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*
Institut für Anthropologie und Humangenetik, Ludwig-Maximilians-Universität München, Munich, Germany;
Institut für Medizinische Mikrobiologie und Hygiene, Universität Regensburg, Regensburg, Germany; and
Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain/ß2m dimer with TAP, which is believed
to ensure effective peptide loading of the empty class I molecule. We
have previously demonstrated impaired intracellular transport of the
class Ib molecule HLA-E in mouse myeloma cells cotransfected with the
genes for HLA-E and human ß2m, which is most likely
attributable to inefficient intracellular peptide loading of the HLA-E
molecule. We therefore analyzed the ability of HLA-E in the
transfectant cell line to bind synthetic peptides by means of their
ability to enhance cell surface expression of HLA-E. Peptide binding
was confirmed by testing the effect on the thermostability of soluble
empty HLA-E/human ß2m dimers. Two viral peptides binding
to HLA-E were thus identified, for which the exact positioning of the N
terminus appeared critical for binding, whereas the contribution of the
length of the C terminus seemed to be minor, allowing peptides as short
as seven amino acids and up to 16 amino acids to exhibit considerable
binding activity. Furthermore, we demonstrate that HLA-E interacts with
TAP and that this interaction can be prolonged by the proteasome
inhibitor
N-acetyl-L-leucyl-L-leucyl-L-norleucinal,
which reduces the intracellular peptide pool. The presented data
indicate that HLA-E is capable of presenting peptide ligands similar to
the repertoire of HLA class Ia molecules. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using the similarity of amino acid sequence to class Ia molecules, a
second group of class Ib molecules can be defined that contains genes
encoded within and outside the MHC and is heterogeneous with regard to
its size in different species as well as to the tissue expression
pattern and the degree of polymorphism of its members. The function of
these class Ib molecules remains largely unknown, although a role for
these molecules in Ag presentation is most likely (for review, see 13 . In the mouse the majority of these molecules are encoded in the Q,
T, and M subregions of the MHC (14, 15). They comprise the Qa-2
molecules that bind nonameric peptides (16), the Qa-1 Ag that is
capable of interacting with mycobacterial heat shock protein 65 (17),
and a Glu50-Tyr50 copolymer (18), as well as
the H-2 M3 Ag that selectively presents bacterial peptides with unique
chemical properties (19, 20). In contrast, the thymus leukemia Ag is
recognized by T cells expressing 
TCRs in the absence of
detectable bound peptides (21, 22). In man, class Ib molecules comprise
the low polymorphic HLA-E, -F, -G, and CD1 Ags in addition to the
highly polymorphic MIC-A and -B molecules (23). Of these, HLA-G was
shown to present nonameric peptides, whereas CD1b has been implicated
in the recognition of mycobacterial lipid and lipoglycan compounds by
CD4-CD8-ß+ T cells (24).
HLA-E is the only class Ib gene that is transcribed in all human
tissues and cell lines (25, 26), and we have previously reported that
the HLA-E Ag is weakly expressed at the cell surface upon
cotransfection with the gene for human ß2m
(hß2m) into the mouse myeloma cell line P3X63Ag8.653
(X63) (26). In these transfectants the intracellular transport of the
HLA-E molecule was impaired, most likely due to inefficient peptide
loading within the ER (27). In this report we address the question of
whether HLA-E binds peptides, in particular exclusively special
peptides, possibly present only in low abundance within the cell.
Moreover, we wanted to know whether the peptide binding groove of HLA-E
is only accessible for exactly processed peptides that might reduce its
efficient loading. Finally, we investigated whether a disturbed
interaction of HLA-E with TAP might reduce its chance of encountering
appropriate peptides.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Abs used for immunofluorescence and immunoprecipitation were mAb B9.12.1 (IgG2a) (28), which is specific for HLA class I complexed with ß2m and which was used as a supernatant of the hybridoma; mAb W6/32 (IgG2a, American Type Culture Collection, Rockville, MD), which is specific for HLA class I complexed with ß2m and was applied purified; mAb A1.4 (IgG2b; United Biomedical, Hauppauge, NY), which is specific for HLA class I heavy chains and was used as hybridoma supernatant; mAb 27-11-13s (IgG2a), which is specific for H-2Dd and Db (provided by F. Kievits, Amsterdam, The Netherlands) as ascites; and two antisera, D90, which is specific for rat TAP1 and cross-reactive with mouse TAP1, and 116/3, an antiserum specific for rat TAP2 and cross-reactive with mouse TAP2 (both provided by Dr. Jonathan Howard, Cologne, Germany). All the Abs were raised in mice, with the exception of D90 and 116/3, which are rabbit antisera. As secondary reagents, dichlorotriazinyl aminofluorescein-conjugated goat anti-mouse IgG F(ab')2 (Jackson ImmunoResearch Laboratories, West Grove, PA) were used for immunofluorescence, and alkaline phosphatase-labeled goat anti-mouse Ig or biotin-labeled goat anti-rabbit Ig (both from Tropix, Bedford, MA) were applied for immunodetection of Western blots.
