From the Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom
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ABSTRACT |
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Retention of soluble proteins in the endoplasmic reticulum is dependent on their interaction with the KDEL (Lys-Asp-Glu-Leu) receptor in the Golgi apparatus and their subsequent retrieval back to the endoplasmic reticulum. We have studied the three-dimensional organization of the human KDEL receptor using site-directed mutagenesis and sulfhydryl-specific labeling. We have identified four amino acid residues, Arg-5, Asp-50, Tyr-162, and Asn-165, which we suggest participate in the formation of the ligand binding pocket. Arg-5 and Asp-50 are shown to be located on the lumenal side of the membrane and are inaccessible from the cytoplasm. In addition, our results strongly support a topology of the KDEL receptor similar to the family of G-protein-coupled receptors with seven transmembrane domains. Furthermore, Asp-50 plays a crucial role in the binding of His/Lys-Asp-Glu-Leu ligands, but is not required for Asp-Asp-Glu-Leu binding, suggesting that this residue forms an ion pair with the positively charged amino acid residue positioned 4 residues from the carboxyl terminus of the ligand.
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INTRODUCTION |
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The precise sorting of proteins along the secretory pathway is crucial for the maintenance of organelle integrity. The localization of chaperones and other soluble proteins to the ER1 is achieved by their continuous retrieval from post-ER compartments by the KDEL receptor, a membrane protein localized in the Golgi apparatus. Binding of soluble ER proteins to the receptor is dependent on a conserved tetrapeptide sequence at their carboxyl terminus, which is usually KDEL in animal cells and HDEL in Saccharomyces cerevisiae (see Ref. 1 for review). The HDEL receptor was originally isolated from yeast; it is the product of the ERD2 gene and has subsequently been cloned from other organisms (2-6). Erd2 determines both capacity and specificity of the H/KDEL retrieval system (7), and a direct interaction between KDEL ligands and the human receptor has recently been demonstrated (8, 9). Hydrophobicity analyses and the "positive inside rule" (10) suggest seven primarily hydrophobic membrane-spanning domains with the amino terminus in the lumen and the carboxyl terminus in the cytoplasm (11, 12). The topology of the KDEL receptor would thus be similar to the organization of G-protein-coupled receptors, although no amino acid sequence homology is observed. Based on studies using fusions to N-linked glycosylation sites, a different topology has been proposed with six transmembrane domains and the amino terminus located in the cytoplasm (13).
Although the KDEL recycling system is well established, several significant questions have remained unanswered. For example, the sorting of KDEL proteins is believed to be controlled by the pH difference between the Golgi apparatus and the ER (8). However, it is unclear whether the pH difference between these two compartments, which is estimated to be approximately 0.5 pH units (8), is sufficient to ensure efficient binding of KDEL proteins in the Golgi apparatus and their release in the ER. In addition, ligand binding was suggested to induce retrograde transport of the receptor-ligand complexes, involving a signal transduction mechanism across the membrane (14). Neither the mechanism of signal transduction nor any components of the machinery apart from the KDEL receptor have been identified. It has been shown that the KDEL receptor, presumably together with bound ligand, is selectively recruited into COPI-coated vesicles from Golgi membranes, but this process is not well characterized (15, 16). As a better understanding of these events requires detailed analyses of the receptor structure, we set out to identify amino acid residues involved in ligand binding and to investigate the topology of the human Erd2.1 protein, one of two related KDEL receptors identified in humans (17). We undertook a sulfhydryl-specific labeling approach, creating site-directed mutants with unique cysteine residues engineered at different positions in the receptor, followed by chemical labeling with thiol-specific reagents. We have investigated the effect of derivatization of single cysteine residues on ligand binding and the sidedness of these residues by labeling of receptor mutants with membrane-permeant and -impermeant sulfhydryl-specific compounds.
