Biochemie-Zentrum Heidelberg (BZH), Im Neuenheimer Feld 328, 69120 Heidelberg, Germany
Author for correspondence (e-mail:
walter.nickel{at}urz.uni-heidelberg.de)
Accepted 9 December 2002
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Summary |
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Key words: CA125, Galectin, Counter receptor, Nonclassical export, Cell-surface expression, Tumor antigen
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Introduction |
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Only recently was the primary structure of CA125 elucidated, demonstrating
that CA125 represents a giant mucin-like glycoprotein
(O'Brien et al., 2001;
Yin and Lloyd, 2001
). On this
basis, CA125 has been termed Muc16 to reflect the nature of CA125 as a new
member of the protein family of mucins
(Yin and Lloyd, 2001
).
Full-length CA125 contains more than 11,000 amino acids that form the
proteinaceous core structure (O'Brien et
al., 2001
; Yin and Lloyd,
2001
). CA125 is both N- and O-glycosylated in its N-terminal
extracellular domain (Zurawski et al.,
1988
; Nagata et al.,
1991
; Lloyd et al.,
1997
; Lloyd and Yin,
2001
), which is composed of a stalk domain next to the
transmembrane span, more than 60 repeat structures (each of which consists of
156 amino acids) and an N-terminal extension
(O'Brien et al., 2001
;
Yin and Lloyd, 2001
). Towards
the C-terminus, CA125 contains a putative transmembrane span and a short
cytoplasmic tail (O'Brien et al.,
2001
). The release of soluble fragments of CA125 into the
extracellular space appears to be triggered by serine/threonin-and/or
tyrosine-dependent phosphorylation within the cytoplasmic domain
(Fendrick et al., 1997
;
Lloyd and Yin, 2001
).
Tumor-specific cell-surface expression concomitant with the release of
extracellular fragments suggests a role for CA125 in the regulation of cell
proliferation and/or tumor progression. Owing to its nature as a glycoprotein,
potential ligands of CA125 include lectins of the extracellular matrix (ECM)
such as the ß-galactoside-specific family of galectins
(Perillo et al., 1998;
Rabinovich et al., 2002
).
Intriguingly, galectin-1 and galectin-3 expression is upregulated in various
cancer-derived cell lines compared with benign tissue
(Iurisci et al., 2000
;
Lahm et al., 2001
;
Lloyd, 2001
;
Armstrong et al., 2002
).
Moreover, one member of this family, galectin-3, has been reported to
represent a ligand of a colon cancer mucin
(Bresalier et al., 1996
).
At the molecular level, the best-characterized members of this protein
family are galectin-1 and -3 (Hughes,
1997; Hughes,
1999
). Owing to its dimeric character, cell-surface recruitment of
galectin-1 is thought to affect the conformation and oligomeric status of
glycosylated protein domains by forming intra- or intermolecular bridges that,
in turn, might exert a cellular response
(Perillo et al., 1998
).
Biological counter receptors for galectin-1 include laminin, fibronectin, lamp
1 and 2, GM1 glycolipid (reviewed by
Perillo et al., 1998
) as well
as cell-type-specific molecules such as the T-cell glycoproteins CD43 and CD45
(Perillo et al., 1995
;
Nguyen et al., 2001
). The
pattern of oligosaccharide chains presented on the cell surface of individual
cells is likely to influence the way galectin-1 interacts with their surface.
Therefore, the way a particular cell type responds to galectin-1 might also be
regulated by variations in the activity of glycosyl transferases and/or
glycosidases (Perillo et al.,
1998
). Known cellular responses to the cell-surface recruitment of
galectin-1 include a change in proliferation activity, regulation of cell
survival and regulation of cell adhesion. Interestingly, depending both on the
cellular context and its local concentration, galectin-1 exerts both
inhibitory and stimulatory effects on these processes
(Perillo et al., 1998
).
Galectin export from mammalian cells has been shown to occur in a
nonclassical manner independent of the function of the endoplasmic reticulum
(ER) and the Golgi (Cooper and Barondes,
1990; Cleves et al.,
1996
). Consistently, galectins lack a conventional signal peptide
for translocation into the ER (Cleves,
1997
; Hughes,
1999
). The balance between cytoplasmic and extracellular
populations appears to be tightly regulated
(Hughes, 1999
). For example,
galectin-1 export from muscle cells is developmentally regulated as increased
export is observed upon differentiation from myoblasts to myotubes
(Cooper and Barondes, 1990
).
Moreover, it has been reported that galectin-1 externalization can be
triggered upon differentiation of K562 leukemia-derived cells induced by
erythropoietin (Lutomski et al.,
1997
).
