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J Biol Chem, Vol. 275, Issue 3, 1581-1586, January 21, 2000
From Tumor Immunology, Lund University, Solvegatan 21, s-22362 Lund, Sweden
The transporter associated with antigen
processing (TAP) binds peptides in its cytosolic part and subsequently
translocates the peptides into the lumen of the endoplasmic reticulum
(ER), where assembly of major histocompatibility complex (MHC) class I
and peptide takes place. Tapasin is a subunit of the TAP complex and
binds both to TAP1 and MHC class I. In the absence of tapasin, the
assembly of MHC class I in the ER is impaired, and the surface expression is reduced. To clarify the function of tapasin in the processing of antigenic peptides, we studied the interaction of peptide
and TAP, peptide transport across the membrane of the ER, and
association of peptides with MHC class I molecules in the microsomes
derived from tapasin mutant cell line 721.220, its sister cell line
721.221 expressing tapasin, and their HLA-A2 transfectants. The binding
of peptides to TAP in tapasin mutant 721.220 cells was significantly
diminished in comparison with 721.221 cells. Impaired peptide-TAP
interaction resulted in a defective peptide transport in tapasin mutant
721.220 cells. Interestingly, despite the diminished peptide binding to
TAP, the transport rate of TAP-associated peptides was not
significantly altered in 721.220 cells. After transfection of tapasin
cDNA into 721.220 cells, efficient peptide-TAP interaction was
restored. Thus, we conclude that tapasin is required for efficient
peptide-TAP interaction.
Major histocompatibility complex
(MHC)1 class I molecules
present antigenic peptides to cytotoxic T cells (1-4). The processing of MHC class I-presented peptides includes degradation of cytosolic proteins, translocation of generated peptides into the lumen of the
endoplasmic reticulum (ER), and the assembly of MHC class I with
peptides (2, 5). The transporter associated with antigen processing
(TAP) (2, 5) transports peptides across the membrane of the ER. TAP was
first discovered as a heterodimeric complex composed of two proteins,
TAP1 and TAP2, each consisting of C-terminal hydrophilic domains that
bind ATP and peptides and N-terminal multimembrane-spanned hydrophobic
domains (5). The feature of TAP1 and TAP2 proteins were revealed as
members of the ABC transporter family (5). TAP1 and TAP2 bind short
peptides of 7-12 amino acids and have broad specificity (6-8). The
efficient binding of peptides requires expression of both TAP1 and TAP2 (8). The interaction of TAP and peptides is ATP-independent (6-9). The
addition of ATP dissociates peptides from TAP and stimulates the
assembly of peptide and MHC class I in the lumen of the ER indicative
of translocation of TAP-released peptide across the membrane of the ER
(6-9). Immunoprecipitation with anti-TAP1 antiserum demonstrated that
TAP associates with MHC class I heavy
chain- The importance of MHC class I-TAP interaction in peptide loading was
opposed by recent findings, in which the ER luminal domain of tapasin
(soluble tapasin) was expressed in 721.220 cells. Soluble tapasin
associated with class I but did not interact with TAP (16). In the
absence of interaction with TAP, soluble tapasin restored surface
expression of MHC class I and the presentation of viral peptides to CTL
(16). The conclusion from this study was that tapasin-MHC class I
interaction, but not tapasin-TAP interaction, was required for peptide
loading (16). However, there are conflicting reports concerning the
importance of the tapasin-MHC class I interaction in promoting peptide
loading. It has been reported that HLA-B27 and HLA-A2 could assemble
with peptide in tapasin mutant cells, although HLA-B27 and HLA-A2 could interact with tapasin in wild type cells (17, 18). In addition, a
murine mutant MHC class I Dd (E222K) was discovered having a Glu to Lys
mutation at residue 222 of the heavy chain. This mutation caused
deficiency in interaction with tapasin (19). Significant peptide
loading onto Dd (E222K) was observed (19). These results indicate that
the interaction of tapasin and MHC class I is not essential for peptide loading.