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EXPERIMENTAL PROCEDURES |
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Materials--
The baculovirus transfer vector pVL1393 and
linearized baculovirus DNA were from PharMingen, Ni-NTA resin was from
Qiagen, NEM and 3-(N-maleimidylpropionyl)biocytin (biotin
maleimide) were from Sigma,
4-acetamido-4-maleimidylstilbene-2,2
-disulfonic acid (AMS) was from
Molecular Probes, and a streptavidin-horseradish peroxidase conjugate
was from Pierce.
Plasmids--
Site-directed mutagenesis was performed according
to Kunkel (18) in a pBluescript vector containing the human Erd2.1
cDNA modified at the 3-end to add a Myc and a hexahistidine tag
(hErd2-Myc-His, see Ref. 9). All sequences were confirmed by dye
terminator cycle sequencing using an Applied Biosystems model 377 sequencer. The cDNAs encoding receptor mutants were cloned into a
SmaI/BglII-digested baculovirus transfer vector
pVL1393. Mutants are named as (wild-type residue and number)
(mutant residue).
Preparation of Virus and Expression of Recombinant Proteins-- Recombinant baculoviruses containing mutant Erd2.1 cDNAs under the control of a polyhedrin promoter were created using the Baculogold kit from PharMingen according to the information provided by the manufacturer. Isolation of virus clones and infection of Sf9 cells were carried out as described (19).
Membrane Preparations and Ligand Binding Assays-- Preparation of a crude membrane fraction from infected cells and ligand binding assays were carried out as described previously (9). Briefly, standard binding assays contained membranes derived from infected insect cells expressing wild-type or mutant KDEL receptors, approximately 180,000 cpm of 125I-YTSEKDEL, 100 ng of YTSEKDELGL (nonfunctional ligand) in binding assay buffer (20 mM NaCl, 250 µg/ml bovine serum albumin, and 50 mM sodium cacodylate pH 5.0). Unless otherwise mentioned, all binding data are shown without background subtraction. Competition assays contained 180,000 cpm 125I-YTFEHDEL and variable amounts of competitor peptide. All binding assays were done under nonsaturating conditions.
Treatment of Membranes with Thiol-specific Maleimides--
To
assess the effect of alkylation of individual cysteine residues on the
binding activity of receptor mutants, 1-10 µg of crude membranes
were incubated with NEM or biotin maleimide in 10 mM Hepes,
pH 7.0, for 10 min on ice and were then directly used in the peptide
binding assay. For analysis by Western blotting, 100 µg of crude
membranes were incubated with 100 µM biotin maleimide for
10 min on ice in phosphate buffer (50 mM sodium phosphate, pH 7.0), quenched with 10 mM -mercaptoethanol and
solubilized with 0.5% SDS at room temperature. The soluate was spun at
14,000 × g for 20 min, and the receptor was bound to
Ni-NTA resin by incubation of the soluble detergent extract with 40 µl of resin under rotation for 1 h at room temperature. Unbound
proteins were washed off and bound receptor protein eluted with 1 M imidazole, pH 7.0. All buffers during the purification
contained 0.5% SDS. Biotin-labeled KDEL receptor was visualized by
immunoblotting using a streptavidin-horseradish peroxidase conjugate
(Pierce, 1 µg/ml in 1% bovine serum albumin) and the ECL
chemoluminescence detection system (Amersham International, United
Kingdom).
