From the Department of Pathology and Laboratory Medicine,
University of Wisconsin Medical School, Madison, Wisconsin 53706
Fibronectin matrix assembly is thought to involve
binding interactions between the amino-terminal I1-5
repeats and the first type III repeat (III1). Here we
report that a third site, located within the III12-14
repeats of the carboxyl-terminal heparin II domain of fibronectin, is
also involved in fibrillogenesis. Heparin II fragments inhibited fibril
formation and binding of 125I-labeled fibronectin and/or
70-kDa fragments to the cell surface, deoxycholate-insoluble matrix,
and adsorbed 160-kDa cell adhesion fragments of fibronectin. The
inhibitory effects of heparin II fragments were as large or up to 20 times larger than those of a 44-kDa fibronectin fragment containing the
III1 repeat. Under physiological conditions, amino-terminal
fragments of fibronectin containing the I1-5 repeats
interacted preferentially with proteolytically derived heparin II
fragments and a recombinant III12-14 peptide both in
solution and in solid phase, indicating that matrix assembly may
involve direct interactions between I1-5 and
III12-14 repeats. Interactions between the
I1-5 repeats and 160-kDa fragments containing the
III12-14 and III1 repeats could be inhibited
by
90% by either an anti-III13-14 monoclonal
antibody (mAb) (IST-2) or an anti-III1 mAb (9D2),
suggesting that cooperative interactions between III12-14
and III1 repeats may also promote binding of the
I1-5 repeats. Neither mAb IST-2 nor mAb 9D2, alone or in
combination, inhibited binding of 125I-labeled 70-kDa
fragments to cycloheximide-treated cells plated on the 160-kDa
substrate, suggesting that additional I1-5 binding sites,
independent of the III1 and III12-14 repeats, may be involved in fibrillogenesis.
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INTRODUCTION |
Fibronectin is required for normal growth and development (1) and
plays an important role in regulating cell attachment and movement,
wound healing, and tumorigenesis (for review, see Refs. 2 and 3). It is
a 500-kDa disulfide-bonded dimer consisting of similar subunits and is
found as a soluble glycoprotein in blood and other body fluids and as
an insoluble fibrous matrix component in tissues. Each subunit of
fibronectin consists of three different types of repeating sequences,
called types I, II, and III, which are arranged into discrete
structural and functional modules.
Assembly of dimeric fibronectin into the extracellular matrix involves
multiple consecutive binding interactions with integrin receptors, with
itself, and with matrix components such as type I collagen (for review,
see Refs. 4-6). Although the
5
1 integrin appears to be the primary fibronectin receptor involved in matrix assembly (7-12), at least two other integrins,
IIb
3 and
v
3, can also support fibronectin
fibrillogenesis (13, 14). High affinity binding interactions between
these integrins and the RGD site in the 10th type III repeat
(III10) of fibronectin are thought to promote
fibrillogenesis by exposing appropriate self-assembly sites in
fibronectin. Such sites may become exposed through local integrin-induced conformational changes in III10
repeats (15) or through integrin-mediated stretching (reversible
unfolding) of one or a whole array of type III repeats in fibronectin
in response to cell movements (13, 16).
Self-assembly of fibronectin dimers into fibrils is currently thought
to involve primarily interactions between the first five type I repeats
(I1-5) and the first type III (III1) repeat
(17, 18). The I1-5 repeats are critical for matrix assembly, i.e. peptides including these repeats block
assembly of fibronectin into fibrils, and fibronectin dimers lacking
these repeats will not be incorporated into fibrils (19-22). The
III1 repeats are also important for fibril formation, and
either anti-III1 monoclonal antibodies or peptides derived
from III1 repeats can block assembly of fibronectin into
matrix (23, 24). The mechanism, however, by which
I1-5-III1 interactions affect matrix assembly remains controversial. For example, Hocking et al. (25) have shown that III1 repeats will interact not only with
I1-5 repeats but also with heat-denatured
III10 repeats. They have proposed that the latter
interaction activates the III1 repeat thereby allowing it
to function as a receptor for the amino termini of a second fibronectin
dimer. In contrast, Sechler et al. (26) have shown that
fibronectin dimers lacking III1-7 repeats are readily
polymerized into fibrils. In fact, the mutated dimers are rendered
deoxycholate-insoluble more rapidly than intact dimers, suggesting that
III1 repeats do not promote, but rather inhibit matrix
assembly by keeping fibronectin in a compact form through intramolecular interactions with I1-5 repeats.
Earlier studies by Homandberg and Erickson (27) had indicated that
another complementary binding site of the I1-5 repeats is
located within the III12-14 repeats of the
carboxyl-terminal heparin II binding domain of fibronectin, but the
functional significance of this site was never investigated. Here we
report that the heparin II domain plays an important role in matrix
assembly, and we demonstrate that peptides containing the
III12-14 repeats specifically block binding of fibronectin
and its 70-kDa amino-terminal fragments to cells and their matrix. We
also present evidence indicating that the inhibitory effects of the
heparin II domain may involve direct interactions between the
I1-5 and the III12-14 repeats and that
binding of the I1-5 repeats may depend on cooperative
interactions between the III12-14 and III1 repeats. Finally, the data presented here confirm our earlier report
(12) and show that the amino terminus participates in additional
binding interactions that are independent of either the
III1 or the III12-14 repeats.
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MATERIALS AND METHODS |
Cell Culture and Binding Assays--
Neonatal human skin
fibroblasts were maintained in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum. Binding assays in confluent
cultures of matrix-deprived cells were performed as described before
(12). Briefly, cells were harvested with trypsin and EDTA, washed in
the presence of soybean trypsin inhibitor, and kept in suspension for
1 h at 21 °C in binding buffer (Dulbecco's modified Eagle's
medium containing 25 µg/ml cycloheximide, 2 mg/ml
BSA,1 100 units/ml penicillin
G, 5 µM streptomycin sulfate, and 25 mM
Hepes, pH 7.4). Cells were then plated in 96-well polystyrene microtiter wells precoated with 40-120 µg/ml of the 160-kDa or 5 µg/ml of the 75-kDa cell adhesion fragments of fibronectin. These
coating concentrations had been shown previously to be optimal for cell
spreading and binding of 70-kDa fragments (12). Plates were blocked
overnight with 2 mg/ml heat-denatured BSA (5 min at 80 °C). In some
experiments, antibodies were added to the cell suspension just before
plating. After 3 h at 37 °C, cells were incubated with
125I-labeled fibronectin (1 × 107
cpm/pmol) or 70-kDa fragments (1.5-3 × 106 cpm/pmol)
for 1 or 2 h at 37 °C, and wells were washed, separated, and
counted.