In the current study, we identify CA125 as a novel counter receptor for galectin-1. Using affinity chromatography and mass spectrometry, as well as immunological analyses, galectin-1 is shown to bind specifically to CA125 in a direct manner. A comparison with the second most-abundant family member, galectin-3, indicates that CA125 exhibits specificity towards galectin-1. A C-terminal fragment of CA125, CA125-C-TERM (defined by NCBI clone AK024365) retains the ability to integrate into secretory membranes and, like full-length CA125, is shown to be transported to the cell surface. Cell-surface delivery of CA125-C-TERM is demonstrated to occur by ER/Golgi-dependent vesicular transport. CA125-C-TERM is found to bind to galectin-1 with twofold higher efficiency compared with galectin-3 when expressed in HeLa cells, and with sevenfold higher efficiency compared with galectin-3 when expressed in CHO cells. These results demonstrate that CA125 represents a novel counter receptor for galectins and has binding characteristics that can be regulated by the cellular background in which it is expressed.
In order to investigate the functional significance of the interaction reported, we compared tumor-derived, CA125-expressing HeLa cells with non-tumor-derived, CA125-deficient CHO cells with regard to various galectin-1 parameters. Although we find that galectin-1 expression and cell-surface binding capacity for galectin-1 is similar in HeLa and CHO cells, we demonstrate that HeLa cells contain more than ten times as much cell-surface galectin-1 compared with CHO cells. Our results suggest that CA125 might allow tumor cells to interact differentially with the ECM in a galectin-1-dependent manner.
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Materials and Methods |
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The mAb anti-CA125 antibody OC125 was purchased from Zymed. Secondary antibodies used for western blotting were from Bio-Rad, those used for FACS sorting and indirect immunofluorescence confocal microscopy were from Molecular Probes.
Galectin-1 affinity matrix and binding experiments employing
subcellular fractions from HeLa cells
To conduct affinity purification of galectin-1-interacting proteins,
GSTgalectin-1 and GSTgalectin-3 fusion proteins, as well as GST
as a control, were expressed in E. coli BL21(DE3) cells. Cells were
resuspended in PBS containing 1 mM DTT, 0.1% (w/v) Triton X-100, 10% (w/v)
glycerol and protease inhibitor tabs (Roche), followed by homogenization using
a cell disruptor (Avestin). A 100,000 gav
supernatant was obtained and incubated with glutathione beads for 2 hours at
4°C on a rotating wheel. Following extensive washing using homogenization
buffer (see above), 250 µl of beads containing 250 µg of coupled protein
were used per binding experiment.
S-HeLa cells (ATCC CCL-2.2) were cultured in spinner flasks according to
standard procedures. Typically, cultures were grown to a density of about
6-7x105 cells per ml. Cells were collected by centrifugation
and resuspended in HeLa homogenization buffer (25 mM Tris, pH 7.5; 130 mM KCl;
protease inhibitor tabs) at 1 g cells per ml. Following cell breakage using a
Balch homogenizer (Balch and Rothman,
1985), the homogenate was sequentially centrifuged twice at 1000
gav and twice at 3500
gav. The resulting supernatant was subjected to
centrifugation at 100,000 gav. Following separation
of supernatant and sediment, the soluble fraction was diluted with PBS
(supplemented with 1 mM DTT and protease inhibitor tabs) to give a final
protein concentration of about 0.25 mg/ml. Typically, when starting with 5 g
of cells, the soluble fraction was adjusted to a final volume of 50 ml. The
corresponding sediment was then resuspended in 50 ml of PBS supplemented with
1 mM DTT, protease inhibitor tabs and 1% (w/v) NP-40 (Roche). Per experimental
condition, 25 ml of the soluble or the membrane fraction, respectively, were
incubated with 250 µl of GSH beads containing about 250 µg
GSTgalectin-1, GSTgalectin-3 or GST, respectively. Bound
proteins were eluted sequentially with 100 mM lactose and 25 mM glutathione,
respectively. Further details are given in the corresponding figure
legends.
Protein identification employing MALDI-TOF mass spectrometry
In order to identify individual proteins eluted from the galectin-1
affinity matrix, the eluates were separated on 10% Novex Bis-Tris gels
(Invitrogen) followed by protein staining using the SilverQuest system
(Invitrogen). After excision of gel pieces containing individual proteins,
in-gel trypsin digestion allowed extraction of tryptic peptides. Proteins were
identified based on the masses of the peptides obtained in this way by
employing MALDI-TOF mass spectrometry
(Wilm et al., 1996).
