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*
Institute of Biochemistry, Swiss Federal Institute of Technology (Eidgenössiche Technische Hochschule), Zurich, Switzerland; and
Medical Research Council, Human Immunology Unit, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The pathway that yields HLA-E-binding epitopes derived from MHC class I
signal sequences is not known. During translocation of proteins through
the translocons at the endoplasmic reticulum
(ER)5 membrane, signal
sequences are usually cleaved off from the pre-protein by signal
peptidase (8). At this stage, some liberated signal
peptides are thought to span the ER membrane at their central
hydrophobic region, with the N terminus facing the cytosol and the C
terminus exposed toward the ER lumen (9). The conserved
HLA-E binding epitope of polymorphic MHC class I molecules is located
in the N-terminal portion of their signal sequences (see Fig. 1
A). We therefore hypothesized
that the liberated N-terminal MHC class I signal peptides are
initially released from the ER membrane toward the cytosol. This model
would fit with the requirement of a functional TAP transporter for
HLA-E cell surface expression (2, 10).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The SalI/HindIII fragment of
pBK-CMV/HLA-A*0301 was transferred into pSV-Sport1 (Life Technologies,
Carlsbad, CA) under the control of the SP6 promoter to give
pSV-Sport1/HLA-A*0301. To generate the signal sequence mutant
(HLA-Aspmt, see Fig. 2 for sequence),
codons 14, 15, 16, 18, and 20 of the coding region were exchanged by
the Quick Change Site-Directed Mutagenesis kit (Stratagene, La Jolla,
CA) using the sense primer
5'-CTCCTCCTGCTACTCGTGGTGCTCCTGCTCCTGCGCCAGACCTGGGCGGG-3' and the
antisense primer
5'-CCCGCCCAGGTCTGGCGCAGGAGCAGGAGCACCACGAGTAGCAGGAGGAG-3'. It
resulted in pSV-Sport1/HLA-Aspmt. To generate a mutant of HLA-A*0301
with an extended signal peptide (HLA-Aspext, see
Fig. 1
for sequence) the coding region of pSV-Sport1/HLA-A*0301 was
amplified by PCR using the sense primer
5'-AGTCAGGTCGACCATGGGCAAGAACAGCAAGGTGGCCGTCATGGCGCCCCG-3',
which included a SalI restriction site and codons for the
six additional amino acids (underlined). A standard T7 primer was used
as reverse primer. The SalI/HindIII fragment of
the resulting PCR product was transferred into pSV-Sport1 to generate
pSV-Sport1/HLA-Aspext. For stable transfections,
the insert of pSV-Sport1/HLA-Aspmt was subcloned into the
BamHI restriction site of pCDNA3 (Invitrogen, San
Diego, CA).
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To prepare mRNA coding for HLA-A*0301/124, HLA-Aspmt/124, and the signal peptides of HLA-A*0301, HLA-Aspmt, and HLA- Aspext, the respective coding region was amplified with PCR using Pfu DNA polymerase (Stratagene), SP6 primer, and a reverse primer, starting with 5'-NNNNNNNNNCTA, to introduce a TAG stop codon at the desired position. PCR-amplified DNA fragments were transcribed in vitro with SP6 RNA polymerase at 42°C in the presence of 500 µM m7G(5')ppp(5')G CAP analog (New England Biolabs, Beverly, MA) (12).
Translations of mRNA coding for HLA-A*0301/124 and HLA-Aspmt/124 were performed in 25 µl of reticulocyte lysate (Promega, Madison, WI) containing [35S]methionine (Amersham Pharmacia Biotech, Little Chalfont, U.K.) and, where indicated, two equivalents of nuclease treated rough microsomes prepared from dog pancreas and N-glycosylation inhibitor N-benzoyl-Asn-Leu-Thr-methyamide (13) and 5 µM (Z-LL)2-ketone (11). Samples were incubated for 15 min at 30°C. Microsomes were extracted with 500 mM KOAc and prepared for SDS-PAGE as described previously (11). For extraction with alkali, KOAc-extracted microsomes were treated with 100 mM Na2CO3, pH 11.3 (13). Translations of mRNA coding for the signal peptides were translated in 25 µl of wheat germ extract at 25°C for 15 min (13).