Peptides
Peptides were either obtained RP-HPLC purified from A. McMichael (Oxford, U.K.), G. Pape (Munich, Germany), S. Modrow (Regensburg, Germany), B. Schoel (Ulm, Germany), P. Robinson (London, U.K.), H. Zweerink (Rahway, NJ), and S. Shawar (Houston, TX) or were synthesized using a 9050 PepSynthesizer (Milligen, Burlington, MA) and F-moc coupling technology as described previously (29). The latter were further purified by reverse phase chromatography on a PepRPC 15 µm HR 16/10 column (Pharmacia, Piscataway, NJ) using a Pharmacia fast protein liquid chromatography system. The peptides E-1L6 and E-2F6 were custom synthesized and RP-HPLC purified by Affiniti Research Products (Nottingham, U.K.).
Transfectants
The transfection of P3X63Ag8.653 (X63) with human ß2m (X63M), human ß2m and HLA-E*01033 (X63EM), or human ß2m and HLA-B*2705 (X63BM) has been described previously (26). The RMA-S cell line was obtained from Dr. Marika Pla (Institut National de la Santé et de la Recherche Médicale (INSERM), Hôpital St. Louis, Paris, France). Cells were cotransfected by standard electroporation procedures (Gene Transfector 300, BTX, San Diego, CA) with 15 µg of a BamHI-linearized 15-kb BamHI/SalI subclone of the human ß2m-gene in pUC19 (28) together with 5 µg of a SalI-linearized derivative of the COS-203 vector (30), in which the EBV sequences and COS site had been deleted by Eca digestion and which contained either no insert or a HindIII/BglII fragment of cosmid cd3.14 (26) encoding the HLA-E*01033 allele. Cells were maintained in 5% CO2 at 37°C in RPMI 1640 medium containing 2 mM L-glutamine (BioWhittaker, Walkersville, MD) and supplemented with 10% FCS (Serva, Heidelberg, Germany), 1 mM sodium pyruvate, 50 U/ml penicillin, and 50 µg/ml streptomycin (all from BioWhittaker). To select and grow the transfectant clones of the RMA-S and X63 cells, 0.4 mg/ml hygromycin B (Boehringer Mannheim, Indianapolis, IN) and 1 mg/ml G418 (Life Technologies, Gaithersburg, MD), respectively, were added to the culture medium.
For expression of soluble HLA-E molecules in Drosophila
melanogaster cells, a truncated cDNA that encodes the signal
peptide and 1-3 domains of the HLA-E*0101 allele was generated by
PCR and cloned into the pRMHa-3 expression vector (31). This construct
was cotransfected with a hß2m cDNA cloned into pRMHa-3
and the pUCshsneo vector (32) as a selection marker. Transfection of
SC2 D. melanogaster tissue culture cells and selection of
transformants have been described previously (33). Transfectants were
maintained in Schneider medium (Life Technologies) supplemented with
10% FCS and 0.5 mg/ml G418 at room temperature. For the induction of
soluble HLA-E/hß2m dimer expression, cells were
transferred into serum-free Insect Xpress medium (BioWhittaker) at a
density of >107 cells/ml, and 1 mM CuSO4 was
added for 48 h.
Thermal stability assay for soluble HLA-E molecules
The supernatant of transfected SC2 D. melanogaster cells that were induced to express soluble HLA-E/hß2m dimers was diluted 1/20 with PBS and either supplemented with Nonidet P-40 to 1% (w/v) or used directly. To 1 ml of this dilution either peptides (50 and 100 µM) or purified hß2m (Serologic Reagents, East Grinstead, U.K.; 2 µM) were added. The solution was incubated overnight at 4°C and then heated to 32°C for 1 h. Then, 5 µg of mAb W6/32 was added for 40 min at 4°C, followed by a 20-min incubation at 4°C in the presence of 20 µl of swollen protein A-Sepharose CL-4B (Pharmacia). The immunoprecipitates were recovered by centrifugation, washed five times with 0.1% Nonidet P-40 in PBS, separated by 10% SDS-PAGE, and transferred onto nitrocellulose membranes (Schleicher and Schuell, Keene, NH) using standard Western blotting techniques. HLA-E heavy chains were visualized using the Western-Light (Tropix) chemiluminescent detection system according to the manufacturer’s instructions. In brief, blocked membranes were incubated for 1 h at room temperature in 12 ml of a 1/20 dilution of A1.4 hybridoma supernatant in 0.1% (v/v) Tween-20/PBS (TPBS), washed twice for 5 min each time at room temperature in TPBS, and then incubated in 14 ml of a 1/14,000 dilution of the secondary anti-mouse antiserum conjugated with alkaline phosphatase in TPBS for 45 min at room temperature. After washing three times for 10 min each time at room temperature in TPBS, membranes were processed as recommended using CSPD (Tropix, Bedford, MA) as the chemiluminescent substrate.