Binding interactions between ligand and substrate which occur in the
absence of cells were also measured in microtiter wells as described
previously (12). For some of these experiments (see Figs. 6 and 7), the
pH of the binding buffer was adjusted to 6.6 with HCl. Nonspecific
binding was measured either in wells that had been coated only with the
blocking agent (2 mg/ml heat-denatured BSA) or in the presence of a
>100-fold excess of unlabeled ligand. Nonspecific binding measured in
the presence of excess competitor represented 10-20% and 20-50% of
total binding in the presence and absence of cells, respectively, and
was independent of the addition of any of the antibodies.
Matrix Assembly Assay--
Cells were plated at confluence and
maintained in the presence of 10% fetal bovine serum for 3 days. Cells
plated in 35-mm dishes were then incubated with 0.1 nM
125I-fibronectin (1 × 106 cpm/ml) in 0.25 ml of binding buffer, pH 7.4, for 2 h at 37 °C with gentle
rocking. Cells plated in 60-mm dishes were incubated with 0.75 nM 125I-labeled 70-kDa fragments (2 × 106 cpm/ml) in 1 ml of binding buffer, pH 7.4, for 4 h
at 37 °C with gentle rocking. The cells were washed five times with
binding buffer and lysed at 4 °C with 1% deoxycholate in hypotonic
buffer (1.5 ml/dish 50 mM Tris-HCl, pH 8.3, containing 1 µg/ml pepstatin, 2 µg/ml leupeptin, and 2 µg/ml aprotinin) and
scraped from the dishes. The deoxycholate-soluble cellular fraction
(pool I) and insoluble matrix fraction (pool II) were separated by
centrifugation at 35,000 × g as described (28). The
deoxycholate-insoluble material was washed with 1% deoxycholate in
hypotonic buffer and recentrifuged. All operations were done at
4 °C.
Isolation of Adherent Matrices--
Adherent matrices were
prepared from fibroblast cultures that had been plated at confluence in
60-mm dishes and maintained in the presence of 10% fetal bovine serum
for 3 days. Cells were lysed with 1% deoxycholate in hypotonic buffer
for 5 min (28). The lysates in the plates were treated with DNase I (50 units/ml; Sigma), MgCl2, and CaCl2 (5 mg/ml
each) in hypotonic buffer for 15 min, and the plates were rinsed with
hypotonic buffer followed by binding buffer. The fibrous matrices,
prepared in this way at 21 °C, remained firmly attached to the
dishes in subsequent binding assays. Binding assays using isolated
matrices were done as described above for intact cell layers.
Binding Interactions in Solution--
Binding reactions in
solution were carried out at 21 °C by mixing 0.3-1.8 pmol of the
125I-ligand (3-16 × 105 cpm/pmol) with
25 pmol of biotinylated 70-kDa fragments in 240 µl of 50 mM sodium phosphate, pH 7.4, containing 0.08 M
NaCl (PBS) in the absence or presence of 1 mg/ml bis(sulfosuccinimidyl)
suberate (BS3; Pierce). After 30 min, the cross-linker was
inactivated by adding 27 µl of 1.5 M glycine, pH 7.4. Twenty min later 11 µl of 50 mM BSA was added. From each
reaction vial, 250 µl was transferred to a new vial containing 6 µl
of packed streptavidin-conjugated agarose beads (1.2 mg
streptavidin/ml; Sigma) and 40 µl of packed Sepharose CL-4B
(Pharmacia Biotech Inc.). The agarose beads and Sepharose were
preblocked with 2 mg/ml BSA in PBS to reduce nonspecific binding.
Binding to the beads was carried out at 21 °C for 1 h on an
end-over-end rotator. The beads were then sedimented, washed three
times with 1 ml of PBS containing 0.05% Tween 20 and counted in a
counter. In each case, BS3-dependent as well as
BS3-independent binding increased linearly with the
concentration of the ligand (data not shown).
Proteolytic Fragments of Fibronectin and Antibodies--
Fig.
1 illustrates the fragments used in this
study. Human plasma fibronectin and its 29-kDa (type I1-5
repeats), 40-kDa (type I6-I9 repeats), 70-kDa
(type I1-I9 repeats), and 160-kDa (type
III1-III14 repeats) proteolytic fragments were
prepared as described before (29-31).

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Fig. 1.
Schematic diagram of fibronectin indicating
the fibronectin fragments and antibodies used. The diagram
represents a monomer of plasma fibronectin consisting of type I
(rectangles), type II (ovals), type III
(numbered squares) repeats, and the variable connecting
sequence (CS). The two main heparin binding domains
(Hep I, Hep II) and the binding site of the
5 1 integrin (RGD) are also
indicated. The location of the epitopes recognized by the monoclonal
antibodies used in this study are indicated above. The location of the
proteolytic fragments used in this study are indicated below together
with their molecular mass. The proteolytically derived heparin
II-binding fragments consisting of five peptides and their weighed
average molecular mass (50 kDa) are indicated by a solid
line. Some of these fragments may extend further as indicated by
the dotted line.
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The carboxyl-terminal heparin II-binding fragments were isolated from a
catheptic D digest of plasma fibronectin, from which gelatin-binding
fragments (notably the 70-kDa fragment) had been removed by first
passing the digest over on a gelatin-Sepharose column. The flow-through
from the gelatin-Sepharose column was then passed through a
heparin-agarose column, and the bound material was eluted with
increasing concentrations of NaCl (70-600 mM in 10 mM Tris-HCl, pH 7.4). SDS-polyacrylamide electrophoresis
and Western blots indicated that the carboxyl-terminal heparin-binding fragments were included in the last peak eluted from the column. This
peak contained five fragments (65, 46, 43, 41, and 39 kDa). None of
these fragments contained the COOH-terminal disulfide bond.
Calculations of molar concentrations are based on the weighed average
molecular mass of these fragments (50 kDa).
