From the Department of Cell and Developmental
Biology, Oregon Health Sciences University, Portland, Oregon
97201-3098, the
Department of Biochemistry, Cornell University
College of Medicine, New York, New York 10021, and ¶ Bristol
Meyers Squibb, Syracuse, New York 13201
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ABSTRACT |
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The soluble human transferrin receptor (TfR) found in blood is the result of a proteolytic cleavage occurring in the ectodomain of the receptor close to the transmembrane domain at Arg-100. We have discovered another cleavage site between Gly-91 and Val-92 even closer to the transmembrane domain. Cleavage at Gly-91 differs markedly from the normal cleavage site. It occurs when the entire cytoplasmic portion or the proximal 31 amino acids of the transmembrane domain are deleted. A soluble disulfide-bonded dimer of the TfR is released into the medium in contrast to the cleavage at Arg-100 where a dimer lacking intersubunit disulfide bonds is released. Whereas the cleavage at Arg-100 is generated by cycling through the endosomal system, pulse-chase experiments indicate that cleavage at Gly-91 occurs predominantly during the biosynthesis of the receptor. Pulse-chase analysis of the biosynthesis of mutant TfRs that lack the membrane-proximal cytoplasmic domain show that they exit the endoglycosidase H-sensitive compartment at a slower rate than the wild type TfR. These results suggest that the cytoplasmic domain influences the trafficking of the TfR either by influencing the folding of the ectodomain or by providing a positive signal for its transport through the biosynthetic pathway.
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INTRODUCTION |
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The transferrin receptor (TfR)1 mediates cellular iron accumulation by binding the iron transport protein transferrin. Iron is released from transferrin in the acidic environment of endosomes, and transferrin and its receptor return to the cell surface where the cycle is repeated 100-200 times during the lifetime of the receptor. The number of TfRs on the cell surface regulates the amount of iron taken into cells. In turn, TfR numbers are regulated at the synthetic level by the stability of its mRNA, which is sensitive to intracellular iron pools. It is regulated at the level of degradation by the release of TfR from the cell by proteolytic cleavage and presumably the lysosomal degradative pathway (1).
The soluble form of the TfR found in blood has been characterized and studied extensively (for review, see Refs. 1 and 2). The TfR is a type II membrane protein with the NH2 terminus in the cytoplasm and the COOH terminus in the ectodomain, and NH2-terminal sequencing of the soluble TfR revealed the cleavage site to be Arg-100 (3). It is released as a homodimer composed of 80-kDa subunits. Under denaturing but nonreducing conditions it migrates as a monomer, indicating a lack of intersubunit disulfide bonds (3, 4). Cleavage occurs during its transit through endocytic compartments after the TfR reaches the cell surface (5). The extent of TfR released into the medium varies with cell type (6).2 In both rats and humans the amount of soluble TfR in the blood correlates directly with erythropoiesis, indicating that cleavage of the TfR may be the major way of down-regulating the TfR during the differentiation of red blood cell precursors (2, 7).
While examining the trafficking of various mutated TfRs, we discovered a different cleavage site in the ectodomain of the TfR which results in the release of a soluble disulfide-bonded dimer of the TfR. This soluble TfR was sequenced, and the cleavage site was determined to be after Gly-91. Cleavage at Gly-91 occurs in TfRs in which 20 amino acids are deleted in the cytoplasmic domain of the TfR proximal to the membrane (amino acids 29-59). No detectable cleavage of wild type TfR at Gly-91 is detected. We present evidence that the protease that cleaves the TfR at Gly-91 is a resident of the early biosynthetic pathway, further distinguishing this cleavage from the previously characterized endosomal cleavage at Arg-100. Importantly, the extent of cleavage at Gly-91 correlates with the transit rate of the TfR through the ER-Golgi compartments. The longer the TfR spends in the endo H-sensitive compartment (e.g. ER/cis-Golgi), the greater the extent of cleavage at Gly-91. These results imply that the region of the cytoplasmic domain of the TfR proximal to the membrane is required for rapid transit through the early biosynthetic compartments. The deletion of amino acids 29-59 increases cleavage of the TfR by slowing its transit through the early compartments, thereby increasing the time the TfR is in the compartment containing the protease.
