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*
Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110; and
Department of Newborn Medicine, Children’s Hospital, St. Louis, MO 63110
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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2-microglobulin (2m)
and peptide are allowed to egress from the ER and transit to the cell
surface. Although several members of this class I peptide-loading
complex have been identified, their selective roles and importance for
different class I molecules remain controversial. Initially, nascent H
chains are detected in association with calnexin, a lectin-like
chaperone associated with various incompletely assembled oligomeric
glycoproteins (2). After assembly with
2m (3), class
I/2m heterodimers next associate with
calreticulin (4, 5, 6), another lectin-like, general
chaperone for assembly of oligomeric glycoproteins (7).
Subsequently, class I/2m heterodimers are
detected in physical association with TAP (8, 9, 10, 11), the
class I-dedicated peptide transporter, as well as the MHC-encoded
48-kDa glycoprotein, tapasin (4, 12, 13). More recent
studies have shown that the thiol-dependent reductase ERp57 is also a
component of the peptide loading complex (14, 15, 16). Once
the peptide binds, fully assembled class I molecules dissociate from
the peptide-loading complex and transit to the cell surface.
Importance of tapasin in the expression of various human class I
alleles is based largely on studies of tapasin-deficient LCL721.220
(.220) cells (4, 10, 12, 17, 18). Class I molecules in
.220 cells are not detected in association with TAP. Furthermore,
at least certain alleles were found to be more peptide accessible in
cell lysates and had reduced surface expression. Although
2m and TAP are expressed in .220 cells, no
functional tapasin protein was detected. However, due to a single
nucleotide change in the tapasin gene and a resulting frame shift, .220
cells express a small amount of a truncated tapasin protein missing the
last 8 amino acids of the signal peptide and the first 49 amino acids
of the N terminus (19). Expression of various human class
I alleles in .220 cells suggested that different alleles may display
different tapasin dependency for surface expression and Ag presentation
(17). This result could imply that different alleles may
bind to tapasin with different affinities. Alternatively, the available
peptide pool capable of binding to each class I allele may influence
its observed tapasin dependency. Thus, the nature of the reported
differences in the expression of various human class I alleles in .220
cells and the implications of these differences on tapasin function are
unclear.
The observation that the association of class
I/2m complexes with TAP is dependent upon
tapasin, suggested that tapasin might bridge class I with TAP
(4). Thus one of the proposed functions of tapasin is to
bring class I molecules into physical proximity with the peptide
source, TAP (8, 9). Although it remains to be proven, this
physical association of class I with TAP could promote peptide binding,
at least to certain class I alleles. Alternatively, tapasin appears to
have chaperone functions that are independent of promoting physical
association with TAP. For example, the removal of the transmembrane and
cytoplasmic domains of tapasin resulted in a secreted molecule that no
longer facilitated class I binding to TAP (20, 21).
Interestingly, this truncated tapasin increased surface expression and
Ag presentation of class I (20). From these findings it
was concluded that the association of tapasin with class I was
sufficient to facilitate class I folding. In another study, mouse
tapasin was shown in Drosophila cells to retain
peptide-empty mouse Kb molecules in the absence
of TAP (22). These authors concluded that tapasin
increases the expression of fully assembled class I molecules by
retaining empty class I molecules until they bind peptide. Furthermore,
using another insect expression system (Lepidoptera), Lauvau
et al. (23) concluded that tapasin facilitates assembly of
peptide with class I independently of mediating their retention in the
ER. Finally, other recent studies have suggested that tapasin may be
involved in peptide editing of class I in a manner analogous to the
role of DM with MHC class II molecules (24, 25, 26). Thus the
potential functions of tapasin include: 1) localizing class I near the
peptide supply, 2) facilitating peptide binding to class I and perhaps
peptide editing, and 3) ER retention/release of class I upon peptide
binding. Although there is little doubt that tapasin is a class
I-dedicated chaperone, the relative importance of each of these alleged
tapasin functions for different class I alleles remains to be
elucidated.