The 44- and 75-kDa fragments were obtained by digesting reduced and
alkylated plasma fibronectin with 10 µg of tosylphenylalanyl chloromethyl ketone-treated trypsin/mg of fibronectin for 30 min at
37 °C and separating the resulting fragments on a Sephacryl S-300
column (Pharmarcia) equilibrated with 20 mM Tris-HCl,
0.15 M NaCl, pH 7.4. The 44-kDa fragments were purified
further on a heparin-agarose column to remove traces of
contaminating material reacting with mAb IST-2.
All fragments were characterized by SDS-polyacrylamide gel
electrophoresis, Western blots, and direct or competitive enzyme-linked immunoassays. The large 160-kDa cell adhesion fragment was recognized by mAbs 9D2 (anti-III1), C6F10
(anti-III8-11), and IST-2 (anti-III13-14) but
not by IST-7 (anti-III15), whereas the smaller 75-kDa cell
adhesion fragment was recognized by C6F10 only.
The 44-kDa fragment was recognized by 9D2 but not by L8 (anti-I9-III1) or
C6F10. None of the four antibodies recognized the amino-terminal 29-, 40-, and 70-kDa fragments. All of the COOH-terminal heparin-binding fragments, except for the 43-kDa fragment, were recognized by mAbs IST-2 and IST-7. None of these COOH-terminal fragments was recognized by the mAbs
C6F10 and 9D2. All fragments were iodinated
with carrier-free Na125I by the chloramine-T method (32).
These radioiodinated probes were also used to measured adsorption and
desorption of fibronectin fragments in microtiter wells. Biotinylated
70-kDa fragments were prepared using Enzotin (NZM Biochem, New York) as
described by the manufacturer.
The mAbs L8, 9D2, and C6F10 were provided by
Dr. Deane Mosher, University of Wisconsin. The mAbs IST-2 and IST-7
were obtained from Dr. Luciano Zardi, Instituto Nationale per La Ricera
Sul Cancro. The antibodies were concentrated by precipitation with 30%
ammonium sulfate and/or solvent extraction with flakes of polyethylene
glycol followed by dialysis against 20 mM sodium phosphate,
pH 7.4, containing 0.15 M NaCl. IgG concentrations were
determined by using a mouse IgG kit (Boehringer Mannheim).
Preparation of Recombinant Type III12-14
Peptide--
Human full-length fibronectin cDNA, pFH100 (33), was
used as a template for polymerase chain reaction amplification (34) of
a DNA sequence (bases 5072-5923) encoding the III12-14 repeats of fibronectin (amino acid Glu1687 through
Pro1970). The DNA was generously provided by Dr. Deane
Mosher (who received it from Dr. Jean Paul Thiery, CNRS URA, Paris).
The bases are numbered starting with the G before the first codon of
the first amino acid of the mature protein (GenBankTM
accession no. X02761), and the amino acids are numbered starting with
the pyroglutamic acid (35). This region spans the entire length of the
III12-14 repeats and also includes 3 amino acids upstream
of the III12 repeat (QST) as well as 10 amino acids downstream of the III14 repeat (DELPQLVTLP). The sense
primer, 5
-GAATTCCAGTCCACAGCTATTCCTG, generated an
EcoRI site (in boldface), whereas the antisense primer,
5
-CTCGAGCTATGGAAGGGTTACCAGTTG, generated an
XhoI site (in boldface) and also introduced a stop codon
(underlined). The polymerase chain reaction-amplified DNA was purified
using the WizardTM PCR Preps DNA Purification System
(Promega, Madison, WI) and subsequently ligated into the pGEM-T vector
(Promega) according to the manufacturer's instructions. The pGEM-T
vector containing the insert was transfected through electroporation
(ElectroCell Manipulator 600 BTX Electroporation System) into JM109
bacteria. The pGEM-T vectors containing the appropriate sized insert
were digested with EcoRI and XhoI, and the
cDNA fragment encoding the III12-14 repeats was gel
purified using the WizardTM PCR Preps DNA Purification
System. This purified DNA fragment was then cloned in-frame into the
bacterial expression vector pGEX-4T1 (Pharmacia) and electroporated as
described above. Expression of the glutathione S-transferase
fusion peptide (GST-III12-14 repeats) was confirmed using
Western blot analysis with mAb IST-2.
The GST-III12-14 fusion peptide was purified using
modifications of the procedure described previously (36). The
modifications included adding 1 mM Pefabloc®SC (Boehringer
Mannheim) and lysing the cells with a French press followed by
sonication (three cycles of 10 s each). The fusion peptide was
adsorbed to glutathione-Sepharose 6B beads (Pharmacia) and digested
overnight at 4 °C with thrombin (10 NIH units/500 ml of original
culture). The beads were removed by centrifugation at 250 × g for 5 min at 4 °C. The supernatant containing the
cleaved peptide was incubated with p-aminobenzamidine Sepharose 6B beads (Sigma) for 20 min at 4 °C to remove thrombin. The beads were removed by centrifugation at 250 × g
for 5 min, and the recombinant III12-14 peptide in the
final supernatant was concentrated on flakes of polyethylene glycol and
dialyzed into PBS. SDS-polyacrylamide gel electrophoresis and Western
blots showed that the recombinant III12-14 peptide
migrated as a doublet (~29-kDa) and was recognized by the mAb IST-2
(data not shown). Western blot analysis using a goat anti-GST serum (gift from Dr. James Tracy, University of Wisconsin, Madison) showed
that the recombinant peptide did not contain any uncleaved fusion
peptide or GST (data not shown).
Immunofluorescence Microscopy--
Teflon® coated 12-well
slides (Polysciences, Warrington, PA) were precoated for 2 h at
21 °C with 20 µg/ml of the 160-kDa fragment of fibronectin in
Hanks' balanced salt solution. Fibroblasts suspended in Dulbecco's
modified Eagle's medium containing 7.5% fetal bovine serum, 100 units/ml penicillin G, and 5 µM streptomycin sulfate were
plated at a density of 3 × 105 cells/well and
incubated at 37 °C to allow the cells to attach. After 1 h, the
medium was replaced with serum-free medium (Dulbecco's modified
Eagle's medium, 2 mg/ml BSA, 25 µg/ml cycloheximide, 100 units/ml
penicillin G, and 5 µM streptomycin sulfate), and the
cells were allowed to spread for 3 h at 37 °C. The medium was
removed again and replaced with fresh serum-free medium containing 1 µg/ml human plasma fibronectin. In some wells, either the 70-kDa (93 µg/ml) or heparin II-containing fragments (200 µg/ml) were added as
inhibitors. Fibroblasts were kept overnight at 37 °C, washed with
Hanks' balanced salt solution, and fixed with 4% paraformaldehyde, 0.1 M sodium phosphate buffer at pH 7.4 for 30 min at
21 °C. Fibronectin fibrils were labeled with a polyclonal
anti-fibronectin serum as described previously (37) and viewed by
epifluorescence with a Nikon Optiphot microscope. Images were digitized
using a Photometrics Image Point®CCD camera and Image Pro Plus®
program.
