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*
Gwen Knapp Center for Lupus and Immunology Research, Committee on Immunology and Department of Pathology, University of Chicago, Chicago, IL 60637;
Department of Microbiology and Immunology, Howard Hughes Medical Institute, Vanderbilt University School of Medicine, Nashville, TN 37232;
Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110; and
Center for Immunology, University of Texas Southwestern Medical Center, Dallas, TX 75390
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-microglobulin
(2m)3
(1). The function of MHC class I molecules is to bind
antigenic peptides of 8–10 residues in the endoplasmic reticulum (ER)
and transport them to the cell surface where they are displayed to
cytotoxic T cells (2). Two types of MHC class I molecules
are classified based on their polymorphism and levels of expression.
The highly expressed polymorphic class Ia molecules bind a diverse set
of peptides derived from the cytosol, whereas the less abundant,
nonpolymorphic or oligomorphic class Ib molecules bind a more
restricted set of peptides (3). The process involved in
the assembly of MHC class Ia-peptide complexes is highly orchestrated;
however, it is unclear whether a similar process and regulation applies
to the assembly of MHC class Ib-peptide complexes.
Assembly of the MHC class Ia-peptide complex occurs in the ER and is
assisted and controlled by several molecular chaperones. The majority
of peptides bound to class Ia molecules are generated by cleavage of
cytosolic proteins by proteasome. The resulting peptides are
translocated into the ER by TAP (4, 5). In mouse cells,
nascent MHC class Ia molecules dimerize with
2m with the aid of two molecular chaperones,
calnexin and calreticulin (6, 7). Before binding to
peptide, class Ia-2m heterodimers are found in
association with calreticulin, Erp57 (8), tapasin
(9), and TAP in a large loading complex. Upon peptide
binding, fully assembled class Ia molecules dissociate from the loading
complex and are transported to the cell surface.
Calnexin and calreticulin are highly conserved lectin-like ER-resident chaperones. They associate with many newly synthesized glycoproteins and are thought to facilitate their folding and assembly in multisubunit complexes (10, 11, 12). Calnexin and calreticulin share striking sequence similarity in their central and C-terminal domains. Because surface expression and peptide loading of class I molecules is unaffected in a calnexin-deficient cell line (CEM.NKR), it has been suggested that calreticulin and calnexin may have redundant functions in class Ia assembly (13). However, several distinct features between calnexin and calreticulin, including their ability to interact with different protein and glycan determinants during the assembly of class I molecules, suggest that these chaperones have nonredundant functions as well (14, 15).
Unlike calnexin and calreticulin, which are involved in the
biosynthesis of many glycoproteins, TAP and tapasin appear to be
dedicated solely to the assembly of class I-peptide complexes. TAP is
composed of two subunits, TAP1 and TAP2, that belong to the ATP-binding
cassette transporter family (16, 17). TAP preferentially
transports peptides of 
8–15 aa residues into the lumen of the ER in
an ATP-dependent fashion (18, 19). In TAP-deficient cells,
the majority of class I molecules are retained in the
ER/cis-Golgi and degraded (20, 21, 22). A recent
study showed that of
Kb-2m complexes in
TAP-deficient cells adopt a distinct conformation from those in
TAP-positive cells. These findings support that TAP acts as a molecular
chaperone to stabilize the class I-2m
heterodimer in an immature conformation before binding of high-affinity
peptide (23).
Tapasin is a 48-kDa type I transmembrane glycoprotein that resides in
the ER (9). In a mutant human cell line (721.220) that
lacks tapasin, class I-2m dimers fail to
associate with TAP. In turn, this defect impairs class I peptide
loading and surface expression (9, 24). Thus, one of the
proposed functions of tapasin is to bring class I molecules into
physical proximity with the peptide source, TAP. However, studies with
a soluble form of tapasin that fails to interact with TAP suggest that
association of tapasin with class I is sufficient to promote class I
peptide loading (25, 26). Recent studies have implicated
several functions of tapasin in the assembly of class I-peptide
complexes. Tapasin expression enhances the stability of TAP1-TAP2
complexes, increasing the overall peptide transport into the ER
(25, 27). In insect cells, tapasin was shown to retain
empty class Ia molecules in the ER (28). Likewise, tapasin
was shown to prevent premature release of Kb
molecules from the ER of 721.220 cells, suggesting that tapasin may
play a role in the retention of class I heterodimers in the ER until
optimal ligand selection is completed (29). Studies with
tapasin-deficient (tapasino) mice showed that
most Kb and Db molecules
exit the ER of these cells in the absence of stably bound peptides.