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MATERIALS AND METHODS |
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Cell Culture, Transfection, and Metabolic Labeling--
TRVb
cells, a Chinese hamster ovary cell line deficient in endogenous TfR
(8), were grown in Ham's F-12 medium (Sigma) with 2 g/liter
glucose and 5% fetal bovine serum or in HyQ CCM5 serum-free medium
(HyClone, Logan, UT). TRVb cells were transfected with the DNA-encoding
mutant transferrin receptors P59A TfR and 29-59 TfR in the
expression plasmid pcDNA3 (Invitrogen, San Diego) by the calcium
phosphate method (9) and selected for resistance to Geneticin as
described previously (10). TRVb cells expressing the C20A23 TfR, the
3-28 TfR, the
3-59 TfR, and the T104D TfR were generated
previously (5, 8, 10-13). Cells were metabolically labeled with
[35S]Met/Cys (Tran35S-label, ICN, Irvine, CA)
or [3H]palmitate (NEN Life Science Products) in Ham's
F-12 Met/Cys-deficient medium (Life Technologies, Inc.) with 5% fetal
bovine serum.
Mutant Transferrin Receptors--
The P59A TfR and the 29-59
TfR mutants were made by PCR overlap extension (14). The TfR cDNA
in pcDTR1 (15) was cut out with EcoRV and XbaI,
eliminating the 5'- and 3'-noncoding regions, and was inserted into
pcDNA3, in which the HindIII site in the multiple
cloning site was destroyed by digestion with HindIII and
filled in with Klenow DNA polymerase. PCR mutagenesis was carried out
using the outside primers to the T7 promoter
(5'-TAA TAC GAC TCA CTA TAG GG-3') and to nucleotides 975-995 of
the TfR (5'-ATG TCC AAA GAA TGA AAGTTC-3'). The inside,
overlapping, and mutagenic primers for creating the P59A TfR were
5'-AAG GCC AAT GTC ACA AAA GCA AAA AGG TGT AGT-3' and 5'-TTT
TGT GAC ATT GGC CTT-3', and for creating the
29-59 TfR they
were 5'-AGC CTG GCT CGG CAA AAA AGG TGT AGT GGA AGT-3' and
5'-TTG CCG AGC CAG GCT-3'. The first round of PCRs, using 3 units
of Vent polymerase, from an outside primer to an inside primer created
two overlapping fragments, one containing the mutation. These fragments
were gel purified by electroelution, ethanol precipitated, and
resuspended for the second round of PCR, with 3 units of Taq polymerase (Life Technologies, Inc.), the products from the first PCR
round, and the two outside primers, to create a full-length mutated
fragment. The fragment was gel purified, digested with EcoRV
and HindIII, and ligated into the pcDNA3 TfR plasmid,
which had been digested with EcoRV and HindIII.
The resulting plasmids were transformed into DH5'
bacteria, and
colonies were screened for the correct plasmid, which was then
sequenced along the entire length that had been subjected to PCR, about
900 nucleotides. No errors were found. The T104D
3-59 was made by
loop-out mutagenesis of the T104D construct using the Amersham
site-directed mutagenesis kit and the oligonucleotide
5'-CAG TTC AGA ATG ATG AAA AGG TGT AGT GGA-3'.
Gel Electrophoresis-- Samples were processed for electrophoresis and Western blots as described previously (16). Western blots were developed by incubation with a sheep anti-TfR serum (16) (1:10,000), horseradish peroxidase-conjugated swine anti-sheep (1:10,000) and were visualized by enhanced chemiluminescence ECL (Amersham Pharmacia Biotech).
