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*
Department of Microbiology and Immunology and
School of Dental Science, University of Melbourne, and
Biomolecular Research Institute, Parkville, Victoria, Australia;
Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, Facultad de Ciencias, Madrid, Spain;
¶ Queensland Institute of Medical Research, Brisbane, Queensland, Australia; and
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Department of Immunology, Allergy, and Arthritis, Flinders University of South Australia, Bedford Park, South Australia, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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2-microglobulin. This indicated that a proportion of
suboptimal ligands were associated with B27, and they were lost during
the purification process. Notwithstanding this failure to recover these
suboptimal peptides, there was substantial overlap in the repertoire
and biochemical properties of peptides recovered from B27 complexes
derived from tapasin-positive and -negative cells. Although many
peptides were preferentially or uniquely isolated from B*2705 in
tapasin-positive cells, a number of species were preferentially
recovered in the absence of tapasin, and some of these peptide ligands
have been sequenced. In general, these ligands did not exhibit
exceptional binding affinity, and we invoke an argument based on
lumenal availability and affinity to explain their tapasin
independence. The differential display of peptides in tapasin-negative
and -positive cells was also apparent in the reactivity of
peptide-sensitive alloreactive CTL raised against tapasin-positive and
-negative targets, demonstrating the functional relevance of the
biochemical observation of changes in peptide repertoire in the
tapasin-deficient APC. Overall, the data reveal that tapasin
quantitatively and qualitatively influences ligand selection by class I
molecules. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-microglobulin (2m)
associates with the hc to form class I heterodimers, calnexin is
exchanged for calreticulin. At this stage other chaperones combine to
form the class I loading complex, which consists of the class I
heterodimer, calreticulin, ERp57, and a specialized chaperone called
tapasin (1). Tapasin is a 48-kDa glycoprotein that bridges
peptide-receptive class I heterodimers to TAP molecules (2, 3). This interaction is thought to facilitate the loading
of antigenic peptides into the binding cleft of class I
molecules before their exit from the ER and transport to the cell
surface. In addition to a bridging function, tapasin is thought to
stabilize the peptide-receptive state of the class I complex
(4), influence expression of TAP (4, 5), and
increase peptide binding to the TAP heterodimer (6). It
has also been demonstrated that tapasin retains empty MHC class I
molecules in the ER of insect cells (7). We have also
shown that tapasin prevents premature release of class I molecules from
the ER of mammalian cells, suggesting that tapasin may also play a role
in the retention of suboptimally loaded class I molecules
(8). These functions may be accomplished directly by
tapasin or indirectly through the concerted actions of other chaperones
recruited into the class I loading complex in the presence of
tapasin. Tapasin appears to be highly specific to the class I assembly pathway, while many of the other chaperones involved in class I assembly are more generic and involved in the folding and assembly of most glycoproteins in the ER (9). Therefore, it has been speculated that tapasin may play a role in class I ligand selection and optimization. Several features of tapasin make its putative editorial role attractive; these include the ability to retain empty or suboptimally loaded class I molecules in the ER (7, 8) and the observation that class I molecules that are expressed in tapasin-deficient cells are largely thermolabile and are loaded with a proportion of suboptimal ligands (10, 11, 12). Furthermore, recent studies have implied that tapasin interacts with regions of the class I hc that are sensitive to the presence of peptide in the Ag binding cleft (13, 14), reflecting the potential for tapasin to discriminate between peptide-loaded and empty molecules.
Recently, two groups have independently generated tapasin knockout mice
(15, 16). The class I molecules expressed in these mice
(H-2Kb and H-2Db)
demonstrate pronounced tapasin-dependence for their efficient surface
expression. The defect is not absolute, however, with evidence that
some pathogen-derived peptides are presented by these class I molecules
during infection (16) and that endogenous peptide
presentation is sufficient to provide limited positive selection in the
CD8+ T cell compartment and to induce tolerance
(15). In humans, the tapasin-deficient cell line 721.220
(17) has been used to study the behavior of HLA class I
molecules under these conditions. HLA A and HLA B alleles display
marked variation in their tapasin dependence for both efficient surface
expression and presentation of antigenic determinants to CTL in 721.220
transfectants (12, 18, 19, 20). For example, tapasin is not
required for high levels of surface expression of HLA-B*2705 on 721.220
cells or presentation of viral determinants to CTL by these molecules
(12). In contrast, functional Ag presentation and surface
expression of HLA-B*4402 are highly dependent on tapasin, whereas
HLA-B*0801 lies between these alleles in the spectrum of tapasin
dependence (12). This variation in surface expression
among class I alleles in tapasin-deficient cells most likely reflects
their differential abilities to load endogenous peptides in the absence
of tapasin. Other factors may also contribute to high levels of surface
expression on tapasin-deficient cells. For example, some class I
molecules egress to the cell surface in the absence of a functional
interaction with tapasin that results either from species
incompatibility between the class I molecule and components of the
class I loading complex (8) or from mutations in the class
I heavy chain itself (11). Thus, the murine class I
molecule H-2Kb is expressed at high levels on the
surface of the human 721.220 cells despite poor surface expression in
tapasin knockout mice (15, 16). This is probably due to 1)
greater efficiency of assembly of Kb/human
2M complexes despite suboptimal ligand selection, and 2)
defective retention of Kb molecules by the human
class I loading complex (8). Similarly, the HLA A2 mutant
T134K is not properly retained in the ER by the class I loading complex
and so expresses at high levels at the cell surface (11).
However, neither Kb expressed in 721.220 nor the
HLA A2 T134K mutant are likely to reflect physiological examples of a
tapasin-independent loading pathway, since both function very poorly in
Ag presentation and appear to be loaded with suboptimal peptide
repertoires (8, 11, 20). By comparison, HLA B*2705 is
remarkably efficient at Ag presentation and ligand selection in the
absence of tapasin, suggesting that a tapasin-independent pathway of
B27-peptide loading might operate at some level even under normal
conditions.