These findings suggested a critical role for tapasin in ER retention of
class Ia molecules that are empty or loaded with suboptimal peptides
(30). It is noteworthy that the dependency of tapasin for
class I surface expression varies significantly among class Ia alleles
(31, 32, 33), which may reflect the complexity of tapasin
function.
M3 is a murine class Ib molecule with a high affinity for N-formylated peptides (34, 35). The unique binding specificity of M3 restricts its ligands to peptides derived from the amino terminus of mitochondrial and bacterial proteins. Previously, we developed a mAb against M3 and showed that M3 surface expression is undetectable in most cell types due to the lack of endogenous Ags (36). The majority of M3 is retained in the ER but quickly trafficks to the cell surface after addition of exogenous N-formylated peptide. Peptide-induced M3 expression is most efficient on APCs and is inhibited by brefeldin A and phenylarsin oxide (an inhibitor of endocytosis), which suggests a requirement for exogenous peptides to be endocytosed and transported to the ER before loading onto M3. Unlike the expression of class Ia molecules, M3 surface expression cannot be induced by incubation at low temperature. Yet the intracellular pool of empty M3 is maintained, suggesting a role for chaperones in their stabilization. The unique intracellular source of M3-binding peptides and the unusual trafficking behavior of M3 make it an attractive model to investigate the roles of individual components of the ER peptide-loading complex in the assembly of functional class Ib molecules.
In this study, we investigate the interactions between M3 and several ER chaperones, and examine the role of TAP and tapasin in the assembly and intracellular transport of M3. Our results demonstrate that both TAP and tapasin are required for efficient assembly of M3-N-formylated peptide complexes. In addition to transporting N-formylated peptides into the ER, TAP is essential for maintaining the intracellular pool of M3, possibly by providing low-affinity cytosolic peptides that prevent M3 degradation. Unlike Kb and Db, tapasin is not required for ER retention of empty M3 molecules. However, it is important for facilitating the loading of N-formylated peptides onto M3. Collectively, our data indicate that similar ER-resident chaperones participate in the assembly of class Ia and class Ib molecules. However, class Ia and class Ib molecules differ in their dependence for distinct chaperones.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following mAbs were used in this study: mAb130 (anti-M3, hamster IgG) (36), 34-2-12S (anti-Dd, mouse IgG2a) (37), and Y3 (anti-Kb, mouse IgG2b) (38). Rabbit serum against TAP1 was described previously (39). Rabbit sera against calnexin and calreticulin were purchased from StressGen (Victoria, BC, Canada). Rabbit serum against tapasin was a gift from Dr. G. Schoenhals (R. W. Johnson Pharmaceutical Research Institute, San Diego, CA) (40). FITC-conjugated anti-Kb (AF6-88.5), anti-hamster IgG, and anti-mouse IgG were purchased from BD PharMingen (San Diego, CA). Synthetic peptides were purchased from Research Genetics (Huntsville, AL). All peptides were >90% pure as determined by mass spectrometry. Peptides were dissolved in DMSO at concentrations of 10 µM.
Mice
The generation of TAP1 knockout (TAPo) and tapasin knockout (tapasino) mice has been described (30, 41). KbDb knockout (KboDbo) mice on the B6 background were a gift from Dr. F. A. Lemonnier (Institut Pasteur, Paris, France) (42). Littermate mice heterozygous for both the TAP1 and tapasin gene were used as a control. All mice used were at the age of 6–12 wk.
Cell lines and cell cultures
An M3-transfected P388 macrophage cell line (P388-M3) was generated by transfection of the parental line with M3 cDNA under control of the CMV promoter using Lipofectin (Life Technologies, Gaithersburg, MD). Stable transfectants were selected with 1 mg/ml geneticin (Life Technologies). Both P388 and P388-M3 were maintained in DMEM (Mediatech, Herndon, VA) with 10% FBS, 2 mM L-glutamine, 20 mM HEPES, 50 mM 2-ME, penicillin, and streptomycin (DMEM-10). LPS blasts and Con A blasts were prepared by culturing splenocytes with 5 µg/ml LPS and 3 µg/ml Con A, respectively, in RPMI 1640 with 10% FBS (RPMI-10) for 48 h at 37°C.
