Calreticulin is an endoplasmic reticulum resident
molecule known to be involved in the folding and assembly of major
histocompatibility complex (MHC) class I molecules. In the present
study, expression of calreticulin was analyzed in human peripheral
blood T lymphocytes. Pulse-chase experiments in
[35S]methionine-labeled T cell blasts showed that
calreticulin was associated with several proteins in the endoplasmic
reticulum and suggested that it was expressed at the cell surface.
Indeed, the 60-kDa calreticulin was labeled by cell surface
biotinylation and precipitated from the surface of activated T cells
together with a protein with an apparent molecular mass of 46 kDa. Cell surface expression of calreticulin by activated T lymphocytes was
further confirmed by immunofluorescence and flow cytometry, studies
that showed that both CD8+ and CD4+ T cells expressed calreticulin in
the plasma membrane. Low amounts of cell surface calreticulin were
detected in resting T lymphocytes. By sequential immunoprecipitation
using the conformation independent monoclonal antibody HC-10, we
provided evidence that the cell surface 46-kDa protein co-precipitated
with calreticulin is unfolded MHC I. These results show for the first
time that after T cell activation, significant amounts of calreticulin
are expressed on the T cell surface, where they are found in physical
association with a pool of Calreticulin is a highly conserved and widely tissue distributed
calcium-binding protein with a C-terminal KDEL endoplasmic reticulum
(ER)1 retrieval sequence
(1-3). However, calreticulin has also been found outside the ER, such
as within the secretory granules of cytotoxic lymphocytes, the cell
surface of melanoma cells and virus-infected fibroblasts, and the
cytosol and nucleus of several cell types (Ref. 4-8, reviewed in Ref.
9). Given its lectin-like properties, calreticulin is considered to be
an ER chaperone involved in the assembly and folding of nascent
glycoproteins (10-13).
Mature MHC class I molecules are composites of a 44-49-kDa polymorphic
heavy chain and a 12-kDa light chain ( Recent studies on the biosynthesis of TcR·CD3 complexes suggest that
some chaperones such as calnexin can escape the ER retention mechanisms
and be expressed on the cell surface of immature thymocytes in
association with TcR Cells and Reagents--
Fresh human peripheral blood mononuclear
cells were obtained from buffy coats of regular healthy blood donors
after centrifugation over Lymphoprep (Nycomed, Norway). Partially
purified resting peripheral blood T lymphocytes (usually >80%) were
obtained by conventional adherence techniques and referred to as PBLs
as described previously (33). T cell blasts were obtained after
stimulation of PBLs in RPMI complete media (1% fetal calf serum, 1%
penicillin/streptomycin, 1% glutamine, 25 mM HEPES) with 5 µg/ml phytohemagglutinin (Sigma). After 5 days in a incubator at
37 °C, 5% CO2, and 99% humidity, T cell blasts
(usually >95% CD3+ cells) were processed for subsequent studies.
Antibodies--
The following antibodies were used in the
present study. W6/32 (Dakopatts, Copenhagen, Denmark) is a mouse
monoclonal Ab that recognizes a monomorphic epitope on all HLA heavy
chains, dependent on the presence of Cell Staining and Immunofluorescence--
Staining steps of
resting and activated T cells for flow cytometry studies were performed
at 4 °C for 30 min in staining solution (PBS, 0.2% bovine serum
albumin, 0.1% NaN3) in round-bottom microtiter plates
(Greiner, Nürtingen, Germany) with ~0.5 × 106
cells/well as described previously (38). After staining, the cells were
washed three times in staining solution and analyzed in a FACScan
(Becton and Dickinson, Mountain View, CA). For each sample 10,000 viable lymphocytes were acquired using forward and side scatter
characteristics and subsequently analyzed using the Lysys II software.
For immunofluorescence studies, cytospins of resting and activated T
cells (100,000 cells/cytospin) were air-dried and fixed with 3.7%
formaldehyde. Cells were blocked for 1 h at room temperature with
5% nonfat dry milk in PBS. Cytospins were incubated for 1 h with
PA3-900 (1:100 dilution) followed by fluorescein isothiocyanate-conjugated secondary Ab (1:20 dilution) for 1 additional h. Preparations were mounted with Vectastain H1000 mounting media (Vector Laboratories, USA).