Despite achieving normal steady state levels of surface expression, there is evidence that the assembly of HLA B*2705 with peptide is less efficient in the absence of functional tapasin. For example, a delay in the kinetics of B27-restricted viral peptide presentation to CTL was observed in tapasin-deficient 721.220 cells (12). Moreover, surface class I peptide complexes appear less stable in the absence of tapasin, as revealed by more rapid surface decay (10) and an increase in the expression of denatured HLA B*2705 molecules on the surface of 721.220 transfectants (12). Tapasin also appears to influence the peptide repertoire of HLA B*2705 qualitatively, as highlighted by the ability of certain peptide-sensitive mAbs to differentiate between B*2705 molecules expressed in the presence and the absence of tapasin (10, 12). This differential epitope expression suggests that the peptides presented by HLA B*2705 in the presence and the absence of tapasin might not be identical. Thus, we have chosen to study the repertoire of peptides captured by HLA B*2705 in the presence and the absence of tapasin for the following reasons: 1) a comparison of the peptides presented in the presence and the absence of tapasin provides insights into the function of tapasin; and 2) HLA B27 exhibits redundancy in assembly and Ag presentation pathways (12, 21, 22), and there are grounds to believe that loading of some B27 molecules uses a tapasin-independent pathway in normal cells. Therefore, the principles of repertoire selection under these circumstances are important. 3) The relative tapasin independence of B27 molecules appears unusual among HLA class I alleles, most of which are dependent upon tapasin for high level surface expression and Ag presentation; and 4) any unusual properties or functions of B27 are of great interest because of its strong association with ankylosing spondylitis. Peptides were eluted from B*2705 molecules expressed on the surface of transfected 721.220 (B27.220) cells and from the same cells reconstituted with human tapasin (B27.220.hTsn), then were analyzed by a combination of reverse phase HPLC (RP-HPLC) and mass spectrometry. Our findings demonstrate that tapasin not only enhances peptide loading quantitatively, but its expression is associated with qualitative changes in the repertoire of endogenous ligands presented for T cell recognition. We have determined the amino acid sequence of some ligands that are loaded into the cleft of HLA B27 in the presence and the absence of tapasin. The protein source and binding to HLA B*2705 of such peptides reveal that neither cellular abundance nor B27 peptide affinity explain their tapasin independence. The ability of HLA B27 to acquire antigenic peptides via this tapasin-independent pathway is discussed in the context of the association of this allele with inflammatory arthropathies.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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721.220 is a human lymphoblastoid cell line in which HLA A and B genes have been deleted and a nonfunctional tapasin protein is expressed (17, 23). The transfection of HLA B*2705 and wild-type human tapasin into 721.220 has been described previously (12). The human TAP-deficient cell line T2 transfected with HLA B*2705 (T2.B27) has been described previously (24). All cells were grown in RF-10 (RPMI 1640, Life Technologies, Gaithersburg, MD) supplemented with 2 mM glutamine (Life Technologies), antibiotics, and 10% FBS (Commonwealth Serum Laboratories, Melbourne, Australia). For the peptide elution experiments cells were grown in miniPERM bioreactors (Hereaus, Hanau, Germany) and sequentially conditioned into hybridoma serum-free media (Life Technologies) containing 1% FBS. Flow cytometry and Western blots were used to establish that B*2705 and tapasin were stably expressed in the transfected cell lines during expansion as previously described (25).
Alloreactive CTL and 51Cr release assay
721.220, B27.220, and B27.220.hTsn cells were examined for recognition by a panel of HLA B27-specific alloreactive CTL. These CTL comprised specific alloreactive clones raised against normal B27-positive cell lines that were selected on their ability to recognize B*2705 in a peptide-dependent manner (26, 27). Target cells were grown in RF-10, labeled for 90 min at 37°C with 50 µCi of 51Cr, washed four times, and resuspended in the same medium with 1% FBS. Target cells were then seeded into 96-well plates and coincubated with effector CTL for 4 h at 37°C at given E:T cell ratios. Supernatants of this coculture were harvested and subjected to gamma counting. The percent specific 51Cr release was calculated as ((experimental lysis - spontaneous lysis)/(maximum release - spontaneous lysis)) x 100.
Generation of B27-restricted, peptide-dependent CTL against allogeneic tapasin-deficient APC
The SB and NK series of clones were raised in this study by stimulating PBMC from healthy B27-negative donors with the tapasin-deficient B27.220 cell line as follows. PBMC (2 x 106) from donor SB (HLA A2 A30 B35 B57) or NK (HLA A2 A2 B14 B44) were stimulated in 2 ml of RF-10 with gamma-irradiated (8000 rad) B27.220 stimulator cells at a responder to stimulator ratio of 25:1. After 3 days cells were dispersed and seeded in 0.35% agarose (Seaplaque, FMC BioProducts, Rockland, ME) containing 55% RPMI 1640, 20% FCS, 25% supernatant from MLA-144 cultures, and 30 U/ml rIL-2. Colonies were harvested after an additional 4 days and were amplified in culture with biweekly restimulation with rIL-2, MLA-144 supernatant, and irradiated B27.220 cells.
Preparation of W6/32 affinity matrix
An affinity matrix comprised of 10 mg/ml W6/32 cross-linked to protein A-Sepharose resin was prepared using a modification of published methods (28). Briefly, W6/32 was bound (10 mg/ml resin) to protein A-Sepharose, and the resin was washed extensively in borate-based buffers. A solution of 40 mM dimethylpimelimidate (Sigma, St. Louis, MO) in 0.2 M triethanolamine, pH 8.0, was used to cross-link the Fc region of W6/32 to the protein A matrix, enhancing the spatial distribution and conformational integrity of the immobilized Ab. Cross-linking proceeded for 1 h at room temperature, and the reaction was terminated by addition of ice-cold 40 mM ethanolamine, followed by extensive washing steps.
Metabolic labeling of cells and isolation of HLA B*2705-bound peptides
For metabolic labeling studies, 1 x
108 cells were washed in PBS and transferred into
arginine-free RPMI (RPMI SelectAmine kit, Life Technologies)
supplemented with 10% dialyzed FBS and 2 mM glutamine. The cells were
incubated at 37°C for 1.5 h in arginine-free medium,
metabolically labeled with 750 µCi of
[3H]arginine (arginine L-[2, 3,
4-3H]monohydrochloride, AMRAD, Melbourne,
Australia) for 12 h at 37°C. Cells were harvested and lysed in
0.5% Nonidet P-40 (Sigma), 150 mM NaCl, and 10 mM Tris (pH 8.0)
supplemented with Complete protease inhibitor cocktail (Roche,
Mannheim, Germany). An aliquot of this lysate was retained to normalize
for label uptake. HLA B*2705-peptide complexes were immunoprecipitated
using W6/32-protein A-Sepharose at a ratio of 50 µl
resin/108 cells. The resin was washed
extensively, and peptides were dissociated from the immobilized B*2705
molecules by treating the resin with 10% acetic acid. The eluate was
subsequently passed through a Microcon-3 (Millipore, Bedford, MA)
centrifugal filter to separate the peptides from
2m and hc. The retentate was analyzed by
SDS-PAGE and was shown to contain 2m and hc.