Flow cytometric analysis
One million cells were incubated overnight in RPMI-10 with or without peptides at 37°C. Cells were harvested and washed three times with PBS before cell surface staining experiments. M3 staining was detected by adding 100 µl hybridoma supernatant (mAb130; hamster IgG) followed by mouse anti-hamster IgG FITC. Staining with each reagent was performed for 30 min on ice in HBSS (Life Technologies) containing 2% FBS and 0.1% sodium azide (Sigma, St. Louis, MO), followed by washing with the same buffer.
For intracellular staining, single-cell suspensions were fixed with 1% paraformaldehyde followed by washing with PBS and permeabilization buffer (0.15% saponin, 1% FCS, 0 and 0.1% sodium azide in PBS, pH 7.4). Cells were then stained with the relevant Abs in the permeabilization buffer at 4°C for 30 min, followed by washing with the same buffer and resuspending in PBS. All stained cells were analyzed by flow cytometry using a FACSCalibur with CellQuest software (BD Biosciences, Mountain View, CA).
CTL lines and cytotoxicity assays
ND1
-specific, M3-restricted CTL clones (B6 and 4E3)
(43) were provided by Dr. K. Fischer Lindahl (University
of Texas Southwestern Medical Center, Dallas, TX). For CTL assays, one
million target cells (Con A blasts from TAPo,
tapasino, and control mice) were incubated
overnight in RPMI-10 with or without 10 µM ND1 peptide (fMFFINIL)
(44). Cells were washed free of excess peptide and labeled
with 100 µCi of 51Cr for 1 h at 37°C.
Target cells (1 x 104) were added to
round-bottom microtiter wells containing effector cells. After a 4-h
incubation at 37°C, 100 µl of supernatant from each well was
collected and assayed for 51Cr release. Percent
specific lysis = (experimental release - spontaneous
release)/(maximal release - spontaneous release) x
100.
Cell labeling, immunoprecipitation, and SDS-PAGE analysis
LPS blasts from TAPo, tapasino, and littermate control mice and cell lines were labeled overnight with 35S-Translabel (ICN Pharmaceuticals, Costa Mesa, CA) in the presence or absence of 10 µM LemA (fMIGWII) peptide (45). Labeled cells were lysed in buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 0.5% Nonidet P-40, 20 mM iodoacetamide, 1 mM PMSF, and 10 µg/ml aprotinin. Radiolabeled lysates were precleared successively with protein A-Sepharose and normal hamster sera bound to protein A-Sepharose at 4°C overnight. One milliliter of various mAb supernatants coupled to protein A-Sepharose was used for immunoprecipitation with precleared cell lysate at 4°C overnight. Immune complexes were washed with a buffer containing 1% Nonidet P-40, 5 mM PMSF, 10 mM Tris (pH 8.0), 150 mM NaCl, 5 mM KI, and 5 mM EDTA. After extensive washing, the immunoprecipitates were eluted by boiling for 5 min in SDS sample buffer containing 0.6% SDS and 1% 2-ME. Eluates were then diluted 1:5 with distilled water and split into two equal aliquots, one of which received 2 mU of endoglycosidase H (Endo H; Roche Molecular Biochemicals) at pH 5.5, followed by overnight incubation at 37°C. Samples were separated by 12.5% SDS-PAGE and visualized by autoradiography.
For pulse-chase experiments, 5 x 106 cells were used for each time point. After starvation in 3 ml of methionine/cysteine-free medium (ICN Pharmaceuticals) for 2 h, cells were pulsed with 0.5 mCi/ml 35S-Translabel for 20 min and then chased in complete medium for various periods of time in the presence or absence of 10 µM LemA peptide. Aliquots of cells for each chase point were lysed in lysis buffer. Then the lysates were precleared and M3 molecules were immunopurified and analyzed as described above.