Cell Labeling, Cell Lysis, and Immunoprecipitation--
Western Blots--
Activated human PBLs were metabolically labeled with
[35S]methionine for 15 min and chased in
methionine-supplemented media at different times. Brij 96 detergent
lysates were immunoprecipitated with anti-calreticulin Abs, and the
immunoprecipitates were analyzed by SDS/PAGE. Numerous ER proteins
co-precipitated with the 60-kDa calreticulin at time 0 after the 15-min
pulse (Fig. 1). Among those, several have
been previously identified as transporter-associated protein (70-72
kDa), tapasin (48 kDa), and MHC class I (46 kDa) (21). Additional bands
corresponding to unidentified proteins of ~97, 50, 42, and 20 kDa
were also visible. Interestingly, most of the pulse-labeled proteins,
including calreticulin, rapidly disappeared 1 h after the initial
pulse, with the marked exception of the 46-kDa band. Thus, 4 h
after the pulse, although barely detectable labeled calreticulin was
observed, a strong 46-kDa labeled protein was still co-precipitated by
anti-calreticulin Abs (Fig. 1). Four h after the initial pulse, all
proteins associated with calreticulin that are targeted to the cell
surface would have reached the plasma membrane unless they were
endocytosed and/or degraded. Among these, the survival of surface MHC
class I molecules in activated human T cells is known to be increased (39). Therefore, we suspected that newly synthesized "unlabeled" calreticulin was present in the cell surface of activated T cells in
association with the mature 46-kDa MHC class I heavy chain.
To address this question, resting and activated human PBLs were cell
surface-labeled with biotin, lysed in Brij 96, and immunoprecipitated with the PA3-900 and W6/32 Abs. PA3-900 immunoprecipitated the 60-kDa
calreticulin from lysates of biotinylated resting and activated PBLs
(Fig. 2A). The amount of
precipitated calreticulin was, however, approximately 3-fold higher in
activated PBLs. To note, a protein of 46 kDa similar to the one
immunoprecipitated by W6/32 Abs, which recognize mature
Laboratory of Molecular Immunology,
Department
of Hematology,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-free MHC class I molecules.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2m) complexed with
a cytosolic-processed peptide that are expressed on the plasma membrane
of almost every nucleated cell (14). A number of ER resident molecular
chaperones, such as calnexin, BiP, and transporter-associated protein,
are involved in the assembly of the different composites (15-18).
Recently, calreticulin has also been shown to function as a chaperone
in the assembly and folding of MHC class I molecules in the ER
(19-22). Contrary to calnexin, calreticulin binds to MHC class
I-
2m dimers and to transporter-associated protein via another chaperone, tapasin (19, 21). After peptide loading and
deglucosylation of N-linked glycans, calreticulin
dissociates from the MHC class I-
2m dimers, thus
allowing the final transport of mature MHC class I molecules to the
cell surface (20).
chains (23-27). On the contrary, studies on
the biosynthesis of MHC class I molecules have never reported associations of ER chaperones with MHC class I molecules outside the ER
and Golgi compartments (19-22, 28-30). Given that calreticulin displays multiple functions and has long been associated with several
chronic diseases (31, 32), we undertook a study of calreticulin
expression on human peripheral blood T lymphocytes to gain new insights
into possible biological functions of this molecular chaperone. In the
present study, we report the finding that calreticulin is expressed at
the cell surface of activated human peripheral blood T lymphocytes,
where it is physically associated with a pool of unfolded MHC class I molecules.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2m (34). PA3-900
(Affinity Bioreagents, Golden, CO) is a polyclonal rabbit serum that
recognizes the molecular chaperone calreticulin (35). HC-10 is a mouse
monoclonal Ab that reacts preferentially with unfolded HLA B and C
heavy chains not associated with
2m (36). Anti-C1q-R Ab
is a rabbit polyclonal serum that recognizes the cC1q receptor (37). A
rabbit serum against the intracellular tyrosine kinase FYN was a gift
of Dr. Chris E. Rudd (Dana Farber Cancer Institute, Boston, MA). A
rabbit serum against the intracellular tyrosine phosphatase SHP-1 was a
gift of Dr. R. J. Matthews (Howard Hughes Medical Institute, St.
Louis, MO). Mouse anti-human CD8-PE and CD4-Cy5PE and swine anti-rabbit
fluorescein isothiocyanate (F(ab')2 fraction) were from Dakopatts.
