From the The Wistar Institute, Philadelphia, Pennsylvania 19104
Received for publication, June 1, 2000, and in revised form, October 26, 2000
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ABSTRACT |
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The GA733-2 antigen (GA733) is a homotypic
calcium-independent cell adhesion molecule (CAM) present in most
normal human epithelial cells and gastrointestinal carcinomas. Because
oligomerization of some CAMs regulates cell adhesion and signal
transduction, the correlation between GA733 oligomeric state and
cell-cell adhesion was investigated. Sedimentation equilibrium studies
showed that full-length (-FL) GA733 exists as dimers and tetramers in
solution, whereas the GA733 extracellular domain (-EC) is a monomer.
The Kd of GA733-FL is less than 10 nM
for the monomer-dimer association, whereas the dimer-tetramer
association is about 1000-fold weaker (Kd ~10
µM). Chemical cross-linking of purified GA733-FL in
solution resulted in a major product corresponding to GA733 dimers, and
minor amounts of trimers and tetramers. However, GA733-EC cross-linked
under the same conditions was consistently a monomer. Chemical
cross-linking of dissociated colon carcinoma cells produced
predominantly GA733 dimers, whereas cross-linking of cells in
monolayers yielded some tetramers as well. GA733-FL retained its
cell-cell adhesion function as shown by inhibition of cell aggregation,
whereas monomeric GA733-EC was inactive. These data show that GA733
exists predominantly as high affinity noncovalent
cis-dimers in solution and on dissociated colon carcinoma cells. The lower affinity association of dimers to form tetramers is
most likely the head-to-head interaction between GA733
cis-dimers on opposing cells that represents its cell-cell
adhesion activity.
Cell-cell adhesion is a dynamic process essential for the normal
development and function of multicellular organisms and is known to
play an important role in tumor progression and metastasis. The GA733-2
(Ep-CAM/EGP40/17-1A antigen/KSA/ESA) cell surface glycoprotein was
initially defined by the CO17-1A monoclonal antibody (mAb)1 (1) and GA733 mAb (2).
More recently, it was shown to function as a
Ca2+-independent homotypic cell adhesion molecule (CAM)
(3). However, it appears to belong to a novel class of CAMs distinct,
by its structural features, from the four known superfamilies of CAMs (immunoglobulin-type, selectins, cadherins, and integrins) (for review
see Refs. 4 and 5). The sequence of GA733-2 indicates no significant
homology with other known proteins except with GA733-1 (Trop-2),
previously cloned by Linnenbach et al. (6). The amino acid
sequences of GA733-2 and GA733-1 are ~50% identical.
GA733-2 (referred to hereafter as GA733) is synthesized as a 32.8-kDa
nonglycosylated precursor, but migrates in SDS-PAGE gels as a 40-kDa
band. The extracellular portion of GA733 contains three potential
N-linked glycosylation sites and consists of a cysteine-rich
N-terminal region followed by a cysteine-poor region. GA733 has a short
transmembrane domain and a cytoplasmic tail of 26 amino acids with
potential sites of phosphorylation on tyrosine and serine residues.
GA733 is present in most epithelial cells and is overexpressed in the
vast majority of gastrointestinal tumors (7-10). Moreover, emerging
evidence correlates the increase of GA733 expression with other types
of cancer lesions, notably in cervical squamous epithelia (11). The
increase of cell proliferation in cervical squamous epithelia, which
are normally GA733-negative, correlates with the expression of GA733.
An increase of the number of positive cells as well as in levels of
GA733 protein was observed during progression of cervical lesions from
cervical intraepithelial neoplasia grade I to grade III (11).
The significance of increased GA733 expression in cancer lesions
remains mysterious. Surprisingly, a recent study from our group showed
that GA733 inhibits invasion of tumor cells in vitro when
transfected into CT-26 mouse colon carcinoma cells (12). In the same
study, the mouse homologue of GA733, mEGP (a homotypic CAM with 82%
amino acid sequence identity with GA733) expressed by transfected mouse
CT-26 cells (that are mEGP-negative) significantly decreases cell
growth in vitro and inhibits metastasis in vivo. Although the mechanism(s) responsible for these two phenomena remain
unclear, the strong intercellular adhesion profile of CT-26-mEGP cells
(clumping and clustering observed in vitro) may inhibit cell
dispersion and limit their migration capacity (12). The same mechanism
was proposed to explain decreased metastases in nude mice by human
breast carcinoma cells overproducing another homotypic adhesion
molecule, E-cadherin (13). Alternatively, the decrease of cell growth
may be due to the activation of certain mEGP-associated signal
transduction pathways that down-regulate cell proliferation, when
activated by increased homotypic engagement of mEGP. Although very
little is known about the biochemical properties of GA733 and its
murine homologue, mEGP, other than their role as homotypic CAMs, it is
likely that they participate in "inside-out" and "outside-in"
cell signaling pathways controlling cell growth and adhesion. This
hypothesis is supported by a recent report showing that GA733-1
(Trop-2) is a calcium signal transducer (14).
Several clinical trials targeting GA733 by anti-idiotypic antibodies
and recombinant protein vaccines have shown induction of humoral and
cellular immunity in colorectal cancer patients (15-20). More
importantly, CO17-1A mAb against GA733 enhances survival of patients
with colon carcinoma in a phase II randomized control study (21, 22).
Furthermore, a recombinant adenovirus expressing GA733 significantly
enhanced survival of mice bearing established CT-26-GA733 tumors (23).
The inhibitory effect of GA733 on cancer cell growth and invasion, its
increased expression in human cancer lesions, and the beneficial
treatment with mAbs against GA733 in experimental animals and patients
appear to be paradoxical. Clearly, further characterization of the
biochemical and functional properties of this protein and its role in
tumor progression is needed.
Recent studies have suggested that, in addition to mediating homotypic
associations between opposing cell membranes, cis
interactions between some types of receptors on the same cell surface
can play an important role in regulating cell adhesion, signal
transduction, and migration (24). For example, it has been shown that
C-CAM (25), PECAM-1 (26), and ICAM-1 (27, 28) exist as
cis-dimers in the cell membrane. Furthermore, dimeric forms
of recombinant soluble ICAM-1 have significantly higher affinities for
its purified ligand, LFA-1 (CD11a/CD18), whereas the monomeric form
shows no binding (27, 28), illustrating the central role CAM
oligomerization can play in cell-cell adhesion.
The molecular organization of GA733 within normal epithelia and colon
carcinoma cells remains unknown, despite its use with some success as a
target for passive and active immunotherapy of colon cancer in human
clinical trials (15-22). Elucidating the cis-oligomerization of GA733 on the cell membrane and the
mechanism of GA733-mediated cell adhesion should contribute to a better understanding of GA733 function and may lead to substantial
improvements of GA733 targeting in immunotherapy of colon cancer.
