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INTRODUCTION |
Fibronectin (Fn)1 is a
plasma and extracellular matrix protein which interacts with other
macromolecules and with cells via specific binding sites present in
well defined structural and functional domains (reviewed in Ref. 1). Fn
contains two main cell adhesion domains located in the central and
COOH-terminal regions, respectively. The active sites in the central
domain are RGD in repeat III10 and its synergistic sequence PHSRN in repeat III9 (Refs. 1 and 2, see Fig. 1). These sites bind mainly
5
1 integrin although RGD is also a ligand for activated
4
1
(3). The COOH-terminal cell-binding region comprises the active sites
CS-1 and CS-5 within the alternatively spliced segment IIICS, as well
as H1 (IDAPS) in the high affinity heparin-binding domain or Hep II
(Fig. 1). CS-1, CS-5, and H1 are ligands for
4
1 integrin
(4-9).
Besides the H1
4
1-binding site, the Hep II domain contains
several well characterized sequences which bind heparin and cell surface proteoglycans (PG) (10-12). One of these sequences is
WQPPRARITGY (peptide FN-C/H V) (12) which mediates cell adhesion via PG and promotes focal adhesion formation (13, 14). There is now extensive
evidence showing that PG may modulate the function of
4
1 and that
cell adhesion to the COOH-terminal region of Fn involves the
cooperation between both types of receptors (11, 15-18). It is also
well established that integrin function can be up-regulated by external
factors including the divalent cation Mn2+ and certain
anti-
1 mAbs such as TS2/16 (19). Whether these reagents are
mimicking the effects of physiologic regulators such as PG remains to
be determined.
These previous studies have clearly established an important role for
the Hep II domain of Fn in the adhesion of many cell systems including
melanoma (11), lymphoid (5, 20), hematopoietic precursors (15, 18), and
neural crest cells (21). However, it is now becoming evident that other
heparin-binding regions also contribute to cell adhesion. Fn contains
2-3 additional heparin-binding domains located at the
NH2-terminal (Hep I) and central (Hep III) part of the
molecule (see Fig. 1). These domains differ in their binding affinity
and sensitivity to divalent cation regulation (22-24). The Hep I
domain was recently shown to induce cell adhesion via interaction with
the
5
1 integrin (25). It is not known if this region also
interacts with cell surface PG, although it binds several
uncharacterized molecules at the cell surface (26).
We have also recently reported that a recombinant fragment containing
the Fn III5 repeat, which is part of the Hep III domain, mediates
lymphoid cell adhesion due to the interaction of the KLDAPT sequence
with
4
1 and
4
7 integrins previously activated with TS2/16
mAb or Mn2+, respectively (27). Our previous observations
therefore reveal a novel function for this domain and highlight the
importance of heparin-binding regions for the overall cell binding
activity of Fn.
In the present study we have further characterized the heparin and cell
binding properties of the Hep III domain of Fn. By preparing
recombinant fragments containing type III repeats from this region, we
show that adhesion of Jurkat T cells to a fragment containing Fn
III4-III5 repeats involves the cooperation of activated
4
1
integrin and chondroitin sulfate (CS) but not heparan sulfate (HS) PG.
We also show that the contribution of each receptor depends on whether
Mn2+ or TS2/16 are used to activate
4
1 and that CSPG
is crucial when Mn2+ is the reagent of choice. Furthermore,
we have identified a novel 20-amino acid sequence in repeat III5,
structurally similar to FN C/H V, which binds heparin and induces cell
adhesion via CSPG exclusively. These results therefore establish novel
interactions that regulate cell adhesion to the Hep III Fn domain.
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EXPERIMENTAL PROCEDURES |
Fibronectin Fragments and Synthetic Peptides--
Recombinant
fragments containing type III homology repeats 4-5-6 (FN-III4-5-6),
4-5 (FN-III4-5), 4 (FN-III4), and 5 (FN-III5) were produced by
polymerase chain reaction amplification using UlTma DNA-polymerase
(Perkin-Elmer), cDNA from FN-III1/22-11 clones and
appropriate primers as described previously (27). Polymerase chain
reaction products were cloned into pQE-12 or pQE-3/5 vector using the
QIAexpress kit and expressed in Escherichia coli. All cloned
cDNAs were sequenced using a Sequenase 2.0 DNA sequencing kit
(U. S. Biochemicals Corp., Cleveland, OH). Fragments were purified
by immunoaffinity chromatography using the specific mAbs (see below)
IST-3 (FN-III4, FN-III4-5, and FN-III4-6) or IST-4 (FN-III5)
conjugated to Sepharose 4B (Pharmacia Biotech, Uppsala, Sweden). None
of the fragments contained a 6xHis tag. Purity of recombinant fragments
was confirmed by SDS-polyacrylamide electrophoresis gels (15%
acrylamide; see Fig. 1). The recombinant fragment H0, comprising the
Hep II domain (repeats III12-14) and repeat III15 was prepared exactly
as described (28).