For
cell surface biotinylation, resting or activated T cells
(107 cells/ml) were incubated with 0.5 mg/ml
sulfosuccinimidyl 6-biotinamido-hexanoate (EZ-LinkTM
Sulfo-NHS-LC-biotin) (Pierce) in PBS for 10 min at room temperature
followed by four washes in PBS. For metabolic labeling, activated T
cells were cultured in methionine-deficient media (Sigma) for 1 h
and pulse-labeled with 100 µCi of [35S]methionine
(Amersham Pharmacia Biotech) for 15 min. Cells were chased at different
times, as indicated in the results, after removing the labeling media
and adding complete RPMI media. Labeled cells were lysed in lysis
buffer (20 mM Tris, pH 7.6, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, and 1% Brij) for 30 min
on ice. In some experiments, Triton X-100 was used in the lysis buffer (see Fig. 2B legend). The lysates were centrifuged at 12,000 ×g to remove cell debris and precleared for 30 min by
end-over-end rotation with protein-A-Sepharose CL-4B beads (Amersham
Pharmacia Biotech). Precleared detergent lysates corresponding to
107cells, unless otherwise indicated, were
immunoprecipitated with the proper antibodies and protein-A-Sepharose
CL-4B beads for 2 h at 4 °C by end-over-end rotation. In some
experiments, biotinylation of whole cell lysates was performed as
follows. Resting or activated T cells were lysed in lysis buffer (20 mM Tris, pH 7.6, 150 mM NaCl, 1 mM
phenylmethylsulfonyl fluoride, and 1% Brij) for 30 min on ice and
processed as above. Precleared detergent lysates were incubated with
0.5 mg/ml N-hydroxysuccinimide sulfobiotin (Pierce) in PBS
for 10 min at room temperature followed by centrifugation at 12,000 ×g. Supernatants were used for subsequent
immunoprecipitations as described above. Washed immunoprecipitates were
boiled for 5 min in 1% SDS sample buffer and resolved by SDS/PAGE.
Polyacrylamide gels from 35S-labeled samples were fixed and
processed for autoradiography using Biomax MR-1 films (Eastman Kodak
Co.). For re-precipitation experiments, the primary immune complexes
were boiled for 5 min in 2% SDS and diluted 10-fold with lysis buffer.
The beads were spun down, and the supernatants were recovered and
precleared for 30 min with protein A-Sepharose CL-4B beads. Proteins
were precipitated with the proper Abs plus protein A-Sepharose CL-4B beads overnight. Immunoprecipitates were washed three times, boiled for
5 min in 1% SDS buffer, and resolved by SDS/PAGE.
Polyacrylamide gels from biotin-labeled
samples were electroblotted to nitrocellulose membranes (Hybond
C-super, Amersham Pharmacia Biotech) and blocked with 5% nonfat dry
milk in Tris-buffered saline, 0.1% Tween. Proteins were visualized
after incubation with ExtrAvidin-conjugated horseradish peroxidase
(Sigma) and exposure to Biomax MR-1 film using enhanced
chemoluminiscence (ECL or ECL+, Amersham Pharmacia Biotech). For
immunodetection of MHC class I heavy chains in blocked nitrocellulose
filters, the HC-10 Ab was used, followed by incubation with horseradish peroxidaseconjugated goat anti-mouse antibody (Transduction
Laboratories, Lexington, UK).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (68K):
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Fig. 1.
Co-precipitation of calreticulin and a 46-kDa
protein from lysates of [35S]methionine-labeled activated
human PBLs. Five-day-activated human PBLs (40 × 106 cells) were metabolically labeled for 15 min with 100 µCi of [35S]methionine and chased for the periods
indicated in hours. At each time point, 107 cells were
solubilized in 1% Brij 96 lysis buffer, and lysates were
immunoprecipitated with PA3-900. Precipitates were boiled in 1% SDS
sample buffer, and aliquots were separated on 12% SDS, PAGE under
denaturing conditions. Gels were dried and exposed to Biowax MR-1 film
for 7 days. Calreticulin (CRT) and the co-precipitated
46-kDa protein band are indicated by arrows. Molecular mass
markers in kDa are indicated on the left.
