From the Department of Biochemistry and Molecular Biology,
University of Texas M. D. Anderson Cancer Center,
Houston, Texas 77030
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INTRODUCTION |
Heparin/heparan sulfate
(HP/HS)1 and heparan sulfate
proteoglycans (HSPGs) participate in diverse biochemical and
physiological processes via interactions with a variety of proteins.
HP/HS and HSPGs bind and modulate activities of growth factors
(3), maintain extracellular matrix integrity (4, 5), modulate
hemostasis (6), participate in cell adhesion, growth, and
differentiation (7), and regulate smooth muscle cell proliferation (8,
9). Studies in our laboratory have demonstrated that HP/HS and HSPGs are involved in the initial stages of mouse embryo implantation. HSPGs
can be detected as early as the two-cell stage (10), and HSPG
expression on cell surfaces of mouse blastocysts increases 4-5-fold at
the peri-implantation stage (11-13). Inhibition of HSPG synthesis or
enzymatic removal of HS from blastocyst surfaces inhibits embryo
attachment (12, 14). Mouse embryo attachment to fibronectin, laminin,
and isolated mouse uterine epithelial cells is
HP/HS-dependent (11). Complementary specific, high affinity
HP/HS sites on the cell surface of mouse uterine epithelial cells also
have been characterized (15). Thus, it is suggested that HSPGs and
their corresponding binding sites are involved in the initial
attachment of mouse embryos to uterine epithelium.
The initial attachment of the human trophoblastic cell line, JAR, to
RL95 cells, a human uterine epithelial cell line, is HP/HS-dependent (16). Furthermore, a single class of highly specific, cell surface HP/HS-binding sites has been identified on RL95
cell surfaces (17). Cell surface, HP/HS-binding peptides were isolated
from RL95 cells, and partial NH2-terminal amino acid
sequence was obtained. A full-length cDNA sequence corresponding to
one of these peptides was identified (1). Predicted peptide sequence
from this cDNA revealed an antigenic sequence that also has
features expected for a HP/HS-binding motif (18). Polyclonal antibodies
directed against this synthetic peptide recognize a novel
HP/HS-interacting protein (HIP)
with a Mr on SDS-polyacrylamide gel
electrophoresis of 24,000. HIP is expressed by a variety of epithelial
and endothelial cells and cell lines and on RL95 cell surfaces (1, 2).
In addition, HIP is highly expressed by human cytotrophoblast at the
fetal-maternal interface throughout pregnancy in juxtaposition to a
potential HSPG ligand, perlecan (19). Previous studies (20) suggested
that a large fraction of the HP/HS binding and cell adhesion promoting
activity of intact HIP is attributable to this same peptide
sequence.
In this paper, we describe functional studies on the synthetic peptide
corresponding to this putative HP/HS-binding motif: CRPKAKAKAKAKDQTK,
referred to as HIP peptide. The current studies demonstrate that HIP
peptide is a highly selective HP/HS-binding peptide, recognizes certain
forms of HP and cell- and extracellular matrix-associated HS expressed
by human cell lines, and can support the HS-dependent
attachment of a human trophoblastic cell line. Therefore, this peptide
motif can account, at least in part, for the HS/HP binding and adhesion
promoting activities of HIP.
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EXPERIMENTAL PROCEDURES |
Materials--
Heparin, bovine kidney heparan sulfate, bovine
intestinal mucosa heparan sulfate, chondroitin sulfates A and C,
dermatan sulfate, keratan sulfate, hyaluronic acid, heparin
disaccharides, chondroitinases AC and ABC, heparinases I, II and III,
BSA, and CHAPS were purchased from Sigma. [3H]HP (0.44 mCi/mg) and [35S] sulfate (43 Ci/mg) were purchased from
NEN Life Science Products and ICN Biochemical Inc. (Irvine, CA),
respectively. [3H]HP was labeled by reduction with sodium
[3H]borohydride. [6-3H]Glucosamine was
purchased from ARC, Inc. (St. Louis, MO). Trypsin/EDTA solution, tissue
culture media, and supplements were from Irvine Scientific (Santa Ana,
CA). Fetal bovine serum, and Dulbecco's phosphate-buffered saline
(PBS) were from Life Technologies, Inc.. Guanidine hydrochloride was
purchased from U. S. Biochemical Corp. Imject activated immunogen
conjugation kits were purchased from Pierce. All chemicals used were
reagent grade or better.
Cell Culture--
JAR and RL95 cells were grown in a 1:1 mixture
of Dulbecco's modified Eagle's medium (DMEM) and Ham's F-12 medium
(F-12) containing 10% (v/v) heat-inactivated fetal bovine serum, 15 mM Hepes, pH 7.4, 50 units of penicillin/ml, and 50 µg of
streptomycin sulfate/ml.
Peptide Synthesis, Conjugation to Maleimide-activated BSA, and
HIP Peptide Affinity Chromatography--
A synthetic peptide (HIP
peptide) derived from HIP, a novel cell surface HS/HP-binding protein
(1, 2), CRPKAKAKAKAKDQTK, and a randomly scrambled peptide,
CQKAKTRAKAAKPDKK, were synthesized on a Vega 250 peptide synthesizer
using Fmoc (N-(9-fluorenyl)methoxycarbonyl) methodology
(21). This synthetic peptide was conjugated to maleimide-activated BSA
(Pierce) through the sulfhydryl group of cysteine in the peptide following the coupling protocol provided by the manufacturer. HIP
peptide affinity matrix was formed by cross-linking the BSA-conjugated HIP peptide to cyanogen bromide-activated Sepharose (Sigma) in the
presence of N-acetylated HP. Inclusion of acetylated HP was adopted to produce a more stable affinity matrix by shielding the
HP-binding sites of the HIP peptide complex from cross-linking to the
Sepharose beads. Acetylation of HP was performed following the method
described previously (22). In a parallel study, three distinct affinity
classes of HP, peaking at 0.15 M NaCl, 0.3 M NaCl, and 2.2 M NaCl, respectively, were separated by HIP
peptide affinity chromatography using gradient elution (23). Therefore, in most cases, stepwise elution of HIP peptide affinity chromatography was employed. The HIP peptide affinity matrix was packed into a 3-ml
column and equilibrated with 0.15 M NaCl-PBS.