The synthetic peptides KLDAPT (H2), WTPPRAQITGYRLTVGLTRR (HBP/III5),
WTPQARPITGYRLTVGLTRR (scrambled HBP/III5 or HP/III5.sc), WTPPRAQITGY
(HBP/III5-N11), WTPPRAQITGYRLT (HBP/III5-N14), YRLTVGLTRR (HBP/III5-C10), and YQLTVGLTRR (HBP/III5-C10.sc) were synthesized on an
automated multiple peptide synthesizer (AMS 422, ABIMED, Langenfeld,
Germany) using standard solid phase procedures and purified by reverse
phase high performance liquid chromatography. Peptides were covalently
conjugated to keyhole limpet hemocyanin (KLH, Calbiochem-Novabiochem
Int., La Jolla, CA) with glutaraldehyde (29) by mixing KLH and peptide
at a molar ratio of 1:3000 in phosphate-buffered saline. Glutaraldehyde
was added dropwise to the peptide-KLH solution and the mixture
incubated at room temperature for 2 h with gentle stirring; after
extensive dialysis against phosphate-buffered saline, peptide coupling
efficiency was determined by amino acid analysis after hydrolysis using
a Biochrom 20 analyzer (Pharmacia).
Antibodies and Enzymes--
IST-3 and IST-4 mAbs reactive with
FN-III4 and FN-III5 repeats, respectively, were produced as reported
(30). Activating anti-
1 mAb T2/16 (purified Ab) and function
blocking anti-
4 HP2/1 (culture supernatant) were generously donated
by Dr. Francisco Sánchez-Madrid (Hospital de la Princesa, Madrid,
Spain). Chondroitinase ABC (EC 4.2.2.4) and heparinase III
(heparitinase I, EC 4.2.2.8) were purchased from Sigma. Chondroitinase
AC II (EC 4.2.2.5) and heparitinase (a mixture of 95% heparitinase I
and 5% heparitinase II, EC 4.2.2.8) were purchased from Seikagaku
America Inc. through ams Biotechnology (Oxon, United Kingdom).
Cells and Cell Cultures--
The human T cell line Jurkat was
obtained from Dr. Margarita López-Trascasa (Hospital La Paz,
Madrid). Cells were maintained in RPMI 1640, 10% fetal bovine serum
(ICN Pharmaceuticals, Costa Mesa, CA), and 24 µg/ml gentamycin (Life
Technologies, Inc., Middlesex, UK).
Cell Adhesion Assays--
Flat bottom 8-well strips with
N-oxysuccinimide amine binding surface (Costar Co.,
Cambridge, MA) were coated overnight with recombinant fragments or
peptides diluted in 0.1 M sodium borate, pH 8.5. Adhesion
assays were carried out as described (3, 5, 20) for 3 h at
37 °C; attached cells were stained with toluidine blue and the
absorbance at 620 nm was determined on a Multiskan Bichromatic plate
reader (Labsystems, Helsinki, Finland). Quantitation of cell attachment
was done using calibration curves as described (31) and optical density
at 620 nm was found to be practically a linear function of the number
of cells attached. Total cellular input was calculated in these assays
by spinning wells with the original number of cell aliquots, then
fixing, staining, and measuring optical density. Integrin activation
was performed by incubating the cells (total volume 250 µl) with 5 µl of TS2/16 mAb (0.28 mg/ml) in RPMI 1640, 10 mM Hepes,
1% bovine serum albumin, or 10 mM Tris, 150 mM
NaCl, 1% bovine serum albumin, 2 mM Mn2+, pH
7.2, for 15 min at 37 °C prior to the attachment assay. For inhibition experiments, cells were incubated in a total volume of 300 µl, with appropiate dilutions of HP2/1 supernatant (1:5 or 1:50),
chondroitinase ABC (0.3 or 1.0 Sigma units/ml), chondroitinase AC II (1 Seikagaku units/ml), heparinase III (0.3 or 1 Sigma units/ml), heparitinase (2 Seikagaku milliunits/ml), or synthetic peptides (0.5 mg/ml) for 30 min at 37 °C on a rotary shaker; the cell suspension was then diluted to 7 × 105 cells/ml and 100 µl
were added to each substrate-coated well. The assay was then continued
as explained above. To rule out the possibility of protein degradation
by protease contaminants present in the enzyme preparations, digestions
were performed in the presence of 2 mM phenylmethylsulfonyl
fluoride, 0.36 mM pepstatin A, 2.5 µg/ml leupeptin, 0.1 mg/ml soybean trypsin inhibitor, and 10 µg/ml aprotinin.
