From the Department of Chemical and Biomolecular
Engineering, ** Department of Biomedical Engineering, and
¶ Molecular Biophysics Program, The Johns Hopkins University,
Baltimore, Maryland 21218
Received for publication, December 27, 2002, and in revised form, January 8, 2003
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ABSTRACT |
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P-selectin expressed on activated platelets and
vascular endothelium mediates adhesive interactions to
polymorphonuclear leukocytes (PMNs) and colon carcinomas critical to
the processes of inflammation and blood-borne metastasis,
respectively. How the overall adhesiveness (i.e. the
avidity) of receptor/ligand interactions is controlled by the affinity
of the individual receptors to single ligands is not well understood.
Using single molecule force spectroscopy, we probed in situ
both the tensile strength and off-rate of single P-selectin molecules
binding to single ligands on intact human PMNs and metastatic colon
carcinomas and compared them to the overall avidity of these cells for
P-selectin substrates. The use of intact cells rather than
purified proteins ensures the proper orientation and preserves
post-translational modifications of the P-selectin ligands. The
P-selectin/PSGL-1 interaction on PMNs was able to withstand forces up
to 175 pN and had an unstressed off-rate of 0.20 s Cellular adhesion mediated by biological macromolecules and
their respective ligands plays an essential role in a number of diverse
biological phenomena including inflammation and cancer metastasis.
Leukocyte recruitment to sites of infection is regulated by highly
specific receptor/ligand interactions that allow leukocytes to first
tether and roll on activated endothelium under hydrodynamic shear and
then firmly adhere prior to extravazation into the tissue space. These
stages are mediated via three distinct classes of adhesion molecules:
the selectins, integrins, and immunoglobulins (1-3). Accumulating
evidence suggests that tumor cell arrest in the microcirculation is
also mediated through receptor/ligand interactions between tumor cells
and the vascular endothelium in a manner analogous to leukocyte
recruitment (4-6). Both processes involve highly regulated molecular
events that rely on the local circulatory hemodynamics and the
mechanical and kinetic properties of participating adhesive molecular
groups, which have yet to be characterized at the single molecule level.
The involvement of P-selectin is critical within immune system
functioning. P-selectin, a cell-surface glycoprotein expressed on
activated endothelial cells and platelets, supports leukocyte tethering
and rolling in response to inflammatory signals by interacting with its
counter-receptor, P-selectin glycoprotein ligand-1
(PSGL-1),1 located on
leukocyte microvilli (7). Recently, specific P-selectin-tumor cell
interactions have been revealed providing direct evidence of its
participation in metastasis as well. The most compelling evidence for
the role of P-selectin in the metastatic process is the pronounced
inhibition of metastasis in P-selectin-deficient mice compared with
wild-type controls in a colon carcinoma cell model (8, 9). Several
lines of evidence suggest that the P-selectin ligands on a variety of
tumor cell lines are sialylated molecules distinct from PSGL-1
(10-12). Along these lines, enzymatic removal of these PSGL-1 distinct
P-selectin ligands from the carcinomas results in a marked reduction of
experimental metastasis (9).
Biophysical parameters of P-selectin/ligand binding have previously
been obtained by quantifying leukocyte tethering duration (13, 14) and
rolling velocity (15) on purified P-selectin substrates. These
techniques may not effectively differentiate avidity from the affinity
of a single receptor/ligand pair and, most importantly, rely on
broad assumptions to estimate the forces on receptor/ligand bonds.
Previous single molecule work on the characterization of
P-selectin/PSGL-1 binding was performed using purified adhesion
molecules rather than intact target cells (16). However, subtle
differences between native and recombinant forms of PSGL-1 (17) can
impact biophysical measurements. Here we employ single molecule force
spectroscopy to probe the tensile strength and unstressed off-rate of
P-selectin/PSGL-1 binding on intact human polymorphonuclear leukocytes
(PMNs), a technique that preserves the orientation and
post-translational modifications of PSGL-1. This methodology was
extended to characterize P-selectin/ligand binding on intact metastatic
colon carcinoma cells. Macroscopic studies performed using a
parallel-plate flow chamber reveal that PSGL-1-mediated PMN recruitment
to P-selectin substrates is more efficient than tumor cell-P-selectin
interactions under dynamic flow conditions and results in stable
versus transient rolling interactions (10-12). Therefore,
we aimed to provide a mechanistic interpretation at the molecular level
for the differential abilities of PMNs and tumor cells to roll on
P-selectin by evaluating and comparing the affinity of single
P-selectin/ligand bonds. The LS174T human colon adenocarcinoma cell
line was chosen as a model because it has been used in a number of
diverse assays ranging from characterization of surface adhesion
molecules to cell-substrate and cell-cell interaction studies (5, 10,
18, 19). Finally, using highly specific enzymes and glycoconjugate
biosynthesis inhibitors, we have characterized the biochemical nature
of the putative P-selectin ligand on LS174T colon carcinomas.