Retroviral transduction of MCAT-expressing HeLa and CHO cells
In order to transduce target cells with the cDNA of CA125-C-TERM, the ORF
was cloned into the vector pFB (Stratagene). This vector promotes constitutive
expression of the cDNA in question. Retroviral particles were generated by
conventional transfection of HEK293T cells employing the VPack vector system
(Stratagene). In this context, an envelope protein with an ecotropic host
range was used, encoded by the vector pVPack-eco (Stratagene). Retroviral
particles harvested from the medium of triple-transfected HEK293T cells were
added to the medium of the target cells. As target cells, HeLa and CHO cells
were used that stably express the murine cation amino acid transporter MCAT-1
(Albritton et al., 1989;
Davey et al., 1997
) and a
doxicycline-dependent transactivator
(Urlinger et al., 2000
). These
cell lines were designated HeLaMCAT-TAM2 and
CHOMCAT-TAM2, respectively
(Engling et al., 2002
).
FACS
HeLaMCAT-TAM2 and CHOMCAT-TAM2 were cultured
according to standard procedures. Further details are given in the
corresponding figure legends. In order to detach the cells from the culture
plates without using protease-based protocols, cell-dissociation buffer (Life
Technologies) was used to generate a cell suspension devoid of cell
aggregates. Cell-surface antigens were detected with the primary antibodies
indicated, followed by decoration with secondary antibodies coupled to either
Alexa-488 or allophycocyanine (APC; Molecular Probes), respectively. Antibody
incubations were performed on a rotating wheel for 1 hour at 4°C (primary
antibody) and 30 minutes at 4°C (secondary antibody), respectively. Wash
procedures were carried out by sedimenting the cells at 200 g
for 3 minutes at 4°C. Where indicated, propidium iodide (1 µg/ml) was
added prior to the FACS analysis in order to detect damaged cells.
Flow cytometric measurements were performed using a Becton Dickinson FACSCalibur system. Autofluorescence was determined by measuring trypsinized cells that were otherwise treated identically compared with the positive controls. Alexa-488-derived and APC-derived fluorescence can be measured simultaneously on a FACSCalibur two-laser system without the need of channel compensation.
Confocal microscopy
Cells were grown on glass cover slips to about 75% confluency. Following a
wash with PBS, the cells were further processed by paraformaldehyde fixation
(3% w/v, 20 minutes at 4°C), with or without permeabilization, employing
0.5% (w/v) Triton X-100. Antibody processing was achieved as indicated in the
corresponding figure legends. For double-staining procedures, Alexa-488- and
Alexa-546-coupled secondary antibodies (Molecular Probes) were used in all
experiments. The specimens were mounted in Fluoromount G (Southern
Biotechnology Associates) and viewed with a Zeiss LSM 510 confocal
microscope.
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Results |
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In addition to these known interaction partners of galectin-1, we
identified 16 tryptic peptides from protein band #1 whose masses were
consistent with corresponding tryptic fragments of a potential ORF defined by
cDNA clone AK024365 (NCBI database; Fig.
1D, boxed sequences indicate peptides identified by mass
spectrometry). In order to verify whether band #1 is the product of a gene
corresponding to AK024365, we generated a polyclonal antiserum against an
artificial recombinant protein corresponding to the N-terminal part (amino
acids 1-356; Mr: 39 kDa) of AK024365. As shown in
Fig. 1B, immunoreactive
material with a broad high-molecular-weight migration behavior was detected in
lanes 2, 4, 6 and 8, which correspond to the various eluates (see above) of
the GSTgalectin-1 affinity matrix. No signal could be observed under
control conditions. Binding of immunoreactive material to galectin-1 appeared
to be mediated by a galactoselectin interaction as more than 90%
(determined by a quantitative analysis using Bio-Rad® QuantityOne®
Software) eluted upon treatment of the affinity matrix with lactose. About 80%
of the total immunoreactive material was recovered from the soluble fraction,
with the remaining population derived from the membrane fraction.
More recently, the AK024365 ORF was found to represent a C-terminal
fragment of 1148 amino acids in length of a giant mucin-like glycoprotein
(O'Brien et al., 2001;
Yin and Lloyd, 2001
).