In this study, we used reporter peptides in which the Cells--
721.220 and 721.221 cell lines and 721.220-HLA-A2 and
721.221-HLA-A2 cell lines were kindly provided by Drs. J. C. Solheim and T. Elliott, respectively. The 721.174 cell line was a gift from Dr. S. Kvist The cell lines were cultured in RPMI 1640 medium (Life Technologies, Inc.), supplemented with 10% heat-inactivated fetal bovine serum, 100 IU/ml penicillin, 100 mg/ml streptomycin, and 2 mM glutamine at 37 °C in a 5% CO2 atmosphere.
The cDNA encoding wild type human tapasin was constructed into
pCEP4 expression vector (CLONTECH). The constructs
were transfected into 721.220 cells by electroporation. Transfected
cells were selected in hygromycin-containing medium (Life Technologies,
Inc.) and then cloned by limiting dilution. Expression of transfected tapasin was measured by immunoblotting with anti-human tapasin antiserum.
Antibodies--
The monoclonal antibody BB7.2, specific for
HLA-A2, and rabbit antiserum to human MHC class I (R425) were kindly
provided by Dr. S. Kvist. Rabbit antisera against human TAP1 and
tapasin were described previously (9, 14). The polyclonal antibodies were affinity-purified before use.
Metabolic Labeling, Immunoprecipitation, and
Immunoblotting--
Cells were washed twice with phosphate-buffered
saline and incubated for 15 min at 37 °C in methionine-free RPMI
1640 medium containing 3% dialyzed fetal bovine serum. Then 0.2 mCi/ml
[35S]methionine (Amersham Pharmacia Biotech) was added,
and the incubation was continued for 60 min. At the end of labeling,
cells were washed three times with ice-cold phosphate-buffered saline
and lysed in 1% digitonin (Sigma) or 1% Nonidet P-40 lysis buffer
containing 0.15 M NaCl, 25 mM Tris-HCl, pH 7.5, 1.5 mg/ml iodoacetamide, and a mixture of protease inhibitors (2 mM phenylmethylsulfonyl fluoride, 10 mg/ml leupeptin, 30 mg/ml aprotinin, 10 mg/ml pepstatin). The cleared lysates were added to
antibodies previously bound to protein A-Sepharose beads (Amersham
Pharmacia Biotech, Uppsala, Sweden). After washing, the
immunoprecipitates were analyzed by SDS-PAGE. Western blotting and FACS
analysis were performed as described previously (9).
Peptides and Peptide Modification--
All peptides were
synthesized in a peptide synthesizer (Applied Biosystems, model 431A),
using conventional Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry. Peptides were subsequently purified by high pressure liquid
chromatography and dissolved in phosphate-buffered saline. The
Preparation of Microsomes and Photocross-linking--
Microsomes
from cell lines were prepared and purified according to Saraste
et al. (20). For photocross-linking,
125I-labeled and ANB-NOS-modified peptide was mixed with 20 µl of microsomes (concentration of 60 A280/ml)
to a final concentration of 100 nM, in RM buffer (250 mM sucrose, 50 mM triethanolamine-HCl, 50 mM KOAc, 2 mM MgOAc2, 1 mM dithiothreitol). This mixture was then kept at 26 °C
for 5 min UV irradiation was subsequently carried out for 5 min on ice
at 366 nm. Microsomal membranes were recovered by centrifugation
through a 0.5 M sucrose cushion in RM buffer containing 1 mM cold peptide (unlabeled peptide without ANB-NOS modification). The microsomal membranes were washed once with cold RM
buffer, lysed by 1% digitonin or 1% Nonidet P-40, and subjected to
immunoprecipitation. Cross-linked microsomal proteins were
immunoprecipitated with specific antiserum. The precipitates were
analyzed by SDS-PAGE or quantitated by a Interaction of Peptides with TAP in Microsomes of Tapasin Mutant
721.220 Cells and Wild Type Cells--
It was previously reported that
the peptide transport across the ER membrane is a stepwise process
including an ATP-independent interaction of peptide and TAP and a
subsequent ATP-dependent translocation of peptide (6, 9).