The flow-through (<3 kDa) was then fractionated by RP-HPLC using a
SMART system HPLC (Pharmacia Biotech, Uppsala, Sweden). Peptides were
separated using a µRPC
C2/C18 (2.1 mm (inside
diameter) x 10-cm column; Pharmacia Biotech, Uppsala, Sweden)
operating initially with a linear gradient from buffer A (0.1%
trifluoroacetic acid (TFA)) to 40% B (acetonitrile/0.09% TFA),
1.3%/min at a flow rate of 200 µl/min. Following this gradient a
more rapid 40% B to 60% B, 4%/min gradient was employed at a flow
rate of 200 µl/min. Individual HPLC fractions (150 µl) were
collected, and the incorporated [3H]arginine
was measured by a beta liquid scintillation counter (Packard,
Meriden, CT).
Purification of HLA B*2705 and peptide analysis
Purification of HLA B*2705 from various cell lines was performed based on the method of den Haan et al. (29). B27.220 or B27.220.hTsn cells were grown in miniPERM bioreactors in hybridoma serum-free medium supplemented with 1% FBS. For accurate comparison these cells were processed in tandem; 5 x 109 cells from each line were lysed at 4°C in 0.5% Nonidet P-40, 20 mM Tris, and 150 mM NaCl (pH 7.4) supplemented with Complete protease inhibitor cocktail (Roche). Cell lysates were clarified by two rounds of centrifugation, and the supernatant was filtered and passed over a Tris-blocked Sepharose 4B precolumn. The precleared lysates were then applied to columns containing 2 ml of W6/32 affinity matrix, and the columns were washed in buffer containing 50 mM Tris, 150 mM NaCl, and 0.005% Nonidet P-40 (pH 8.0). The columns were subsequently washed extensively with 50 mM Tris and 150 mM NaCl (pH 8.0), a high salt buffer, to remove nonspecifically bound material (50 mM Tris and 500 mM NaCl (pH 8.0)) and finally into 50 mM Tris (pH 8.0). Bound HLA-B*2705 peptide complexes were eluted with 10% acetic acid, which also facilitates dissociation of the B*2705-bound peptides. The eluate was then passed through a Centricon 3 membrane (Millipore), and the flow-through was concentrated by vacuum centrifugation to a final volume of 300 µl. This material was then subjected to further purification with RP-HPLC using the column and HPLC system described above.
Eluted peptides were resolved from contaminating detergent polymers by employing a rapid gradient from 0 to 60% acetonitrile in 0.1% aqueous TFA (12%/min, 200 µl/min). This material was then subjected to pool Edman sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS; as described below). Further purification of the eluted B*2705-restricted peptides was afforded using an optimized gradient with the same HPLC system and column. A linear gradient from buffer A (0.1% TFA) to 40% B (acetonitrile/0.09% TFA; 1.3%/min), then 40% B to 60% B (4%/min) at a flow rate of 200 µl/min was used to separate peptides. Fractions (150 µl) were retained and analyzed by MALDI-TOF MS. Identical conditions were used for the purification of B*2705-restricted peptides from both B27.220 and B27.220.hTsn to allow comparison of equivalent fractions from the two cell lines. Importantly, the entire peptide elution experiment was replicated to examine the reproducibility of specific peptide recovery.
Peptide synthesis, sequencing, and MS
Synthetic peptides were purchased from Chiron Technologies
(Clayton, Australia) and synthesized with free carboxyl and amino
termini using PIN-based techniques (30). N-terminal
automated Edman sequencing was performed on a Hewlett-Packard G1000A
protein sequencer (Hewlett-Packard, Palo Alto, CA) using standard Edman
chemistries. MALDI-TOF MS was performed using a Bruker Reflex mass
spectrometer (Bruker-Franzen Analytik, Bremen, Germany) operated
exclusively in the reflectron mode as described previously
(31). Aliquots of fractions (1–2 µl) were mixed with an
equal volume of 
-cyano-4-hydroxycinnamic acid (10 mg/ml in
acetonitrile/ethanol, 1/1, v/v), spotted onto a target, and dried for
analysis. For comparison of HPLC fractions, identical laser irradiance
and repetitions were used to ionize each sample. Care was taken to
ensure that uniform matrix/sample crystals were deposited onto the
target, ensuring minimal variation in sample ionization between
different regions on a given target position and between replicate
samples. These measures improve the quantitative nature of the MALDI
data. Post-source decay (PSD) experiments were performed using 14
stepwise decrements in the reflectron potential and increasing the
laser irradiance to optimize the production of fragment ions at each
voltage. Assembly of the individual spectra on to a continuous mass
scale was performed using FAST software routines within the Bruker XTOF
software package. Identification of fragmented ion species was
determined by manually assigning C- and N-terminal ion series and
comparing parent m/z and fragmentation data to database entries using
MS-FIT routines available through the protein prospector program
(http://prospector.ucsf.edu). Accurate parent ion mass and
fragmentation data allowed assignment of peptide sequence or sequences
in several instances. Peptide sequencing by ion trap electrospray
ionization MS was performed as previously described (32).
Briefly, dried samples were resuspended in 3 µl of methanol/water
(1/1) containing 0.1% formic acid. One microliter of each sample was
subjected to peptide sequencing in an LCQ electrospray/ion trap mass
spectrometer (Finnigan Thermoquest, San Jose, CA). Collision energy and
precursor ion resolution were individually optimized for each peptide
to obtain the optimum fragmentation spectra. Putative peptide sequences
were obtained by database comparison of the fragmentation spectra using
the PEPSEARCH program (Bioworks package, Finnigan Thermoquest) followed
by manual assignment of expected fragments from the highest score
sequences. In several instances the authenticity of these sequence
assignments was confirmed by comparing the fragmentation spectrum with
that of the corresponding retrospectively synthesized peptide.
Competitive HLA B*2705 peptide binding assay
For measurement of relative B27 peptide binding affinities we
developed a competition assay in which test peptide was titrated
against a fixed amount of labeled reporter peptide and coincubated with
T2.B*2705 cells (33). The reporter peptide was synthesized
with a biotinylated lysine residue chosen on the basis of predicted
solvent accessibility at amino acid position P3, P4, or P5 of the bound
peptide ligand in crystal structures (34) or theoretical
models (35) of HLA B*2705 complexes. A peptide based on
residues 284–292 of the GroEL protein from Chlamydia
trachomatis (RR[N
-biotinyl-lysine]AMFEDI) was used
in the assay based on the signal intensity observed. The assay
proceeded as follows; 3 x 105 T2.B*2705
cells were seeded into each well of a 24-well plate and incubated at
26°C overnight in an atmosphere of 5% CO2. The
reporter peptide RRK*AMFEDI was added to each
well to a final concentration of 10 µM, and graded concentrations of
test peptide (0–50 µM) were added to individual wells, incubated for
1 h, then shifted to a 37°C incubator for an additional 2 h
to allow equilibrium to be established at physiological temperature.