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RESULTS |
Heparin II-containing Fragments of Fibronectin Inhibit Assembly of
Fibronectin into Matrix--
Earlier studies by Homandberg and
Erickson (27) had indicated that the heparin II binding domain of
fibronectin may contain an amino-terminal binding site. Because an
amino-terminal binding site in the III1 repeat of
fibronectin had been shown to play an important role in the assembly of
fibronectin into fibrils (17, 18, 25, 26), we wanted to know whether
the heparin II binding domain of fibronectin also participates in
fibrillogenesis. To examine this question, cycloheximide-treated
fibroblasts capable of assembling exogenous plasma fibronectin into
fibrils (38) were tested for their ability to assemble plasma
fibronectin into fibrils in the presence, or absence, of
proteolytically derived fragments of fibronectin. In the absence of any
fibronectin fragments, numerous fibronectin fibrils were observed in
these cultures (arrowheads, Fig.
2a). If, however, fragments of
fibronectin containing the heparin II domain were added, fibril
formation was inhibited completely (Fig. 2c), indicating
that the heparin II domain may indeed play a role in fibrillogenesis.
The 70-kDa amino-terminal fragment of fibronectin, as expected (19,
39), also blocked incorporation of exogenous fibronectin into
matrices (Fig. 2b).

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Fig. 2.
Inhibition of fibronectin fibrillogenesis in
the presence of the 70-kDa or heparin II-containing fragments.
Cycloheximide-treated fibroblasts plated on the 160-kDa fragment of
fibronectin were incubated overnight with plasma fibronectin
(panel a), plasma fibronectin and the 70-kDa amino-terminal
fragment (panel b), or plasma fibronectin and heparin
II-containing fragments (panel c) as described under
"Materials and Methods." At the concentrations used, 70-kDa (1.3 µM) and heparin II fragments (4.4 µM)
inhibited binding of 125I-fibronectin by 50 and 40%,
respectively (see Fig. 4A). A phase image of the cell layer
is shown in panel d. All cultures were fixed and labeled
with an anti-fibronectin serum followed by an anti-rabbit IgG
conjugated to rhodamine as described under "Materials and Methods."
Fibronectin fibrils (arrowheads) can be found only in
cultures incubated with plasma fibronectin alone. Cultures incubated
with either fibronectin fragment did not form fibrils. Bar = 50 µm.
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The inhibitory effect of the heparin II domain on fibrillogenesis was
confirmed biochemically using the matrix assembly assay described by
McKeown-Longo and Mosher (19, 28). In this assay, incorporation of
125I-fibronectin into the deoxycholate-insoluble matrix
(pool II) was inhibited with increasing concentrations of unlabeled
70-kDa fragments (Fig. 3). The
IC50 of the inhibition was 1.0 µM. At 10-fold
higher concentrations, fragments containing the III12-14 repeats were equally effective inhibitors (IC50 = 10 µM). The 44-kDa fragment containing the
III1-4 repeats was also inhibitory at these higher
concentrations, however it never blocked incorporation of
125I-fibronectin by more than 40%. In contrast, 75-kDa
fragments containing the III5-10 repeats or BSA had little
(20%) or no inhibitory effects.

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Fig. 3.
The incorporation of fibronectin into
deoxycholate-insoluble matrix is inhibited by various fibronectin
fragments. The incorporation of 125I-fibronectin into
matrix in confluent cell layers was measured as described under
"Materials and Methods." Increasing concentrations of unlabeled
amino-terminal 70-kDa fragments ( ), heparin II-binding fragments
( ), 44-kDa fragments containing the III1 repeat ( ), 75-kDa cell adhesion fragments ( ), or BSA ( ) were added to cell layers in the presence of 125I-fibronectin. Incorporation
of fibronectin is expressed as percent of controls. The controls had
incorporated 0.5 ± 0.02 fmol/dish 125I-fibronectin
over the 2-h labeling period. The data are means of two or three
measurements; bars indicate the S.E. Curves are second order regression lines.
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Fragments containing the heparin II binding domain also inhibited
binding of 125I-fibronectin to the cell surface in
confluent cell layers of matrix-deprived cycloheximide-treated
fibroblasts plated on the 75-kDa cell adhesion fragment of fibronectin
(12). As shown in Fig. 4A, the
70-kDa fragment was again the most effective inhibitor (IC50 = 0.1 µM) followed by the heparin II
(IC50 = 3 µM) and the 44-kDa
(IC50
20 µM) fragments. The 75-kDa
fragment and BSA had little or no inhibitory effect. Thus, under
physiological conditions, III12-14-containing fragments
inhibited binding of 125I-fibronectin to cell layers at
least 10 times more efficiently than either the III1-4- or
III5-10-containing fragments. These studies clearly
establish that besides the amino-terminal I1-5 repeats,
the type III repeats contained in the carboxyl-terminal heparin
II-binding fragments are the most effective inhibitor of fibronectin
matrix assembly and that they are capable of inhibiting the initial
binding of fibronectin to the cell surface as well as its subsequent
incorporation into fibrils.

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Fig. 4.
Inhibition of the binding of
125I-labeled fibronectin or 70-kDa fragments to
matrix-deprived fibroblasts by various fibronectin fragments.
Cycloheximide-treated fibroblasts (3 × 104
cells/well) were plated for 3 h at 37 °C in microtiter wells coated with 0.4 pmol of the 75-kDa fragment of fibronectin. Cultures were incubated for 2 h with either 0.9 nM (1 × 107 cpm/well) 125I-fibronectin (panel
A) or 2.8 nM (5 × 106 cpm/well)
125I-labeled 70-kDa fragments of fibronectin (panel
B) in the presence or absence unlabeled 70-kDa fragments ( ),
heparin II-containing fragments ( ), 44-kDa fragments containing type
III1 repeats ( ), 75-kDa cell adhesion fragment ( ), or
BSA ( ). None of the added peptides had any discernible effects on
the appearance of the cells or the cell density. All data represent
means of triplicate measurements. Bars indicate the S.E.