To investigate the involvement of tapasin in TAP function, three
reporter peptides (see "Materials and Methods") were modified with
cross-linker and labeled with 125I as described previously
(7) and incubated with microsomes derived from the tapasin mutant cell
721.220, its sister cell line 721.221 with wild type tapasin, and a TAP
mutant cell line 721.174 in the absence of ATP. After incubation, the
microsomes were irradiated by UV to induce cross-linking. The
peptide-bound TAP molecules were precipitated by anti-TAP1 antiserum.
Analysis of precipitates revealed efficient binding of all three
reporter peptides to TAP in microsomes from 721.221 (Fig.
1A, lanes
2, 4, and 6). Significantly diminished
peptide-TAP interaction was observed in tapasin mutant microsomes (Fig.
1A, lanes 3, 5, and 7). The expression level of TAP1 and TAP2 in 721.221 and
721.220 was very similar, as indicated by immunoprecipitation with
anti-TAP1 antiserum (Fig. 1B, lanes
2-3). TAP mutant 721.174 served as a negative control in
both cross-linking and immunoprecipitation experiments (Fig. 1,
lane 1, in A and B). A
similar experiment performed with microsomes of tapasin-transfected
721.220 cells demonstrated restoration of an efficient peptide binding
to TAP (Fig. 2). These results indicate
that tapasin is required for an efficient peptide-TAP interaction.
Low Affinity Peptide-TAP Interaction in Tapasin Mutant 721.220 Cells--
To assess the binding affinity of peptide to TAP in 721.221 or 721.220 cells, 100 nM of cross-linker-modified reporter
peptide, 125I-OVA-ANB-NOS, was incubated with 721.221 or
721.220 microsomes in the presence of native and unlabeled OVA peptide
at different concentrations. In order to dissect the peptide binding
from the peptide translocation, the assay was done in the absence of
ATP as previously reported (9). Again a significant deficiency of
peptide-TAP interaction was detected in 721.220 microsomes in the
absence of competing peptide (Fig. 3,
lane 1). The peptide binding to TAP in 721.220 microsomes was completely competed by a lower concentration (400 nM) of native peptide (Fig. 3), whereas in 721.221 microsomes, more than 1.6 µM concentration was required for completely competing away reporter peptide binding (Fig. 3). These
data indicate that the affinity for peptide binding to TAP in tapasin
mutant cells is much lower than in wild type cells.
Peptide Transport Efficiency Is Not Reduced in Tapasin Mutant
Cells--
After having demonstrated a lower peptide binding to TAP in
tapasin mutant cells, we examined the transport efficiency by measuring
the time for translocation of 50% TAP-bound peptides into the
microsomes of wild type, 721.221A2, or tapasin mutant cells, 721.220A2,
in the presence of ATP. After incubation of 125I-MP-ANB-NOS
peptide with microsomes from 721.220A2 or 721.221A2 cells, the excess
125I-MP-ANB-NOS peptides were washed off. Peptide-loaded
microsomes were then resuspended in transport buffer with 100 µM ATP and incubated for different periods of time. After
incubation, the microsomes were lysed in 1% Nonidet P-40 lysis buffer
and subsequently precipitated with anti-TAP1 or anti-HLA-A2 antibodies.