2m-associated MHC I, was again co-precipitated with
calreticulin in activated but not resting PBLs (Fig. 2A). These results provided evidence that the ER resident chaperone calreticulin was expressed in the cell surface of human PBLs, that its
expression increased after T cell activation, and that it was
associated with a 46-kDa protein corresponding to the mobility of the
MHC I heavy chain. The use of more stringent conditions during the cell
lysis procedure (i.e. 2% Triton X-100), although decreasing
markedly the amount of the co-precipitated 46-kDa protein, did not
completely disrupt the association (Fig. 2B). The observed difference between detergents did not reflect less protein extraction by Triton X-100, as both detergents extracted similar amounts of
calreticulin (PA3-900 Ab) and MHC I heavy chain (W6/32 Ab). These
results suggest that this association might be of physiological importance and rules out the possibility that the co-precipitation of
the 46-kDa protein with the PA3-900 Ab is the consequence of an
artifact resulting from cross-reactivity. It should be emphasized that
there was some variation in the amount of the 46-kDa protein co-precipitated with calreticulin between the individuals studied.
View larger version (37K):
[in a new window]
Fig. 2.
Co-precipitation of calreticulin and a 46-kDa
protein from the surface of biotinylated resting and activated human
PBLs. A, resting and 5 day-activated human PBLs were
surface-biotinylated. Cells were solubilized in 1% Brij 96, and cell
lysates corresponding to 107 cells immunoprecipitated with
beads only (NONE), with anti-MHC class I heavy chain Abs
(W6/32), and with anti-calreticulin Abs (PA3) are
shown. Precipitates were boiled in 1% SDS and separated on 10%
SDS/PAGE under denaturing conditions. Proteins were transferred to
nitrocellulose filters, and biotinylated proteins were visualized by
enhanced chemoluminiscence (ECL). B, Five-day-activated
human PBLs were surface-biotinylated and solubilized either in 1% Brij
96 or 2% Triton X-100. Cell lysates corresponding to 107
cells were immunoprecipitated with anti-calreticulin Abs
(PA3-900) and anti-MHC class I heavy chain Abs
(W6/32). Calreticulin (CRT) and the
co-precipitated 46-kDa protein are indicated by arrows.
Molecular mass markers in kDa are indicated on the left.
Calreticulin or calreticulin-like proteins have been described in
almost every compartment of the cell. Recent reports have shown that
human T cells express receptors for the C1q protein and that one of
those receptors (cC1q-R) shows homology with calreticulin (40, 41). To
rule out the possibility that our PA3-900 antibody was recognizing the
cC1q receptor, we performed depletion experiments. As shown in Fig.
3, depletion of T cell surface
calreticulin by four rounds of immunoprecipitation with the PA3-900 Ab
did not abolish immunoprecipitation of the cC1q receptor. These data
provide evidence that the calreticulin expressed in the surface of
activated human T lymphocytes is apparently different from the C1q
receptor. Next, we wanted to rule out the possibility that the
calreticulin we were detecting on the cell surface of human PBLs could
be result of intracellular biotinylation because of background labeling during the handling of the cells. Using activated human PBLs, immunoprecipitation of Brij 96 cell lysates from cell
surface-biotinylated cells allowed the detection of cell
surface-expressed molecules, such as calreticulin, but not of
intracellular molecules such as the protein-tyrosine kinase FYN and the
protein-tyrosine phosphatase SHP-1 (Fig.
4, left panel). On the
contrary, immunoprecipitation of biotinylated Brij 96 cell lysates
allowed the detection of both sets of molecules, together with a number
of co-precipitated proteins (Fig. 4, right panel).
Interestingly, antibodies against the intracellular tyrosine kinase FYN
were able to co-precipitate a strong band of similar mobility to the
MHC class I heavy chain (Fig. 4, right panel).
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To further characterize calreticulin expression on human PBLs,
immunofluorescence microscopy and flow cytometry were performed. For
that purpose, cytospins of resting and activated human PBLs were
stained with PA3-900 followed by a F(ab')2 fraction of swine anti-rabbit fluorescein isothiocyanate conjugated Ab to avoid nonspecific binding to Fc receptors in activated T cells. The results
demonstrated a faint cell surface expression of calreticulin in resting
PBLs that markedly increased in activated T cells (Fig. 5A), which is in agreement
with the immunoprecipitation results (Fig. 2). Permeabilized PBLs,
either resting or activated, showed a conspicuous cytoplasmic staining
(data not shown). To determine whether calreticulin was differentially
expressed by the two major peripheral blood T cell subsets, two color
flow cytometry studies were performed. The results showed that both
CD8+ and CD4+ activated T cells expressed calreticulin at similar
levels (Fig. 5B).