In this study we compare the expression of three different mouse class
I molecules Ld, Kd, and
Kb, in tapasin-deficient .220 cells to that in
tapasin-positive .221 cells. A novel strategy was employed whereby a
serological epitope (64-3-7) specific for open forms of
Ld was introduced into Kd
and Kb molecules (27). This 64-3-7
epitope is located on a loop in the 
1 domain that connects
strand with helical structure. Furthermore, it has been speculated that
the region defining the 64-3-7 epitope constitutes a hinge region that
changes conformation when peptide binds (27). The transfer
of the 64-3-7 epitope to other class I molecules allows their
respective open forms to be compared using the same mAb, thus
facilitating analytical comparisons between expression of these three
mouse class I molecules in the presence or absence of functional
tapasin. Our findings clearly show that tapasin greatly facilitates
intracellular peptide binding to Ld,
Kd and Kb molecules, since
new synthesis of 8- to 12-fold more class I was required in the absence
of tapasin to attain comparable surface expression of each of these
alleles. In addition a higher percentage of each of these class I
molecules was detected in the open conformation at the cell surface
when expressed in tapasin-deficient .220 cells compared with
tapasin-positive .221 cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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The .220 cell line is a human B lymphoblastoid cell line that does not express tapasin (10) and was kindly provided by Dr. Thomas Spies (Fred Hutchinson Cancer Research Center, Seattle, WA). The LCL721.221 (.221) cell line is a closely related cell line that does express tapasin (28, 29). L-Ld cells were made by introducing the Ld gene into Ltk- DAP-3 fibroblast (30). To produce a site-directed mutant of Kb expressing the 64-3-7 epitope, a Kb cDNA was kindly provided by Dr. Larry Pease (Mayo Clinic, Rochester, MN). It was subcloned into the mammalian expression vector RSV5.neo (31). Site-directed mutagenesis was performed using the Quik Change mutagenesis kit from Stratagene (La Jolla, CA) according to the manufacturer’s instructions. The synthetic oligodeoxynucleotides used for the reaction were as the forward primer: 5'-GGA GAA TCC GAG ATA TGA GCC GCA GGC GCC GTG GAT GGA GCA GGA GGG GC-3' and the reverse primer: 5'-GCC CCT CCT GCT CCA TCC ACG GCG CCT GCG GCT CAT ATC TCG GAT TCT CC-3'. L cells were transfected with the cDNAs using LipoFectin and selected in 0.6–1.0 mg/ml geneticin, both from Life Technologies (Gaithersburg, MD). Transfections of .220 and .221 cells were done by electroporation using the Gene Pulser II system from Bio-Rad (Hercules, CA). The construction and expression of epitope-tagged Kd was previously reported (27).
Peptide inhibition and flow cytometry
Peptides were synthesized using Fmoc solid-phase chemistry (32) on an Applied Biosystems (Foster City, CA) model 432A peptide synthesizer. All peptides described in this study were readily soluble in water at neutral pH and in cell culture media at physiological pH. Purity was >98% as assessed by reverse-phase HPLC and mass spectrometry. To test peptides for their ability to inhibit mAb 64-3-7 binding to Ld, peptides were diluted in 100 µl HBSS containing 0.2% BSA and 0.1% sodium azide and incubated at 4°C for 30 min in the wells of round-bottom microtiter plates with 20 µl culture supernatant containing mAb 64-3-7 or control mAbs. L-Ld cells (400,000/well) were then added and the peptide-mAb-cells incubation continued at 4°C for 1 h. The cells were then washed and incubated with FITC-conjugated, Fc-specific, affinity-purified F(ab')2 of goat anti-mouse IgG (ICN Pharmaceuticals, Cappel, Costa Mesa, CA). Viable cells, gated by forward and side scatter, were analyzed an a FACScalibur (Becton Dickinson, San Jose, CA) equipped with an argon ion laser tuned to 488 nm and operating at 150 mW. The data are expressed as linear fluorescence values obtained from log-amplified data using CellQuest Software (Becton Dickinson). Cells incubated only with the fluorescent Ab were used as negative controls.
