I-Restricted Presentation Processing

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This information is current as
of February 11, 2017.
Exogenous Peptides Delivered by Ricin
Require Processing by Signal Peptidase for
Transporter Associated with Antigen
Processing-Independent MHC Class
I-Restricted Presentation
Daniel C. Smith, Awen Gallimore, Emma Jones, Brenda
Roberts, J. Michael Lord, Emma Deeks, Vincenzo Cerundolo
and Lynne M. Roberts
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Copyright © 2002 by The American Association of
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J Immunol 2002; 169:99-107; ;
doi: 10.4049/jimmunol.169.1.99
http://www.jimmunol.org/content/169/1/99
The Journal of Immunology
Exogenous Peptides Delivered by Ricin Require Processing by
Signal Peptidase for Transporter Associated with Antigen
Processing-Independent MHC Class I-Restricted Presentation1
Daniel C. Smith,* Awen Gallimore,† Emma Jones,† Brenda Roberts,* J. Michael Lord,*
Emma Deeks,* Vincenzo Cerundolo,† and Lynne M. Roberts2*
rotective CD8⫹ CTL that recognize viral or tumor peptides in association with MHC class I are deemed valuable
in controlling pathogen spread and, potentially, in reducing tumor progression. In recent years, several avenues have been
investigated to pursue the challenge of artificially inducing protective CTL (1–3). These methods have used both live and nonlive vectors to deliver Ags to the cytosol for subsequent presentation of peptides that correspond to epitopes recognized by CTL.
Indeed, several protein toxins constitute a class of non-live vectors
whose intracellular trafficking and/or membrane translocation abilities can be exploited to allow delivery of peptides for the induction of a CTL response (4 –9).
In a previous study we showed that the A subunit of a
catalytically defective Shiga-like toxin 1 (SLT1)3 could be
engineered so that the holotoxin could successfully deliver a
viral peptide into the conventional MHC class I processing and
P
*Department of Biological Sciences, University of Warwick, Coventry, United Kingdom; and †Molecular Immunology Group, Nuffield Department of Medicine, John
Radcliffe Hospital, Oxford, United Kingdom
Received for publication January 17, 2002. Accepted for publication April 29, 2002.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by a Wellcome Trust Program Grant and a U.K. Biotechnology and Biological Sciences Research Council project grant (to L.M.R. and
J.M.L.), a grant from the Wellcome Trust (to A.G.), and a grant from Cancer Research
U.K. (to V.C.).
2
Address correspondence and reprint requests to Dr. Lynne M. Roberts, Department
of Biological Sciences, University of Warwick, Coventry CV4 7AL, U.K. E-mail
address: [email protected]
3
Abbreviations used in this paper: SLT1, Shiga-like toxin 1; ER, endoplasmic reticulum; ERAD, ER-associated protein degradation; EndoH, Endo-␤-N-glucosaminidase H; RTA, ricin A chain; RTB, ricin B chain; BFA, brefeldin A; LCMV, lymphocytic choriomeningitis virus; NP, peptide 366 –374 of the influenza nucleoprotein;
gp33, peptide 33– 41 of the LCMV glycoprotein.
Copyright © 2002 by The American Association of Immunologists, Inc.
presentation pathway (7). However, due to the restricted cellular expression pattern of CD77 (10), the receptor for SLT1, the
use of this protein as a generalized delivery vehicle may be
limited. By contrast, the plant cytotoxin ricin has the capacity to
bind to and be endocytosed into a vast number of different cell
types (11). Ricin, like SLT1, has an unusual endocytic routing
that takes it from the cell surface via the Golgi complex to the
endoplasmic reticulum (ER) lumen (12–14). In a productive
intoxication of the cell, the fraction of toxin that reaches the ER
is somehow perceived as a substrate for ER-associated protein
degradation (ERAD). This arm of the ER quality control process normally apprehends newly synthesized but aberrant proteins or orphan subunits of oligomers, and triggers their passage
through Sec61 channels to the cytosol. In a tightly coupled
sequence of events, such dislocated proteins are normally deglycosylated, certain lysyl residues become ubiquitinated, and
the protein is then degraded by proteasomes (reviewed in Ref.
15). In the ER lumen, the catalytic subunit of ricin, though
structurally native and endocytosed from the exterior of the cell,
appears able to exploit this process to enter the cytosol (16).