Heparin-Sepharose Affinity Chromatography--
Recombinant Fn
fragments were applied to columns containing heparin-Sepharose
(Pharmacia) in 25 mM Tris, 30 mM NaCl, 0.5 mM Na2EDTA, pH 7.4, buffer. After washing the
columns with this buffer, bound fragments were eluted with a linear
gradient ranging from 30 to 450 mM NaCl. Synthetic peptides
(1 mg each) were dissolved in 0.4 ml of 25 mM Tris, 50 mM NaCl, 0.5 mM Na2EDTA, pH 7.6, buffer and incubated with 0.3 ml of heparin-Sepharose matrix for 1 h at room temperature under mild shaking. Unbound material was
determined by reading the absorbance of the solution at 280 nm after
centrifugation. The beads were washed with the same buffer until the
absorbance was zero and bound peptides were eluted using 25 mM Tris, 500 mM NaCl, 0.5 mM EDTA,
pH 7.6, buffer and monitored by optical density at 280 nm. Further
identification of the bound material was achieved by amino acid
analysis and mass spectrometry using a REFLEX time-of-flight instrument
(Bruker-Franzen Analytik, Bremen, Germany) operated in the positive mode.
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RESULTS |
Heparin Binding Characteristics of Recombinant Fragments Containing
Repeats III4-5-6 of Fn--
Previous studies have identified a low
affinity heparin/DNA-binding domain (Hep III) in the central region of
Fn (1, 22-24). To further analyze the binding properties of this
domain, we prepared recombinant fragments spanning Fn repeats III4-6,
III4-5, III4, and III5, respectively (Fig.
1), and tested their ability to bind to
heparin-Sepharose affinity matrices. As shown in Table
I, all fragments bound to heparin but the
conditions for elution from the matrix were different. The FN-III4-5
fragment showed the highest heparin-binding avidity and bound more
strongly than the larger FN-III4-5-6 fragment. Full activity of
FN-III4-5 apparently requires both repeats to be present since
recombinant fragments containing single repeats (FN-III4 and FN-III5)
bound to heparin with lower affinity (Table I).

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Fig. 1.
A, schematic drawing of the human Fn
molecule showing the three types of internal homology units with type
III repeats numbered. Shaded regions indicate the
alternatively spliced segments (ED-B, ED-A, IIICS) only present in
certain Fn subunits. The location of relevant sequences involved in
cell adhesion via integrin or PG receptors, including the novel site
(HBP/III5) reported here is indicated. The heparin-binding domains (Hep
I, II, and III) of Fn are also shown. B, drawing and
SDS-polyacrylamide gel electrophoresis analysis (15% acrylamide) of
the recombinant fragments prepared and used in the present study.
Numbers indicate the molecular mass (kDa) of known standars.
(Modified from Moyano et al. (27)).
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The FN-III4-5 Fragment Mediates Cell Adhesion by Binding
4
1
Integrin and CSPG, Role of Each Receptor Depends on the Stimulus Used
to Activate
4
1--
We have recently shown that the FN-III5
fragment mediates cell adhesion via interaction of the KLDAPT sequence
(H2) with activated
4 integrins (27). To establish a possible
correlation between the heparin binding activity of FN-III4-5 repeats
and cell binding, we first tested whether the FN-III4-5 fragment also
mediated cell adhesion. As shown in Fig.
2, resting Jurkat T cells did not bind to
this fragment; however, upon incubation with 2 mM
Mn2+ or the activating anti-
1 mAb TS2/16, cells attached
to this fragment in a dose dependent manner. This suggested the
implication of a
1 integrin as the receptor for FN-III4-5, possibly
4
1 which is the receptor for FN-III5 (27). Since FN-III4-5 bound heparin with high avidity (Table I), we studied the contribution of
both,
4
1 integrin and cell surface PG for attachment to this fragment.

