| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Department of Molecular Immunology, German Cancer Research Center (Deutsches Krebsforschungszentrum), Heidelberg, Germany;
Basel Institute for Immunology, Basel, Switzerland; and
Department of Immunology, Hebrew University, Ein-Karem, Jerusalem, Israel
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2-microglobulin and the TAP peptide
transporter. The function of this TAP-associated complex was unclear
because it was reported that soluble Tpn that has lost TAP interaction
would be fully competent in terms of peptide loading and Ag
presentation. We found, however, that only wild-type human Tpn (hTpn),
but not three soluble hTpn variants, a transmembrane domain point
mutant of hTpn (L410
F), wild-type mouse Tpn, nor a mouse-human Tpn
hybrid, fully up-regulated peptide-dependent Bw4 epitopes when
expressed in Tpn-deficient .220.B*4402 cells. Consistent with
suboptimal peptide loading, the t1/2 of
class I molecules was considerably reduced in the presence of soluble
hTpn, hTpn-L410F, and murine Tpn. Furthermore, eluted peptide spectra
and the class I-mediated inhibition of NK clones showed distinct
differences to the hTpn transfectant. Only wild-type hTpn efficiently
recruited HC and calreticulin (Crt) into complexes with TAP and
endoplasmic reticulum p57 (ERp57). The L410F mutant was defective in
TAP association, but bound to class I molecules, Crt, and ERp57. Mouse
Tpn associated with human TAP and ERp57 on the one hand, and with HC
and Crt on the other, but failed to recruit normal amounts of HLA class
I molecules into the TAP complex. We conclude that the loading with
peptides conferring high stability requires the Tpn-mediated
introduction of HC into the TAP complex, whereas the mere interaction
with Tpn is not sufficient. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2-microglobulin (2m)
(4, 5). Subsequently, the HC/2m
dimer exchanges Cnx for another lectin-like chaperone, Crt (6, 7). In mouse cells, the interaction of class I HC with Cnx was
also reported to persist after binding of 2m
until late stages of class I maturation in the ER (4, 8, 9). Association of Crt with class I HC is not detected in the
absence of 2m (6), nor in the
absence of Tpn (10, 11, 12).
The thiol oxidoreductase ERp57 interacts with class I HC in the
Cnx-associated stage and joins class I/2m
dimers into the TAP-associated complex (10, 13, 14, 15). ERp57
is thought to mediate the formation/isomerization of disulfide bonds
and thereby ensure a proper folding of class I HC in conjunction with
Cnx and Crt (3, 15). In the absence of
2m or Tpn, ERp57 does not associate with HC,
Crt, or TAP (10, 11).
While an association of Tpn with HC/2m has
also been observed in TAP1/2-deficient cells (6, 16), the
prime function of Tpn is, however, to bridge
HC/2m, Crt, and ERp57 to the peptide
transporter (6, 17, 18, 19, 20, 21, 22). Similar to Crt, Tpn requires
HC/2m dimers for association with class I
molecules. In human cells lacking expression of
2m, only small amounts of class I HC can be
detected in association with TAP (11, 20), whereas in
2m-deficient mouse cells, a HC:Crt:Tpn:TAP
subcomplex seems to assemble more readily (13). Specific
peptide is required to dissociate class I molecules from the Tpn:TAP
complex (8, 23, 24) as well as from Tpn in TAP-deficient
cells (16). Therefore, a lack of suitable peptides can
lead to prolonged association with TAP (9, 25, 26). A
large proportion of the total amount of HC/2m
dimers in the ER is found in complexes with TAP (21, 27).
Early studies using the mutant human B cell line .220 have demonstrated a down-regulation of HLA-A and HLA-B products on the cell surface that considerably varied among HLA alloforms (28). Due to aberrant splicing of a mutated Tpn gene, .220 cells express only minute quantities of a nonfunctional, N-terminally truncated Tpn molecule, but no wild-type Tpn (6, 29). In .220 cells, class I HC associates normally with Cnx, whereas only trace amounts of class I HC form a complex with TAP (17, 30). The functional consequences of Tpn deficiency in .220 cells are unstable class I molecules and diminished ER egress due to a partial failure of loading with stabilizing peptides (30, 31) as well as impaired Ag presentation, which could be restored by transfection of wild-type Tpn (18, 32). Equivalent results were obtained with a H-2Dd mutant that fails to assemble with Tpn (21). By contrast, wild-type Kb molecules expressed in .220 or in Tpn-deficient insect cells were found to exit the ER more rapidly than in the presence of Tpn (12, 33). For different HLA alleles, a hierarchy was noted in the dependence on Tpn for loading of peptides recognized by CTL (32), and also mouse class I alleles differed in the amounts of peptide-loaded forms that were detectable on the cell surface in the absence of Tpn (34). Thus, from the studies employing .220 transfectants, it can be concluded that Tpn is not absolutely required for the loading of TAP-dependent peptides. Recent investigations provided, however, evidence for a quantitative and qualitative shaping of the class I-bound peptide repertoire by Tpn (12, 35, 36). Available evidence suggests that mouse Tpn (mTpn) is able to functionally replace human Tpn (hTpn) in HLA-transfected .220 cells (37, 38). On the other hand, certain human class I alloforms insufficiently assemble with peptides when expressed in presentation-competent mouse cells (32, 38). The structural reasons for this distinct species incompatibility of mTpn remained, however, elusive.
The analysis of Tpn-deficient mice clearly demonstrated that Tpn is indispensable for a proper function of the class I Ag presentation pathway (35, 39). Tpn mutant mice show strongly reduced class I surface expression and stability of surface class I molecules. The presentation of cytosolic Ags by Tpn-/- cells can be significantly impaired (35). Not only were defects in the development of CD8+ T cells and immune responses against some viruses noted, but also an altered NK cell repertoire was observed in Tpn-/- mice (35, 39).
Deletion of the N-terminal 50 residues ablated a proper interaction of Tpn with class I HC, Crt, and ERp57 (11). Another study suggested the full competence of Tpn lacking the transmembrane (TM) and cytoplasmic domains in terms of rescuing peptide loading and surface expression of class I molecules in .220.B8 cells, although the interaction with TAP was abolished (40). If soluble Tpn could generally replace membrane-anchored Tpn, this result would, however, challenge the physiological significance of the TAP:Tpn complex.