Coimmunoprecipitations and Western blot analysis
P388-M3 cells and LPS blasts from tapasino and wild-type (WT) control mice were lysed in buffer containing 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS; Boehringer Mannheim, Indianapolis, IN) in TBS (pH 7.4), 0.2 mM PMSF (Sigma), and 20 mM iodoacetamide. The lysates were precleared and M3 molecules were immunopurified as described above, except that immune complexes were washed four times with a buffer containing 0.1% CHAPS, 5 mM PMSF, 10 mM Tris (pH 8.0), 150 mM NaCl, 5 mM KI, and 5 mM EDTA. After washing, immunoprecipitates were run on 10% SDS-PAGE gels and transferred to Immobilon P membranes (Millipore, Bedford, MA) by electroblotting. After overnight blocking (5% skim milk powder in PBS/0.05% Tween 20) at 4°C, membranes were incubated with relevant Abs for 2 h, washed three times with PBS/0.05% Tween 20, and incubated for 1 h with biotin-conjugated goat anti-rabbit IgG (Caltag, South San Francisco, CA). Following three washes with PBS/0.05% Tween 20, membranes were incubated for 1 h with HRP-conjugated streptavidin (Zymed, San Francisco, CA). After three washes with PBS/0.3% Tween 20, membranes were incubated with chemiluminescent reagents (Amersham, Arlington Heights, IL) and exposed to ECL film for varied lengths of time.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2m and ER-resident chaperones
Biochemical analyses of M3 are limited by the low levels of M3
transcripts in all cell types (46). To overcome this
limitation, we transfected a macrophage cell line (P388) with an
expression plasmid containing full-length M3 cDNA under control of the
CMV promoter. Stable M3 transfectants were identified by intracellular
staining with mAb130, which recognizes the M3 H chain and is
insensitive to the conformational change induced by peptide binding
(36). Fig. 1
A
illustrates that the intracellular pool of M3 increased significantly
in M3-transfected P388 cells (P388-M3). However, despite the presence
of a larger pool of intracellular M3, surface levels of M3 on P388-M3
were as low as on untransfected cells. After overnight incubation with
LemA peptide, an N-formylated M3-binding peptide, M3 surface
expression was enhanced 3- and 20-fold on P388 and P388-M3,
respectively. In addition, intracellular staining revealed that the
size of the intracellular M3 pool increased in peptide-treated cells,
suggesting that peptide treatment increases the M3 stability.
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B, mAb130 precipitates substantially fewer M3
H chains from untreated cells, and 2m is
barely detectable. In contrast, no difference in the amount of
H-2Dd H chains and 2m is
detected from the untreated and peptide-treated cells using a
Dd-specific Ab. Most of the M3 in the
peptide-treated cells acquire Endo H resistance, whereas the majority
of the M3 in the untreated cells remain Endo H sensitive (Fig. 1B). Taken together, these data suggest that peptide
enhances the stability of M3 by increasing the affinity between M3 and
2m. Furthermore, association with
2m may be a prerequisite for M3 to egress from
the ER/cis-Golgi compartment. This conclusion is further
supported by the observation that M3 remains Endo H sensitive in
LemA-treated 2m-deficient cells (data not
shown).
To assess association of M3 with ER chaperones that are involved in the
assembly of class Ia molecules, lysates of P388-M3 cells were used in
coimmunoprecipitation experiments with mAb130 or
anti-Dd Ab. Immunoprecipitates were blotted
to detect the presence of various ER chaperones. As shown in Fig. 2, comparable levels of calnexin,
calreticulin, TAP, and tapasin are detected in precipitates of
anti-M3 and anti-Dd. Thus, M3 associates
with several chaperones that are known to assist the assembly of class
Ia molecules.
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Of the ER chaperones that associate with M3, TAP and tapasin are
dedicated to the class I assembly. To determine the function of TAP and
tapasin for the assembly of M3-N-formylated peptide
complexes, we first examined M3-restricted Ag presentation in the
TAPo, tapasino, and WT mice
by CTL assays. Two M3-specific alloreactive CTLs (B6 and 4E3), which
recognize mitochondrial ND1 peptide in the context of M3, failed to
recognize Con A-activated splenocytes from both types of knockout mice,
while they efficiently lysed Con A blasts from WT mice (Fig. 3A). This result indicates
that TAP and tapasin are required for the expression and proper
assembly of M3 with endogenous mitochondrial peptides. To determine
whether lack of TAP and tapasin also influence the expression and
assembly of M3 with exogenous N-formylated peptides, we
performed CTL assays using ND1 peptide-treated Con A blasts from
TAPo, tapasino, and control
mice as targets. Although CTLs can recognize peptide-treated
splenocytes from all three strains of mice, the sensitivity to CTL
lysis differed among these targets. At a low E:T ratio, the reactivity
of M3-specific CTLs to TAPo and
tapasino splenocytes was significantly lower than
that of WT splenocytes (Fig. 3A), indicating that TAP and
tapasin are also involved in the assembly of M3 with exogenous
N-formylated peptides.