In this study, we investigated the oligomeric state of
membrane-associated and soluble forms of GA733 and their ability to block cell-cell adhesion. We show that GA733-FL, which inhibits cell
aggregation, self-associates to form high affinity
cis-dimers in solution and in intact colon carcinoma cell
membranes, whereas the soluble recombinant extracellular domain
(GA733-EC) is a monomer and does not inhibit cell aggregation.
Reagents and Antibodies--
Chemicals were from Sigma Chemical
Co. (St. Louis, MO). The membrane-impermeable bis(sulfosuccinimidyl)
suberate (BS3) and membrane-permeable disuccinimidyl
glutarate (DSG) cross-linkers were purchased from Pierce (Rockford,
IL). GA733 mAb, which recognizes the extracellular segment of GA733
(2), was produced and affinity-purified using a protein A-Sepharose
column (Amersham Pharmacia Biotech, Uppsala, Sweden) as described
previously (29). GA733 polyclonal antibody (GA733 pAb) was obtained
from rabbits immunized with GA733-EC and purified on a Protein
A-Sepharose column.
Protein Purification--
Recombinant GA733-FL and GA733-EC were
produced in insect cells using the baculovirus system as described
previously (30, 31). GA733-FL was essentially produced as described for
GA733-EC (31), except that a full-length GA733 cDNA was used (32). All purification steps were performed at 0-4 °C. For recombinant GA733-FL, typically 500 ml of baculovirus-transfected Sf9 insect cells (~12 × 108 cells) were harvested by
centrifugation at 48-h post-infection, and the cell pellet was
resuspended in lysis buffer (50 µl/106 cells) containing
100 mM Tris-Cl (pH 7.6), 10 mM EDTA, 10 mM EGTA, 1 mM
During the purification process, some covalent cross-linking of GA733
occurs presumably due to the activity of insect cell transglutaminases,
enzymes that catalyze the formation of
The secreted GA733-EC was purified from the culture supernatant of
baculovirus-infected Hi Five insect cells using a GA733 mAb-Sepharose
affinity column. The column was washed with PBS, and the bound proteins
were eluted with 100 mM triethylamine, pH 11.5, purchased
from Pierce.
Protein Assay--
Protein samples were concentrated using
Centriprep 10 concentrators (Amicon, Beverly, MA). GA733-EC was
concentrated in PBS, whereas GA733-FL was concentrated in PBS
containing either 0.5 mM octyloxyethylene dodecyl ether
(C12E8) or 10 mM pentaoxyethylene octyl ether (C8E5) (Sigma). GA733-FL protein
concentrations were determined using the bicinchoninic acid protein
assay (Pierce). The concentration of GA733-EC was determined by
spectrophotometry at 280 nm in a 1-cm path length using an extinction
coefficient of 0.926 (mg/ml) HPLC Gel Filtration--
For analytical ultracentrifugation
experiments, recombinant proteins were further purified by HPLC gel
filtration to remove trace contaminants and aggregates. GA733-EC was
concentrated as described previously, injected into two TSK columns,
G3000SWXL and G2000SWXL (Toso-Haas, Japan),
connected in series, and separated at 0.8 ml/min using PBS. GA733-FL
was concentrated in PBS containing 0.5 mM
C12E8 and chromatographed on a Superose 12 column (Amersham Pharmacia Biotech) at 0.5 ml/min using PBS containing
0.15 mM PMSF and 0.5 mM
C12E8.
Mass Spectrometry--
Matrix-assisted laser
desorption/ionization-time of flight (MALDI-TOF) mass spectrometry was
carried out on a Voyager DE-PRO mass spectrometer (PE Biosystems,
Framingham, MA). Recombinant protein solutions (2 µl) were mixed 1:1
with a saturated solution of sinapinic acid in 33% acetonitrile
and 0.1% trifluoroacetic acid and allowed to air dry on the sample
target before analysis. Several 2-fold serial dilutions of the protein
samples were tested, and protein A was used as an external and internal standard.
Analytical Ultracentrifugation--
Experiments were performed
in an Optima XL-I analytical centrifuge. Sedimentation equilibrium runs
were performed with three initial concentrations of the protein sample.
Absorbance or fringe displacement data was collected every 4-6 h until
equilibrium was reached, as determined by comparison of successive
scans using the MATCH program.
Sedimentation equilibrium experiments were performed using either the
interference optics (for GA733-EC) or the absorbance optics (for
GA733-FL) to measure the protein concentration gradient. For
experiments with GA733-EC, three cells were assembled with double-sector 12-mm centerpieces and sapphire windows. The cells were
loaded with 110 µl of HPLC gel filtration buffer as the reference, and 110 µl of sample at concentrations of 2.0, 1.0, or 0.5 mg/ml. A
blank scan of distilled water was taken before the run, to correct for
the effects of window distortion on the fringe displacement data (34).
Experiments were performed at 4 °C or 30 °C, at 23,000-30,000 rpm. Fringe displacement data was collected every 4-6 h until equilibrium was reached.
Sedimentation equilibrium experiments on GA733-FL were performed in the
presence of either C8E5 or
C12E8 nonionic detergents. For experiments
using C8E5, affinity-purified GA733-FL was
concentrated in PBS containing 0.15 mM PMSF and 10 mM C8E5 and dialyzed against the
same buffer for 24 h at 4 °C. For experiments in the presence of C12E8, GA733-FL was chromatographed using
HPLC gel filtration as described above. Fractions containing purified
GA733-FL were then concentrated to 0.1-0.4 mg/ml and dialyzed for at
least 24 h at 4 °C against PBS containing 0.15 mM
PMSF and 0.5 mM C12E8 in 22%
D2O (v/v). In the presence of detergent, the protein is part of a protein-detergent complex that has a buoyant molecular mass,
Mc(1
For all experiments, attainment of sedimentation equilibrium was
determined by comparison of successive scans using the MATCH v.7
program, and the data were edited using the REEDIT v.9 program (both
programs kindly provided by David Yphantis). Nonlinear regression fitting of the sedimentation equilibrium data with various models was
performed using the NONLIN program (38). The reduced molecular weight,
Cell Culture--
The colon carcinoma cell lines Caco-2 (clone
C2BBe1) and Colo-205 were purchased from ATCC (Manassas, VA). Caco-2
cells were cultured in Dulbecco's modified Eagle's medium (Life
Technologies, Inc., Grand Island, NY) with 4.5 g/liter glucose, 1 mM sodium pyruvate, 0.01 mg/ml human transferrin, and 10%
of fetal bovine serum (HyClone, Logan, UT). Colo-205 cells were
cultured in RPMI 1640 medium (ATCC) adjusted to contain 10 mM HEPES, 1 mM sodium pyruvate, 2 mM L-glutamine, 4.5 g/liter glucose, 1.5 mg/liter sodium bicarbonate, and 10% fetal bovine serum. The cells
were incubated in 5% CO2, 95% air at 37 °C. All media
were antibiotic/antimycotic free. Cells at 80-90% confluence were
subcultured every 7 days. Caco-2 cells were dissociated with 0.2 g/liter EDTA (Life Technologies, Inc.) containing 0.01% trypsin,
whereas Colo-205 cells were dissociated with EDTA only.