In this study, we investigated the function of wild-type hTpn, soluble or point-mutated hTpn, as well as mTpn during the peptide loading of HLA-B*4402 molecules. Only wild-type hTpn was able to facilitate binding of an optimized spectrum of peptides due to distinct failures of all other Tpn variants to mediate assembly of the TAP-associated loading complex.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
hTpn cDNA was cloned from a human leukocyte Marathon-Ready cDNA
library (Clontech, Heidelberg, Germany) using as 5' primer the Marathon
AP1 primer and as 3' primer the Tpn-specific oligonucleotide
5'-CAGAGATGATGGTGGCTTCC-3'. The PCR product was cloned into the
EcoRI site of pCR2.1 (Invitrogen, Groningen, The
Netherlands) and sequenced. The coding sequence differs from the
published hTpn sequence AF009510 (18) at codons 161 (GTT
instead of GTC) and 260 (ACA instead of AGA). mTpn cDNA was cloned from
a BALB/c spleen Marathon-Ready cDNA library (Clontech) using as 5'
primer the Marathon AP2 primer and as 3' primer the Tpn-specific
oligonucleotide 5'-TGGAGGGAGAAGAAGAGAAGAAGG-3'. The PCR product was
cloned into the EcoRV site of pBluescript II
KS+ (Stratagene, Amsterdam, The Netherlands) and
sequenced. The coding sequence differs from the published murine Tpn
(mTpn) sequence AF043943 (37) at codons 17 (GTC instead of
TTC), 79 (GTG instead of CTG), 274 (GCT instead of GGT), and 419 (CTA
instead of CTG). A chimeric mouse-human Tpn cDNA was constructed by
using the unique XhoI site in the mTpn cDNA, which is
conserved in the hTpn sequence. For hTpn lacking the C-terminal 33 or
49 residues, StuI or filled-up BstEII sites were
ligated with filled-up XbaI (SpeI) polylinker
sites, to create stop codons directly after hTpn residues
G395 or T379, respectively.
For the hTpn-
C53KDEL construct, the hTpn
Eco47III site was ligated into a vector with an ER retention
sequence, creating the C-terminal sequence NSEKDEL* after
hTpn residue S375. To generate the hTpn point
mutant L410F, an oligonucleotide containing TTC instead of CTG at codon
410 was inserted between the unique StuI and AccI
sites. All Tpn constructs were cloned into the episomal expression
vector pREP4 (Invitrogen).
Cell lines and transfections
The Tpn-deficient, human B-lymphoblastoid mutant cell line LCL .220 (28) was kindly provided by R. DeMars (University of Wisconsin, Madison, WI). HLA-B*4402-transfected .220 cells were granted by C.-A. Peh and J. McCluskey (Melbourne University, Melbourne, Australia). The .220.B*4402 cells were transfected with Tpn cDNAs constructs in pREP4 by electroporation at 220 V, 4 pulses, 4 ms, using a BTX electroporator (BTX, San Diego, CA). Following selection, transfectants were maintained in RPMI 1640 (Life Technologies, Freiburg, Germany) supplemented with 0.3 mg/ml hygromycin B (Roche Diagnostics, Mannheim, Germany), 0.5 mg/ml geneticin (Sigma, Taufkirchen, Germany), 2 mM glutamine, 0.2% glucose, and 10% FCS (Biochrom, Berlin, Germany).
Antibodies
Rabbit antisera against N-terminal residues 2–20 (Rb
PAV) and
C-terminal residues 418–428 (RbSTC) of hTpn were raised against the
keyhole limpet hemocyanin-coupled, cysteine-modified peptides
PAVIECWFVEDASGKGLAK-C and C-STCKDSKKKAE, respectively, by G.
Moldenhauer (DKFZ, Heidelberg, Germany). Rabbit anti-mTpn
N-terminal peptide antiserum, Ra2223 (41), was a
gift of T. Hansen (Washington University, St. Louis, MO). Mouse mAbs
specific for Crt (FMC75), ERp57 (MaP.Erp57), and anti-Crt rabbit
antiserum (SPA-600) were from StressGen/Biomol (Hamburg, Germany).
Mouse anti-TAP1 mAb 148.3 (42) was provided by R.
Tampé (University of Marburg, Marburg, Germany), and rabbit antiserum
raised against the cytoplasmic domain of human TAP1 fused to GST
(43) was a gift of J. Neefjes (Netherlands Cancer Center,
Amsterdam, The Netherlands). The Cnx-specific mAb AF8 (5)
was provided by M. Brenner (Harvard Medical School, Boston, MA). mAbs
HC-10, preferentially reacting with 2m-free
HLA-B and HLA-C HC (44), and W6/32 recognizing HLA-A,
B, C HC/2m dimers (45) as well as
the HLA-Bw4-specific mAbs TÜ109 (46) and TT4-A20
(47) have been described. The Bw4 mAb T116-5-28 was from
Saxon Europe (Newmarket, Suffolk, U.K.).
Immunoprecipitation
Cells were lysed in TBS (150 mM Tris-HCl, 50 mM NaCl, 5 mM
MgCl2, pH 7.4) supplemented with 1% digitonin
(Riedel-de Haen, Seelze, Germany) and Complete protease inhibitor
cocktail (Roche) for 2 h at 4°C. After centrifugation at
15,000 x g to remove nuclei, samples were incubated
for 1 h at 4°C with Ab prebound to protein A-Sepharose beads
(Amersham Pharmacia, Freiburg, Germany), or protein A/G PLUS agarose
beads (Santa Cruz Biotechnology, Heidelberg, Germany) in case of
MaP.Erp57. Beads were washed thrice with TBS/0.1% digitonin, followed
by heating at 95°C for 5 min with SDS-containing sample buffer and
analysis by 10% SDS-PAGE. For the determination of class I
HC/2m stability in lysates, cells were lysed
in TBS containing 1% Nonidet P-40 (Sigma). The postnuclear supernatant
was incubated on ice or at 37°C for 1 h before
immunoprecipitation with W6/32-conjugated Sepharose 4B beads.
Western blot analysis
To determine the expression of transfected Tpn constructs and TAP, cells were lysed in TBS containing 1% Nonidet P-40 for 30 min at 4°C. To analyze the ER egress of Tpn constructs, samples were digested overnight with 10 mU endoglycosidase H (Endo H; Roche) at 37°C in 50 mM NaAc, pH 5.5. Samples were separated on 10% SDS-PAGE gels and transferred onto polyvinylidene difluoride membranes (Millipore, Eschborn, Germany). Membranes were blocked with 1% skim milk powder (Sigma) in PBS with 0.05% Tween 20 (Sigma) and incubated with the primary Ab for 1 h. After extensive washing with PBS, membranes were incubated with goat anti-mouse IgG or anti-rabbit IgG HRP-conjugated second Abs at appropriate dilutions (Dianova, Hamburg, Germany) for 1 h. Following the development with SuperSignal West Dura ECL substrate (Pierce/KMF, Sankt Augustin, Germany), bands were visualized and quantified using the Lumi-Imager system and LumiAnalyst 3.x software (Roche).