[3H]HP or [35S]HS resuspended in 0.15 M NaCl-PBS was loaded into the column. The column then was
washed sequentially with 0.15 M NaCl-PBS (run-through (RT)-HP (or HS)), 0.45 M NaCl-PBS (low affinity (LA)-HP (or
HS)), and 3.0 M NaCl-PBS (high affinity (HA)-HP (or HS)).
Radioactivity of each fraction was determined by liquid scintillation
counting.
Metabolic Labeling and Isolation of Different Fractions of
Cell-expressed HS--
Near confluent cultures of RL95 or JAR cells
were rinsed several times with serum-free medium consisting of RPMI
1640 (minus sulfate) supplemented with 3.3 mM
MgCl2, 1.2 g/liter NaHCO3, 15 mM
Hepes, pH 7.2, 2.5 units/ml penicillin, and 2.5 µg/ml streptomycin sulfate. Streptomycin sulfate served as the sole source of
nonradioactive sulfate in this medium. The cells were incubated
overnight in the same low sulfate medium described above containing 0.5 mCi/ml H2[35S]O4. For generation
of 3H-labeled HS, RL95 cells were labeled overnight in
media containing 25 µCi/ml [3H]glucosamine. The
following day, the medium was collected and the cell layers were rinsed
several times with ice-cold PBS. The cell layers then were incubated
for 30 min on ice with PBS containing 50 µg/ml trypsin to release
cell surface proteoglycans. Cells were not released from the tissue
culture surfaces under these conditions, nor was cell viability
compromised as indexed by trypan blue exclusion. The material released
into the "trypsinate" was collected, the cell layers were rinsed
several more times with ice-cold PBS, and the cells scraped from the
tissue culture surface with a rubber policeman into ice-cold water. The
trypsinate was placed in a boiling water bath for 2-3 min and
immediately cooled on ice to inactivate the trypsin. Isolation of
35S-labeled extracellular matrix was performed following
the method described (24). Briefly, approximately 50% confluent
cultures of RL95 cells were labeled with the same
H2[35S]O4-containing low sulfate
medium as described above for 3 days. Cells then were washed with PBS
twice, and dissolved with PBS containing 0.5% (v/v) Triton X-100 and
20 mM NH4OH for 5 min at room temperature.
Afterward, the dish was washed four times with PBS, and the
extracellular matrix was collected using a rubber policeman. All
fractions then were precipitated with 10% (w/v) trichloracetic acid,
3% (w/v) phosphotungstic acid as described previously (25). The
pellets were resuspended in 1-2 ml of 0.5 M urea, 20 mM Tris-HCl, pH 8.0, 0.01% (w/v) octylglucoside, 0.02% (w/v) sodium azide and fractionated by Mono Q anion exchange
chromatography exactly as described (25). The material eluting from the
column with 2-4 M NaCl constituted 90% or more of the
total radioactivity in all cases and was pooled and extensively
dialyzed against PBS. [35S]HS or [35S]DS
was isolated following
-elimination as described (11) and used for
HIP peptide binding assays. The size of RL95 cell surface-derived
35S-labeled HS used for the solid phase binding assays was
determined as described (26). Specific activity was calculated based on the specific activity used for metabolic labeling (50 µCi/nmol) and
assuming an average sulfate content of 1.5 sulfate residues/HS disaccharide unit. Where indicated, the fractions were further analyzed
by digestion with glycosaminoglycan lyases or nitrous acid as described
in detail previously (16). HS was defined as sensitive (>95%) to
nitrous acid digestion and insensitive to chondroitinase AC or ABC
digestion. Dermatan sulfate was defined as sensitive (>95%) to
chondroitinase ABC digestion, but insensitive to chondroitinase AC or
nitrous acid digestion.
Structural Characterization of HS
Fractions--
35S- or 3H-labeled HS fractions
from RL95 cells were prepared as described above and exhaustively
digested with Pronase and subjected to mild alkaline hydrolysis to
remove associated peptides as described (25). Residual chondroitin and
dermatan sulfate contaminants were removed by digestion with
chondroitinase ABC as described previously (25). RT-HS, LA-HS, and
HA-HS were prepared by stepwise salt elution from a HIP peptide
affinity matrix as described above. HS disaccharides were prepared by
digestion at room temperature with 1 unit/ml each of heparinase I, II,
and III (Sigma) in 20 mM Tris acetate (pH 7.0) plus 2 mM CaCl2 and 2 mM
MgCl2. After 24 h an additional 1 unit/ml amount of
each enzyme was added. Mono Q anion exchange liquid chromatography was
performed on these fractions using 2 M urea, 20 mM Tris acetate (pH 7.0), 0.02% (w/v) sodium azide as
buffer A and the same buffer containing 4 M NaCl as buffer
B. The column was run at room temperature at a flow rate of 1 ml/min.
The gradient conditions were: 3 min of buffer A followed by a 25 min
linear gradient to 100% buffer B, followed by 2 min during which 100%
buffer B was maintained. Fractions were collected every minute and
analyzed by liquid scintillation counting. Recoveries of radioactivity
were routinely >95%. Superose 12 liquid chromatography was performed
exactly as described previously (25). Strong anion exchange liquid
chromatography was performed to analyze HS disaccharides and was
patterned after Tekotte et al. (27) with several
modifications. A 4.6 mm (inner diameter) × 25-cm Hydropore-5-AX HPLC
column (Rainin Instrument Co., Inc., Woburn, MA) was equilibrated with
0.2 M NaCl (pH 3.5) as buffer 1 and 2 M NaCl
used as buffer 2. The column was eluted at a flow rate of 1 ml/min at
room temperature. The gradient conditions were 100% buffer 1 for 4 min, followed by a 4-min linear gradient to 25% buffer 2, maintenance
of 25% buffer 2 for 12 min, followed by a step increase to 55% buffer
2, which then was maintained for 16 min followed by a 3-min linear
gradient to 100% buffer 2 to elute any residual material. Every sample
run included the presence of 10 µg of a heparin disaccharide mix
containing equal amounts of eight heparin disaccharide standards
described in the legend to Fig. 5. Elution of these internal standards
was monitored at 232 nm using a Beckman model 163 flow-through variable
wavelength spectrophotometer. Fractions were collected every 0.5 min,
and elution of radioactivity was determined by liquid scintillation counting. Recoveries of radioactivity exceeded 95% in all cases.
