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INTRODUCTION |
The fibronectins (FNs)1
comprise a group of extracellular matrix proteins that mediate cell
adhesion, migration, proliferation, and differentiation (1). FNs play
significant roles in embryonic development and are prominent components
of the provisional matrix following tissue injury in adults (2, 3). The
fundamental importance of the FNs is substantiated by the observation
that homozygous mutations in either the FN gene or in the
5 integrin, a FN-specific receptor, are lethal (4, 5).
The FNs are disulfide-linked, dimeric glycoproteins with structural
domains that bind cells, collagen, proteoglycans, and fibrin. Each FN
consists of homologous repeats, either type I, II, or III. Individual
type III repeats within FN exhibit high sequence similarity between
species (greater than 90% identity (6)). Despite variations in protein
sequence identity between different type III repeats within FN
(20-40% (6)), these repeats show a high degree of structural homology (7-11). X-ray crystallographic studies demonstrate that each type III
repeat consists of two
sheets, made up of four strands (G, F, C,
C') and three strands (A, B, E) respectively, folded into a
sandwich (7). This structural arrangement is also conserved in other
proteins, including growth hormone (12), tenascin (13), neuroglian
(14), tissue factor (15), and chitinases (16).
Diversity in the FNs occurs by alternative splicing in two type III
repeats termed EIIIA (or ED-A) and EIIIB (or ED-B) and one
non-homologous repeat called V (or IIICS) (1). The EIIIA and EIIIB
segments are either entirely included or excluded, whereas the V region
may be included, excluded, or partially included in FN. An additional
splicing variant lacking the V region, the 10th type I repeat
(I10), and the 15th type III repeat (III15) has
recently been reported (17). Despite the present understanding of the
structures within type III repeats and the mechanisms controlling the
alternative splicing of FN mRNA (18-26), the roles of the
alternatively spliced domains on overall FN protein structure and
cellular function remain unclear. Alterations in FN structure, which
occur as a consequence of splicing, may influence the function of
adjacent domains. For example, insertion of an alternatively spliced
domain may change the conformation of the adjacent type III domains by re-adjusting interdomain rotations and tilts (7, 13). This is supported
by the observation that a small rotation occurs between FN-III9 and FN-III10 and places the RGD loop of
FN-III10 and the synergy site of FN-III9 on the
same surface of the FN molecules (7). Alternatively, the inclusion of
an extra type III domain could have longer range effects on the overall
conformation of the FN molecule by rotating the N-terminal portion of
the FN molecule relative to the C-terminus. Such a structural change
could enhance the accessibility of a functional domain, such as the RGD
loop, in the cell-binding domain or of sites involved in fibril
assembly (8, 27, 28). Finally, an alternatively spliced segment may interact directly with cells.
The FNs display a wide range of physiological functions, which have
been mapped to specific segments of FN. In some instances, the reacting
sequences have been localized to short stretches of amino acids. For
example, synthetic peptides that include the Arg-Gly-Asp (RGD) sequence
from the FN-III10 block interactions between FN and
integrins (29, 30). Although identified first in FN, the RGD sequence
has been identified in numerous proteins and mediates cell adhesion
(31). However, key peptide sequences often function best in the context
of the whole type III repeat. For example, a short stretch of amino
acid residues (Pro-His-Ser-Arg-Asn, PHSRN) in the 9th type III domain
(FN-III9), termed the synergy site, of FN has been found to
enhance cell adhesion to the RGD sequence in FN (32-35).
The function of the EIIIA and EIIIB sequences is largely unknown. These
extra domains are present at specific stages of embryonic development
and organogenesis (36-41), whereas most normal adult tissues express
much lower amounts of EIIIA and EIIIB (42). However, during specific
pathological conditions, such as wound healing (43, 44), lung, liver,
and kidney fibrosis (45-47), vascular intimal proliferation (48, 49)
and cardiac transplantation (50), the expression of EIIIA and EIIIB
domains is significantly up-regulated. Several lines of evidence,
including studies utilizing the mAb IST-9 to block function, have shown
that the EIIIA segment may play roles in promoting cell adhesion,
regulating cell proliferation, and in promoting the differentiation of
lipocytes and fibroblasts into myofibroblasts (27, 47, 51-54). Another
EIIIA-specific mAb, DH1, has recently been shown to block
chondrogenesis in chicken embryos (55). The differentiation of
myofibroblasts is observed during morphogenetic processes, wound
healing, organ fibrosis, and the stromal reaction to carcinomas
(56-58). Uncontrolled myofibroblast differentiation has been suggested
to be the leading cause of several fibrotic diseases as well (59).
Under pathological conditions, TGF-
1 has been shown to be a potent
inducer of the myofibroblast phenotype and can increase the expression
of collagen, FN, and certain integrins by fibroblasts (60-62). Tissue
fibrosis may result from the disregulation of TGF-
expression (63).
Recent data demonstrate a potential role for the EIIIA segment of FN in
the regulation of TGF-
's action on dermal fibroblasts (53).