Molecular cloning of the corresponding gene revealed that this new mucin is
identical to the ovarian cancer antigen CA125, a putative integral membrane
protein present on the cell surface of tumor cells that has originally been
defined by the mAb OC125 (Bast et al.,
1981
). Therefore, we analyzed the various eluates from the
galectin-1 affinity matrix with regard to immunoreactivity based on OC125. As
shown in Fig. 1C, the pattern
of immunoreactive bands detected with OC125 is strikingly similar to the
pattern detected with the polyclonal anti-AK024365 antiserum (from now on
referred to as anti-CA125-C-TERM1-356 antiserum) described above
(Fig. 1B). Since CA125 was
reported to represent an integral membrane protein with a single membrane span
that is cleaved in the extracellular domain in order to release soluble
fragments, we conclude that the pattern of immunoreactive bands eluted from
the galectin affinity matrix represents both soluble and membrane-anchored
fragments of CA125. From now on, the 1148 amino acid, C-terminal part of CA125
(defined by cDNA clone AK024365) will be termed CA125-C-TERM.
Specificity of CA125-mediated galectin binding
In order to analyze whether CA125 preferentially binds to certain
ß-galactoside-specific lectins, we compared CA125 binding efficiency for
galectin-1 with that for galectin-3, the second most prominent member of this
protein family of lectins (Barondes et al.,
1994; Perillo et al.,
1998
; Hughes,
1999
; Rabinovich et al.,
2002
). As demonstrated in Fig.
2, HeLa-derived fragments of CA125 bind to galectin-1 twice as
efficiently compared with galectin-3 (Fig.
2A,B; compare lanes 1 and 2 as well as 3 and 4, respectively).
This difference is significant because comparable amounts of galectin-1 and
galectin-3 fusion proteins were used (Fig.
2A, compare lanes 9 and 10) and because the total patterns of
lactose-eluted molecules reveal proteins that specifically bind to galectin-1
(labeled with ), galectin-3 (labeled with
), as well as proteins
that bind equally to both galectin-1 and galectin-3 (labeled with
).
This demonstrates that differential binding efficiencies can be detected under
the experimental conditions applied (Fig.
2C).
|
When CA125-C-TERM was expressed in both HeLa and CHO cells by retroviral
transduction, a more defined protein band was observed
(Fig. 3). Interestingly,
CA125-C-TERM retains the ability of full-length CA125 to bind galectin-1, an
observation consistent with the fact that the translation product of this
construct contains both the stalk domain of CA125 [which is supposed to
represent the part of the full-length CA125 molecule that contains the
majority of N-linked sugars (O'Brien et
al., 2001)] and almost three CA125 repeat structures that are
heavily O-glycosylated (O'Brien et al.,
2001
). Intriguingly, CA125-C-TERM expressed in HeLa cells exhibits
the same characteristics as endogenous full-length CA125 with regard to
galectin interactions as it binds galectin-1 about twice as efficiently as
galectin-3 (Fig. 3). By
contrast, CA125-C-TERM expressed in CHO cells binds galectin-1 more than seven
times as efficiently as galectin-3 (Fig.
3). These results demonstrate that, besides N- and/or O-linked
sugar moieties of CA125, the proteinaceous core structure of CA125 contributes
to the specificity of galectin recruitment. Moreover, we have established
cell-type-dependent galectin-binding characteristics of CA125.
|
In order to provide evidence for a direct interaction between CA125-C-TERM and galectin-1, crosslinking experiments were conducted where CA125-C-TERM bound to GSTgalectin-1 beads was treated with the crosslinking reagent disuccinimidyl glutarate (DSG, Pierce) (Fig. 3C). Following elution with SDS sample buffer, crosslinking products (labeled with a square bracket) with an apparent molecular mass of about 160-180 kDa (corresponding to the approximate molecular mass of CA125-C-TERM plus GSTgalectin-1 in a 1:1 complex) can be detected that react with both anti-galectin-1 and anti-CA125 antibodies. The products display a smear-like appearance as expected for a glycoprotein-containing crosslinking product and can only be observed in the presence of DSG. Larger crosslinking products (>180 kDa), which would be indicative of an indirect interaction of CA125-C-TERM with the galectin-1 affinity matrix, cannot be observed.
CA125-C-TERM binding to galectin-1 largely depends on O-linked
ß-galactose-terminated oligosaccharide chains
To characterize further the molecular mechanism of galectin-1 binding to
CA125-C-TERM, we conducted interaction studies using cell lysates derived from
CA125-C-TERM-expressing CHO cells grown in the presence of tunicamycin
(Fig. 4). Under control
conditions (Fig. 4A, lanes
1-3), about 40% of CA125-C-TERM could be recovered on GSTgalectin-1
beads as calculated based on the input amount shown in lane 1 of
Fig. 4. This value was set to
100% (Fig. 4B) and compared
with the galectin-1-binding efficiency of CA125-C-TERM derived from
tunicamycin-treated cells (Fig.