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Fig. 2.
Adhesion of Jurkat cells to increasing
concentrations of FN-III4-5 fragment. Untreated cells (resting)
or cells previously treated with Mn2+ or TS2/16 mAb were
incubated for 3 h on wells coated with the indicated
concentrations of FN-III4-5. Attached cells were quantitated as
described under "Experimental Procedures." Values are the means of
at least three different experiments with variability of <10%.
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For these experiments, Jurkat cells were treated with either 2 mM Mn2+ or TS2/16 mAb and then incubated with
anti-
4 mAb HP2/1, chondroitinase ABC, heparinase III, or the
combination of mAb and enzymes prior to the attachment assay. As shown
in Fig. 3, the contribution of
4
1
and PG to cell adhesion to FN-III4-5 or FN-III5 fragments was
different depending on whether the cells had been treated with
Mn2+ or TS2/16. For cells incubated with Mn2+,
HP2/1 (1:5 dilution) had little effect on adhesion to either FN-III4-5
(27% inhibition) or FN-III5 fragments (8% inhibition). However,
treatment with chondroitinase ABC (1.0 units/ml) completely inhibited
adhesion to both fragments (92 and 95% inhibition, respectively). Treatment with heparinase III (0.3 or 1 units/ml) or heparitinase (2 milliunits/ml, not shown) had no effect. Since chondroitinase ABC also
degrades dermatan sulfate, we tested the effect of chondroitinase ACII
which is specific for CS. At 1.0 units/ml, this enzyme inhibited adhesion to FN-III4-5 (91% ± 8.3) and FN-III5 (93% ± 2.1, mean of
four experiments, data not shown), therefore indicating that the
receptors for these fragments were CSPG.

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Fig. 3.
Effect of HP2/1 mAb and PG-degrading enzymes
on cell adhesion to FN-III4-5 and FN-III5 fragments.
Mn2+- or TS2/16-treated Jurkat cells were preincubated for
30 min with HP2/1 (anti- 4), chondroitinase ABC
(Chond.), heparinase III, or mixtures of HP2/1 (1:50
dilution), and enzymes (0.3 unit/ml chondroitinase, 1 unit/ml
heparinase III), and added to wells coated with FN-III4-5 or FN-III5
(38 µg/ml). After 3 h, attached cells were quantitated. Values
represent percentage of control (no inhibitor) and are the means of at
least five separate experiments. Standard deviation is indicated by
bars.
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Fig. 3 also shows that further dilutions of HP2/1 (1:50) or
chondroitinase ABC (0.3 unit/ml), used individually, did not affect adhesion to either fragment (0-3% inhibition) of cells incubated with
Mn2+. Interestingly, when both reagents were combined at
these concentrations, they inhibited cell adhesion to FN-III4-5 (65%)
and FN-III5 (98%), thus suggesting a cooperation between both types of
receptors. The residual binding to FN-III4-5 could not be inhibited
under these conditions. As expected, the combination of HP2/1 (1:50) and heparinase III had no effect (Fig. 3).
Incubation of Jurkat cells with TS2/16 mAb resulted in a different
pattern of adhesion to both fragments. As shown in Fig. 3, HP2/1 (1:5
dilution) efficiently inhibited (92%) adhesion to FN-III4-5 fragment
and induced partial inhibition (62%) at 1:50 dilution. Chondroitinase
ABC had a minor effect at 0.3 unit/ml (10% inhibition) and produced
60% inhibition at 1.0 unit/ml. Similar results were obtained with
chondroitinase ACII (not shown). As observed for cells treated with
Mn2+, treatment with heparinase III (or heparitinase) did
not affect adhesion. The combination of HP2/1 (1:50) and chondroitinase
ABC (0.3 unit/ml) completely inhibited (99%) adhesion to the
FN-III4-5 fragment while the combination of HP2/1 and heparinase III
had no effect (Fig. 3).
The HP2/1 mAb also completely (100%, 1:5 dilution) or partially (45%,
1:50 dilution) inhibited adhesion to FN-III5 as we had previously
reported (27). Chondroitinase ABC (1.0 unit/ml) had little effect in
attachment to this fragment (22% inhibition) but when combined with
1:50 dilution of HP2/1 mAb, produced 100% inhibition (Fig. 3).