The observation that an EIIIA-specific mAb (IST-9) and a soluble form
of the EIIIA segment markedly reduce TGF-
-mediated myofibroblast
differentiation suggests that structural features of EIIIA are
important in fibrosis. Here, we present an epitope map of EIIIA for two
function-blocking mAbs, IST-9 and DH1, and another EIIIA-specific mAb,
3E2. To do so, we carried out systematic mutation of bacterial EIIIA
fusion proteins, including deletion mapping and alanine-scanning
mutagenesis. Our results clearly identify two amino acid residues of
the EIIIA domain that are located at a loop region between two
strands and constitute the IST-9 and DH1 epitopes. These data provide
the foundation for the identification of a functional motif in the
EIIIA domain.
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EXPERIMENTAL PROCEDURES |
Materials--
Restriction enzymes were obtained from Promega
(Madison, WI), Roche Molecular Biochemicals, and Amersham Pharmacia
Biotech. The anti-EIIIA mAbs, IST-9 (64), DH1 (65), and 3E2, were
purchased from Harlan Bioproducts (Indianapolis, IN), Locus Genex
(Helsinki, Finland), and Sigma, respectively. BCA Protein Assay Kit,
Gel Code Blue, SuperBlock, Super Signal, and horseradish
peroxidase-conjugated goat anti-mouse IgG were from Pierce. GST gene
fusion vectors and U.S.E. Mutagenesis kit were obtained from Amersham
Pharmacia Biotech. Qiagen Plasmid Maxi kit, QIAprep 8 Miniprep kit,
QIAprep 8 M13 kit, and QIAquick Gel Extraction kit were from Qiagen
Inc. (Valencia, CA). Glutathione (reduced form) and
glutathione-immobilized agarose were obtained from Sigma. AEBSF
hydrochloride was from Calbiochem. Synthesized oligonucleotides were
purchased from Life Technologies, Inc. All other reagents were at least
reagent grade and obtained from standard suppliers.
Polymerase Chain Reaction (PCR) and Subcloning of the EIIIA
cDNAs--
Full-length wild type rat EIIIA cDNA was amplified
using PCR in a 50-µl reaction mixture containing 1 ng of a rat
cDNA (clone 74T, kindly provided by R. O. Hynes, Massachusetts
Institute of Technology) (6), 50 mM KCl, 10 mM
Tris-HCl, pH 8.3, 1.5 mM MgCl2, and 0.001%
(w/v) gelatin, 200 µM each dNTP, 0.5 µM
primers (EIIIA-sense and EIIIA-antisense, Table
I), and 2.5 units of Taq DNA
polymerase (Perkin-Elmer). The samples were placed in a DNA thermal
cycler 480 (Perkin-Elmer) programmed for a temperature-step cycle of 94 (45 s), 55 (1 min), and 72 °C (1 min) for 30 cycles. After the final
cycle, the reaction was maintained at 72 °C for an additional 7 min.
The final reaction products were resolved on a 1% agarose gel
containing ethidium bromide (0.5 µg/ml). Amplified DNA fragments were
purified from the gel, digested with EcoRI, and subcloned
into the pGEX-2T vector. The recombinant plasmid (EIIIA-pGEX-2T) was
isolated from liquid bacterial cultures using QIAfilter Plasmid Maxi
kit or QIAprep Spin Miniprep kit and subjected to DNA sequencing.
Various deletion constructs of rat EIIIA cDNA were generated by
using PCR. The amplification was performed in a 25-µl reaction volume
containing 1 ng of EIIIA-pGEX-2T plasmid DNA, 10 mM
Tris-HCl, pH 9.0, 50 mM KCl, 0.1% Triton X-100, 1.5 mM MgCl2, 200 µM each dNTP, 0.5 µM sense strand and antisense strand primer, and 2.5 units of Taq DNA polymerase (Promega, Madison, WI). The
sequences of 5' and 3' primers are listed in Table I. Constructs were
then subcloned and propagated as described above. Deletion of EIIIA cDNA was confirmed by DNA sequencing analysis.
Site-directed Mutagenesis of the EIIIA Expression
Plasmid--
Point mutations were selectively introduced into the wild
type rat EIIIA expression construct, EIIIA-pGEX-2T, by a procedure called unique site elimination (66) using the U.S.E. Mutagenesis kit.
Synthesized oligonucleotides (300 pmol each; Table I) were phosphorylated in a 30-µl reaction mixture containing One-Phor-All Buffer PLUS (10 mM Tris acetate, 10 mM
magnesium acetate, 50 mM potassium acetate, pH 7.5), 1 mM ATP, and 10 units of T4 polynucleotide kinase.
Phosphorylation reactions were incubated (37 °C, 30 min), terminated
by heating to 65 °C for 10 min, and used directly in the mutagenesis reactions.
Mutagenesis mixtures consisted of 0.025 pmol of plasmid DNA, 1.25 pmol
of U.S.E. selection primer (Table I), 1.25 pmol of target mutagenic
primer, and One-Phor-All Buffer PLUS in a total volume of 20 µl.