Reagents--
The chimeric form of P-selectin-IgG Fc
(P-selectin) consisting of the lectin, epidermal growth factor, and
nine consensus repeat domains for human P-selectin linked to each arm
of human IgG1 was a generous gift from Dr. Ray Camphausen of Wyeth
External Research (Cambridge, MA) (20). All of the other reagents were purchased from Sigma unless otherwise stated.
Cell Culture--
LS174T human colon adenocarcinoma cells were
obtained from the American Type Culture Collection (Manassas, VA) and
cultured in the recommended medium. Cells were detached from culture
flasks using 0.25% trypsin, EDTA for 2 min at 37 °C (5, 10, 21, 22). For force spectroscopy experiments, 20 µl of 1 × 107 cells/ml LS174T cell suspension was layered on 35-mm
tissue culture dishes and subsequently incubated overnight at 37 °C
to allow adhesion to the culture dish and regeneration of surface
glycoproteins (5, 10). This procedure resulted in ~20% cellular
confluency as assessed by phase-contrast microscopy. Prior to use in
force spectroscopy experiments, non-adherent LS174T cells were removed by gentle rinsing with D-PBS, and the standard medium was
replaced with serum-free medium containing Hank's salts, which help to stabilize the pH outside of the 5% CO2 environment of an
incubator. In flow-based adhesion assays, the trypsinized LS174T cell
suspension was incubated for 2 h at 37 °C to regenerate surface
glycoproteins and was used immediately thereafter (5, 10, 21, 22).
Cell Treatments--
In selected experiments, LS174T cell
suspensions were incubated for 30 min at 37 °C with 20 µg/ml
trypsin to cleave cell surface glycoproteins (21) and washed once
before use in flow-based assays. For metabolic inhibitor studies,
LS174T cells were cultured for 48 h at 37 °C in medium
containing either 2 mM
benzyl-2-acetamido-2-deoxy- PMN Isolation and Monolayer--
Human PMNs were isolated from
citrate phosphate dextrose-anticoagulated venous blood of
healthy volunteers as described previously (21, 22), resuspended at
1 × 106 cells/ml in
Ca2+/Mg2+-free D-PBS (0.1% BSA),
and stored at 4 °C for no more than 3 h before use in all
experiments. To immobilize PMNs, 200 µl of the isolated PMN cell
suspension was allowed to incubate on a 35-mm tissue culture dish for 5 min at RT (23). To prevent further activation, a 1%
formalin-D-PBS solution was added to the cell culture dish
and maintained at RT for 10 min (23). The PMN monolayer was rinsed and
refilled with D-PBS containing
Ca2+/Mg2+. This procedure resulted in a PMN
monolayer of ~40% confluency.
Cantilever Functionalization--
To provide a surface that
readily binds soluble proteins, molecular force probe (MFP) cantilevers
(TM Microscopes, Sunnyvale, CA) were silanized with 2%
3-aminopropyltriethoxysilane (10). The cantilevers were then incubated
in a 3.3-µg/ml solution of anti-human IgG Fc mAb in D-PBS
containing 50-fold molar excess of the cross-linker
bis(sulfosuccinimidyl) suberate (BS3, Pierce) for 30 min followed by quenching with Tris buffer. Cantilevers were
subsequently incubated with dilute P-selectin-IgG Fc chimera protein in
D-PBS for 2 h at RT followed by immersion in 1% BSA to block nonspecific interactions. Binding the IgG Fc portion of the
P-selectin chimera to the immobilized anti-IgG Fc mAb on the cantilever
maintains its proper functional orientation. Concentrations of the
anti-human IgG Fc and P-selectin chimera solutions were optimized to
result in a low percentage of binding events during force spectroscopy
experiments (~30 binding events/100 cell contacts). 1.8 and 3.0 µg/ml P-selectin-IgG Fc solutions were used for PMN and LS174T cell
experiments, respectively.
Single Molecule Force Spectroscopy--
Experiments were
conducted using an MFP (Asylum Research Inc., Santa Barbara, CA). Two
triangular-shaped cantilevers with nominal spring constants of 10 and
40 picoNewton/nm were calibrated using thermal noise amplitude,
and their deflection was measured by laser reflection onto a split
photodetector. Measurements were carried out using serum-free
medium for the LS174T cells or D-PBS containing
Ca2+/Mg2+ for PMNs. The 35-mm culture dish
containing the adherent cellular monolayer immersed in either medium or
D-PBS was placed on the MFP stage and positioned so that
the cantilever was directly above a single cell. The distance between
the cantilever and the cell was adjusted so that each approach cycle
resulted in a slight depression force on the cell before reproach. The
reproach velocity was varied from 0.5 to 30 µm/s, and the dwell time
between the cantilever and the cell was set to 0.001 s to minimize the
occurrence of multiple events (24). Rupture forces were derived from
force versus distance traces using IgorPro 3.11 software
(Wavemetrics, Inc., Lake Oswego, OR). Histograms representing ~350
approach/reproach cycles were compiled for each reproach velocity, and
the mean rupture force of a P-selectin/ligand interaction was
evaluated. Prior work has shown that the mean and the mode of the
rupture force approach one another over the higher loading rate regime, and the mean can accurately be used to estimate Bell model parameters (25).