Signal peptide processing with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate-solubilized microsomal membrane proteins was performed as described elsewhere (11), except that the signal peptidase inhibitor N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone (250 µM; Sigma-Aldrich, St. Louis, MO) was added to the reaction mixture to prevent cleavage of the HLA-A signal peptides at a cryptic signal peptidase cleavage site.
Electrophoresis
Proteins and peptides were analyzed by SDS-PAGE using Tris-bicine gels (14). Membrane pellets and proteins precipitated with (NH4)2SO4 or trichloroacetic acid were dissolved in sample buffer containing 360 mM bis-Tris, 160 mM bicine, 1% SDS, 50 mM DTT, 15% sucrose, and 0.004% Serva blue. All samples were incubated for 15 min at 65°C. Proteins were finally separated on 15% T, 5% C, 8 M urea acrylamide gels (70 x 80 x 1 mm). Labeled proteins were visualized by a STORM PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
HLA-E cell surface expression
To prepare stably transfected cells expressing mutant HLA-A*0301 (HLA-Aspmt), 721.221 were electroporated with 30 µg of the respective plasmid DNA at 270 V with a capacitance of 1500 µF (15). Stable transfected clones were obtained after 3 wk. Surface expression of HLA-A*0301 and HLA-E was monitored by flow cytometry using GAP-A3 and DT9 Abs followed by PE-labeled anti-mouse (Fab')2 (Sigma-Aldrich) (2).
TAP transport
721.221 cells were permeabilized according to Jadot et al. (16), except that digitonin (0.006%) was used instead of saponin. For TAP transport (17), 3 x 105 permeabilized cells were incubated in 25 µl of 50 mM HEPES-KOH (pH 7.6), 150 mM KOAc, 5 mM Mg(OAc)2, 250 mM sucrose, and 1 mM DTT, and 60 nM 125I-labeled RRYQNSTEL (9 Ci/mmol) and 2 µl of ATP mix (12.5 mM ATP, 3.5 U/µl creatine kinase, and 110 mM creatine phosphate). ATP was depleted by the addition of 0.3 U of hexokinase and 20 µmol of glucose during the assay. After the reaction, salt concentration was raised to 500 mM KOAc, and cells were separated by a 3-min centrifugation through a 100-µl sucrose cushion (50 mM HEPES-KOH (pH 7.6), 500 mM KOAc, 2 mM MgOAc2, and 500 mM sucrose) at 48,000 rpm and 4°C in a Beckman TLA100 rotor (Beckman Coulter, Fullerton, CA). 125I-Labeled peptide in the membrane fraction was quantified by gamma-counting and analyzed by SDS-PAGE as previously described (11). All values were determined by three independent assays.
Peptide binding to HLA-E
HLA-E-binding assays were conducted as described previously (1). 721.221 cells were starved for 60 min in methionine-free medium followed by labeling with 100 µCi of [35S]methionine (Amersham Pharmacia Biotech.) per 107 cells for 1 h. Cells were then washed in ice-cold PBS and lysed for 20 min at 4°C in lysis buffer (40 mM Tris-HCl (pH 7.6), 5 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40, 5 mM iodoacetamide, and 2 mM PMSF) in the presence or absence of the indicated concentrations of peptide or with the conformation-dependent Ab W6/32. Supernatants were heated for 2 min at 44°C before preclearing by the addition of 10% Staphylococcus aureus cells (Pansorbin; Calbiochem, San Diego, CA) overnight. Peptide-HLA-E complexes were recovered by immunoprecipitation using W6/32 and protein A-Sepharose beads (Sigma-Aldrich). Samples were analyzed by 1D-IEF (18) followed by autoradiography. Peptide binding was quantified by densitometry of the HLA-E H chain band using a FLA-2000 Image Analyzer (Raytek Scientific, Sheffield, U.K.). Results are expressed as a ratio of ODs (ODpeptide - ODPBS/ODW6/32 - ODPBS) x 100 and referred to as a percentage of binding to HLA-E. Each peptide was tested in three independent assays.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although MHC class I molecules are highly polymorphic, their
signal sequences are relatively conserved. This is consistent with
their role in providing a conserved peptide to HLA-E. To characterize
how these peptides are generated, we investigated in vitro the fate of
a representative MHC class I molecule, HLA-A*0301. mRNA coding for the
signal sequence plus 100 additional residues of HLA-A*0301 (HLA-A/124)
was translated in reticulocyte lysates in the presence of ER-derived
rough microsomes (Fig. 1B). Microsomes were subsequently
isolated and analyzed for
[35S]methionine-labeled translation products.