Immunoprecipitation
Cells were preincubated for 60 min at 37°C in culture media that lacked methionine, after which 125–250 µCi/ml of [35S]methionine was added and the cells were radiolabeled for 5–10 min. The cells were then washed three times in PBS containing 20 mM iodoacetamide (Sigma, St. Louis, MO) and lysed in buffer that contained 1% digitonin (Wako, Richmond, VA) and 0.5 mM freshly added PMSF. The lysis buffer was supplemented with a saturating amount of mAb or rabbit Ab before addition to pelleted cells. After incubation for 30 min on ice, nuclei were removed by centrifugation, and lysates were incubated with protein A-Sepharose beads (Pharmacia, Piscataway, NJ). The beads were washed four times with 0.1% digitonin in TBS (pH 7.4) and the samples were eluted by boiling in 0.125 M Tris (pH 6.8)/2% SDS/12% glycerol/2% bromophenol blue. For autoradiography, gels were treated with Amplify (Amersham, Boston, MA), dried, and exposed to BioMax MR film (Eastman Kodak, Rochester, NY) at -70°C for varied lengths of time. For Western blots, SDS-PAGE gels were transferred to Immobilon P membranes (Millipore, Bedford, MA). After overnight blocking, membranes were incubated in a dilution of Ab for 2 h, washed three times with PBS/0.05% Tween 20, and incubated for 1 h with biotin-conjugated goat anti-mouse or anti-rabbit IgG (Caltag Laboratories, San Francisco, CA). Following three washes with PBS/0.05% Tween 20, membranes were incubated for 1 h with streptavidin-conjugated HRP (Zymed, San Franscisco, CA), washed three times with PBS/0.3% Tween 20, and incubated with enhanced chemiluminescent reagents (Amersham).
51Cr release assay
The Ld-alloreactive, p2Ca-specific clone 2C was a generous gift from Herman Eisen (Massachusetts Institute of Technology, Cambridge, MA). 2C was maintained in 24-well plates at 5 x 105 cells/well and stimulated weekly with 5 x 106 irradiated (2000 rad) BALB/c splenocytes/well in sensitization medium (RPMI 1640 medium supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 100 U/ml penicillin/streptomycin and 10% FBS) and 10 U/ml murine rIL-2. Target cells (1 x 106) were labeled for 1 h with 150 µCi 51Cr (Na51CrO4; NEN, Boston, MA; 1 Ci = 37 GBq) in 200 µl RPMI 1640 medium plus 10% bovine calf serum at 37°C in 5% CO2. Effector cells were plated at various concentrations into 96-well U-bottom microtiter plates, and 5 x 103 washed target cells per well were added. The plates were centrifuged at 50 x g for 1 min and incubated for 4 h at 37°C in 5% CO2. Radioactivity in 100 µl of supernatant was measured in an Isomedic counter (ICN Biomedicals, Huntsville, AL). The mean of triplicate samples was calculated and the percentage of 51Cr release was determined according to the following equation: % 51Cr release = 100 x ((experimental 51Cr release - control 51Cr release)/(maximum 51Cr release - control 51Cr release)), where experimental 51Cr release represents counts from target cells mixed with effector cells; control 51Cr release represents counts from target cells incubated in medium alone (spontaneous release); and maximum 51Cr release represents counts from target cells lysed with 5% (v/v)Triton X-100 (Sigma).
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Ld molecules expressed at high levels in the absence of tapasin have a higher percentage of peptide-free forms at the cell surface
Studies of the synthesis and chaperone association of the Ld allele have been greatly facilitated by the use of mAb 64-3-7 that is specific for open forms (33, 34). For example, addition of specific peptide ligands to cell lysates showed that 64-3-7+ Ld conformers are precursors of folded Ld molecules as detected by a mAb such as 30-5-7. Furthermore, folded Ld molecules at the cell surface were found to transit through a 64-3-7+ conformation after peptide dissociation (34), and acid stripping results in the emergence of 64-3-7+ Ld molecules (Y.Y.L.Y., unpublished observations). Thus, mAb 64-3-7 is clearly capable of detecting Ld molecules that are truly empty, although it might also detect Ld molecules with ligands incapable of inducing a completely folded, native conformation. In any case, 64-3-7 can specifically detect both open forms of nascent Ld molecules awaiting peptide as well as open forms of Ld at the surface arising after peptide dissociation (34). Given these findings, we considered the ability to discriminate open from folded Ld molecules a novel approach to assess the role of tapasin in the expression of this allele.