[3H]HP or [35S]HS Binding to HIP
Peptide-Solid Phase Assay--
[3H]HP binding to HIP
peptide was performed in 96-well microassay plates (Corning, Corning,
NY). Fifty µl of BSA-conjugated HIP peptide in PBS (100 µg/ml), as
well as the same amount and concentration of other controls, was added
to each well and dried at 37 °C overnight. The next day, each well
was rinsed with 200 µl of PBS three times, and 100 µl of 0.1%
(w/v) heat-denatured BSA (80 °C for 30 min) was added and incubated
in a 37 °C incubator for at least 1 h. Afterward, the wells
were rinsed with 200 µl of PBS three times, and then 50 µl of
[3H]HP (or [35S]HS)-containing reaction
mixture ([3H]HP typically was added at a concentration of
1.5 × 105 dpm/well (approximately 300 nM)
along with 0.1% (w/v) BSA and 0.1% (w/v) CHAPS in PBS) was added, and
incubated in a 37 °C incubator overnight. There were no differences
in the amount of [3H]HP bound after two to four washes
under these conditions. Therefore, the amount of bound radioactivity
detected after three washes was considered to represent a functional
equilibrium and was used to calculate
KD(app). Where indicated, unlabeled HP, bovine kidney HS, bovine intestinal mucosa HS, chondroitin sulfates A
and C, dermatan sulfate, keratan sulfate, or hyaluronic acid were
included as competitors. At the end of each experiment, unbound [3H]HP (or [35S]HS) was removed with 200 µl of PBS three times. Bound [3H]HP (or
[35S]HS) was extracted from each well by overnight
incubation at 37 °C with 100 µl of extraction buffer: 4 M guanidine HCl, 25 mM Tris-HCl, pH 8.0, 2.5 mM EDTA, and 0.02% (w/v) sodium azide. Half of the extract
was counted in a Beckman scintillation counter.
Cell Attachment Assay of HIP Peptide--
Cell attachment assays
employed 24-well tissue culture plates (Costar). Each well was coated
either with 200 µl of BSA-conjugated HIP peptide (100 µg/ml) or
with the same amount of other controls. The plates were incubated in a
37 °C incubator. The next day, each well was rinsed with 0.5 ml of
PBS three times. Subsequently, 500 µl of 0.1% (w/v) heat-denatured
BSA in PBS was added to each well and incubated at 37 °C for at
least 1 h. Each well was rinsed again with 0.5 ml of PBS three
times prior to the addition of cells. JAR cells were grown to
approximately 90% confluence. Cells were detached with trypsin/EDTA
solution for 5 to 10 min at 37 °C, washed with DMEM/F-12 (1:1) plus
0.1% (w/v) BSA three times, and resuspended in DMEM/F-12 (1:1) plus
0.1% (w/v) BSA. This cell suspension (5-10 × 104
cells/well) was then added to the wells coated as described above, and
incubated in a 37 °C incubator for the time periods indicated in
each experiment. At the end of each experiment, unattached cells were
gently rinsed away with 0.5 ml of PBS three times, and the relative
cell attachment was determined using the hexosaminidase assay as
described (28).
Glycosaminoglycans (GAG) Competition and the Effects of GAG
Lyases on JAR Cell Attachment to HIP Peptide--
A variety of GAGs,
including unlabeled HP, bovine kidney HS bovine intestinal mucosa HS,
chondroitin sulfates A and C, dermatan sulfate, keratan sulfate, and
hyaluronic acid, were used as competitors in assays of cell attachment
to HIP peptide. Different GAGs (100 or 200 µg/ml as indicated) were
included in the incubation medium for cell attachment assay in the
competition studies. To determine if cell surface HP/HS or other GAGs
of JAR cells mediate JAR cell attachment to HIP peptide, some cell
attachment assays were performed in the presence of different GAG
lyases (500 milliunits/ml), including heparinases I, II, and III,
chondroitinases AC and ABC, with the addition of a protease inhibitor
solution (11).
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RESULTS |
[3H]HP Binding to HIP Peptide--
Solid phase
assays were employed to determine the HP/HS binding activity of HIP
peptide. Preliminary studies established that a coating concentration
at 100 µg/ml HIP peptide in PBS gave optimal binding for both
[3H]HP and cell attachment. Furthermore,
[3H]HP did not bind to BSA or BSA conjugated to an
irrelevant peptide (CSSLSYTNPAVAATSANL) corresponding to the
cytoplasmic tail region of the mucin, MUC1 (29). Therefore, 100 µg/ml
HIP peptide was used typically in [3H]HP or
[35S]HS binding and cell attachment studies. In addition,
solid-phase assays also were performed using HIP peptide not conjugated
to maleimide-activated BSA as coating material, and similar results were obtained as described above for BSA-conjugated HIP peptide (data
not shown). Collectively, these results demonstrate HP binding was to
HIP peptide and not the BSA carrier. [3H]HP binding also
was time-dependent, reaching maximal values within 10-15
min (data not shown).