4A, lanes 4-6). As depicted in
Fig. 4B, binding efficiency was
reduced to about 65% compared with control conditions. When CA125-C-TERM was
expressed in CHOclone 13 cells that are incapable of translocating
UDP-galactose into the lumen of the Golgi and, therefore, neither form
galactosylated glycoproteins nor galactosylated glycolipids
(Deutscher and Hirschberg,
1986), the binding capacity of CA125-C-TERM to
GSTgalectin-1 was almost abolished
(Fig. 4A, lanes 14-16) at about
10% residual binding efficiency (Fig.
4B). It is of note that antigenicity towards OC125 and/or the
expression level in both tunicamycin-treated CHOMCAT-TAM2 cells and
CHOclone 13 cells was observed to be lowered compared with
untreated CHOMCAT-TAM2 cells. Under all experimental conditions,
CA125-C-TERM binding to galectin-1 was established to be specific
(Fig. 4B).
|
In order to analyze whether CA125-C-TERM binding to galectin-1 depends on O-linked galactose-terminated oligosaccharide chains in vivo, we studied binding of exogenously added recombinant GSTgalectin-1 to untreated CHOMCAT-TAM2 cells, tunicamycin-treated CHOMCAT-TAM2 cells and CHOclone 13 cells using FACS (Fig. 5). As expected, untreated CHOMCAT-TAM2 cells displayed a high galectin-1-binding activity (Fig. 5, dark-blue curve), which was not saturated under the conditions used. This binding activity was significantly reduced when cells were pre-treated with tunicamycin (Fig. 5, red curve). As expected, GSTgalectin-1 binding to the cell surface was almost abolished in CHOclone 13 cells (Fig. 5, dark green curve), which allowed us to determine whether expression of CA125-C-TERM under these conditions (i.e. in a background deficient for galactosylation of glycoproteins and glycolipids) is capable of binding galectin-1. As demonstrated in Fig. 5, CA125-C-TERM cell-surface expression (data not shown, see also next section) does not alter cell-surface binding capacity for galectin-1 (Fig. 5, compare the dark green and the light green curves), demonstrating that galectin-1 binding to CA125-C-TERM requires galactosylation. The combined data shown in Figs 3, 4 and 5 suggest that the interaction between CA125-C-TERM and galectin-1 is direct.
|
Despite lacking a signal peptide, CA125-C-TERM is transported to the
cell surface of CHO and HeLa cells
Endogenous CA125 is expressed on the cell surface of tumor cells
(Bast et al., 1981). However,
based on available primary structure information
(O'Brien et al., 2001
;
Yin and Lloyd, 2001
), an
obvious signal peptide is not present at the N-terminus of either full-length
CA125 or CA125-C-TERM. In order to initiate studies on the molecular mechanism
of CA125 cell-surface expression, we first asked whether the C-terminal
fragment of CA125 (CA125-C-TERM) used in this study is also able to reach the
cell surface. CA125-C-TERM was expressed in MCAT+ CHO and HeLa
cells (Engling et al., 2002
)
using retroviral transduction. Cell-surface expression was assessed by a FACS
analysis using the monoclonal anti-CA125 antibody OC125
(Fig. 6). Autofluorescence of
CHO (Fig. 6A) and HeLa cells
(Fig. 6B) was determined using
trypsin-treated cells (red curves). Whereas CHO cells treated with retroviral
control particles did not present endogenous CA125 on their cell surface (A,
green curve), HeLa cells treated under identical conditions did contain small
but significant amounts of endogenous CA125 on their surface (B, green curve).
Upon retroviral expression of CA125-C-TERM, cell-surface staining strongly
increased for both CHO and HeLa cells (Fig.
6A,B; blue curves). In the case of CHO cells, retroviral
transduction with CA125-C-TERM was more than 90% efficient whereas about 60%
of HeLa cells were found to be transduced under the conditions applied. The
vast majority of this signal disappeared when cells were treated with trypsin
prior to the FACS analysis (data not shown). Therefore, despite lacking a
conventional signal peptide at the N-terminus, CA125-C-TERM is transported to
the cell surface.
|
CA125-C-TERM is transported to the cell surface via the
ER/Golgi-dependent secretory pathway
To analyze whether CA125-C-TERM enters the classical (i.e.