Chemical Cross-linking--
Prior to cross-linking, the cells
were washed twice with PBS. Adherent or detached Caco-2 and Colo-205
cells were overlaid with PBS, and cross-linking was initiated by the
addition of either DSG or BS3 at different concentrations
(0.1, 0.2, 0.5, 1, and 2 mM). After 5 min at room
temperature, the reaction was quenched for 15 min with Tris-Cl, pH 7.6, at a final concentration of 50 mM. Cells were lysed for 20 min at 4 °C in a buffer containing 0.5% Triton X-100, 20 mM Tris-Cl (pH 7.6), 5 mM EDTA, 1 mM
For purified proteins, cross-linking was performed using 16 µg of
GA733-FL or GA733-EC in 100 µl of PBS containing different detergents
(i.e. 0.1% or 0.5% Triton X-100, 0.5 mM
C12E8, 10 mM C8E5, 0.1% Tween 20, 0.5% CHAPS, 1%
n-octyl glucoside) with 0.1 mM BS3
or DSG for 5 min at room temperature. After quenching with 50 mM Tris-Cl, pH 7.6, for 15 min, samples were frozen on dry
ice, lyophilized, and separated by SDS-PAGE, and the protein bands were
visualized using Coomassie Blue R-250.
Immunoprecipitations, SDS-PAGE, and Western Blots--
GA733 was
immunoprecipitated from supernatants of cell lysates using GA733
mAb-Sepharose. The supernatants were first precleared for 1 h with
a bovine serum albumin-Sepharose resin, and proteins were adsorbed to
25 µl of GA733 mAb-Sepharose for 1 h at 4 °C with gentle
rotation. Precipitates were washed five times with 400 µl of cold
lysis buffer, and bound proteins were eluted with 200 µl of 0.5% SDS
using 0.22-µm filter microcentrifuge tubes (Millipore,
Bedford, MA). Bound proteins (20 µl) were boiled for 2 min in 1%
Laemmli sample buffer and separated by 6% Tris-Tricine SDS-PAGE under
nonreducing and reducing conditions according to Schägger and Von
Jagow (39).
Western blotting was performed after electroblotting proteins to
polyvinylidene difluoride Immobilon-P membranes (Millipore, Bedford,
MA), using a Trans-Blot cell (Bio-Rad, Hercules, CA) at 250 mA for
2 h, as described by Mozdzanowski et al. (40). Membranes were blocked overnight with 5% nonfat dry milk and probed either with GA733 mAb or GA733 pAb, followed by an appropriate second
step antibody and detection was performed using either the
5-bromo-4-chloro-3-indolyl phosphate-nitro blue tetrazolium (Promega,
Madison, WI) or ECL chemiluminescence reagents (Amersham Pharmacia
Biotech, Buckinghamshire, United Kingdom).
Flow Cytometry Analysis--
FACS analysis was performed as
described previously (41). Briefly, 106 cells in 1 ml per
condition in PBS containing 1% BSA (PBS-B) were incubated for 20 min
on ice with 20 µg of GA733 mAb. After two washes with 5 ml of PBS-B,
a rabbit anti-mouse secondary antibody coupled to FITC (Calbiochem, San
Diego, CA) was added to the cells for another 20 min on ice (dilution
1:40). Excess antibody was removed by washing three times with PBS-B.
Cells were analyzed on an EPICS XL flow cytometer (Coulter Corp.,
Hialeah, FL) using forward scatter and side scatter to exclude dead
cells. For the negative control condition, the cells were labeled with
the secondary antibody-FITC only. Data analysis was performed using the
WinMDI software available on the web from the Scripps Institute.
Cell Aggregation Assay--
Cells were detached either with EDTA
containing 0.01% trypsin (for Caco-2) or with EDTA alone (for
Colo-205) as described above for subcultivation. The aggregation assay
was carried out in 24-well plates according to Litvinov et
al. (3) with minor modifications. To prevent cell adhesion to the
bottom of the wells, 1 ml of 1% agarose was poured in each well and
allowed to solidify. Typically, 5 × 105 cells in 2 ml
of HCMF buffer (Hanks' buffer containing 100 mM Hepes, 1%
bovine serum albumin, 100 µg/ml DNase I, and 0.35 g/liter NaHCO3) without Ca2+ and Mg2+ were
placed in each well and incubated in a rotating platform (100 rpm) at
37 °C and 5% CO2, for different time periods. For inhibition studies, recombinant GA733 proteins were added at time zero
to the single cell suspension at different concentrations. The cells
were allowed to aggregate for 2 h and 500-µl samples were
analyzed in a Coulter Counter "CC" (Coulter Corp.) to determine the
number of particles. The extent of aggregation was represented by the
degree of aggregation (D) calculated according to Shimoyama et al. (42) as D = (N0 Purification and Characterization of Recombinant GA733-EC and
GA733-FL--
Recombinant proteins were expressed and purified as
described under "Materials and Methods". Both recombinant proteins
exhibited moderate covalent cross-linking to dimers and higher
oligomers during expression in insect cells and affinity purification,
presumably resulting from transglutaminases in the insect cells. Fig.
1A shows a Coomassie Blue stained gel of
GA733-EC after immunoaffinity chromatography and after HPLC gel
filtration. The retention time of GA733-EC (MW ~ 28 kDa) in the
HPLC column falls between those of protein standards with molecular
weights of 44 and 17 kDa (Fig. 2A),
suggesting that GA733-EC is possibly a monomer. After HPLC gel
filtration, the minor amount of cross-linked dimers was efficiently separated from the uncross-linked GA733-EC as shown in Fig. 1A.