Stable transfectants of Ld expressed in .220 and
.221 cells were selected that showed comparable surface expression as
determined using mAb 30-5-7 that detects folded/assembled
Ld molecules. The fact the we were able to detect
matched .220/.221 cell lines demonstrated that tapasin was not an
absolute requirement for Ld surface expression.
Furthermore, these matched cell lines were found to be comparable in
their presentation of the endogenous peptide p2Ca to
Ld-reactive 2C T cells (Fig. 1
). Thus, based on criteria used in
previous studies, namely surface expression and Ag presentation,
Ld would be considered tapasin-independent.
However, when the amount of open Ld
(64-3-7+) was compared on .220 vs .221 cells, a
striking difference was observed. As shown in Fig. 2A,
.220-Ld cells express more open
Ld than folded Ld at the
cell surface, whereas .221-Ld cells expressed
more folded Ld than open
Ld. Indeed, the percentage of open
Ld molecules is about 2-fold higher on
.220-Ld cells compared with
.221-Ld cells. We have compared the ratio of open
to folded forms of Ld in independent assays of
the same cell lines and found them to be remarkably constant.
Furthermore, the ratios of independently derived
.220-Ld or .221-Ld cell
lines were found to be very similar (data not shown). When the ratios
of open to folded forms on .220-Ld vs
.221-Ld were compared, they were found to be
significantly different (p < 0.05, Tukey’s
multiple comparison test). Thus measurements of the amount of open to
folded class I molecules on the cell surface is a reliable method to
analytically compare the quality of class I expression.
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). Thus Ld appears to productively interact with
either human or mouse tapasin to increase the fraction of
Ld molecules expressed in the folded
conformation. Surface expression of open forms of Ld on both .221 and .220 cells is ablated by culture with exogenous peptides
To compare the peptide accessibility of open forms of
Ld molecule expressed in the presence or
absence of tapasin, .220-Ld and
.221-Ld cells were cultured overnight with
the Ld-binding CMV peptide, YPHFMPTNL
(37). As shown in the top panels of Fig. 3A, the level of open
Ld as detected by 64-3-7 was sharply reduced on
both .221-Ld and .220-Ld
cells. Published studies suggest that exogenous peptide can either bind
peptide-empty surface class I molecules (33) or,
alternatively, peptide can be transported into cells and bind nascent
class I molecules in the ER (38). However, regardless of
mechanism, the reduction of the expression of
64-3-7+ Ld molecules in the
presence of peptide provides further evidence that
64-3-7+ molecules are indeed peptide empty.
Furthermore, these findings show that open Ld
forms expressed in the presence or absence of tapasin can be eliminated
by incubation with exogenous peptide. In summary, these comparisons of
.220-Ld and .221-Ld
demonstrate that high levels of surface Ld
expression can be achieved in the absence of tapasin, but such
molecules display a higher percentage of open forms than
Ld molecules expressed in the presence of mouse
or human tapasin.
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To determine whether our findings regarding the tapasin
dependencies of Ld could be extended to
additional mouse class I alleles, we introduced the 64-3-7 epitope into
Kd and Kb molecules. In a
previous study we determined that 64-3-7 recognition of
Ld was determined largely by glutamine at
position 48 and proline at position 50 (27). Furthermore,
peptide inhibition of 64-3-7 binding to Ld showed
that the epitope was contained within the 21 amino acid sequence
corresponding to residues 35–55. To further define the 64-3-7 epitope,
length variants were tested and a 10-mer peptide (residues 46–55) was
found to be as potent as the 21-mer (data not shown). We next truncated
the 10-mer peptide at both termini and determined that the 7-mer
peptide(EPQAPWM) was the minimal length peptide that gives maximal
inhibition of 64-3-7 binding to Ld (Fig. 4A). To define critical
residues within this 7-mer peptide, peptides with alanine substitutions
were tested. As shown in Fig. 4B, residues at positions 48,
50, and 51 were critical for 64-3-7 inhibition. Within the 7-mer
sequence comprising the 64-3-7 epitope, Kd only
differs from Ld by the single amino acid R vs P
at residue 48, and Kb only differs by the two
amino acids R vs Q at 48 and R vs P at 50 (Fig. 5, top left panel). Thus, to
transfer the 64-3-7 epitope to Kd, the single
substitution R48Q was introduced (previously shown in Ref.