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B). Culturing the splenocytes in the presence of 10 µM
LemA peptide increases M3 surface expression in both WT and
TAPo splenocytes, although, consistent with our
previous observation, induction is less efficient in
TAPo splenocytes (30% of WT levels)
(36). Notably, peptide-induced M3 surface expression was
more severely impaired in tapasino splenocytes.
Similar results were obtained when other M3-binding peptides were used
(data not shown). For comparison, surface expression of
Kb class Ia molecules was examined using a
conformation-sensitive Kb-specific Ab (AF6-88.5).
Kb expression is not affected by incubation with
LemA peptide. Unlike M3, surface Kb expression is
detected on tapasino splenocytes, although at
reduced levels (20% of WT levels). The expression of
Kb is undetectable on TAPo
splenocytes. Differential roles of TAP and tapasin in the assembly of M3 with N-formylated peptides
Impaired peptide-induced M3 surface expression in
TAPo and tapasino
splenocytes could be due to instability of the intracellular M3 pool
and/or inefficient assembly of M3-peptide complexes. The intracellular
pool of M3 in TAPo,
tapasino, and WT splenocytes was compared by
intracellular staining. As shown in Fig. 4A, in the absence of
exogenous peptide supply, the amount of M3 in
TAPo splenocytes was barely detectable, whereas
the M3 pool from tapasino splenocytes was
comparable to that of WT splenocytes. Thus, TAP, but not tapasin, is
essential for maintaining the intracellular pool of M3. Treatment with
LemA peptides increases the M3 pool in WT and
TAPo splenocytes, but has no effect on the pool
of M3 in tapasino splenocytes (Fig. 4A). Lack of peptide-induced M3 expression detected both by
surface and intracellular staining in tapasino
splenocytes indicates that tapasin plays a critical role in
facilitating the binding of N-formylated peptides to M3.
Metabolic labeling experiments confirmed that significant amounts of M3
are retained in the ER (Endo H sensitive) of
tapasino splenocytes, even in the presence of
LemA peptide (Fig. 4B). In addition,
tapasino cells lack M3 molecules that are stably
associated with 2m, suggesting that these M3
molecules are "empty" (or lack stably bound peptide). The
observation that M3 is still actively retained in the ER of
tapasino cells indicates that other ER
chaperone(s) participate in the retention of empty M3 molecules in
the ER.
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To further examine the effect of TAP and tapasin on the stability
of the intracellular M3 pool and the kinetics of M3 trafficking, LPS
blasts from TAPo, tapasino,
and WT splenocytes were subjected to pulse-chase radiolabeling with or
without LemA peptide. In the absence of LemA, most of the M3 molecules
in the TAPo splenocytes were degraded within
2 h of chase. In sharp contrast, significant amounts of Endo
H-sensitive M3 molecules were detected in
tapasino and WT splenocytes, even after 20 h
of chase (Fig. 5). In the presence of
LemA peptide, a significant portion of M3 molecules from
TAPo cells acquired Endo H resistance after
2 h of chase (Fig. 5). The kinetics of M3 maturation in
TAPo cells are similar to those observed in the
WT control cells, except that the intensity of the M3 H chain is
reduced in TAPo cells. These data suggest that
the time course for delivery of N-formylated peptides to the
ER by the TAP-independent and TAP-dependent pathways are quite
comparable. In tapasino splenocytes, M3 remained
in an immature state for at least 20 h, even in the presence of
LemA peptide (Fig. 5). This result further supports the conclusion that
lack of tapasin severely impairs the assembly of
M3-N-formylated peptide complexes and the kinetics of M3
trafficking.