Following incubation at 100 °C for 5 min, the reaction mixtures were
cooled in ice for 5 min and then incubated at room temperature for 30 min. Subsequently, 7 µl of Nucleotide Mix (2.86 mM each
dATP, dCTP, dGTP, and dTTP, 4.34 mM ATP, 1.43× One-Phor-All Buffer PLUS) and 3 µl of Reaction Mix (0.83-1.17 kilounits/ml FPL T4 DNA ligase, 0.83-1.67 kilounits/ml FPL T4 DNA
polymerase, and 0.2-0.28 mg/ml T4 Gene 32 protein) were added into the
reaction mixtures. The reaction mixtures were further incubated at
37 °C for 1 h, followed by heating at 85 °C for 15 min.
Before being transformed into bacterial host cells, the mutagenesis reaction mixture was digested with 10 units of PstI in a
final volume of 50 µl, and transformation was carried out as
described by the manufacturer. Transformed cells were incubated in 4 ml of L-broth with 100 µg/ml of ampicillin at 37 °C overnight with shaking at 250 rpm. Plasmid DNAs were prepared from the overnight cultures using QIAprep 8 Miniprep kit (Qiagen) and were subjected to a
second restriction digestion by PstI. One µg of digested DNA from the second round of restriction enzyme selection was transformed into Escherichia coli competent cells, followed
by plating of the transformed cells onto LB plates containing 100 µg/ml ampicillin. The plates were incubated at 37 °C overnight, and individual transformant colonies were selected to prepare plasmids
for sequencing to verify the presence of the desired point mutations.
DNA sequencing was performed on subcloned fragments in multicopy
plasmids, PCR amplimer fragments using Taq polymerase in a
dideoxy dye-terminator reaction (67). The sequencing reactions were
resolved on an Applied Biosystems ABI 377 Sequencer (DNA Sequencing
Core Facility, Department of Molecular Biology, Massachusetts General
Hospital). The sequencing results were assembled and analyzed using the
GCG Software Package (version 9.0, the University of Wisconsin and
Genetics Computer Group), which includes "Pileup," "Bestfit,"
and "Pretty."
Production and Purification of Bacterially Expressed EIIIA
Proteins--
A 500-ml L-broth culture containing 100 µg/ml ampicillin was inoculated with 50 ml of an overnight culture of
the recombinant E. coli strain and grown at 37 °C for
2 h. Protein expression was then induced by the addition of
isopropyl-
-D-thiogalactoside to a final concentration of
1 mM. A protease inhibitor, AEBSF, was included in the
induction culture medium with a final concentration of 1 mM. After 4 h induction, cells were harvested by
centrifugation at 5,000 × g for 30 min. The cell
pellets were washed with PBS and used for protein purification or
stored at
80 °C until ready to use.
For protein purification, cell pellets were resuspended in 10 ml of PBS
with 1 mM AEBSF (PBS/AEBSF) and sonicated on ice using a
Sonifier 450 (Branson Ultrasonics Corp., Danbury, CT) with microtip at
full power for 1 min. Subsequently, 100 µl of Triton X-100 was then
added into the sonicated suspension, and incubation at 4 °C was
carried out for 30 min. Cell debris was removed by centrifugation at
14,000 × g for 30 min. The clarified supernatant was
collected and mixed with 1 ml of glutathione-agarose (50% slurry
pre-equilibrated with PBS/AEBSF) at 4 °C for 2 h. Protein-bound
agarose beads were collected by centrifugation at 1,000 × g for 1 min and washed with 10 ml of PBS/AEBSF 5 times.
Washed beads were mixed with 1 ml of elution buffer (25 mM
glutathione, 120 mM NaCl, and 100 mM Tris-HCl,
pH 8.0) at 4 °C for 10 min to elute the GST fusion proteins,
followed by centrifugation at 1,000 × g for 1 min. The elution step was repeated twice, and the eluted fractions were pooled
and dialyzed against PBS. Purified proteins were quantitated using the
BCA protein assay reagent and stored at
80 °C.
SDS-Polyacrylamide Gel Electrophoresis and Western Blot
Analysis--
Purified recombinant EIIIA proteins (10 µg or as
specified elsewhere) were mixed with an equal volume of 2× SDS sample
buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 0.005%
bromphenol blue, 20% glycerol, 2% dithiothreitol, and 5%
-mercaptoethanol) and boiled at 100 °C for 5 min. Samples were
then loaded onto a pre-cast Tris glycine polyacrylamide gel (10%)
(Novex, San Diego, CA) and resolved with 25 mA/gel in an XCell II
Mini-Cell system (Novex) containing SDS running buffer (24 mM Tris base, 192 mM glycine, and 0.1% SDS)
(68). Gels were then processed either for Western blot analysis or
stained using Gel Code Blue for 1 h at room temperature, followed
by extensive washes in distilled water.