Affinity purification of GA733-FL was more problematic and several
procedures gave poor yields and resulted in more extensive cross-linking of the protein (up to 50% as determined by SDS-PAGE) with the presence of several low and high molecular weight
contaminants. Extensive washes of protein bound to the mAb column using
buffer with 0.05% Triton X-100 substantially reduced the amount of
most contaminants. However, a 220-kDa band, identified as myosin, was difficult to eliminate totally (Fig. 1B). The amount of cross-linked protein could be reduced to about 5% (Fig. 1B) by using 100 mM Tris buffer with 10 mM EDTA, 10 mM EGTA throughout the affinity purification as described
under "Materials and Methods". HPLC gel filtration of GA733-FL in
the presence of 0.5 mM C12E8 lead to a high protein recovery (Fig. 1B) compared with the use of other
detergents (10 mM C8E5, 0.1%
Triton X-100, 0.1% Tween 20, 1% n-octyl glucoside) (data not shown).
As shown in Fig. 1B, myosin and minor low molecular weight contaminants
were efficiently separated from GA733-FL on the HPLC gel filtration
column, whereas cross-linked and uncross-linked GA733-FL coeluted.
MALDI mass analysis of GA733-EC after HPLC gel filtration
chromatography is shown in Fig. 3A. Two
broad peaks were observed, with average molecular masses of about
28,331 and 29,348 Da. Since the sequence molecular weight of the
extracellular domain is 27,372 Da after removal of the signal peptide,
these results suggest that the molecule is heterogeneously
glycosylated, resulting in two major populations with about 960 and
1,977 Da carbohydrate, with substantial mass heterogeneity within these
peaks as reflected by their shape. Attempts to obtain GA733-FL mass
spectra with affinity-purified, detergent-free protein or protein after
HPLC gel filtration in 0.5 mM C12E8
were not successful. The data shown in Fig. 3B was obtained using
affinity-purified protein concentrated in the presence of 10 mM C8E5 and shows two peaks with
average masses of 34,291 and 35,962 Da. Comparison of these masses with the expected sequence mass for GA733-FL (32,675 Da) suggests
heterogeneous glycosylation, which contributes ~1,616 and 3,287 Da to
the mass, although some of this mass difference might be contributed by tightly bound detergent or lipid molecules, or other uncharacterized post-translational modifications.
Sedimentation Equilibrium Analyses of GA733 Recombinant
Proteins--
To systematically evaluate the oligomeric state of
GA733-EC, sedimentation equilibrium experiments were performed at
4 °C and 30 °C, and the concentration versus radius
data was fitted with various models using nonlinear regression (38). At
both temperatures, the data were described well by a single ideal
species model; representative 30 °C data is shown in Fig.
4A. The estimated molecular mass obtained
by the fitting program was 28,705 Da, in excellent agreement with the
average molecular mass of the two-glycosylated species observed using
mass spectrometry (28, 839 Da). The data clearly demonstrates that the
extracellular domain is monomeric up to a concentration of at least 6 mg/ml.
Sedimentation equilibrium analyses of GA733-FL were initially performed
on samples in the presence of 10 mM
C8E5. This detergent is a useful alternative to
C12E8 for sedimentation equilibrium as it has a
partial specific volume of 0.993 cm3/g (43), eliminating
the need for density correction of the solvent with D2O
(44). Affinity purified samples could not be further purified by HPLC
gel filtration due to poor sample recovery in this detergent, and
therefore the affinity purified protein was used directly for
sedimentation equilibrium analyses. Table I summarizes the analyses of GA733-FL
using different conditions. Various models were used to fit the
sedimentation equilibrium data. For all six experiments performed in
C8E5, the data was fitted well with models
where the predominant species was a dimer. However, in four experiments
there was also a small proportion of a large molecular weight species
(n = 8 to 40) that was not in chemical equilibrium with
the dimer and apparently represented irreversible aggregates. The
presence of this large species interfered with unambiguous
identification of other species that may have been present. However, in
one experiment with no detectable aggregate, the data was fitted well
by a reversible monomer-dimer-tetramer model, with estimated
Kd's of 98 nM for the monomer-dimer association and 49 µM for the dimer-tetramer
association.
In the presence of 0.5 mM C12E8,
the small proportion of aggregates and minor contaminants in the
affinity-purified GA733-FL could be removed by HPLC gel filtration. In
contrast to the experiments on the affinity purified protein, three
sedimentation equilibrium experiments with GA733-FL further purified by
HPLC gel filtration, consistently fitted well to models containing only
dimer and tetramer species, as shown in Fig. 4B and 4C. Models that
included monomer or oligomers other than tetramer (e.g.
trimer, hexamer, octamer) gave significantly poorer fits as judged by
the size of the variance and randomness of the residuals. When the six
data sets shown in Fig. 4B and 4C (representing three different loading
concentrations of protein and two rotor speeds) were fitted
simultaneously, the data was fitted well by a model describing a
reversible dimer-tetramer association, with an estimated
Kd of 10 µM. When the data sets were
fitted individually, allowing a different equilibrium constant for each
set, the randomness of the residuals improved slightly, and
Kd's ranged between 5 µM and 14 µM, but the improvement in the variance was not
significant at a 67% confidence level. Thus the data is consistent
with a predominantly reversible association of GA733-FL dimers to
tetramers with a Kd ~10 µM. Two
other experiments on different preparations of GA733-FL were also
fitted well by the same model and returned a Kd for
the dimer-tetramer association of ~7 µM. The slight
improvement in the fit with individual equilibrium constants suggests
that a very small amount of one or more species is unable to associate or dissociate. This is most likely due to the small proportion of
covalently cross-linked dimer present in most samples of GA733-FL. The
association of GA733-FL monomers to dimers appears to be very strong,
with no detectable dissociation observed in all samples in
C12E8, and very little dissociation observed in
C8E5 with Kd ~ 98 nM. The absence of detectable monomer in the
C12E8 experiments is consistent with a
Kd < 10 nM for the monomer-dimer association. The slightly weaker association in
C8E5 (Kd ~ 98 nM) could be a detergent effect, but is more likely due to the presence of contaminating low molecular weight proteins in these
less pure samples.
Chemical Cross-linking of GA733 Recombinant Proteins in
Solution--
The oligomeric states of GA733 recombinant proteins,
GA733-EC and GA733-FL, in the presence of various detergents were
evaluated using chemical cross-linking. Fig.