From our understanding of these events, we reasoned that genetic fusions of immunodominant epitopes with toxins such as
SLT1 and ricin could be introduced directly into the MHC class I
processing pathway after their retrotranslocation to the cytosol
from the ER. In the present study we have investigated the potential of using a disarmed version of ricin to deliver exogenous
epitopes and found that, in contrast to SLT1 and most other toxin
carriers, delivery and presentation of viral peptides is not dependent
on proteasome activity or TAP. We present evidence for release of
viral peptides from the toxin carrier by ER signal peptidase due to the
presence of a potential signal peptidase cleavage site in the surfaceexposed N-terminal segment of mature ricin A chain (RTA). Therefore, use of engineered ricin may be of particular value in delivering
0022-1767/02/$02.00
In this study we demonstrate that a disarmed version of the cytotoxin ricin can deliver exogenous CD8ⴙ T cell epitopes into the
MHC class I-restricted pathway by a TAP-independent, signal peptidase-dependent pathway. Defined viral peptide epitopes
genetically fused to the N terminus of an attenuated ricin A subunit (RTA) that was reassociated with its partner B subunit were
able to reach the early secretory pathway of sensitive cells, including TAP-deficient cells. Successful processing and presentation
by MHC class I proteins was not dependent on proteasome activity or on recycling of MHC class I proteins, but rather on a
functional secretory pathway. Our results demonstrated a role for signal peptidase in the generation of peptide epitopes associated
at the amino terminus of RTA. We showed, first, that potential signal peptide cleavage sites located toward the N terminus of RTA
can be posttranslationally cleaved by signal peptidase and, second, that mutation of one of these sites led to a loss of peptide
presentation. These results identify a novel MHC class I presentation pathway that exploits the ability of toxins to reach the lumen
of the endoplasmic reticulum by retrograde transport, and suggest a role for endoplasmic reticulum signal peptidase in the
processing and presentation of MHC class I peptides. Because TAP-negative cells can be sensitized for CTL killing following
retrograde transport of toxin-linked peptides, application of these results has direct implications for the development of novel
vaccination strategies. The Journal of Immunology, 2002, 169: 99 –107.
100
TAP-INDEPENDENT DELIVERY OF PEPTIDE USING RICIN VECTOR
peptides to MHC class I molecules in TAP-deficient cells, e.g., in
tumors where there is a down-regulation of TAP (17).
Materials and Methods
Reagents
Synthetic peptides ASNENMDAM (peptide 366 –374 of the influenza
nucleoprotein (NP)) and KAVYNFATC (LCMV glycoprotein (gp33)), encompassing the 366 –374 residues of influenza nucleoprotein and the
33– 41 residues of lymphocytic choriomeningitis virus (LCMV), respectively, were obtained from Alta Bioscience (Birmingham, U.K.) and stored
at ⫺20°C in PBS. Lactacystin (Calbiochem, La Jolla, CA), brefeldin A
(BFA; Sigma, Poole, U.K.), chloroquine (Sigma), decanoyl-Arg-Val-LysArg-chloromethylketone (Bachem, St. Helens, U.K.) were used for inhibition studies at the concentrations indicated in the figures.
Cell lines
Production of recombinant RTA-peptide fusions
DNAs encoding nNP-RTA and nNP-6K A chain proteins were constructed
by PCR mutagenesis using synthetic oligonucleotides containing the mutations of choice in an overall PCR strategy as described previously (7).
This resulted in the fusion of the NP sequence (encoding residues 366 –374
of the influenza nucleoprotein) to the mature 5⬘ termini of two RTA cDNA
variants encoding RTAR180H (18) and RTA6K-R180H (19), respectively. The
claNP-RTA was constructed by inserting annealed complementary oligonucleotides encoding the NP sequence into the ClaI restriction site (at base
position ⫹338) in the RTA cDNA (20). PCR-directed mutagenesis was
used, as described previously (7), to introduce arginyl point mutations
into the DNA coding for the nNP-6K RTA variant. All variants were
fully sequenced before expression and fusion constructs were then ligated into a pUC18 vector modified to optimize RTA expression in
Escherichia coli (21).
Expression and purification of RTA fusions and reassociation
into ricin
Recombinant RTA fusions were expressed in a 1-L culture of E. coli
JM101 cells and purified by cation exchange chromatography as previously
described (18). The purified fusions were reassociated to plant-derived ricin B chain (RTB; Vector Laboratories, Burlingame, CA) by dialysis
against PBS, following mixing 100 ␮g of each chain in the presence of the
reducing agent 2-ME (2% in PBS containing 100 mM lactose), as described previously (22).
EndoH treatment
EndoH (Boehringer Mannheim, Indianapolis, IN) digestions were performed by resuspending the pellets of protein A-Sepharose-bound immunoprecipitates in 30 ␮l of 50 mM sodium citrate buffer (pH 5.5) containing
0.2% SDS and heating to 95°C for 5 min before addition of 3 U (1.5 ␮l)
of EndoH and incubation overnight at 37°C.
ER import assays
In vitro transcription of mRNAs and translation in rabbit reticylocyte lysate
(Promega, Madison, WI), in the presence or absence of canine pancreatic
microsomes, was performed as described (24). Cotranslational import into
microsomes and cleavage by signal peptidase was determined after a 40min posttranslational treatment with 0.5 mg/ml proteinase K on ice (⫾ 1%
Nonidet P-40) and visualizing bands by SDS-PAGE and fluorography.