Heparinase III, either alone or combined with HP2/1 (1:50 dilution),
did not affect adhesion to the FN-III5 fragment (Fig. 3). To confirm
that the heparinase enzymes were active in our assays, we tested their
effect on cell adhesion to the H0 Fn fragment. H0 contains the Hep II
domain and interacts with
4
1 integrin, CSPG, and HSPG (11, 28,
32). In results not shown, heparinase III (1 unit/ml) or heparitinase
(2 milliunits/ml) had a minor effect when used alone but when combined
with 1:100 dilution of HP2/1 mAb (which did not inhibit by itself) they
produced >95% inhibition (mean of three experiments), indicating that
the enzymes were active under the conditions used. Altogether these results indicate that adhesion to FN-III4-5 or FN-III5 fragments involves the cooperation of
4
1 and the glycosaminoglycan (GAG) chains of CSPG. CSPG play an important role when
4
1 is activated with Mn2+ while this integrin is the major receptor when
activation is induced with TS2/16 mAb.
Identification of a 20-Residue Amino Acid Sequence in Fn III5
Repeat That Contains Heparin and Cell Binding Activities--
The
preceding results indicated that the FN-III4-5 fragment binds heparin
and CSPG. To further define the specific sites(s) involved in these
interactions, we compared amino acid sequences in repeats III4-III5
with previously characterized heparin or PG-binding sites in Fn. The
sequence WTPPRAQITGY in III5 was highly homologous to WQPPRARITGY or
FN-C/H V, previously shown to bind heparin and HSPG (12, 13). We
therefore prepared several synthetic peptides spanning the WTPPRAQITGY
sequence in III5 (Fig. 4) and tested
their capacity to bind to heparin-Sepharose matrices and to mediate
cell adhesion.

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Fig. 4.
Synthetic peptides containing sequences from
Fn type III5 repeat prepared and used in the present study.
Sequences are aligned with the previously identified peptide FN-C/H V
(12). The H2 sequence was recently described as an active site in III5
(27). HBP, heparin-binding peptide; N or
C refer to the NH2- or COOH-terminal halves of
HBP/III5, respectively; sc, scrambled.
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As shown in Fig. 5A, the
synthetic peptides WTPPRAQITGY (HBP/III5-N11) and WTPPRAQITGYRLT
(HBP/III5-N14) did not bind to heparin. However, the 20-residue peptide
WTPPRAQITGYRLTVGLTRR (HBP/III5) as well as HBP/III5.sc, in which the
sequence PPRAQIT was replaced by PQARPI, bound heparin very efficiently
and >90% of the applied material was recovered in the 0.5 M NaCl fraction (Fig. 5A). Furthermore, peptide
HBP/III5-C10, containing the C-terminal half of HBP/III5 (see Fig. 4),
retained the heparin binding ability and approximately 41% of peptide
eluted in the bound fraction (Fig. 5A). Substitution of the
first arginine of C-10 by glutamine (peptide HBP/III5-C10.sc, Fig. 4)
reduced the amount of bound peptide to 23% (Fig. 5A). The
identity of peptides HBP/III5 and HBP/III5-C10 as the heparin-bound material was confirmed by amino acid analyses (not shown) as well as
mass spectrometry analyses (Fig. 5B) of the 0.5 M NaCl eluted fractions. These results therefore show that
the heparin-binding site contained in HBP/III5 resides in the
C-terminal sequence YRLTVGLTRR and that all three arginine residues
appear to be important for the interaction.

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Fig. 5.
Heparin binding properties of HBP/III5
synthetic peptide and smaller related constructs. A,
elution profile from heparin-Sepharose affinity matrices of the
indicated peptides. 0.4-ml fractions were collected and monitored by
absorbance at 280 nm. B, mass spectrometry analysis of the
0.5 M NaCl eluted material from assays corresponding to
peptides HBP/III5 and HBP/III5-C10.
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To determine whether the HBP/III5 peptide also mediated cell adhesion,
resting, Mn2+-treated, or TS2/16-treated Jurkat cells were
added to wells containing increasing concentrations of peptide
covalently coupled to KLH. As shown in Fig.