Data were normalized to the amount of ligand binding seen in the
absence of any additions (for 125I-fibronectin (panel
A): 0.26 ± 0.01 fmol/well, n = 15; for
125I-labeled 70-kDa fragments (panel B):
0.63 ± 0.02 fmol/well, n = 12). All
curves are third order regression lines.
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Heparin II-containing Fragments Inhibit Binding of
125I-Labeled 70-kDa Fragments to Cell Layers, 160-kDa
Fibronectin Fragments, and Matrix Fibrils--
Because the assembly of
fibronectin into fibrils is thought to be mediated by the
amino-terminal I1-5 repeats (19, 20), we also examined
whether the heparin II-binding fragments could interfere directly with
binding of 125I-labeled 70-kDa fragments to cell layers. As
shown in Fig. 4B, unlabeled 70-kDa (IC50 = 0.006 µM), heparin II (IC50 = 2 µM), and 44-kDa (IC50 = 20 µM) fragments inhibited
binding of 125I-labeled 70-kDa fragments to cells in almost
exactly the same manner as they inhibited binding of
125I-fibronectin (see Fig. 4A). Thus, all of the
binding interactions of fibronectin in cell layers which can be
specifically inhibited by these fibronectin fragments appear to be
I1-5-dependent binding interactions. In
control experiments, high concentrations of BSA elevated the binding of
125I-labeled 70-kDa fragments slightly.
To test whether the inhibitory effects of heparin II fragments involve
direct fibronectin-fibronectin binding interactions independent of
cells, we measured the ability of these fragments to inhibit direct
binding of 125I-labeled 70-kDa fragments to 160-kDa cell
adhesion fragments of fibronectin adsorbed to plastic wells (12). This
reaction was specifically inhibited by unlabeled 70-kDa
(IC50 = 0.02 µM), heparin II
(IC50 = 0.1 µM), and 44-kDa fragments
(IC50 = 2 µM), whereas the 75- and 160-kDa
cell adhesion fragments had the same nonspecific effect as high
concentrations of BSA (Fig. 5). Both the
heparin II fragments and the 44-kDa fragments thus inhibited binding of
the 125I-labeled 70-kDa ligand to the 160-kDa substrate 10 times more efficiently than they inhibited binding of 70-kDa fragments
to the cell surface. This suggests that the III12-14
repeats, like the III1 repeats, may preferentially affect
I1-5-dependent fibronectin-fibronectin
interactions in the extracellular matrix.

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Fig. 5.
Inhibition of the binding of
125I-labeled 70-kDa fragments to adsorbed 160-kDa substrate
by fibronectin fragments containing the III12-14 or the
III1 repeats. Microtiter wells, which had adsorbed 2.9 pmol of 160-kDa fragments, were labeled with 2.8 nM
125I-labeled 70-kDa fragments (5 × 105
cpm/well) in 60 µl of binding buffer, pH 7.4, for 2 h at
37 °C in the absence or presence of unlabeled 70-kDa fragments
( ), fragments containing the III12-14 repeats ( ), or
44-kDa fragments containing the III1 repeat ( ) at the
indicated concentrations. Nonspecific effects caused by increasing the
protein concentration were measured by supplementing the binding buffer
with additional BSA ( ). All data represent means of triplicate
measurements (bars indicate the S.E.) normalized to the
binding of 125I-labeled 70-kDa fragments seen in the
absence of any additions (0.90 ± 0.07 fmol/well;
n = 9). Curves are third order regression lines.
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To examine this possibility, we compared the ability of heparin II
fragments to block the binding of 125I-labeled 70-kDa
fragments to the cell surface (the deoxycholate-soluble pool I) and to
the matrix (deoxycholate-insoluble pool II) in confluent cell layers.
Earlier experiments had shown that compared with intact fibronectin,
70-kDa fragments were poorly incorporated into pool II (19). We found,
however, that over a 4-h period, as much as 44% of the total
125I-labeled 70-kDa fragments binding to confluent normal
cell layers could be recovered in pool II (Table
I); under the same conditions, ~60% of
125I-fibronectin entered pool II (data not shown). We also
found that both fibronectin and its 70-kDa fragment could bind directly to isolated matrices, provided that the deoxycholate-insoluble extracted cell layers were first treated with DNase to remove any DNA
that was released during cell lysis and had coated the adherent
matrices. Direct binding to such matrices over a 4-h period amounted to
nearly 70% of the binding of 125I-labeled 70-kDa fragments
(Table I) or 125I-fibronectin (data not shown) in intact
cell layers. Incorporation of 125I-labeled 70-kDa fragments
into pool I, pool II, and isolated matrices was equally inhibited with
unlabeled 70-kDa fragments (~35% inhibition with 0.8 µM). The incorporation of 125I-labeled 70-kDa
fragments to pool I, pool II, and isolated matrices was also
significantly (p > 0.05) inhibited by the addition of unlabeled heparin II-binding fragments. At the concentration chosen (4 µM), heparin II fragments inhibited 15, 25, and 29% of
the total binding or 42, 69, and 88% of the specific binding of
125I-labeled 70-kDa fragments into pool I, pool II, and
isolated matrices, respectively. We conclude, therefore, that the amino terminus of fibronectin can interact directly with the matrix in cell
layers and that the heparin II-binding fragment can interfere with such
binding significantly. Presumably, the ability of the heparin II
fragments to inhibit the binding interaction between the 70-kDa ligand
and the adsorbed 160-kDa substrate (Fig. 5) is physiologically
significant in that it reflects this aspect of fibronectin matrix
assembly.
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Table I
Inhibition of amino-terminal binding to cell layers and isolated
matrices by fragments containing the III12-14 repeats
Deoxycholate soluble (pool I) and insoluble fractions (pool II) and
isolated fibronectin matrices were prepared as described under
"Materials and Methods." Percent inhibition of specific binding
refers to the percent inhibition by the heparin II containing fragments
(hep II) relative to the percent inhibition by the 70-kDa fragment (0.8 µM). All data represent the means of four
measurements ± S.E. The differences between controls and dishes
treated with the inhibitor were significant at the 95% confidence
level in each of the seven experiments (t test; comparison
of means).