Flow Cytometry--
Expression levels of the P-selectin ligand
on LS174T cells and human PMNs were quantified by indirect single color
immunofluorescence and flow cytometry. To this end, P-selectin chimera
solution was preincubated with fluorescein isothiocyanate-labeled goat
anti-human IgG Fc mAb for 1 h at RT before incubation of the
mixture with either LS174T or PMN cell suspensions at 1 × 106 cells/ml in D-PBS, 0.1% BSA (8). After
incubation at 4 °C for 1 h, cells were washed with
D-PBS and then fixed using 2% formalin (v/v) (8). Cells
were washed again and resuspended in D-PBS containing 0.1%
BSA. Background levels were determined by incubating with conjugated
protein in the presence of 30 mM EDTA (8).
Flow-based Adhesion Assays--
P-selectin-coated surfaces were
prepared by incubating 1-4 µg/ml anti-human IgG Fc mAb in
D-PBS on untreated 35-mm polystyrene culture dishes
overnight at 4 °C. Equally diluted solutions of P-selectin-IgG Fc
protein were then layered on the dish and allowed to incubate for
2 h at RT before rinsing with D-PBS and blocking with
1% BSA for 2 h (10). Suspensions of either LS174T cells or PMNs
at a final concentration of 1 × 106 cells/ml were
perfused over the P-selectin-coated dishes using a parallel-plate flow
chamber (250-µm-channel depth, 5.0-mm-channel width) for 3 min at
37 °C (26). Rolling velocities were computed as the distance
traveled by the centroid of the translating cell divided by the time
interval (10, 26).
Statistical Analysis--
Data are expressed as the mean ± S.E. for at least three independent experiments. Statistical
significance of differences between the means was determined by ANOVA.
Whether the means were shown to be significantly different
(p < 0.05), multiple comparisons were performed by the
Tukey test.
Single Molecule Force Spectroscopy Measurements--
An MFP was
used to determine the tensile strength and unstressed off-rate of
P-selectin binding to its ligands on intact human PMNs and LS174T colon
carcinomas at the single molecule level. To this end, force-distance
traces (Fig. 2a) were generated by lowering the
P-selectin-functionalized cantilever to the immobilized cellular
monolayer (Fig. 1), maintaining
P-selectin/cell contact for a set period of time (0.001 s) to allow
establishment of receptor/ligand binding and subsequently retracting
the cantilever from the cell surface at a constant prescribed velocity.
Upon retraction of the cantilever, the force was recorded as a function
of the vertical displacement until dissociation of P-selectin/ligand
bonds occurred (Fig. 2a).
Under the conditions of this study, receptor/ligand unbinding at any
given reproach velocity predominantly involved single rather than
multiple steps (24) as shown in the representative force-distance
trace. Additionally, rupture force histograms at a given reproach
velocity always indicated a single peak distribution for both LS174T
cell and PMN experiments (Fig. 2b), suggesting the rupture
of a single P-selectin/ligand complex (24). A single peak distribution
also indicates that nonspecific interactions did not play a significant
role in these measurements.
To ensure that most binding events were mediated by a single
receptor/ligand pair, a low frequency of binding events (an average of
30 per 100 contacts) was achieved by decorating cantilevers with
sufficiently dilute P-selectin chimera solutions. The concentration of
P-selectin on the cantilever was greater for the LS174T cell (3 µg/ml) versus PMN (1.8 µg/ml) experiments, a result of
the differing expression levels of P-selectin ligands on the cell surfaces (Table I). The percentage of
binding events in this study ranged from 18 to 40% with 30%
being the targeted average value for both PMN and LS174T cells. Based
on Poisson distribution statistics, when 30% of cantilever to cell
contacts lead to P-selectin/ligand binding, 83% of those successful
adhesions are probably because of a single bond (Nb = 1) (25, 27). Moreover, statistics predict that there will be 15%
double bonds (Nb = 2) and <3% will have
Nb of >2.
Two different control experiments were performed to demonstrate the
specificity of P-selectin/ligand binding. First, the addition of EDTA
to the tissue culture dish at a final concentration of 0.5 mM consistently abrogated binding (Fig. 2a), a
finding that is in accord with the calcium dependence of
selectin/ligand binding (7, 10). Second, incubating the
P-selectin-coated cantilever with a function-blocking anti-P-selectin
mAb AK4 (50 µg/ml, BD Biosciences) drastically reduced the frequency
of binding events from ~30% to <5% (Fig. 2a), an
observation that is consistent with other receptor/ligand measurements
at the single molecule level blocked by mAbs (25, 28). These
observations were valid for both LS174T and PMN cells.
Calculation of Bell Model Parameters and Monte Carlo
Analysis--
To construct a plot of rupture force versus
loading rate, rupture forces and corresponding loading rates for
hundreds of events were tabulated off-line and compiled into histograms
for each reproach velocity and mean rupture forces were calculated
(25). Histograms from a representative P-selectin/PSGL-1 experiment over a range of reproach velocities are presented in Fig.