27 , and shown in this study for comparison) and, to
transfer the epitope to Kb, the double
substitution of R48Q,R50P was introduced (Fig. 5, bottom
panels). As shown in Fig. 5, these respective substitutions
rendered a subset of the Kd and
Kb molecules positive with mAb 64-3-7 when
expressed in L cells. These epitope-tagged forms, subsequently referred
to as etKd and etKb, were
tested extensively for peptide binding and T cell recognition and were
found to be indistinguishable from wild type molecules (Ref.
27 and our unpublished data). Thus epitope transfer did
not interfere with peptide binding or T cell interaction, a finding
consistent with its location on the 3D structure of the folded class I
molecule (27). Indeed, residues 48 and 50 are on a loop in
the 1 domain that connects the last strand with the beginning of
the helix. These residues point out and away from the peptide
binding groove of the folded class I molecule, and it has been proposed
that this region displays conformational flexibility when peptide binds
(27, 39). In any case, epitope tagging offers a unique
opportunity to identify open forms of Kd and
Kb molecules, and better determine the role of
tapasin in their expression.
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To determine the role of tapasin in the expression of
Kd and Kb molecules,
epitope-tagged forms of these class I alleles were expressed in .221
and .220 cells lines. As shown in the middle panel of Fig. 2, a matched
pair of .221-etKd and
.220-etKd cells that have about the same level of
expression of folded Kd as detected by mAb
SF1–1.1.1 (40) were selected for comparison. Similarly, a
matched pair of .221-etKb and
.220-etKb cells that have about the same level of
expression of folded Kb as detected with mAb
B8-24-3 (41) were selected for comparison (Fig. 2, right panel). Thus, tapasin is clearly not an absolute
requirement for the expression of folded Kd or
Kb molecules. It is important to note that
Kd and Kb molecules
expressed on L cells have about the same ratio of folded to open forms
as Kd and Kb molecules
expressed respectively on .221 cells (Fig. 2). This result suggests
that Kd and Kb alleles can
functionally interact with either mouse or human peptide-loading
complex (i.e., tapasin, calreticulin, and Erp57). By contrast
Kd and Kb molecules
expressed in the .220 cells showed about 2- to 3-fold more empty forms,
compared with Kd and Kb
molecules respectively expressed in .221 cells. Therefore, the
expression of open forms of Ld,
Kd, and Kb is increased
about two to three times in the absence of functional tapasin, thus
defining a common tapasin dependency of these three mouse class I
alleles. However, interestingly, there were significantly fewer open
forms of Kd and Kb than
Ld in either the presence or absence of tapasin
(Fig. 2). This higher level of open Ld forms is
consistent with the relatively weak peptide binding characteristic of
this class I molecule (42).
Surface expression of open forms of Kd and Kb on both .221 and .220 cells is reduced by culture with exogenous peptide
To compare the peptide accessibility of open forms of surface
Kd and Kb molecules
generated in the presence or absence of tapasin, transfected cells were
cultured overnight in the presence of known peptide ligands. For the
Kd allele we used the CW3 peptide RYLKNGKETL
(43) as a known Kd ligand, and a
length matched control peptide YASNENMETM (NP) as a
non-Kd binder. As shown in the middle
panels of Fig. 3A, culture of either
.221-etKd or .220-etKd
cells overnight with exogenous CW3 peptide resulted in a
peptide-specific, dose-dependent drop in 64-3-7 expression. For the
Kb molecule we used the OVA peptide SIINFEKL
(44) as a positive control and the CW3 peptide as a
negative control. As shown in the bottom panels of Fig. 3A, overnight incubation with peptide resulted in the
dose-dependent decrease in open forms of etKb as
detected with mAb 64-3-7. However, this decrease was complete with
.221-etKb cells, but less pronounced with
.220-etKb cells. To extend these findings,
.220-etKb and .221-etKb
cells were treated with a wider range of OVA peptide concentrations up
to 500 µM. As shown in Fig. 3B treatment with exogenous
SIINFEKL resulted in complete elimination of open forms of
etKb on .211 cells. By contrast about 1/3 of the
open forms of etKb on .220 cells remained after
treatment with high concentrations of OVA peptide. Thus a fraction of
the open forms of surface Kb molecules are more
refractory to peptide binding when expressed in the absence vs presence
of tapasin.