As expected, HLA-A/124 was translocated into the microsomes and yielded
HLA-A/100 after cleavage of the signal sequence (Fig. 1B,
lane 2). The 24-residue-long signal peptide was not found in
the membrane fraction, suggesting it had been further processed.
We have recently reported that in vitro the liberated signal peptide of
p-Prl was rapidly processed by SPPase, and signal peptide fragments
were released from the membrane. However, when SPPase was inhibited by
the new specific inhibitor (Z-LL)2-ketone, the
unprocessed signal peptide remained associated with the microsomes
(11). To test whether SPPase was also involved in the
processing of MHC signal sequences, the SPPase inhibitor was added to a
reaction with HLA-A/124. The signal peptide of HLA-A*0301 was found to
be associated with the microsomes in the presence of the SPPase
inhibitor (Fig. 1B, lane 3). Extraction with
sodium carbonate revealed a tight interaction of the signal peptide
with the lipid bilayer similar to typical transmembrane proteins (Fig. 1B, lanes 4 and 5) (19).
These findings indicate that the HLA-A*0301 signal peptide liberated
from the pre-protein is further processed by SPPase, inducing the
release of signal peptide fragments from the membrane.
In an attempt to locate the SPPase cleavage site, a protease assay was
performed with in vitro-translated signal peptide and
detergent-solubilized, partially purified SPPase (11). The
HLA-A*0301 signal peptide was cleaved (Fig. 1C, lane
2), but cleavage products were not detected, most likely because
the expected short peptide could not be fixed on the gel. When
(Z-LL)2-ketone was added, processing of the
signal peptide was inhibited (Fig. 1C, lane 3).
To identify cleavage products on the gel, an extended signal peptide
(SPext) with six additional residues
(MGKNSKVAVM...) at the N terminus was applied in the
assay. SPext was processed by SPPase
like the wild-type (wt) signal peptide (Fig. 1C, lane
5). The observed cleavage product, which was labeled by the
methionine residues in the N-terminal portion, had an electrophoretic
mobility similar to a peptide corresponding to the N-terminal 20
residues of SPext (Fig. 1C, lane
7). This result indicates that SPPase cleaves the peptide in the
center of the hydrophobic region where the helix-breaking serine and
glycine residues are located (Fig. 1A). The result is
consistent with the previous finding that the signal peptide of p-Prl
is cleaved by SPPase in the center of the hydrophobic region at the
helix-breaking serine and asparagine residues (Fig. 1A)
(11).
The generation of HLA-E epitopes requires signal peptide processing by SPPase
To test whether signal peptide processing by SPPase is an
essential step in HLA-E epitope generation, we prepared an HLA-A*0301
signal sequence mutant (HLA-Aspmt) that cannot be processed by SPPase.
Systematic studies with p-Prl and HLA-A*0301 signal sequence mutants
revealed that positively charged residues flanking the hydrophobic core
inhibit signal peptide processing, and central helix-breaking residues
are essential for proteolysis in the transmembrane region (M. K.
Lemberg and B. Martoglio, unpublished data). The helix-breaking motif
in the center and the small residues in the C-terminal portion of the
hydrophobic region were therefore replaced by amino acids with long
hydrophobic side chains and an arginine was introduced at the end (Fig. 2A). In the in vitro translation/translocation experiment,
HLA-Aspmt/124 was translocated into microsomes and cleaved by signal
peptidase like wt HLA-A/124 (Fig. 2B, lane 2).