Detection of TAP association
X63EM, X63BM, and X63M transfectant cells (7 x 107) were lysed for 30 min at 4°C in 0.6 ml of lysis buffer (1% (w/v) digitonin (Sigma), 0.1 mM PMSF, and 1% (v/v) Trasylol (Bayer, Pittsburgh, PA; equivalent to 14 µg/ml aprotinin) in PBS). After removal of cellular debris by centrifugation, 200 µl of B9.12.1 supernatant was added, followed by incubation for 40 min at 4°C. After addition of 20 µl of swollen protein A-Sepharose CL-4B (Pharmacia) and incubation for 40 min at 4°C, immunoprecipitates were recovered by centrifugation and washed five times with 0.1% digitonin in PBS. Two aliquots, each equivalent to 3 x 107 cells, and one aliquot equivalent to 1 x 107 cells were separated in different lanes by 10% SDS-PAGE and blotted onto nitrocellulose membranes (Schleicher and Schuell) by standard Western blotting techniques. Membranes with the transfer of 3 x 107 cell equivalent immunoprecipitates were incubated for 1 h at room temperature in 5 ml of TPBS with either the TAP1-specific antiserum D90 (1/1000) or the TAP2-specific antiserum 116/3 (1/2000). After washing twice for 5 min each time at room temperature with TPBS, the membranes were processed using Western-LightPlus (Tropix) chemiluminescent detection according to the manufacturer’s instructions. The resulting immune complexes were labeled with a biotinylated secondary Ab and streptavidin alkaline phosphatase and were visualized using CSPD as the chemiluminescent substrate. HLA class I heavy chains were visualized on membranes with the immunoprecipitates of 1 x 107 cells/lane using A1.4 as first Ab and the Western-Light (Tropix) chemiluminescent detection system as described above.
Pulse-chase experiments and immunoprecipitations
X63EM and X63BM transfectants were kept at 1.2 x 106/ml in methionine- and cysteine-free DMEM (BioWhittaker) for 1 h at 37°C and 5% CO2. During this period, X63EM transfectants were either treated with LLnL (Sigma) at 250 µM or left untreated. Thereafter, Trans35S-label (ICN, Costa Mesa, CA) was added to the cells at 100 µCi/ml. After 20 min of labeling, a 100-fold molar excess of cysteine and methionine was added; an aliquot of 1.2 x 106 cells was removed, washed, and placed on ice; and the remaining 1.2 x 106 cells were chased at 37°C for 90 min. The cells were lysed for 30 min at 4°C in 500 µl of PBS supplemented with 1% digitonin, 1 mM PMSF, and 1% (v/v) Trasylol. After removal of debris by centrifugation, lysates were precleared overnight at 4°C by adding 4 µl of a purified IgG2a mAb (ICN) and 20 µl of protein A-Sepharose. Precleared lysates were immunoprecipitated by the addition of 2 µl of the 116/3 antiserum followed by incubation for 40 min at 4°C and then addition of 30 µl of protein A-Sepharose followed by incubation for 20 min at 4°C. Immunoprecipitates were recovered by centrifugation, washed five times with 0.1% digitonin/PBS, resuspended in 30 µl of 2% SDS and 0.5 mM PMSF in PBS, and boiled for 5 min. After addition of 600 µl of PBS containing 1% Nonidet P-40, 1 mM PMSF, and 1% (v/v) Trasylol (Bayer), the solution was kept for 30 min at room temperature and for another 30 min at 4°C before the addition of 150 µl of A1.4 hybridoma supernatant and incubation for 45 min at 4°C. After an additional incubation for 20 min at 4°C with 20 µl of protein A-Sepharose, the immunoprecipitates were recovered by centrifugation and washed five times with 0.1% Nonidet P-40/PBS before analysis by 10% SDS-PAGE. Gels were treated with Amplify (Amersham, Arlington Heights, IL), dried, and fluorographed at -80°C.