For Western blot analysis, separated proteins were transferred
electrophoretically to a polyvinylidene difluoride membrane (Bio-Rad)
for 2 h at 32 V using Xcell II Blot Module (Novex) filled with
Transfer Buffer (190 mM Tris, 25 mM glycine,
and 20% methanol). The membrane was treated with Blocking Buffer (5%
non-fat dry milk, 0.05% Tween 20 in PBS) at room temperature
(overnight). Following brief washes, the membrane was reacted with an
EIIIA-specific monoclonal antibody (1:500 diluted in SuperBlock) for
1 h at room temperature and washed with PBST (0.05% Tween 20 in
PBS) three times (5 min each wash). Subsequently, the membrane was
incubated with horseradish peroxidase-conjugated goat anti-mouse IgG
(Pierce) (1:5000 in SuperBlock), followed by three washes in PBST. The immunoblot was then incubated with Supersignal chemiluminescence substrate for 10 min and exposed to a phosphor cassette. Images of the
blots and gels were processed with the Molecular Image System GS-525
and the Fluor-S MultiImager, respectively, using Multi-Analysis
software version 1.1 (Bio-Rad).
Enzyme-linked Immunosorbent Assays--
The reactivity of mutant
EIIIA-GST fusion proteins was tested by enzyme-linked immunosorbent
assays (ELISA). Microtiter plates (96-well) were coated with 100 µl/well of 10 µg/ml purified EIIIA-GST proteins in coating buffer
(100 mM NaHCO3, pH 8.6) at 4 °C in a
humidified chamber overnight. The plates were briefly rinsed in washing
buffer (0.1% Tween 20 in PBS), blocked with 300 µl/well of blocking
buffer (3% bovine serum albumin in coating buffer) at 37 °C for
1 h, and rinsed again in washing buffer. Serial dilutions (1:60-1:2 × 1010) of monoclonal antibodies (mAbs)
were prepared in washing buffer. Diluted mAbs (100 µl) were added to
the wells, and the reactions were incubated at room temperature for
1 h. Wells were washed as above, incubated (1 h, room temperature)
with diluted horseradish peroxidase-conjugated goat anti-mouse IgG
(1:5000 in washing buffer, 100 µl/well), washed again, and incubated
(30 min, room temperature) with substrate solution (200 µl/well, 0.4 mg/ml o-phenylenediamine dihydrochloride, 0.4 mg/ml urea
hydrogen peroxide, and 50 mM phosphate-citrate buffer). The
absorbance of individual reactions was then measured (420 nm, ThermoMax
microplate reader, Molecular Devices). Titer evaluations of antibody
dilutions were done by logarithmic curve fitting. The x
value corresponding to 50% of the highest absorbance along the
sigmoidal curve is defined as titer for the reactivity of mAbs to
specific EIIIA proteins.
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RESULTS |
Protein Sequence Comparison and Antibody Reactivities Suggest That
the C-C'-E Segment of EIIIA Encompassing the His44 Residue
Constitutes Epitopes--
Protein sequences, derived from mRNAs,
for human, mouse, rat, chicken, and frog EIIIA segments show extensive
sequence similarity (Fig. 1A).
All are 90 amino acids in length, and the consensus sequence for these
five species is 70% conserved. The EIIIA protein sequences for mouse,
rat, chicken, and frog display 96.7, 94.4, 85.6, and 80% identity,
respectively, to the human EIIIA protein (Fig. 1B). All
sequences conform to a domain structure in which seven
-strands
(denoted by A, B, C, C', E, F, and G) are conserved in the type III
repeat crystal structure (7).

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Fig. 1.
Sequence comparison of the EIIIA segment of
FN from different species. A, peptide sequences of
EIIIA proteins from various sources (and corresponding
GenBankTM accession numbers), including human (X02761)
(81), mouse (P11276) (82), rat (X15906) (83), chicken (P11722) (40),
and Xenopus (M77820) (84). Amino acids conserved in all five
species are shown in capital letters. A consensus sequence
is included in the alignment, and the dashes indicate the
positions where the aligned amino acids are only partially conserved.
Rectangular boxes and letters above each box
highlight the structural domains of anti-parallel sheets (7). The
arrow denotes His44 which is conserved in human,
rat, and chicken EIIIA but not in mouse and Xenopus.
Underlined amino acids, Asp53 and
Glu54, of mouse EIIIA sequence that we obtained differ from
the Glu53-Asp54 in the published mouse EIIIA
sequence (82). B, a dendrogram of the aligned polypeptide
sequences in A. The numbers represent the percent
identity of the corresponding sequence relative to the human EIIIA.
Optimized multiple sequence alignment was generated using the Pileup
program.
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The mAb IST-9, raised against human cellular fibronectin (cFN),
specifically recognizes the EIIIA segment in rat and human cFN (64).