As shown in Fig. 1A,
[3H]HP binding to HIP peptide is
concentration-dependent with a 50% saturation at
approximately 245 nM when these data are analyzed as a
Scatchard plot (Fig. 1A, inset). Four separate
experiments yielded an average 50% saturation point of 300 nM. A peptide with the same amino acid composition as HIP peptide, but with a scrambled sequence, CQKAKTRAKAAKPDKK, bound [3H]HP with a 10-20-fold lower affinity than HIP peptide
(data not shown). Therefore, the peptide sequence, rather than
composition, was crucial to obtain high affinity binding. In a parallel
study, HIP peptide affinity chromatography of commercial
[3H]HP revealed three affinity classes of HP (RT-HP,
LA-HP, and HA-HP) with regard to binding to HIP peptide (23). To
determine the affinity of HA-HP binding to HIP peptide, HA-HP was
collected and subjected to the solid-phase HIP peptide binding assay.
Fig. 1B shows that HIP peptide binds HA-HP with higher
affinity (50% saturation at approximately 10 nM) than bulk
HP. Thus, these results suggest that HIP peptide can recognize and bind
certain forms of HP better than other forms of HP.

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Fig. 1.
Concentration dependence of
[3H]HP binding to HIP peptide. A, the
indicated concentrations of bulk commercially available
[3H]HP were added to wells coated with HIP peptide in the
presence ( ) or absence ( ) of 1 mg/ml unlabeled HP, and incubated
at 37 °C for 2 h. At the end of the experiment, unbound
[3H]HP was removed and the bound [3H]HP was
determined. Results are means ± S.D. of triplicate
determinations. Inset, kinetic analyses using assumptions
for Scatchard analysis of the data indicating an apparent
KD = 245 nM. B, bulk
[3H]HP was fractionated by HIP peptide affinity
chromatography as described under "Experimental Procedures." The
HIP peptide affinity-purified [3H]HP also was subjected
to solid phase assays as described in A. The
inset shows that Scatchard analyses of the data and
demonstrates an apparent KD = 10 nM.
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Selectivity of [3H]HP Binding to HIP
Peptide--
Selectivity of [3H]HP binding to HIP
peptide was determined using different GAGs, including unlabeled
heparin (HP), bovine kidney HS (HS-BK), bovine
intestinal mucosa HS (HS-BIM), chondroitin sulfate A
(CS-A), dermatan sulfate (DS), chondroitin
sulfate C (CS-C), keratan sulfate (KS), and
hyaluronic acid (HA), as competitors. As shown in Fig.
2, HP most effectively inhibited
[3H]HP binding to HIP peptide. Dermatan sulfate at 100 µg/ml inhibited [3H]HP binding to HIP peptide; however,
other GAGs were ineffective in this regard (Fig. 2). We noticed that
commercially available HS (HS-BIM and HS-BK) did
not significantly inhibit HP binding to HIP peptide. As shown below,
this agrees with other lines of evidence, indicating that only certain
forms of HS bind to HIP peptide. A dose dependence study demonstrated
that HP was approximately 50-fold more effective than dermatan sulfate
at inhibiting [3H]HP binding to HIP peptide (Fig. 2,
inset). Collectively these studies demonstrated that HIP
peptide binds HP much more selectively than other GAGs.

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Fig. 2.
Selectivity of glycosaminoglycan binding to
HIP peptide. Equal amounts of [3H]HP (150,000 dpm/well) were added to wells coated with HIP peptide in the presence
of 100 µg/ml unlabeled glycosaminoglycans (C, no
competitor control; HP, unlabeled heparin; HS-BK,
bovine kidney heparan sulfate; HS-BIM, bovine intestinal
mucosa heparan sulfate; CS-A, chondroitin sulfate A;
DS, dermatan sulfate; CS-C, chondroitin sulfate
C; KS, keratan sulfate; HA, hyaluronic acid) and
incubated at 37 °C for 2 h. At the end of the experiment,
unbound [3H]HP was removed with PBS and the bound
radioactivity was determined. The inset is a comparison of
[3H]HP binding to HIP peptide in the presence of the
indicated concentrations of HP ( ) and DS ( ) and demonstrates that
HP is at least 50-fold more effective than DS in inhibiting
[3H]HP binding. Results are means ± S.D. of
triplicate determinations in all cases.
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Cell Surface and Extracellular Matrix-associated HS Binds to HIP
Peptide--
Fig. 3 shows that, in
solid-phase assays, [35S]HS isolated from RL95 cell
surfaces binds to HIP peptide in a concentration-dependent and saturable fashion. Previous studies established the median molecular weight of cell surface HS chains of RL95 cells (26). Assuming
the specific activity of the [35S]sulfate in these HS
preparations was similar to that of the sulfate precursor used for
metabolic labeling, a 50% saturation point of approximately 0.2 nM was estimated. Inclusion of 100 µg/ml unlabeled HP
inhibited almost all of this binding (>90%). Furthermore, HS isolated
from cell surfaces of various breast cancer cell lines also
specifically binds to HIP peptide (30). Collectively, these data
illustrate that certain forms of HS expressed at the cell surface or
the extracellular matrix by human cells binds to HIP peptide.

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Fig. 3.
Concentration dependence of
[35S]HS binding to HIP peptide. The indicated
amounts of [35S]HS derived from cell surfaces of RL95
cells, were added to wells coated with HIP peptide in the presence
( ) or absence ( ) of 100 µg/ml unlabeled HP, and incubated at
37 °C for 2 h. At the end of the experiment, unbound
[35S]HS was removed and bound [35S]HS was
determined. The data shown are the averages ± range of duplicate
determinations in each case.
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Given the ability of dermatan sulfate to partially compete for
[3H]HP binding in the solid phase assays as well as in
JAR cell adhesion assays (see Ref. 16, and studies below),
[35S]O4-labeled HS and dermatan sulfate
were isolated from either RL95 or JAR cells and tested for HIP peptide
binding by HIP peptide affinity chromatography. Representative HIP
peptide affinity column elution profiles of [35S]DS and
[35S]HS are shown in Fig. 4
(A and B, respectively). These experiments demonstrate that a much higher percentage of cell-associated
glycosaminoglycans bound to HIP peptide than secreted forms. In most
fractions, bound glycosaminoglycans eluted between 0.2 and 0.3 M NaCl; however, cell surface HS contained higher affinity
species that required more than 0.4 M NaCl for elution.