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Direct Binding Interactions between I1-5 and
III12-14 Repeats--
To determine whether direct
interactions between I1-5 and III12-14
repeats could account for the inhibitory effects of heparin II-binding
fragments in fibrillogenesis, 70-kDa fragments adsorbed to plastic
wells were tested for their ability to bind proteolytically derived and
recombinant peptides containing the III12-14
repeats. As shown in Fig. 6,
125I-labeled III12-14-containing
peptides bound to absorbed 70-kDa fragments in a
concentration-dependent manner. On a molar basis, the
recombinant peptide was bound three to four times more efficiently than
the proteolytic fragments. Binding of radioiodinated recombinant and
proteolytic fragments was inhibited by ~50% in the presence of a
300-400-fold molar excess of unlabeled heparin II-binding fragments.
No further inhibition could be achieved by increasing the concentration
of the competitor (data not shown). Presumably, this was because of the
self-association of the heparin II-binding fragments at the higher
concentrations (27). Binding interactions between the heparin
II-binding fragments and the 70-kDa fragments involve the
amino-terminal I1-5 repeats. As shown in Fig.
7, binding of 125I-labeled
amino-terminal fragments to increasing concentrations of adsorbed
heparin II fragments is associated exclusively with the 29-kDa
fragments containing the I1-5 repeats and not with the
40-kDa gelatin binding domain. In the experiments reported in Figs. 6
and 7, binding reactions were carried out at pH 6.6 because binding was
considerably enhanced at this pH compared with the binding seen at pH
7.4 (data not shown).

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Fig. 6.
Binding of recombinant and proteolytically
derived III12-14-containing peptides to adsorbed 70-kDa
fragments. Microtiter wells that had been coated with the
indicated amounts of the 70-kDa fragment were labeled for 4 h at
pH 6.6 and 37 °C with 0.86 pmol/well 125I-labeled
recombinant III12-14 peptide (2.7 × 105
cpm/well) ( , ) or 0.57 pmol/well 125I-heparin
II-binding proteolytic fragments (4.6 × 105
cpm/well)( , ) in the absence (filled symbols) and in
the presence of 240 pmol/well unlabeled 50-kDa fragments (open
symbols). Data represent means of triplicate measurements. S.E.
(data not shown) were all <0.8 fmol/well. Binding of the recombinant
fragment was normalized to the input of the proteolytic fragment.
Nonspecific binding to BSA-coated wells was subtracted.
Curves are third order regression lines.
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Fig. 7.
Comparison of 29-, 40-, and 70-kDa
amino-terminal fragments binding to adsorbed heparin II-binding
fragments. 125I-Labeled 29-kDa (18.3 nM or
2.3 × 105 cpm/well) ( ), 70-kDa fragments (11.9 nM or 5.3 × 105 cpm/well) ( ), or
40-kDa gelatin-binding fragments (12.3 nM or 3.7 × 105 cpm/well) ( ) were incubated at pH 6.6 for 4 h
at 37 °C in microtiter wells coated with increasing concentrations
of heparin II-binding fragments. The data have been normalized to a
ligand input of 10 nM, and they represent the means of
triplicate measurements. Bars indicate the S.E.
Curves are third order regression lines.
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Adsorbed 70-kDa fragments clearly bound recombinant and proteolytically
derived III12-14-containing peptides in preference to
other fibronectin fragments such as the 44-, 75-, or 40-kDa fragments
(Fig. 8A). Such preferential
binding was not merely an artifact caused by denaturation of the
adsorbed 70-kDa fragment, but could also be demonstrated in solution.
As shown in Fig. 8B, soluble biotinylated 70-kDa fragments
bound the recombinant and the proteolytically derived
III12-14-containing peptides two or three times more
efficiently than they bound the 44-, 75-, or 40-kDa fragments, both in
the absence and the presence of a homobifunctional cross-linker
(BS3). These studies leave little doubt that direct
I1-5-III12-14 interactions contribute to the
inhibitory effects of the heparin II-binding fragments. The modest
preferential binding interactions between the heparin II and 70-kDa
fragments seen in solution, however, suggest that direct binding
interactions cannot readily account for the fact that the inhibitory
effect of the heparin II fragments may be 20 times larger than those of
the 44-kDa fragments (see Fig. 5). Thus, the III12-14
repeats may also affect binding interactions of the I1-5
repeats by other mechanisms.

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Fig. 8.
Binding interactions between biotinylated
70-kDa fragments and various other fragments of fibronectin.
Binding reactions were done in a solid phase assay (panel A)
or in solution (panel B). In panel A, the
proteolytically derived heparin II fragment (7.4 × 105 cpm/pmol), the recombinant III12-14
peptide (3.3 × 105 cpm/pmol), the 44-kDa fragment
containing the III1 repeat (4.8 × 105
cpm/pmol), the 75-kDa cell adhesion fragment (8.1 × 105 cpm/pmol), or the 40-kDa gelatin-binding fragment of
fibronectin (8.2 × 105 cpm/pmol) was bound to
biotinylated 70-kDa fragments (30 µg/ml) absorbed to microtiter
wells. Binding was done for 4 h at 37 °C in PBS, pH 7.4, containing 2 mg/ml BSA. Binding to uncoated BSA-blocked wells was
subtracted from the data. Nonbiotinylated 70-kDa fragments (adsorbed at
30 µg/ml) bound the various ligands in exactly the same way as the
biotinylated substrate (data not shown). In panel B, soluble
biotinylated 70-kDa fragments were incubated with the various
125I-fibronectin fragments as described under "Materials
and Methods." Binding was measured in the presence (hatched
bars) and the absence (open bars) of the cross-linker
BS3. In both panels A and B, the data
represent the means of triplicate measurements. Bars
indicate the S.E.
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mAbs IST-2 and 9D2 Inhibit Binding of Amino-terminal Fragments of
Fibronectin to the 160-kDa Substrate but Not to Cells--
As shown in
Figs. 3-5, maximal inhibition of the binding of
125I-fibronectin and 125I-labeled 70-kDa
fragments by either the heparin II or the 44-kDa fragments does not
plateau and may not be limited at all (note a possible exception in
Fig. 3,
). This suggests that the inhibitory effects of the
III12-14 and III1 repeats may not be
independent of each other. To examine this possibility, we compared the
roles of the III12-14 and III1 repeats in the
binding of 125I-labeled 70-kDa fragments with the 160-kDa
substrate using anti-III13-14 (IST-2) and
anti-III1 (9D2) monoclonal antibodies.