Results
Ricin is a heterodimeric protein composed of two subunits, RTA
and RTB, that are normally disulfide bonded together. RTA has a
rRNA-specific N-glycosidase activity and is therefore capable of
inactivating cytosolic ribosomes should it encounter them, while
RTB is a galactose-specific lectin able to bind the holotoxin to the
target cell surface before endocytic uptake. We created genetic
fusions of DNA for the H-2Db-restricted peptides, influenza nucleoprotein NP 366 –374 peptide (ASNENMDAM) (25), or
LCMV glycoprotein 1 (gp33) peptide, residues 33– 41 (KAVYN
FATC) (25), to either the 5⬘ end of the cDNA encoding mature
RTA (20) or spliced into an internal ClaI site that permits exposure
on the toxin surface (26) or onto the 3⬘end (data not shown). These
proteins were expressed in and purified from E. coli as described
previously (18) before being reassociated with glycosylated RTB
purified from Ricinus communis (22). For most experiments, a catalytic site mutant (R180H) was used (18), and in one case a variant
NP-ricin fusion was made using a lysine-enriched RTAR180H that
is known to be more effectively degraded by proteasomes (19).
The NP fusion proteins are schematically depicted in Fig. 1.
Ag presentation assays
Cells were pulsed with ricin fusions at the concentrations and durations
stated in the text. Target cells (2 ⫻ 106) were radiolabeled with 100 ␮Ci of
51
Cr for 2 h. Target cells were infected with 5 PFU/cell of recombinant
vaccinia virus expressing the relevant viral protein for 90 min before labeling, or pulsed with 500 nM free peptide (NP or gp33) for 2 h during the
51
Cr labeling. Labeled cells were plated at 104 cells/well and CTL were
mixed with the targets at the E:T ratios shown in the figures. Supernatants
were harvested after 4 h. The percentage of specific lysis was calculated as
described previously (7). To study the effect of various inhibitors and reagents on the processing, cells were treated at the concentrations and durations stated in the figures before performing the Ag presentation assay.
Immunoprecipitations
RMA-S cells (20 ⫻ 106) were treated with toxin (500 ng/ml) or mocktreated for 90 min at 37°C. Cells were washed and starved for 60 min in 1
ml methionine-free RPMI 1640 medium supplemented with 10% (v/v)
FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 ␮g/ml streptomycin
at 37°C. They were then metabolically labeled in 500 ␮l fresh methioninefree RPMI 1640 medium with L-[35S]methionine (Amersham Pharmacia
Biotech, Little Chalfont, U.K.) at a concentration of 10 ␮Ci/␮l for 30 min
FIGURE 1. Schematic representation of the ricin fusion proteins. A
MHC class I-restricted antigenic peptide (NP) corresponding to residues
366 –374 (ASNENMDAM) from the influenza nucleoprotein was made as
a set of recombinant fusions with RTA. Three different fusion proteins are
shown, in all of which RTA contained an active site mutation (R180H).
The purified recombinant fusions were associated to plant-derived RTB.
RMA cells and the corresponding TAP-deficient RMA-S cell line, both
derived from a Rauscher virus-induced T cell lymphoma, and MC57 cells,
a C57BL/6-derived fibrosarcoma, were grown in RPMI 1640 (Life Technologies, Rockville, MD) containing 10% (v/v) FCS, 2 mM L-glutamine,
100 U/ml penicillin, and 100 ␮g/ml streptomycin. For IFN-␥ stimulation,
cells were incubated in the presence of 10 U/ml murine rIFN-␥ (PeproTech, London, U.K.) for 48 h. CTL lines were maintained in RPMI 1640
containing 10% (v/v) FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100
␮g/ml streptomycin, 1 mM sodium pyruvate, and 50 ␮M 2-ME by weekly
restimulation using peptide-pulsed spleen cells.
at 37°C. Cells were lysed for 30 min on ice in lysis buffer (50 mM Tris-HCl
(pH 7.5) containing 1% (v/v) Triton X-100 and 150 mM NaCl), and their
nuclei were removed by centrifugation for 15 min at 15,000 ⫻ g. For
aliquots to be lysed in the presence of stabilizing peptide, 10 ␮M NP366 –374
was added at this stage. Cell lysates were precleared overnight with 100 ␮l
fixed Staphylococcus aureus (Sigma) in PBS, then incubated with the B22
H-2Db conformational-specific Ab (23) for 90 min before adding a small
volume of protein A-Sepharose for 45 min. All steps were performed at
4°C. Immunoprecipitates were washed three times in lysis buffer and subjected to Endo-␤-N-glucosaminidose H (EndoH) digestion overnight (see
EndoH treatment) and resolved by SDS-PAGE. Fluorographs were analyzed using a densitometer and ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
101
FIGURE 2. Ricin-mediated delivery of the
NP366 –374 results in specific lysis by NP-restricted CTL. NP or gp33-specific CTL were
tested for their ability to lyse target cells
treated as indicated along the x-axis. MC57
target cells, with or without IFN-␥, were
pulsed with control peptides, infected with
vaccinia virus-expressing full-length Ags, or
treated with the NP-ricin fusions. The percentage of specific lysis at three E:T ratios is
shown. The data are representative of at least
two similar experiments.