2b. The loading rate (pN/s) for each reproach velocity was
determined by first evaluating the slope of the force versus
distance trace just before each rupture event (Fig. 2a) and
multiplying this number (pN/µm) by the reproach velocity (µm/s).
The Bell model parameters, namely the unstressed off-rate
k
To validate the accuracy of the calculated Bell model parameters, the
experimental data were modeled over the entire range of loading rates
by using the probability density function for bond rupture (25, 31) as
shown in Equation 1,
To further validate the agreement of our data with the Bell model,
Monte Carlo simulations of receptor/ligand bond rupture under constant
loading rates were performed. Given a
k
Taken together, our data indicate that the tensile strength of
P-selectin binding to its ligand on LS174T colon carcinoma is
significantly lower than that of P-selectin/PSGL-1 binding at any given
loading rate (Fig. 3a). The differences in their respective
tensile strengths as well as the unstressed off-rates and ligand
densities provide a mechanistic basis for the differential rolling
behavior of LS174T colon carcinomas and PMNs on P-selectin substrates
observed in flow-chamber experiments (Table I).
Biochemical Characterization of P-selectin Ligands on LS174T Colon
Carcinomas--
We have recently shown that the P-selectin ligand on
LS174T colon carcinomas is a novel sialylated molecule functionally
distinct from the previously identified P-selectin ligands PSGL-1,
glycoprotein Ib/IX, and CD24 (10). Further experiments on the
biochemical nature of the target ligand on LS174T cells reveal that it
is a protease-sensitive glycoprotein rather than a glycosphingolipid (Fig. 4a). This was
demonstrated by the marked inhibition of trypsin-treated LS174T cells
to tether to purified P-selectin as opposed to cells cultured in the
presence of a glycosphingolipid biosynthesis inhibitor threo-PPPP (21),
which retained their ability to bind to P-selectin underflow (Fig.
4a).
We next examined whether critical P-selectin binding determinants on
LS174T colon carcinoma cells are presented on O-linked and/or N-linked glycans. To this end, LS174T cells were
cultured in the presence of either benzyl-GalNAc known to inhibit
O-linked glycosylation or DMJ (which disrupts
N-linked processing) before use in adhesion assays. The
results indicated that LS174T cell tethering to P-selectin-coated
surfaces was drastically inhibited by benzyl-GalNAc but not by DMJ
(Fig. 4a). Similar results were obtained using single
molecule force spectroscopy. More specifically, the treatment of LS174T
cells with benzyl-GalNAc reduced the frequency of binding from 32 to
<8%, whereas DMJ impacted neither the force distribution nor the
frequency of events (Fig. 4b). Collectively, these data
reveal that the P-selectin ligand is a novel O-linked, sialylated protease-sensitive structure and does not require
N-glycans for binding.
The interaction of PSGL-1 with the actin cytoskeleton is known to
regulate the strength of leukocyte adhesion to P-selectin as evidenced
by increased resistance to shear-induced detachment forces upon PMN
treatment with cytochalasin B, a compound that caps the growing end of
actin filament (32). Therefore, we wished to examine whether
P-selectin-mediated LS174T binding is also dependent on actin
cytoskeleton. However, the treatment of LS174T cells with cytochalasin
B affected neither the extent nor the pattern of cell tethering to
immobilized P-selectin chimera at a wall shear stress of 1 dyne/cm2 (data not shown). Furthermore, latrunculin
A (1 µM), a compound that prevents actin polymerization
by irreversibly binding to actin monomers (33), failed to affect
P-selectin-mediated LS174T cell adhesion (data not shown), although it
nearly abrogated PMN binding to P-selectin under flow (control samples
(156 ± 15 tethered PMNs/mm2) versus
lantriculin-treated samples (7 ± 1 PMNs/mm2)).
Cumulatively, these data provide evidence for the absence of actin
cytoskeleton involvement in the regulation of P-selectin/LS174T P-selectin ligand interactions unlike P-selectin/PSGL-1 (32).
Using single molecule force spectroscopy, we measured
the biophysical parameters of P-selectin binding to ligands on intact LS174T colon carcinomas and PMNs at a single molecule resolution. By
carefully controlling the concentration and orientation of immobilized
P-selectin chimeras on the surface of an MFP cantilever and regulating
its contact time and depression force on the immobilized cells, we
evaluated the affinity of single P-selectin/ligand interactions under
conditions that preserve all appropriate post-translational ligand
modifications. In this study, we determined that the tensile strength
of P-selectin/PSGL-1 binding is substantially greater than
P-selectin/LS174T P-selectin ligand binding for all loading rates.
Additionally, the unstressed off-rate for P-selectin/PSGL-1 (0.20 s The kinetics of P-selectin to PSGL-1 binding has previously been
studied by several different techniques including neutrophil tethering
lifetime as assessed in flow-chamber experiments (13, 14, 34), plasmon
resonance (35), and atomic force microscopy (16). To extract the
biophysical parameters (tensile strength and unstressed off-rate) from
neutrophil tethering measurements, several assumptions are required.