Purification of Soluble TfR and Amino Acid
Sequencing--
Soluble TfR was purified from 5.5 liters of
conditioned medium from TRVb cells expressing the 3-59 TfR using a
5-ml transferrin-agarose column as described previously (10). Amino
acid sequencing was carried out as described previously (10). Samples
were subjected to SDS-PAGE and blotted onto polyvinylidene difluoride,
which was subsequently Coomassie stained and destained. Protein bands were excised and subjected to automated Edman degradation sequence analysis in either an Applied Biosystems Inc. 476A or 477A system using
standard sequencing cycles. Amino acid determinations were made by
visual inspection of resulting chromatograms.
Biotinylation of Cell Surface Proteins-- Cells (35-mm dishes) were washed with phosphate-buffered saline and cooled on ice. They were incubated with 1 ml of 3.4 mM sulfosuccinimidyl biotin (Pierce) dissolved in phosphate-buffered saline just before use to label cell surface proteins. After 30 min on ice they were washed three times with medium and incubated in 2 ml of medium overnight (12-17 h) at 37 °C in 5% CO2. The medium was collected, and the cells were washed and lysed with 1 ml of 50 mM Tris, 5 mM EDTA, 150 mM NaCl, pH 7.5, 1% Triton X-100. The TfR was isolated from the lysates and media with 100 µl and 200 µl of transferrin-agarose, respectively. It was eluted with 2 × Laemmli buffer (17) lacking reducing agents. Samples were subjected to SDS-PAGE under nonreducing conditions.
Pulse-Chase with [35S]Met/Cys-- Cells (35-mm dishes) were pulsed for the indicated times with [35S]Met/Cys in medium lacking Met as described previously (18). After labeling they were washed twice with complete medium and incubated at 37 °C, 5% CO2. At the indicated times the medium was collected, and the cells were washed twice with phosphate-buffered saline and lysed with 0.05 M Tris-Cl, 0.15 M NaCl, 5 mM EDTA, pH 7.5, 1% Triton X-100. The TfR was isolated from the medium with transferrin-agarose and from the lysate by immunoprecipitation with anti-TfR serum (5, 6, 10, 16).
Endo H Treatment-- Cells labeled with 100 µCi of [35S]Met/Cys for 10 min were washed, solubilized, and immunoprecipitated with anti-TfR serum. Half of the immunoprecipitate was digested for 2 h at 37 °C with 50 milliunits of endo H in sodium citrate buffer, pH 6, according to manufacturer's instructions (New England Biolabs). The other half was mock digested under the same conditions. The TfR was eluted from the immunoprecipitate with 2 × Laemmli buffer and subjected to SDS-PAGE under reducing conditions.
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RESULTS |
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Characterization of Two Juxtamembrane Cleavage Sites in the Ectodomain of the TfR-- We have shown previously that the soluble TfR found in blood is generated by cleavage of the wild type TfR at Arg-100 by an unidentified protease (3). The soluble TfR fragment migrates as a monomer on nonreducing SDS gels, indicating that it does not contain any intersubunit disulfide bonds. Elimination of the O-linked carbohydrate by mutating the Thr at position 104 to any number of amino acids potentiated the cleavage of the TfR at Arg-100 (10). Cleavage is affected by the composition of the O-linked carbohydrate four amino acids away and is generated in endocytic vesicles (5, 6, 10).
The cleavage pattern of a TfR in which the cytoplasmic domain has been deleted (
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Cleavage of the TfR at Gly-91 Is Not Correlated with Time Spent on
the Cell Surface--
Previous studies indicated that cleavage of the
TfR at Arg-100 occurs when the TfR cycles through the endocytic
compartments (5). The TfR contains the endocytic sequence YTRF in the
cytoplasmic domain at amino acids 20-23. The 3-59 TfR is defective
in endocytosis and accumulates on the cell surface (13, 19, 26).
