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of June 17, 2017.
Interaction of Murine MHC Class I Molecules
with Tapasin and TAP Enhances Peptide
Loading and Involves the Heavy Chain α3
Domain
Woong-Kyung Suh, Michael A. Derby, Myrna F.
Cohen-Doyle, Gary J. Schoenhals, Klaus Früh, Jay A.
Berzofsky and David B. Williams
J Immunol 1999; 162:1530-1540; ;
http://www.jimmunol.org/content/162/3/1530
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Copyright © 1999 by The American Association of
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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References
Interaction of Murine MHC Class I Molecules with Tapasin
and TAP Enhances Peptide Loading and Involves the Heavy
Chain a3 Domain1
Woong-Kyung Suh,2* Michael A. Derby,† Myrna F. Cohen-Doyle,* Gary J. Schoenhals,‡
Klaus Früh,‡ Jay A. Berzofsky,† and David B. Williams3*
M
ajor histocompatibility complex (MHC) class I molecules signal the presence of intracellular pathogens
such as viruses by binding antigenic peptides and displaying them at the cell surface to cytotoxic T cells. Assembly of
the class I heavy chain (H chain)4 and b2-microglobulin (b2m)
subunits with peptides of approximately 8 –10 residues occurs in
the endoplasmic reticulum (ER), and the resulting heterotrimeric
complex is transported along the secretory pathway to the cell
surface. Whereas the H chain and b2m are cotranslationally translocated into the ER lumen, the bulk of class I binding peptides are
generated in the cytosol and then are delivered into the ER via
TAP. TAP is a member of the ATP-binding cassette family of
transporters, and it preferentially transports peptides of approximately 8 –15 residues in an ATP-dependent manner (reviewed in
Refs. 1 and 2).
Numerous studies have focused on proteins that participate in
class I assembly. In mouse cells, newly synthesized class I H
*Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada;
†
Metabolism Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; and ‡R. W. Johnson Pharmaceutical Research Institute, San Diego, CA 92121
Received for publication May 6, 1998. Accepted for publication October 26, 1998.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by a grant from the National Cancer Institute of Canada
with funds from the Canadian Cancer Society (to D.B.W.) and by a Steve Fonyo
studentship from the National Cancer Institute of Canada (to W.-K.S.).
2
Current address: Department of Immunology and Infectious Diseases, Harvard
School of Public Health, Boston, MA 02115.
3
Address correspondence and reprint requests to Dr. David B. Williams, Department
of Biochemistry, Medical Sciences Building, University of Toronto, Toronto, Ontario,
Canada, M5S 1A8. E-mail address: [email protected]
Abbreviations used in this paper: H chain, class I heavy chain; b2m, b2-microglobulin; ER, endoplasmic reticulum; Z-L3VS, carboxybenzyl-leucyl-leucyl-leucine vinyl
sulfone; pfu, plaque-forming units; Met, methionine; endo H, endoglycosidase H.
4
Copyright © 1999 by The American Association of Immunologists
chains associate with calnexin (3–5), a membrane-bound chaperone of the ER, and this interaction facilitates H chain folding and
promotes assembly with b2m (6). Calnexin remains bound to the
H chain following b2m association and participates in retaining
both the free H chain and H chain-b2m assembly intermediates in
the ER (7–9). Calreticulin, a soluble homologue of calnexin, has
also been shown to associate with H chain-b2m heterodimers but
not free H chains in mouse cells (10, 11). The relative abundance
of calnexin- vs calreticulin-associated class I heterodimers seems
to be variable in different murine cells (9 –11). Calnexin also binds
to free H chains in human cells (4, 7, 12), enhancing H chain
folding (13) and retaining this assembly intermediate in the ER
(14). However, in contrast to the situation in mouse cells, calnexin
largely dissociates upon H chain-b2m assembly and is replaced by
calreticulin (15, 16). Despite the demonstrated functions of calnexin, it is not essential in class I biogenesis, since class I assembly
occurs normally in a calnexin-negative cell line (17, 18). However,
it is likely that other ER chaperones, particularly calreticulin, can
replace calnexin under these conditions. At present, the functions
of calreticulin in class I biogenesis are poorly understood, although
it probably participates in ER retention of peptide-deficient assembly intermediates (19). Furthermore, calnexin and calreticulin appear to play a major role in recruiting another protein, ERp57, into
assembling class I complexes (see below).
In addition to chaperone interactions, H chain-b2m heterodimers,
but not free H chains, associate with TAP in both mouse and human
cells (20, 21) forming large complexes that contain calnexin or calreticulin, H chain, b2m, and TAP (9, 15). Stoichiometric analysis has
revealed that as many as four H chain-b2m heterodimers (and associated chaperones) may bind to a single TAP molecule (22). Very
recently, a resident ER protein termed ERp57 has been detected in
these complexes as well (23–25). ERp57 possesses both thiol oxidoreductase and cysteine protease activities, and it has been found
associated with diverse, newly synthesized proteins in the ER (26,
0022-1767/99/$02.00
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In human cells the association of MHC class I molecules with TAP is thought to be mediated by a third protein termed tapasin.
We now show that tapasin is present in murine TAP-class I complexes as well. Furthermore, we demonstrate that a mutant H-2Dd
molecule that does not interact with TAP due to a Glu to Lys mutation at residue 222 of the H chain (Dd(E222K)) also fails to bind
to tapasin. This finding supports the view that tapasin bridges the association between class I and TAP and implicates residue 222
as a site of contact with tapasin. The inability of Dd(E222K) to interact with tapasin and TAP results in impaired peptide loading
within the endoplasmic reticulum. However, significant acquisition of peptides can still be detected as assessed by the decay
kinetics of cell surface Dd(E222K) molecules and by the finding that prolonged viral infection accumulates sufficient target
structures to stimulate T cells at 50% the level observed with wild-type Dd. Thus, although interaction with tapasin and TAP
enhances peptide loading, it is not essential. Finally, a cohort of Dd(E222K) molecules decays more rapidly on the cell surface
compared with wild-type Dd molecules but much more slowly than peptide-deficient molecules. This suggests that some of the
peptides obtained in the absence of an interaction with tapasin and TAP are suboptimal, suggesting a peptide-editing function for
tapasin/TAP in addition to their role in enhancing peptide loading. The Journal of Immunology, 1999, 162: 1530 –1540.
The Journal of Immunology
tween TAP and class I molecules (15). Transfection of tapasin
cDNA into 721.220 cells restores TAP-class I association, normal
cell surface class I expression, and presentation of viral Ag to
CTL, thereby establishing the functional importance of tapasin in
the TAP-class I interaction and providing support for the view that
an association with TAP enhances peptide loading (22). Recently,
this view has been called into question by a study in which the
soluble, ER luminal domain of tapasin was expressed in 721.220
cells (38). Soluble tapasin retained the ability to associate with
class I, but did not appear to interact with TAP. Remarkably, in the
apparent absence of a TAP-class I interaction, soluble tapasin restored normal cell surface class I expression and the presentation
of viral Ag to CTL. The authors concluded that tapasin itself is
sufficient to promote peptide loading of class I molecules (38). It
is noteworthy that soluble tapasin-class I complexes were unstable
in detergent lysates and required chemical cross-linking for their
visualization. Since a relatively small portion of TAP is predicted
to reside within the ER lumen, the possibility remains that the
inability to detect soluble tapasin-TAP complexes by cross-linking
may reflect the lack of appropriately oriented functional groups
rather than the absence of these complexes in vivo.