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Two possible explanations may account for the instability of the
intracellular M3 pool in TAPo mice. First, TAP
may stabilize the intracellular M3 pool by providing low-affinity
binding peptides for M3. Alternatively, TAP may stabilize M3 through
physical interactions. If the latter were true, one has to assume that
TAP can bind to M3 in the absence of tapasin since the intracellular
pool of M3 is stable in the tapasino cells. To
test this possibility, we performed coimmunoprecipitation experiments
of molecular chaperones with M3 in tapasino and
WT splenocytes. As shown in Fig. 6, M3
failed to associate with TAP in tapasino cells,
whereas M3 retained its ability to interact with calnexin and
calreticulin in these cells. These results indicate that tapasin
mediates the association between M3 and TAP, but that this interaction
is not required for maintenance of the stable pool of M3 in the
ER.
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Since M3 and class Ia molecules require the same ER chaperones for
proper assembly, it remains possible that competition with more
abundant MHC class Ia molecules for access to ER chaperones limits
M3 expression. Thus, we compared M3 surface expression on splenocytes
derived from KbDb-deficient
(KboDbo) and WT mice. Fig. 7 shows that
KboDbo and WT controls have
similar basal levels of M3 surface expression. In addition, there is no
significant difference between
KboDbo and WT splenocytes
in the levels of M3 surface induction by several
N-formylated peptides. Our data suggest that access to
molecular chaperones is not the limiting factor for M3 surface
expression.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Impaired Ag presentation and heightened instability of M3 in
TAPo cells imply that TAP contributes to M3
expression at two distinct steps. First, TAP enhances the transport of
N-formylated peptides into the ER, which promotes surface
expression of mature M3-N-formylated peptide complexes.
Second, TAP transports low-affinity (nonformylated) cytosolic peptides
into the ER. It is likely that some of these peptides have sufficient
affinity for M3 to promote its folding into a stable conformation while
insufficient for egress of M3 from the ER. In this way, a steady pool
of M3 molecules can be maintained in the absence of high-affinity
N-formylated peptides (Fig. 8A).
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Unlike TAPo cells, the intracellular pool of M3
is relatively stable in tapasino cells, but the
assembly of M3 with N-formylated peptides is defective. How
does tapasin influence loading of N-formylated peptides onto
M3? Several proposed functions of tapasin may be involved in M3
assembly. First, tapasin may serve as a linker between TAP and M3
molecules and enhance peptide loading by localizing M3 near the peptide
supply. Second, tapasin may directly interact with M3 H chains and
facilitate peptide binding or peptide exchange. Third, tapasin may
promote assembly of M3-peptide complexes by retaining empty M3
molecules in the ER until they bind N-formylated peptides.
Our pulse-chase experiments clearly demonstrate that empty M3 is
retained in the ER in tapasino cells, suggesting
that tapasin is not responsible for ER retention of immature M3
molecules. As loading of exogenous N-formylated peptides to
M3 proceeds normally in the absence of TAP (Figs. 4 and 5), lack of
peptide-induced M3 surface expression in tapasino
splenocytes cannot be explained solely by the bridging function of
tapasin. Therefore, it is likely that tapasin has a direct role in
promoting the binding of N-formylated peptides with M3 (Fig. 8B). A putative tapasin binding site on MHC class I
molecules includes amino acid residues 128–136 (53, 54, 55),
which form a loop in the 2 domain. This region is conserved in M3.
Therefore, it is conceivable that the interaction of tapasin with this
region of M3 modulates its Ag-binding groove, which in turn influences
the binding of N-formylated peptides. Alternatively, but not
mutually exclusive, tapasin may catalyze the exchange of low-affinity
(unformylated) peptides for high-affinity N-formylated
peptides. However, we cannot exclude the possibility that tapasin is
required for efficient recruitment of a novel ER chaperone to the
M3-peptide loading complex.