First, the rolling cell is assumed to be a rigid sphere decorated with
immobile adhesion molecules. However, leukocytes are covered with
deformable microvilli where cellular adhesion molecules are
concentrated, and experiments confirm that their rolling velocity over
P-selectin substrates is greatly influenced by the processes of
microvilli elongation and cytoskeletal rearrangement (15).
Additionally, the applied force on a receptor/ligand bond is estimated
from the wall shear stress and approximate geometric relationships (13,
15), because the actual force exerted on the receptor/ligand pair is
unknown. Here, we have experimentally measured each bond rupture force and rigorously determined its respective loading rate by evaluating the
slope of the force-distance trace just before the rupture event.
The highest value for a P-selectin/PSGL-1 rupture force
that we determined approached 175 pN, a value similar to that found by
Fritz et al. (16) using single molecule force spectroscopy, although our results were obtained at substantially higher reproach velocities. The relative loading rates for the two studies, which would
allow direct comparisons to be made, are unknown. The unstressed off-rate for P-selectin/PSGL-1 calculated in this study was 0.20 s The impact of undesirable multiple events during single molecule force
spectroscopy has been evaluated using Monte Carlo simulations (25). At
30% binding/cell/cantilever contact, it is possible that 17%
approach/reproach cycles may involve multiple adhesions, which may
impact the lifetime of bound complexes and inflate rupture force
values. However, it has been found that multiple events only very
modestly reduce the theoretically attainable Bell model parameters
found from simulations that involve strictly single events (25). In the
case of 40% binding, which means that 23% multiple bonds are
expected, the k To effectively use PMNs in an MFP experiment, PMNs must be
firmly immobilized on a substrate and subsequently fixed to prevent further morphological changes (23). The fixation of PMNs has been shown
to abrogate microvillus elasticity, causing them to behave as
PSGL-1-coated rigid microbeads but without impairing the molecular
interaction between P-selectin and PSGL-1 (15). The process of fixing
PMNs decreases the likelihood that the applied load on the
P-selectin/PSGL-1 complex will be partially dissipated by viscous
deformation during microvilli extension. Thus, the rupture forces for
fixed PMNs may be higher at all loading rates than for unfixed PMNs,
because nearly the entire applied load is placed on the receptor/ligand
pair and any cytoskeletal contributions have been eliminated.
We also examined the effects of fixation with 1% formalin on the
biophysical parameters of P-selectin/ligand binding on LS174T cells.
Our experiments reveal that neither the tensile strength at all loading
rates nor the Bell model parameters were appreciably impaired by the
fixation process (see Supplemental Fig. 1), a finding that may be
partially attributed to the lack of actin cytoskeleton interaction with
P-selectin ligand on LS174T cells. Thus, under experimental conditions,
in which orientation and post-translational modifications of P-selectin
ligands are preserved and the cytoskeletal contributions have been
minimized in both cell types, the tensile strength and affinity of
P-selectin/PSGL-1 interactions are higher than those of
P-selectin/LS174T selectin ligand binding. Although we have not
identified the putative P-selectin ligand on the LS174T cell surface,
our data indicate that it is a novel sialylated, protease-sensitive
molecule that displays O-glycan-dependent
binding activity. Moreover, the existence of a single peak distribution
of rupture forces (Fig. 4b) suggests the presence of one
major LS174T P-selectin ligand or, alternatively, the existence of more
than one ligand with very similar properties.
In conclusion, we have employed single molecule force
spectroscopy to probe in situ the biophysical parameters of
P-selectin binding to ligands on intact human cells at the single
molecule level. The differential abilities of PMNs and colon carcinomas to roll on P-selectin substrates are attributed in part to intrinsic differences in receptor/ligand binding affinity as well as varying densities of ligands on their respective cell surfaces. Given the
direct role of P-selectin in hematogenous spread (8, 9), characterizing
the biochemical and biophysical properties of functional P-selectin
ligands on carcinomas will provide guidelines to engineer novel
therapeutic agents that will selectively block ligand function and thus
interfere with metastatic spread. Such a strategy would offer specific
anti-metastatic efficacy without impairing other important
P-selectin-mediated pathophysiological processes. The in
situ technique presented here provides a direct means for
evaluating the efficacy of potential adhesion-blocking therapeutic agents.
1. The
tensile strength of P-selectin binding to a novel O-linked, sialylated protease-sensitive ligand on LS174T colon carcinomas approached 125 pN, whereas the unstressed off-rate was 2.78 s
1. Monte Carlo simulations of receptor/ligand bond
rupture under constant loading rate for both P-selectin/PSGL-1 and
P-selectin/LS174T ligand binding give distributions and mean rupture
forces that are in accord with experimental data. The pronounced
differences in the affinity for P-selectin/ligand binding provide a
mechanistic basis for the differential abilities of PMNs
and carcinomas to roll on P-selectin substrates under blood flow
conditions and underline the requirement for single molecule affinity measurements.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside (benzyl-GalNAc) to inhibit O-linked glycosylation (21) or 1 mM deoxymannojirimycin (DMJ) to disrupt N-linked
processing (21), or for 96 h, LS174T cells were cultured with 5 µM
D,L-threo-1-phenyl-2-amino3-morpholino-1-propanol hydrochloride (threo-PPPP, Matreya, Inc., State College, PA), which
blocks the transfer of UDP-glucose to ceramide, thereby blocking the
synthesis of glycosphingolipids having a glucosylceramide core (21).