The peptide-bound TAP or HLA-A2 molecules were quantitated by a
The Binding of Peptides to HLA A2 in Microsomes from 721.221A2 and
721.220A2 Cells--
As shown above, the peptide-TAP interaction is
deficient in tapasin mutant cells. To further investigate MHC class I
assembly in the absence of tapasin, intact microsomes of 721.221A2 and 721.220A2 cells were mixed with 125I-MP-ANB-NOS peptide,
which binds to HLA-A2, in the presence of ATP. The peptide
translocation was monitored by 125I-MP-ANB-NOS peptide
cross-linked HLA-A2. After cross-linking, microsomes were lysed with
1% digitonin. The cleared lysates were aliquoted and precipitated with
anti-TAP1 and anti-HLA-A2, respectively. Consistent with the results
shown in Fig. 1, anti-TAP1 recovered peptide-bound TAP and HLA-A2 in
721.221A2 microsomes (Fig. 5, lane 3), but only a weak peptide cross-linked TAP
band was detected in 721.220A2 microsomes (Fig. 5, lane
2). With anti-HLA-A2 antibody, less peptide-cross-linked
HLA-A2 was recovered in 721.220A2 microsomes (Fig. 5, lane
6) in comparison with 721.221A2 microsomes (Fig. 5,
lane 4). In addition, a weak peptide-cross-linked
tapasin was also detected (Fig. 5, lane 4), which
corresponds to our previous findings (14, 21). The less
peptide-cross-linked HLA-A2 in 721.220A2 microsomes (Fig. 5,
lane 6) was correlated with reduced HLA-A2
surface expression (Fig. 6). However, in
721.220A2 microsomes, anti-HLA-A2 could still precipitate a significant
amount of peptide-bound A2 (Fig. 5, lane 6),
which was compatible with the amount of peptides bound to TAP (Fig. 5,
lane 2) and with the amount of HLA-A2 expressed on the cell surface (Fig. 6).
These results suggest that the reduced surface expression and
translocated peptides in the ER of 721.220A2 cells result from deficient peptide-TAP interaction. Moreover, despite the deficiency of
peptide-TAP interaction in 721.220A2 cells, TAP still can transport peptides in the presence of ATP, which is compatible with previous findings (12).
Interaction of Peptide-loaded HLA-A2 with Tapasin--
Previously,
it was reported that the assembly of peptide and MHC class I in tapasin
mutant cells was defective (8). We have previously demonstrated that
murine tapasin associated with peptide-bound H-2Kb (21). To
further confirm the interaction of peptide-bound MHC class I with human
tapasin, purified microsomes from 721.220A2 and 721.221A2 were
incubated with 125I-MP-ANB-NOS peptide in the presence of
ATP. Anti-tapasin antiserum precipitated peptide-bound HLA-A2 and TAP
in 721.221A2 but not in 721.220A2 microsomes (Fig.
7, lanes 1 and
2). This result clearly indicates that tapasin does not
exclusively bind to peptide free MHC class I.
The function of TAP to mediate peptide transport into the ER is
well established (2, 5, 8). TAP interacts with peptides at its
cytosolic part and with MHC class I at its luminal part (6-9). The
importance of a direct interaction between peptide and TAP for peptide
translocation was clearly demonstrated by the findings in which the
herpes simplex virus ICP47 protein inhibits the MHC class I antigen
presentation pathway by occupying the peptide binding site on TAP
(22-24). The association of MHC class I and TAP is mediated by tapasin
(13-15, 18, 21). Tapasin is a type I ER membrane protein and bridges
the association of MHC class I with TAP (13, 14). Cells with mutated
tapasin have a defective surface expression of MHC class I (12), and
this defect can be corrected by transfection of wild type tapasin
cDNA (13). Tapasin was suggested to function by promoting the
peptide loading onto MHC class I (8). In the present study, we
systematically examined the peptide interaction with TAP, the peptide
translocation into the ER, and the peptide assembly with MHC class I in
microsomes purified from tapasin mutant cells and wild type cells as
well. A severe defect in peptide-TAP interaction was revealed in the tapasin mutated cell 721.220. In contrast, the peptide translocation across the membrane of the ER was intact, as indicated by the same off
rate of TAP-associated peptides in tapasin mutated and wild type
microsomes in the presence of ATP. The translocated HLA-A2-binding
peptide could bind to HLA-A2 molecules in both tapasin mutated and wild
type microsomes, although the amount of peptide-associated HLA-A2 in
721.220-A2 cells was much less than that in its sister cell line
721.221-A2 expressing wild type tapasin. This reduction was due to the
deficient interaction between peptide and TAP.