Although the presence of tapasin in human cells is well established, it has been difficult to detect in murine TAP-class I complexes (9, 21). In this study we demonstrate that tapasin is indeed
associated with TAP and class I molecules in mouse cells. In addition, we showed previously that a Glu to Lys mutation at residue
222 in the a3 domain of H-2Dd (designated Dd(E222K)) abrogates
association with TAP without affecting H chain-b2m assembly (9).
We now show that Dd(E222K) fails to associate with tapasin,
thereby supporting a role for tapasin in bridging the TAP-class I
interaction and implicating residue 222 as a site of contact with the
tapasin/TAP complex. Furthermore, the lack of association with
tapasin and TAP impairs peptide loading and reduces expression
of Dd(E222K) at the cell surface. However, a significant degree of
peptide loading was retained, as evidenced by the decay kinetics of
surface Dd(E222K) molecules and by the ability to accumulate
target structures for CTL recognition. Thus, although interaction
with tapasin/TAP promotes efficient peptide loading, it is not essential for acquisition of TAP-transported peptides.
Materials and Methods
Transfection and cloning
The pSV2neo plasmids containing genomic constructs encoding the wildtype H-2Dd H chain or mutant Dd containing a Glu to Lys mutation at
residue 222 (Dd(E222K)) were provided by Dr. Terry A. Potter (National
Jewish Center for Immunology and Respiratory Diseases, Denver, CO)
(39). The genomic constructs were transfected into murine thymoma
BW5147 cells (H-2k haplotype) by electroporation. Transfected cells were
selected in geneticin-containing medium (Life Technologies, Gaithersburg,
MD) and then cloned by limiting dilution. Cells exhibiting a single symmetrical peak by flow cytometry using anti-Dd mAb 34-5-8S were judged
to be clonal. Clones synthesizing similar levels of wild-type and E222K
mutant Dd molecules were chosen for further study and were designated
BW.Dd and BW.Dd(E222K), respectively. Bulk geneticin-resistant cells
transfected with the pSV2neo plasmid were used as a negative control
(BW.neo).
Cells, Abs, and other reagents
BW5147 transfectants were maintained in RPMI 1640 medium supplemented with 2 mM glutamine, 10% FCS, and antibiotics. L cells expressing
wild-type or E222K mutant Dd molecules (gifts from Dr. Terry. A. Potter)
were maintained in DMEM supplemented with 2 mM glutamine, 10%
FCS, and antibiotics. The CD8-negative T cell hybridoma, B4.2.3, recognizes a peptide Ag derived from HIV-1 gp160 (residues 318 –327; sequence RGPGRAFVTI) in an H-2Dd-restricted manner (40). B4.2.3 cells
were maintained in RPMI 1640 medium supplemented with 2 mM glutamine, 10% FCS, and antibiotics.
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27). It is likely that the association of ERp57 with class I-TAP complexes is mediated primarily through calnexin and calreticulin, since
specific binding of ERp57 to both chaperones has been demonstrated
(28) (M. Leach and D. B. Williams, unpublished observations), and
the interaction of ERp57 with newly synthesized proteins (including
class I) is prevented by treatment with castanospermine, an oligosaccharide processing inhibitor that abrogates the binding of calnexin and
calreticulin to most glycoprotein substrates (23, 25–27). ERp57 has
been proposed to participate in the oxidation or interchange of HC
disulfide bonds, in the trimming of peptide ligands for class I within
the ER, or in the reduction and degradation of misfolded or incompletely assembled class I molecules (23–25). The final step in class I
assembly is the binding of high affinity peptides to the TAP-associated
HC-b2m heterodimer. This appears to be a critical event leading to
dissociation of these large complexes and subsequent export of fully
assembled class I proteins out of the ER (5, 9, 11, 20, 21).
It has been proposed that the TAP-class I interaction may enhance peptide loading onto class I molecules, possibly by providing a high local concentration of peptides (20, 21). However, there
are conflicting reports concerning the importance of the TAP-class
I interaction in promoting efficient assembly of class I molecules.
It has been reported that both membrane-bound and soluble forms
of HLA-G molecules contain similar sets of endogenous peptides
despite the fact that membrane-bound HLA-G, but not the soluble
form, can be coimmunoprecipitated with TAP (29). Furthermore,
class I molecules encoded by various HLA-B alleles appear not to
associate with TAP as assessed by coimmunoprecipitation and display no apparent defects in their abilities to present antigenic peptides (30). Although these findings argue against a role for the
TAP-class I interaction in enhancing peptide loading, it is uncertain whether the absence of coimmunoprecipitable TAP-class I
complexes in detergent lysates always reflects a lack of TAP-class
I interaction in the cell. The opposite conclusion was reached in
two studies using a mutant HLA-A2.1 molecule containing a Thr
to Lys mutation at residue 134 in the a2 domain (T134K) that
cannot associate with TAP (31, 32). Although T134K was fully
capable of binding b2m and peptide in vitro, b2m-associated forms
of the molecule were unstable in detergent lysates and were expressed at the cell surface at about 20% the level of wild-type
HLA-A2.1. These characteristics are similar to those of peptidedeficient class I molecules produced in cells lacking a functional
TAP transporter (33–35). Furthermore, the T134K mutant was
substantially impaired in its ability to present peptides derived
from cytosolically expressed viral proteins to CTL (;0 –25% relative to HLA-A2.1). These findings appear to underscore the importance of the TAP-class I interaction in peptide loading, but
interpretation of the data is complicated by the recent finding that
the T134K mutant molecule has also lost the ability to associate
with the ER chaperone, calreticulin (19). Consequently, it is not
clear whether the impairment in peptide loading of T134K is due
solely to its inability to associate with TAP.
Compelling support for the importance of the TAP-class I interaction in Ag presentation is based on studies of a mutant human
lymphoblastoid cell line, 721.220. Surface levels of a variety of
different class I molecules expressed in these cells are 20 –25% of
normal, and nascent class I molecules are labile in detergent lysates unless stabilized by exogenously added peptides (22, 36, 37).
Furthermore, the ability to present cytosolically produced viral
peptides to T cells is dramatically reduced (22). Although these
cells have normal TAP function, the TAP-class I interaction is
abolished due to the lack of tapasin, a 48-kDa type I membrane
glycoprotein (15, 16, 22). Since complexes of tapasin can be isolated with class I in the absence of TAP or with TAP in the absence
of class I, tapasin has been proposed to bridge the interaction be-
1531
1532
CLASS I ASSOCIATION WITH TAPASIN AND TAP IN MURINE CELLS
The following mAb were used in this study: 34-5-8S and 34-4-20S,
which recognize b2m-associated Dd molecules (41, 42); mAb 34-2-12S,
which recognizes the a3 domain of Dd molecules regardless of b2m-association (41, 43); and mAb 16-3-1N which is specific for b2m-associated Kk
molecules (44). Rabbit antisera specific for TAP1, TAP2, and calnexin
have been described previously (8, 21). Anti-tapasin antiserum was raised
in rabbits against a peptide corresponding to the carboxyl-terminal 20
amino acids of murine tapasin (sequence: SKEKATAASLTIPRNSKKSQ)
(45). Rabbit anti-calreticulin antiserum was purchased from Affinity Bioreagents (Golden, CO).
Carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone (Z-L3VS) is a synthetic protease inhibitor that modifies the active site Thr of the catalytic b
subunits of the proteasome in a highly specific manner in vitro and in vivo
(46). It was provided by Dr. Hidde Ploegh (Massachusetts Institute of
Technology, Cambridge, MA). The following class I binding peptides were
used in this study: tum2, a Dd-binding cellular peptide originally designated tum2 35B-F9 (sequence NGPPHSNNF) (47); p18-I10, a Dd-binding
peptide corresponding to residues 318 –327 of the HIV-1 envelope protein
gp160 (sequence RGPGRAFVTI) (48, 49); and Flu NP Y367–374, a Dbbinding peptide corresponding to residues 367–374 of the influenza A/PR/
8/1934 nucleoprotein with an additional tyrosine at the amino terminus
(sequence YSNENMETM) (50).
Metabolic radiolabeling of L cell or BW5147 transfectants without or with
chase incubation was performed as described previously (9, 21). For isolation of Dd molecules, 5 3 106 cells were lysed on ice for 30 min in 0.5
ml of Nonidet P-40 lysis buffer (1% Nonidet P-40 in PBS (pH 7.4), 10 mM
iodoacetamide, 1% aprotinin, and 0.25 mM PMSF). Following centrifugation to remove nuclei and cell debris, the supernatant fraction was incubated on ice for 2 h with anti-Dd mAb, and then immune complexes were
collected by shaking for 1 h with protein A-agarose. Immunoisolated proteins were eluted from protein A-agarose beads either directly or after
endoglycosidase H (endo H) digestion and then were analyzed by SDSPAGE (12.5% gel) followed by fluorography (8). For quantitation of bands,
x-ray films were pre-exposed to white light to enhance the detection of
weak bands and then exposed to the dried gel for durations chosen to
ensure that the intensities of bands to be quantified were within the linear
range of the film. Films were scanned using an EPSON 1000C scanner and
were analyzed using National Institute of Health Image software. Background was subtracted by integrating a blank area of the film corresponding
in size to that of the band to be quantified.
For detection of tapasin- or TAP-associated Dd molecules, cells were
lysed in digitonin lysis buffer (0.5% digitonin in 10 mM HEPES (pH 7.2),
25 mM CaCl2, 10 mM iodoacetamide, 1% aprotinin, and 0.25 mM PMSF)
and then subjected to sequential immunoisolation as described previously
(9). Briefly, class I complexes with tapasin or TAP that were recovered in
an initial round of immunoisolation with either anti-tapasin or anti-TAP2
antiserum were dissociated by heating at 40°C for 1 h in PBS, pH 7.4,
containing 0.2% SDS. The solution was adjusted to contain 2% Nonidet
P-40 and 5% skim milk powder and then was subjected to a second round
of immunoisolation with mAb 34-2-12S. Immune complexes were washed
only once with 0.5% Nonidet P-40, 10 mM Tris (pH 7.4), 150 mM NaCl,
and 1 mM EDTA before elution and analysis by SDS-PAGE.
Flow cytometric analysis
To assess the levels of cell surface class I molecules, 1 3 106 cells were
incubated with mouse anti-class I Abs (7 mg of mAb 34-5-8S for Dd or
mAb 16-3-1N for Kk) for 30 min at 4°C in 0.25 ml of HBSS, 1% BSA, and
0.01% NaN3 (FACS buffer). After incubation, cells were washed once with
FACS buffer and then were incubated with 5 mg of fluorescein-conjugated
goat anti-mouse IgG (H1L chain specific; Jackson ImmunoResearch Laboratories, West Grove, PA) in 0.25 ml of FACS buffer for 30 min at 4°C.
Cells were washed twice with wash buffer (HBSS and 0.01% NaN3) and
then fixed in 1% paraformaldehyde in PBS, pH 7.4. Subsequent flow cytometric analysis was performed using an EPICS Elite flow cytometer
(Coulter, Hialeah, FL).
For analysis of decay kinetics of cell surface class I molecules, cells
were harvested at 4°C and washed with ice-cold protein-free RPMI 1640
medium. Cells were then resuspended at 106/ml in prewarmed (37°C) protein-free RPMI 1640 medium containing 10 mg/ml brefeldin A (Sigma, St.
Louis, MO). A 1-ml aliquot was immediately removed and diluted into 2
ml of ice-cold FACS buffer. After centrifugation, the cells were processed
at 4°C for flow cytometry as described above. The remaining cells were
placed in a 37°C water bath in a CO2 incubator, and at various times 1-ml
aliquots were removed and processed for flow cytometry.
The ability of cells to present exogenously loaded peptide Ag or HIV-1
gp160-derived Ag to T cells was evaluated by the amount of IL-2 produced
by T cell hybridoma B4.2.3 cells upon recognition of its target structure
(40, 51). Ltk2 cells and L cell transfectants expressing wild-type Dd or
Dd(E222K) were trypsinized, washed with PBS, and then either loaded
with exogenous peptide or infected with recombinant vaccinia viruses. For
peptide loading, cells were incubated with or without 1 mM p18-I10 peptide in RPMI 1640 medium for 4 h at 37°C. For viral infection, cells were
incubated with either vSC8 (control vaccinia virus containing the lacZ
gene) (52) or vPE16 (vaccinia virus containing the HIV-1 gp160 gene) (53)
at 37°C in a CO2 incubator at either 5 or 20 pfu/cell. Excess viruses were
washed out after 1 h and then incubated further for either 4 h (20 pfu/cell
infection) or 16 h (5 pfu/cell infection). All cells were subsequently fixed
by incubating in 5 mg/ml Psoralen (Sigma) for 10 min at room temperature
followed by irradiation at 365 nm for 5 min. After washing, cells were
plated in quadruplicate in a U-bottom 96-well plate at 5 3 104 cells/well
and then were incubated overnight in a CO2 incubator with B4.2.3 cells
(1 3 104 cells/well). The relative amount of IL-2 released into the supernatant fraction by B4.2.3 cells was measured by quantitating [3H]thymidine incorporation into the IL-2-dependent T cell line CT.EV during overnight culture (51).
Results
Dd(E222K) molecules do not bind to TAP or tapasin and exhibit
reduced expression at the cell surface
Using transfected L cells, we showed previously that Dd molecules
possessing a Glu to Lys mutation at residue 222 fail to associate
with the TAP peptide transporter (9). To help clarify the basis for
this loss of association, we examined the interaction of wild-type
Dd and Dd(E222K) with tapasin. As shown in Fig. 1A (lane 1),
when a digitonin lysate of L cells expressing wild-type Dd was
subjected to immunoisolation with anti-tapasin antiserum, a major
band was recovered with the expected electrophoretic mobility of
tapasin (48 kDa) as well as additional bands with mobilities corresponding to TAP1 and TAP2 subunits, class I H chain, and b2m.
