remained unchanged after treatment (Fig. 3C).
These results suggest that in the absence of tapasin, HLA-G cannot be
transported to the cell surface, despite an abundant supply of high
affinity peptides. Somehow, HLA-G exits the ER, but instead of
transport to the cell surface, HLA-G is retrieved back to the ER.
View larger version (61K):
[in a new window]
Fig. 3.
Effect of tapasin on retrieval of HLA-G.
NIH3T3.h 2m cells (A and C) and
NIH3T3.h
2m.hTpn cells (B and D)
were transfected with the minigene encoding high affinity peptide
ligands for HLA-G (C and D) or with the empty
vector (A and B). The cells were incubated
in the presence (+Nz) or absence (
Nz) of 20 µM nocodazole for 5 h. Cells were then fixed,
permeabilized, and labeled with the G233 antibody. The samples were
analyzed for intracellular localization by immunofluorescence
microscopy.
View larger version (26K):
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Fig. 4.
Intracellular fate of HLA-G in the absence of
tapasin. NIH3T3.h 2m (tapasin-negative)
(A and B) and NIH3T3.h
2m.hTpn
(tapasin-positive) (C and D) cells were
transfected with HLA-G alone (A and C) or HLA-G
and the minigene encoding high affinity peptides (B and
D). Cells were fixed, permeabilized, and
double-immunostained with the K455 antibody for HLA-G (left
column) and with anti-GM130 for endogenous cis-Golgi marker proteins
(middle column). Co-localization of HLA-G or
HLA-G/E114H with marker proteins was analyzed by confocal laser
microscopy. The right column shows the merged
images.
-COP, a
subunit of coatomer, as functions of tapasin and availability of high
affinity peptides. Proteins can be retrieved to the ER by retrograde
transport from the Golgi complex by COPI-coated vesicles (30). The
-COP association of HLA-G and HLA-G/E114H with high affinity
peptides was weaker than the
-COP association of these molecules
without high affinity peptides (Fig. 5).
In the absence of tapasin and high affinity peptides,
immunoprecipitation of HLA-G coprecipitated a substantial amount of
-COP (Fig. 5A, lane 3), whereas
much less
-COP coprecipitated with HLA-G/E114H (Fig. 5A,
lane 4). This differential binding between HLA-G
or HLA-G/E114H and
-COP was more evident in cells expressing high affinity peptides but no tapasin (Fig. 5B, lanes
3 and 4). In the presence of both high affinity
peptides and tapasin, a negligible amount of
-COP was
coimmunoprecipitated for both HLA-G and HLA-G/E114H (Fig.
5B, lanes 5 and 6). Taken
together, these results indicate that tapasin is essential for HLA-G
molecules to load high affinity peptides, escape retrieval, and
transport forward to the cell surface.
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Fig. 5.
Interaction of HLA-G with
-COP as a function of tapasin and high affinity
peptide ligands. 721.220 and 721.220.Tpn cells were transfected
with DNA encoding the indicated heavy chains alone (A) or
with DNAs encoding the heavy chains and high affinity peptide
ligands (B). Cells were lysed by 1% digitonin, and lysates
were immunoprecipitated (I.P.) with the indicated
antibodies. The immunoprecipitated proteins were separated by SDS-PAGE
and blotted (I.B.) with anti-
-COP antibody. As
a loading control, the same membranes were reblotted with the K455
antibody (bottom panels).
2m. Such unfolded class I
molecules cannot be recognized by the
conformation-dependent mAb W6/32. Hence, we performed
temperature stability assays on cell lysates prepared in pulse-chase
experiments. No class I heterodimers for both HLA-G and HLA-G/E114H
could be immunoprecipitated from cells expressing ICP47, a herpes
simplex virus protein that stops peptide translocation by the TAP
heterodimer (34), after the lysates were incubated at 37 °C for
1 h at both chase time points (Fig.
6, A and B,
lanes 9-16). Surprisingly, even in the absence
of both ICP47 and tapasin, not only tapasin-independent HLA-G/E114H but
also tapasin-dependent HLA-G molecules acquired thermostability even at the 0-min chase point, suggesting that "tapasin-dependent" HLA-G molecules are loaded with
endogenous peptides immediately after synthesis.
View larger version (62K):
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Fig. 6.
Effect of tapasin on thermostability of HLA-G
complexes. 721.220 (A) and 721.220.Tpn cells
(B) were transfected with DNA encoding the indicated heavy
chains alone or with DNAs encoding the indicated heavy chains and
ICP47. Cells were metabolically labeled for 15 min and chased for 30 min. After lysis in 1% Nonidet P-40, lysates were divided into two
aliquots and either kept on ice or incubated at 37 °C for 1 h
prior to immunoprecipitation with the mAb W6/32.
View larger version (66K):
[in a new window]
Fig. 7.
ER-mediated degradation of empty HLA-G
molecules. 721.220 and 721.220.Tpn cells were transfected with DNA
encoding the indicated heavy chains alone (B) or with DNAs
encoding the indicated heavy chains and ICP47 (A). Cells
were metabolically labeled for 15 min and chased for the indicated time
periods. The cells were lysed in 1% Nonidet P-40, and the resulting
lysates were immunoprecipitated with the K455 antibody.
Immunoprecipitates were analyzed by 12% SDS-PAGE.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2m. If
2m is absent, class I heavy chain molecules fail to
transport to the cell surface and are degraded (38). MHC class I
complexes that are expressed in mutant cell lines lacking TAP function
are devoid of peptides and are also degraded in the ER or in the
ER-Golgi compartment (39, 40). In corroboration of these observations,
in ICP47-expressing cells, the resulting peptide-deficient HLA-G is
strictly retained and degraded in the ER. Given the fact that the
correct assembly of the class I heavy chain with
2m and
a peptide is necessary for transport of the complex out of the ER to
the cell surface, class I molecules that leave the ER in tapasin
knockout mice (4) could be fully conformed with peptide, but these
peptides would be of generally lower affinity. These results suggest
that tapasin, although not required for assembly of MHC class I
molecules, provides a gating mechanism for reducing the expression of
class I molecules containing suboptimal peptides. However, the
suboptimal peptides might play an important role in preventing HLA-G
from the otherwise prolonged ER retention that could increase the risk
of recognition by a quality control system in the ER. In this way,
HLA-G molecules might have multiple chances to bind high affinity
peptides in a single lifetime.
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ACKNOWLEDGEMENTS |
---|
We are very grateful to Drs. P. Cresswell and Y. Loke for providing materials.
![]() |
FOOTNOTES |
---|
* This work was supported by a grant from the 21C Frontier for Functional Genome Analysis of the Human Genome.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Graduate School of
Biotechnology, Korea University, 1, 5-ka, Anam-dong, Sungbuk-Gu, Seoul
136-701, Korea. Tel.: 82-2-3290-3445; Fax: 82-2-927-9028; E-mail:
ksahn@korea.ac.kr.
Published, JBC Papers in Press, February 11, 2003, DOI 10.1074/jbc.M212882200
2 B. Park and K. Ahn, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
ER, endoplasmic
reticulum;
2m,
2-microglobulin;
TAP, transporter associated with antigen processing;
MHC, major
histocompatibility complex;
mAb, monoclonal antibody;
PBS, phosphate-buffered saline;
endo-H, endoglycosidase-H;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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REFERENCES |
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