Immunofluorescence staining and flow cytometric analysis
X63 transfectant cells (1–2 x 105) were incubated in a 96-well microtiter plate well with peptides at 25, 50, or 100 µM for 6 h at 37°C in culture medium before immunofluorescent staining. Alternatively, subconfluent transfectants were incubated in culture medium for 4 h at room temperature. After two washes with RPMI 1640, 1 to 2 x 105 cells were incubated in a 96-well microtiter plate well with peptides at 100 µM for 1 h at room temperature in RPMI 1640. After incubation at 37°C for 1 h, cells were stained for flow cytometric analysis with mAbs B9.12.1 and A1.4 as described previously (27).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and A1.4 (data not shown). None
of the peptides enhanced staining with these mAbs of the negative
control X63 cells expressing either HLA-B27 and hß2m
(X63BM) or hß2m alone (X63M), even at 100 µM (data not
shown). The effect of the peptides added at 25 µM on HLA-E cell
surface expression was considerably less than that of exogenous
hß2m added at 12 µg/ml (
1 µM), which served as a
positive control and which we considered to produce the maximum
increase in HLA-E cell surface expression achievable in this system.
Moreover, BZLF-139–51 enhanced HLA-E cell surface
expression slightly more than did InflM57–68K62. We
therefore performed a titration experiment using increasing peptide
concentrations. As shown in Figure 2,
increasing peptide concentrations resulted in the same enhancement of
HLA class I staining as that obtained upon stabilization with
hß2m. The slight differences in enhancement maxima were
within the range of experimental variation. In this experiment, no
increase in HLA-E cell surface expression with 25 µM
InflM57–68K62 could be observed. BZLF-139–51
enhanced the HLA-E cell surface expression at lower concentrations than
InflM57–68K62. This enhancement leveled off at 50 µM,
whereas the maximum effect of InflM57–68K62 was reached
only at 100 µM. No effect was observed when peptides were tested on
X63BM or X63M cells (data not shown).
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) and A1.4 (data not shown). We thus
tested whether the peptides were able to sustain the enhancement of
HLA-E cell surface expression obtained during the preincubation of
cells at room temperature. Peptides capable of stabilizing HLA-E cell
surface expression were considered to bind to HLA-E. As negative
controls, the same protocol was performed on X63M and X63BM cells,
which did not result in enhanced B9.12.1 or A1.4 staining of either
cell line with any of the peptides used (data not shown). Figure 3
shows exemplary results. A carboxyl-terminal truncation of the
initially identified BZLF-139–51 peptide, resulting in
the nonameric BZLF-139–47 peptide, stabilized HLA-E at
least as well as BZLF-139–51 itself. In contrast,
InflM57–68K62 could not maintain the enhanced HLA-E cell
surface expression caused by the preincubation (data not shown),
presumably due to the absence of extracellular processing, whereas
a decamer (InflM59–68K62) and a nonamer
(InflM59–67K62) obtained by N-terminal truncation of
InflM57–68K62 could stabilize HLA-E. A further
single amino acid truncation of the InflM59–68K62 peptide
at the N terminus, giving InflM60–68K62, abrogated its
stabilizing effect on HLA-E. The results of the assay are summarized
qualitatively in Table I. The exact
positioning of the peptides’ N termini appears to be critical for
their binding to HLA-E. Extension or truncation of the N terminus by a
single amino acid absolutely abrogated peptide binding to HLA-E in the
temperature shift assay, although some of the peptides
(BZLF-132–46, BZLF-134–46, and
InflM57–68K62) containing an extended N terminus enhanced
HLA-E cell surface expression when X63EM cells were incubated with them
for 6 h at 37°C in the presence of FCS. Thus, it is possible
that appropriate N termini of these peptides were generated during the
incubation by extracellular processing. In contrast, in the thermal
shift assay, C-terminal length variations of the peptides were
tolerated without considerable alteration to the peptides’ stabilizing
effect on HLA-E (Fig. 4), although
maximum stabilization was achieved with the 9 mer
BZLF-139–47. Only when the 7 mer,
BZLF-139–45, was truncated to give the 6 mer,
BZLF-139–44, was the stabilizing ability of the peptide
almost undetectable (although it was still visible in experiments in
which the maximum enhancement of HLA-E cell surface expression reached
higher levels than those shown in Fig. 4). The only internal
substitution abrogating the peptide’s effect on HLA-E that we
identified to date exchanged P at amino acid position 4 of
BZLF-139–47 for a Q. Since this position tolerates amino
acids as different as K and F in the InflM peptide without affecting
binding activity to HLA-E, it seems unlikely that position 4 is a
dominant anchor for HLA-E binding peptides. Substitution of small
hydrophobic amino acids at the C terminus of the peptides by K did not
affect peptide binding. In the case of BZLF-140–47 and
InflM59–68, these C-terminal substitutions with the
hydrophilic K rendered the peptides soluble in water, which were
otherwise only soluble in DMSO, and thus increased their effective
concentrations in the assay. Since InflM59–68K68
stabilizes HLA-E, the lack of detectable binding of
InflM59–68 is most likely attributable to its insolubility
in water. In contrast, BZLF-140–47 does not bind to HLA-E,
even when its solubility in water is increased by C-terminal K
substitution.