This mAb exhibits function-blocking activities in these species (47,
53). We tested the reactivity of IST-9 and 3E2 against the EIIIA
segment in chicken, frog, and mouse FN by either immunofluorescence,
immunoblotting, or ELISA. IST-9 reacted with chicken cFN (data not
shown), but not appreciably with either mouse or frog EIIIA (see
below). Likewise DH1, which binds chicken FN (55), also does not react
with mouse or frog EIIIA (see below). 3E2 reacts with chicken (data not
shown) and mouse (41) but not frog EIIIA (see below). Comparisons of
these reactivities with the protein sequences (Fig. 1) revealed that an
amino acid residue (His44) within the EIIIA segment is
conserved in rat, human, and chicken but is not in either mouse or frog
(arrow, Fig. 1). As a preliminary test of reactivity with
mouse EIIIA, we prepared a mutation that replaces the conserved
His44 residue of rat EIIIA segment with the arginine found
in mouse EIIIA. This resulted in a significant reduction of IST-9
reactivity (Fig. 2). A potential
polymorphism at residue Glu54 suggests that this residue
might also play a role. Based on the sequence comparison and our
immunological evidence, we hypothesized that the conformational domain
C-C'-E of rat EIIIA encompassing the His44 residue is
crucial for constituting the IST-9 epitope.

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Fig. 2.
Immunochemical studies of the EIIIA segment
of FN. Western blot of bacterial EIIIA fusion proteins reacting
with IST-9. Lane 1, wild type rat EIIIA protein; lane
2, a mutant rat EIIIA protein (rat EIIIA-H44R) in which the
conserved His44 residue found in rat FN was replaced by an
arginine present in mouse FN. Proteins (3 µg per lane) were loaded,
resolved, and blotted as described under "Experimental Procedures."
The His to Arg mutation significantly reduced the IST-9 reactivity of
the rat EIIIA-H44R mutant protein.
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The "Native" Conformation of Full-length Rat EIIIA Protein Is
Required for Its IST-9 Reactivity--
To generate sufficient material
for mapping the IST-9 binding epitope, the wild type sequence of rat
EIIIA was cloned into a bacterial expression vector, pGEX-2T, and then
used to generate various deletion constructs of rat EIIIA by PCR (Fig.
3). All of these deletion mutants retain
the hypothesized epitope sequence of domain C-C'-E. Wild type and
deletion mutant constructs were expressed as GST fusion proteins in
E. coli and purified by glutathione-affinity chromatography.

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Fig. 3.
Deletion mutants of rat EIIIA protein.
Map of various N-terminal, C-terminal, or internal peptides
(arrows) derived from wild type (WT) rat EIIIA
(rEIIIAWT, 90 amino acids). Deletion constructs were
generated by PCR and subcloned into the pGEX-2T vector as described
under "Experimental Procedures." Arrows indicate the
length of individual deletion constructs relative to the wild type
sequence that is shown on the top of the figure. A
solid circle above the peptide sequence of rEIIIA-WT
highlights the conserved His44. The amino acids included in
truncated mutant rat EIIIA proteins are numbered.
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Antibody reactivities of wild type rat EIIIA and the derived deletion
mutants were tested by ELISA, and the dilution yielding 50% binding
(titer) for each mAb was determined. Three EIIIA-specific mAbs, IST-9,
3E2, and DH1, were included in these analyses. When reacted with wild
type rat EIIIA, the titers of IST-9, 3E2, and DH1 were 5 × 104, 4 × 103, and 1 × 104, respectively (Fig. 4).
However, unlike the strong reactivity exhibited by rat EIIIA toward
these mAbs, none of the six deletion mutants displayed any detectable
antibody reactivity (insets, Fig. 4). These results ruled
out the possibility that the C-C'-E domain alone of the EIIIA segment
was sufficient to constitute the antibody recognition epitope.
Nevertheless, the amino acid sequence of C-C'-E domain could still be
crucial for the reactivity with mAbs and may need to be maintained in a
specific conformation. This antibody-reactive conformation may only
react with these mAbs when the full-length EIIIA polypeptide sequence
is intact.

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Fig. 4.
The reactivity of rat EIIIA-GST deletion
mutants with three monoclonal antibodies. The deletion mutants
were tested for reactivity in ELISAs with monoclonal antibodies as
described under "Experimental Procedures." The sigmoid plot in each
panel represents the reactivity of wild type rat EIIIA
(rEIIIA-WT) with IST-9 (A), 3E2 (B),
or DH1 (C). Dilution folds are expressed in log
scale. Bar graph insets in each panel illustrate the titer
(in percentage) of each deletion protein relative to rEIIIA-WT. The
points of OD420 at various dilution folds
represent the average of quadruples; standard deviations greater than
0.1 OD are shown as bars.
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Thr35, Tyr36, Ser37,
Glu40, and Asp41 Residues of Rat EIIIA Are
Important for Maintaining an Optimal Conformation for Antibody
Binding--
To test whether and to what extent the C-C'-E domain of
EIIIA protein is crucial for antibody reactivity, we generated a series of rat EIIIA double mutants in which two adjacent amino acids were
simultaneously replaced by alanines and then tested for reactivity to
mAbs by ELISA. In some preliminary screenings, antibody reactivities were determined by Western blotting. We found that most of the double
mutants retain some or all of the antibody reactivity of the wild type
rat EIIIA protein (Table II). Among these
was a double mutant, rat EIIIA-D53A/E54A, representing a potential
polymorphism in mouse EIIIA (Fig. 1 and Table II). No loss of
reactivity to IST-9 and 3E2 and a minimal effect on DH1 binding were
observed (Fig. 5, Table II), indicating
that His44 rather than Glu54 was most important
for reactivity. However, some double mutants sharply reduced antibody
reactivity (Fig. 5). One of these mutants, rat EIIIA-V34A/T35A,
completely lost its ability to react with all three mAbs tested.