Tapasin is a subunit of the TAP complex as indicated by the consistent
and stoichiometric association of tapasin with TAP1 and TAP2 (14).
Previously, it was reported that peptide transport in tapasin mutant
cells was not altered (12). In that study, translocation of peptides in
721.220 and wild type cells was analyzed by recovery of a glycosylated
reporter peptide in streptolysin O-permeabilized cells. Since
TAP-dependent peptide translocation was intact in tapasin
mutant cells, it was conceivable that accumulation of translocated
peptides in the ER might result from the decay kinetics of transported
peptides in tapasin mutant cells. Restored peptide-TAP binding and
peptide translocation in tapasin-transfected 721.220 cells clearly
demonstrated that tapasin is required for efficient peptide binding to
TAP. In agreement with our results from tapasin-transfected 721.220, a
study of 721.220B8 tapasin transfectant also revealed a more than
4-fold increase in peptide translocation after expressing tapasin in
721.220B8 cells (16). In addition, it has recently been reported that
the C-terminal region of tapasin was identified as a binding site of
TAP (25). Transfection of the C-terminal region of tapasin enhanced the function of TAP in tapasin mutant cells (25).
In previous studies (14, 21) as well as the present studies (Fig. 5,
lane 4), we found a weak interaction of peptides and tapasin in the presence of ATP but not in the absence of ATP. Since
the peptide-TAP interaction is independent of ATP, it is not likely
that tapasin is involved in the peptide binding site on the TAP
complex. Both TAP1 and TAP2 are required for efficient peptide binding,
certain peptides binding preferentially to TAP1 and others to TAP2
(10). Although peptide binding to human TAP is relatively promiscuous,
the difference of binding affinity of various peptides can be as great
as 3 orders of magnitude, depending on both the terminal and the
internal sequence of the peptides (7, 26). Among three peptides tested
in this study, similar deficiency in their ability to bind to TAP of
721.220 cells suggested that the requirement of tapasin for peptide-TAP interaction is not based on the sequence of the peptides.
Searching for peptide-binding sites, the regions of the TAP subunits to
which the photoactive peptides bind were mapped by proteolysis of the
TAP proteins after photocross-linking and immunoprecipitation with
antisera specific for distinct hydrophilic regions (27, 28). The
results suggest that the binding site is composed of multiple regions
of TAP1 and TAP2. Interaction of the same photoreactive peptide to
multiple regions of TAP1 and TAP2 may indicate a large binding pocket
(27, 28). This study also suggested that the binding regions are very
close to or part of the transmembrane domains. The interaction of
tapasin with TAP may largely regulate the peptide binding site.
Recently, the ER luminal domain of tapasin was expressed as a soluble
form of tapasin in 721.220 cells (16). Soluble tapasin bound to MHC
class I, but not to TAP. The peptide transport was measured in 721.220, 721.220 transfected with soluble tapasin, and 721.220 with wild type
tapasin. Results showed that wild type tapasin increased the efficient
peptide translocation in 721.220 cells, but soluble tapasin
transfectants retained their deficient peptide transport (16). Taken
together, these results and our own indicate that the interaction of
tapasin and TAP is critical for efficient peptide transport. Tapasin
may function to stabilize the peptide-binding site of TAP.
Since tapasin directly interacts with MHC class I heavy chain and
We thank Drs S. Kvist and T. Elliott for
providing the 721.174 cell line and the 721.220A2 and 721.221A2 cell
lines, respectively; Dr. J. C. Solheim for providing the 721.220 and 721.221 cell lines; and Dr. S. Kvist for generous gifts of antisera.