Flow cytometry
For surface immunofluorescence stainings, 1–2 x 106 cells were washed once in ice-cold Dulbecco’s PBS (D-PBS)/0.05% NaN3/2% FCS, followed by incubation with a saturating amount of the primary Ab for 30 min at 4°C. After three washes with D-PBS, cells were incubated with goat anti-mouse IgG-FITC (Dianova) for 30 min at 4°C. Cells were washed twice and resuspended in 300 µl D-PBS/0.1% propidium iodide (Sigma). FACS analysis was performed using FACScan flow cytometers (BD Biosciences, Heidelberg, Germany). To measure the stability of surface class I molecules, cells were cultured for 10 h in the presence of 20 µg/ml brefeldin A (Sigma) before staining with W6/32. Loading with the natural HLA-B44 ligands EBNA-6 657–666 VEITPYKPTW (see "SYFPEITHI" database, http://syfpeithi.bmi-heidelberg.com), Hsp90 427–436 AEDKENYKKF, or the HLA-A11-binding peptide ASYDKAKLK as control was performed at a peptide concentration of 100 µM in PBS/0.1% NaN3 for 2 h at room temperature prior to extensive washing to remove excess exogenous peptide and staining with the Bw4-specific mAbs TT4-A20 or TÜ109. Peptides were synthesized by -moc chemistry in the peptide synthesis unit of the DKFZ and >95% pure by mass spectrometrical analysis.
Pulse-chase radiolabeling
Cells were preincubated for 1 h with either the proteasome inhibitor lactacystin (Calbiochem, Schwalbach, Germany) at a concentration of 100 µM or DMSO for control. After washing with warm D-PBS, cells were starved in methionine/cysteine-free RPMI (ICN, Eschwege, Germany) supplemented with 3% dialyzed FCS for 1 h at 37°C. In case of the lactacystin-treated probe, fresh lactacystin (100 µM) was added after each washing step. Pulse radiolabeling of the cells was performed with 1 mCi [35S]Met/Cys Pro-mix (Amersham Pharmacia) per 5 x 106 cells/ml for 10 min. After washing with warm RPMI 1640, cells were chased in complete RPMI 1640/10% FCS. Harvested aliquots were washed with ice-cold D-PBS and lysed in TBS/1% Nonidet P-40. Postnuclear supernatants were subjected to immunoprecipitation with W6/32-conjugated Sepharose beads. Precipitates were analyzed by 10% SDS-PAGE and autoradiography. Class I HC bands were quantified using the Lumi-Imager.
Analysis of class I-associated peptides
HLA-B*4402/Cw1-associated self peptides were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MS), as described (48). In brief, 1–2 x 107 cells were lysed in 1% n-octylglucoside (Sigma), and HLA molecules were precipitated with W6/32-conjugated Sepharose beads. Beads were transferred into an Ultrafree ultrafiltration tube with a 5-kDa cutoff (Millipore, Volketswil, Switzerland), washed with 10 vol of 20 mM Tris, pH 7.8, 0.1% Zwittergent 3-12 (Calbiochem) and 20 vol of bidestilled water. Peptides were eluted by incubation in 0.1% trifluoroacetic acid in bidestilled water for 30 min, followed by ultrafiltration and lyophilization. Spectra were recorded on a Reflex III mass spectrometer (Bruker Analytik, Rheinstetten, Germany) and collected by averaging the ion signals from 50–100 individual laser shots.
Isolation of NK clones and cytotoxicity assays
NK cell lines and clones were isolated from PBL using the human NK cell isolation kit and the autoMACS instrument (Miltenyi Biotech, Rishon Le Zion, Israel). NK cells were cultured as previously described (49). The cytotoxic activity of NK cells against the various targets was assayed in 5-h 35S release assays, as described (49). The specific release was calculated as follows: percentage of lysis = ((cpm experimental well - cpm spontaneous release)/(cpm maximal lysis - cpm spontaneous release)) x 100. For all target cells used, the spontaneous release in medium was 15–16% of the maximal release in 0.1 M NaOH.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To assess the function of membrane-anchored vs soluble Tpn for the
loading of class I molecules with peptides, we generated C-terminal
deletion constructs on the basis of a hTpn cDNA clone and expressed
them stably in .220.B*4402 cells. In the hTpn-C49 and hTpn-C33
constructs, the mature protein of 428 residues was truncated after aa
379 and 395, respectively, which are located above the TM domain
(18). These Tpn truncations are thus expected to be
soluble and secreted. In the construct
Tpn-C53KDEL, a KDEL ER retention signal
(50) was engineered C-terminal of hTpn residue 375. In the
point mutant hTpn-L410F, a leucine residue that participates in a
putative leucine zipper motif (L396/L403/L410/L417) in the TM domain
was substituted by phenylalanine. In addition, mTpn as well as a
mouse-human chimera (m
hTpn) containing the N-terminal 149 aa
of mTpn fused to the corresponding C-terminal portion of hTpn were
expressed in .220.B*4402 cells. The Tpn variants used in this study are
schematically depicted in Fig. 1
A.
|
PAV) or 418–428
(RbSTC), respectively. These blots demonstrated similar levels of
expression of the constructs (Fig. 1
B). We noted that the
RbPAV serum recognized mTpn somewhat less efficiently than hTpn,
probably because four residues in mTpn 2–20 vary from the respective
hTpn sequence. Therefore, an immunoblot of the same number of
.220.B*4402/mTpn cells with the genuine anti-mTpn
(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) serum Ra2223 (41) is shown in
addition. Lysates of selected Tpn transfectants were subjected to Endo
H treatment to monitor ER retention (Fig. 1C). Wild-type
hTpn and mTpn molecules were fully Endo H sensitive, consistent with
complete ER retention. Unexpectedly, a fraction of Tpn-L410F molecules
reproducibly displayed Endo H resistance. This suggests that, in
addition to the canonical KKAE ER retention sequence at the C terminus
of Tpn (18, 50), a signal within the TM domain may play a
role in its ER retention. Soluble Tpn-C33 molecules in the total
cellular lysate showed partial Endo H resistance, indicating release
into the secretory pathway. The KDEL-tagged soluble Tpn construct
remained, however, fully Endo H sensitive, proving that the KDEL
retention signal was functional. Tpn TM point mutant pinpoints an interaction site with TAP
Next, we assessed the capacity of hTpn, mTpn as well as
hTpn-L410F, mTpn to stabilize steady state levels of the TAP peptide
transporter (11, 40). Immunoblots of whole cell lysates of
equal numbers of .220.B*4402 transfectants were probed with the
TAP1-specific mAb 148.3, or anti-Cnx as a loading control. In cells
transfected with mTpn (Fig. 1D, see also Fig. 5B) and mhTpn (data not shown), we detected 80–100% of
the TAP levels present in hTpn transfectants, whereas in the presence
of the Tpn-L410F mutant only 
10% of the normal TAP1 amounts were
detected (Fig. 1D). Thus, the point mutation apparently
impaired an interaction of Tpn and TAP located in the membrane-spanning
domain. As shown in Fig. 5B (anti-TAP1 blot), hTpn-L410F
transfectants retained a residual capacity to stabilize TAP as compared
with Tpn-deficient .220 cells.
|
The surface expression of class I molecules on .220.B*4402
Tpn transfectants was analyzed using mAb W6/32 recognizing a
monomorphic HC/2m combinatorial determinant on
all HLA-A, B, C products, including the transfected B*4402 and the
endogenously expressed Cw1 molecules (28, 32, 45, 51). The
level of HLA-B*4402/Cw1 expression in the absence of functional Tpn is
indicated by the stainings of .220.B*4402/pREP4 control transfectants.