Treated LS174T cells were washed twice before use in experiments, and
cell viability was consistently >97% as detected by the trypan blue
exclusion assay.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
MFP schematic indicating the use of intact
cells to measure in situ cellular adhesion
forces. The cantilever was positioned directly above a cell and
was allowed to approach until it touched and slightly deformed the cell
membrane before reproaching at a set velocity. This approach/reproach
cycle was repeated hundreds of times to obtain a statistically
significant value for the P-selectin/ligand rupture force at that
velocity.
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Fig. 2.
a, force versus vertical
displacement traces taken from an experiment using LS174T colon
carcinomas in the absence (I) and presence of either EDTA
(II) or blocking anti-P-selectin mAb (III).
b, histograms of P-selectin to PSGL-1 binding on human PMNs
at six different reproach velocities representing at least 300 cell
contacts at each velocity.
Expression levels of P-selection ligands and average rolling velocities
of PMNs and LS174T colon carcinomas on P-selectin substrates
were
tabulated (Table II) by a least squares
fit to the linear region of the rupture force versus
logarithm of loading rate and extrapolating to zero force (Fig.
3a) (25, 29, 30). At some lower limit of the loading rate, the force versus logarithm
of loading rate plot begins to deviate from linearity or develop another linear region (25, 31). This phenomenon may indicate a change
of unbinding mechanism and transition states at lower loading rates
(29), but predictably the curve should approach the origin as zero
loading rate provides zero force. This lower limit of linearity was
reached at ~100 pN/s for P-selectin to PSGL-1 and 200 pN/s for
P-selectin to the ligand on LS175T cells (Fig. 3a). Table II
gives the values for k
for both cell types over the higher loading
rate regions. As can be seen, the unstressed off-rate is dramatically
higher for P-selectin/LS174T ligand (2.78 s
1) than for
P-selectin/PSGL-1 (0.20 s
1), whereas the values for
x
are similar.
Bell model parameters for P-selectin/PSGL-1 and
P-selectin/LS174T cell interactions
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Fig. 3.
a, molecular force versus
loading rate for P-selectin binding to PMN/LS174T cells. The data shown
comprise at least five independent experiments for each cell type
conducted on separate days to validate reproducibility. The
superimposed solid line indicates the non-linear least
squares fit over the entire range of experimental loading rates found
from the probability density function for bond rupture and
corresponding Bell model parameters. b, Monte Carlo
simulation for P-selectin/PSGL-1 binding at a reproach velocity of 25 µm/s.
where p(t, f) is the probability
of bond rupture at time t, rf is the
loading rate, kb is the Boltzmann's constant, and
T is the absolute temperature. The mean rupture force,
<fb>, from this distribution (25, 31) is given by
Equation 2.
(Eq. 1)
The Bell model parameters,
k
(Eq. 2)
, were estimated by a non-linear least
squares fit of the above equation to the experimental data over the
entire range of loading rates (Fig. 3a). As shown in Table
II, the values of Bell model parameters obtained by the non-linear fit
are in good agreement with those obtained by the linear fit of rupture
force versus logarithm of loading rate over the high loading
rates regime.
in each simulation, we calculated the
rupture force (Frup = rf × n
t) for which the probability of bond rupture
Prup is greater than Pran, a random number between zero and one,
where
(Eq. 3)
t is the interval and
n
t is the time step. The distributions and
means of rupture forces obtained in our experiments are in accord with
these simulations as shown for a representative experimental condition
and Monte Carlo simulations for P-selectin/PSGL-1 binding at a retract
velocity of 25 µm/s (Fig. 3b).
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Fig. 4.
a, LS174T cell tethering/rolling
over purified P-selectin at a wall shear stress of 1 dyne/cm2. Data are expressed as the percentage of untreated
(control) LS174T cells that interacted with purified P-selectin
throughout the 3-min experiment. Immobilized P-selectin was incubated
with an anti-P-selectin mAb (AK4, 20 µg/ml) for 10 min prior to
LS174T cell perfusion. LS174T cells were incubated with trypsin (20 µg/ml) for 1 h at 37 °C. Alternatively, untreated cells were
cultured with threo-PPPP (5 µM) or benzyl-GalNAc (2 mM), an O-linked glycan extension inhibitor, or
DMJ (1 mM), an N-linked glycan extension
inhibitor. b, P-selectin binding to LS174T tumor cells as
measured by single molecule force spectroscopy. Shown are rupture force
histograms for control LS14T cells and cells cultured in media
containing benzyl-GalNAc and DMJ.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1) was significantly different from the value for
P-selectin/ligand on LS174T cells (2.78 s
1). Thus, the
differential avidity of PMNs and LS174T colon carcinomas for P-selectin
substrates as assessed by rolling assays is attributed in part to
intrinsic differences in receptor/ligand binding affinity as well as
varying densities of ligands on their respective cell surfaces.