The identities of the TAP subunits and the Dd H chain were confirmed by dissociating the anti-tapasin immunoprecipitate in SDS
and recovering either TAP1 and TAP2 or Dd in a second round of
immunoprecipitation (Fig. 1A, lane 2, and Fig. 1B, lane 2, respectively). Faint bands corresponding to calnexin and calreticulin
were also identified by this technique (Fig. 1A, lane 1, and data not
shown). These findings are consistent with results obtained using
human cells (15, 16, 22) and confirm that in murine cells tapasin
is present in complexes with TAP and class I molecules. Based on
its mobility, the band migrating slightly faster than calreticulin
probably corresponds to ERp57 (23, 24); the major band of about
100 kDa has not yet been identified (Fig. 1A, asterisk).
To compare the interaction of wild-type Dd and Dd(E222K)
molecules with tapasin, anti-tapasin immunoprecipitates of lysates
from L cells expressing Dd or Dd(E222K) were dissociated in SDS
and subjected to a second round of immunoprecipitation with antiDd mAb (Fig. 1B, lanes 2 and 6) or with an isotype-matched irrelevant mAb (lanes 3 and 7). Although the resolution of this gel
was somewhat poorer than that shown in A, wild-type Dd H chains
were clearly recovered from the anti-tapasin immunoprecipitate,
whereas Dd(E222K) H chains could not be detected. Thus, as assessed by coimmunoisolation, the Glu to Lys substitution in the a3
domain of Dd prevents association with tapasin in addition to TAP.
HLA-A2.1 and H-2Ld molecules possessing point mutations at
H chain residues 134 and 227, respectively, do not form complexes
with TAP and, in addition, they have lost the ability to associate
with calreticulin (11, 31, 32). Consequently, it was important to
determine whether the failure of Dd(E222K) to associate with tapasin and TAP might be an indirect consequence of altered binding
to an ER chaperone, specifically calnexin or calreticulin. To test
this possibility, radiolabeled lysates of L cells expressing Dd or
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Metabolic radiolabeling and immunoisolation
T cell activation assay
The Journal of Immunology
1533
Dd(E222K) were subjected to a first round of immunoprecipitation
with anti-calnexin or anti-calreticulin Abs, and then the immunoprecipitates were dissociated in SDS and subjected to a second
round of immunoprecipitation with anti-Dd mAb. As shown in Fig.
1C, lane 3, wild-type Dd molecules could readily be recovered
from anti-calnexin immunoprecipitates, indicating interaction with
this chaperone. By contrast, no association with calreticulin was
observed (Fig. 1C, lane 5). Only upon prolonged exposure (2–3
wk) could faint bands corresponding to wild-type Dd molecules be
detected from anti-calreticulin immunoprecipitates (data not
shown). We also examined chaperone binding by immunoprecipitating Dd molecules with either b2m-dependent (34-5-8S) or b2mindependent (34-2-12S) mAbs and then immunoblotting the precipitates with anti-calnexin or anti-calreticulin Abs. Again,
calnexin was readily detected in both Dd immunoprecipitates, but
only trace amounts of calreticulin were observed (data not shown).
This predominant interaction of wild-type Dd with calnexin, even
following b2m association, differs from the results of other studies
in which murine Ld and Kb molecules were shown to bind to both
calnexin and calreticulin (10, 16). Presumably, the calreticulin
band we detected in anti-tapasin immunoprecipitates (Fig. 1A, lane
1) arises from its association with endogenous k-haplotype class I
molecules present in L cells. Most importantly, mutant Dd(E222K)
molecules exhibited a pattern of chaperone interaction similar to
that observed for wild-type Dd. As shown in Fig. 1C, lanes 8 and
10, mutant molecules associated with calnexin but not with calreticulin. However, trace interaction with calreticulin could be detected upon prolonged exposure (data not shown). Identical results
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FIGURE 1. The E222K mutation abolishes association with tapasin and TAP but does not affect interactions with calnexin or calreticulin. A, Analysis
of tapasin-associated proteins. L cells expressing wild-type Dd were radiolabeled for 30 min with [35S]Met, lysed in digitonin lysis buffer, and then subjected
to a first round of immunoisolation with anti-tapasin antiserum. Recovered proteins were either analyzed by SDS-PAGE directly (lane 1) or dissociated
by heating at 40°C in 0.2% SDS in PBS. The SDS-treated sample was adjusted to 2% Nonidet P-40 and then subjected to a second round of immunoisolation
with a mixture of anti-TAP1 and anti-TAP2 antisera (lane 2). HC, Dd heavy chain; cnx, calnexin; crt, calreticulin. B, Detection of tapasin-Dd complexes.
L cells expressing Dd or Dd(E222K) were radiolabeled and lysed as described in A, and then were subjected to a first round of immunoisolation with anti-Dd
mAb 34-2-12S or with anti-tapasin antiserum as indicated. Immune complexes were either analyzed directly (lanes 1 and 5) or were dissociated in SDS
and subjected to a second round of immunoisolation with mAb 34-2-12S or with an isotype-matched irrelevant mAb, MK-D6 (anti-I-Ad), as indicated.
Lanes 2, 3, 6, and 7 were exposed fivefold longer than the other lanes. The presence of tapasin in second round immunoprecipitates reflects either incomplete
dissociation of the initial anti-tapasin immunoprecipitates in SDS or renaturation of anti-tapasin Abs in Nonidet P-40 (lanes 2, 3, 6, and 7). This is a
consequence of the low temperature (40°C) employed in the SDS-dissociation step that was required for preservation of the mAb 34-2-12S epitope in the
second round of immunoprecipitation. C, Interaction of Dd and Dd(E222K) with calnexin and calreticulin. L cells expressing Dd or Dd(E222K) were
radiolabeled and lysed as described in A and then were subjected to a first round of immunoisolation with anti-Dd mAb 34-2-12S or with anti-calnexin or
anti-calreticulin antisera as indicated. Immune complexes were either analyzed directly (lanes 1 and 6) or were dissociated in SDS and subjected to a second
round of immunoisolation with mAb 34-2-12S or with mAb, MK-D6 (anti-I-Ad), as indicated. Lanes 2–5 and 7–10 were exposed threefold longer than lanes
1 and 6.
1534
CLASS I ASSOCIATION WITH TAPASIN AND TAP IN MURINE CELLS
were obtained by immunoprecipitating Dd(E222K) and then immunoblotting with anti-calnexin or anti-calreticulin Abs (data not
shown). Therefore, the inability of Dd(E222K) to bind tapasin and
TAP cannot be attributed to altered association with either
chaperone.
To facilitate further investigation of the phenotype resulting
from the lack of tapasin/TAP association, wild-type Dd or
Dd(E222K) was expressed in BW5147 murine thymoma cells (H2k), a nonadherent cell line that is more amenable to flow cytometric analysis and the production of large scale cultures. Cloned
cells were chosen that synthesize similar levels of wild-type and
mutant Dd (designated BW.Dd and BW.Dd(E222K)). Immunoprecipitation of cell lysates with anti-TAP2 or anti-tapasin antisera
followed by SDS dissociation of immunoprecipitates and subsequent treatment with anti-Dd mAb confirmed that wild-type Dd,
but not Dd(E222K), formed complexes with tapasin and TAP in
these cells (data not shown).
We next examined to what extent the loss of tapasin/TAP association affects the cell surface level of Dd molecules. It is well
established that in cells lacking a functional TAP transporter the
surface level of class I molecules is reduced 10- to 20-fold relative
to that in parental cells (33, 35). The decrease in surface expression
is due to the creation of “empty” class I molecules (H chain-b2m
heterodimers devoid of peptide) that are largely retained in the ER.