Cells were harvested on ice and washed three times in wash buffer (1%
FBS and 0.02% sodium azide in PBS). The cells were stained with
streptavidin-PE (Vector, Burlingame, CA), followed by a biotinylated
anti-streptavidin mAb (Vector) and finally with a second round of
streptavidin-PE. The cells were then analyzed on a FACSort flow
cytometer (Becton Dickinson, San Jose, CA). Competition was visualized
as the percent binding of the reporter peptide (background corrected)
observed in the absence of competitor or in the presence of 50 µM
irrelevant peptide (EEFGRAFSF). The inhibitory concentration at which
reporter peptide binding was reduced by 50%
(IC50) was derived for each test peptide.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate the influence of tapasin on peptide loading, class
I molecules were immunoaffinity purified from 5 x
109 B27.220 or B27.220.hTsn cells using a matrix
consisting of the pan class I mAb W6/32 cross-linked to protein
A-Sepharose. Acid-eluted peptides were isolated over a Centricon 3
membrane and crudely separated by RP-HPLC to remove traces of
detergent. The retentate on the Centricon 3 filter was analyzed further
by SDS-PAGE and revealed equivalent yields of class I hc and
2m from both cell types (Fig. 1
A), consistent with the
similar levels of surface HLA B*2705 expressed by these cells
(12). Importantly, the relative intensities of the HLA B27
hc and 2m bands were similar in both
retentates, suggesting that the immunoaffinity-purified material was
not substantially composed of altered forms of HLA B27. Furthermore,
these gels were electrophoresed under nonreducing conditions, and there
was no evidence of B27 hc homodimers (
90 kDa) (36),
consistent with our findings that cell surface hc homodimer is not
reactive with W6/32 (C. A. Peh, unpublished observations).
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B) consistent with the
known B*2705 binding motif (37). The contributions of
other amino acids to subdominant anchor positions, notably at P1, P3,
and P8/9, were also similar for both eluates (data not shown). Thus,
the absence of tapasin does not alter the structural constraints that
confer the well-recognized binding motif for HLA B*2705. Also evident
in Fig. 1B is the reduced yield of peptides containing
arginine at P2 derived from tapasin-deficient cells. 721.220 cells lack
functional tapasin and have lost HLA A and B loci, but retain low
levels of HLA Cw*0102. This allele also has a preferred P2 anchor
residue, in this case for alanine (38). Therefore, direct
comparison of arginine and alanine yields from cycle 2 of Edman
sequencing reflects the relative contributions of peptides derived from
HLA B*2705 and Cw*0102 to the peptide pool. In the tapasin-deficient
cell line no increase above background was observed for alanine at P2,
while a small, but significant, alanine peak was observed in the
peptides recovered from the B27.220.hTsn cell line. The other amino
acids in this cycle do not significantly deviate from background
levels. These data indicate a minor contribution of Cw1 peptides to the
eluate from tapasin-reconstituted cells.
We next exploited the preserved P2 arginine motif of B*2705 ligands in
tapasin-deficient cells to further assess the quantitative affects of
tapasin on the recovery of peptides associated with HLA B*2705. HLA
B*2705 transfected cells were metabolically labeled with
[3H]arginine, the radiolabeled peptide ligands
were isolated and separated by RP-HPLC, and individual fractions were
analyzed on a liquid scintillation counter. As shown in Fig. 1C, significantly more labeled peptide was recovered from
the B27.220.hTsn cell line compared with B27.220 or the control 721.220
cell line transfected with human tapasin alone (220.hTsn). Peptides
recovered from the B*2705 transfectants with and without tapasin
exhibited similar broad distributions of labeled species throughout the
chromatographic separation, while some hydrophobic species were
observed in the immunoprecipitated material from 220.hTsn cells,
reflecting low yields of endogenous HLA Cw1-bound peptides. The reduced
yield of peptides eluted from B27.220 was reproducible in a number of
independent experiments and is probably due to loss of poorly bound
B*2705 ligands during the metabolic labeling and purification process.
This hypothesis is consistent with decreased yield of arginine in cycle
2 of Edman degradation and the reduced surface stability of the
B*2705-peptide complexes expressed by tapasin-deficient cells
(10). Therefore, both the current study and previous
studies indicate the selection of a proportion of suboptimal ligands by
HLA B*2705 in tapasin-deficient cells.
Tapasin quantitatively and qualitatively alters the B*2705-bound peptide repertoire
The biochemical identity of bound peptides contained in the
eluates derived from B27.220 and B27.220.hTsn cells was further
analyzed by MALDI-TOF MS. Identical ionization conditions
were used for each sample to allow accurate comparison of the peptides
present in both samples. In addition, care was taken to mix analyte and
matrix components identically and to prepare a uniform coverage of
sample/matrix fine crystals over the target. This resulted in minimal
variation in signal when the analyte was analyzed at different regions
on the same target or on replicate target positions. As shown in Fig. 2A, the overall ionization
intensity of species within the expected m/z range of 8–12
mer (800–1300 Da) was higher in the peptide eluate from cells
expressing wild-type tapasin (spectra of positive polarity). This
observation was highly reproducible and is consistent with the reduced
recovery of radiolabeled peptides from cells lacking tapasin (Fig. 1C). Comparison of MALDI spectra of B*2705-bound peptides
derived from both cell lines revealed a high degree of spectral
overlap, with strong coincidence of species with the same
m/z values identified in both cell lines. This is
particularly evident in Fig. 2B, which represents a portion
of the spectra shown in Fig. 2A with an exploded
m/z axis. Thus, the majority of bound species isolated from
tapasin-positive and -negative cell lines shared m/z values
and presumably represent the same peptides. The signal obtained for
some species (Fig. 2B), however, differed significantly
between tapasin-deficient and reconstituted cells. For example, the
signal from the species with m/z of 1098.5 was 2.5-fold stronger in
cells expressing wild-type tapasin. Similarly, species with
m/z values of 1090.4, 1115.4, and 1108.4 were all more
intense in eluates from tapasin-positive cells. In contrast, species
with m/z of 1094.4 and 1095.4 (arrowed) were present at
equivalent intensities in eluates from the two cell lines. Although
MALDI-TOF MS is not a strictly quantitative tool, these differential
effects on signal intensities strongly suggested differential
presentation and recovery of B27-peptide ligands in the presence and
the absence of tapasin. Overall, the findings indicate that most, but
not all, of the peptides loaded into HLA B*2705 in the presence of
wild-type tapasin are also loaded in the absence of this chaperone, but
generally with lower efficiency. In addition, a spectrum of tapasin
dependency was observed for the recovery of individual species. These
observations were highly reproducible, with essentially identical
results obtained from two independent peptide elution experiments (data
not shown).