Table I summarizes the results of HIP
peptide affinity chromatography of [3H]HP and different
fractions of [35S]HS or [35S]DS
isolated from RL95 or JAR cells. Some variability (46-63%) was
observed in binding with different [3H]HP preparations.
Most (78-93%) extracellular matrix HS from RL95 cells bound to HIP
peptide affinity matrix at physiological salt concentrations.
Cell-associated [35S]HS of JAR cells, either accessible
or nonaccessible to trypsin release, also bound to HIP peptide, albeit
a lower percentage than the corresponding fractions from RL95 cells. In
contrast, secreted [35S]HS from both cell lines displayed
very little binding to HIP peptide. A similar pattern was observed for
JAR cell DS, i.e. a fraction of cell-associated forms,
either accessible or inaccessible to trypsin release, bound to HIP
peptide whereas secreted DS bound relatively poorly. It was concluded
that cell- or extracellular matrix-associated forms of HS or DS were
capable of binding to HIP peptide under physiological conditions;
secreted HS or DS bound poorly.

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Fig. 4.
HIP peptide affinity chromatography of JAR
cell dermatan sulfate and HS fractions. Glycosaminoglycan
fractions were prepared from JAR cells and HIP peptide affinity
chromatography performed as described under "Experimental
Procedures." Typically, 1-6 × 105 dpm of
35S-labeled glycosaminoglycan was loaded. For simplicity,
the elution profiles of the run-through, i.e. not bound at
0.15 M NaCl, have been omitted and only the elution
profiles of the bound material is shown. Profiles of cell surface,
i.e. trypsin-releasable ( ), cell-associated but not
trypsin-releasable ( ), and secreted ( ) are shown for both
dermatan sulfate (A) and HS (B). The salt
gradient (0.15-2.0 M NaCl) is indicated by the
dashed line. Fractions of 0.45 ml were collected in each
case.
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Table I
Binding of HS and DS Fractions to HIP peptide
HS and DS fractions from either RL95 or JAR cells were prepared as
described under "Experimental Procedures." In each case, the
glycosaminoglycan fraction was applied to the column and eluted in PBS
until only background levels of radioactivity were detected in the
eluate. Then the resin was either subjected to linear or step gradient
elution with NaCl up to 3.0 M. The total bound
radioactivity is shown in each case. A range of values is shown for
[3H]HP to indicate the variability observed with different HP
preparations. With exception of the JAR cell surface dermatan sulfate
fraction, more than 100,000 dpm were used in each analysis.
Approximately 18,000 dpm of JAR cell surface dermatan sulfate was used.
Secreted refers to glycosaminoglycans isolated from the medium, ECM
refers to glycosaminoglycans isolated from extracellular matrix
preparations, cell surface refers to glycosaminoglycans isolated from
material released from intact cell layers by mild trypsinization, and
trypsin-resistant refers to glycosaminoglycans isolated from material
remaining in the cell-associated fraction after mild trypsinization.
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Characterization of HIP Peptide-binding HS
Fractions--
[3H]glucosamine- or
[35S]O4-labeled HS were prepared from RL95
cells representing secreted or released, cell surface (cell-associated and trypsin-releasable), intracellular (cell-associated, but not trypsin-releasable) and extracellular matrix forms. Each fraction was
further separated by affinity on a HIP-peptide affinity matrix into
three additional fractions. These fractions were: 1) material that did
not bind at physiological salt concentrations (RT-HP); 2) material that
bound at physiological salt concentrations, but could be eluted with
0.45 M NaCl (LA-HP); and 3) material that remained bound at
0.45 M NaCl, but could be eluted with 3 M NaCl (HA-HP). Each fraction was characterized further by several methods. Anion exchange liquid chromatography (Fig.
5A) demonstrated that cell
surface-derived LA- and HA-HS had a slightly higher negative charge
density than RT-HS; however, there were no detectable differences in
this regard between LA- and HA-HS. These differences between RT-HS and
the other HS fractions were found not only in cell surface HS fractions
(shown in figure), but also in all other HS fractions examined (data
not shown). Molecular exclusion chromatography (Fig. 5B)
demonstrated that LA- or HA-HS fractions that bound to HIP peptide were
generally larger (median mass > 70 kDa relative to polysaccharide
standards) than RT-HS (median mass < 70 kDa). HA-HS was skewed
toward lower molecular mass forms relative to LA-HA. These observations
were consistently observed in HS derived from the cell surface (shown
in figure) as well as secreted, extracellular matrix-associated, and
intracellular forms of HS (data not shown).

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Fig. 5.
Molecular exclusion and anion exchange
chromatography of HS fractions.
[35S]O4-Labeled derived from RL95 cell
surfaces were prepared and fractionated by stepwise HIP-peptide
affinity chromatography as described under "Experimental
Procedures." These fractions then were analyzed either by anion
exchange chromatography on Mono Q (A) or molecular exclusion
chromatography on Superose 12 (B) as described under
"Experimental Procedures." Fractions were collected every minute in
each case and the profile of the total radioactivity eluting in each
fraction determined by liquid scintillation counting is shown.
RT-HS ( ) or run-through HS refers to HS that did not bind
to HIP peptide at physiological salt concentrations; LA-HS
( ) refers to low affinity HS or HS that bound at 0.15 M
NaCl, but could be eluted with 0.45 M NaCl;
HA-HS ( ) or high affinity HS refers to HS that remained
bound at 0.45 M NaCl, but could be eluted with 3 M NaCl. The dashed line in A shows
the salt gradient used. The elution positions of the following size
markers is shown in B: Vo, blue dextran
with molecular weight > 2 × 106;
531K, dextran with a median molecular weight of 531,000;
71K, dextran with a median molecular weight of 71,000;
12K, commercial HP with a median molecular weight of 12,000;
Vt, potassium dichromate.