5 shows a representative cross-linking experiment of GA733 recombinant proteins in the presence of 0.5% Triton X-100. GA733-FL was affinity-purified and eluted in the presence
of 0.5% Triton X-100, as described under "Materials and Methods",
to minimize artifactual cross-linking during purification. Triton X-100
was added to the GA733-EC preparations after purification to have
consistent conditions for both proteins. Both membrane permeable (DSG)
and membrane impermeable (BS3) cross-linkers were evaluated
(see intact cell experiment below) and an optimal concentration of 0.1 mM was determined in preliminary experiments. As seen in
Fig. 5, cross-linking of GA733-FL produces predominantly dimers with
additional bands at the trimers and tetramers positions. On the other
hand, no cross-linking of GA733-EC was observed under similar
conditions, confirming that it is a monomer in both the presence and
absence of detergents. Similar results to those shown for Triton X-100
were obtained using BS3 and DSG in the presence of other
detergents (0.1% Triton X-100, 0.5 mM
C12E8, 10 mM
C8E5, 0.1% Tween-20, 0.5% CHAPS, 1%
n-octyl glucoside), as well as with the thiol-cleavable
cross-linkers (0.1 and 0.2 mM dithiobis [succinimidyl
propionate], 0.1 and 0.2 mM 3, 3'-dithiobis
[sulfosuccinimidylpropionate]) (data not shown).
GA733 Oligomeric State in Intact Colon Carcinoma Cells--
To
characterize the molecular organization of GA733 in intact cell
membranes, chemical cross-linkers (BS3 and DSG) were added
to colon carcinoma cells either on monolayers or in single cell
suspension. Two human colon carcinoma cell lines expressing high levels
of GA733 were evaluated: 1) a homogeneous clone of Caco-2 epithelial
cells (C2BBe1; passages 45-76), which forms tight and polarized
monolayers, with an apical brush border morphologically comparable to
that of the human colon (45, 46) and; 2) the colorectal adenocarcinoma
cell line Colo-205 (47), which is characterized by reduced cell-cell
and cell-substrate adhesion. When cultured in tissue culture flasks,
Colo-205 cells exhibit a minimally adherent morphology, form small
aggregates, and a substantial proportion of the cells grows in suspension.
Treatment of intact Caco-2 cells with the membrane-impermeable
BS3 cross-linker at different concentrations, followed by
protein extraction, immunoprecipitation, and Western blots (Fig.
6, A and B)
resulted in appearance of multiple cross-linked species with a major
band at ~80 kDa in GA733 mAb immunoprecipitates. When Caco-2 cells in
monolayer are treated with high cross-linker concentrations (Fig.
6A), a 160-kDa band corresponding to GA733 tetramers is
observed. When single cell suspensions are cross-linked using the same
conditions (Fig. 6B), a strong dimer band is still observed
but the tetramer band is substantially reduced. Quantification of
tetramer/dimer ratios using densitometry showed a ratio of 0.4 versus 0.1 for cells in monolayer and single cell
suspensions, respectively, when 0.5 mM BS3 was
used. Similarly, tetramer/dimer ratios were 0.8 versus 0.2 at 2 mM BS3 for cells in monolayer and single
cell suspension, respectively. The noncross-linked controls
(Cont) only showed the monomeric GA733 band (Fig. 6).
Similar cross-linking results were obtained for cells in monolayers and
cell suspensions using the membrane-permeable DSG cross-linker (data
not shown), suggesting that GA733 was not cross-linked to cytoplasmic
proteins under these conditions.
Similarly, Colo-205 colon carcinoma cells were cross-linked either
in situ, in a tissue culture flask (Fig. 6C) or
in single cell suspensions (Fig. 6D). Addition of
BS3 to nondetached cells resulted in a decrease in the
intensity of the GA733 monomer band in a
concentration-dependent manner and the appearance of one
major band with the molecular weight of a GA733 dimer (Fig.
6C). GA733 pAb also recognized minor bands with approximate
molecular sizes of 120 and 160 kDa, presumably corresponding to GA733
trimers and tetramers. Compared with adherent Caco-2 cells, the ratio
of tetramers to dimers for Colo-205 was very low, i.e. 0.07 at 0.5 mM BS3 and 0.12 at 2 mM
BS3. When single cell suspensions were cross-linked using
the same conditions, dimers but not tetramers were observed (Fig.
6D). Cross-linking was also performed using DSG at the same
concentrations and produced similar results both in detached and
nondetached cells (data not shown).
GA733 in polarized monolayers of Caco-2 was less extensively
cross-linked compared with Colo-205 (compare Fig. 6A with
6C), even when high concentrations of cross-linkers were
used. The decreased cross-linking efficiency on Caco-2 cells is likely
due to reduced accessibility of the reagent to GA733 protein complexes in these cells. GA733 is known to be predominantly present at the
cell-cell boundaries of epithelial cells and carcinoma cells (lateral
membranes) and absent from the exposed apical surface (3, 48, 49),
which is consistent with the observed reduced accessibility of the
cross-linkers to GA733 molecules in Caco-2 cells compared with Colo-205
cells in these experiments.
Trypsin and EDTA Treatments Have No Effect on GA733 Expression at
the Surface of Colon Carcinoma Cells--
Before testing the ability
of GA733 recombinant proteins to inhibit cell-cell aggregation, we
evaluated whether EDTA or trypsin/EDTA treatments had any effect on
GA733 expression at the cell surface. Nose et al. (50)
showed that trypsin/EDTA treatment, but not trypsin/CaCl2
treatment affected the membrane expression of cadherins, which were
temporarily removed from the cell surface most likely by
internalization. Furthermore, Litvinov et al. (3) reported that GA733 was internalized in the absence of calcium on some human
mammary carcinoma cells. To test whether total cellular GA733 levels
were affected by trypsin and/or EDTA treatments, single cell
suspensions of Caco-2 cells (dissociated with trypsin/EDTA) and
Colo-205 cells (dissociated with EDTA alone) were lysed as described
under "Materials and Methods" and GA733 cellular protein levels
were compared with the corresponding cell line lysed in monolayers
(without dissociation) by Western blot using GA733 mAb. As shown in
Fig. 7A, quantitative Western
blot reveals a single major GA733 band at the expected molecular mass
(~40 kDa), the intensity of which is not appreciably affected by
trypsin/EDTA (Caco-2) or EDTA (Colo-205) treatments.
To further confirm that the GA733 molecules detected by Western blots
are on the surface of detached colon carcinoma cell lines, flow
cytometry analysis using GA733 mAb was performed. Both dissociated
Caco-2 and Colo-205 cells were highly and homogeneously positive for
GA733 expression (Fig. 7B). These results are consistent with data obtained with L cells transfected with GA733 (3), where the
authors showed that both trypsin/EDTA and trypsin/CaCl2 treatments are not able to remove GA733 molecules expressed at the
surface of these transfectants.
GA733-FL Inhibits Cell Aggregation, Whereas GA733-EC Has No
Effect--
Recent work from our group showed that GA733-FL was active
and GA733-EC was inactive in solid-phase binding assays, where the
recombinant protein was immobilized on nitrocellulose-coated plates
(12). Although its validity was previously demonstrated for GA733 (12)
and other adhesion molecules (51), the solid-phase binding assay is not
fully representative of adhesion of two living cells. Furthermore, a
truncation mutant of GA733 lacking the 26-amino acid cytoplasmic tail
was not able to mediate aggregation when transfected into L cells,
although adhesion of this transfectant to solid-phase-adsorbed GA733
remained unaffected (48).