Therefore we wanted to test whether the Gly-91 cleavage was occurring
at the plasma membrane because cell surface metalloproteases such as
TACE have been implicated in the cleavage of an increasing number of
proteins such as the tumor necrosis factor
, receptors, and cell
adhesion molecules (27-30). Mutation of two critical amino acids,
Tyr-20
Cys and Phe-23
Ala (C20A23 TfR), or deletion of the
endocytic signal (
3-28 TfR) results in TfRs equally as defective in
endocytosis as the
3-59 TfR (11, 19, 20). Thus if cleavage was on
the cell surface then these cytoplasmic mutations should result in the
release of a soluble disulfide-linked TfR dimer into the medium. No
detectable amounts of cleaved TfR above that of the wild type TfR could
be detected in the medium even when large amounts of medium were
collected and the blots were exposed longer than normal (Fig.
3). These results indicate that a cell
surface protease was not responsible for the cleavage of the TfR at
Gly-91. They differ from the generation of the soluble TfR cleaved at
Arg-100. In the latter case, elimination of the endocytic signal
resulted in less soluble TfR being produced (5). Because cleavage at
Gly-91 is seen when the entire cytoplasmic domain is missing and not
when the region from amino acids 3-28 is missing, this suggests that
the region 29-59 may be important for generating the soluble
disulfide-linked TfR.
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Cleavage of the TfR at Gly-91 Is Potentiated by Deletion of the
Juxtamembrane Region of the Cytoplasmic Domain--
The TfR with a
deletion in the juxtamembrane region was generated to determine whether
this sequence is important in the generation of the soluble
disulfide-linked TfR dimer. A soluble TfR containing intersubunit
disulfide bonds is generated from 29-59 TfR (Fig. 4). This process is not as efficient as
in the
3-59 TfR (Fig. 3). The cleavage of the TfR missing the
NH2-terminal 26 amino acids in the cytoplasmic domain
(
3-28 TfR) was compared with the cleavage of the TfR missing the 31 transmembrane-proximal amino acids (
29-59 TfR) (Figs. 3 and 4). The
3-59 TfR produces a greater amount of soluble disulfide-bonded
dimer than does the
29-59 TfR. Because the
29-59 TfR and the
3-28 TfR have similarly sized cytoplasmic domains, the composition
of the cytoplasmic domain is likely to be more important than the
length of the cytoplasmic domain in regard to the production of this
form of the soluble TfR.
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Cleavage of the TfR at Gly-91 Occurs during the Biosynthesis of the
TfR--
Earlier studies indicated that generation of the soluble TfR
by cleavage at Arg-100 occurred after the TfR had reached the plasma
membrane and while it was cycling through endocytic compartments in the
cell (5). Two methods were used to determine that most of the soluble
TfR cleaved at Gly-91 is generated during the biosynthesis of the TfR
before it reaches the cell surface. In one set of experiments cells
were labeled with [35S]Met/Cys for 1 h, washed, and
chased for up to 24 h. Nonreducing SDS-PAGE analysis of newly
synthesized 3-59 TfR indicates that the soluble receptor that is
cleaved at Gly-91 (dimer on nonreducing SDS gels) is generated within
the first 2 h of its synthesis, and very little additional dimer
is generated with a longer chase. The cleavage at Arg-100 (monomer on
nonreducing SDS gels) takes place continuously and linearly during the
lifetime of the TfR (Fig. 6). In another
set of experiments, cells were labeled overnight with
[35S]Met/Cys, washed to remove soluble TfR generated soon
after labeling, and chased with nonradioactive medium. Most of the
radioactivity is in the mature fully glycosylated form of the TfR and
less in immature forms under these conditions. Production of soluble
disulfide-linked dimer from mature TfR is reduced compared with that
from newly synthesized TfR. The results from these experiments indicate
that the soluble disulfide-linked dimer is generated during the
biosynthesis of the TfR. A precursor-product relationship of dimer
being converted to monomer is not seen because the amount of dimer
remains stable instead of decreasing. These results are consistent with
the hypothesis that the soluble disulfide-linked TfR cleaved at Gly-91
is generated in the biosynthetic pathway, and the soluble TfR cleaved
at Arg-100 is generated from the mature TfR.