The relative refractory nature to exogenous peptide of
etKb molecules synthesized in the absence of
tapasin is intriguing. We know epitope tagging Kb
does not influence its ability to bind SIINFEKL or other known
Kb ligands (e.g., Fig. 3B, upper
panel and data not shown). Furthermore,
etKb-SIINFEKL complexes were found to stimulate a
T cell hybridoma (not shown). Thus, the relative refractory nature of
etKb molecules synthesized in the absence of
tapasin does not reflect aberrant peptide binding. Alternatively, this
refractoriness may reflect a unique structural feature of
Kb or the manner by which it interacts with the
human proteins in LCL721-derived cell lines (i.e., .220 and .221).
Indeed, the high level of expression of Kb in
human TAP-deficient T2 cells and not mouse TAP-deficient RMA-S cells
has been proposed to result from the high affinity of
Kb for human vs mouse 2m
(45). The proposed model was that a higher affinity
interaction with human 2m could help
Kb better form stable complexes with peptides and
thus attain a higher level of surface expression. However, it should be
mentioned that Ld and Kd
have also been reported to bind human 2m
better than mouse 2m (46). Thus,
the refractoriness of open forms of Kb to bind
exogenous peptide, relative to Ld and
Kd (Fig. 3), cannot easily be explained by it
having a higher affinity for human vs mouse
2m. Furthermore, it warrants noting that all
three of these mouse class I molecules had the very similar percentage
of open forms when each was respectively expressed on the surface of L
cells (mouse 2m+) vs
.221 cells (human 2m+;
Fig. 2). Thus, using the approach reported in this study, we detected
no differences in the expression of Ld,
Kd, or Kb in the presence
of mouse vs human 2m.
Comparable surface expression of Ld, Kd, or Kb in the absence of tapasin requires strikingly more newly synthesized H chains
The above findings demonstrate that Ld,
Kd, and Kb can be expressed
at high levels on .220 cells, implying there is no strict requirement
for tapasin for each of these alleles. However, to compare the
efficiency of expression of these mouse class I molecules in the
presence or absence of tapasin, we quantified the amount of newly
synthesized class I molecules in each pair of cell lines matched to
have comparable levels of surface expression, i.e.,
.221-Ld vs .220-Ld,
.221-etKd vs .220-etKd, and
.221-etKb vs .220-etKb. To
compare the levels of newly synthesized class I molecules, cells were
pulse labeled for 5 min and precipitated with mAb 64-3-7. Labeled H
chain bands were resolved by SDS-PAGE and precipitin bands were then
quantified by densitometry. For each mouse class I molecule studied,
the .220 partner cell line had about 6–12 times the level of newly
synthesized H chains compared with the .221 partner (Fig. 6, top panels). The
implication of these findings is that comparable surface expression of
Ld, Kd, or
Kb requires 6- to 12-fold higher de novo H chain
synthesis. This observation thus provided evidence using mammalian
cells that tapasin greatly facilitates the assembly of these three
mouse class I alleles. To estimate the steady-state levels of open H
chains in each of the matched cell lines, mAb 64-3-7 was used to stain
cells intracellularly and whole cell lysates were precipitated and
Western blotted with mAb 64-3-7. In both assays we estimated the
steady-state level of open forms of each allele was 2- to 3-fold higher
in .220 vs .221 cells. Thus, the relative difference in the
steady-state level of opens forms on the matched .220/.221 appears to
closely reflect what is on the cell surface (i.e., 2–3 to 1). These
combined findings imply that to achieve comparable surface expression
of Ld, Kd, or
Kb in the absence of tapasin, more H chains need
to be synthesized, and many of these are rapidly turned over.
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Consistent with our previous findings with
Ld (11) and
etKd (27),
64-3-7+ etKb molecules were
detected in association with TAP (Fig. 6, middle panels).