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We were particularly interested in the possibility that some
peptides might be captured selectively in the absence of tapasin, but
replaced or removed by other peptides in the presence of tapasin. To
examine this possibility, eluted peptides were further fractionated
using an optimized RP-HPLC separation protocol, and the resultant
fractions analyzed were extensively by MALDI-TOF MS. The mass spectra
of six representative RP-HPLC fractions derived from both B27.220 and
B27.220.hTsn eluates (fractions 26, 27, 28, 30, 34, and 36) are shown
in Fig. 3. The high degree of overlap in
the spectrum of peptides derived from tapasin-deficient and proficient
cells was still evident in the mass spectra of individual RP-HPLC
fractions, but a greater number of discordant species was now obvious
(see arrowed species). Consistent with the mass spectra on the peptide
pools, the majority of species were recovered more efficiently from
tapasin-positive cells (as depicted in the spectra derived for fraction
30); however, there were several notable exceptions. For example, some
species were found only in the presence of tapasin, see m/z
1058.4 (arrowed) or m/z 1197.5 in fraction 36, while other
species were recovered preferentially from the tapasin-deficient cells,
such as the peptide with m/z 1378.8 (arrowed) from fraction
26. This peptide was isolated in greater abundance (2- to 5-fold) from
tapasin-deficient cells in two independent experiments. Likewise,
species with m/z 1169.3 in fraction 27 and 1094.1 and 1797.4
in fraction 28 (arrowed) were all detected at a significantly higher
signal in tapasin-deficient cells. Another example is found in fraction
34, where a species of m/z = 1019.4 (arrowed) was found
uniquely in tapasin-deficient eluates, while in the same fraction the
species with m/z 1027.4 was detected with similar intensity
in both cell lines. Importantly, in these same fractions some other
species were recovered more abundantly in the tapasin-positive eluates,
as is obvious for numerous species in fraction 26 and the species of
m/z = 1031.1 (arrowed) in fraction 28.
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and 3 were
derived from HLA Cw1 we eluted peptides from 5 x
109 220.hTsn cells in a manner identical with
that used for the B27 transfectants. Only a few dominant species were
observed in the mass spectrum of these eluted peptides (data not
shown), and none had masses corresponding to those species observed in
the mass spectrum of the B27 eluted material. Therefore, combined with
the Edman sequencing and metabolic labeling studies, these observations
indicate that B27-bound peptides overwhelmingly contribute to the
peptide eluates examined in this study. Thus, our data indicate that
most peptides are loaded into HLA B*2705 less efficiently in the
absence of functional tapasin. However, contrary to the majority of
peptides, some species recovered from tapasin-deficient cells are
missing from the repertoire or are less abundant in tapasin-proficient
cells. This reflects a tapasin-dependent, although not necessarily
directly tapasin-mediated, editing mechanism and provides the first
direct biochemical evidence that tapasin qualitatively affects the
composition of species bound to class I molecules. Biochemical characterization of B*2705 ligands selectively presented by tapasin-positive cells
The MALDI-TOF MS analysis of 40 matched RP-HPLC fractions from
tapasin-deficient and tapasin-reconstituted eluates (i.e., 80 fractions
in total) revealed qualitative differences in the peptide repertoire
for numerous species. Several approaches were taken to further
investigate the nature of some of these qualitative changes in HLA
B*2705 peptide repertoire. Firstly, a number of previously reported
B*2705 ligands (39) were synthesized, and their RP-HPLC
retention behavior was modeled. Once the retention time of these
species was ascertained under the appropriate elution conditions, the
corresponding fractions from the micropreparative separation of natural
B*2705 ligands were examined for the presence of corresponding ions by
MALDI-TOF MS. Candidate ions within 1000 ppm of the expected mass were
observed for the majority of the synthetic peptides tested. The mass
accuracy of synthetic peptides and their putative natural analogues was
well within the range seen for peptides that were subsequently
sequenced by Edman degradation or mass spectrometry (
Da = -1.0
to 0.8). We conclude that several of the matched species are likely to
correspond to the modeled peptides; some of these candidates are listed
in Table I.
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The majority of modeled peptides representing previously characterized
B*2705 ligands appeared to be dependent on tapasin for efficient
presentation (see Table I). Notably, one peptide identified by LCQ
electrospray ion trap MS was only observed in eluates derived from
cells expressing tapasin, with no detectable recovery of this ligand
from tapasin-deficient cells. This species
(15GRYSGRKAV23) has not
been reported as a B*2705 ligand before and is derived from the 60S
ribosomal protein L27.
Biochemical identification of B*2705 ligands present equally in tapasin-positive and tapasin-negative cells
Although most ligands appeared to be recovered more efficiently
from tapasin-positive cells, some peptides were present at similar
levels in the eluates from both tapasin-positive and -negative cells.
Four peptides whose levels were similar in the eluates from both cell
types were sequenced using electrospray ion trap MS. Two of these
peptides have been previously characterized as B*2705 ligands:
173GRIDKPILK181, derived
from the 60S ribosomal protein L8 (41), and
36GRLTKHTKF44 ,
derived from the L36 ribosomal protein (42, 43). A third ligand,
299GRHGVFLEL307 (a
previously characterized ligand (39) derived from the
proteasome regulatory subunit S2), was initially recovered more
abundantly in the eluate from tapasin-deficient cells. This species
represents one of the prominent species (m/z = 1027.4
Da) contained in fraction 34 of the tapasin-deficient eluate, and its
relative abundance in the tapasin-deficient and reconstituted eluates
is shown in Fig. 3. However, in a second independent experiment, this
peptide was recovered in similar amounts from both cell lines, this
species was thus classed as a tapasin-independent ligand. The fourth
peptide,
379GRFGTKGLAITF390, is a
newly identified ligand derived from the ATP-dependent RNA helicase P47
protein. The recovery of this unusually long peptide from both
tapasin-positive and -negative cells suggests that tapasin does not
generically bias the size of peptides bound to the B*2705 molecule.
Similarly, analysis of the overlaid MALDI-TOF spectra of peptides
eluted from tapasin-positive and -negative cells did not reveal a
significant difference in the capture of longer peptides (>10 aa) by
B*2705 molecules in either cell line. The data also imply that tapasin
is unlikely to act as a trimming enzyme in the ER.
Biochemical characterization of B*2705 ligands selectively presented by tapasin-negative cells
We were particularly interested in identifying the nature of those
peptides that were recovered more abundantly in tapasin-deficient
cells. Edman sequencing of a fraction derived from tapasin-deficient
B27.220 cells identified one such peptide. Interestingly, this peptide
was derived from the B*2705 hc itself and has previously been reported
as a B*2705 ligand in normal cells (42). As shown in Fig. 3 (fraction 26), the intensity of the
169RRYLENGKETL179 peptide
(m/z 1378.9) derived from the B*2705 hc was significantly
higher in the tapasin-deficient cells (negative spectra) compared with
cells expressing wild-type tapasin (positive spectra). This result was
highly reproducible, and this species was preferentially recovered from
tapasin-deficient eluates in two independent peptide elutions. A number
of other species were identified in this study that also were
reproducibly recovered in greater abundance from the tapasin-deficient
cells (see, for example, the species of m/z 1169.3 observed
in fraction 27, m/z 1094.1 and 1797.4 in fraction 28, and
m/z 1019.4 and 1027.4 in fraction 34 depicted in Fig. 3).