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RESULTS |
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Mutagenesis of Endogenous Cysteines-- The human KDEL receptor Erd2.1 contains four cysteine residues, none of which is strictly conserved in all receptor homologues. There are no cysteines in the S. cerevisiae receptor, indicating that cysteine residues are not strictly required for receptor function (2). However, Cys-29 in the human sequence (Fig. 1) is conserved in six out of nine receptor homologues, and a change to alanine at this position reduces the binding activity of the human KDEL receptor approximately 2-3-fold, implying that a mutation at this position has indirect affects on the receptor structure (12). The first objective was to test whether any individual cysteine residue was strictly required for structure or function of the human KDEL receptor. Therefore, site-directed mutagenesis was used to change all four cysteines to serines, and Cys-less and wild-type receptors were expressed in insect cells. We used the baculovirus expression system, as we have recently demonstrated that it can be used to produce large amounts of functional human KDEL receptor (9). We expressed a both Myc- and hexahistidine-tagged version of the human Erd2.1 protein (hErd2-Myc-His, see "Experimental Procedures"), since the addition of these tags to the carboxyl terminus of the receptor does not alter the affinity of the receptor for KDEL ligand and Myc-His-tagged receptor can be easily purified in a single step by nickel affinity chromatography (9). Crude membranes containing equal amounts of wild-type receptor and a cysteine-less receptor mutant were analyzed for KDEL peptide binding. The binding activity of Cys-less human KDEL receptor is significantly higher than the wild-type receptor (Fig. 2A). This is not due to a change in ligand affinity and thus suggests an increase in the amount of functional receptor, which was estimated to be approximately 2.3-fold as judged by Scatchard analysis (data not shown). Together with earlier data, which showed around 40% of the human KDEL receptor to be functional when expressed in insect cells (9), this suggests that more than 90% of the Cys-less KDEL receptor in these preparations is functional. To determine which cysteine residue is responsible for this effect, we mutated three cysteines at a time to serine, leaving one of the endogenous cysteine residues. Fig. 2A shows that a receptor containing only Cys-29, which is predicted to be in the first cytoplasmic loop, shows a binding activity similar to wild-type receptor. Thus, it is evidently the presence of Cys-29 that causes some of the overexpressed wild-type receptor in insect cells to accumulate in an inactive state. Immunoblotting showed an increase in the amount of receptor dimers for the Cys-29-containing mutant compared with Cys-less receptor (data not shown), suggesting that oxidation of Cys-29 may play a role in receptor inactivation.
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Construction and Activity of Cysteine Mutants--
We reintroduced
individual cysteine residues into the Cys-less KDEL receptor sequence
at 25 different positions by site-directed mutagenesis to probe their
function and location (Fig. 1). In the choice of the residues, we were
guided by an earlier study, which used mutational analysis to identify
amino acid residues crucial for ligand binding (12). Although this
approach could not be used to distinguish between residues directly
contacting the ligand or ones that merely altered the receptor
structure, thus affecting ligand binding indirectly, it allowed us to
predict which residues might be involved in forming the binding pocket. We concentrated on charged residues or residues predicted to be on the
same side of a potential -helix as charged or polar residues (Fig.
1). Recombinant baculoviruses encoding receptor mutants were used to
infect insect cells, and crude membrane preparations were subsequently
analyzed for their ability to bind ligands in vitro (Table
I). The replacement of charged residues
by cysteines had a significant effect on KDEL binding activity,
consistent with earlier findings (12). The first half of transmembrane domain 1 was particularly sensitive to mutations, even a replacement of
Gly by Cys (Gly-8
Cys) strongly affected ligand binding, whereas
less conserved residues in the same region (Leu-10, Ser-11, and Leu-13)
could be changed without significant effect (Table I). In general,
mutation of less conserved residues or nonpolar residues had only minor
effects on KDEL binding. Surprisingly, a minor change in the fifth
transmembrane domain (Ser-123
Cys) greatly reduced KDEL binding,
whereas an identical amino acid mutation in the same transmembrane
domain (Ser-128
Cys) has no effect on the binding activity (see
below).
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Effect of Treatment of Cysteine Mutants with NEM and Biotin
Maleimide--
Crude membranes containing wild-type or mutant
receptors were treated with NEM or biotin maleimide (BCM), and then
KDEL binding activity was determined (see Fig.