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, lane 9). When 1 ml
of this diluted supernatant was heated to 32°C for 1 h, the
amount of HLA-E/hß2m dimers detectable by W6/32
immunoprecipitation and immunoblotting was greatly diminished (Fig. 5, lane 1). In the presence of 2 µM
hß2m, the immunoprecipitated amounts of
HLA-E/hß2m dimers equaled those in the positive control
experiment (Fig. 5, lane 8). The two peptides,
InflM57–68K62 and BZLF-139–51, were able to
stabilize the HLA-E/hß2m dimers in a similar manner,
although less effectively than hß2m, as can be deduced
from the weaker intensity of the HLA-Esol
-chain-specific bands of the immunoblot (Fig. 5, lanes
2–5). The 13 mer gag47–59 peptide that was
negative with regard to enhancement of HLA-E cell surface expression of
X63EM cells in the previous experiments did not increase the
thermostability of the HLA-E/hß2m complexes (Fig. 5, lanes 6 and 7). There was no apparent
difference between the abilities of InflM57–68K62 and
BZLF-139–51 to stabilize HLA-E/hß2m dimers
in this assay, although only the latter was positive in the thermal
shift assay using X63EM cells. Thus, in the presence of Nonidet P-40,
the conformation of HLA-E does not select for an optimal trimming of
the N terminus of peptides for binding.
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). Although detection with A1.4
showed a far lower abundance of HLA -chains in X63EM cells than in
X63BM cells (Fig. 6, upper panel), TAP1 and TAP2 were
only detectable in association with HLA class I molecules in X63EM
cells (Fig. 6, middle and lower panels).
Only after prolonged exposure were TAP1 and TAP2 molecules associated
with HLA-B27 detectable (not shown), whereas the lane of X63M cells
remained negative for TAP1 or TAP2 signals. Therefore, under steady
state conditions HLA-E is complexed with TAP to a much greater extent
than HLA-B27. TAP1 and TAP2 are both required to form a functional
peptide transporter and are both associated with HLA-E. Unfortunately,
the experimental design did not allow us to test for possible
disturbances in the stoichiometry of the complexes, which might affect
the function of the peptide transporter itself.
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). Immediately
after the labeling, HLA-B27 Ag associated with TAP was detected;
this association was lost entirely after a 90-min chase. HLA-E
-chains, coprecipitating with TAP, gave a very faint signal at time
zero. However, this did not reflect a weak association of HLA-E with
TAP, but was due to a weak labeling efficiency of HLA-E in the X63EM
cells (27). After a 90-min chase, no TAP-associated HLA-E -chains
were detectable. The large differences in the intensities of
TAP-associated HLA -chains between X63EM and X63BM cells did not
allow us to make assumptions about differences in the association
kinetics of the class I molecules with TAP. In particular, it was not
possible to demonstrate a prolonged association of HLA-E with TAP that
might explain the differences in TAP association between HLA-E and
HLA-B27 in the steady state. To determine whether TAP association of
HLA-E is dependent on peptides and thus whether HLA-E is loaded with
peptides in the ER, we performed a parallel experiment in which
proteasomes were inhibited by the addition of 250 µM LLnL (Fig. 7, lanes 1 and 2). In the presence of this
inhibitor, the absolute amount of HLA-E associated with TAP increased,
and the signals from TAP-associated HLA-E -chains showed identical
intensities after 0- and 90-min chase, the latter pointing to a peptide
dependency for HLA-E’s TAP association and indirectly to the peptide
loading of HLA-E in the ER.