Since most peptides are detected at equivalent or higher abundance in
tapasin-proficient cells, it is unlikely that the prominence of these
ligands in eluates from tapasin-deficient cells reflects any bias in
the recovery process or differential ionization of recovered ligands.
The increased recovery of the
169RRYLENGKETL179 peptide
derived from the B*2705 hc could be due to increased turnover of HLA
B*2705 in the absence of tapasin. However, this does not explain the
preferential recovery of other B*2705-bound peptides in the
tapasin-deficient cells (Fig. 2A and data presented above).
It is also worth noting that the
1MRVTAPRTL9 peptide is
derived from the B*2705 hc signal sequence, and our modeling data
suggest that this peptide is preferentially recovered from
tapasin-positive cells (Table I). The MRVTAPRTL peptide binds to B*2705
with a relative affinity comparable to that of the hc 169–179 peptide
(see Fig. 4) and thus might also be
expected to be preferentially presented in the absence of tapasin if
there were increased HLA B27 expression and turnover in
tapasin-deficient cells. Furthermore, it is unlikely that the other
peptides preferentially recovered from the tapasin-deficient cells
correspond to B27-derived peptides. The B*2705 hc sequence was scanned
for potential peptide fragments corresponding to molecular masses of
1094.1 and 1169.3. Although some B27-derived oligopeptides of
appropriate mass could potentially be liberated from the B27 hc, they
did not appear to be appropriate as B27 ligands based on their lack of
a canonical arginine 2 anchor motif and the presence of unfavorable
amino acids at this position.
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There may be several explanations for the ability of certain
peptides to be presented efficiently in the absence of tapasin. These
include continued presentation due to high cellular abundance or high
affinity for HLA B*2705 relative to tapasin-dependent ligands. The
source and abundance of the peptide ligands identified in this study
can be inferred from the sequence identification shown in Table I. Of
interest, several of these peptides were derived from ribosomal
proteins, with four peptides derived from proteins associated with the
60S subunit and two peptides derived from proteins associated with the
40S subunit. Even ligands derived from the same ribosomal subunit
displayed divergent tapasin dependence (e.g., GRHGVFLEL vs GRYSGRKAV).
Although the exact stoichiometries of these proteins within ribosomes
is not fully understood, we speculate that similar levels of these
ribosomal proteins would be available in the cytoplasm for epitope
liberation. Therefore, our data suggest that general cytoplasmic
abundance is not a dominant factor contributing to tapasin dependency
of these peptide ligands. Other sources of peptides sequenced in this
study include the B27 hc itself, the S2 proteasome regulatory subunit,
and an RNA helicase. Since the B27 hc was introduced as a transgene
into these cells, and some B27 molecules might still fail to bind a
ligand in the absence of tapasin, it is possible that increased
turnover of B27 in tapasin-deficient cells could explain the
preferential recovery of the hc 169–179 peptide.
The relative binding efficiency of the peptides isolated from HLA
B*2705 was inferred from a competition assay using binding of a
biotinylated reporter peptide to temperature-stabilized B*2705
molecules on the surface of transfected human TAP-deficient cell line
T2 (22). Representative data for some of the
tapasin-independent peptides are shown, and the
IC50 values for all peptides tested are listed in
Fig. 4. A broad range of IC50 values was observed
for both tapasin-independent and -dependent ligands (2.5–15 µM).
These values were comparable to those obtained for well-documented HLA
B*2705-restricted viral ligands from influenza and EBV (44, 45) that displayed IC50 values in the
range of 7–25 µM in this assay. As expected, the longer peptides
displayed poorer IC50 values relative to
nonameric ligands. Thus, neither enhanced binding to class I
heterodimers nor cytoplasmic abundance appears to correlate with the
tapasin dependency of the B27 ligands.
Peptide dependent-alloreactive T cell clones discriminate between B*2705 molecules expressed on tapasin-positive and -defective cell lines
To confirm that tapasin qualitatively alters the peptide map
presented on the cell surface we examined the functional CTL
recognition of HLA B*2705 expressed by tapasin-positive and -negative
cell lines. Most T cell allorecognition is thought to depend upon
presentation of endogenous self peptides that participate either
directly or indirectly in TCR interactions (32, 46, 47).
We used two panels of B*2705-specific alloreactive T cell clones to
examine the functional presentation of peptide ligands by HLA B*2705.
The first of these CTL was raised against normal B27-positive cell
lines, and all react with B*2705 targets (26). The second
panel of CTL was raised against tapasin-deficient B*2705 targets (i.e.,
the same clone used for peptide elution studies) during the course of
these studies. Both panels of CTL are known to be either
peptide-sensitive or peptide-specific, since they recognize the
TAP-deficient cell line T2.B*2705 only when sensitized with specific
fractions containing peptides eluted from B*2705-bearing cells
(27). The natural allo-ligand is known for a number of
these CTL. 27S69 recognizes HLA B*2705 in the context of an octamer
derived from the proteasome C5 subunit, RRFFPYYV (32).
SB52 was generated during this study toward the B27.220 cell line and
recognizes B*2705 bound to a nonamer derived from the 26S proteasome
regulatory subunit S2 (GRHGVFLEL). The peptide dependence of these CTL
clones was used to probe the peptide repertoire constitutively
presented by the B27.220 and B27.220.hTsn cell lines by comparing the
patterns of alloreactivity on both cell lines (see Fig. 5, A and B). As
expected, none of the clones tested demonstrated lysis of the parental
721.220 cell line. The CTL clones raised against normal B27-positive
cells recognized tapasin-proficient cells either more efficiently or at
least as well as tapasin-deficient cells. For instance, the CTL clones
37GRK, 18DLH, and 36DLH recognized targets with wild-type tapasin more
efficiently (at least a 30% increase in specific
51Cr release) than cells with defective tapasin
(error bars represent SDs from the mean of multiple experiments). In
contrast, CTL clones 27S69 and 58GRK recognized tapasin-positive and
-negative targets equivalently. These patterns of allorecognition were
further evident when the lysis of B*2705-positive targets was examined
at different E:T cell ratios, as shown in Fig. 5C for
selected clones.
|
B) either recognized the
tapasin-positive and tapasin-negative cells equivalently (SB3, SB5,
NK8, SB14, SB15) or recognized the tapasin-deficient cells more
efficiently (SB8). These data are consistent with MALDI-TOF MS data
that demonstrated higher levels of most, but not all, peptide ligands
presented by B*2705 in tapasin-positive vs tapasin-deficient cells.