the endosomal system becomes processed by endoprotease(s) to
release the NP peptide such that it is able to interact with either
surface recycling MHC class I proteins (28) or with MHC class I
complexes that are being secreted. To investigate the possibility of
ricin fusions being processed in the early endocytic pathway, we
first studied the effect of inhibiting the ubiquitous furin-related
serine proteases of the subtilisin family using the competitor inhibitor
decanoyl-Arg-Val-Lys-Arg-chloromethylketone-CMK. Members of
the furin family are frequently involved in activating proproteins in
FIGURE 3. Presentation of ricin-delivered peptide is not dependent on
functional proteasomes. NP-specific CTL were tested for their ability to
lyse MC57 target cells treated as indicated along the x-axis. MC57 cells
were incubated with either nNP-ricin or nNP-6K for 16 h. Where indicated,
cells were pretreated with IFN-␥ and subsequently with 20 ␮M clastolactacystin-␤-lactone (␤L) in 0.2% DMSO for 24 h before the addition of
toxin. MC57 cells were used as target cells. Controls included MC57 cells
infected with a vaccinia virus-encoding influenza nucleoprotein (VacNP),
or pulsed with either influenza nucleoprotein (NP pep) or LCMV glycoprotein
(gp33 pep) peptides. The percentage of specific lysis at three E:T ratios is
shown. The data are representative of at least two similar experiments.
Ricin was able to deliver NP peptide to MC57 cells for processing and presentation by MHC class I molecules, because cells
treated with nNP-ricin or nNP6K-ricin were sensitized for specific
lysis by NP366 –374-restricted CTL, but not by the irrelevant gp33restricted CTL (Fig. 2). Controls show that these cells were able to
process influenza nucleoprotein expressed in the cytosol from a
vaccinia virus construct (VacNP). However, for both nNP-ricin or
nNP6K-ricin, significant lysis was observed only in toxin-pulsed
MC57 cells following pretreatment for 48 h with IFN-␥. Such
treatment is known to up-regulate gene expression of MHC class
I proteins, several proteasomal subunits, and the proteasome regulator PA28 (27). The fact that IFN-␥ is not known to up-regulate
extracellular proteases and that presentation of the NP peptide was
only seen after IFN-␥ treatment of MC57 cells suggests that extracellular processing of toxin is not occurring. Supporting this
conclusion, peptide presentation was blocked when the nNP-ricin
was prevented from binding and subsequently entering cells by
pretreating the toxin with 100 mM lactose (data not shown). Interestingly, both the claNP-ricin fusion (Fig. 2) and a C-terminally
located NP-ricin fusion (data not shown) failed to promote specific
lysis. Therefore, it was decided to focus on the N-terminally located NP-ricin fusions.
Surprisingly, however, peptide presentation was not dependent
on active proteasomes (Fig. 3). When MC57 cells were treated for
24 h with clasto-lactacystin-␤-lactone, such that processing and
presentation of NP peptides from vaccinia-expressed nucleoprotein was significantly reduced (Fig. 3), surface presentation still
occurred under these conditions when cells were challenged with
free NP peptide or the fusions nNP-ricin or nNP-6K-ricin. We also
observed that the extent of specific lysis between nNP-ricin and the
normally more degradable nNP-6K-ricin, in either the presence or
absence of proteasome inhibition, was consistently similar. Most
strikingly of all, processing and presentation of ricin-delivered NP
was independent of functional TAP (Fig. 4). The TAP-deficient
RMA-S cells were significantly lysed when ricin delivered NP
peptide via endocytosis. Furthermore, this presentation was not
dependent on IFN-␥ (Fig. 4). Taken together, these data suggest
that the processing of NP366 –374 from ricin was occurring within
the endomembrane system of the cell rather than at the cell surface
or within the cytosol.
It was of interest to locate the site of peptide processing and
MHC class I loading within the endomembrane system. Ricin is a
promiscuous toxin that, on its journey, can harmlessly enter all
compartments of the endosomal/lysosomal/trans-Golgi network
system of the cell. Therefore, it is conceivable that ricin entering
102
the late secretory pathway, and they have been shown able generate
class I peptides (29 –31). However, the furin inhibitor did not block
the processing of the N-terminally placed NP peptide from ricin (Fig.
5A). Likewise, presentation was not sensitive to the disruption of endosome/lysosome pH by the lysomotropic amine, chloroquine, which
affects furin activity (32) and which would be predicted to indirectly
FIGURE 5. Presentation of toxin-derived NP peptide is both furin and chloroquine independent but is reliant on a functional secretory pathway. RMA-S
cells (6 ⫻ 106) were incubated with nNP-6K ricin (nNP-6K) at 500 ng/ml for 2 h, following a 2-h pretreatment with increasing concentrations of the specific
furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone (A), chloroquine (B), or BFA (C). Controls include RMA-S cells pretreated with the various
inhibitors for 2 h, then pulsed with free peptide from either influenza nucleoprotein (NP pep) or LCMV glycoprotein (gp33 pep). The percentage of lysis
is shown for three E:T ratios. The data are representative of at least two similar experiments.