1, a result that lies in between the value obtained by
Fritz et al. (0.02 s
1) (16) and the studies
using flow-chambers (~1 s
1) (13, 14, 34). Differences
between our Bell Model parameters for P-selectin/PSGL-1 and those
calculated by Fritz et al. (16) may originate from slight
differences between the recombinant form of PSGL-1 and the natural form
on PMNs or any molecular orientation modifications that occur when
PSGL-1 is nonspecifically immobilized. Interestingly, if the Bell model
parameters are found by fitting a line to the low loading rate regime
(up to 100 pN/s), the off-rate at 0.03 s
1 approaches that
by Fritz et al. (16). However, we have chosen to calculate
the Bell model parameters from a linear fit to data within the higher
loading rate regime (100-10000 pN/s), a decision also made by others
using single molecule-spectroscopic techniques (25). Using a Monte
Carlo simulation, Tees et al. (25) concluded that for
selectin-carbohydrate binding, values for Bell model parameters are
correctly estimated from the steeper, higher loading rate region. The
lower loading rate regime does not in fact represent another set of
parameters but is consistent with a single model spanning low to
intermediate loading rates. In addition, estimations for the
physiological loading rates exerted on cellular adhesion molecules
range from ~100-10000 pN/s (25). It is noteworthy that the Bell
model parameters calculated using Equation 2 spanning the entire range
of loading rates showed excellent agreement with those obtained from
linear fit at the high loading rate regime. Furthermore, Monte Carlo
simulations using these parameter values yield rupture force
distributions in accord with those observed experimentally.
parameters were reduced by only 7 and 3%,
respectively (25).
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Ray Camphausen (Wyeth External Research) for the generous gift of P-selectin Fc IgG chimera protein. We also acknowledge fruitful discussions with Jason Cleveland (Asylum Research, Inc.) and with Dr. Ronald L. Schnaar (The Johns Hopkins University School of Medicine).
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FOOTNOTES |
---|
* This work was supported by a Whitaker Foundation grant and National Science Foundation Grant CTS0210718.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.
The on-line version of this article (available at
http://www.jbc.org) contains Supplemental Fig. 1.
§ Both authors contributed equally to this work.
To whom correspondence may be addressed. Dept. of Chemical and
Biomolecular Engineering, The Johns Hopkins University, 3400 N. Charles
St., Baltimore, MD 21218. Tel.: 410-516-6290, Fax: 410-516-5510;
E-mail: kkonsta1@jhu.edu (K. K.) or Tel.: 410-516-7006; Fax:
410-516-5510; E-mail: wirtz@jhu.edu (D. W.).
Published, JBC Papers in Press, January 8, 2003, DOI 10.1074/jbc.M213233200
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ABBREVIATIONS |
---|
The abbreviations used are:
PSGL-1, P-selectin glycoprotein ligand-1;
D-PBS, D-phosphate-buffered saline;
BSA, bovine serum albumin;
benzyl-GalNAc, benzyl-2-acetamido-2-deoxy--D-galactopyranoside;
mAb, monoclonal antibody;
DMJ, deoxymannojirimycin;
threo-PPPP, D,L-threo-1-phenyl-2-amino3-morpholino-1-propanol
hydrochloride;
PMN, polymorphonuclear leukocyte;
MFP, molecular
force probe;
RT, room temperature;
ANOVA, analysis of variance.
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REFERENCES |
---|
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---|
1. | McEver, R. P. (2002) Curr. Opin. Cell Biol. 14, 581-586[CrossRef][Medline] [Order article via Infotrieve] |
2. | Konstantopoulos, K., Kukreti, S., and McIntire, L. V. (1998) Adv. Drug Deliv. Rev. 33, 141-164[CrossRef][Medline] [Order article via Infotrieve] |
3. | Springer, T. A. (1995) Annu. Rev. Physiol. 57, 827-872[CrossRef][Medline] [Order article via Infotrieve] |
4. | Giavazzi, R., Foppolo, M., Dossi, R., and Remuzzi, A. (1993) J. Clin. Invest. 92, 3038-3044[Medline] [Order article via Infotrieve] |
5. | Mannori, G., Crottet, P., Cecconi, O., Hanasaki, K., Aruffo, A., Nelson, R. M., Varki, A., and Bevilacqua, M. P. (1995) Cancer Res. 55, 4425-4431[Abstract] |
6. | Mannori, G., Santoro, D., Carter, L., Corless, C., Nelson, R. M., and Bevilacqua, M. P. (1997) Am. J. Pathol. 151, 233-243[Abstract] |
7. | Moore, K. L., Patel, K. D., Bruehl, R. E., Li, F., Johnson, D. A., Lichenstein, H. S., Cummings, R. D., Bainton, D. F., and McEver, R. P. (1995) J. Cell Biol. 128, 661-671[Abstract] |
8. |
Borsig, L.,
Wong, R.,
Hynes, R. O.,
Varki, N. M.,
and Varki, A.
(2002)
Proc. Natl. Acad. Sci. U. S. A.