CTL recognition of B27.220 and B27.220.hTsn was similar for a number of
CTL clones, including SB52, 102DRF, 58GRK, and 27S69, which is also
consistent with the peptide elution studies showing that some peptides
are presented to the same degree in the presence and the absence of
tapasin. The peptide ligand recognized by SB52 is presented by both
tapasin-positive and -negative cells at similar levels, and, as
expected, this CTL recognized B27.220 and B27.220.hTsn with similar
efficiency (Fig. 5C). The peptide specificity of this clone
is also shown in Fig. 5C using GRHGVFLEL-pulsed T2.B*2705
cells at different E:T cell ratios. These same cells pulsed with other
B27-binding peptides or irrelevant peptides were not recognized by this
CTL (data not shown). Overall, these data confirm a significant overlap
in the repertoire of peptides recognized by CTL when presented by HLA
B*2705 expressed in the presence and the absence of wild-type tapasin.
Importantly, consistent with the observation that some peptides were
recovered in greater abundance from tapasin-deficient APC, the clone
SB8 demonstrated preferential recognition of B27.220 cells,
demonstrating the functional consequences of preferential peptide
presentation in tapasin-deficient APC.
|
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Patterns of alloreactivity using peptide-dependent HLA B*2705-restricted CTL essentially mirrored the biochemical analyses of B*2705 peptide eluates from tapasin-positive and -negative cells. Examples of peptide-dependent allo-CTL that were clearly able to discriminate between tapasin-positive and -negative cells and others that did not probably reflects their dependence on tapasin-dependent or tapasin-independent ligands, respectively. In addition, specific stimulation of allogeneic PBMC from healthy B27-negative donors yielded clones that were not only peptide dependent, but were also peptide specific. One such example is clone SB52, which recognizes the tapasin-positive and -negative cell lines with similar efficiency and was shown in this study to be specific for the GRHGVFLEL peptide. The ability of this clone to kill both targets with equal efficiency directly correlates with the similar levels of GRHGVFLEL peptide recovered from the two cell lines and clearly defines the tapasin independence of this ligand. Furthermore, the clone SB8 demonstrated greater lysis of tapasin-deficient APC, confirming that some peptides are presented more abundantly on the surface of tapasin-deficient cells. The peptide ligand recognized by clone SB8 was not defined in this study. SB8 did not recognize T2.B*2705 pulsed with known B27 ligands or any of the peptides defined in this study, reflecting the peptide-dependent and peptide-specific manner by which this clone recognizes HLA B*2705.
Tapasin has a number of distinct functions that offer some clues to the mechanism of its role in repertoire modulation. These functions include colocalization of class I and TAP molecules, increasing the level of TAP expression, and possibly affecting peptide translocation from the cytosol into the lumen of the ER (5, 6). In addition, as highlighted from studies with soluble tapasin (4), this chaperone may stabilize class I complexes and influence peptide loading independently of TAP association. Our data suggest that an alternative pathway of peptide acquisition by HLA B*2705 operates when tapasin is limiting or when TAP association is abrogated, as with the T134K HLA A2 mutant (11) and RT1.1u molecules expressed on the TAPa background (48). However, TAP-translocated peptides remain the primary source of the peptides selected by HLA B*2705 under these alternative conditions (49). Under normal circumstances it is generally assumed that peptide-receptive MHC class I heterodimers acquire antigenic peptide when bridged to the TAP (1, 50). However, it is possible that some B*2705 molecules might initially acquire peptides via an alternative tapasin-independent pathway and then subsequently be localized to TAP via tapasin binding. Physical association with TAP may allow a degree of peptide exchange with less abundant or shorter half-life peptides as well as peptides of better fit. This idea is consistent with some peptides having an absolute requirement for tapasin for class I loading through a process that may involve direct interactions among tapasin, the class I complex, the TAP molecule itself, and perhaps the peptide. Such an arrangement could allow a direct role for tapasin in the editing process. There is also ample scope for tapasin to exert an indirect influence on this putative editing process; tapasin modulates TAP function by enhancing the translocation of peptides and therefore might alter the competitive advantage of certain peptides. The presence of tapasin is also required for efficient recruitment of other components of the class I loading complex, which may exert individual or concerted effects on peptide editing (1).
How tapasin exerts its influence on the peptide repertoire is still
unclear, though obviously tapasin is important in the assembly of
nascent class I heterodimers with peptide and in retaining these
complexes in the ER until a suitable peptide ligand is bound. Other
opportunities for fine-tuning the bound ligand may also exist. Recent
studies indicate that class I molecules associate stably with the TAP
for periods of up to several minutes (51) and are still
associated with tapasin before dissociating from the TAP complex
(52). These studies also revealed that once dissociated
from the TAP complex, peptide-loaded molecules did not egress to the
cell surface immediately, suggesting that a window of opportunity for
additional editing of bound peptides may exist after TAP-mediated
loading. A putative tapasin binding site on class I MHC molecules
includes amino acid residues 128–136, which form a loop in the 2
domain, and conceivably interaction with this region of the class I
molecule could influence peptide binding by modulating cleft
conformation (13, 14). Combined with our data we propose
an integral role for tapasin in both peptide loading of class I
molecules and ligand optimization. From our small dataset it does not
appear that tapasin-independent ligands possess enhanced binding
affinity for HLA B*2705, nor does the cellular source of the ligands
indicate that cytoplasmic abundance necessarily correlates with tapasin
dependency. We propose that the ER lumenal availability of the peptide
plays an important role in dictating class I loading in
tapasin-deficient cells. Lumenal availability is conceivably a product
of TAP-dependent translocation of peptides into the ER, the resistance
of the peptides to ER protease, and their requirement for processing or
trimming following translocation from the cytoplasm into the ER lumen.
Further analysis of the peptides defined here and in future studies
will explore whether further rules regarding the selection of peptide
ligands by tapasin exist.
Recent studies with tapasin knockout mice suggest a generalized defect in Ag presentation in tapasin-deficient cells (15, 16). However, these studies observed a low level of Ag presentation in knockout mice sufficient for some positive selection of CD8+ T cells and tolerance induction. This is entirely consistent with our biochemical findings described here that some peptides continue to be presented for T cell recognition in the absence of tapasin. We suggest that the extent of Ag presentation will be highly dependent on the class I allele and ligand availability. It is clear from several studies using the 721.220 cell line that human class I alleles are differentially affected by tapasin deficiency and that HLA B*2705 is perhaps the most successful allele in overcoming such defects. A prediction of our study is that in normal APC some degree of tapasin-independent loading of HLA B*2705 may operate and that a proportion of B27 molecules may be more receptive to exogenous ligand due to the acquisition of suboptimal peptides (10, 12). Indeed, such peptide receptivity has been observed for B27 molecules under normal conditions (22). The physiological significance of these phenomenon remains unclear, but it is interesting to speculate that this property of HLA B*2705 may contribute to this allele’s very strong association with ankylosing spondylitis and other inflammatory arthropathies.