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HS disaccharides generated by exhaustive digestion with a heparinase
mixture revealed additional structural differences in HS species that
interacted with HIP peptide. A representative disaccharide profile is
shown in Fig. 6. The data obtained from all HS fractions are summarized in Table
II. In general, RT-HS fractions from all
cellular compartments were poorly sulfated with a very high fraction of
nonsulfated disaccharides (53-97%). Nonsulfated disaccharides also
represented a higher proportion of the total in LA-HS (43-53%) than
HA-HS (24-38%). In contrast, HA-HS species were greatly enriched for
di- and trisulfated disaccharide species (35-55%). LA-HS species had
a larger proportion of di- and trisulfated disaccharides (22-30%)
than RT-HS and displayed intermediate proportions of di- and
trisulfated disaccharides (18-22%) relative to HA-HS. LA-HS fractions
also were generally enriched for monosulfated species relative to RT-HS
(10-22% versus 3-15%, respectively). Collectively, these
studies indicated that there were structural differences not only
between the HS species that bound or did not bind to HIP peptide, but
also between the species that bound with low versus high
affinity.

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Fig. 6.
Anion exchange liquid chromatography of HS
disaccharides. [3H]Glucosamine-labeled HS derived
from RL95 cell surfaces was prepared and fractionated by stepwise
HIP-peptide affinity chromatography as described under "Experimental
Procedures." These fractions then were digested to completion with a
heparinase mixture and analyzed by strong anion exchange liquid
chromatography on a Hydropore-5-AX column as described under
"Experimental Procedures." Fractions were collected every 0.5 min,
and the profile of the radioactivity eluting in each fraction as
determined by liquid scintillation counting is shown. The
numbers above the peaks refer to the elution positions of
the following disaccharides used as internal controls and monitored by
measuring A232 using a flow-through
spectrophotometer (where UA represents
4-deoxy- -L-threo-hex-4-enopyranosyluronic
acid, GlcNAc represents 2-acetamido-2-deoxy-D-glucose, and
GlcNS represents 2-deoxy-2-sulfamido-D-glucose):
1, UA GlcNac; 2, UA GlcNS;
3, UA GlcNAc-6-O-sulfate; 4,
UA GlcNAc; 5, UA GlcNS-6-O-sulfate;
6, UA-2-O-sulfate GlcNS; 7,
UA-2-O-sulfate GlcNAc-6-O-sulfate;
8,
UA-2-O-sulfate GlcNS-6-O-sulfate.
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JAR Cell Attachment to HIP Peptide--
JAR cells, a human
trophoblastic cell line derived from a placental choriocarcinoma (31),
was used in a detailed examination of cell attachment to HIP peptide.
As described above, preliminary studies determined that 100 µg/ml
peptide was an optimal coating concentration and was used for routine
JAR cell attachment assays. Kinetic studies showed that JAR cell
attachment to HIP peptide was time-dependent with near
maximal attachment observed between 4 and 8 h of incubation at
37 °C (data not shown). Thus, the routine JAR cell attachment assays
were performed using 4-6-h incubations. Table
III shows that JAR cells attached to HIP
peptide, and more than 90% of this attachment was inhibited in the
presence of 1 mg/ml HP. Heat-denatured BSA, used to block nonspecific
binding sites after coating, did not support JAR cell attachment. JAR cells attached well to fibronectin (FN); however, attachment to FN was
not inhibited by 1 mg/ml HP. CT-1 peptide, a peptide sequence corresponding to a cytoplasmic region of the human epithelial mucin,
MUC1 (29), conjugated to maleimide-activated BSA also was used as a
nonspecific peptide control as well as a control for the activated BSA
used in HIP peptide conjugation, and did not support JAR cell
attachment. Thus, JAR cells specifically attached to HIP peptide in a
HP-inhibitable fashion. The calculated pI of HIP peptide is relatively
high (
9.9). Therefore, it was considered that JAR cell attachment to
HIP peptide was due to a nonspecific charge effect; however, lysozyme
(pI = 10.5-11.0), failed to support JAR cell attachment.
Furthermore, JAR cells also failed to attach to a peptide containing a
randomly scrambled sequence of the same amino acid composition and
therefore, identical pI, as the HIP peptide. As an additional test of
the specificity of cell attachment to HIP peptide, surfaces were coated
with HIP peptide and soluble HIP peptides or a scrambled peptide were
used as competitors. As shown in Table
IV, soluble HIP peptide at 100 µg/ml
effectively inhibited cell attachment; however, the scrambled sequence,
even at 500 µg/ml, had little effect in this regard. Consequently,
cell recognition of HIP peptide appeared to be sequence-specific.
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Table III
HIP peptide specifically supports JAR cell attachment in a
HP-inhibitable fashion
JAR cells (1 × 105 cells/well) were added to the wells
coated with HIP peptide-BSA, heat-denatured BSA (D-BSA), fibronectin,
CT-1-peptide-BSA, or lysozyme as described under "Experimental
Procedures," and incubated at 37 °C for 5 h. Unattached cells
were rinsed off with PBS at the end of experiment, and cell attachment
was determined by hexosaminidase assay (28). Data are expressed as
percentages of untreated control, i.e. attachment to HIP
peptide in the absence of HP. Results represent the means ± S.D. of triplicate determinations in each case.
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Fig. 7 is a representative picture of JAR
cell attachment to HIP peptide-, FN-, or heat-denatured BSA-coated
surfaces after cells were incubated at 37 °C for 24 h and
unattached cells rinsed away with PBS. JAR cells attached to both HIP
peptide and FN, but only spread on FN. JAR cells did not attach to
heat-denatured BSA. JAR cell attachment to HIP peptide in the presence
of HP was similar to that of the heat-denatured BSA. Collectively,
these results demonstrate that HIP peptide supports JAR cell
attachment, but not spreading, in a HP-inhibitable fashion.

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Fig. 7.
JAR cells attach but do not spread on HIP
peptide. JAR cells were added to wells coated with fibronectin
(FN), HIP peptide (HIP Pep), or heat-denatured
BSA (D-BSA), respectively, and incubated at 37 °C for
24 h. Unbound cells were removed with PBS at the end of the
experiment, and cell attachment was determined by hexosaminidase assay.