Furthermore, association of each of these mouse class I molecules with
TAP is dependent upon tapasin. Indeed this tapasin-dependency is rather
striking. Despite the higher levels of H chain synthesized by each
.220-transfected cell line compared with its matched .221-transfected
cell line, class I molecules only displayed prominent TAP association
in the presence of functional tapasin (Fig. 6). The implication of the
combined findings in this figure is that association of each of these
mouse class I alleles with TAP/tapasin facilitates peptide binding and
surface expression of fully assembled class I molecules. In support of
this conclusion, each of these three mouse class I molecules displayed
a higher rate of surface turnover, when expressed in the absence of
tapasin (.220 cells) compared with their expression in the presence of
tapasin (.221 cells) (data not shown).
Peptide preferentially folds mouse class I molecules in association with TAP/tapasin in cell lysates
For this analysis we initially used L-etKb
cells. L-etKb cells were metabolically labeled
for 10 min and lysates were incubated with the OVA peptide or the
non-Kb binding peptide, CW3. As shown in Fig. 7A, mAb 64-3-7 precipitated
substantial levels of etKb molecules, consistent
with its detection of nascent class I molecules awaiting peptide.
Furthermore, the addition of OVA peptide led to a modest (maximal 25%
by densitometry) increase in the detection of folded
etKb molecules as detected with mAb B8-24-3. A
commensurate loss of 64-3-7+
Kb was detected with addition of OVA peptide (as
determined by densitometry of a significantly lighter exposure than the
autoradiograph shown in Fig. 7A). Interestingly, the
2m-associated 64-3-7+
etKb molecules disappeared upon the addition of
peptide, demonstrating that peptide was preferentially folding
2m-associated Kb
molecules. Immunoprecipitates of open and folded
etKb molecules were also tested for TAP
association by Western blotting. OVA peptide eliminated
etKb molecules associated with TAP (Fig. 7A, lower panel) and tapasin (data not shown).
This result implied that OVA peptide was preferentially binding to
2m-assembled, peptide-empty forms of
Kb in physical association with TAP/tapasin.
Similar findings were also obtained with
.221-etKb cells (data not shown), demonstrating
that mouse vs human tapasin functioned similarly in this assay. Using a
reciprocal approach peptide was added to cell lysates, TAP was
precipitated and etKb molecules were blotted with
64-3-7. As shown in Fig. 7B, OVA peptide induced a
dose-dependent release of Kb from TAP. Thus, in
cell lysates, peptide preferentially binds Kb
molecules assembled with 2m and induces their
release from association with TAP. Furthermore, as shown in Fig. 7C peptide-induced folding of etKb was
significantly less efficient in tapasin-deficient .220 cell lysates
compared with tapasin-positive .221 cell lysates. These findings thus
strongly support the direct involvement of tapasin/TAP in facilitating
peptide binding to class I molecules as previously suggested using
disparate approaches (47). Furthermore, these findings
provide additional evidence that 64-3-7+ forms of
class I are peptide receptive while associated with the peptide-loading
complex, thus further highlighting the utility of the 64-3-7
epitope.
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Acknowledgments |
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Footnotes |
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2 N.B.M and M.R.H. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Ted H. Hansen, Department of Genetics, Box 8232, Washington University School of Medicine, St. Louis, MO 63110.
4 Abbreviations used in this paper: ER, endoplasmic reticulum; 2m, 2-microglobulin; et, epitope tag: .220, LCL721.220; .221, LCL721.221.
Received for publication April 10, 2000. Accepted for publication August 22, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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2-microglobulin complexes associate with the TAP transporter before peptide binding. Nature 368:864.[Medline]
2-microglobulin, class I heavy chain conformation, and tapasin in the interactions of class I heavy chain with calreticulin and the transporter associated with antigen processing. J. Immunol. 158:2236.[Abstract]
3 domain. J. Immunol. 162:1530.2-microglobulin mutants with MHC class I heavy chains and structural analysis demonstrate allele-specific interactions. Mol. Immunol. 36:56.
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) of Ld,
etKd, and etKb were detected using mAb 64-3-7
(
) were detected using
mAb 30-5-7 (