3 for examples). Biotin maleimide is a
bulky maleimide-derivative, which was shown to be membrane-permeant at
higher concentrations (20). This reagent was used to determine whether
the size of the modifying reagent is of importance, and it also
facilitated detection of labeled receptor. We have identified four
receptor mutants (Arg-5 Cys, Asp-50
Cys, Tyr-162
Cys, and
Asn-165
Cys), which upon derivatization with NEM or BCM show
significantly reduced binding activity (Fig. 3 and Table II). Binding of KDEL peptides to
wild-type or Cys-less KDEL receptor was not affected by derivatization
(Fig. 3). Arg-5
Cys, Asp-50
Cys, Tyr-162
Cys, and Asn-165
Cys already show a significant defect in binding (Table I), which
was enhanced when the cysteine residue was alkylated; note that the
binding experiments in Fig. 3 used a larger amount of Arg-5
Cys
mutant than wild-type receptor to compensate for its lower binding
activity. We consider it to be unlikely that these mutations have a
severe effect on folding of the receptor protein, the insertion into
the bilayer or protein stability, as their expression levels are
comparable with that of wild-type or Cys-less Erd2 (data not shown).
Labeling with biotin maleimide was visualized by purification of the
KDEL receptor using nickel affinity chromatography followed by
immunoblotting and probing with a streptavidin-peroxidase conjugate.
Biotin maleimide is specific for cysteine residues under our
conditions, since we were unable to detect reaction of BCM with the
Cys-less receptor (Fig. 4A).
Arg-5
Cys, Asp-50
Cys, Tyr-162
Cys, and Asn-165
Cys
could be labeled with both BCM and NEM, where labeling with NEM was
visualized by its ability to prevent biotinylation (Fig. 4A).
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Topological Studies-- We investigated the location of the amino acid residues identified above relative to the membrane by testing the ability of a membrane-impermeant maleimide, stilbenemaleimide disulfonic acid (AMS), to prevent derivatization with biotin maleimide. These experiments crucially depended on intact membrane vesicles with the receptor being oriented in its natural way with the ligand binding side facing the lumen of the vesicles. To test the orientation of the receptor, binding assays were performed in the presence or absence of low amounts of detergent. The addition of 0.3% CHAPS stimulated the binding of 125I-YTSEKDEL to wild-type receptor and all mutants examined at least 4-fold (data not shown). This suggests that 80% of the KDEL binding sites are inaccessible to the hydrophilic peptide without permeabilization of the membrane. We assume that a small fraction of the membrane vesicles are either inverted or slightly leaky, allowing access of the peptide to the ligand binding side in the absence of detergent.
AMS has been reported to be a membrane-impermeant thiol-specific compound (20) and to confirm its physical properties we chose two mutants, Thr-85
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Ligand Specificity--
The tetrapeptide signal used for the
retention of soluble ER proteins is conserved among different species,
although some variations can be found. On most major ER proteins in
higher organisms, KDEL acts as the signal (1), whereas S. cerevisiae uses HDEL and Kluyveromyces lactis uses both
HDEL and DDEL (7). Intriguingly, one of the residues described above,
Asp-50, which we propose to be involved in ligand binding is located in
a region which was shown earlier to be important in determining ligand
specificity in the K. lactis and S. cerevisiae
receptors (11). The same region in the human receptor (amino acids
50-56) was shown to be crucial for binding of KDEL ligands (8, 14). To
determine whether a change of Asp-50 affects ligand specificity, we
tested the binding activity of KDEL and DDEL peptides to the Asp-50 Cys mutant. Octapeptides were shown to bind with higher affinity than
tetrapeptides,2 presumably
because sequences upstream of the tetrapeptide signal contribute to the
interaction with the receptor. To eliminate these additional effects,
we used tetrapeptides as competitors for binding of radioactively
labeled YTFEHDEL. Binding of 125I-YTFEHDEL to the Cys-less
receptor was competed efficiently with the peptide KDEL, whereas the
peptide DDEL competed poorly (Fig. 6A), consistent with earlier
findings (8). Binding of DDEL peptides to Asp-50
Cys was comparable
with binding to the Cys-less receptor; a concentration of approximately
250 µM DDEL was required to compete 50% of
125I-YTFEHDEL bound to the Cys-less mutant or Asp-50
Cys (Fig. 6). Strikingly, KDEL was a substantially worse competitor
with the Asp-50
Cys mutant than with the Cys-less receptor (Fig. 6B). The same effect, which was specific for Asp-50
Cys,
was observed for HDEL and RDEL peptides. These results suggest that the
efficient binding of peptides with a positive charge at position
4
from the carboxyl terminus of the ligand (HDEL, KDEL, RDEL) requires
the aspartic acid at position 50, presumably involving a direct ionic
interaction.