However, the liberated signal peptide of HLA-Aspmt was not processed by
SPPase and remained entirely associated with the membrane in a
carbonate-resistant manner (Fig. 2B, lanes
2–5).
To test whether the mutation of the HLA-A*0301 signal sequence affects
HLA-E cell surface expression, the HLA-A-, -B-, -C-, and -G-negative
cells 721.221 were stably transfected and selected to express an
identical level of either wt HLA-A*0301 or mutant HLA-Aspmt (Fig. 3) (20). HLA-E surface
expression, as measured by flow cytometry using the mAb DT9
(2), was only observed with cells expressing wt HLA-A*0301
(Fig. 3). HLA-E surface expression could not be detected with cells
expressing HLA-Aspmt, whose signal peptide cannot be processed by
SPPase (Fig. 3). Cleavage by SPPase in the C-terminal portion of the
hydrophobic region is therefore essential for the generation of
HLA-E-binding epitopes.
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HLA-E presents nonameric peptides derived from the signal sequence
of classical HLA molecules, e.g., residues -22 to -14 of HLA-A*0301
(Fig. 1A) (1, 2, 10). To generate the nonamer
from the 
14-residue-long N-terminal signal peptide fragment produced
by SPPase, the N and C termini have to be trimmed either before or
after the peptide binds to HLA-E. HLA-E expression has been found to be
TAP dependent (2, 10), suggesting that the N-terminal
signal peptide portion of HLA-A*0301 is released toward the
cytosol.
To assess where the trimming occurs, a series of truncated N-terminal
HLA-A*0301 signal peptide fragments was synthesized and their transport
by TAP and binding to HLA-E were tested. TAP transport into
digitonin-permeabilized 721.221 cells was assayed using the
125I-labeled reporter peptide RRYQNSTEL with
unlabeled synthetic HLA-A*0301 signal peptide fragments as competitors
(Fig. 4A) (17).
The peptide corresponding to the nonameric epitope (VMAPRTLLL) was
transported most efficiently and competed transport of the reporter
peptide with an IC50 value of 0.4 µM (Fig. 4B). With the exception of the 14-residue-long signal
peptide fragment ending with a serine, all other peptides tested were
transported efficiently as well and reached IC50
values of 0.7–3 µM in the competition assay (Fig. 4B).
The slightly reduced transport efficiency of the 14-mer
(IC50 8 µM) is most likely due to the
C-terminal serine residue, which is known to reduce the binding
affinity of peptides to TAP (21, 22). These results
indicate that potential N-terminal signal peptide fragments of
HLA-A*0301 are all good substrates for TAP, but N- and C-terminal
trimming can increase the efficiency of transport.
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The synthetic HLA-A*0301 signal peptide fragments were next tested
for binding to HLA-E as determined by stabilization of HLA-E molecules
in cell lysates (Fig. 5)
(1). Extension by two residues at the N terminus of the
nonamer peptide did not significantly affect peptide binding affinity
(Fig. 5, peptide VII). By contrast, extension at the C terminus did.
Peptides with additional residues at the C terminus bound to HLA-E at
30 µM as previously shown (1). However, binding at the
lower concentrations of 3 and 0.3 µM (Fig. 5B, peptides
III–VI) was not significantly higher than binding of the negative
control VTAPRTLLL, the epitope known to fail to up-regulate HLA-E at
the cell surface (Fig. 5, peptide II) (1, 2). These
results show that the expected 14-residue-long N-terminal HLA-A*0301
signal peptide fragment produced by SPPase has to be trimmed at its C
terminus for efficient binding to HLA-E. Nevertheless, because the
nontrimmed peptide can bind to HLA-E with low affinity, it remains
open, whether some trimming can occur after binding and during
transport of the peptide-HLA-E complex to the cell surface, as it was
described for other MHC class I Ags (23).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The functions of a signal sequence in protein targeting and membrane insertion are well established (24) but the fate of signal peptides after they have been cleaved off from the pre-protein by signal peptidase is hardly understood (25). As shown here in the in vitro translation/translocation system, the liberated signal peptide is anchored in the microsomal membrane in a carbonate-resistant manner, like a membrane protein. Cleavage within the hydrophobic transmembrane region by the SPPase promotes the release of signal peptide fragments from the lipid bilayer. This mechanism is reminiscent of the regulated intramembrane proteolysis described for an increasing number of eukaryotic or prokaryotic membrane proteins involved in a variety of cellular pathways (for review, see Refs. 26, 27).