ER/Golgi-dependent) secretory route or whether it, in a similar manner to the
galectins, makes use of a so-far-uncharacterized nonclassical secretory
pathway we determined its subcellular distribution in permeabilized CHO and
HeLa cells using confocal microscopy (Figs
7,
8). In nonpermeabilized cells
(Fig. 7A-D), CA125-C-TERM was
detected on the cell surface of both CHO (B) and HeLa (D) cells. Specificity
of the observed staining pattern was established using retroviral control
particles (A and B, respectively). CA125-C-TERM cell-surface staining was
found not to be homogenous but rather appeared in subdomains with significant
parts of the plasma membrane not stained at all.
|
|
Permeabilization of HeLa cells prior to anti-CA125 antibody treatment
revealed that CA125-C-TERM expression results in its incorporation into
membranes of the classical secretory pathway (Figs
7,
8). At low magnification,
intracellular CA125-C-TERM could be detected in a perinuclear region
(Fig. 7E-H), where it was
colocalized with the Golgi marker p27
(Füllekrug et al., 1999;
Jenne et al., 2002
). Again,
this signal was established to be specific as it could not be observed when
cells were treated with retroviral control particles
(Fig. 7E). Additionally,
high-resolution confocal microscopy revealed CA125-C-TERM+ staining
of the nuclear envelope (Fig.
8B,D), which is indicative for ER localization. This was confirmed
by double-labeling experiments using antibodies directed against the ER marker
calreticulin (Fig. 8A)
(Sönnichsen et al.,
1994
). Whereas most of the calreticulin staining was found to be
ER associated, only low amounts of CA125-C-TERM were found in the ER compared
with high amounts in the Golgi (Fig. 8,
compare A and B). These results indicate that, following insertion
into the ER membrane, CA125-C-TERM is efficiently transported in an
anterograde direction from the ER to the Golgi. In order to rule out the
possibility that CA125-C-TERM+ perinuclear structures represent
endosomal compartments localized at the microtubule organizing center, we
treated CA125-C-TERM-expressing HeLa cells with brefeldin A, a drug that
disrupts the Golgi apparatus and, therefore, inhibits biosynthetic secretory
transport (Lippincott-Schwartz et al.,
1989
; Orci et al.,
1991
). As shown in Fig.
8F, the compact perinuclear staining of CA125-C-TERM
(Fig. 8D) disappears following
brefeldin A treatment. The resulting staining pattern matches brefeldin
A-induced redistribution of an established marker protein of the cis-Golgi,
the KDEL receptor (Fig. 8C,E)
(Lewis and Pelham, 1990
;
Lewis and Pelham, 1992
;
Füllekrug et al., 1997
).
These results provide proof for the presence of CA125-C-TERM in the ER and the
Golgi apparatus.
In order to characterize functionally the mode of intracellular transport of CA125-C-TERM, we conducted in vivo cell-surface expression experiments in the presence or absence of brefeldin A based on FACS (Fig. 9). CA125-C-TERM-expressing HeLa cells were grown to about 70% confluency, followed by incubation for 90 minutes in the presence of brefeldin A. The cells were then trypsinized to remove pre-existing cell-surface CA125-C-TERM, spread onto new culture plates at the same cell density and were then further incubated in the presence or absence of brefeldin A for 4 hours at 37°C. As a control, cells were applied to the same protocol without adding brefeldin A at any time point of the experiment. The amount of CA125-C-TERM transported to the cell surface within 4 hours in the absence of brefeldin A was set to 100% (Fig. 9A, light green curve; Fig. 9B, lane 2). When compared with the level of cell-surface CA125-C-TERM under steady-state conditions (Fig. 9A, red curve; Fig. 9B, lane 1), more than 50% of the cell-surface population recovers after trypsinization within 4 hours of incubation (Fig. 9B, lane 2). When cells were treated with brefeldin A before trypsinization, followed by incubation for 4 hours in the absence of brefeldin A, the level of cell-surface CA125-C-TERM was reduced by about 60% (Fig. 9A, dark green curve; Fig. 9B, lane 3). When cells were treated with brefeldin A throughout the course of the experiment, cell-surface transport of CA125-C-TERM was reduced by about 90% (Fig. 9A, blue curve; Fig. 9B, lane 4).
|
These data combined with the morphological analysis presented in Fig. 8 establish that CA125-C-TERM is transported to the cell surface via conventional secretory transport involving the ER and the Golgi apparatus.
Correlation of endogenous CA125 expression with increased
cell-surface expression of endogenous galectin-1 in CHO and HeLa cells
Our observation that CHO cells do not express detectable amounts of
endogenous CA125 as opposed to HeLa cells
(Fig. 6) is consistent with the
fact that CHO cells are not derived from a tumor
(Puck et al., 1958), whereas
HeLa cells were isolated from a cervix carcinoma
(Gey et al., 1952
). Therefore,
we were interested to compare CA125-deficient CHO cells with CA125-expressing
HeLa cells for various parameters with regard to galectin-1. Using FACS
analysis to analyze CHO and HeLa cells for the amount of cell-surface
expression of galectin-1, we found that HeLa cells contain more than ten times
as much galectin-1 on their surface compared with CHO cells
(Fig. 10). For this purpose,
autofluorescence of CHO and HeLa cells was determined with trypsin-treated
cells and adjusted to the same value for both cell lines
(Fig. 10A,B; red curves).