6, in all three cases Jurkat cells
attached to HBP/III5 in a dose-dependent manner. Cells also
attached similarly to the HBP/III5.sc peptide but not to the shorter
HBP/III5-N11 or HBP/III5-N14, and attached only minimally (<20%) to
HBP/III5-C10 and HBP/III5-C10.sc peptides (results not shown).

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Fig. 6.
Jurkat cell adhesion to increasing
concentration of HBP/III5 peptide. Resting or treated cells (with
Mn2+ or TS2/16) were incubated for 3 h on wells coated
with the indicated concentrations of HBP/III5 coupled to KLH. Values
are the mean of at least three experiments with variability of
<10%.
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Cell adhesion to HBP/III5 was completely inhibited by chondroitinase
ABC and ACII (1.0 unit/ml) and by soluble HBP/III5 peptide (0.5 mg/ml),
regardless of the stimulus used for activation, while heparinase III (1 unit/ml), heparitinase (2 milliunits/ml, not shown), HP2/1 mAb (1:5
dilution) or 0.5 mg/ml of soluble H2 peptide (see Fig. 4) had no effect
(Fig. 7). These results indicate that the
full 20-residue sequence contained in HBP/III5 is necessary for an
efficient adhesion and that adhesion to this sequence is exclusively
mediated by CSPG.

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Fig. 7.
Inhibition of cell adhesion to
HBP/III5-KLH by chondroitinase ABC and chondroitinase
ACII. Resting or treated (with Mn2+ or TS2/16) Jurkat
cells were preincubated for 30 min with HP2/1 (1:5 dilution),
chondroitinase ABC or ACII (1.0 unit/ml), heparinase III (1 unit/ml),
or soluble HBP/III5 or H2 peptides (0.5 mg/ml each), and added to wells
previously coated with HBP/III5-KLH (19 µg/ml). Attached cells were
quantitated after 3 h. Values are expressed as percentage of
control (no inhibitor) and are the mean of five different
experiments.
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Functional Role of HBP/III5 and H2 Sites in Cell Adhesion to
FN-III5 and FN-III4-5 Fragments--
The preceding results had
identified a novel sequence in Fn III5 repeat, HBP/III5, which mediates
cell adhesion via CSPG. Since III5 contains another sequence, KLDAPT
(peptide H2) which binds activated
4 integrins (27), we studied
whether both sets of interactions acted in a coordinate manner to
produce cell adhesion. The attachment of Jurkat cells to the previously
described H2 peptide was first studied. As shown in Fig.
8, adhesion of Mn2+-treated
cells to H2-KLH was completely inhibited by mAb HP2/1 or soluble H2
peptide while heparinase III had no effect. Interestingly, chondroitinase ABC and ACII also completely inhibited adhesion to H2
while soluble HBP/III5 peptide was a poor inhibitor (20% inhibition).
For cells treated with TS2/16 mAb, adhesion to H2 was completely
blocked by HP2/1 or soluble H2 peptide (Fig. 7) in agreement with our
previous report (27), but not by soluble HBP/III5 (22% inhibition).
Chondroitinases ABC and ACII in this case had little effect (15 and 8%
inhibition, respectively) and heparinase III did not inhibit adhesion.
These results suggest that for Mn2+-treated cells there is
an interdependence of
4
1 and CSPG receptors for recognition of
the H2 sequence. For TS2/16-treated cells, however, adhesion is almost
exclusively dependent on
4
1 integrin.

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Fig. 8.
Effect of HP2/1, PG-degrading enzymes, and
soluble peptides on cell adhesion to the H2 peptide.
Mn2+- or TS2/16-treated cells were preincubated for 30 min
with the indicated reagents and added to wells coated with H2-KLH (19 µg/ml). Attached cells were quantitated after 3 h. Values are
expressed as percentage of control (no inhibitor) and are the
mean of five different experiments.
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To determine the contribution of the CSPG or
4
1-binding sites to
cell attachment to FN-III4-5 or FN-III5 fragments, Mn2+-
or TS2/16-treated cells were incubated with soluble HBP/III5 or H2
peptides prior to the adhesion assay. As shown in Fig.
9, adhesion to the FN-III5 fragment was
clearly dependent on
4
1 interaction with the H2 sequence since
soluble H2 peptide almost completely blocked adhesion of
Mn2+- or TS2/16-treated cells (90 and 93% inhibition,
respectively). Soluble HBP/III5 peptide had a minor effect (8-15%
inhibition) although in combination with H2 increased the inhibition to
100% in both cases.