In higher organisms, MHC class I molecules present 8–10 residue peptides on the surface of virtually every nucleated cell, where they can serve as target Ags for cytotoxic T lymphocytes (28). The major proteolytic activities required for the generation of these peptides are the proteasome in the cytosol for protein fragmentation, and in some cases aminopeptidases in the cytosol or ER lumen for peptide trimming (29, 30, 31, 32, 33, 34). Although it is possible to prevent the generation of most epitopes through the use of proteasome inhibitors (35, 36), others remain resistant to their effects. This suggested that nonproteasomal proteases might be responsible for the generation of a fraction of MHC class I ligands (37, 38, 39). It is speculated that the proteasome is not involved in the generation of HLA-E-binding peptides, as far as it can be deduced from experiments with the mouse functional homologue of HLA-E, Qa-1 (40). However, we show that the SPPase is required to release the peptide fragment containing the HLA-E epitope from the ER membrane. The peptide requires further trimming at both N and C termini to produce the nonamer epitope. Because extension at the C terminus dramatically reduces the peptide-binding affinity to HLA-E, it is likely that C-terminal trimming occurs in the cytosol. This would be consistent with previous reports suggesting that the generation of the Qdm peptide binding to Qa-1 involves cytosolic C-terminal trimming (41) and that ER-resident proteases can trim peptides at their N terminus and not at the C terminus (31, 32, 33, 36, 42). Conversely, N-terminal trimming could occur in the cytosol or the ER and may even take place after binding of the peptide to HLA-E since N-terminal extensions do not appear to affect peptide binding (30, 31, 32, 33, 36, 43).
TAP is also required for the cell surface expression of signal peptide-derived HLA-E epitopes (2, 10). TAP dependency is consistent with the results presented here, which indicate that SPPase promotes the release of the epitope-containing signal peptide portion from the ER membrane toward the cytosol. SPPase apparently produces TAP substrates from membrane-anchored signal peptides in analogy to the proteasome, which produces TAP substrates from cytosolic proteins (29). The generation of MHC class I epitopes via signal peptide processing may thus be an alternative route to the more common proteasome-dependent pathway of epitope production, and may guarantee a close correlation between the number of HLA-E-peptide complexes and synthesized MHC class I molecules. One aspect that still needs to be investigated is whether SPPase can generate both TAP-dependent and TAP-independent signal peptide fragments capable of binding to MHC class I molecules. Interestingly, removing the charged residue (Arg at position 7) in the N-terminal region of the mouse HLA class I signal sequence alters its insertion and induces TAP-independent presentation of the Qdm peptide (41, 44). The human cytomegalovirus glycoprotein UL40 (HCMV gpUL40) also provides such signal peptide in a TAP-independent manner (45, 46). Preliminary studies have however ruled out a role for SPPase in its generation (V. M. Braud, B. Martoglio, and collaborators, manuscript in preparation). It also remains to determine whether SPPase cleaves only a discrete number of signal peptides, which may have specific properties and functions beyond processing, or whether signal peptide processing is part of a default pathway to clear the ER membrane from the unwanted peptides by analogy to the involvement of the proteasome in the clearance of defective ribosomal products (47). The latter function seems more likely, but stresses the question of the fate of the released peptides. Are signal peptide fragments released into the cytosol generally substrates for TAP, as proposed above, or are they substrates for cytosolic proteases? Is there a selection, for example, HLA-E epitope-containing peptides and how are liberated signal peptides protected from degradation? One can speculate that chaperone molecules may be involved in such a process (25). Clearly the detailed characterization of the SPPase responsible for the cleavage of signal sequences and its role in peptide fragmentation and epitope generation will be a challenge for the future.
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Acknowledgments |
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Footnotes |
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2 M.K.L. and F.A.B. contributed equally to this work.