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Finally, we wanted to confirm the identity of the 46-kDa protein
co-precipitated with calreticulin. Because the W6/32 Ab was unable to
co-precipitate a protein of similar mobility with calreticulin, we
decided to include in our studies the conformation-independent Ab
HC-10, which detects 2m-free MHC class I molecules. As
shown in Fig. 6A, calreticulin
Abs were able again to co-precipitate low but significant amounts of a
protein with 46 kDa. Re-precipitation of the primary immunoprecipitates
with HC-10 demonstrated that the 46-kDa protein co-precipitating with
calreticulin was indeed the MHC class I heavy chain (Fig.
6B). Immunodetection using the HC-10 Ab further confirmed
that the 46-kDa band co-precipitating with calreticulin corresponds to
the MHC class I heavy chain (data not shown). On the other hand, HC-10
but not W6/32 Abs were able to co-precipitate a protein of the same
mobility as calreticulin from the surface of activated human PBLs (Fig.
6A). Re-precipitation of the primary immunoprecipitates with
the PA3-900 Ab confirmed that calreticulin could be re-precipitated
from HC-10 but not W6/32 primary immunoprecipitates (Fig.
6C), therefore demonstrating that calreticulin is associated
with a pool of unfolded
2m-free MHC class I molecules on
the cell surface of activated human PBLs.
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DISCUSSION |
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Recent studies on calreticulin are disclosing the key role played by this molecular chaperone in the folding and assembly of MHC class I molecules in the ER (19-22). However, a number of studies indicate that calreticulin is involved in a number of biochemical and physiological functions unrelated with its ER chaperone role. These studies have shown that calreticulin can be found in different locations within the cell, from the nucleus to the plasma membrane of several cell lines (reviewed in Ref. 31). How calreticulin can escape the ER retention mechanisms and move into the cytoplasm, nucleus, or plasma membrane is an enigma that challenges current biology. In addition, calreticulin is thought to be involved in a number of diseases, all sharing an immunological basis (32, 42). All these findings have led to the suggestion that calreticulin may represent a family of highly homologous proteins.
The present study addressed the characterization of calreticulin expression in human peripheral blood T lymphocytes. Our interest in studying calreticulin relies 1) on our previous work demonstrating that T lymphocytes and classical MHC class I molecules play a regulatory role in tissue damage under excessive iron accumulation caused by a primary defect in an MHC class I like molecule (reviewed in Ref. 43) and 2) that calreticulin has also been described as an iron-binding protein (44). The few studies of calreticulin in human T lymphocytes have shown that the levels of calreticulin mRNA and protein increase after mitogenic stimulation and that calreticulin is a constituent of lytic granules in lymphokine-activated killer cells (4, 45). The present results extend these studies by showing that calreticulin is expressed on the plasma membrane of peripheral blood human T lymphocytes and that cell surface expression increases after T cell activation.
Previous studies have shown that calreticulin or calreticulin-like molecules are expressed on the plasma membrane of transformed cell lines of different origin. These studies raised the question of how calreticulin can be expressed at the cell surface because it lacks a transmembrane domain. A likely explanation is that cell surface calreticulin is anchored to other(s) surface molecules (46, 47). By cell surface biotinylation and immunoprecipitation, we have provided evidence that calreticulin co-precipitates with several T cell surface molecules, and we have identified MHC class I molecules as one of these T cell surface molecules. Escape of ER resident molecular chaperones to the plasma membrane has previously been reported in several cell types including thymic lymphoma cells (24, 27, 31, 42). It has been argued that this may result from changes in the relative availability of KDEL receptors present in post-ER compartments and known to be involved in the retrograde transport of proteins containing this C-terminal retrieval sequence (48-50). Because we have detected calreticulin on the surface of normal human T lymphocytes, it is not unlikely to anticipate that other KDEL-containing ER chaperones such as BiP and GRP94 could also be expressed at the T cell surface as well.
The association of calreticulin with MHC class I molecules on the
surface of activated human T cells is intriguing considering the
properties displayed by these polymorphic molecules. T cell surface MHC
class I molecules are subjected to spontaneous cycles of
endocytosis/re-expression via coated pits (39, 51-53). Moreover, endocytosed 2m-associated MHC class I molecules are
re-expressed at the cell surface as
2m-free unfolded MHC
class I molecules (54, 55). On the other hand, recent studies have
shown that calreticulin preferentially associates with unfolded MHC
class I molecules in the ER and that the oligosaccharide moiety in the
1 domain and a residue within the
3 domain are critical for interaction with calreticulin (29, 30). Considering that the calreticulin-MHC I association was detected in activated but not resting PBLs, it is interesting to notice that the increase in the
translocation of ER calreticulin to the plasma membrane observed in
activated human PBLs parallels the increase in the amount of unfolded
MHC class I molecules that arise after T cell activation (Fig.