The potential for viruses or intracellular pathogens to target tapasin as a mechanism for evading CTL recognition has been highlighted in a study that examined the role of adenovirus protein E19 in preventing class I presentation (53). HLA alleles that encode tapasin-independent molecules may have evolved in response to such selective pressures and permit the host CTL compartment to respond to pathogens that specifically target this aspect of class I assembly. It is interesting to note that the HLA B27 group of alleles is evolutionarily successful, being distributed in nearly all ethnic groups, and is associated with slow progression of HIV infection (54, 55). Our data would suggest that if tapasin function was abrogated there is potential for the up-regulated presentation of poorly tolerized self-peptides. If this occurs in an inflammatory context this may also elicit autoimmune responses, since these peptides would not normally be presented at high density to the immune system by tapasin-proficient cells. It is interesting to note that the B27 hc peptide 169–179 that is preferentially presented by tapasin-deficient cells has previously been espoused as a potential arthritogenic peptide (42). The exact role of this peptide in disease is unclear, since this peptide is prominent in peptide pools derived from some of the B27 subtypes reported not to be associated with ankylosing spondylitis (56) (our unpublished observations). Moreover, widespread reactivity toward self-peptides (vasoactive intestinal peptide 1 receptor 400–408 and LMP2236–244) has been observed in patients with ankylosing spondylitis (57), highlighting the importance of the repertoire of self-peptides in ankylosing spondylitis and presumably other B27-associated inflammatory arthropathies. Given the qualitative and quantitative influence of tapasin on the self-peptide repertoire it will be of great interest to determine whether other ligands that are preferentially presented in the absence of tapasin are involved in autoimmunity.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Anthony W. Purcell, Department of Microbiology and Immunology, University of Melbourne, Parkville 3052, Victoria, Australia.
3 Abbreviations used in this paper: ER, endoplasmic reticulum; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; MS, mass spectrometry; RP-HPLC, reverse phase HPLC; hc, heavy chain; K*, N-biotinyl lysine; PSD, post-source decay; TFA, trifluoroacetic acid.
4 A. W. Purcell and J. J. Gorman. The use of post source decay in matrix assisted laser desorption-ionization mass spectrometry to delineate T cell determinants. Submitted for publication.
Received for publication May 1, 2000. Accepted for publication October 18, 2000.
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W. A. Macdonald, A. W. Purcell, N. A. Mifsud, L. K. Ely, D. S. Williams, L. Chang, J. J. Gorman, C. S. Clements, L. Kjer-Nielsen, D. M. Koelle, et al. A Naturally Selected Dimorphism within the HLA-B44 Supertype Alters Class I Structure, Peptide Repertoire, and T Cell Recognition J. Exp. Med., September 2, 2003; 198(5): 679 - 691. [Abstract] [Full Text] [PDF]
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A. L. Ackerman and P. Cresswell Regulation of MHC Class I Transport in Human Dendritic Cells and the Dendritic-Like Cell Line KG-1 J. Immunol., April 15, 2003; 170(8): 4178 - 4188. [Abstract] [Full Text] [PDF]
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B. Park and K. Ahn An Essential Function of Tapasin in Quality Control of HLA-G Molecules J. Biol. Chem., April 11, 2003; 278(16): 14337 - 14345. [Abstract] [Full Text] [PDF]
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B. Park, S. Lee, E. Kim, and K. Ahn A Single Polymorphic Residue Within the Peptide-Binding Cleft of MHC Class I Molecules Determines Spectrum of Tapasin Dependence J. Immunol., January 15, 2003; 170(2): 961 - 968. [Abstract] [Full Text] [PDF]
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N. S. Dangoria, M. L. DeLay, D. J. Kingsbury, J. P. Mear, B. Uchanska-Ziegler, A. Ziegler, and R. A. Colbert HLA-B27 Misfolding Is Associated with Aberrant Intermolecular Disulfide Bond Formation (Dimerization) in the Endoplasmic Reticulum J. Biol. Chem., June 21, 2002; 277(26): 23459 - 23468. [Abstract] [Full Text] [PDF]
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K. M. Paulsson, M. J. Kleijmeer, J. Griffith, M. Jevon, S. Chen, P. O. Anderson, H.-O. Sjogren, S. Li, and P. Wang Association of Tapasin and COPI Provides a Mechanism for the Retrograde Transport of Major Histocompatibility Complex (MHC) Class I Molecules from the Golgi Complex to the Endoplasmic Reticulum J. Biol. Chem., May 17, 2002; 277(21): 18266 - 18271. [Abstract] [Full Text] [PDF]
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M.-E. Paquet and D. B. Williams Mutant MHC class I molecules define interactions between components of the peptide-loading complex Int. Immunol., April 1, 2002; 14(4): 347 - 358. [Abstract] [Full Text] [PDF]
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T. Pentcheva, E. T. Spiliotis, and M. Edidin Cutting Edge: Tapasin Is Retained in the Endoplasmic Reticulum by Dynamic Clustering and Exclusion from Endoplasmic Reticulum Exit Sites J. Immunol., February 15, 2002; 168(4): 1538 - 1541. [Abstract] [Full Text] [PDF]
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P. Tan, H. Kropshofer, O. Mandelboim, N. Bulbuc, G. J. Hammerling, and F. Momburg Recruitment of MHC Class I Molecules by Tapasin into the Transporter Associated with Antigen Processing-Associated Complex Is Essential for Optimal Peptide Loading J. Immunol., February 15, 2002; 168(4): 1950 - 1960. [Abstract] [Full Text] [PDF]
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) and B27.220 (
)
eluates. C, W6/32 immunoprecipitated class I molecules
from 1 x 108 cells metabolically labeled with
[3H]arginine were treated with acid, and dissociated
peptides were fractionated by RP-HPLC. Individual peptide fractions
from B27.220.hTsn (heavy line), B27.220 (light line), and 721.220.hTsn
(dashed line) were counted by liquid scintillation counting. To control
for label incorporation, aliquots of labeled cells were washed in PBS
and lysed in 0.1% TFA, and the incorporated [3H]arginine
was measured by liquid scintillation counting. All cells examined
incorporated equivalent amounts of [3H]arginine (data not
shown).
), GRIDKPILK (
), RRYLENGKETL (•), and GRHGVFLEL
(
), B27.220 (
), and B27.220.hTsn (
), B27.220 (