3 Current address: Institut de Pharmacologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, 06560 Valbonne, France.
4 Address correspondence and reprint requests to Dr. Bruno Martoglio, Institute of Biochemistry, Swiss Federal Institute of Technology (Eidgenössiche Technische Hochschule), 8092 Zurich, Switzerland. E-mail address: bruno.martoglio{at}bc.biol.ethz.ch
5 Abbreviations used in this paper: ER, endoplasmic reticulum; p-Prl, preprolactin; SPPase, signal peptide peptidase; wt, wild type.
Received for publication August 1, 2001. Accepted for publication October 4, 2001.
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 K. Okamoto, K. Moriishi, T. Miyamura, and Y. Matsuura Intramembrane Proteolysis and Endoplasmic Reticulum Retention of Hepatitis C Virus Core Protein J. Virol., June 15, 2004; 78(12): 6370 - 6380. [Abstract] [Full Text] [PDF]
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A. C. Nyborg, A. Y. Kornilova, K. Jansen, T. B. Ladd, M. S. Wolfe, and T. E. Golde Signal Peptide Peptidase Forms a Homodimer That Is Labeled by an Active Site-directed {gamma}-Secretase Inhibitor J. Biol. Chem., April 9, 2004; 279(15): 15153 - 15160. [Abstract] [Full Text] [PDF]
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 T. Kambayashi, J. R. Kraft-Leavy, J. G. Dauner, B. A. Sullivan, O. Laur, and P. E. Jensen The Nonclassical MHC Class I Molecule Qa-1 Forms Unstable Peptide Complexes J. Immunol., February 1, 2004; 172(3): 1661 - 1669. [Abstract] [Full Text] [PDF]
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 B. Martoglio and T. E. Golde Intramembrane-cleaving aspartic proteases and disease: presenilins, signal peptide peptidase and their homologs Hum. Mol. Genet., October 15, 2003; 12(90002): R201 - 206. [Abstract] [Full Text] [PDF]
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F. A. Bland, M. K. Lemberg, A. J. McMichael, B. Martoglio, and V. M. Braud Requirement of the Proteasome for the Trimming of Signal Peptide-derived Epitopes Presented by the Nonclassical Major Histocompatibility Complex Class I Molecule HLA-E J. Biol. Chem., September 5, 2003; 278(36): 33747 - 33752. [Abstract] [Full Text] [PDF]
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J. D. Miller, D. A. Weber, C. Ibegbu, J. Pohl, J. D. Altman, and P. E. Jensen Analysis of HLA-E Peptide-Binding Specificity and Contact Residues in Bound Peptide Required for Recognition by CD94/NKG2 J. Immunol., August 1, 2003; 171(3): 1369 - 1375. [Abstract] [Full Text] [PDF]
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A. Weihofen, M. K. Lemberg, E. Friedmann, H. Rueeger, A. Schmitz, P. Paganetti, G. Rovelli, and B. Martoglio Targeting Presenilin-type Aspartic Protease Signal Peptide Peptidase with gamma -Secretase Inhibitors J. Biol. Chem., May 2, 2003; 278(19): 16528 - 16533. [Abstract] [Full Text] [PDF]
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D. C. Smith, A. Gallimore, E. Jones, B. Roberts, J. M. Lord, E. Deeks, V. Cerundolo, and L. M. Roberts Exogenous Peptides Delivered by Ricin Require Processing by Signal Peptidase for Transporter Associated with Antigen Processing-Independent MHC Class I-Restricted Presentation J. Immunol., July 1, 2002; 169(1): 99 - 107. [Abstract] [Full Text] [PDF]
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A. Weihofen, K. Binns, M. K. Lemberg, K. Ashman, and B. Martoglio Identification of Signal Peptide Peptidase, a Presenilin-Type Aspartic Protease Science, June 21, 2002; 296(5576): 2215 - 2218. [Abstract] [Full Text] [PDF]
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),
MAVMAPRTLLLLL (
), MAVMAPRTLLLL (
), MAVMAPRTLLL (
), and
VMAPRTLLL (
). Values of transport with ATP and without ATP,
respectively, are indicated in the bar graph.