As shown in Fig. 9, the mAb IST-2
inhibited binding of 70-kDa fragments in a
concentration-dependent manner by at least 80% and in some
experiments by >90% (Fig. 9;
). In contrast, mAb C6F10, recognizing the III8-10
repeats, inhibited binding by no more than 20% over a wide range of
concentrations (Fig. 9;
). The difference is even more striking
because at equal IgG concentrations mAb C6F10
binds to the 160-kDa fragment at least 100-fold more efficiently than
mAb IST-2 (data not shown). Nonimmune mouse IgG is noninhibitory in
this system (Fig. 9;
). Apparently, mAb IST-2 interferes with
binding interactions of the type I1-5 repeats because it
inhibits binding of 125I-labeled 29-kDa fragments as
efficiently as it inhibits binding of 125I-labeled 70-kDa
fragments (data not shown). If mAb 9D2 was used, binding of the 70-kDa
ligand to the 160-kDa substrate was also blocked in a
concentration-dependent manner by > 90% provided that the antibody concentrations were increased
10-fold over the effective IST-2 concentrations. Thus, either of the antibodies could substitute for the other (see below, Fig. 11,
).

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Fig. 9.
Inhibition of binding interactions between
125I-labeled 70-kDa fragments and adsorbed 160-kDa
substrate by mAb IST-2. Microtiter wells, which had been coated
with 5.3 ( , ) or 3.2 pmol/well ( ) of the 160-kDa cell adhesion
fragment, were incubated for 4 h at 37 °C with mAbs IST-2
( ), C6F10 ( ), or with nonimmune mouse IgG
( ) at the indicated IgG concentrations. Labeling with the
125I-labeled 70-kDa fragment (1.8 × 105
cpm/well or 0.1 pmol/well) was done during the last hour. All data
represent specific ligand binding normalized to specific binding
measured in the absence of antibodies (0.8 fmol/well; nonspecific
binding was 0.2 fmol/well). Points are means of five ( , ) or two
( ) independent determinations. Bars represent the S.E.
The smooth curve is a third order regression line.
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When cycloheximide-treated cells were plated on the 160-kDa substrate,
binding of 125I-labeled 70-kDa fragments in the absence of
the antibodies was equal to the binding to the substrate alone
(e.g. in Fig. 10, specific binding was 0.5 fmol/well in the presence as well as in the absence of
cells), or it was enhanced up to 3-fold (12). Surprisingly, none of the
binding of 125I-labeled 70-kDa fragments to these cell
layers was inhibited by either the mAb IST-2 (Fig. 10), the mAb 9D2
(data not shown), or when both antibodies were used together (Fig.
11;
), even though both antibodies
could inhibit binding to the substrate (Fig. 11,
). Instead, binding
to cells was enhanced by 30-40% in the presence of either the mAb
IST-2 (0.1-10 µg/ml) or the mAb 9D2 (data not shown). Binding to
cell layers was unaffected by the mAb C6F10 or
nonimmune IgG (Fig. 10). In all of these experiments, antibodies were
present from the time cells were plated to the end of the labeling
period. This procedure ensured that the antibodies had access to the
160-kDa substrate in the confluent cell layers (12). In conclusion,
binding of 70-kDa fragments to cell surfaces appears to be independent
of the type III12-14 and the type III1 repeats, whereas each of these domains proved to be critical for nearly
all of the binding of the 70-kDa fragment to the 160-kDa substrate.

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Fig. 10.
Binding of 125I-labeled 70-kDa
fragments to cell layers is not inhibited by mAb IST-2.
Suspensions of cycloheximide-treated cells were mixed with mAbs IST-2
( ), C6F10 ( ), or with nonimmune mouse IgG
( ) at the indicated IgG concentrations. Cells were plated in
microtiter wells that had been coated with 3.2 pmol/well of the 160-kDa
cell adhesion fragment of fibronectin. Cell attachment and spreading
were unaffected by any of the antibodies (not shown). After 3 h,
the confluent monolayers (3 × 104 cell/well) were
labeled for 1 h with the 125I-labeled 70-kDa fragment
(2 × 105 cpm/well or 0.1 pmol/well). In the same
experiment, cell-independent binding of the 70-kDa fragment to the
160-kDa substrate was measured in the presence of mAb IST-2 as
described in Fig. 3 ( ). All data are means of triplicate
measurements of specific binding normalized to specific binding seen in
the absence of antibodies (0.4 fmol/well in the presence or absence of
cells). Bars represent the S.E. Smooth curves are
third order regression lines.
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Fig. 11.
Binding of 125I-75-kDa fragments
to cell layers is not inhibited by the mAbs IST-2 and 9D2. Binding
of the 125I-labeled 70-kDa fragments to cell layers ( )
and to substrate alone ( ) was measured at increasing concentrations
of mAb 9D2 in the presence of a fixed amount of mAb IST-2 (1.6 µg/ml). Suspensions of cycloheximide-treated cells were mixed with
mAb 9D2 and/or mAb IST-2 and plated in culture wells that had been
coated with 3.2 pmol/well of the 160-kDa cell adhesion fragment. Cell
attachment and spreading were unaffected by any of the antibody
treatments (not shown). After 3 h, the confluent monolayers
(3 × 104 cells/well) were labeled for 1 h with
125I-labeled 70-kDa fragments (2 × 105
cpm/well or 0.1 pmol/well). Binding to substrate alone was measured at
the same time. Data represent means of triplicate measurements of
specific binding normalized to specific binding seen in the absence of
antibodies (0.5 fmol/well in the presence or absence of cells).
Bars represent the S.E.
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DISCUSSION |
In this paper we demonstrate that the carboxyl-terminal heparin II
binding domain of fibronectin is involved in the assembly of
fibronectin into fibrils. Using binding assays and immunofluorescence microscopy, we show that proteolytic fragments of fibronectin containing the III12-14 repeats inhibit binding of
fibronectin, or its amino terminus, to cell layers and assembly of
fibronectin into fibrils. Until now, the III1 repeat was
the only type III repeat known to inhibit fibronectin fibril formation
(23, 24). Under physiological conditions, the heparin II-binding
fragments inhibited binding of fibronectin and amino-terminal 70-kDa
fragments to cells
10 fold more efficiently than a 44-kDa
fragment containing the III1 repeat. We conclude therefore
that assembly of fibronectin fibrils involves at least two separate and
distinct type III repeats.