Therefore, cell-cell aggregation assays were performed to further
confirm the biological activity of the GA733 recombinant proteins
analyzed above. Specifically, the inhibitory effect of GA733
recombinant proteins on cell-cell aggregation was correlated with their
oligomeric states using Caco-2 and Colo-205 cells. Cell-cell
aggregation is observed as early as 30 min, and the size of the cell
aggregates increases progressively with time. An optimal aggregation
time of 2 h was chosen for counting particles and was used for
most experiments.
The photographs in Fig. 8A,
show representative fields of Caco-2 cell aggregation under different
conditions. In the absence of added protein, the cells were extensively
aggregated after 2 h (Fig. 8A, b).
Aggregation was greatly reduced in the presence of GA733-FL
(c) but was unaffected by addition of GA733-EC
(d). Similar results were observed when Colo-205 cells were
tested (data not shown). As shown in Fig. 8B, addition of
affinity purified GA733-FL to the cells inhibits aggregation, whereas
GA733-EC has no effect, indicating that dimers, but not monomers, are
capable of blocking cell-cell adhesion. Fig. 8C shows the
concentration-dependent effects of GA733-FL and GA733-EC on
the inhibition of Caco-2 cell aggregation. Even at concentrations of
~1 µM, GA733-EC does not significantly inhibit cell
aggregation, whereas 50% inhibition by GA733-FL occurred at less than
0.5 µM. Cell-cell adhesion is a complex and poorly
understood process involving multiple CAM systems. In addition, other
proteins that may interact with GA733, including cytoskeletal proteins,
may affect GA733-mediated cell-cell adhesion in vivo.
Nonetheless, the inhibition of cell-cell adhesion by GA733-FL in the
low µM range as shown in Fig. 8C is consistent with the Kd~10 µM measured for the
dimer-tetramer association using the analytical ultracentrifuge.
Intercellular interactions mediated by cell surface CAMs are known
to be involved in a wide variety of dynamic processes, including cell
movement, proliferation, and differentiation. The control of these
different processes plays critical roles in embryogenesis, wound
healing, maintenance of normal tissue morphogenesis, and tumor
progression. GA733 is a transmembrane glycoprotein that mediates
homotypic Ca2+-independent cell-cell adhesion. GA733
targeting has shown promising results in the immunotherapy of colon
cancer (21, 22), and recent data demonstrated an inhibitory effect of
GA733 on tumor invasion in vitro (12). Yet, the precise
function of GA733-mediated cell-cell adhesion, its role in normal
epithelial cells, and its involvement in colon cancer are poorly understood.
In the present study, we explored the oligomeric state of GA733 and the
mechanism of GA733-mediated cell-cell adhesion. Our data show: (i)
recombinant GA733-EC is a monomer in solution and is inactive in
cell-cell aggregation assays; (ii) recombinant GA733-FL forms high
affinity dimers (Kd < 10 nM) and moderate affinity tetramers (Kd ~ 10 µM) in solution and exhibits inhibitory activity in cell
aggregation assays in the low micromolar range; and (iii) GA733 in
human colon carcinoma cells exists primarily as noncovalent
cis-dimers in single cell suspensions. These results
demonstrate that GA733 dimerization is essential for GA733-mediated
cell-cell adhesion. The monomeric nature of GA733-EC indicates that the
cytoplasmic and/or the transmembrane domain of GA733 are needed for
this cis-dimer formation. Further studies with truncated
recombinant GA733 proteins will be needed to map the specific regions
of these domains responsible for controlling dimer formation.
The extent of cross-linking to dimers, in both Caco-2 and Colo-205
colon carcinoma cells, was similar for adherent monolayers versus single cell suspensions. This indicates that GA733
dimerization occurs by cis-interactions within the membranes
of individual cells, rather than trans-interactions between
GA733 monomers present in two different cells. Other CAMs exhibit
similar interactions, e.g. the carcinoembryonic antigen was
found to form noncovalent dimers (52), and there are a growing number
of other CAMs that require dimer formation for ligand binding.
Dimerization of PECAM-1, E-cadherin, VCAM-1, and ICAM-1 seems to be
important for their adhesion function and may represent an important
regulatory mechanism of signal transduction pathways (26-28, 53, 54).
The small amount of GA733 tetramers detected when Caco-2 cells were
cross-linked in monolayers are most likely due to the head-to-head
association between GA733 dimers on opposing cells, because GA733 is a
homotypic CAM (see model in Fig. 9). The
dimer-tetramer association is a moderate affinity interaction
(Kd ~ 10 µM), which is consistent
with reversible intercellular adhesion.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol, 2% Triton
X-100, 40 mM sodium chloride, 0.15 mM
phenylmethylsulfonyl fluoride (PMSF), 2 mM leupeptin, and
10 µg/ml pepstatin, pH 7.4. The lysate was sonicated, centrifuged for
20 min at 16,000 × g, and the supernatant was filtered
through a 0.22-µm filter and loaded onto a column containing 30 mg of affinity-purified GA733 mAb coupled to Sepharose CL-4B by CNBr activation. The column was washed with a buffer containing 100 mM Tris, pH 7.4, with 0.05% Triton X-100, 10 mM EDTA, 10 mM EGTA, 0.15 mM PMSF,
2 mM leupeptin, and 10 µg/ml pepstatin, washed with the
same buffer without detergent and bound proteins were eluted with 50 mM glycine, pH 2.5. The pH of the peak fractions was
immediately neutralized, followed by dialysis against PBS (10 mM sodium phosphate, 130 mM NaCl, pH 7.4)
supplemented with 0.15 mM PMSF, 5 mM EDTA, 0.02% sodium azide. The quality of purifications was monitored by
SDS-PAGE and Western blot analysis using the GA733 mAb.
-glutamyl-
-lysine bonds.
Oligomers of GA733 (up to tetramers, but primarily dimers) could be
detected by Coomassie Blue-stained SDS gels and on Western blots.
Maintaining 100 mM Tris, with 10 mM EDTA and 10 mM EGTA throughout the washes, substantially minimized this
artifactual cross-linking and was used in most purifications.
Alternatively, we were able to completely eliminate this artifactual
cross-linking by using 0.5% Triton X-100 in the wash and elution
buffers. However, such preparations were used only for chemical
cross-linking experiments, because Triton X-100 could not be
efficiently removed by dialysis and attempts to use ion-exchange
chromatography to remove the detergent were unsuccessful, making these
preparations unsuitable for either cell aggregation assays or
analytical ultracentrifugation studies.