Employing an affinity-purified anti-galectin-1 rabbit antiserum, a relatively
small but significant population of endogenous galectin-1 (A; green curve)
could be detected on the surface of CHO cells when the cells were not treated
with trypsin prior to the FACS analysis. This observation is consistent with
various studies that demonstrated cell-surface expression of endogenous
galectin-1 in CHO cells (Cho and Cummings,
1995
; Lutomski et al.,
1997
). However, HeLa cells that do express endogenous CA125 in
appreciable amounts contain more than ten times the amount of endogenous
galectin-1 on their surface (B; blue curve) compared with CA125-deficient CHO
cells.
|
We next investigated whether this effect was due to (1) different total galectin-1 expression levels, (2) different cell-surface binding capacities for galectin-1 or (3) different regulation of galectin-1 export in CHO and HeLa cells, respectively. As shown by a western blot analysis (Fig. 1C), similar signals for galectin-1 were obtained from CHO and HeLa cells when the amount of SDS-lysed cells was titrated (20,000, 50,000 and 150,000 cells, respectively) and analyzed with affinity-purified anti-galectin-1 antibodies. Thus, CHO and HeLa cells do not differ to a significant extent in the total amount of galectin-1 expression. Cell-surface binding capacity for galectin-1 was analyzed using FACS by titrating increasing amounts of a recombinant GSTgalectin-1 fusion protein into cultures of CHO and HeLa cells, respectively. The total binding capacity for galectin-1 was found to exceed the amount of endogenous galectin-1 present on the cell surface of CHO and HeLa cells by a factor of more than 50-fold, with CHO cells being the cell type with an even higher galectin-1-binding capacity compared with HeLa cells (data not shown). Therefore, the strikingly different amounts of endogenous cell-surface galectin-1 on CHO versus HeLa cells (Fig. 6A,B) cannot be due to a lower galectin-1-binding capacity of CHO cells.
On the basis of these experimental observations, we conclude that CA125-expressing HeLa cells possess a more active galectin-1 export pathway than CA125-deficient CHO cells. These results are discussed in the following section with regard to the origin of HeLa and CHO cells as tumor- and non-tumor-derived cell lines, respectively.
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Discussion |
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Recently, two research groups independently succeeded in cloning the gene
that encodes CA125 (O'Brien et al.,
2001; Yin and Lloyd,
2001
), showing that CA125 is a giant mucin-like glycoprotein that
consists of more than 11,000 amino acids. Full-length CA125 is suggested to
represent a type I transmembrane protein with a single membrane-spanning
domain close to the C-terminus. The extracellular domain contains repeat
structures that are likely to be heavily O-glycosylated
(O'Brien et al., 2001
).
Besides its putative nature as an integral membrane protein, soluble fragments
of CA125 have been observed (Fendrick et
al., 1997
; Lloyd and Yin,
2001
). Apparently, phosphorylation of the cytoplasmic domain
causes extracellular cleavage of the N-terminal domain, which results in the
release of soluble fragments into the extracellular space
(Fendrick et al., 1997
;
Lloyd and Yin, 2001
).
In the current study, we conducted proteinprotein and
proteincarbohydrate interaction studies in order to affinity-purify
proteins that interact with galectin-1. Tryptic peptides were analyzed by mass
spectrometry, which identified one of the proteins shown to interact
specifically with galectin-1 as CA125. This conclusion was drawn from the fact
that 16 tryptic peptides could be identified as parts of the translation
product of cDNA clone AK024365 (NCBI) that, based on sequence information
reported by the laboratories of Lloyd and O'Brien
(O'Brien et al., 2001;
Yin and Lloyd, 2001
), encodes
the 1148 C-terminal amino acids of CA125 (CA125-C-TERM). These results were
confirmed by immunological identification of CA125-derived antigens by both
the original anti-CA125 antibody OC125
(Bast et al., 1981
) and a
rabbit antiserum directed against the N-terminal 356 amino acids of
CA125-C-TERM. Since the majority of the material bound to the galectin-1
affinity matrix was elutable with lactose, we concluded that this interaction
is galactose dependent. These data were confirmed by experiments demonstrating
that the interaction between CA125-C-TERM and galectin-1 both in vitro and in
vivo is almost completely abolished when CA125-C-TERM was expressed in a CHO
mutant that is incapable of galactosylation of glycoproteins or glycolipids
(Deutscher and Hirschberg,
1986
). Interestingly, CA125-C-TERM binding to galectin-1 is only
partially inhibited when cells were grown in the presence of tunicamycin, a
drug that inhibits N-glycosylation. These data establish that the interaction
of CA125-C-TERM with galectin-1 largely depends on O-linked
ß-galactose-terminated oligosaccharide chains. This conclusion appears to
be even more significant under physiological conditions for full-length CA125,
which is likely to be characterized by a much higher ratio of O- to
N-glycosylation compared with CA125-C-TERM.