This staining is mostly due to B*4402 molecules since .220/pREP4 or
.220 cells were significantly less strongly labeled with W6/32 (data
not shown). On .220.B*4402 cells transfected with hTpn-C49,
hTpn-C33 (Fig. 2A),
hTpn-C53KDEL (Fig. 2C), hTpn-L410F,
mTpn, and mhTpn (Fig. 2B), the labeling with W6/32 was
increased to levels that were similar to .220.B*4402/hTpn cells,
suggesting that all Tpn constructs were equally competent to promote
the formation of stable, presumably peptide-loaded
HC/2m dimers that migrate to the cell surface.
Apparently, the low amounts of TAP that were detected in the presence
of hTpn-C49, hTpn-C33 (data not shown), and hTpn-L410F (Fig. 1D) were not limiting during the assembly of enhanced
amounts of W6/32-reactive HC/2m dimers.
|
1 domain (52). The Bw4 epitope
recognized by mAb TÜ109 has been reported to be sensitive to the
peptide cargo of HLA-B*5101 molecules (53). Except for a
slightly weaker staining of .220.B*4402/hTpn-C49 cells, hTpn mutants
and mTpn strongly induced the Bw4 epitope recognized by T116-5-28 to
levels that equalled or even exceeded wild-type Tpn (Fig. 2, A and B), thus confirming the results obtained
with W6/32. In clear contrast, stainings with TT4-A20 and TÜ109
revealed distinct defects of soluble hTpn constructs to generate Bw4
determinants (Fig. 2A). This failure could not be rescued by
ER retention of soluble Tpn, as indicated by the strongly reduced
TÜ109 staining of the hTpn-C53KDEL
transfectant (Fig. 2C). Also, the hTpn-L410F point mutant,
mTpn, and mhTpn showed pronouncedly reduced stainings with TT4-A20
(Fig. 2B) and TÜ109 (data not shown). From the
observed defect of the mouse-human Tpn hydrid, we conclude that the
N-terminal 149 residues of mTpn are responsible for the failure of mTpn
to produce wild-type amounts of these Bw4 epitopes. Lower stability of class I molecules in the presence of Tpn mutants and mTpn
Using biochemical assays, we analyzed the stability of class I
molecules in lysates and on the cell surface, which is a measure for
the stabilizing properties of the spectrum of loaded peptides. First,
we determined the thermostability of W6/32-precipitable class I
molecules in lysates (51). Quantification of Western blots
stained with HC-10 revealed that 75% of HC/2m
dimers from .220.B*4402/hTpn cells survived an incubation at 37°C for
1 h (Fig. 3A). In
contrast, only 30% and 40% of HC/2m dimers
in lysates of .220.B*4402/hTpn-C49 and .220.B*4402/mTpn cells,
respectively, survived this treatment, suggesting suboptimal
stabilization by endogenous peptides in these cases.
|
B). BFA
treatment reduced the mean fluorescence intensity (MFI) of
surface-stained class I molecules on .220.B*4402/hTpn cells by 27%. In
contrast, on hTpn-C49, hTpn-C33, mTpn, and hTpn-L410F
transfectants, the MFI was reduced by 38–41%, pointing to a lower
average stability and survival of surface B*4402 molecules.
This finding was further substantiated in a pulse-chase
experiment with metabolically labeled Tpn transfectants (Fig. 3C). Quantification of class I molecules that were
precipitable with W6/32 from Nonidet P-40 lysates after chase periods
up to 18 h showed considerable differences in their
t1/2. While metabolically labeled
class I molecules had an approximate
t1/2 of 20 h in .220.B*4402/Tpn
cells, the t1/2 were reduced to 4–8 h
in .220.B*4402/mTpn, mhTpn, hTpn-C49, and hTpn-L410F cells. Class
I molecules in Tpn-deficient .220.B*4402/pREP4 cells showed the lowest
stability with an approximate t1/2 of
2 h.
Peptide- and Tpn-dependent ER egress of class I molecules
A more rapid ER egress of HLA-B8 molecules in the presence
of soluble Tpn was reported previously (40). Since
premature ER release might be accompanied by an incomplete loading of
class I molecules with peptides, we examined whether in the presence of
Tpn mutants HLA-B*4402 and -Cw1 molecules were more readily allowed to
leave the ER without peptide (or loaded with suboptimal peptide) than
in the presence of wild-type Tpn. To this end, we analyzed the
acquisition of Endo H resistance of class I HC, being indicative of
transport to and beyond the Golgi apparatus, in the presence of the
proteasome-specific inhibitor lactacystin, which is known to cause a
reduced class I maturation due to limited supply with cytosolic
peptides (55). In the absence of lactacystin, we noted
similar kinetics in the formation of Endo H-resistant class I molecules
expressed in .220.B*4402/hTpn, hTpn-C49, and mTpn cells (Fig. 3D, left). The maturation of class I in
.220.B*4402/pREP4 cells was significantly slower. In the presence of
lactacystin, the overall amounts of W6/32-precipitable
HC/2m dimers and their maturation kinetics
were concomitantly reduced in all transfectants (Fig. 3D,
right). Under conditions of limited peptide supply, the
lower stability of HC/2m dimers in C49-,
mTpn-, and particularly in pREP4-transfected cells was apparent already
after 120 min of chase, as indicated by decreased amounts of
W6/32-precipitated material. Thus, this experiment provided no
indication for an increased peptide-independent or more rapid ER egress
of the studied class I alloforms in the presence of mTpn or hTpn
mutants.
Altered class I-associated peptide repertoire in the presence of mTpn and hTpn mutants
To investigate whether differential peptide loading in the
presence of soluble hTpn mutants leads to the defects in the formation
of Bw4 determinants, we incubated .220.B*4402 cells expressing
wild-type hTpn, hTpn-C49, or hTpn-C33 with known B44-binding
peptides before surface labeling with either TT4-A20 or TÜ109.