FIGURE 4. Ricin-dependent peptide presentation is more efficient in
TAP-deficient RMA-S cells. MC57, RMA, and RMA-S cells were incubated with nNP-ricin or nNP-6K (50 ng/ml) for 16 h. NP-specific CTL
were tested for their ability to lyse target cells treated as indicated along the
x-axis. Controls include cells pulsed with free peptide from either influenza
nucleoprotein (NP pep) or LCMV glycoprotein (gp33 pep). The percentage
of lysis at three E:T ratios is shown. The data are representative of at least
two similar experiments.
reduce the proteolysis normally occurring within acidic compartments
(Fig. 5B). To further investigate any processing in the early endocytic
pathway, and to specifically rule out presentation by surface recycling
MHC class I proteins, we studied the effect of disrupting both anterograde and retrograde traffic in the Golgi stack with BFA. Such treatment would prevent the toxin moving along its productive entry pathway to the ER and concomitantly block exocytosis of newly
assembled MHC class I complexes from the ER. Fig. 5C shows that
surface presentation of the NP peptide delivered into cells by ricin is
extremely sensitive to BFA. These data support the idea that processing and loading were occurring in the early secretory pathway.
To more precisely investigate whether peptide processed from
ricin interacted with MHC proteins as they were being transported
through the early secretory pathway, or whether it interacted to
stabilize MHC complexes in the ER, we looked at the loading of
newly synthesized, empty MHC class I H chains (Fig. 6). This was
achieved by radiolabeling RMA-S cells with a short pulse of
[35S]methionine after treating cells with or without ricin or the
ricin-peptide fusion, followed by detergent lysis, immunoprecipitation of any peptide-loaded H-2Db complexes using the conformation-specific mAb B22, and EndoH treatment. Samples were
compared with non-EndoH-treated controls. One hundred percent
relative loading was seen when cell lysates were incubated with
free NP peptide, while the background level of correctly assembled
complexes was ⬃10%. The pulse-labeled MHC H chains were
EndoH sensitive as judged by the faster migration of bands compared with a non-EndoH-treated sample (Fig. 6A). When cells
were pretreated with nNP-6K ricin, the amount of immunoprecipitable peptide-loaded MHC complexes increased by ⬃180% relative to untreated or ricin-only controls (Fig. 6, B and C). A structural-based algorithm (33) used to predict any H-2Db MHC class
I epitopes present in the nNP-6K ricin primary sequence identified
a total of 23 putative epitopes. Of these only 12 had an energy
score to indicate good binding interactions, with the NP epitope
ranked first. Indeed, as shown in Fig. 6, B and C, there was no
increase above the background level in the loading of newly synthesized MHC proteins when the 6K-ricin lacking the NP epitope
was tested. Collectively, the experimental and theoretical data in-
dicated that the increased loading seen in TAP-negative RMA-S
cells of newly synthesized, EndoH-sensitive MHC proteins could
be attributed solely to the presence of the NP epitope within the
ricin construct, rather than to processing and loading of internal
RTA peptides. The significant increase in loading of newly synthesized, EndoH-sensitive MHC proteins when TAP-deficient
cells were treated with nNP-ricin supported the idea that the ricin
fusions were being processed in the ER or cis-Golgi.
Interestingly, examination of the N terminus of the nNP-RTA
revealed the existence of two potential signal peptidase cleavage
sites that fit the consensus defined by von Heijne et al. (34) (Fig.
7A) but that are never used in the biosynthesis of the toxin in the
producing plant (35). Theoretical prediction analysis indicated that
the second site (AGA2TV) would be a more effective cleavage
site than the first (IFP2KQ). When these sequences were engineered into the secretory protein preprolactin to replace the natural
preprolactin signal peptidase cleavage site, they were indeed able
to serve as cleavage sites during in vitro translation in the presence
of canine microsomal membranes. After cotranslational import, a
posttranslational incubation with protease showed that the lower,
signal peptide-cleaved band was protected within the vesicles, unless the protease was added together with a membrane-disrupting
detergent (Fig. 7B). As predicted, the first site, where incomplete
cleavage was seen, was not as effective a substrate as the second
site (Fig. 7B, ⴱ). To exclude the possibility that simply any sequence from the N terminus of RTA can serve to provide a signal
peptidase cleavage site, a stretch of residues between the two putative cleavage sites (PIINF) was also engineered into preprolactin.
Because the translation product in this case was resistant to proteinase K (i.e., protected within the membrane vesicles) but remained a full-length protein, we conclude that the sequence was
not able to provide a cleavage site for signal peptidase (Fig. 7B).