When we compared the CA125-C-TERM binding efficiency of galectin-1 with that of galectin-3, the second most-abundant member of the galectin family, we found that galectin-1 is the primary ligand for CA125. Thus, the interaction observed does not appear to represent a simple carbohydratelectin interaction but rather depends on additional aspects of specificity based on the proteinaceous environment. Moreover, as CA125-C-TERM expressed in CHO cells showed an even higher preference towards binding of galectin-1, we conclude that the cellular background in which CA125 is expressed also has a significant impact on its binding specificity for members of the galectin family. On the basis of experiments presented in this study we, therefore, conclude that CA125 represents a specific galectin counter receptor with galectin-1 as the primary ligand. Since CA125 expressions appears to be largely restricted to tumor cells, it appears likely that tumor cell attachment to the ECM can be modulated in a galectin-1-dependent manner.
CA125-C-TERM encodes about three O-glycosylated repeat structures and the
N- and O- glycosylated stalk structure of the extracellular domain, the
transmembrane span and the cytoplasmic domain of full-length CA125. As
discussed above, these structural features are consistent with our finding
that CA125-C-TERM retains binding activity towards galectin-1. Intriguingly,
expression of this construct in both CHO- and HeLa cells resulted in
CA125-C-TERM cell-surface expression. Besides the lack of an N-terminal signal
peptide in both full-length CA125 and CA125-C-TERM, we demonstrate that
CA125-C-TERM cell-surface expression is mediated by ER/Golgi-dependent
secretory transport. Thus, CA125 represents a classical secretory cargo
protein whose molecular mechanism of insertion into the membrane of the ER
will be interesting to study in future experiments. As
single-peptide-independent mechanisms of protein insertion into the ER have
been described (Kutay et al.,
1995), it will also be of interest to analyze how CA125 ER
insertion compares with these known processes. Moreover, given its huge size
of more than 11,000 amino acids plus mucin-like levels of glycosylation,
questions arise about the mode of intracellular transport on its way to the
cell surface.
In order to initiate studies investigating the relevance of the interaction
reported with regard to a comparison between non-tumor- and tumor-derived
cells, we made use of CHO cells, a non-tumor-derived cell line
(Puck et al., 1958) and HeLa
cells, a cervix carcinoma cell line (Gey
et al., 1952
; Scherer et al.,
1953
). While we isolated fragments of endogenous CA125 from the
HeLa cell line, we were not able to detect CA125 fragments bound to the
galectin-1 affinity matrix when CHO cells were used as starting material (data
not shown). This observation is consistent with the detection of endogenous
cell-surface CA125 in HeLa cells and the lack of cell-surface CA125 in CHO
cells based on flow cytometry. Moreover, these data are in line with studies
that suggest that CA125 is expressed primarily in tumor tissue
(Bast et al., 1981
). Employing
a novel in vivo assay that allows us to assess quantitatively galectin-1
export from CHO and HeLa cells by flow cytometry, we have now demonstrated
that HeLa cells are characterized by more than tenfold higher levels of
cell-surface galectin-1 compared with CHO cells. As we show that CHO and HeLa
cells do not differ with regard to total expression levels of galectin-1, as
well as cell-surface binding capacity for galectin-1, we conclude that HeLa
cells are significantly more active in the non-conventional export of
galectin-1 compared with CHO cells.
To this end, the link between CA125 expression by tumor cells concomitant
with the increased cell-surface expression of galectin-1 remains correlative
and, therefore, future studies must elucidate whether CA125 expression has a
direct impact on the non-conventional export route of galectin-1. However, the
current study provides the first evidence for a potential functional link
between CA125 and galectin-1 which, in the light of the fact that both
molecules represent well-characterized tumor markers
(Bast et al., 1981;
Bast et al., 1983
;
Bon et al., 1996
;
Perillo et al., 1998
;
Rabinovich et al., 2002
),
might proof to be of significant biomedical importance in the future.
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Acknowledgments |
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Footnotes |
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References |
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