6A and data not shown). Therefore, if under T cell
activation large amounts of calreticulin escape the ER retention
mechanisms and are translocated to the cell surface, interaction with
unfolded MHC class I molecules would be expected. Indeed, we have shown that T cell surface calreticulin is associated with unfolded but not
folded MHC class I molecules, as determined using the HC-10 Ab. This is
in agreement with previous studies showing that W6/32 Abs do not
co-precipitate calreticulin in the ER (19) and with the fact that large
amounts of unfolded MHC class I molecules can be precipitated from the
surface of activated human PBLs (Fig. 6 and data not shown). The low
amount of calreticulin co-precipitated with the HC-10 Ab observed in
this study may reflect the preferential reactivity of the HC-10 Ab with
unfolded MHC class I molecules of the B and C locus (36). Consequently,
any possible calreticulin associated with unfolded MHC class I
molecules of the A locus would not be detected. This is important,
taking into consideration that in the ER, calreticulin appears to show
different patterns of association with MHC class I molecules,
associating strongly with alleles of the A locus, such as A29 and A3,
but weakly with alleles of the B locus (19).
The similarities between the behavior of the calreticulin-MHC class I
association in the ER and the T cell surface of activated human T cells
raise intriguing questions regarding the possible physiological
significance of this association. Although in the ER the association
appears to be related with the folding and assembly of immature MHC
class I molecules, in the plasma membrane it could be related to an
important previously uncharacterized biological function related to the
proliferative status of the cells. In this context, the present
findings could be of particular relevance to the recent demonstration
that calreticulin-bound peptides obtained from tumors can elicit a
peptide-specific CD8+ T cell response (56). Calreticulin has also been
described as an Fe3+ binding protein that is found in
endocytic vesicles containing iron-transferrin/transferrin receptor
complexes, where it becomes labeled with iron released from transferrin
(57). On the other hand, surface MHC class I molecules are known to
regulate endocytosis of several receptors, such as the transferrin
receptor, a finding consistent with earlier studies demonstrating that
MHC class I molecules and transferrin receptors co-localize in
endocytic vesicles in T lymphocytes (58, 59). In this context, it is
tempting to suggest that T cell surface calreticulin, by means of its
association with MHC class I molecules, may be an intermediary in the
process of iron uptake and transfer to intracellular compartments in
dividing T lymphocytes, a hypothesis that can be easily tested. The
recent demonstration that the hemochromatosis gene product, an MHC
class I-like protein, associates with the transferrin receptor must be
taken into consideration (60-62). Alternatively, T cell surface calreticulin may also perform a chaperone function by mediating the
refolding of unfolded MHC class I molecules that arise as a result of T
cell division (63), a function that may uncover a new role for
calreticulin in protein quality control.
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ACKNOWLEDGEMENTS |
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We thank the personnel of the Blood Bank of the Hospital Geral Santo António in Porto for their cooperation and support in collecting blood samples. We thank Dr. Hidde Ploegh (Harvard Medical School, Boston, MA) and Dr. Bob Sim (Oxford University, Oxford) for providing the HC-10 and C1q-R antibodies, respectively. We also thank Dr. Robert A. Clark (University of Texas) for helpful comments during the revision of this manuscript.
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FOOTNOTES |
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* The American Portuguese Biomedical Research Fund supported this work.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.
§ Funded by a Ph.D. fellowship from Fundação para a Ciência e a Technologia (PRAXIS BD 3209/94). To whom correspondence should be addressed: Laboratory of Molecular Immunology, Institute for Molecular and Cell Biology, Rua do Campo Alegre, 823, 4150, Porto, Portugal. Tel.: 351-2-6074956; Fax: 351-2-6098480; E-mail: farosa{at}ibmc.up.pt.
¶ Present address: Ludwig Cancer Research Institute, Norfolk Place, London, W2 1PG UK.
** Funded by a post doctoral fellowship from Fundação para a Ciência e a Technologia (PRAXIS BPD 6010/95).
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ABBREVIATIONS |
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The abbreviations used are:
ER, endoplasmic reticulum;
MHC I, major histocompatibility complex class I;
2m,
2 microglobulin;
PBL, partially purified human
peripheral blood T lymphocyte;
Ab, antibody;
PBS, phosphate-buffered
saline;
PAGE, polyacrylamide gel electrophoresis.
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