*
This study was supported by Crafoordska Stiftelsen Grant
990589, Swedish Cancer Society Grant 3975-B98-02XBB, a grant from the
Medical faculty, Lund University, and grants from the Foundation for
Strategic Research (Infection and Vaccionlogy Grant 36/98 and
Inflammation Research Program Grant 99).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.
The abbreviations used are:
MHC, major
histocompatibility complex;
TAP, transporter associated with antigen
processing;
ER, endoplasmic reticulum;
tapasin, TAP-associated
glycoprotein;
PAGE, polyacrylamide gel electrophoresis;
FACS, fluorescence-activated cell sorting;
MP, membrane peptide;
OVA, ovalbumin;
ANB-NOS, N-(5-azido-2-nitrobenzoyloxy)succinimide.
Tapasin Is Required for Efficient Peptide Binding to Transporter
Associated with Antigen Processing*
,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2-microglobulin dimer (10, 11). The importance of
the TAP-MHC class I interaction for the assembly of MHC class I and
peptides was suggested by the finding in which deficiency in MHC class
I surface expression was found in a cell line, 721.220, lacking
interaction of MHC class I and TAP (12). With anti-TAP1 antiserum, a
48-kDa protein (tapasin) was coprecipitated, and this protein was
missing in 721.220 cells, indicative of requirement of tapasin in the
interaction of MHC class I and TAP (13). cDNA cloning of tapasin
revealed a type I membrane protein with a cytoplasmic tail containing a
double lysine motif known to maintain membrane proteins in the ER (14,
15). Immunoprecipitation with anti-TAP1 or anti-tapasin antisera
demonstrated a consistent and stoichiometric association of tapasin and
TAP1 and TAP2 (14). The importance of tapasin in MHC class I antigen
presentation was demonstrated by restored MHC class I surface
expression and class I-TAP interaction in 721.220 cells after
transfection with tapasin cDNA (15), suggesting that either tapasin
directly regulates the assembly of MHC class I and peptide or the
association of MHC class I with TAP enhances peptide loading.
-amino group
of lysine was covalently modified by coupling a chemical cross-linker
(ANB-NOS), and the tyrosine was labeled by iodination (125I). These modifications allowed photocross-linking of
the reporter peptides to TAP and MHC class I and enabled us to monitor
the peptide binding to TAP, peptide translocation, and assembly of peptide and MHC class I in purified microsomes derived from tapasin mutant cells, 721.220, and its sister cells, 721.221, as well as their
HLA-A2 transfectants. Our results clearly indicate that tapasin is
required for efficient peptide-TAP interaction.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-amino group of lysine in peptides of influenza-A virus
nucleoprotein, NP383-391R389K (SRYWAIKTR); influenza-A matrix protein
(MP), M58-64G58YF62K (YILGKVFTL); and OVA 257-264I259Y (SIYNFEKL)
were covalently modified by a photoreactive cross-linker as described
previously (7). An aliquot (1 µg) of the peptide was labeled by
chloramine T-catalyzed iodination (125I). The modification
and labeling experiments were performed in the dark. The cross-linker
modified and 125I-labeled peptides are referred to as
125I-OVA-ANB-NOS or 125I-MP-ANB-NOS or
125I-nucleoprotein-ANB-NOS.