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Fig. 9.
Effect of soluble peptides on cell adhesion
to FN-III4-5 and FN-III5 fragments. Mn2+- or
TS2/16-treated Jurkat cells were preincubated for 30 min with HBP/III5
or H2 peptides (0.5 mg/ml) and added to wells coated with FN-III4-5 or
FN-III5 (38 µg/ml). After 3 h, attached cells were quantitated.
Values are expressed as percentage of control (no peptide) and are the
mean of five different experiments.
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In the case of FN-III4-5, the HBP/III5 peptide alone produced a 13%
inhibition regardless of the activation stimulus used; the H2 peptide
did not inhibit adhesion (in fact it increased it slightly) of
Mn2+-treated cells but produced 71% inhibition on
TS2/16-activated cells (Fig. 9). Interestingly, the combination of both
peptides effectively inhibited (60%) the adhesion of
Mn2+-treated cells and increased the inhibitory effect of
H2 for TS2/16-treated cells to 86% (Fig. 9). The residual adhesion
observed in both conditions could not be inhibited by these reagents.
These results indicate that both sites H2 and HBP/III5 participate in
adhesion to the FN-III4-5 fragment when
4
1 is activated with
Mn2+, but H2 is prevalent when the integrin is activated
with TS2/16 mAb.
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DISCUSSION |
The function of the Hep III domain of Fn has been largely unknown
mainly because proteolytic fragments corresponding to this region do
not bind heparin (or DNA) at physiological salt concentrations (22-24). We have recently shown that a recombinant fragment containing repeat III5, within the Hep III domain induced cell adhesion via interaction of the H2 site (KLDAPT) with activated
4 integrins, thus
establishing a novel function for this region of Fn (27).
In this report we have further characterized the heparin and cell
binding activities of this domain. We show that a recombinant fragment
containing repeats III4-III5 (FN-III4-5) bound heparin at
physiological NaCl concentrations and with higher avidity than the
larger fragment FN-III4-6 (see Table I). One possible explanation for
this is that the site(s) contained in repeats III4-III5 is partially
cryptic and removal of repeat III6 fully exposes it and increases the
avidity of the interaction. Fn contains several cryptic sites including
those involved in self-association (33) and chemotaxis (34). A recent
study has shown that physical tension (such as that produced by tissue
injury) exposes cryptic sites in the Fn molecule (35), thus confirming
the biological relevance of these "latent" regions. These regions
may also become functional upon proteolytic degradation of Fn at sites
of tissue damage.
The FN-III4-5 fragment also induced cell adhesion and this involved
the cooperation of
4
1 integrin and the GAG chains of CSPG. Early
studies had shown that HSPG binding to fragments containing the Hep II
domain was necessary for focal adhesion and stress fiber formation of
fibroblasts attached to the central cell-binding domain of Fn (36).
Other reports have demonstrated that melanoma cell adhesion to
fragments or peptides from the Hep II domain involves the cooperation
of CSPG and
4
1 integrin (11, 15-18), that CSPG can modulate the
function of
4
1 (11), and that this regulation may be exerted by
direct interaction of CSPG with
4 via a newly identified functional
GAG-CS-binding site in this integrin subunit (17).
Our present results on the Hep III domain are in agreement with these
previous studies on the COOH-terminal region of Fn and show important
differences between cell adhesion to the Hep II and Hep III Fn domains.
First, adhesion to FN-III4-5 and FN-III5 fragments required previous
activation of
4
1 with Mn2+ or TS2/16. Second and most
importantly, the role of
4
1 and CSPG was clearly dependent on
whether
4
1 had been activated with Mn2+ or TS2/16
mAb. We and others have previously shown that these two agents induce
high affinity forms in
4
1 and it was generally assumed that both
lead to similar activation states (19, 20, 37). However, careful
examination of these previous reports reveals subtle differences with
respect to the effects of Mn2+ and TS2/16. For example,
Masumoto and Hemler (37) showed that in cells with constitutively low
4
1 activity, Mn2+ stimulated adhesion to CS-1 and
VCAM-1, whereas TS2/16 only induced adhesion to VCAM-1. We have also
reported (20) that TS2/16 was 2-3-fold more effective than
Mn2+ in inducing recognition of the Hep II domain of Fn by
monocytic cells. Likewise, TS2/16 but not Mn2+ enhanced the
constitutive adhesion of these cells to a Fn fragment containing CS-1
(20).