Signal peptidase is the best-characterized endoprotease within
the ER. To investigate any Ag processing function of this protease,
the two potential cleavage sites within the nNP-6K ricin were separately rendered noncleavable by replacing the ⫺3 and ⫺1 positions with arginine. As described previously (36), such changes
would preclude processing by signal peptidase. Indeed, replacement of the preprolactin cleavage site with either RFRKQ or
RGRTV, followed by in vitro translation in the presence of canine
microsomal membrane, confirmed that such substitutions prevented cleavage (data not shown). Therefore, these mutant proteins
carrying the NP peptide were purified from E. coli. They were
shown to be structurally sound because the modified RTAs readily
reassociated with RTB, and when the catalytically active RTA
equivalent was used the reassembled holotoxins exhibited native
FIGURE 7. Sites in the first 20 residues of mature RTA can function as substrates for signal peptidase. A, The first 20 amino acids of mature RTA contain
two potential signal peptidase cleavage sites (in boldface and underlined, where arrowheads indicate potential cleavage sites). These sequences, along with
a control sequence (boldface only), were inserted into preprolactin (ppl) to replace its natural signal peptide cleavage site. B, In vitro transcripts encoding
preprolactin wild type, preprolactin-IFPKQ, preprolactin-PIINF, and preprolactin-AGATV were translated in vitro in rabbit reticulocyte lysates in the
absence or presence of canine pancreas microsomes (RMs). Some samples were then treated with proteinase K (PrK) in the absence or presence of the
detergent Nonidet P-40 (NP-40) as indicated. Translation products were separated by SDS-PAGE and visualized by fluorography.
FIGURE 6. Toxin-derived NP peptide stabilizes newly synthesized
H-2Db molecules. A, RMA-S cells (20 ⫻ 106) were incubated for 90 min
with medium alone (lanes 1-3), nNP-6K ricin (nNP-6K; lanes 4 and 5), or
6K ricin (lanes 6 and 7) at 500 ng/ml. Following radioactive labeling for
30 min, cell lysates were precleared in the absence (lanes 3, 5, and 7) or
presence (lanes 1, 2, 4, and 6) of a saturating concentration (20 ␮M) of a
stabilizing influenza nucleoprotein peptide (NP pep). Peptide-loaded
H-2Db molecules were recovered by immunoprecipitation using the conformation-specific Ab B-22 that detected only loaded H chains, and immunoprecipitates were treated with EndoH (lanes 2-7) or were left untreated (lane 1). B, Resolved bands were quantified and expressed as the
percentage of relative loading of newly synthesized H-2Db molecules. Integrated optical band intensities, expressed as a percentage of radiolabeled
unstabilized to radiolabeled stabilized immunoprecipitations, were taken as
a measure of the loading of peptide onto newly synthesized H-2Db molecules. C, The percentage of increase in loading upon incubation with nNP6K, compared with the average of the mock-treated and the 6K ricintreated controls, is shown. The data are means of duplicate samples and are
representative of two similar experiments.
103
104
Discussion
Ricin is an opportunistic toxin that can promiscuously bind to cell
surfaces via the galactoside-binding RTB and be internalized by
endocytosis using a multiplicity of pathways to reach endosomes,
lysosomes, the Golgi complex, and the ER lumen (37). Only the
FIGURE 8. Signal peptidase is involved in Ag processing from ricinmediated delivery. RMA-S cells (6 ⫻ 106) were incubated with 500 ng/ml
nNP-6K ricin (nNP-6K), nNP(RFRKQ)-6K ricin (nNP-RFRKQ-6K), or
nNP(RGRTV)-6K ricin (nNP-RGRTV-6K) for 16 h. NP-specific CTL
were tested for their ability to lyse target cells treated as indicated along the
x-axis. Control RMA-S cells were pulsed with free peptide from either
influenza nucleoprotein (NP pep) or LCMV glycoprotein (gp33 pep). The
percentage of lysis at three E:T ratios is shown. The data are representative
of at least two similar experiments.
FIGURE 9. Ricin-mediated delivery and TAP-independent surface presentation is not epitope specific. RMA-S cells (6 ⫻ 106) were incubated
with either 500 ng/ml nNP-6K ricin (nNP-6K) or 500 ng/ml gp33-6K ricin
(gp33-6K) for 16 h. NP- and gp33-specific CTL were tested for their ability
to lyse target cells treated as indicated along the x-axis. Control RMA-S
cells were pulsed with free peptide from either influenza nucleoprotein (NP
pep) or LCMV glycoprotein (gp33 pep). The percentage of lysis is shown
for three E:T ratios. The data are representative of at least two similar
experiments.
fraction of toxin that reaches the ER will retrotranslocate to the
cytosol (14), where a small proportion evades proteasomal degradation to inactivate ribosomes (19). We reasoned that, because the
productive intoxication pathway involves toxin dislocation from
the ER as a pseudo-substrate of the ERAD pathway and an encounter with the ubiquitin/proteasome machinery, it may be possible to use ricin to deliver Ags directly into the MHC class I
processing pathway. Our data now show that nanomolar amounts
of a catalytically crippled form of ricin can indeed be used as a
delivery vehicle in vitro. However, only peptides genetically fused
at the N terminus of RTA were successfully processed and presented (Fig. 2), and processing was not dependent on active proteasomes (Fig. 3). Indeed, CTL-induced lysis was most efficient
when ricin fusions were internalized by cells of a TAP-deficient
cell line in the absence of IFN-␥ (Fig. 4). So, although ricin can
retrotranslocate from the ER of TAP-deficient cells (known because the native version can kill RMA-S cells (data not shown))
processing of the fused peptide from nNP-ricin is clearly not dependent on this step or other downstream events within the cytosol.