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DISCUSSION |
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This paper describes an investigation of the three-dimensional organization of the human KDEL receptor Erd2.1, using a site-directed sulfhydryl labeling approach. We show that all four endogenous Cys residues of the human KDEL receptor can be removed without affecting the ligand binding activity. Furthermore, the removal of cysteine 29 leads to the expression of almost homogeneously active receptor protein in Sf9 cells. Together with an earlier study, in which we describe the high level expression and the purification of functional receptor protein (9), this will prove to be a significant step toward the determination of the receptor structure by two-dimensional crystallization and cryo-electron microscopy.
We have investigated the topology of the KDEL receptor by testing the
sidedness of amino acids relative to the lipid bilayer using
membrane-permeant and -impermeant thiol-specific reagents. This was of
particular interest since the topology of the amino-terminal half of
the KDEL receptor has been controversial (12, 13). Using this method,
we have demonstrated that both Arg-5 Cys and Asp-50
Cys are not
accessible to membrane-impermeant reagents, indicating that these
residues are located in the lumen of the membrane compartments and
contribute to the formation of a hydrophilic binding site for KDEL
ligands, which is not accessible from the cytoplasmic side of the
membrane. Furthermore, Cys-29 was shown to be located on the
cytoplasmic side of the membrane. This suggests that the amino terminus
is located in the lumen as proposed previously (12). Together with the
location of Thr-85
Cys and Thr-114
Cys, which were shown to be
located on the cytoplasmic and lumenal side of the membrane,
respectively, these findings strongly point toward a two-dimensional
organization of the KDEL receptor analogous to the family of
G-protein-coupled receptors with seven membrane-spanning segments.
By determining the binding activity of single cysteine mutants of the
human KDEL receptor after reaction with thiol-specific reagents, we
have identified four residues that we believe contribute to the
formation of a hydrophilic binding pocket for KDEL ligands (Arg-5,
Asp-50, Tyr-162, and Asn-165). The KDEL binding activity of these
mutants is considerably decreased compared with the Cys-less receptor
and alkylation of these cysteines further inhibits KDEL binding. This
suggests that these four residues play a critical role in KDEL binding
and that the alkylation of the cysteine replacing the wild-type residue
leads to steric hindrance of ligand binding. We do not believe that
these mutations cause a significant change in receptor conformation, as
these mutants retain substantial KDEL binding activity and no
differences in expression levels could be observed compared with the
wild-type receptor. Furthermore, mutations of these residues described
earlier (Arg-5 Gln, Asp-50
Asn, and Asn-165
Ala) do not
interfere with the proper localization of these mutants to the Golgi
apparatus and no accumulation of the mutants in the ER, indicative of
misfolded proteins, was observed (12).
The reaction of Tyr-162 Cys and Asn-165
Cys with biotin
maleimide was less efficient compared with Arg-5
Cys and Asp-50
Cys, presumably because they are located closer to the inside of the
hydrophilic binding pocket, making their side chains less accessible to
a bulkier reagent such as BCM, whereas a smaller reagent, NEM, was
still efficient in inhibiting binding of YTSEKDEL. Arg-5
Cys was
equally well labeled by both NEM and biotin maleimide, and residual
KDEL binding to Arg-5
Cys was efficiently inhibited by both
reagents. Strikingly, all four residues are predicted to be located at
the lumenal end of the transmembrane domains based on a model with
seven transmembrane domains (see Fig. 1).