Incubation with exogenous B44 ligands significantly increased the MFI
of TT4-A20 staining of hTpn-C49 cells as compared with cells
incubated with an irrelevant A11-binding peptide (Fig. 4A, left). The
B44-binding peptides were also able to augment the staining by
TÜ109 of both hTpn-C49 and hTpn-C33 transfectants (Fig. 4A, right). These findings indicate that the
epitopes recognized by TT4-A20 and TÜ109 mAbs are influenced by
HLA-B*4402-bound peptides, and suggest that B*4402 molecules expressed
in the presence of soluble Tpn constructs contain altered peptide
spectra because they appear to be more receptive for exogenous peptides
than B*4402 in the presence of wild-type Tpn.
|
B. For comparison, class I-deficient .221 cells as well as
.221/Cw3 and .221/Cw4 transfectants were included. Some NK clones were
inhibited by .221/Cw3, but not by .221/Cw4 cells, suggesting that they
employed KIR-2DL2 or KIR-2DL3 receptors (58), which should
also be inhibitable by HLA-Cw1 molecules expressed on the .220 cells.
Other NK clones that were inhibited by .220.B*4402/Tpn transfectants,
but not by .221/Cw3 or .221/Cw4, probably engaged the Bw4-reactive
KIR-3DL1 receptor (58). The pREP4 control transfectant was
efficiently killed by essentially all NK clones consistent with the
comparably low surface staining with W6/32. Most NK clones were equally
well inhibited by .220.B*4402 cells transfected with hTpn, hTpn-C33,
hTpn-C49, or mTpn. A number of clones displayed, however, clear
differences in their inhibition by Tpn transfectants. Clones 13 and 14
are examples for clones that were more efficiently inhibited by
transfectants expressing C33/C49 (and mTpn) than by the hTpn
transfectant. On the other hand, clones 31 and 49 were not
significantly inhibited by the mTpn transfectant, but were blocked by
the transfectants expressing full-length or soluble hTpn. These results
suggest that certain NK clones were sensitive to specific
conformational changes in HLA-peptide complexes expressed in the
presence of the different Tpn variants. In the light of similar total
amounts of surface class I molecules (see Fig. 2A), this is
consistent with the concept that peptide loading of B*4402 and Cw1
molecules is differentially modulated by the Tpn constructs
investigated in this study.
To gain direct insight into the composition of class I-associated
peptide spectra, peptides were acid eluted from immunopurified class I
molecules and analyzed by MS (Fig. 4C). On immunoblots
probed with HC-10, we determined the total amount of HLA-Cw1 in .220
cells to be <5% of HLA-B*4402 and Cw1 molecules expressed in
.220.B*4402 cells (data not shown). Thus, the vast majority of eluted
peptides were derived from B*4402 molecules. Peptides from
.220.B*4402/hTpn cells showed a broad distribution in the range of
950-1350 Da with a relative abundance of masses between 1100 and 1200
Da. In the spectrum derived from .220.B*4402/mTpn cells, peptides in
the range of 950-1050 Da were more prominent than in the profile from
hTpn-expressing cells, whereas peptide species at about 1150 Da were
less abundant. Independent repeats confirmed the differences between
these spectra. In another MS analysis, the peptide profile also
produced from .220.B*4402/C49 cells showed a high frequency of
peptides in the 950- to 1050-Da range (data not shown). We noted a few
prominent peaks of relatively high masses that were underrepresented in
the mTpn profile and, on the other hand, a few peptide species of
relatively low masses that were underrepresented in the hTpn profile.
In the peptide profile eluted from .220.B*4402/hTpn-L410F cells, some
prominent peaks of the hTpn profile were missing, while the overall
distribution of masses was quite similar.
Proper assembly of the human TAP-associated loading complex requires wild-type hTpn
To investigate the molecular basis for the defective formation of
Bw4 epitopes, we analyzed complexes containing ER components of the
class I pathway by coprecipitation
experiments that are summarized in Table I. In Fig. 5A, Crt and Crt-associated
molecules were immunoprecipitated from digitonin lysates of equal
amounts of .220.B*4402 transfectants expressing hTpn, mTpn,
hTpn-C49, hTpn-C33, hTpn-C53KDEL, or
hTpn-L410F, respectively. Western blots of SDS-PAGE-separated
precipitates were probed with Abs specific for Crt, TAP1, ERp57, class
I HC, and Tpn. The Crt immunoblot showed similar amounts of Crt,
proving that precipitations from all cell lines had been equally
efficient. Striking differences were visible for the Crt-associated
proteins. Only in the presence of hTpn, all components of the human
TAP-associated complex could efficiently be coprecipitated with Crt.
Little TAP1 and ERp57, but normal or only slightly reduced amounts of
HC and Tpn were associated with Crt in the presence of hTpn-L410F. Also
in mTpn transfectants, Crt:HC and Crt:Tpn complexes were readily
detectable. However, the quantities of TAP1 coprecipitated through Crt
were clearly diminished, and Crt:ERp57 complexes were undetectable in
this and repeating experiments. In cells expressing soluble hTpn
variants, Crt:HC complexes were strongly reduced or absent, and no Tpn,
TAP1, and ERp57 were detected in Crt immunoprecipitates. Thus,
defective peptide loading in the presence of hTpn mutants and mTpn
seems to be correlated with distinct failures during the formation of a
complete HC:Crt:Tpn:ERp57:TAP complex.
|
B). Low amounts of these components could be coisolated
with TAP1 in the presence of hTpn-L410F, which is probably due to the
reduced TAP levels present in this transfectant (compare Fig. 1D). In digitonin lysates of .220.B*4402/mTpn cells,
normal amounts of Tpn and ERp57 were found to be associated with TAP,
whereas the complex formation of TAP with Crt and HC was
significantly disturbed. Thus, although mTpn recruited ERp57 into the
TAP complex and independently participated in a Tpn:Crt:HC complex (see
Fig. 5A), it apparently did not properly bridge HC and Crt
to TAP.