JAR cells attached to both HIP peptide and FN, but only spread on FN.
JAR cells did not attach to denatured BSA. JAR cell attachment to
HIP peptide in the presence of 1 mg/ml HP showed the similar
pattern as that of denatured BSA (data not shown). Original
magnification, ×200.
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HP Sensitivity and Glycosaminoglycan Selectivity of JAR Cell
Attachment to HIP Peptide--
GAG selectivity of JAR cell attachment
to HIP peptide was determined using different GAGs as competitors. Fig.
8 shows that HP was the most effective
inhibitor among all GAGs tested. Dermatan sulfate and keratan sulfate
also displayed inhibitory activity at a concentration of 200 µg/ml,
but other GAGs did not significantly inhibit cell attachment to HIP
peptide. Further studies on the inhibitory effect of different
concentrations of HP, dermatan sulfate, and keratan sulfate
demonstrated that both of the latter polysaccharides failed to inhibit
JAR cell attachment activity at concentrations below 50 µg/ml,
i.e. about 100-fold higher concentrations than that required
for HP (Fig. 8, inset). In fact, very low concentrations (1 µg/ml) of dermatan or keratan sulfate consistently stimulated JAR
cell attachment to HIP peptide, although the reason for this is
unclear. Additional studies demonstrated that JAR cell attachment to
HIP peptide was inhibited more than 90% in the presence of 0.5 µg/ml
(approximately 50 nM) of HP. Therefore, JAR cell attachment to HIP peptide is highly selective for HP versus other
GAGs.

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Fig. 8.
HP most effectively inhibits JAR cell
attachment to HIP peptide. Equal numbers of JAR cells (100,000 cells/well) were added in the presence of the indicated concentrations
of GAGs to wells coated with HIP peptide, and incubated at 37 °C for
5 h. At the end of the experiment, unattached cells were removed
with PBS, and cell attachment was determined by hexosaminidase assay.
Results represent the means ± S.D. of triplicate determinations
in all cases. In the inset, JAR cells were added in the
presence of the indicated concentrations of HP ( ), keratan sulfate
(KS, ), and DS ( ) to HIP peptide-coated wells, and
cell attachment was determined as described above. Data are expressed
as percentages of cell binding observed in the absence of GAG
competitor. Abbreviations are as described for Fig. 2.
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To determine if HS on JAR cell surfaces mediated JAR cell attachment to
HIP peptide, JAR cell attachment to HIP peptide was performed in the
presence of different GAG lyases. GAG lyases were added to HIP
peptide-coated wells in the presence of protease inhibitors (Fig.
9). Hep 3 (a mixture of heparinases I,
II, and III) inhibited JAR cell attachment to HIP peptide by
approximately 90%. In contrast, chondroitinase ABC inhibited
attachment to a lesser extent (55%); however, chondroitinase AC was
ineffective in this regard. Consequently, consistent with previous
studies (16), cell surface dermatan sulfate, but not chondroitin
sulfate, appeared to participate in JAR cell binding to HIP peptide as well as HS. In addition, Fig. 9 shows that soluble HIP peptide as well
as HP both effectively inhibited JAR cell attachment to HIP peptide.
Collectively, these observations indicate that cell surface HS and, to
a lesser extent, dermatan sulfate expressed by JAR cells mediate
binding to HIP peptide.

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Fig. 9.
Glycosaminoglycan lyases inhibit JAR cell
attachment to HIP peptide. Equal numbers of JAR cells (60,000 cells/well) were added in the presence of the indicated enzymes,
soluble HIP peptide, or HP to HIP peptide-coated wells, and incubated
at 37 °C for 5 h. At the end of the experiment, unattached
cells were removed with PBS, and cell attachment was determined by
hexosaminidase assay. Results are means ± S.D. of triplicate
determinations in each case. Abbreviations: Hep 3, mixture
of heparinases I, II, and III; ABC, chondroitinase ABC;
AC, chondroitinase AC; PICS, protease inhibitor
mixture solution.
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 |
DISCUSSION |
Multiple lines of evidence support the hypothesis that HSPGs and
their corresponding binding sites participate in the initial attachment
of murine blastocyst and human trophoblastic cell lines to the uterine
epithelial cells and cell lines. Highly specific cell surface
HS/HP-binding sites have been identified on RL95 cells, and a partial
amino-terminal amino acid sequence was obtained for HP binding, tryptic
peptides derived from RL95 cell surfaces (17). A full-length cDNA
has been obtained corresponding to one of these fragments using
approaches of reverse transcription-polymerase chain reaction and
library screening (1). Structural analysis of the predicted peptide
sequence derived from this cDNA revealed a region that is composed
of highly positive-charged amino acids separated by hydrophobic amino
acids, a motif found in various HS/HP-binding proteins and suggested to
function as a HS/HP-binding domain (18). This region is hydrophilic and
likely to be exposed at the exterior surface of the protein, where it
might participate in HS/HP-binding events. Antibodies directed to this
peptide react with RL95 cell surfaces and recognize a protein that
binds HP in an 125I-HP overlay assay and binds to
HP-agarose with high affinity (2). In the present studies, a synthetic
peptide was synthesized corresponding to this putative HP/HS-binding
region for functional studies. HP/HS binding to this simple peptide
also was highly selective for certain forms of HP and HS and of
comparable affinity to various intact HP/HS-binding proteins (32, 33).
The results confirm the prediction that this sequence binds HS/HP.
[3H]HP binding to HIP peptide was both time- and
concentration-dependent as well as saturable. More than 90% of
this binding was blocked by unlabeled HP at concentrations as low as 10 µg/ml. HIP peptide bound bulk [3H]HP with an apparent
KD of 300 nM. Among all the GAG competitors tested, HP was the most effective inhibitor of
[3H]HP binding to HIP peptide and was approximately
50-fold more effective than dermatan sulfate, the next most active GAG.