-counter. Cross-linking reactions with transport buffer containing 1 mM ATP were
performed as described previously (7, 14). For peptide competition, 100 nM of the 125I-labeled and ANB-NOS-modified
peptide was mixed with a 10-fold molar excess or with the
concentrations indicated in Fig. 3 of unlabeled and unmodified peptide
before the cross-linking reaction.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Binding of peptides to TAP in microsomal
membranes derived from tapasin mutant cells, 721.220, and cells
expressing wild type tapasin, 721.221. Three cross-linker-modified
peptides (OVA, nucleoprotein (NP), and MP) were labeled with
125I and incubated with microsomes from 721.220 (A, lanes 3, 5, and
7), from 721.221 (A, lanes
2, 4, and 6), and from 721.174 (A, lane 1) at 25 °C for 5 min and
subsequently irradiated under UV for 2 min on ice. After centrifugation
and washing, the membranes were solubilized. The cleared lysates were
precipitated by anti-TAP1 antiserum. The precipitates were analyzed on
SDS-PAGE. In order to quantitate the TAP proteins in these cell lines,
the cells were metabolically labeled with
[35S]methionine. TAP1 and -2 were precipitated by
anti-TAP1 antiserum from cellular lysates and analyzed on SDS-PAGE
(B).
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Fig. 2.
Restoration of efficient peptide-binding
capacity to TAP in 721.220 tapasin transfectant.
Cross-linker-modified OVA peptide was labeled with 125I and
incubated with microsomal membranes from 721.220 (A,
lane 1) or 721.220 tapasin transfectant
(A, lane 2). After cross-linking, the
cleared lysates were precipitated with an anti-TAP1 antiserum. The
precipitates were analyzed on SDS-PAGE. The expression of transfected
tapasin in 721.220 tapasin transfectant was detected by Western
blotting with anti-tapasin antiserum (B).
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Fig. 3.
Low affinity of peptide-TAP binding in
tapasin mutant 721.220 cells. Cross-linking was carried out with
microsomes and cross-linker-modified and 125I-labeled OVA
reporter peptide, 125I-OVA-ANB-NOS, in the absence or
presence of various concentrations of native and cold OVA peptide as
indicated. After cross-linking, the microsomes were lysed in 1%
Nonidet P-40 and subsequently precipitated with anti-TAP1 antiserum.
The precipitates were analyzed on SDS-PAGE.
-counter. In the presence of ATP, peptide rapidly dissociated from
TAP in both 721.220A2 and 721.221A2 microsomes, despite the fact that
the amount of peptide-bound TAP1 in 721.220A2 cells was lower (Fig. 4, upper panel).
Fifty percent dissociation of peptides from TAP was detected at an
early time point in both 721.220A2 and 721.221A2 microsomes (Fig. 4,
upper panel). Moreover, 50% of translocated peptides were recovered by anti-HLA-A2 antibody at the same time point
in both 721.220A2 and 721.221A2 microsomes, although a much lower
amount of peptide-bound A2 was obtained in 721.220A2 microsomes (Fig.
4, lower panel). These results indicated that
mutation of tapasin greatly affected peptide interaction with TAP but
affected the peptide translocation much less or not at all.
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Fig. 4.
Kinetics of peptide binding to TAP and HLA-A2
in 721.220As and 721.221A2 microsomes. Microsomes of 721.221A2 or
721.220A2 were incubated with cross-linker-modified and
125I-labeled HLA-A2 binding peptide in the absence of ATP.
After removing the excess peptide, the peptide-loaded microsomes were
resuspended in transport buffer in the presence of ATP (see
"Materials and Methods"). Aliquots of incubated microsomes were
pelleted at different time points as indicated, irradiated under UV,
and lysed by 1% Nonidet P-40 lysis buffer. The peptide-cross-linked
TAP and HLA-A2 molecules were recovered by anti-TAP1 or anti-HLA-A2
antibody. The precipitates were quantitated by a -counter. The
presented results are from one representative experiments out of three
performed.
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Fig. 5.
Reduced peptide assembly in 721.220A2 is
correlated with deficiency of peptide-TAP interaction. The
cross-linker-modified HLA-A2 binding peptide, MP, was incubated with
microsomes derived from 721.221A2 or tapasin mutant 721.220A2 in the
presence of ATP. After cross-linking, the cleared lysates were
precipitated by anti-TAP1 (lanes 1-3) and
anti-HLA-A2 (lanes 4-7), respectively. The
precipitates were analyzed on SDS-PAGE. In lanes
5 and 7, a 10-fold excess of native and unlabeled
MP peptide was added in the reaction. Microsomes of 721.174 were used
as a negative control (lane 1).