Another double mutant, rat EIIIA-Y36A/S37A, displayed a complete loss
of reactivity to IST-9 and 3E2 but still retained low (~1%)
reactivity to DH1, as compared with wild type EIIIA. An additional
double mutant, rat EIIIA-E40A/D41A, showed a dramatic reduction in
reactivity to IST-9 (3% of rat EIIIA-WT) and less significant effects
on the reactivities of 3E2 and DH1. On the other hand, rat
EIIIA-P50A/D51A had no significant effect on IST-9 reactivity and only
minor effects on the reactivities of 3E2 and DH1.
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Table II
Epitope mapping of three rat EIIIA-specific monoclonal antibodies using
deleted and mutagenic rat EIIIA proteins
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Fig. 5.
Reaction of mAbs with rat EIIIA double
mutants. Recombinant EIIIA protein was mutagenized simultaneously
at two amino acid residues replacing with alanines. The specificity of
mAbs for mutated rat EIIIA (rEIIIA) fusion proteins was
determined by ELISA, and the titer of each mAb to mutant rat EIIIA was
determined relative to wild type rat EIIIA (rEIIIA-WT).
Solid, gray, and striped bars represent the
relative titer of IST-9, 3E2, and DH1, respectively. Amino acids in the
EIIIA protein are indicated by single-letter code followed by the
residue number representing the mutated position relative to the
beginning of the EIIIA segment.
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The reduction in antibody reactivity exhibited by rat EIIIA-V34A/T35A
and rat EIIIA-Y36A/S37A could result directly from disruption of the
antibody-recognition epitope or indirectly by changing the tertiary
structure of the EIIIA segment, which in turn alters the conformation
of the epitope. To distinguish these possibilities, we prepared single
mutants that contained only one alanine replacement at
Thr35, Tyr36, Ser37,
Glu40 and Asp41, respectively. These were
tested for their reactivities with mAbs. Despite our observation that
the double mutants (rat EIIIA-V34A/T35A, rat EIIIA-Y36A/S37A, and rat
EIIIA-E40A/D41A) showed either a complete loss or a dramatic reduction
in antibody reactivity, we found that the single mutants, rat
EIIIA-T35A, rat EIIIA-Y36A, rat EIIIA-S37A, rat EIIIA-E40A, and rat
EIIIA-D41A, exhibited antibody reactivity levels comparable to those of
wild type rat EIIIA (Fig. 6 and Table
II), implicating the amino acid residues, Thr35,
Tyr36, Ser37, Glu40 and
Asp41, in maintaining an optimal conformation of the
epitope.

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Fig. 6.
Alanine scanning mutagenesis of rat EIIIA and
reactivity to mAbs. Mutagenized rat EIIIA (rEIIIA)
fusion proteins were generated and purified as described under
"Experimental Procedures." The replaced amino acid in each mutant
is indicated. The titer values are given in percentage (%) using
results obtained with wild type rat EIIIA (rEIIIA-WT) as the
100% reference value. Solid, gray, and striped
bars indicate the relative titer of IST-9, 3E2, and DH1,
respectively, as described in Fig. 5.
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Ile43 and His44 of Rat EIIIA Segment Are
Critical for IST-9 Binding--
Site-directed mutagenesis of the rat
EIIIA segment was conducted to generate a panel of single mutants.
These mutants were tested for reactivity to IST-9, 3E2, and DH1 (Fig. 6
and Table II). In one mutant, rat EIIIA-H44R, an arginine residue was
substituted for histidine to mimic the Arg44 (H44R) in the
mouse EIIIA segment. A striking loss of reactivity for IST-9 and DH1
was observed (solid and striped bars, Fig. 6). By
contrast, reactivity with 3E2 was retained (gray bar, Fig. 6), consistent with the reported use of this mAb in
immunohistochemistry of mouse tissues (41).
When alanine-scanning mutagenesis was carried out from
Arg33 through Pro48, the majority of single
mutants retained reactivity to IST-9, 3E2, and DH1 (Fig. 6 and Table
II). As observed for H44R, when alanine was substituted for arginine
(H44A), a marked decrease in the reactivity of mAbs IST-9 and DH1 was
observed. By contrast, 3E2 retained reactivity to H44A as it did to
H44R. When the adjacent isoleucine residue was mutated to alanine
(I43A), IST-9 reactivity was markedly reduced. These results indicated
that Ile43 and His44 were required for IST-9
and DH1 binding. These data also indicated that IST-9, 3E2, and DH1
recognized distinct but overlapping epitopes.