Asp-50 Cys is a particularly interesting mutant. KDEL binding,
which is substantially reduced compared with the Cys-less receptor,
could only be inhibited by reaction with biotin maleimide, although
labeling of this mutant with both NEM and biotin maleimide was
efficient. This suggests that this residue is located closer to the
opening of the binding pocket than Arg-5
Cys, and a large group
added to the cysteine side chain is required to perturb peptide
binding. Furthermore, we have studied the effects of this mutation on
the specificity of ligand binding. As mentioned above, the yeast
K. lactis uses both HDEL and DDEL as retention signals, whereas the S. cerevisiae receptor only recognizes HDEL.
Studies in yeast suggest that amino acids 51-57 in the K. lactis receptor determine ligand specificity in vivo
(11). Similar results were obtained when the corresponding residues
(amino acids 50-56) in the human receptor were replaced with the
K. lactis sequence, which significantly decreased the
ability of the human receptor to bind KDEL ligands but not DDEL
ligands, both in vitro and in vivo (8, 14). We
have demonstrated that Asp-50 in the human receptor sequence is
required for the efficient binding of KDEL ligands; the binding of KDEL
(and other peptides with a positive charge at the position
4 such as
HDEL and RDEL) to Asp-50
Cys was strongly reduced, whereas the
binding of DDEL peptides was unaffected. This suggests that DDEL, at
least partly, interacts with different amino acids than H/KDEL as
proposed previously (11). The fact that binding of peptides with a
positive charge (His, Lys, or Arg) is specifically weakened by a
mutation in the receptor that changes a negatively charged residue to a
polar residue implicates a direct interaction. Close proximity between this residue and the ligand is also supported by the finding that ligand binding to Asp-50
Cys was inhibited by alkylation of the
cysteine side chain (see above). Strikingly, Asp-50 is conserved in all
Erd2 sequences known except for the K. lactis receptor. The
importance of this residue is further demonstrated by the fact that one
of the receptor mutants originally isolated in S. cerevisiae
was localized to the equivalent residue in the yeast receptor, changing
the aspartate to an asparagine (2). However, there must be other
interactions significantly contributing to ligand binding, in
particular to the binding of HDEL peptides; the K. lactis
receptor, which has an asparagine residue at the corresponding
location, binds HDEL ligands in vivo, but not KDEL ligands.
In addition, the binding of HDEL was substantially worsened by the
Asp-50
Cys mutation, but it still binds considerably tighter to
Asp-50
Cys than all other peptides tested, suggesting additional
interactions of the histidine residue both in the human and the
K. lactis receptor. Furthermore, our competition experiments were performed with tetrapeptides. They bind with lower affinity to the
receptor than the corresponding octapeptides, indicating that
interactions with residues upstream of the conserved tetrapeptide contribute to ligand binding, although no sequence conservation among
different ER proteins can be observed (1). Additionally, soluble ER
proteins have been identified without a positively charged residue at
position
4 (1).
In conclusion, our study provides insight into the three-dimensional
organization of the KDEL receptor; we present evidence for the
existence of a hydrophilic ligand binding pocket formed by conserved
amino acid residues of the transmembrane domains. Together with earlier
findings (12, 13), our data suggest a secondary structure of the KDEL
receptor with seven membrane-spanning domains, similar to the family of
G-protein-coupled receptors. Furthermore, we have shown that Asp-50 is
involved in determining the ligand specificity, presumably by directly
pairing with a positively charged amino acid at position 4 from the
COOH terminus of the ligand.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of grants from the German Academic Exchange Service (HSP
II) and the European Commission.
§ To whom correspondence should be addressed. Tel.: 44-1223-402290; Fax: 44-1223-412142.
1
The abbreviations used are: ER, endoplasmic
reticulum; AMS, 4-acetamido-4-maleimidylstilbene-2,2
-disulfonic
acid; BCM, 3-(N-maleimidylpropionyl)biocytin; NEM,
N-ethylmaleimide.
2 A. Scheel, unpublished observation.
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REFERENCES |
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