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Fig. 6.
Reduced surface expression of HLA-A2 in
721.220A2 cells. The cells of 721.220A2 or 721.221A2 were
incubated with BB7.2 antibody and subsequently with fluorescein
isothiocyanate-conjugated goat anti-mouse Ig antibody. After washing,
the BB7.2-labeled cells were analyzed by flow cytometry on a
FACScan.
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Fig. 7.
Tapasin associated with peptide-loaded
HLA-A2. The microsomes of 721.221A2 (lane 1)
and 721.220A2 (lane 2) were incubated with HLA-A2
binding peptides as described in the legend to Fig. 5. The cleared
lysates were precipitated with anti-tapasin antibody. The precipitates
were analyzed on SDS-PAGE.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2-microglobulin dimer, it was suggested that this
interaction enhances peptide loading onto class I molecules, possibly
due to a high local concentration of peptides or to the requirement of
tapasin itself for peptide-MHC class I assembly. Support for the
importance of MHC class I-TAP interaction was evidenced by the finding
of a deficient TAP-dependent peptide assembly of a mutant
HLA-A2.1 (29, 30). A point mutation of theronine 134 to lysine resulted
in HLA-A2.1 incapable of interacting with TAP (29, 30). Moreover, the
deficient assembly of HLA-A2.1 (T134K) was corrected by a direct
delivery of peptide to the ER in a TAP-independent manner. Therefore,
it was concluded that the interaction of TAP and MHC class I is
essential for peptide loading. Recently, this notion has been called
into question by the finding that a soluble tapasin restored the
surface expression of HLA-B8 in 721.220B8 cells, despite the lack of
interaction between MHC class I and TAP (16). Notably, soluble
tapasin-MHC class I interaction was unstable in detergent lysates and
required chemical cross-linking for their detection. Since soluble
tapasin lacks the motif of the ER retrieval signal present in the wild
type tapasin, the increased surface expression can be explained as a
lack of control of MHC class I retention in the ER. Tapasin may
regulate the MHC class I release from the ER rather than directly load
peptides on MHC class I. Recently, it was found that HLA-B27 and HLA-A2 presented peptides to cytotoxic T cells in tapasin mutant 721.220B27 or
721.220A2 cells (17, 31), although both alleles were found to associate
with tapasin in wild type cells. Furthermore, a mutant H2-Dd with a Glu
to Lys mutation at residue 222 (Dd E222K) was shown not to be
substantially impaired in its ability to present peptides (18) despite
its loss of ability for interaction with tapasin. Although there is a
lack of direct evidence for the involvement of tapasin in peptide
loading onto MHC class I, a recent study of the HLA-A2 T134K mutant
indicates that the interaction of MHC class I with the TAP complex may
be important for the optimization of MHC class I assembly (32). The
T134K mutant did not bind to tapasin or calreticulin, and it failed to
present endogenous viral peptides to T cells (32). However, the mutant
class I had the ability to bind peptides in vitro (32).
Therefore, it was suggested that the TAP complex is involved in a
quality control stage during MHC class I assembly. This view is
supported by the study of green fluorescence protein-tagged MHC class I
(33), which shows that peptide-loaded MHC class I can also be retained
in the ER for optimizing peptide loading. In the presented study, most
of the peptide-loaded HLA-A2 was found to be associated with tapasin in
721.221A2, which is in line with our previous finding of an association
of peptide-loaded mouse MHC class I with tapasin (21) and indicates
that tapasin may also regulate the transport of assembled MHC class I
to the cell surface.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed. Tel.: 46-46-2227848;
Fax: 46-46-2224606; E-mail: su-ling.li@wblab.lu.se and
ping.wang@wblab.lu.se.
ABBREVIATIONS
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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