In this report we clearly demonstrate functional differences between
activation of
4
1 with Mn2+ or TS2/16, which results
in a higher dependence of GAG-CSPG in the case of Mn2+
treatment. The effect of Mn2+ was not due to a charge
neutralization of the sulfated chains of GAG since other divalent
cations (Ca2+, Mg2+, resting conditions) did
not induce cell adhesion. Assuming that
4
1 and CSPG form a
complex at the cell surface, we can postulate that activation with
Mn2+ results in a partially active
4
1 unable to
support adhesion by itself after disruption of the complex with
chondroitinase ABC. In contrast, TS2/16 would lock the integrin in an
active conformation which would no longer require cooperation by CSPG. In support of this, chondroitinase ABC and chondroitinase ACII completely inhibited adhesion to the synthetic peptide H2 (which does
not bind PG) when
4
1 was activated with Mn2+, but had
a minor effect on TS2/16 activation. These results differ from previous
findings on melanoma cells where activation of
4
1 with either
reagent reverted the effect of chondroitinase ABC (17). An explanation
for this could be a different constitutive activity of
4
1 in
melanoma and lymphoid cells (our study) thus implying a different
regulation by CSPG.
We have also identified a novel amino acid sequence in Fn repeat III5,
WTPPRAQITGYRLTVGLTRR (named HBP/III5), which binds heparin and mediates
cell adhesion via CSPG. Although the NH2-terminal half of
this sequence is highly homologous to the previously described WQPPRARITGY or FN-C/H V located in repeat III14 (12), HBP/III5 required
all 20 amino acid residues for full activity and this was partially
retained in the last 10 residues but not in the NH2-terminal portion of the peptide. This indicates that
the sequence requirements are different for HBP/III5 and FN-C/H V and
that the three arginine residues of the COOH-terminal portion seem to
be crucial for activity of the former. The nature of the GAG chains
that interact with FN-C/H V and HBP/III5 may also be different. In our
study, HBP/III5 as well as fragments FN-III4-5 and FN-III5 clearly
bound CS but not HSPG. Although this conclusion is based on the lack of
effect of heparinase III or heparitinase in the adhesion assays, we
have confirmed that these enzymes were active under identical
conditions when tested on a substrate previously known to interact with
HSPG (32, 36). Peptide FN-C/H V, however, was originally shown to bind
only HSPG (13) although phorbol 12-myristate 13-acetate-treated U937
monocytic cells apparently bind this peptide through both types of PG
(14). It is therefore possible that the differential use of CS or HS
GAG chains for interactions with Fn depends on the nature of the ligand
and/or on the cell type of study.
Based on the present results we can establish that repeat III5 contains
two closely located active sites, the previously described H2 which
binds activated
4 integrin and HBP/III5 which binds CSPG. Both sites
cooperate in mediating cell adhesion to the Hep III domain. Moreover,
we have consistently observed that adhesion to FN-III4-5 (but not to
FN-III5) could not be completely inhibited neither with the combination
of chondroitinase ABC and HP2/1 mAb nor with the mixture of H2 and
HBP/III5 peptides, suggesting that additional active sites may exist in
this fragment. These sites could be located in repeat III4, however, a
recombinant fragment containing only this repeat (FN-III4) did not
mediate adhesion of Jurkat cells (results not shown). This suggests
that additional sites may require the entire III4-III5 region for
activity, a fact that is essential for the high affinity binding of
this domain to heparin (see Table I).
The physiological significance of the Hep III domain is beginning to be
revealed. Besides our previous demonstration of the cell binding
activity of repeat III5 and the results presented here, other authors
have shown that: 1) the region encompassing repeats III1-III7 may
regulate Fn matrix formation (38); 2) a mAb recognizing an epitope in
repeat III5 inhibited fibroblast-mediated collagen gel contraction
(39), suggesting a role for this region in interactions with collagen.
Although further work is necessary to completely understand the
function of this domain, it is possible that repeats III4-III5 are
involved in the process of Fn matrix formation by interacting with
cells as well as with other macromolecules. In this regard, it was
recently shown that repeats III12-14 in the Hep II domain participate
in Fn polymerization (40). The III4-III5 region may also constitute an
important cell attachment domain for activated leukocytes at
inflammatory sites and injured tissues, where proteolytic degradation
or conformational changes of Fn take place physiologically.