Ile43 and His44 Substitution in Frog EIIIA
Protein Restore Antibody Binding--
To establish whether or not
Ile43 and His44 are sufficient to constitute
the IST-9 binding epitope, we sought to introduce these two residues
into comparable positions of a homologous type III repeat that does not
react with IST-9. We found that neither IST-9, 3E2, nor DH1 reacted
with frog EIIIA (Fig. 7B).
Although frog EIIIA does not react with these mAbs, it displays an 80%
homology in protein sequence to both human and rat EIIIA (Figs.
1B and 7A). Moreover, it is well established that
type III repeats are highly related structurally (7). We prepared a
double (V43I/K44H) and a single (K44H) mutant and observed that IST-9
reacted with these mutants at levels comparable to, or higher than,
wild type rat EIIIA (solid bar, Fig. 7B). Thus,
either Ile43 and His44 together or frog
Val43 with His44 are sufficient to reconstitute
IST-9 reactivity. These results taken with those for rat I43A (Fig. 6)
indicate that specific amino acids at position 43 in conjunction with
His at position 44 are required for IST-9 binding. On the other hand,
both the double (V43I/K44H) and single (K44H) mutants exhibited about
50% of the binding to 3E2 and DH1 relative to wild type rat EIIIA (gray bar and striped bar, Fig. 7B).
In summary, Ile43 and His44 in conjunction with
the conformation of the EIIIA segment, likely involving the C domain,
are critical to the IST-9 epitope and contribute to the epitopes for
3E2 and DH1 (Fig. 8).

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Fig. 7.
Reaction of mAbs with the frog EIIIA fusion
proteins. Wild type frog EIIIA (fEIIIA-WT) fusion
protein with the sequence Val43-Lys44 was
mutated at these two residues (fEIIIA-V43I/K44H) to resemble
rat EIIIA. Alternatively, fEIIIA-WT was mutated only at one position
(fEIIIA-K44H). A, the sequence comparison between
wild type rat EIIIA (rEIIIA-WT) and frog EIIIA is shown in
A where the mutated residues are highlighted. Capital
letters denote the conserved peptide sequences among human, mouse,
rat, chicken, and Xenopus EIIIA proteins. B, the
titer of mAbs to rEIIIA-WT, fEIIIA-WT and mutated fEIIIA fusion
proteins. Solid, gray, and striped bars indicate
the relative titer of IST-9, 3E2, and DH1, respectively. Titer values
are determined by using the results obtained with rEIIIA-WT as 100%
reference value.
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Fig. 8.
The epitopes for IST-9 and DH1 within
EIIIA. The amino acid sequence of wild type rat EIIIA
(rEIIIA-WT) is shown, and the corresponding anti-parallel
-strands are indicated by short heavy lines denoted by
A, B, C, C', E, F, and G, respectively, based on
the x-ray crystallography of human fibronectin (7). The predicted
epitopes of the EIIIA segment that react with two mAbs, IST-9 and DH1,
are highlighted by the shaded box which encompasses the two
critical residues (Ile43 and His44) denoted by
an arrow.
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DISCUSSION |
We report here that two key amino acids, Ile43 and
His44, are a necessary part of the epitope for a
function-blocking monoclonal antibody (IST-9) that reacts with the
EIIIA segment of human, rat, and chicken FN. The Ile43 and
His44 residues lie in a loop between two
strands (C and
C'). The overall conformation of the EIIIA segment, and particularly
that conferred by the C domain, exerts important effects on the IST-9 epitope (Fig. 8). The epitopes for DH1 and 3E2 are also
conformationally dependent and appear to reside in the same loop. The
key residues for DH1 and 3E2 binding overlap with, and yet are distinct
from, those for IST-9. We infer that the loop between the C and C'
strands is critical to the mechanism by which the EIIIA segment regulates cell function.
Emerging evidence suggests that alternatively spliced FNs, prominent in
embryogenesis, are important in the adult tissue response to injury.
The expression of FNs that include the EIIIA or EIIIB segments are
present in rather low amounts in many adult tissues (42). A
stereotypical pattern of response to injury is evident in which FNs
that lack the EIIIA and EIIIB segments (i.e. plasma FN) are
deposited first. Following tissue injury, the EIIIA and EIIIB segments
are included in the FN mRNAs synthesized by wound cells. This
occurs in skin (43, 44), arteries (48, 49, 69), kidneys (70, 71), liver
(47), and heart (50, 72, 73). Such up-regulation occurs both as a
consequence of increases in total FN and increases in the ratios of
inclusion of segments into FN (50, 74). Moreover, the temporal pattern
of appearance of EIIIA and EIIIB differ, suggesting distinct roles for
each segment (47, 50). Splicing in a nonhomologous repeat, the V
region, also occurs following injury. The V95 segment is included in
most FNs, but variations occur in the inclusion of the cell adhesive
portion, termed CS-1, during regeneration of peripheral nerves (75) and
following cardiac transplantation (50).