Empty class I molecules are unstable, and those that do reach the
cell surface denature rapidly at 37°C unless stabilized by exogenously added peptide or by culturing cells at low temperature (54,
55). Distinct from the situation in TAP-deficient cells,
BW.Dd(E222K) should have normal TAP function, but the
Dd(E222K) molecules may be peptide deficient to some degree
due to their inability to associate with tapasin/TAP. Consistent
with this idea, flow cytometric analysis using the conformationsensitive anti-Dd mAb, 34-5-8S, revealed that the surface level of
Dd(E222K) is about 30% that of wild-type Dd (Fig. 2). This reduced
surface expression of Dd(E222K) is not due to defect(s) in the Agprocessing machinery that could have been introduced during trans-
FIGURE 3. Newly assembled Dd(E222K) molecules are peptide deficient. A, Biosynthetic levels of Dd and Dd(E222K). Left panel, BW5147
transfectants were metabolically radiolabeled with [35S]Met for 20 min,
lysed in Nonidet P-40 lysis buffer, and then subjected to immunoisolation
with the b2m-independent anti-Dd mAb 34-2-12S. Right panel, BW.Dd and
BW.Dd(E222K) cells were radiolabeled for 10 min with [35S]Met and then
chased for 10 min in protein-free medium. To maximize the recovery of
peptide-deficient H chain-b2m heterodimers, 2 3 107 cells were lysed for
2 h in 0.5 ml of Nonidet P-40 lysis buffer containing the b2m-dependent
mAbs, 34-5-8S and 34-4-20S. Mobilities of the Dd H chain (H. C.) and of
a 45-kDa standard are shown. B, BW.Dd and BW.Dd(E222K) cells were
radiolabeled for 10 min with [35S]Met and then chased for 10 min in protein-free medium. Cells (5 3 106) were lysed for 30 min in 0.5 ml of
Nonidet P-40 lysis buffer in the absence or the presence of 100 mM Ddbinding peptide (tum2) or control peptide (NP) as indicated. The b2mdependent mAb 34-5-8S was added either immediately after lysis (lanes 1
and 4) or following incubation of lysates at 4°C for 21 h (other lanes).
Isolated proteins were analyzed by SDS-PAGE. The faster mobility of the
Dd(E222K) H chain relative to that of wild-type Dd is presumably a consequence of the change in net charge accompanying the substitution of an
acidic residue by a basic residue. The same phenomenon has been observed
for other H chain mutants possessing similar substitutions, e.g., Dd(E223K)
and Dd(E232K) (9).
fection and cloning because the levels of endogenous H-2Kk were
indistinguishable between BW.Dd and BW.Dd(E222K) cells. It is also
not a consequence of a clonal peculiarity, because the bulk transfectants displayed a similar pattern (data not shown).
Newly assembled Dd(E222K) molecules are deficient in stably
bound peptides
We next sought to determine whether the low cell surface level of
Dd(E222K) is caused by inefficient peptide loading in the ER. To
address this question, cells were radiolabeled with [35S]Met, and
newly assembled Dd and Dd(E222K) molecules were isolated with
the conformation-sensitive mAb, 34-5-8S, which recognizes only
b2m-associated Dd. Even though Dd and Dd(E222K) H chains
were synthesized at similar rates when analyzed with the b2mindependent mAb 34-2-12S (Fig. 3A, left panel), the amount of
34-5-8S-reactive Dd(E222K) was 44% that of Dd (Fig. 3B, compare lanes 1 and 4). This was not due to an inherent defect in the
ability of Dd(E222K) H chains to associate with b2m, since the
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FIGURE 2. Cell surface levels of Dd and Dd(E222K). BW.neo, BW.Dd,
and BW.Dd(E222K) cells were incubated without primary Ab (dotted line)
or with the b2m-dependent mAbs 34-5-8S (Dd; solid line) or 16.3.1N (Kk;
dashed line). The cells were washed, incubated with FITC-conjugated goat
anti-mouse IgG, fixed, and then analyzed by flow cytometry. The mean
fluorescence intensities for BW.Dd and BW.Dd(E222K) cells were 43.4
and 12.9, respectively.
The Journal of Immunology
recovery of b2m-associated Dd(E222K) molecules could be enhanced by increasing the protein concentration of cell lysates and
by adding two b2m-dependent mAbs, 34-5-8S and 34-4-20S, at the
time of cell lysis (Fig. 3A, right panel). These conditions favor the
recovery of unstable H chain-b2m heterodimers (56).
To test the stability of b2m-associated Dd and Dd(E222K) molecules, lysates were incubated for 21 h in the absence or the presence of exogenously added peptides. Approximately 70% of
Dd(E222K) molecules lost the 34-5-8S epitope after prolonged incubation. However, this loss could be prevented at the time of cell
lysis by the addition of a Dd binding peptide (tum2) but not by a
control peptide that binds to Db (NP; Fig. 3B, compare lanes 4, 5,
6, and 7). In contrast, wild-type Dd molecules were stable under
the same conditions (Fig. 3B, compare lanes 1 and 2). The results
suggest that newly synthesized Dd(E222K) molecules are either
peptide deficient or occupied with suboptimal peptides (peptides
that deviate from the canonical length or class I binding motif) that
readily dissociate (57). It is noteworthy that the addition of exogenous Dd binding peptide increased the amounts of mAb 34-5-8S
reactive molecules for wild-type Dd as well as Dd(E222K) (Fig.
3B, compare lanes 1 vs 3 and lanes 4 vs 6). This phenomenon has
been observed previously (34) and indicates that even in wild-type
cell lysates there are either free H chains and b2m or unstable H
chain-b2m heterodimers that can undergo peptide-driven assembly
or stabilization, respectively.
Dd(E222K) molecules are transported inefficiently out of the ER
and are unstable on the cell surface
In cells lacking a functional TAP transporter, empty class I molecules are generally transported inefficiently from the ER and,
upon arrival at the cell surface, denature rapidly (55, 56, 58). To
determine whether Dd(E222K) molecules share these characteristics, the kinetics of ER to Golgi transport of Dd and Dd(E222K)
molecules were examined in a pulse-chase experiment using the
b2m-independent mAb 34-2-12S (Fig. 4). Processing of H chain
oligosaccharides to complex forms resistant to digestion by endo H
was used as a measure of the movement of Dd molecules from the
ER to the medial Golgi. Wild-type Dd was converted quantitatively
to endo H-resistant forms with a t1/2 of approximately 45 min and
remained stable at the cell surface as expected for molecules pos-
sessing bound peptide (Fig. 4, Dd). By contrast, only a small portion of Dd(E222K) molecules was converted to endo H-resistant
forms, which were unstable and disappeared over time (Fig. 4,
Dd(E222K), compare 2 h and 4 h time points). These labile
Dd(E222K) molecules could be stabilized by adding a Dd-binding
peptide and human b2m to the medium, suggesting that
Dd(E222K) molecules reach the cell surface either empty or with
suboptimal peptides that readily dissociate (Fig. 4, Dd(E222K) and
peptide/b2m). However, conversion of Dd(E222K) molecules to
endo H-resistant forms was not quantitative, suggesting that the
majority of endo H-sensitive Dd(E222K) molecules may be degraded intracellularly. This view is supported by the finding that
the rate of disappearance of endo H-sensitive Dd(E222K) (t1/2 ;70
min) could be slowed by treating cells with Z-L3VS, a specific
inhibitor of proteasome-mediated degradation (t1/2 ;155 min; data
not shown). Degradation is not related to the E222K mutation itself, since empty wild-type Dd molecules produced in TAP-deficient LKD8c cells exhibited similar behavior (data not shown).