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we compare the class I
cell surface expression pattern of transfectant clones with the cell
lines RMA-S and X63, which were shown to contain equal amounts of
HLA-E and hß2m transcripts by Northern blot analysis. The
RMA-S cells expressing hß2m and HLA-E (RMA-SEM) stained
slightly less brightly for the HLA-E Ag than control X63EM cells
(histogram shaded in gray). Addition of either exogenous
hß2m (thin lines) or peptide (BZLF-139–47)
enhanced cell surface expression of HLA-E on both cells to a similar
extent. This increase is low compared with the stabilization of
H-2Db expressed by RMA-S cells by adding a
Db-specific NP2 peptide (ASNENMETM). This result shows that
HLA-E molecules can be expressed on the cell surface in a
TAP-independent manner.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Of 79 partially overlapping synthetic peptides, two viral peptides were
demonstrated to bind to HLA-E. These peptides increased the HLA-E cell
surface expression of X63EM cells at 37°C as well as stabilized the
enhancement of HLA-E cell surface expression at 37°C that was
generated by incubating X63EM cells at room temperature (Figs. 1
and 3). Furthermore, these modified peptides increased the thermostability
of soluble HLA-E/hß2m dimers (Fig. 5). For screening we
also included peptides with N-terminal modifications, in particular
N-formylation, which is a major structural requirement for
binding to H-2 M3 (37). Since the low availability of these ligands may
limit the expression of H-2 M3 (38), we wanted to test whether a
similar mechanism operates on the expression of HLA-E. None of these
modified peptides showed any detectable binding activity to HLA-E. As
the N-formylated peptides display a low solubility in water,
it is possible that their effective concentrations in the assay were
too low to detect binding. However, this seems unlikely, since in a
similar assay Vyas et al. (38) could demonstrate that f-Bla-z at
concentrations as low as 5 µM significantly increased the cell
surface expression of M3a-Ld chimeric
molecules. Moreover, the binding of the N terminus of
N-formylated peptides to H-2 M3 is coordinated by residues
in and around the B pocket of the class I molecule, since its A pocket
is occluded by L and F replacing the consensus T-167 and W-171,
respectively (39). In HLA-E the latter positions are identical with the
consensus. Consequently, it does not meet the structural requirements
to bind formylated peptides.
We also tested peptides from heat shock proteins, since, for Qa-1, induction of cell surface expression by either heat shock or incubation with a tryptic digest of Mycobacterium bovis hsp65 has been reported (17). Thus, at least some nonpolymorphic MHC class I molecules are conceptually appreciated as potential components of a first-line immune defense (40). Of 17 peptides representing different hsp65 sequences, none had an influence on HLA-E cell surface expression or had a tryptic digest of purified mycobacterial hsp60 (data not shown). Moreover, heat shock treatment of the X63EM cells did not alter the level of expression of HLA-E at the cell surface, although heat shock has been shown to up-regulate Qa-1 expression (17).
With regard to the peptide binding characteristics of HLA-E, it appears
that an exact positioning of the peptide’s N terminus is crucial for a
stable interaction with HLA-E. This feature was observed when peptides
were tested for the ability to stabilize an increase in HLA-E cell
surface expression of X63EM cells, obtained by preincubation of the
cells at room temperature, but was undetectable when peptides were
tested for their effects on the thermostability of soluble
HLA-E/hß2m dimers in the presence of Nonidet P-40.
Peptides with extended N termini also stabilized HLA-E on X63EM cells
at 37°C in the presence of FCS. One possible explanation might be
that longer peptides are cleaved by proteases present in FCS to produce
an N terminus suitable for binding, although how such a processing
mechanism could generate the exact peptide length in solution is
unclear. Therefore, it is not possible to judge whether the
conformation of the peptide binding cleft of HLA-E present at the cell
surface of X63EM cells at room temperature is a particularly closed one
that can only be accessed by peptides with an appropriate N terminus or
whether the addition of Nonidet P-40 to empty HLA-E/hß2m
dimers as well as culturing X63EM cells at a physiologic temperature
induce an open conformation of the peptide binding groove of HLA-E that
is accessible for peptides that protrude at either end. The second
explanation is supported by the finding that addition of Nonidet P-40
to soluble empty HLA-E/hß2m dimers in solution reduces
their thermostability (data not shown). Furthermore, it has been shown
that class I peptide dissociation kinetics vary according to the
presence or the absence of detergent in the assay (33). In contrast to
other MHC class I molecules for which the stability of the complex has
been shown to depend on the peptides’ N and C termini (41), in our
assay systems the C terminus of the peptide is of only minor importance
for the binding to HLA-E. Only when the peptide is as short as six
amino acids is its binding activity considerably diminished. The
possibility that the stabilizing effect of longer peptides on HLA-E at
the cell surface of X63EM cells is due to the presence of shortened
peptides generated by proteolysis is unlikely, since binding of BZLF-1
peptide activity gradually declines with further C-terminal truncations
of the 9 mer in a serum-free assay, and its 13 mer stabilizes HLA-E
more effectively than the 10 mer (Fig. 4). Matsumura et al. (33) also
report for H-2Kb that the exact positioning of the
N-terminal amino acid of a peptide contributes more to the binding
affinity than does correct positioning of the C-terminal amino acid of
the peptide. Computer modelling of HLA-E predicts an enlarged F pocket,
resulting from the replacement of both T-143 and W-147 by S residues
(42). It should therefore be possible for a peptide to extend the F
pocket of HLA-E. For HLA-A2, the stability of a class I-peptide complex
with the peptide’s C terminus extending the binding cleft has been
shown (43).