In good agreement with these results, the coprecipitation via
anti-ERp57 Abs showed that in .220.B*4402/hTpn or mTpn
transfectants, considerable amounts of TAP1 were associated with ERp57
(Fig. 5C). In the presence of hTpn-C33, hTpn-L410F, or in
the vector control transfectant, ERp57:TAP complexes were not formed or
below the threshold of detection. The assembly of ERp57 with Crt was
clearly reduced in the presence of mTpn, as compared with hTpn, and
barely detectable in C33 and pREP4 control transfectants. In the
ERp57 precipitate from .220.B*4402/hTpn-L410F cells, only a slight
reduction of coisolated Crt was observed. The fact that Crt was more
readily coprecipitated with anti-ERp57 than vice versa (Fig. 5A) may be due to differences in the relative portions of
these ER housekeeping proteins that are engaged with the respective
partner under steady state conditions. Similar, or in other experiments
even increased amounts of mTpn coprecipitated with ERp57 as compared
with hTpn, whereas the interaction of hTpn-L410F with ERp57 appeared
slightly reduced (Fig. 5C). Only small quantities of HC
could be coisolated with ERp57 from .220.B*4402/mTpn cells. These
findings would be consistent with the hypothesis that mTpn fails to
recruit normal amounts of HC and Crt into a properly assembled
Tpn:ERp57:TAP complex. In hTpn-L410F cells, the amount of HC associated
with ERp57 appeared only slightly reduced as compared with wild-type
hTpn cells. As no concomitant association with TAP was detected, this
result suggests that, independently of TAP, ERp57:HC(:hTpn-L410F:Crt)
complexes stably assembled. HC and TAP were, however, not detectably
associated with ERp57 in .220.B*4402/pREP4 and .220.B*4402/hTpn-C33
cells, clearly showing that membrane-anchored Tpn is required to
mediate assembly of ERp57 with HC, and additionally, that an intact TM
region of Tpn is required to associate this complex with TAP.
Recently, it was reported that in human cells a Tpn:TAP subcomplex is
first associated with Cnx, which is then released upon binding of HC
and Crt (22). We asked whether a prevailing association of
Cnx with the TAP complex may be correlated with the apparent failure of
mTpn to bridge HC and Crt to TAP. As shown in Fig. 5D, TAP1
immunoprecipitates of .220.B*4402/mTpn and hTpn cells indeed revealed
that larger amounts of Cnx are TAP associated under steady state
conditions in mTpn- as compared with hTpn-expressing transfectants.
Thus, a partial inability of mTpn to facilitate the displacement of Cnx
from human TAP:Tpn complexes may contribute to the defective adaptor
function.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In the absence of Tpn, B*4402 molecules are poorly expressed on the
cell surface (32, 38 ; this study). The three soluble
hTpn-C mutants examined in this study up-regulated W6/32 surface
staining to the same extent that was observed with full-length hTpn,
suggesting that soluble Tpn variants actively supported peptide loading
and ER egress of HLA-B*4402 (and -Cw1) HC-2m
dimers (Fig. 2). In keeping with published work regarding .220.B8 cells
(40), we found that our hTpn-C mutants also
up-regulated W6/32 staining in .220.B8 as well as in .220 cells (P. Tan
and F. Momburg, unpublished results). We did not observe, however,
faster maturation kinetics of class I molecules in the presence of
soluble Tpn. Soluble Tpn could not be coprecipitated from digitonin
lysates using Crt Abs (Fig. 5A) or
2m Abs (unpublished result). It can thus be
concluded that the interaction of soluble Tpn with HC is weak and
probably short-lived, even if soluble Tpn was retained in the ER by
means of a KDEL sequence. Nevertheless, soluble hTpn variants retained
an albeit reduced capacity to facilitate the formation of precipitable
HC:Crt complexes that, however, did not contain detectable amounts of
ERp57 (Fig. 5, A and C). The quantities of
coprecipitated HC decreased with the length of the C-terminal
truncation, being the largest with the hTpn-C33 construct that
contains the entire connecting sequence between the membrane-proximal
Ig-like domain and the TM domain (59). This suggests that
the connecting sequence of Tpn may directly or indirectly contribute to
the recruitment of Crt to HC that was previously shown to require the
N-terminal 50 residues of Tpn (11).
Through an unknown mechanism, the type I glycoprotein Tpn increases
steady state levels of the heterodimeric TAP peptide transporter, and
thereby increases quantitatively the peptide transport capacity of TAP
(11, 40). At present, it can only be speculated whether
this stabilization is due to the permanent presence of up to four Tpn
molecules (18) per TAP1/TAP2 heterodimer, or whether Tpn
facilitates the assembly of newly synthesized TAP1 and TAP2 subunits in
a chaperone-like fashion. The novel TM point mutant L410F has
essentially lost the capacity of wild-type Tpn to elevate TAP1 steady
state levels. Since this mutation interrupts a putative leucine-zipper
motif in the hTpn TM, such a motif may play a role in the interaction
between the TM of hTpn and TMs to be identified within the
multimembrane-spanning TAP1 and TAP2 molecules. The partial suspension
of ER retention of the L410F mutant may have contributed to the failing
stabilization of TAP. The formation of complexes between Crt,
hTpn-L410F, and HC (Fig. 5A) suggests, however, that the TM
mutation did not significantly affect the interaction of Tpn with HC.
Findings that soluble class I molecules can bind to the TAP complex
(8, 9) support the view that an interaction of the TM
domain of HC with that of Tpn may not be critical for the assembly of
HC and Tpn.
Human .220.B*4402 cells transfected with mTpn exhibited another kind of
assembly defect. Also, mTpn fully reconstituted the class I surface
expression measured by the pan-HLA Ab W6/32 (Fig. 2). Furthermore, it
enabled the formation of subcomplexes containing Crt together with HC
and Tpn (Fig. 5A). We show in this study that ERp57 very
efficiently associated with TAP:mTpn complexes independent of the poor
integration of HC and Crt into this complex (Fig. 5B). This
is in good agreement with the finding that class I-independent
ERp57:Tpn:TAP complexes can assemble in
2m-deficient Daudi cells (22). We
noted a strong interaction of mTpn with human TAP, resulting in the
full up-regulation of TAP steady state levels. To our surprise, this
association of mTpn with hTAP did not result in a similarly productive
accumulation of HC and Crt in the TAP complex (Fig. 5, B and
D). In mTpn-transfected cells, we detected elevated amounts
of Cnx associated with TAP as compared with hTpn transfectants. Cnx was
recently shown to be part of a precursor TAP:Tpn:ERp57 complex,
preceding the entry of Crt and HC into the human TAP complex and the
coincident release of Cnx (22). Alternatively, TAP:Cnx
complexes may in part represent nonfunctional dead-end products.
Whereas earlier work has noted the concomitant presence of Cnx and HC
in murine TAP/Tpn complexes (9, 21), a recent study of
mouse cells demonstrated that the Tpn-dependent assembly of HC with
ERp57, Tpn, and TAP was correlated with the association of Crt rather
than that of Cnx (60). These findings (22, 60) support a model in which the association of Cnx and of
Crt:HC with TAP is mutually exclusive. Therefore, it seems likely that
TAP:Cnx complexes and TAP:HC complexes observed in TAP
immunoprecipitates (Fig. 5D) belonged to separate TAP
subpopulations, and second, that the strongly reduced TAP association
of B*4402/Crt in the presence of mTpn was due to an abnormally low
dissociation rate of human Cnx, possibly caused by a partial inability
of mTpn to release human Cnx.