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Pulse-Chase and Endo H Analysis of Wild Type and Truncated Forms of
the TfR--
Because the cleavage of the TfR at Gly-91 appears to
occur in the biosynthetic pathway of the TfR we examined the transit of
several TfR mutants through this pathway. The loss of endo H
sensitivity was used as a measure of how quickly the wild type and
mutant forms of the TfR transited through the early portions of the
biosynthetic pathway. The mature TfR contains a complex oligosaccharide
at Arg-253, a hybrid oligosaccharide at Arg-319, and a high mannose
oligosaccharide at Arg-727 (33) so that the TfR never becomes
completely resistant to endo H digestion. However, the loss of complete
sensitivity to endo H can be followed. The wild type and 3-28 TfRs
become partially endo H-insensitive within 30-60 min of the chase
period (Fig. 8A). In contrast,
the
29-59 and the
3-59 TfRs are significantly retarded in their
loss of endo H sensitivity, indicating that they pass through the early portion of the biosynthetic pathway slower than the wild type or
3-29 TfRs. Quantitation of the loss of endo H sensitivity confirms
these observations (Fig. 8B). Transit through the
biosynthetic pathway can be affected by the level of expression of
transfected proteins. Overexpressed proteins can saturate the
biosynthetic pathway and slow their transit. All of the transfected
cell lines expressed within a factor of three the same amounts of TfR.
The highest expression level was that of the wild type TfR. This level was within the same range of concentrations of wild type TfR measured in human cell lines (1-3 × 105 surface TfR/cell).
Therefore, the biosynthetic pathway should not be saturated in these
cell lines.
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DISCUSSION |
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Soluble counterparts of a variety of cell surface proteins have
been found in serum. Among these are transforming growth factor (34), kit receptor ligands (35), interleukin-2 receptor (36, 37),
mannose 6-phosphate receptor (38), platelet-derived growth factor
receptor (39), and the transferrin receptor (40). Cleavage occurs,
depending on the protein, at various cellular locations. For example,
cleavage of transforming growth factor
occurs at the plasma
membrane (41), the TfR is cleaved within an endocytic compartment (5),
and the H2a subunit of the asialoglycoprotein receptor is cleaved
during transit through the biosynthetic pathways (42). The
physiological functions of the soluble receptors vary. In the case of
signaling receptors (e.g. interleukin-2 receptor), cleavage
of the receptor provides a mechanism for regulating the signal
responsiveness of cells (37). In most cases the functional significance
of cleavage of nonsignaling receptors is not known, but it could be a
mechanism of rapidly down-regulating the receptor.
The extracellular region of the TfR proximal to the transmembrane domain (~Cys-89 through Arg-120) appears to be relatively unstructured in that it is susceptible to several cellular and extracellular proteases. The soluble form of the TfR found in blood is cleaved at Arg-100 (43, 44) and is partially protected by O-linked glycosylation at Thr-104 (5, 6, 10). When cells are treated with low concentrations of trypsin the TfR is released by cleavage at Arg-120 (45). In this study, we have identified a third cleavage site at Gly-91 when membrane-proximal portion of the TfR cytoplasmic domain is deleted. Because the soluble TfR remains a dimer and is fully capable of binding transferrin, the region between Cys-89 and Arg-120 does not appear to play a critical role in the structure of the TfR.