99,
2193-2198 |
9. |
Borsig, L.,
Wong, R.,
Feramisco, J.,
Nadeau, D. R.,
Varki, N. M.,
and Varki, A.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
3352-3357 |
10. |
McCarty, O. J.,
Mousa, S. A.,
Bray, P. F.,
and Konstantopoulos, K.
(2000)
Blood
96,
1789-1797 |
11. | Goetz, D. J., Ding, H., Atkinson, W. J., Vachino, G., Camphausen, R. T., Cumming, D. A., and Luscinskas, F. W. (1996) Am. J. Pathol. 149, 1661-1673[Abstract] |
12. |
Aigner, S.,
Ramos, C. L.,
Hafezi-Moghadam, A.,
Lawrence, M. B.,
Friederichs, J.,
Altevogt, P.,
and Ley, K.
(1998)
FASEB J.
12,
1241-1251 |
13. | Alon, R., Hammer, D. A., and Springer, T. A. (1995) Nature 374, 539-542[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Ramachandran, V.,
Yago, T.,
Epperson, T. K.,
Kobzdej, M. M.,
Nollert, M. U.,
Cummings, R. D.,
Zhu, C.,
and McEver, R. P.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
10166-10171 |
15. |
Park, E. Y.,
Smith, M. J.,
Stropp, E. S.,
Snapp, K. R.,
DiVietro, J. A.,
Walker, W. F.,
Schmidtke, D. W.,
Diamond, S. L.,
and Lawrence, M. B.
(2002)
Biophys. J.
82,
1835-1847 |
16. |
Fritz, J.,
Katopodis, A. G.,
Kolbinger, F.,
and Anselmetti, D.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
12283-12288 |
17. |
Goetz, D. J.,
Greif, D. M.,
Ding, H.,
Camphausen, R. T.,
Howes, S.,
Comess, K. M.,
Snapp, K. R.,
Kansas, G. S.,
and Luscinskas, F. W.
(1997)
J. Cell Biol.
137,
509-519 |
18. |
Kim, Y. J.,
Borsig, L.,
Han, H. L.,
Varki, N. M.,
and Varki, A.
(1999)
Am. J. Pathol.
155,
461-472 |
19. |
Capon, C.,
Wieruszeski, J. M.,
Lemoine, J.,
Byrd, J. C.,
Leffler, H.,
and Kim, Y. S.
(1997)
J. Biol. Chem.
272,
31957-31968 |
20. | Somers, W. S., Tang, J., Shaw, G. D., and Camphausen, R. T. (2000) Cell 103, 467-479[Medline] [Order article via Infotrieve] |
21. | Jadhav, S., and Konstantopoulos, K. (2002) Am. J. Physiol. 283, C1133-C1143 |
22. |
Jadhav, S.,
Bochner, B. S.,
and Konstantopoulos, K.
(2001)
J. Immunol.
167,
5986-5993 |
23. |
Walcheck, B.,
Moore, K. L.,
McEver, R. P.,
and Kishimoto, T. K.
(1996)
J. Clin. Invest.
98,
1081-1087 |
24. | Benoit, M., Gabriel, D., Gerisch, G., and Gaub, H. E. (2000) Nat. Cell Biol. 2, 313-317[CrossRef][Medline] [Order article via Infotrieve] |
25. |
Tees, D. F.,
Waugh, R. E.,
and Hammer, D. A.
(2001)
Biophys. J.
80,
668-682 |
26. | Burdick, M. M., Bochner, B. S., Collins, B. E., Schnaar, R. L., and Konstantopoulos, K. (2001) Biochem. Biophys. Res. Commun. 284, 42-49[CrossRef][Medline] [Order article via Infotrieve] |
27. |
Chesla, S. E.,
Selvaraj, P.,
and Zhu, C.
(1998)
Biophys. J.
75,
1553-1572 |
28. |
Evans, E.,
Leung, A.,
Hammer, D.,
and Simon, S.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
3784-3789 |
29. |
Schwesinger, F.,
Ros, R.,
Strunz, T.,
Anselmetti, D.,
Guntherodt, H. J.,
Honegger, A.,
Jermutus, L.,
Tiefenauer, L.,
and Pluckthun, A.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
9972-9977 |
30. |
Strunz, T.,
Oroszlan, K.,
Schafer, R.,
and Guntherodt, H. J.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
11277-11282 |
31. | Evans, E., and Ritchie, K. (1997) Biophys. J. 72, 1541-1555[Abstract] |
32. | Sheikh, S., and Nash, G. B. (1998) J. Cell. Physiol. 174, 206-216[CrossRef][Medline] [Order article via Infotrieve] |
33. |
Yarmola, E. G.,
Somasundaram, T.,
Boring, T. A.,
Spector, I.,
and Bubb, M. R.
(2000)
J. Biol. Chem.
275,
28120-28127 |
34. |
Smith, M. J.,
Berg, E. L.,
and Lawrence, M. B.
(1999)
Biophys. J.
77,
3371-3383 |
35. |
Mehta, P.,
Cummings, R. D.,
and McEver, R. P.
(1998)
J. Biol. Chem.
273,
32506-32513 |