It is now recognized that the classical pathway for the presentation of endogenous antigenic peptides is not the only route by
which peptides can reach MHC class I proteins. Indeed, processing
of Ag and loading onto MHC class I can be quite flexible. For
example, cross-presentation of exogenous Ag may occur. This
may happen in some cell lines when exogenous Ags are internalized to intersect the classical processing pathway in the cytosol
after release from endosomes or other intracellular compartments,
or following endosomal degradation and recycling of peptide to
bind cell surface class I molecules (38). However, raising endosomal pH to reduce proteolytic activity or blocking furin-related
endoproteases did not prevent peptide presentation (Fig. 5, A and
B). Furthermore, because protein traffic through recycling endosomes is known not to be affected by BFA, the effect of this drug
in blocking the presentation of peptide delivered via the ricin carrier (Fig. 5C) would again preclude cross-presentation events in
this particular case.
Class I Ag processing in the cytosol is normally mediated by
proteasomes (for an example, see Ref. 39), although this may not
potency (IC50, 0.015 nM) (19). This indicated that the primary
structural changes at the N terminus of RTA did not perturb toxin
trafficking to the ER.
To test that signal peptidase could act posttranslationally to process peptides appended to the N terminus of RTA, we added the
purified mutant proteins described above to TAP-deficient cells
and looked for peptide presentation. We observed that surface presentation of the NP peptide did not occur when the first of the
predicted signal peptidase sites (IFP2KQ) was deliberately disrupted by substitution with arginine (Fig. 8). By contrast, when the
second predicted cleavage site (AGA2TV) was mutated to prevent signal peptidase cleavage, presentation was reproducibly
higher than that seen with the control (nonmutated) nNP-ricin (Fig.
8; for a possible explanation, see Discussion). These findings support the idea that CD8⫹ peptide epitopes positioned at the N terminus of RTA may be cleaved from the endocytosed toxin posttranslationally by signal peptidase (or an undetermined protease
with signal peptidase-like activity), loaded onto MHC class I proteins and subsequently presented, only when the sequence
IFP2KQ is present at the start of mature RTA. Signal peptidase
processing at this site would release the nine-residue N-terminal
NP epitope with a short C-terminal extension of three residues of
mature RTA.
To test whether such processing is confined to one particular
peptide at the N terminus of RTA, we tested an alternative peptide.
When the H-2Db-restricted gp33 peptide from LCMV was positioned at the N terminus of RTA to generate gp33-ricin, specific
lysis by gp33-specific CTL was observed using the standard assay
with RMAS target cells (Fig. 9). The possibility that ricin may
provide a signal peptidase site to allow the processing of N-terminally positioned Ags is addressed in Discussion.
the ricin-producing plant (35). According to theoretical predictions
(34), the second of these sites would be the more effective substrate. Indeed, when the natural signal peptidase cleavage site of a
preprolactin reporter was substituted with either of these sequences
and tested in a translation system supplemented with ER-derived
microsomes, it was clear that they could serve as substrates for
signal peptidase with the predicted efficiencies (Fig. 7). Furthermore, when the first of these sites was replaced with the nonfunctional arginyl sequences in nNP-RTA, peptide presentation in
TAP-deficient cells was completely abolished (Fig. 8). However, a
similar mutation of the second cleavage site in nNP-RTA, predicted to be a more effective signal peptidase substrate, actually
provoked an increase in peptide presentation.
In the absence of a more extensive investigation we can only
speculate as to why this happens. We propose that, although Nterminal peptide fusions to ricin are normally accessible to a signal
peptidase-like activity for the production of both peptides, it is
somehow more favorable to load only the shorter peptide of the
two for subsequent surface presentation. Cleavage at the first site
would release a peptide predicted to contain the nine-residue NP
peptide with an additional three residues at its C terminus (Fig. 7).
By contrast, cleavage at the second site would generate a longer
peptide (the nine-residue peptide epitope with a 16-residue C-terminal extension of the RTA sequence; Fig. 7). This hypothesis is
consistent with a need for further processing to remove the Cterminal extensions, because we consider it unlikely that 12- and
25-mer peptides generated by signal peptidase would be presented
at the cell surface. Although it is known that the NP366 –374
nonamer with a C-terminal extension of three residues can directly
assemble with class I molecules, this species has much faster off
rates than the optimal nonamer (51). Therefore, it is likely that
another enzyme(s) is involved, one that is only able to cut or trim
the shorter 12-mer peptide to optimal size, possibly because the
longer peptide possesses a structure incompatible for further signal
peptidase cleavage and/or subsequent processing. In this scenario,
successful generation and loading of NP366 –374 would normally be
a competitive process. Abrogating cleavage at the second site, as in
nNP-(RGRTV)-6K (Fig. 9), would in effect ensure the production
of only the shorter peptide, rather than a mixed population, and
thereby increase the opportunities for successful loading and
presentation.