Therefore, in the absence of an interaction with tapasin and TAP,
the intracellular transport of Dd(E222K) molecules resembles that
of empty class I molecules produced in TAP-deficient cells. Only
a portion (;20%) reaches the cell surface where it exists transiently; the majority appears to be retained in the ER and degraded.
The retention of the bulk of Dd(E222K) molecules in the absence
of interactions with tapasin and TAP is most likely mediated by
calnexin, since this chaperone associates with endo H-sensitive
Dd(E222K) molecules (Fig. 1C).
Cell surface stabilization of Dd(E222K) molecules
Stabilization of peptide-deficient cell surface class I molecules can
be achieved by exogenous peptides of known class I binding ability or by culturing cells at lowered temperature (54, 59). These
processes are highly dependent upon the presence of exogenous
b2m (provided as bovine b2m in FCS or as purified human b2m)
(59), which drives the equilibrium of H chain interaction with b2m
toward H chain-b2m heterodimers. To assess further the degree of
peptide occupancy of cell surface Dd(E222K) molecules, the extent to which these molecules can be stabilized by peptide or low
temperature was analyzed in comparison with wild-type Dd and
with empty Dd molecules produced in TAP-deficient LKD8c
cells (42).
In LKD8c cells, the basal level of surface Dd molecules was very
low when cells were cultured at 37°C in FCS alone (Fig. 5, condition
9). Consistent with previous observations (42), empty Dd molecules
could be stabilized and the surface level consequently increased by
33-fold after incubating cells with exogenous peptide and b2m at
37°C or by 14-fold following culture at 26°C in the presence of b2m
(Fig. 5, compare condition 9 with conditions 10 and 12). Congruent
results have been reported for empty Kb and Db molecules produced
in the TAP-deficient cell line RMA-S (42, 54).
It has also been demonstrated that peptide or low temperature
combined with b2m enhances the surface expression of class I
molecules in normal cells (54, 55). However, in contrast to empty
Dd molecules on LKD8c cells, the enhancements observed for
wild-type Dd were quite modest following incubation at 37°C with
peptide and b2m or after treatment at 26°C with b2m (1.8- and
2.3-fold, respectively; Fig. 5, compare condition 1 with conditions
2 and 4). The modest enhancement reflects the fact that a substantial portion of wild-type Dd molecules arrives at the cell surface
containing stably bound peptide.
Consistent with the data presented above (Fig. 2), the basal level
of Dd(E222K) surface expression was about 30% that of wild-type
Dd at 37°C (Fig. 5, conditions 1 and 5). This basal expression
could be enhanced in the presence of b2m by either peptide or low
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FIGURE 4. ER to Golgi transport of wild-type Dd and Dd(E222K) molecules. BW.Dd and BW.Dd(E222K) cells were metabolically radiolabeled
with [35S]Met for 10 min and then chased for periods of up to 4 h. In one
experiment (1peptide/b2m), 1 mM tum2 Dd-binding peptide and 10 mg/ml
human b2m were included during radiolabeling and chase incubations. At
the indicated times, aliquots of cells were lysed in Nonidet P-40 lysis buffer
and then subjected to immunoisolation with the b2m-independent mAb
34-2-12S. All samples were digested with endo H and analyzed by SDSPAGE. The mobility of a 45-kDa standard is shown as a solid dot.
1535
1536
CLASS I ASSOCIATION WITH TAPASIN AND TAP IN MURINE CELLS
temperature by 5.6- and 3.8-fold, respectively (Fig. 5, compare
condition 5 with conditions 6 and 8). The fact that these treatments
enhanced the expression of Dd(E222K) molecules to a much lesser
extent than observed for empty class I molecules in LKD8c cells
suggests that a significant portion of Dd(E222K) molecules reach
the cell surface as peptide-bound forms. However, compared with
wild-type Dd, more Dd(E222K) molecules appear to arrive at the
cell surface empty or with suboptimal peptides that readily
dissociate.
Decay kinetics of surface Dd(E222K) molecules suggest the
presence of peptide-containing species
Further evidence for the presence of peptide-containing
Dd(E222K) molecules was obtained by comparing the decay kinetics of cell surface Dd(E222K) with those of wild-type Dd and
empty Dd molecules. BW.Dd, BW.Dd(E222K), and trypsinized
LKD8c cells were grown at 26°C in the presence of human b2m to
stabilize any Dd molecules reaching the cell surface (conditions 4,
8, and 12 in Fig. 5). Cells were then transferred to protein-free
medium containing brefeldin A to block further surface expression
of newly assembled Dd molecules. Following a shift to 37°C, the
levels of b2m-associated Dd molecules on the cell surface were
assessed over a 2-h period by flow cytometry using mAb 34-5-8S.
As shown in Fig. 6A, the decay of Dd(E222K) was faster than
that of wild-type Dd but was substantially slower than the decay of
empty Dd molecules produced in TAP-deficient LKD8c cells. The
decay of Dd(E222K) could be slowed to a rate close to that of
wild-type Dd when BW.Dd(E222K) cells were cultured in the presence of high affinity exogenous peptide and human b2m at 26°C
(Fig. 6A). The intermediate decay rate of Dd(E222K) molecules
stabilized at 26°C is most likely a combination of the decay rates
of as many as three populations: empty molecules, molecules containing peptides that are indistinguishable in nature from those
bound to wild-type Dd, and molecules containing suboptimal peptides that dissociate more rapidly than those bound to wild-type
Dd. Although it is difficult to quantify each population, we tested
for the presence of suboptimal peptides by comparing the decay
kinetics of wild-type Dd and Dd(E222K) molecules on cells that
FIGURE 6. Kinetics of decay of Dd molecules at the cell surface. A,
Cells were incubated for 18 h at 26°C in protein-free RPMI 1640 medium
containing 10 mg/ml human b2m either alone or in combination with 100
mM Dd-binding peptide tum2 as indicated. LKD8c cells were trypsinized
before incubation. After incubation, cells were washed with cold proteinfree medium, resuspended at 37°C in protein-free medium supplemented
with 10 mg/ml brefeldin A, and then transferred to a 37°C incubator. The
level of cell surface Dd molecules was measured by flow cytometry using
the b2m-dependent mAb 34-5-8S and fluorescein-conjugated goat antimouse IgG(H1L). B, BW.Dd and BW.Dd(E222K) cells were cultured at
37°C in RPMI 1640 medium containing 10% FCS. The decay of cell surface Dd molecules was measured as described in A. The results are representative of three independent experiments.
were cultured at 37°C to eliminate empty molecules. As shown in
Fig. 6B, of the Dd(E222K) molecules that remained after culture at
37°C (representing ;27% of the amount present after incubating
at 26°C with b2m; see Fig. 5), a small, but highly reproducible,
portion displayed faster decay kinetics compared with wild-type
Dd at the early phase of the decay, suggesting the presence of
suboptimal peptides. The remainder of the Dd(E222K) molecules
presumably contained high affinity peptides and were relatively
stable. In an effort to visualize peptides bound to Dd(E222K) directly, we immunoaffinity purified Dd and Dd(E222K) molecules,
eluted bound peptides, and analyzed them by reverse phase HPLC.