The poor recruitment of B*4402 into the human TAP complex by mTpn is in contrast with a prolonged association of B*4402 with the murine TAP complex that was recently described for B*4402-transfected mouse J26 cells (38). This prolonged TAP association and the diminished surface expression of B*4402 could be normalized by introduction of hTpn (38). In addition, our own preliminary data on Kd- or Kk-transduced .220 transfectants suggest that mTpn is superior to hTpn in facilitating Kd- or Kk-restricted Ag presentation by this human cell line (M. Lobigs, J. C. S. M. R., A. N. U., Canberra, and F. Momburg, unpublished results). Thus, not only species-specific constraints in the interaction between Tpn and ER chaperones, but also between Tpn and HC may influence the assembly of the TAP complex and successful peptide loading. This partial species barrier should be kept in mind when analyzing the efficiency of Ag presentation by HLA-transgenic mouse cells, or conversely, by H-2-transfected human cells.
The three soluble hTpn variants, the hTpn TM point mutant, and mTpn
failed to assemble a complete TAP-associated complex for different
reasons. Nevertheless, the functional outcome was similar, as none of
these Tpn forms imparted a full-fledged induction of the Bw4 epitopes
detected by mAbs TT4-A20 or TÜ109 as compared with wild-type
hTpn. In accordance with Peh and colleagues (38), a defect
to build up the Bw4 supertypic determinant was, however, not detected
using mAb T116-5-28. This suggests that in the presence of wild-type
hTpn, subtle conformational changes are induced in class I HC
1 helix around residues 80–83, which appear
to be recognized by some Bw4-specific Abs, but not by others (52, 61). Similar observations were made when .220.B8 cells
transfected with wild-type or soluble hTpn variants were examined with
a panel of Bw6-reactive Abs (P. Tan and F. Momburg, unpublished
results). Analysis with TT4-A20 revealed that the failure of mTpn to
elicit the full expression of the Bw4 determinant is a function of the
N-terminal 149 mTpn residues comprising two of the four predicted
ectodomains of Tpn (18, 37, 59). Since 35 aa differ
between hTpn and the mhTpn hybrid, additional studies are required
to exactly delineate those mTpn residues that confer the
incompatibility with the human class I assembly machinery. Clearly,
putative conformational changes depicted by Bw4 reagents in the
presence of hTpn mutants or mTpn escaped detection when labeling with
the monomorphic class I Ab W6/32 that requires
HC/2m heterodimers, but is less sensitive to
the peptide cargo (51). This explains the previous
conclusion that soluble Tpn may be fully functional
(40).
The serological findings with cells expressing hTpn mutants or mTpn
were complemented by biochemical assays that demonstrated the inferior
thermostability of class I molecules in lysates, the reduced stability
of surface class I molecules in the presence of BFA, as well as the
reduced t1/2 of cohorts of
metabolically labeled class I molecules. Consistently, an inferior
thermostability of W6/32-precipitated HLA-B8 molecules was noted in
.220.B8 cells transfected with soluble hTpn constructs (P. Tan and F.
Momburg, unpublished results). These findings all pointed to
qualitative differences in the peptide cargo loaded onto class I
molecules. This view is supported by the result that B*4402-binding
peptides were able to enhance surface staining by TÜ109 and
TT4-A20 on .220.B*4402/hTpn-C33 or C49, but not on
.220.B*4402/hTpn cells (Fig. 4A), suggesting that
peptide-dependent HLA-Bw4 epitopes were incompletely formed in the
presence of soluble Tpn. This is consistent with an earlier study
showing that the TÜ109 epitope was strongly influenced by
residues at position 8 of HLA-B*5101-binding 9-mer peptides
(53). The situation appears similar to
H-2Ld and other mouse class I molecules
displaying more open, peptide-receptive forms on the surface of .220
cells as compared with Tpn-positive .221 cells (34).
Furthermore, it has been shown that negatively or positively charged
residues at peptide position 8 interfere with the recognition by
Bw4-specific KIR-3DL1 inhibitory NK receptors (56). Our
result that certain NK clones can be differentially inhibited by hTpn
transfectants on the one hand, and mTpn or C33(C49) transfectants
on the other hand (Fig. 4B), suggests that these clones may
be sensitive to Tpn-dependent peptides.
In accordance with recent studies by the McCluskey group, who analyzed Kb- and B*2705-associated peptide spectra in the presence and absence of Tpn (12, 36), we noted a considerable overlap in the spectra derived from .220.B*4402 cells transfected with hTpn, mTpn, or hTpn-L410F. The MS analysis of class I-eluted peptides, however, revealed the presence of individual peptides that were bound in the presence of hTpn and not detected in the presence of mTpn and hTpn mutants, or vice versa. Certainly, this does not contradict the previous finding that particular peptides are equally well presented to T cells by .220.B*4402/hTpn and .220.B*4402/mTpn cells (38). Since the t1/2 of HLA class I molecules were more dramatically influenced by Tpn mutants or mTpn than the peptide profiles, it seems possible that the differences in the peptide profiles tend to be underestimated due to the preferential potential loss of low affinity ligands during the immunoisolation of class I molecules.
There is evidence for an enhanced exchange of class I-binding peptides in the ER (62). It remains to be elucidated whether under physiological conditions class I molecules regularly enter the TAP-associated complex in the unloaded form, or whether the Tpn:TAP complex predominantly serves as a peptide editor for class I molecules that may enter the complex with prebound peptides of suboptimal affinity. The latter scenario would resemble peptide editing in the HLA-DR/HLA-DM system (63). Taken together, our findings indicate a predominant function of the TAP-associated complex for the loading of HLA class I molecules with peptides conferring optimal stability. While Tpn is in principle able to modify the peptide cargo in the absence of TAP association, it plays out its crucial role to the full extent only by means of its capacity to recruit class I molecules into the complex with TAP.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Abbreviations used in this paper: ER, endoplasmic reticulum; 2m, 2-microglobulin; BFA, brefeldin A; Cnx, calnexin; Crt, calreticulin; D-PBS, Dulbecco’s PBS; Endo H, endoglycosidase H; HC, MHC class I H chain; Tpn, tapasin; hTpn, human Tpn; KIR, killer cell Ig-like receptor; mhTpn, mouse-human chimera; MFI, mean fluorescence intensity; MS, mass spectrometry; mTpn, mouse Tpn; TM, transmembrane.