Although M3-restricted ND1-specific CTLs cannot recognize
tapasino cells, they can react with ND1
peptide-treated tapasino cells, albeit with
reduced efficiency. This finding suggests that in the presence of high
peptide supply some M3-ND1 complexes assemble independent of
tapasin. In agreement with this observation, flow cytometric analyses
revealed that peptide-induced M3 surface expression can be partially
restored in tapasino cells when high
concentrations (50–100 µM instead of 10 µM) of
N-formylated peptide are used (data not shown). Thus,
increasing the N-formylated peptide pool can partially
overcome the requirement of tapasin for M3 surface expression. These
results support the notion that the available peptide pool capable of
binding to each class I molecule influences its observed tapasin
dependency. Under normal circumstances, the endogenous M3 peptide pool
is extremely small; thus, Ag presentation and surface expression of M3
are completely tapasin dependent. In contrast, Ag presentation and
surface expression of Kb and
Db are only partially impaired in
tapasino cells (30, 56), possibly
due to the presence of a large and complex pool of
Kb- and Db-binding peptides
in the ER.
The surface expression of M3 is enhanced by incubation with exogenous
N-formylated peptides, but not by increasing the level of M3
biosynthesis (Fig. 1) nor by eliminating the expression of MHC class Ia
molecules (Fig. 7). Thus, the supply of N-formylated
peptides is the rate-limiting factor for M3 surface expression. Binding
of N-formylated peptides to M3 appears to induce
conformational changes that stabilize the interaction between M3 and
2m (Fig. 1B). In turn, these
conformational changes in M3 permit the release of the
M3-2m-peptide ternary complex from the
peptide-loading complex (Fig. 8B). Conformational changes of
class I H chain induced by high-affinity peptides have been described
in MHC class Ia molecules (57, 58), which presumably also
strengthen the association between class Ia H chain and
2m. However, unlike some class Ia molecules
(i.e., Db and Ld) that can
express on the cell surface in the absence of
2m (59, 60), the association with
2m is required for M3 to egress from the
ER/cis-Golgi compartment. Empty M3 or M3 bound with
suboptimal peptides are actively retained in the ER, possibly due to
weak interaction with 2m. It remains unclear
which chaperone molecule is responsible for ER retention of empty M3.
In tapasino cells, M3 fails to interact with TAP,
but the interaction of M3 with calreticulin and calnexin remains
intact. As empty M3 is retained in tapasino
cells, it is possible that calreticulin or calnexin retain immature M3
or partially folded M3 in the ER of these cells.
Unlike class Ia molecules that bind a diverse repertoire of peptides, M3 binds a unique and restricted set of peptides. To ensure efficient Ag presentation, M3 depends on the concerted efforts of a variety of molecular chaperones during its biosynthesis. Similar requirements may be imposed upon other class Ib molecules, such as Qa-1, HLA-E, or HLA-F, for effective presentation of a restricted set of peptide Ags.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Chyung-Ru Wang, University of Chicago, Gwen Knapp Center for Lupus and Immunology Research, Room 412, 924 East 57th Street, Chicago, IL 60637-5420. E-mail address: cwang{at}midway.uchicago.edu
3 Abbreviations used in this paper: 2m, 2-microglobulin; ER, endoplasmic reticulum; WT, wild type; TAPo, TAP-deficient or TAP knockout; tapasino, tapasin-deficient or tapasin-knockout; Endo H, endoglycosidase H; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate.
Received for publication April 9, 2001. Accepted for publication June 1, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 domain loop influences interaction with the assembly complex. J. Immunol. 163:4427.2 domain of HLA-A2.1 define a functionally relevant interaction with TAP. Curr. Biol. 6:873.[Medline]
2m associations suggest that 2m facilitates the folding of the 1 and 2 domains during de novo synthesis. J. Immunol. 140:3522.[Abstract]
2m independent presentation of exogenously added foreign peptide and endogenous self-epitope by MHC class I -chain to a cross-reactive CD8+ CTL clone. J. Immunol. 153:4070.[Abstract]This article has been cited by other articles:
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, 
, 
) or with 10 µM ND1
, •, 
) overnight, labeled with 51Cr, and used as
targets in a standard cytotoxicity assay. The E:T ratios are indicated.
B, Splenocytes from the indicated mice were incubated
with DMSO control or with 10 µM LemA peptide and stained for
Kb or M3 expression. Dotted lines, background staining with
isotype control Abs; thin black lines, mAb130 (anti-M3) or AF6-88.5
(anti-Kb) staining of untreated cells; and filled
histograms, mAb130 or AF6-88.5 staining of peptide-treated cells.