In this set of studies we have demonstrated that alteration of the
cytoplasmic domain of the TfR results in its enhanced cleavage. The
cleavage occurs in the ectodomain of the molecule close to the
membrane. Deletion of the portion of the TfR cytoplasmic domain proximal to the transmembrane region correlates with the cleavage of
the extracellular domain. Pulse-chase and biotinylation studies were
used to determine that unlike the cleavage of the TfR at the other
protease susceptible sights, this cleavage occurs in the biosynthetic
pathway. It correlates with the kinetics of the acquisition of endo H
resistance, indicating that it occurs early in the biosynthetic
pathway. The full-length TfR and the 3-29 TfR transit through the
endo H compartments quickly and are not cleaved significantly. The
extent of cleavage of the other TfR mutants,
29-59 TfR and
3-59
TfR, correlates with slowed exit from the endo H-sensitive
compartments. Thus the most cleavage was seen in the mutant TfR lacking
all but three amino acids on the cytoplasmic domain. Mutant TfRs
lacking the membrane-proximal portion of the cytoplasmic domain were
the slowest to exit the endo H-sensitive compartment and the most
susceptible to the protease. Cleavage occurs between two hydrophobic
amino acids consistent the possible involvement of the ER signal
peptidase. In agreement with this hypothesis is an observation by Hoe
and Hunt (46) that a mutated form of the TfR, unable to exit the ER,
was cleaved at Gly-91. A similar cleavage close to the transmembrane
domain was seen with the H2a subunit of the asialoglycoprotein receptor when expressed in the absence of the subunit (42). In this case, the
subunit was retained in the ER and cleaved.
Selective transport of proteins out of the ER was first proposed in the
1980s (47, 48). Different transport kinetics were observed between two
retroviral membrane proteins (47). In hepatocytes, serum proteins
varied widely in the acquisition of endo H resistance, ranging from
1-antitrypsin (t1/2 = 25 min) to transferrin
(t1/2 = 180 min) (48). The time to transit
through the Golgi was relatively uniform (~20 min). Evidence for
concentrative packaging of proteins into transport vesicles and
potential transport receptors that facilitate protein exit was sparse.
Contrary to this hypothesis, Wieland and colleagues (49) presented
evidence that small peptides could transit through the ER quicker than
newly synthesized proteins. These results were interpreted to mean that
protein folding was the rate-limiting step. In the absence of
retention, there was bulk flow from the ER to the Golgi. In keeping
with this idea, proteins that fold at a slower rate are retained in the
ER associated with resident ER chaperone proteins (50). Rose and
Bergmann (51) demonstrated that deletions in portions of the
cytoplasmic domain of vesicular stomatitis virus G (VSV-G) could slow
the exit of this protein from the ER without influencing the folding kinetics. Balch and co-workers (52) have again proposed a specific concentrative step to be involved in the exit of VSV-G from the ER.
This evidence is based on quantitation of the amount of VSV-G in
vesicles budding from the ER by electron microscopy. Most recently Nishimura and Balch (53) demonstrated that a diacidic signal (Asp-X-Glu) was required for efficient exit of VSV-G from
the ER. The transmembrane-proximal portion of the TfR (amino acids 29-59) which appears to participate in efficient transport of the TfR
out of the endo H-sensitive compartment contains the sequence Asp-Glu-Glu-Glu (amino acids 43-48). Thus, both the folding kinetics and cytoplasmic domains of proteins can influence the rate of exit from
the ER.
Future work is aimed at distinguishing between the possibilities that specific signals in the membrane-proximal portion of the cytoplasmic domain influence the concentration of the TfR into vesicles exiting the ER or alter the kinetics of the TfR lumenal domain folding.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants DK40608 (to C. A. E.) and DK52852 (to T. E. M.) and by Postdoctoral National Institutes of Health Award F32DK08910 (to E. A. R.).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.
§ Present address: Markey Molecular Medicine Center, Box 357720, University of Washington, Seattle, WA 98195.
** To whom correspondence should be addressed: Dept. of Cell and Developmental Biology, L215, Oregon Health Sciences University, Portland, OR 97201-3098. Tel.: 503-494-5845; Fax: 503-494-4253; E-mail: ennsca{at}ohsu.edu.
1
The abbreviations used are: TfR, transferrin
receptor; ER, endoplasmic reticulum; endo H,
endo--N-acetylglucosaminidase H (EC 3.2.1.96); PCR,
polymerase chain reaction; ECL, enhanced chemiluminescence; PAGE,
polyacrylamide gel electrophoresis; O-linked, serine/threonine-linked; VSV-G, vesicular stomatitis virus G.
2 E. A. Rutledge, I. Gaston, B. J. Root, T. E. McGraw, and C. A. Enns, unpublished observations.
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REFERENCES |
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