1 calculated from the
GA733-EC sequence according to Pace et al. (33).
'
), containing contributions
from the protein and the bound detergent (35, 36),
where MP is the molecular weight of the
protein,
(Eq. 1)
P and
Det are the
partial specific volumes of the protein and detergent respectively,
Det is the number of grams of detergent bound per gram
of protein, and
is the density of the solvent. Because the partial
specific volume of C12E8 is known (0.973 cm3/g (37)), the density of the solvent can be adjusted to
1/
Det with D2O. In this case the
second term of the equation equals zero, effectively removing the
contribution of the bound detergent. The density of the dialysis buffer
described above was calculated to be 1.0278 g/cm3 at
20 °C using the SEDNTERP program. For experiments on GA733-FL, three
cells were assembled with double-sector 12-mm centerpieces and quartz
windows, and loaded with 110 µl of dialysis buffer as the reference,
and 110 µl of sample at three protein concentrations (typically 0.2, 0.1, and 0.05 mg/ml). Experiments were performed at 4 or 20 °C at
speeds between 14,000 and 23,000 rpm. Absorbance data at 280 or 230 nm
was collected every 4-6 h until equilibrium was reached. For each
scan, data were acquired every 0.001 cm with up to nine replicates in
continuous scan mode.
of a protein is defined as:
= M(1
)
2/RT, where M and
are
the molecular weight and the partial specific volume of the protein,
respectively, R is the gas contant T is temperature in Kelvin, and
the density of the solvent (34). The program SEDNTERP was used to calculate
of GA733-EC and GA733-FL, using the known amino acid sequence of the proteins and an
estimate of the amount of carbohydrate determined from MALDI mass
analysis, which was treated as mannose groups for calculation purposes
of
. At least three data sets from different loading concentrations and/or rotor speeds were fitted simultaneously. Goodness
of fit was determined by examination of the residuals and minimization
of the variance. The association constants returned by NONLIN from
self-association models were converted to the molar scale using the
calculated molar extinction coefficient of the protein.
-mercaptoethanol, 0.5 g/liter deoxycholic acid, 0.1%
SDS, 40 mM sodium chloride, 0.15 mM PMSF, 2 mM leupeptin, and 10 µg/ml pepstatin, pH 7.4. Cell
extracts were centrifuged for 20 min at 16,000 × g,
the pellets were discarded, and the supernatants were saved for
immunoprecipitations, SDS-PAGE, and Western blots.
Nt)/N0, where
Nt is the number of remaining particles
at the time point t = 2 h, and
N0 is the initial number of particles
corresponding to the total number of cells at time zero.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
SDS-PAGE analysis of purified GA733
recombinant proteins. Recombinant proteins GA733-EC (A)
and GA733-FL (B) were separated on 6% Tris-Tricine gels
under reducing conditions and stained with Coomassie Blue, after
affinity purification (left lanes) and after HPLC gel
filtration (right lanes). The positions of standard proteins
in kilodaltons are shown on the left. Arrows
indicate minor amounts of artifactually cross-linked forms of the
protein; these cross-linked products represent less than 5% of the
total protein.
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Fig. 2.
HPLC gel filtration chromatography of GA733
recombinant proteins. Chromatograms of affinity-purified GA733-EC
(A) and GA733-FL (B) separated on HPLC gel
filtration columns. The retention time of GA733-EC falls between
protein standards with molecular masses of 44 and 17 kDa, whereas
GA733-FL elutes between the 158- and 44-kDa standards. The columns and
the conditions used are described under "Materials and
Methods."
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[in a new window]
Fig. 3.
Mass spectra of GA733 recombinant
proteins. MALDI-TOF mass analysis of GA733-EC after HPLC gel
filtration (A) and affinity-purified GA733-FL concentrated
in 10 mM C8E5 (B). For
both proteins, two major species are observed, consistent with
heterogeneous glycosylation.
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Fig. 4.
Representative sedimentation equilibrium
experiments of GA733-EC and GA733-FL. Sedimentation equilibrium
data for three loading concentrations of GA733-EC (A) at
23,000 rpm and 30 °C. The three data sets were fitted simultaneously
using the nonlinear regression program NONLIN as described under
"Materials and Methods." The data were fitted with a model
describing monomers. The raw data (circles) and the global
fit of an ideal single species model (lines) are shown. In the
upper panels, the residuals of the fitted curve to the data
points for the three protein concentrations, from highest to lowest
(top to bottom) are shown. Sedimentation
equilibrium data of GA733-FL at 14,000 rpm (B) and 19,000 rpm (C), at 20 °C. The six data sets were globally fitted
with a model describing high affinity dimers with no detectable
dissociation to monomers and much weaker self-association to tetramers
(Kd = 10 µM), using the nonlinear
regression program NONLIN. The lower panels show the
concentration versus radius data for three loading
concentrations of GA733-FL (circles). The solid
lines represent the calculated fit. The upper panels
show the residuals of the fitted curves to the data points for the
three protein concentrations, from highest to lowest (top to
bottom).
Sedimentation equilibrium experiments performed with GA733-FL in the
presence of C8E5 and C12E8
View larger version (85K):
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Fig. 5.
Representative chemical cross-linking
experiment of GA733 recombinant proteins in solution. A 6% SDS
gel of control (C) and cross-linked (DSG or
BS3) affinity-purified GA733-EC and GA733-FL in the presence
of 0.5% Triton X-100 is shown. The GA733-FL is cross-linked into
dimers with minor bands at the trimer and tetramer positions (indicated
by arrows) when 0.1 mM concentrations of
cross-linkers were used. Under the same experimental conditions,
GA733-EC remains monomeric.
View larger version (86K):
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Fig. 6.
Chemical cross-linking of adherent monolayers
and single cell suspensions of colon carcinoma cells. Caco-2 colon
carcinoma cells were cross-linked with various concentrations (0.1-2
mM) of BS3 in monolayers (A) or in
single cell suspensions (B) after dissociation with
trypsin/EDTA. GA733 was immunoprecipitated with GA733 mAb-Sepharose and
separated by SDS-PAGE. GA733-specific bands were detected by Western
blot analysis using a GA733 pAb. Similarly, Colo-205 cells were
cross-linked either in situ, in culture flasks
(C) or after dissociation with EDTA only (D). The
arrows indicate the positions of GA733 monomers, dimers,
trimers, and tetramers. "Cont" represents the control
noncross-linked condition.
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Fig. 7.