The sequences within the heparin II binding domain which inhibit fibril
formation are unknown. These sequences, like the inhibitory sequences
within the III1 repeat, appear to be cryptic in fibronectin because a 160-kDa fragment of fibronectin containing the
III12-14 and III1 repeats did not inhibit
either the incorporation of fibronectin into the extracellular matrix
or the binding of 70-kDa fragments to absorbed 160-kDa fragments.
The data presented in this paper suggest two alternative mechanisms by
which the heparin II binding domain could inhibit the incorporation of
fibronectin into fibrils. First, the III12-14 repeats
could bind directly to the I1-5 repeats and inactivate the
amino terminus. Second, the heparin II-binding fragments could block
cooperative interactions between the III12-14 and III1 repeats which facilitate binding of the amino
terminus.
Our studies confirm previous studies by Homandberg and Erickson (27)
and show that I1-5 repeats bind preferentially to the
III12-14 repeats, both in solid phase and in solution. This is consistent with the idea that direct
I1-5-III12-14 interactions may contribute to
fibronectin fibrillogenesis. It is also consistent with the idea that
heparin II-containing fragments are inhibitory because they can bind to
the I1-5 repeats in either fibronectin or the 70-kDa
fragment and form a complex that is incapable of binding to cells or
the 160-kDa cell adhesion substrate. It remains uncertain, however, to
which extent direct I1-5-III12-14
interactions can account for the inhibitory effects of the heparin
II-binding fragment because compared with the 44-kDa
III1-containing fragment, III12-14-containing fragments bound soluble 70-kDa fragments only two to three times more
efficiently whereas they could inhibit binding of 70-kDa fragments to
cells or to the 160-kDa substrate
10 times better. This leaves
the possibility that the III12-14 repeats may also
facilitate binding of I1-5 repeats by alternative
mechanisms.
One possible alternative mechanism suggested by our finding is that the
IST-2 as well as the 9D2 antibody could inhibit the binding of the
70-kDa amino terminus to the 160-kDa substrate by
90% even
though these antibodies recognize exclusive epitopes in the type
III13-14 and type III1 repeats, respectively (23, 40). Because this finding seems to be incompatible with the simple
notion that the heparin II domain and the III1 repeat represent two independent I1-5 binding sites, we suggest that binding of the I1-5 repeats to the 160-kDa substrate involves cooperative interactions between the III12-14 and the III1 repeats.
Conceivably, such cooperative interactions might depend on the
III1 and III12-14 repeats coming into close
contact with each other. Thus, binding of type I1-5
repeats would be inhibited by either mAb 9D2 or IST-2, whereas an
antibody like mAb C6F10 (23) would have little
or no effect because it recognizes an epitope in the
III8-11 repeats. Binding of I1-5 repeats
would also be expected to be completely and specifically inhibited by
the heparin II or 44-kDa fragments because they could readily disrupt
the apposition of III12-14 and III1 repeats in
the 160-kDa substrate. Apposition of III1 and
III12-14 repeats might be facilitated by the highly
electronegative decapeptide in fibronectin (residues 722-731) in the
type III2 repeat (41) because this decapeptide, like
heparin, could bind to heparin binding sequences in the
III12-14 repeats (42, 43). In intact fibronectin,
apposition of the III12-14 and III1 repeats
may also involve the binding interaction between the III1 and III15 repeats described recently by Ingham et
al. (44).
Because the heparin II domain falls in between integrin binding sites,
its activity may well be regulated through tensile forces created by
integrin-mediated mechanochemical interactions between the cytoskeleton
and fibronectin dimers (13). Such interactions are thought to promote
reversible unfolding of one or more type III repeats (16) resulting in
the exposure of self-association sites within fibronectin. The
"activated" heparin II domain could then bind directly to the
I1-5 repeats and/or facilitate I1-5 binding
through cooperative interactions with another type III repeat, such as
III1.
Our evidence for direct binding interactions between I1-5
and III12-14 repeats lends further support to the notion that the assembly of a complex fibronectin matrix requires multiple I1-5 binding sites in fibronectin. As predicted before (5, 45), single binding interactions of the I1-5 repeats can only give rise to uniform fibrils, whereas additional interactions would also promote fibril thickening and branching and thereby add
considerable complexity and flexibility to a fibronectin matrix. The
availability of multiple I1-5 binding sites in fibronectin could also reconcile conflicting views of the role of the
III1 repeats in matrix assembly. For example, it now seems
possible that the assembly of fibronectin dimers lacking the
III1-7 repeats (26) into fibrils could proceed entirely
via I1-5-III12-14 interactions.
Although our studies clearly attest to the importance of
III12-14 as well as the III1 repeats in
I1-5-dependent fibronectin-fibronectin binding
interactions during fibrillogenesis, it is also evident that binding of
the 70-kDa amino terminus to cell layers can occur independently of
these two type III domains. As we have shown before (12), the 70-kDa
fragments of fibronectin can bind to cycloheximide-treated cells in the
absence of any III1 or III12-14 repeats. Such
binding could not be attributed to any residual fibronectin left on the
cell surface because the same cells failed to bind any 70-kDa fragments
when they were plated on collagen or vitronectin. In addition, we have
shown here that mAbs IST-2 and 9D2, even when used together, fail to inhibit binding of 70-kDa fragments to cycloheximide-treated cells. The
inability of these antibodies to block binding of the amino terminus to
cell layers cannot be attributed to an inaccessibility of their
epitopes because these antibodies were added at the time cells were
plated and thus had ample access to the substrate (12). Interestingly,
the antibodies were not entirely ineffective in cell layers but
actually enhanced binding of 70-kDa fragments by ~40%. Such
enhancement is seen not only in our cycloheximide-treated cells, but
also in normal fibroblast cultures treated with anti-III1 Fab
fragments (23). The fact that the antibodies fail to block binding
of the amino terminus to cell layers may seem in conflict with our
observation that fibronectin fragments can inhibit such binding
effectively. In this case, however, the fragments, unlike the
antibodies, could simply interact with the ligand in solution.
In conclusion, the heparin II domain in fibronectin, like the
III1 repeat, plays an important role in fibronectin
fibrillogenesis but does not appear to participate in all
I1-5-dependent steps in matrix assembly. The
III12-14 repeats may participate in fibrillogenesis by
binding directly to the amino terminus and/or facilitate binding of the
amino terminus through cooperative interactions with the
III1 repeat.