Recently, it has been demonstrated that HLA-E can bind nonamer peptides
contained in the leader sequences of MHC class I molecules by testing
the ability of peptides to increase the thermoresistance of
W6/32-precipitable endogenous HLA-E Ag present in lysates of the B-LCL
.221 (44). By genetic and 1D-IEF typing we found that this cell line
expresses the HLA-E*0101 allele, which differs from the alleles
(E*01031 and E*01033) we used in our study only at amino acid position
107 (R in E*0101 instead of G in E*01031 and E*01033). Braud et al.
(44) identified positions 2 and 9 as primary anchors occupied by L or M
and L, respectively, and position 7 as an auxiliary anchor. The minimum
sequences of the different peptides identified as binding to HLA-E in
this study and in the report by Braud et al. are aligned in Table II. In our experiments position 9 did not
appear to be important, since there was no considerable difference in
binding activity between the 9 mer BZLF-139–47 and the 7
mer BZLF-139–45 (Fig. 4) or between the 9 mer
InflM59–67K62 and the 8 mer InflM59–66K62.
Moreover, in the InflM peptide, position 9 is occupied by T, and in the
BZLF-1 peptide, this position can be substituted by a K without
affecting binding to HLA-E, although the latter substitution in an
HLA-A2 leader sequence-derived peptide abrogated binding. Position 2,
on the other hand, is occupied in the InflM peptide by L, one of the
anchor residues defined by Braud et al. (44), although a polar Q is
present at this position in the BZLF-1 peptide. Concerning the proposed
auxiliary anchor at position 7, the BZLF-1 and InflM peptides share
polar residues, whereas this position is occupied by a hydrophobic L in
the leader sequence-derived peptides. The dramatic reduction in binding
activity observed after truncation of position 7 (Fig. 4), on the other
hand, might indicate the importance of this position. In contrast to
Braud et al. (44), we found that position 4 shows some degree of side
chain selectivity (K, F, and P are tolerated, whereas Q is not).
Apparently, the structural requirements for peptide binding to HLA-E
cannot be answered conclusively. For example, our screening included
four peptides that contain XL/MX6L but did not stabilize
HLA-E. This might be partly due to possible dominant negative effects
on binding by amino acid residues at auxiliary anchor positions of
the peptide (45). It is also conceivable that the different HLA-E
ligands vary with regard to the anchor positions. For example,
H2-Ld, which preferentially complexes nonameric
peptides through anchors at positions 2 and 9, also binds naturally
processed 8 mers by the interaction with amino acid 7 instead of 2 as
an anchor (46, 47). The differences between our results and those
obtained by Braud et al. (44) may also reflect the influence of the
amino acid exchange at position 107 in the HLA-E allele (HLA-E*0101)
they investigated on peptide ligand specificity. This explanation seems
unlikely, since this substitution is located on the loop connecting the
first and the second ß strand of the 2 domain and thus is outside
the binding groove.
|
) (27). Thus, under steady state conditions, with the majority of
HLA-B27 being stably expressed at the cell surface, an association of
HLA-B27 and TAP is barely detectable. Since the course of transient
association of mouse TAP and HLA-B27 in X63BM cells is similar to the
TAP/HLA class I interaction in human cells, we considered the
transporter to be functional in our system with regard to peptide
loading. The possibility that the lack of HLA-E cell surface expression
in the mouse cell line might be explained by a too high affinity of
HLA-E for mouse TAP preventing its dissociation even in the presence of
appropriate peptide ligands seems unlikely as the HLA-E Ag is not
efficiently expressed on the cell surface of the human cell lines
721.221 and K562 (44) (our unpublished observations). A high degree of
association between human TAP and HLA-E has also been described in
721.221 by Braud et al. (44). However, some peptide transfer onto HLA-E
does occur, as can be estimated from the release of HLA-E from TAP
after 90-min chase, which can be inhibited by the addition of LLnL.