GA733 expression in dissociated colon
carcinoma cell lines. Western blot analysis (A) of
GA733 extracted from cells in situ
(non-dissociated) and dissociated Caco-2 (with trypsin/EDTA)
and Colo-205 (with EDTA alone) cells. The positions of standard
proteins are shown on the left (in kilodaltons). FACS
analysis (B) of cell surface expression of GA733 on colon
carcinoma cells after dissociation with trypsin/EDTA
(Caco-2) or EDTA alone (Colo-205).
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Fig. 8.
Effect of GA733 recombinant proteins on cell
adhesion properties of colon carcinoma cells. In A,
Caco-2 cells were dissociated to single cell suspensions. Cells at time
0 (a) and after aggregation for 2 h in the absence
(b) or the presence of either GA733-FL (c) or
GA733-EC (d) are shown. In B, Caco-2 and Colo-205
cells were counted after being dissociated into single cell suspensions
(at time 0) and then allowed to aggregate for 2 h at 37 °C. The
cells were fixed, total particles were counted, and a degree of
aggregation (D) was calculated as described under
"Materials and Methods." The data shown are the averages of nine
measurements from three independent experiments ± S.D. In
C, the percentage of inhibition of Caco-2 aggregation as a
function of molar concentrations of GA733-EC (triangles) and
GA733-FL (squares) is shown. GA733-FL inhibits aggregation
in a concentration-dependent manner, whereas GA733-EC has
no effect (line represents a least squares fit to the data).
The concentration-dependence of GA733-FL inhibition approximates an
exponential relationship (solid curve).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 9.
Model of GA733 self-association in
vivo. GA733 apparently exists in the cell membrane of colon
carcinoma cells as a high affinity (Kd < 10 nM) noncovalent cis-dimer. GA733 dimers on
opposing membranes can associate via a head-to-head
interaction to form tetramers with moderate affinity
(Kd ~10 µM), consistent with
reversible intercellular associations.
Minor bands at the trimer and tetramer position are observed when Caco-2 cells are cross-linked in suspension, which could result from the fast cell-cell aggregation kinetics of Caco-2 cells during cross-linking. Alternatively, these bands could represent minor cross-linking of GA733 to other proteins. The failure to detect large amounts of GA733 tetramers in Colo-205 colon carcinoma cells cross-linked while attached to the culture flask is not surprising. Despite their normal expression of E-cadherin and catenins as well as GA733, Colo-205 cells do not form monolayers in culture, but grow as small aggregates or dispersed cells with minimal cell-cell contacts, and hence the majority of the surface of individual cells is not engaged in adhesion with other cells. Furthermore, these cells exhibit poor adhesion to extracellular matrix proteins, do not form tight interactions with each other, and dissociate easily in PBS during the cross-linking experiments (data not shown).
Other minor bands are also detected at different positions after cross-linking of Caco-2 and Colo-205 cells, especially a band between the monomer and the dimer at ~60 kDa. This band may be the result of one of the following: (i) gel shift of GA733 due to internal cross-linking and side chain modifications; (ii) coincidental random cross-linking with other proteins due to the high density of GA733 at the surface of colon carcinoma cells (about 106 copies of GA733 per cell (55)); or (iii) specific interactions with other membrane proteins.
The oligomerization of cell surface receptors plays a central role in the regulation of cellular functions by modulating signal transduction pathways. GA733-1 (Trop-2) that shares 50% amino acid identity with GA733 was shown to be involved in cell signaling. A recent study (14) showed that monoclonal antibodies against GA733-1 induce calcium fluxes in MCF-7 and OvCa-432 cells. Another study (56) demonstrated that GA733-1 is phosphorylated on serine 303, and that protein kinase C is involved in phosphorylating this protein in vitro. The exact mechanism by which homotypic GA733 engagement might activate signal transduction pathways in epithelial cells and the nature of these pathways remain intriguing questions for future studies.
The inhibitory effects of GA733 expression on growth and invasion of transfected mouse tumor cells are surprising in view of the beneficial effects of mAbs against GA733 for treating colorectal carcinoma patients. It is tempting to speculate on how these apparently conflicting observations might be reconciled. In addition to tumor cell elimination by the immune system, mainly via the antibody-dependent cellular cytotoxicity pathway, mAbs to GA733 in clinical therapy may have two other beneficial effects: (i) inhibit cancer cell dissociation from the primary tumor by increasing cell-cell association; (ii) decrease cell growth by mimicking GA733 self-ligation and activating signal transduction pathways leading to inhibition of cell proliferation and/or induction of apoptosis. We are currently generating a soluble fusion protein, a covalent GA733 dimer containing the GA733-EC fused to an Fc fragment of an immunoglobulin (EC-Fc), to further study the role of dimerization in homotypic cell-cell adhesion and signal transduction pathways mediated by GA733.
In addition to colon cancer, an increase of GA733 expression was
observed in cervical cancer lesions (11) and a very recent paper
reported increased GA733 levels in lung cancer (57). Understanding the
precise function of GA733 in normal epithelial cells and its role in
the biology of tumors should contribute to the development of new
strategies for cancer treatment or a minima substantially improve the existing GA733-based therapy of colorectal tumors.
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ACKNOWLEDGEMENTS |
---|
We thank Kaye Speicher for performing mass analyses, Peter Hembach and Sandy Harper for assistance in preparation of the figures, and Jeffery Faust for FACS analyses. We also thank Drs. Roland Greimers, Gavin Manderson, Ronen Marmorstein, and Aurélia Venault-Lewis for their useful comments on the manuscript. We are grateful to The Wistar Institute Recombinant Protein Production Facility for providing baculovirus-infected cells.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants CA74294 and CA66671 (to D. W. S.) and by National Cancer Institute Cancer Center Core Grant CA10815.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: Laboratory of Signal Transduction, National
Institute of Environmental Health Sciences, P. O. Box 12233, Research
Triangle Park, NC 27709.
§ Present address: The Victor Chang Cardiac Research Institute, Darlinghurst, Sydney, New South Wales 2010, Australia.
¶ To whom correspondence should be addressed: The Wistar Institute, 3601 Spruce St., Philadelphia, PA 19104. Tel.: 215-898-3972; Fax: 215-898-0664; E-mail: speicher@wistar.upenn.edu.
Published, JBC Papers in Press, October 31, 2000, DOI 10.1074/jbc.M004770200
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ABBREVIATIONS |
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The abbreviations used are: mAb, monoclonal antibody; CAM, cell adhesion molecule; BS3, bis(sulfosuccinimidyl) suberate; C12E8, octyloxyethylene dodecyl ether; C8E5, pentaoxyethylene octyl ether; DSG, disuccinimidyl glutarate; EC, extracellular domain; FL, full-length; HPLC, high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; mEGP, mouse epithelial glycoprotein; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; pAb, polyclonal antibody; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; FACS, fluorescence-activated cell sorting; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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