In addition to data on expression, a growing body of evidence supports
a functional role for the EIIIA segment in modulating the phenotype of
cells. A mAb, IST-9, blocks the conversion of lipocytes to
myofibroblasts (47). Another mAb, DH1, has been shown to block
chondrogenesis (55). Recent studies also demonstrate that IST-9 blocks
the stimulatory activity of TGF-
on smooth muscle cell
-actin
expression during myofibroblast differentiation (53) and that TGF-
controls the expression of extracellular matrix molecules and certain
integrins (60, 61, 76). Thus, disregulation in the TGF-
signaling
pathway may result in tissue fibrosis (63, 77, 78). Because the EIIIA
segment appears to be involved in the TGF-
signaling mechanism in
myofibroblasts, identification of the IST-9 epitope on EIIIA provides
an important first step to probing this mechanism.
In this report we observe that the Ile43 and
His44 residues of EIIIA are necessary for IST-9 binding and
partially constitute the reactive epitopes for DH1 and 3E2. We have
shown that none of the three mAbs react with frog EIIIA unless the
Lys44 is replaced by a His residue found in human, rat, and
chicken FN (Fig. 7). Replacement of the His44 with Arg by
site-directed mutagenesis in rat EIIIA (Fig. 6) or Lys which occurs in
frog FN (Fig. 7) does not suffice for either IST-9 or DH1 binding. In
contrast, mutation at His44 made to either Arg or Ala does
not significantly affect 3E2 binding (Fig. 6). Although full activity
is restored to IST-9 by His44, partial reactivity is
regained with DH1 and 3E2. The adjacent residue, Ile43, is
important to all three mAb epitopes as well. Substitution of an Ala for
Ile43 blocks IST-9 binding completely and markedly reduces
3E2 and DH1 binding (Fig. 6). Reduction in binding was also observed
when Asp41 and Gly42 were mutagenized to Ala.
Gly42 appeared to be quite important for the epitope for
DH1. Taken together, these results indicate that the loop between the C
and C'
-strands encompasses the epitope for these mAbs.
Interestingly, when either the Pro39 or the
Glu45 is replaced by alanine, the IST-9 reactivity is
enhanced. Replacement with alanine at Pro39 may relieve the
steric restriction imposed by the proline residue and hence make the
conformation of the His44-containing C-C' loop more
reactive. On the other hand, the charged Glu45 may provide
electrochemical interaction with the adjacent His44
residue, and this charge-charge interaction could be disrupted by the
replacement of an uncharged residue like alanine, potentially making
the Ile43 and His44 residues more accessible to
IST-9. However, the enhancement of IST-9 reactivity due to mutations
made to Pro39 and Glu45 residues was not
observed with either 3E2 or DH1, indicating that rat EIIIA presents
distinctive epitopes for each mAb.
The reactivities of rat EIIIA deletion constructs to the three mAbs
demonstrate that these epitopes are only active in an intact
polypeptide (Fig. 4). The GST moiety in the purified EIIIA fusion
proteins does not appear to alter the conformation of rat EIIIA because
comparable reactivities are obtained when the GST and EIIIA moieties
are separated by thrombin cleavage (data not shown). The influence of
molecular conformation on rat EIIIA reactivity is further demonstrated
by the antibody reactivities of rat EIIIA double mutants. Mutation of
pairs of amino acids to Ala, either Val34Thr35
or Tyr36Ser37, located in a
-strand upstream
from His44, markedly reduces antibody binding, while these
as single mutants or other mutant pairs tested remain active (Fig. 5,
Fig. 6, and Table II). It is likely that the double mutants at
Val34Thr35 or
Tyr36Ser37 have a greater impact on the
conformation of the
-strand C, which in turn would result in a
significant change in the conformation and antibody reactivity of the
downstream C-C' loop.
The loop regions between
-strands of type III repeats often serve
functional roles, such as cell adhesion and antibody recognition. Indeed, the type III repeat structure is highly conserved in tenascin (13), neuroglian (14), human growth hormone receptor (12), and the
isolated FN-III10 of FN (10, 79), despite relatively low
levels of sequence identity among FN-III repeats. The most notable
sequence differences are observed when comparing the loop regions of
different type III repeats, suggesting that these loops may mediate
functions as well as antibody epitopes (10, 80).
In conclusion, we have demonstrated that the critical residues
mediating antibody recognition by two function-blocking mAbs are
located at the C-C' loop of the EIIIA domain. Because IST-9 is a
function-blocking mAb, these data support a model in which the
functional motif resides within the rat EIIIA domain in the proximity
of the Ile43His44. It is not known yet if
either the EIIIA segment interacts directly with a cell-surface
receptor (53) or influences indirectly the activity of another segment
of FN (27). In either case, the EIIIA+FN-triggered signals may converge
onto the TGF-
-mediated signaling pathway and thus modulate the
stimulatory activity of TGF-
. The detailed characterization of the
EIIIA structure will provide a basis for developing therapeutic
strategies for modulating excessive scarring and tissue fibrosis.