These data also indicate that some TAP-dependent peptide loading of
HLA-E occurs in much the same way as that reported for MHC class Ia
molecules, although interaction of MHC class I molecules with TAP does
not seem to be an absolute requirement for efficient peptide loading
(48). On RMA-S cells cotransfected with HLA-E and hß2m
the HLA-E Ag is detected at the cell surface at similar levels as on
X63EM cells (Fig. 8), indicating either that TAP is not required for
the assembly of cell membrane-expressed HLA-E or that a lack of ER
retention of HLA-E by TAP is responsible for the HLA-E cell surface
expression observed on transfected RMA-S cells. With regard to the
HLA-E peptide ligands derived from MHC class I signal sequences, a
TAP-independent loading onto HLA-E is possible, but the identical
peptides bind to Qa-1 in a TAP-dependent manner (49). Finally, it is
conceivable that the strong association of HLA-E with TAP in X63EM
cells reflects the inability of mouse TAP to supply the appropriate
peptides. Indeed, mouse TAP has been shown to display a higher peptide
selectivity than human TAP (50). At least with regard to HLA-B27, these
differences are apparently not important, since sequencing of
HLA-B27-bound peptides isolated from X63BM and human cells gave
identical results (51). In a model proposed by Sadasivan et al. (12), interaction with TAP is the last step in a series of chaperoning events leading to fully assembled class I complexes that are effectively transported to the cell surface. From these results (and since HLA-E interacts with TAP) we conclude that HLA-E molecules undergo unimpaired preceding assembly events, although the amount of HLA-E molecules metabolically labeled within 20 min in X63EM cells is much less than that of HLA-B27 in X63BM cells despite both transfectants expressing equal amounts of HLA class I mRNA (27). The observed increased detection of labeled HLA-E molecules in the presence of LLnL points to an early degradation of misfolded HLA-E molecules by a proteasome-dependent mechanism under normal circumstances, as has been reported for HLA class I molecules in mutant cells expressing either no functional TAP or ß2m (52).
Although our data imply that HLA-E can present endogenous peptides, this Ag presentation either is not very effective or is highly specific. We have no evidence that HLA-E displays a high degree of peptide selectivity; of the tested 79 peptides, two could be shown to bind to HLA-E. It is therefore likely that other factors reduce HLA-E’s ability to present peptides. In contrast to HLA-B27, HLA-E strictly depends on the presence of hß2m for its expression in mouse X63 cells (our unpublished observations). It is thus conceivable that high affinity peptides have to compensate for the low affinity of ß2m in the formation of a trimeric HLA-E complex that is transported to the cell surface. Interestingly, pulse-chase experiments demonstrated that a large quantity of the HLA-E molecules expressed in X63EM cells is retained within the ER without substantial degradation even after 4 h (27). Therefore, additional molecular mechanisms, such as the association with calnexin or calreticulin, might retain the HLA-E molecule, indicating that it plays a physiologic function within the ER, namely in the Ag presentation by MHC class Ia molecules. In this respect it might be of significance that both BZLF-1 and InflM viral peptides overlap epitopes presented by HLA-A2. Thus, HLA-E possibly operates in an ER resident peptide loading and/or trimming process.
We demonstrated that two peptides present in mature proteins of different viruses bind HLA-E in vitro. In another report leader peptides derived from sequences of MHC class I molecules were described as ligands for HLA-E (44). The intracellular processing of the HLA-E Ag is impaired, and cell surface expression is low due to insufficient peptide supply, much like H-2Ld and Qa-1. Therefore, the HLA-E Ag might have a role in vivo as a restriction element for viral peptides similar to H-2Ld and/or might function similarly to Qa-1. It should now be possible to test whether HLA-E is a restriction element for cytotoxic, virus-specific T cells. The preference of HLA-E to bind hydrophobic and signal sequence-derived peptides supports the assumption that HLA-E might be involved in the control of the T cell response by presenting TCR-derived peptides to CTL as has been implicated for Qa-1 (53).
As cell surface expression of HLA-E seems to be regulated by the cell’s class Ia expression pattern and the supply of appropriate ligands, the HLA-E Ag presented on the cell surface per se might be recognized by effector cells in a possibly peptide-independent manner. Thus, HLA-E might regulate NK cell responses. In the placenta at the feto-maternal interface, the expression of fetus-derived invariant HLA-G may prevent the attack of cytotrophoblast cells by maternally derived NK cells. Since cell surface expression of HLA-E has been reported on amnion cells (54), it is possible that HLA-E fulfills a related function.
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Acknowledgments |
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Footnotes |
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2 Present address: R. W. Johnson Pharmaceutical Research Institute, La Jolla, CA 92121.
3 Address correspondence and reprint requests to Dr. Elisabeth H. Weiss, Institut für Anthropologie und Humangenetik, Richard Wagner Strasse 10/I, 80333 Munich, Germany. E-mail address:
4 Abbreviations used in this paper: ER, endoplasmic reticulum; hß2m, human ß2-microglobulin; LLnL, N-acetyl-L-leucyl-L-leucyl-L-norleucinal; RP-HPLC, reverse phased HPLC; B-LCL, B lymphoblastoid cell line; hsp, heat shock protein.
Received for publication August 7, 1997. Accepted for publication January 5, 1998.
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37°C + 12 µg/ml hß2m) or without
(RT