Consistent with the hypothesis that extensive Ag presentation
can occur within the ER lumen, it has previously been shown that,
in cells devoid of TAP, the embedded NP366 –374 epitope within a
full-length, ER-targeted NP mutant that lacked N-linked glycans
was released for assembly with class I proteins (52). However, a
full-length glycosylated NP targeted to the ER in an identical fashion was not capable of sensitizing TAP-negative target cells for
lysis (44). These results strongly suggest that the tertiary conformation of polypeptides within the ER lumen may influence the
generation of MHC class I epitopes. It should be noted that the
longer peptide predicted to arise from the ricin fusion carries a
glycosylation sequon. Whether the longer peptide becomes posttranslationally glycosylated (14) to similarly preclude further processing, or whether its structure is simply inappropriate for processing, remains unknown.
Although proteases are known to exist in the ER lumen (Refs.
46 – 48 and reviewed in Ref. 53), the exact complement and role of
resident carboxypeptidases, aminopeptidases, and endoproteases
in Ag processing is unclear. Where Ags have been extensively
processed in the ER (44), the enzyme(s) responsible have not usually been identified. One ER endoprotease that is known to be
involved in the generation of HLA-E binding epitopes is signal
be an absolute requirement (40, 41). In this work we have shown
that peptide processing from ricin and surface presentation of peptide can occur in cells where proteasomes are inhibited. Because
presentation can also occur when TAP is absent, these data suggest
that processing is occurring outside the cytosol. Although peptide
transport through TAP is the optimal route by which peptides are
loaded onto empty MHC proteins in the ER, it is now recognized
that a physical interaction with TAP is not required for loading
(42). Indeed, intracellular peptides can be presented on the surface
of TAP-negative cells after delivery to the ER through Sec61
translocons via signal peptides (43, 44) or following direct penetration of exogenous peptides into cell organelles (45). Taken together, our data are consistent with processing and loading within
the secretory pathway.
Where in the secretory pathway could the processing occur? In
addition to the possibility of endosomal processing outlined above,
Ag processing may take place within the secretory pathway by
unknown exoproteases, or by endoproteases of the furin family, for
example. Furin is a member of the subtilisin family of serine proteases that is found predominantly in the TGN from where it cycles
via the cell surface. The TAP-independent generation of MHC
class I peptides by furin has been observed when placed at the C
terminus of the hepatitis B virus core protein (29 –31). However,
the specific furin inhibitor we used had no effect, and the lack of
putative furin sites toward the N terminus of RTA would argue
against a role for this protease in the release of epitopes joined to
the N terminus of RTA. There are, of course, numerous other
endoproteases within the late secretory pathway, most being cell
type specific for the activation of proproteins such as hormones,
growth factors, extracellular enzymes, etc. In our study, we saw a
significant increase in peptide-stabilized MHC complexes that
were still EndoH sensitive when TAP-deficient cells are treated
with the toxin fusions (Fig. 6). This strongly suggested that loading
was occurring within the ER lumen itself. If this were happening
it would clearly make sense for the peptides to be proteolytically
released within this compartment, in the vicinity of empty class I
H chains. Indeed, this may help explain the IFN-␥-independent
processing and presentation in TAP-deficient RMA-S cells (Fig.
4). Treatment of RMA-S cells with IFN-␥ would lead to an increase in MHC class I proteins in the ER lumen (27). However,
such an increase would not be predicted to enhance presentation of
the toxin-delivered NP peptide because, in the absence of other
peptides entering from the cytosol, there is always a high concentration of available class I proteins within the ER. Conversely, in
RMA cells, an IFN-␥-mediated increase in class I molecules
within the ER would permit increased loading of the processed NP
peptide in the face of competition from other peptides entering
via TAP.
The ER lumen is a major site of protein folding in an environment not compatible with protein degradation events. Therefore,
the consequent likely dearth of endoproteases within the ER would
appear to make endoproteolytic release of peptides from ricin unlikely. Nevertheless, some endoproteases do exist within the ER
membrane and lumen (e.g., signal peptide peptidase (46) and the
chaperones ERp60 and ERp72 (47, 48)). The best-characterized of
these is the signal peptidase complex that cleaves short ER-targeting peptide sequences from newly synthesized polypeptides. However, in addition to the removal of signal peptides, this enzyme
may also be responsible for the processing the H-2a subunit of the
mammalian asialoglycoprotein receptor (49) and in the degradation of aberrant membrane proteins in yeast (50).
Close examination of the mature N terminus of RTA revealed
two potential signal peptidase cleavage sites, IFP⫹32KQ and
AGA⫹162TV, although it should be noted that neither is used in
105
106
Acknowledgments
We thank Prof. Tim Elliott (University of Southampton, Southampton,
U.K.) and Prof. Eric Tartour (Institut Pasteur, Paris, France) for helpful
discussions.
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