Therefore, it appears that the interaction of HP with the HIP peptide
is not simply due to generic interactions of negatively charged
polymers with the highly positively charged peptide. This contention is supported further by the observation that [3H]HP binds
relatively poorly to both lysozyme and a scrambled sequence of the HIP
peptide. Interestingly, dermatan sulfate is the only GAG other than HP
that contains iduronic acid in its structure and has a small percentage
of disulfated disaccharide units (34). It has been suggested that the
presence of iduronic acid in the polysaccharide chains provides more
flexibility in polysaccharides which may facilitate their association
with corresponding binding sites (34, 35). HIP peptide binding species
also are enriched for di-and trisulfated disaccharides as compared with nonbinding HS species. HS species that bound to HIP peptide with higher
affinity (HA-HS) also were more enriched, in this regard, than the
species that interacted with lower affinity (LA-HS). The enrichment
with di- and trisulfated disaccharides is consistent with the higher
negative charge density observed for LA-HS and HA-HS compared with
RT-HS by anion exchange chromatography. Collectively, the present
studies demonstrate that HIP peptide binds HP with a high degree of
selectivity and suggest that the presence of iduronic acid and/or
regions with a high degree of sulfation contribute to HP binding to HIP
peptide.
A small fraction (approximately 1-5%) of [3H]HP
isolated from commercially available [3H]HP by HIP
peptide affinity chromatography binds to HIP peptide with a
substantially higher affinity (KD(app)
10 nM) than bulk HP (23). Since HP/HS polysaccharides
possess the highest degree of sequence heterogeneity among GAGs, this
result further suggests that HIP peptide recognizes only a subset of
structures resident within HP/HS chains. In the present study, it was
further demonstrated that certain forms of HS found on cell surfaces
and extracellular matrix of RL95 and JAR cells specifically bound to
HIP peptide. In contrast, very little secreted HS bound to HIP peptide
at physiological salt concentrations (0.15 M NaCl). It is
possible that released forms of HS are depleted of HIP peptide binding
motifs due to the action of heparanase, an enzyme that destroys these
sequences (36). Collectively, these observations strongly suggest that
specific polysaccharide structures recognized by HIP peptide reside
both in commercial HP preparations and certain forms of cell-expressed
HS. Previously, we found that HA-HP binds to antithrombin III with
particularly high affinity (23), suggesting that HIP peptide recognizes
the same well defined HP polysaccharide structure specifically
recognized by antithrombin III (37). Thus, HIP peptide can account, at
least in part, for the HP/HS binding activity of the parental HIP
protein. The HP/HS binding activity of the HIP peptide coupled with the
cell surface disposition of HIP (2) and the affinity of cell surface
and extracellular matrix forms of HS for HIP peptide (Ref. 30, and
present studies) strongly suggest that HIP participates in aspects of
HS-dependent cell adhesion.
JAR cell attachment to HIP peptide was HS-dependent and not
simply due to nonspecific charge effects. JAR cell attachment was much
more sensitive to HP than the other GAGs tested with maximal inhibition
achieved at HP concentrations of 0.5 µg/ml. This concentration is
approximately 50 nM, assuming an average molecular weight
of 10,000 for HP chains and is consistent with the
KD obtained for the high affinity fraction of HP. HS
removal from JAR cell surfaces most efficiently (>90%) inhibited JAR
cell attachment to HIP peptide, indicating that cell surface HS
mediated this binding; however, dermatan sulfate displayed on JAR cell
surfaces also may play a role. Previous studies demonstrated the
presence of dermatan sulfate bearing molecules on JAR cell surfaces and
indicated that this class of GAGs participated to some extent in
adhesion between JAR and RL95 cells (16). In the present studies,
selective removal of dermatan sulfate, but not chondroitin sulfate, as
well as HS from JAR cells also inhibited binding to HIP peptide.
Partial (55%) inhibition was observed after digestion of JAR cells
with chondroitinase ABC, which removes dermatan sulfates from JAR cell
surfaces. In contrast, heparinases reduced binding by 90% or more.
Soluble dermatan sulfate also inhibited JAR cell attachment, but was
much less effective than HP. Collectively, these results demonstrate
that HIP peptide specifically supports JAR cell attachment by binding
HS expressed on JAR cell surfaces. Dermatan sulfate participates in
this process, albeit to a lesser extent.
As discussed above, a number of studies in both mouse and human model
model systems indicate that HSPGs and their corresponding binding
proteins participate in blastocyst attachment to the uterine epithelium
during the initial phases of implantation. HIP is expressed by human
uterine epithelium throughout the menstrual cycle; moreover, the HIP
peptide motif studied here has been shown to be accessible at cell
surfaces (2). Previous studies demonstrated that intact HIP supports
the attachment of human trophoblastic cell lines (20). More recently,
we have determined that human cytotrophoblast not only express, but
also appear to utilize this protein for aspects of trophoblast invasion
as well as attachment to the HSPG, perlecan (19). These same studies
indicate that antibodies to this sequence inhibit trophoblast invasion
in vitro. The interactions of the HIP peptide sequence with
HS motifs important for susceptibility to heparanase action,
anticoagulant activity and trophoblast attachment indicate that HIP
has the potential to play a key role in
HS-dependent processes in developing and adult tissues.
We thank Margaret French, Dr. Andrew Jacobs,
Ruth Pimental, Dr. Gloria Regisford, Dr. Larry Rohde, Dr. Scott Smith,
Dr. Gulnar Surveyor, Dr. Carole Wegner, and Xinhui Zhou for helpful
comments and critical reading of the manuscript. We thank Sharron
Kingston for excellent secretarial assistance and Karen Hensley for
graphics expertise in preparation of this manuscript. The
University of Texas M. D. Anderson Cancer Center Core Facilities,
i.e. Peptide Synthesis Laboratory and Department of
Biomathematics (Computer Analysis of Macromolecular Sequence
Data) are supported by National Institutes of Health Grant NCI
CA-16672.