Received for publication September 17, 2001. Accepted for publication December 6, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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 complexes associate with TAP transporters before peptide binding. Nature 368:864.[Medline]
2 domain loop influences interaction with the assembly complex. J. Immunol. 163:4427.1 helix. J. Immunol. 153:4099.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  S. Lee, B. Park, K. Kang, and K. Ahn Redox-regulated Export of the Major Histocompatibility Complex Class I-Peptide Complexes from the Endoplasmic Reticulum Mol. Biol. Cell, July 15, 2009; 20(14): 3285 - 3294. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Corcoran, X. Wang, and L. Lybarger Adapter-mediated Substrate Selection for Endoplasmic Reticulum-associated Degradation J. Biol. Chem., June 26, 2009; 284(26): 17475 - 17487. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Procko, M. L. O'Mara, W. F. D. Bennett, D. P. Tieleman, and R. Gaudet The mechanism of ABC transporters: general lessons from structural and functional studies of an antigenic peptide transporter FASEB J, May 1, 2009; 23(5): 1287 - 1302. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. C. Verweij, D. Koppers-Lalic, S. Loch, F. Klauschies, H. de la Salle, E. Quinten, P. J. Lehner, A. Mulder, M. R. Knittler, R. Tampe, et al. The Varicellovirus UL49.5 Protein Blocks the Transporter Associated with Antigen Processing (TAP) by Inhibiting Essential Conformational Transitions in the 6+6 Transmembrane TAP Core Complex J. Immunol., October 1, 2008; 181(7): 4894 - 4907. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Roder, L. Geironson, I. Bressendorff, and K. Paulsson Viral Proteins Interfering with Antigen Presentation Target the Major Histocompatibility Complex Class I Peptide-Loading Complex J. Virol., September 1, 2008; 82(17): 8246 - 8252. [Full Text] [PDF]
|
|
|
|
|
|
M. W. Everett and M. Edidin Tapasin Increases Efficiency of MHC I Assembly in the Endoplasmic Reticulum but Does Not Affect MHC I Stability at the Cell Surface J. Immunol., December 1, 2007; 179(11): 7646 - 7652. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Rufer, R. M. Leonhardt, and M. R. Knittler Molecular Architecture of the TAP-Associated MHC Class I Peptide-Loading Complex J. Immunol., November 1, 2007; 179(9): 5717 - 5727. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Tiwari, N. Garbi, T. Reinheckel, G. Moldenhauer, G. J. Hammerling, and F. Momburg A Transporter Associated with Antigen-Processing Independent Vacuolar Pathway for the MHC Class I-Mediated Presentation of Endogenous Transmembrane Proteins J. Immunol., June 15, 2007; 178(12): 7932 - 7942. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Papadopoulos and F. Momburg Multiple Residues in the Transmembrane Helix and Connecting Peptide of Mouse Tapasin Stabilize the Transporter Associated with the Antigen-processing TAP2 Subunit J. Biol. Chem., March 30, 2007; 282(13): 9401 - 9410. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Thammavongsa, G. Raghuraman, T. M. Filzen, K. L. Collins, and M. Raghavan HLA-B44 Polymorphisms at Position 116 of the Heavy Chain Influence TAP Complex Binding via an Effect on Peptide Occupancy. J. Immunol., September 1, 2006; 177(5): 3150 - 3161. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. M. Paulsson, M. Jevon, J. W. Wang, S. Li, and P. Wang The double lysine motif of tapasin is a retrieval signal for retention of unstable MHC class I molecules in the endoplasmic reticulum. J. Immunol., June 15, 2006; 176(12): 7482 - 7488. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. M. Leonhardt, K. Keusekotten, C. Bekpen, and M. R. Knittler Critical Role for the Tapasin-Docking Site of TAP2 in the Functional Integrity of the MHC Class I-Peptide-Loading Complex J. Immunol., October 15, 2005; 175(8): 5104 - 5114. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Sesma, B. Galocha, M. Vazquez, A. W. Purcell, M. Marcilla, J. McCluskey, and J. A. L. de Castro Qualitative and Quantitative Differences in Peptides Bound to HLA-B27 in the Presence of Mouse versus Human Tapasin Define a Role for Tapasin as a Size-Dependent Peptide Editor J. Immunol., June 15, 2005; 174(12): 7833 - 7844. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Petersen, H. D. Hickman-Miller, M. M. McIlhaney, S. E. Vargas, A. W. Purcell, W. H. Hildebrand, and J. C. Solheim A Charged Amino Acid Residue in the Transmembrane/Cytoplasmic Region of Tapasin Influences MHC Class I Assembly and Maturation J. Immunol., January 15, 2005; 174(2): 962 - 969. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Li, B. A. Sullivan, C. J. Aldrich, M. J. Soloski, J. Forman, A. G. Grandea III, P. E. Jensen, and L. Van Kaer Differential Requirement for Tapasin in the Presentation of Leader- and Insulin-Derived Peptide Antigens to Qa-1b-Restricted CTLs J. Immunol., September 15, 2004; 173(6): 3707 - 3715. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Howarth, A. Williams, A. B. Tolstrup, and T. Elliott Tapasin enhances MHC class I peptide presentation according to peptide half-life PNAS, August 10, 2004; 101(32): 11737 - 11742. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Abele and R. Tampe The ABCs of Immunology: Structure and Function of TAP, the Transporter Associated with Antigen Processing Physiology, August 1, 2004; 19(4): 216 - 224. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Ford, A. Antoniou, G. W. Butcher, and S. J. Powis Competition for Access to the Rat Major Histocompatibility Complex Class I Peptide-loading Complex Reveals Optimization of Peptide Cargo in the Absence of Transporter Associated with Antigen Processing (TAP) Association J. Biol. Chem., April 16, 2004; 279(16): 16077 - 16082. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Koch, R. Guntrum, S. Heintke, C. Kyritsis, and R. Tampe Functional Dissection of the Transmembrane Domains of the Transporter Associated with Antigen Processing (TAP) J. Biol. Chem., March 12, 2004; 279(11): 10142 - 10147. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. R. Turnquist, J. L. Petersen, S. E. Vargas, M. M. McIlhaney, E. Bedows, W. E. Mayer, A. G. Grandea III, L. Van Kaer, and J. C. Solheim The Ig-Like Domain of Tapasin Influences Intermolecular Interactions J. Immunol., March 1, 2004; 172(5): 2976 - 2984. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. L. Zarling, C. J. Luckey, J. A. Marto, F. M. White, C. J. Brame, A. M. Evans, P. J. Lehner, P. Cresswell, J. Shabanowitz, D. F. Hunt, et al. Tapasin Is a Facilitator, Not an Editor, of Class I MHC Peptide Binding J. Immunol., November 15, 2003; 171(10): 5287 - 5295. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, .220.B*4402/pREP4; 
,
.220.B*4402/hTpn; 
, .220.B*4402/hTpn-
,
.220.B*4402/mTpn.