Unfortunately, the labile nature of Dd(E222K) resulted in very
poor recovery during affinity chromatography and precluded a
meaningful comparison of the peptide profiles (data not shown).
Taken together, the results suggest that in the absence of an
interaction with tapasin and TAP, most Dd(E222K) molecules
reaching the cell surface are deficient in peptides and denature
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FIGURE 5. Stabilization of cell surface Dd molecules. BW.Dd,
BW.Dd(E222K), and TAP-deficient LKD8c cells were incubated for 17 h
at 37°C either in RPMI 1640 medium containing 10% FCS or in protein
free RPMI 1640 medium containing 100 mM Dd-binding peptide (tum2)
plus 10 mg/ml human b2m. Alternatively, these cells were cultured for 17 h
at 26°C in protein-free RPMI 1640 in the absence or the presence of human
b2m as indicated. Cells were stained with mAb 34-5-8S and fluoresceinconjugated goat anti-mouse IgG and then analyzed by flow cytometry.
The Journal of Immunology
1537
rapidly. The remaining molecules contain mostly high affinity peptides, but also a significant amount of peptides that bind with lower
affinity and hence confer reduced stability.
Ag presentation by Dd(E222K)
FIGURE 7. Impaired Ag presentation by Dd(E222K). A, Dd(E222K)
can bind exogenous peptide and form a T cell target structure. The indicated L cell transfectants were trypsinized and then incubated in the absence or the presence of 1 mM p18-I10 for 4 h. The ability of each cell to
form a target structure for the T cell hybridoma B4.2.3 was assessed using
the T cell activation assay described in Materials and Methods. B, Presentation of the gp160-derived peptide epitope is proteasome dependent. L
cells expressing wild-type Dd were infected overnight with either control
vaccinia virus or HIV-1 gp160-encoding recombinant vaccinia virus at 20
pfu/cell in the absence or the presence of 50 mg/ml of Z-L3VS and then
tested for their ability to activate the T cell hybridoma B4.2.3. C, Impaired
Ag presentation by Dd(E222K). Indicated L cells were infected with either
control vaccinia virus or HIV-1 gp160-encoding recombinant vaccinia virus at 5 pfu/cell (overnight) or 20 pfu/cell (4 h). Excess viruses were
washed out after 1 h and then further incubated overnight or for 4 h before
assaying for T cell activation.
continue for a prolonged period of time; about a twofold difference
was observed after overnight infection (5 pfu/cell; Fig. 7C, left
panel). These findings suggest that the lack of tapasin and TAP
interactions can be partially overcome by a prolonged supply of
antigenic peptide into the ER, leading to the gradual accumulation
of peptide-Dd(E222K) complexes at the cell surface. Therefore,
the tapasin/TAP-class I interaction appears not to be essential for
Ag presentation to T cells but, rather, it enhances the efficiency of
presentation.
Discussion
Previous studies using human cells have implicated tapasin as a
glycoprotein that may bridge the interaction between peptide-deficient class I molecules and TAP (15). However, tapasin has been
much more difficult to detect in association with TAP or class I
molecules in murine cells (9, 21). We now show that tapasin is
indeed associated with both TAP and class I molecules in murine
cells, since anti-tapasin Abs co-isolate TAP and H chain-b2m heterodimers from lysates of L cells or BW5147 thymoma cells expressing H-2Dd. Furthermore, we examined the interaction with
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Finally, we addressed the issue of whether the inability of
Dd(E222K) to interact with tapasin and TAP affects its presentation of endogenously generated peptide Ags to T cells. The E222K
mutation in the Dd H chain has been shown to abrogate CD8 binding as well as TAP association (39). Consequently, a CD8-negative and hence CD8-independent Dd-restricted T cell hybridoma,
B4.2.3, was used in these experiments (40). The optimal peptide
Ag for this T cell hybridoma is a 10-mer peptide termed p18-I10
(sequence: RGPGRAFVTI) corresponding to residues 318 –327 of
the HIV-1 envelope protein, gp160. Intracellular expression of
gp160 protein was accomplished by infecting mouse L cells expressing either Dd or Dd(E222K) with a recombinant vaccinia virus containing the gp160 cDNA (53). L cells were used in these
experiments because the BW5147 transfectants were not readily
infectable with vaccinia virus. Consistent with the results obtained
with the BW5147 transfectants, the cell surface level of
Dd(E222K) was about 15% that of Dd in the L cells, although the
two proteins were synthesized at similar rates (data not shown).
As shown in Fig. 7A, when pulsed with exogenous p18-I10 peptide, uninfected L cells expressing Dd(E222K) were able to stimulate the B4.2.3 hybridoma as efficiently as L cells expressing
wild-type Dd. Thus, Dd(E222K) is fully capable of binding exogenously supplied p18-I10 peptide and forming a TCR target structure that can be recognized by the hybridoma. Indeed, the slightly
higher level of presentation by the mutant-expressing cells may be
due to more homogeneous loading with the p18-I10 peptide in the
absence of as much competition from endogenously loaded peptides. It was also important to establish that under conditions of
viral infection this peptide determinant is generated in the cytosol
and then transported into the ER by TAP. The gp160 is a type I
transmembrane protein that is processed into an extracellular protein, gp120, and a transmembrane protein, gp41, through proteolytic cleavage. Since the antigenic sequence is located in the gp120
portion that passes transiently through the ER, the possibility exists
that the peptide determinant can be generated by proteases within
the ER and thereby bypass cytosolic proteolysis and subsequent
transport by TAP. If this is the case, any advantage of the tapasin/
TAP-class I association might be obscured. Consequently, we
tested whether the presentation of the gp160-derived peptide to the
B4.2.3 hybridoma is dependent on cytosolic proteasome activity.
As shown in Fig. 7B, the B4.2.3 hybridoma specifically recognized
Dd-expressing L cells that were infected by vaccinia virus encoding gp160 but not by control vaccinia virus containing the b-galactosidase gene. Importantly, treatment of cells during viral infection with the specific proteasome inhibitor, Z-L3VS, completely
blocked presentation of the gp160-derived peptide to the B4.2.3
hybridoma, implicating the proteasome in a crucial step of gp160
processing. Thus, the gp160-derived peptide determinant presented to the B4.2.3 hybridoma depends at some stage upon proteasomal cleavage in the cytosol and presumably needs to be transported into the ER by TAP before binding to Dd.
Results from similar T cell assays in which L cells expressing
either Dd or Dd(E222K) were infected with the gp160-encoding
vaccinia virus revealed the importance of the tapasin/TAP-class I
interaction in the presentation of endogenously generated peptide
Ags to T cells. As shown in Fig. 7C, right panel, the level of T cell
activation by Dd(E222K) was only 14% that achieved by wild-type
Dd when infection was conducted for 4 h (20 pfu/cell). The difference was less pronounced if gp160 expression was allowed to
1538
CLASS I ASSOCIATION WITH TAPASIN AND TAP IN MURINE CELLS
tent with the latter possibility is the finding that castanospermine
treatment of C1R cells, which prevents calreticulin and calnexin
binding, inhibits the formation of human class I-TAP
complexes (15).
The functional consequence of the failure of Dd(E222K) to associate with tapasin and TAP is a substantial impairment in the
loading of endogenously generated peptides. This is supported by
several observations that qualitatively resemble the phenotype of
peptide-deficient class I molecules expressed in cells lacking a
functional TAP transporter. First, the level of cell surface
Dd(E222K) is 15% (L cells) to 30% (BW5147 cells) that of wildtype Dd, and expression can be up-regulated to wild-type levels by
the addition of exogenous peptides and b2m. Second, the majority
of nascent Dd(E222K) molecules are labile in detergent lysates
unless stabilized by the addition of exogenous Dd binding peptides.
Third, nascent Dd(E222K) molecules are transported inefficiently
to the cell surface, with the majority being degraded intracellularly
as endo H-sensitive forms. Finally, the ability of Dd(E222K) molecules to present cytosol-derived Ag to T cells is substantially
impaired relative to that of wild-type Dd. Our findings are largely
in agreement with those obtained with the human 721.220 cell line,
in which class I molecules fail to interact with TAP due to the
absence of tapasin (15, 22, 36), and also with studies on a chimeric
Db molecule that does not bind to TAP due to substitution of a3
domain residues 219 –233 (62). The reduced peptide occupancy
observed in all three instances underscores the importance of the
association between class I molecules and tapasin/TAP for efficient
loading of TAP-transported peptides.
It is noteworthy that significant residual peptide loading of the
Dd(E222K) mutant was observed in our study as evidenced by its
decay kinetics at the cell surface (Fig. 6A) and by its ability to
present cytosolically generated viral Ag to T cells (up to 50%
relative to wild-type Dd after 16 h of infection). By contrast, relatively little presentation of Ag to T cells was observed following
infection of tapasin-deficient 721.220 cells expressing HLA-A1,
-B8, or -B*4402 molecules (22, 37) or cells expressing either the
Db (219 –233) chimera (62) or the A2.1(T134K) mutant (32). This
may simply be a consequence of the shorter infection times used in
the latter studies (consistent with our results after only 4 h of
infection; Fig. 7C). Alternatively, it might be argued that the
E222K point mutation permits a weak association with tapasin/
TAP in vivo that cannot be detected by coimmunoprecipitation. If
this is the case, one would expect that a similar spectrum of peptides would be acquired but with reduced efficiency. However,
comparison of the surface decay kinetics of wild-type Dd with the
population of Dd(E222K) molecules that survives culture at 37°C
revealed reproducible differences (Fig. 6B). The data suggest that
some Dd(E222K) molecules possess suboptimal peptides that dissociate more rapidly, thereby arguing against a low level of peptide
loading through weak interaction with tapasin and TAP. Rather,
our findings indicate that in the absence of association with tapasin/TAP, peptides are still acquired but with reduced efficiency,
consistent with the fact that TAP continues to transport peptides
into the lumen of the ER. In agreement with this view, a very
recent report has also documented substantial presentation of Ag to
T cells in tapasin-deficient 721.220 cells following prolonged viral
infection (37).
The presence of suboptimal peptides in surface Dd(E222K) molecules suggests that association with tapasin/TAP provides, in addition to more efficient peptide loading, some type of peptideediting function. While this manuscript was under review,
McCluskey and co-workers reported evidence for acquisition of
altered peptides in the absence of interactions with tapasin/TAP
(37). They found that unlike other class I allotypes, HLA-B*2705
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tapasin of a mutant Dd H chain that possesses a Glu3 Lys substitution at residue 222 in the a3 domain. This mutant, that we had
previously determined not to bind to TAP (9), also fails to associate with tapasin as judged by coimmunoisolation. The observation that a single point mutation affects the binding of both tapasin
and TAP indicates that one protein relies on the other for association with class I. Combined with previous studies showing that
tapasin is required for the association of class I and TAP (15, 36),
this finding supports the view that tapasin acts to bridge the interaction between class I and TAP proteins. It is possible that the
E222K mutation permits some degree of H chain association with
tapasin and TAP in vivo that is not detectable under our conditions
of detergent lysis and immunoisolation. If such a weak association
does exist in vivo it appears not to be functionally relevant, since
the phenotype of Dd(E222K) molecules is similar to that of most
class I molecules expressed in the tapasin-negative cell line
721.220 (see below).
It is unlikely that the E222K mutation causes major structural
alterations that indirectly affect the ability of the Dd molecule to
interact with tapasin. The mutation does not appear to affect the
association of the H chain with b2m (a prerequisite for tapasin
binding) (15), since complexes of the E222K H chain and murine
b2m can be isolated as efficiently as those for wild-type Dd under
conditions where dissociation of peptide-deficient Dd(E222K)
molecules is minimized. Moreover, Dd(E222K) molecules are stabilized at the cell surface at reduced temperature in the presence of
exogenous human b2m. Similarly, the E222K mutation appears
not to affect peptide binding, since Dd(E222K) molecules can bind
peptides in detergent lysates or on the cell surface as evidenced by
the ability of exogenously added peptides (with b2m) to stabilize
surface molecules at 37°C and to create T cell target structures.
Finally, the interaction of Dd(E222K) with calnexin and calreticulin is not detectably altered relative to wild-type Dd, rendering
unlikely the possibility of structural changes due to differences in
molecular chaperone binding. These findings suggest that residue
222 may be present within a segment of the Dd H chain that contacts tapasin directly or that mutation at this site causes relatively
subtle structural changes that affect other H chain-tapasin contact
sites. In this context, it is noteworthy that residue 222 comprises
part of a conserved loop in the a3 domain (amino acids 222–229)
that functions as a major binding site for CD8 (39, 60). The crystal
structure of the CD8-HLA-A2 complex indicates that the two Ig
domains of the CD8aa homodimer bind to the conserved a3 domain loop in the manner of an Ab-Ag complex (61). Since tapasin
has also been reported to contain two potential Ig domains (22), it
is conceivable that binding to the class I a3 domain could occur in
an analogous fashion.
Other studies have implicated the same region of the H chain a3
domain as being important for interaction with TAP. A mutant Ld
molecule possessing an Asp3 Lys point mutation at residue 227
fails to coimmunoprecipitate with TAP (5). Furthermore, a chimeric molecule in which residues 219 –233 of the Db H chain were
replaced by residues 133–147 of the b2 domain of the class II I-Ad
molecule also does not associate with TAP (62). Interaction with
tapasin was not examined in either case. Two additional studies
have pointed to the H chain a2 domain as important for association
with TAP (31, 32). Using transfected human C1R cells, a point
mutation at residue 134 in the HLA-A2.1 molecule (T134K) was
shown to result in loss of TAP association. However, this mutant
as well as the Ld(D227K) mutant described above also lost the
ability to bind to calreticulin; interactions with calnexin were unaffected (11, 19). This raises the question of whether the lack of
TAP association is due to the loss of an important site of contact
with tapasin/TAP or to the loss of calreticulin interaction. Consis-
The Journal of Immunology
Acknowledgments
We thank Drs. Terry Potter and Hidde Ploegh for their generous gifts of
reagents, the Alberta Peptide Institute for peptide synthesis, and Ms. Cheryl
Smith for flow cytometric analyses.
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of TAP.
1539
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CLASS I ASSOCIATION WITH TAPASIN AND TAP IN MURINE CELLS
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