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INTRODUCTION |
Multimeric fibronectin is a major constituent of
extracellular matrices found throughout the body and plays a role in a
wide variety of biological events, including maintenance of endothelial cell integrity, platelet adhesion, and cell migration during blood vessel repair (1-3). Fibronectin circulates at high concentrations in
plasma as a soluble dimeric molecule and exists in an insoluble multimeric form in the extracellular matrix of loose connective tissue,
granulation tissue, and basement membranes (1-3). This insoluble
multimeric form of fibronectin is thought to be the primary functional
form of the molecule (1, 3-5), mediating adhesive and migratory events
associated with wound repair, neovascularization, and embryonic development.
Deposition of fibronectin into the extracellular matrix is a
cell-mediated, multistep process that involves the binding of soluble
fibronectin to specific sites on the surface of substrate-attached cells that have been termed matrix assembly sites (6). Subsequent homophilic binding interactions between fibronectin molecules lead to
the deposition of high molecular mass, disulfide-stabilized multimers
into the extracellular matrix (7-11). Fibronectin that is deposited in
tissues in vivo and in the matrix of cells cultured in
vitro is in the form of high molecular mass multimers (1, 12-14).
This multimeric fibronectin can be converted to monomeric fibronectin
upon treatment with disulfide-reducing agents (12-14), suggesting that
fibronectin in the extracellular matrix is stabilized by disulfide
cross-linking. It has been postulated that this cross-linking event
occurs by a disulfide exchange mechanism involving type I or II modules
in the 70-kDa amino terminus of the molecule (15). However, the regions
involved in disulfide cross-linking of fibronectin in the extracellular
matrix have not been identified (16). In addition, the mechanisms of
this cross-linking are unknown.
A number of proteins involved in disulfide exchange reactions,
including protein-disulfide isomerase and thioredoxin (17-20), contain
the sequence Cys-X-X-Cys in their active sites.
The fifth and sixth cysteines in the twelfth type I module of
fibronectin (I12) are arranged in a similar sequence,
Cys-Asp-Asn-Cys (21), suggesting that fibronectin may contain a
disulfide isomerase activity. To determine whether fibronectin contains
an intrinsic protein-disulfide isomerase activity, we assayed
fibronectin as well as proteolytic and recombinant fragments of
fibronectin for their ability to reactivate reduced and denatured
RNase. Using an established protein refolding assay (22, 23), we found that fibronectin contains a protein-disulfide isomerase- and
thioredoxin-like activity and that this activity is localized to
I12. This is the first demonstration that fibronectin
contains protein-disulfide isomerase activity and suggests that
fibronectin may catalyze disulfide bond rearrangement during its
incorporation into the extracellular matrix.
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EXPERIMENTAL PROCEDURES |
Materials--
Gelatin-Sepharose, Sephadex G-25, and SP Sephadex
C-25 were obtained from Amersham Pharmacia Biotech. Protein-disulfide
isomerase (107 kDa) (EC 5.3.4.1) and thioredoxin (11.7 kDa) were
purchased from Calbiochem-Novabiochem Corp. (La Jolla, CA). The
remaining chemicals were purchased from Sigma unless otherwise indicated.
Fibronectin and Fibronectin Fragments--
Human fibronectin was
purified from a fibronectin and fibrinogen-rich by-product of factor
VIII production as described (24). The 160/180-kDa proteolytic fragment
of fibronectin was generated by limited trypsin digestion of intact
fibronectin essentially as described (25). The 40-kDa (cathepsin and
trypsin) gelatin-binding and 70-kDa (cathepsin) amino-terminal
fragments of fibronectin were the generous gifts of Drs. Paula
McKeown-Longo and Denise Hocking (Albany Medical College, Albany, NY)
and were made as described (6, 26). The 110- and 19-kDa thermolysin
fragments of fibronectin were the generous gifts of Dr. Ken Ingham
(American Red Cross, Bethesda, MD) and were prepared as described
(27-29). A schematic diagram of various fibronectin fragments used in
this study is shown in Fig. 3 (inset).
Generation of I12/pVL1392/His6--
The
baculovirus expression vector pVL1392 (Invitrogen, Carlsbad, CA) was
modified so that genes cloned into the multiple cloning site would be
produced with six histidines at the carboxyl terminus of the protein.
The complementary oligonucleotides
5'-AATTCTTCACCATCACCATCACCATTGATCAG-3' and
5'-GATCCTGATCAATGGTGATGGTGATGGTGAAG-3' (encoding the histidine tag and
containing EcoRI and BamHI overhangs) were
hybridized to each other and then ligated to pVL1392 that was
previously digested with EcoRI and BamHI.
I12 was amplified with the sense primer
5'-CCCAGATCTACTCTCCTCCCATCCACTCAAG-3' and the antisense primer 5'-CCCGAATTCCAGCCCCAGGTCTGCGGCAG-3' using
I12/GE-1 as a template (30). The sense primer has a
BglII restriction enzyme site at the 5'-end (underlined
letters), and the antisense primer has an EcoRI restriction
enzyme site at the 5'-end (underlined letters). The base in boldface
(C) was added to the antisense primer following the
EcoRI site to maintain the correct reading frame. Polymerase
chain reaction-amplified DNA was subcloned into the baculovirus vector
pVL1392/His6. Cloning I12 upstream of the EcoRI site resulted in the addition of Gly-Ile-Leu before
the six-histidine tag. Amplified DNA contains sequences coding for the
fibronectin signal sequence and the I12 coding sequence.
DNA was sequenced (31) to ensure that no DNA mutations were introduced during polymerase chain reaction amplification and to verify the sequence of the modified pVL1392 vector.
Baculovirus
Expression--
I12/pVL1392/His6 was
cotransfected into insect cells with Baculogold DNA (Pharmingen, San
Diego, CA) following the manufacturer's instructions. Recombinant
viruses were prepared using established methods (32, 33). SF21 insect
cells were grown under serum-free conditions using SF900-II (Life
Technologies, Inc.). Conditioned medium containing recombinant
I12 was applied to an SP Sephadex C-25 column equilibrated
with 60 mM NaCl and 100 mM Tris, pH 6.2. The
column was washed with 250 mM NaCl and 100 mM
Tris, pH 6.2, and then eluted with 350 mM NaCl and 100 mM Tris, pH 6.2. Purified I12 was dialyzed into
phosphate-buffered saline or into 30 mM NaCl, 1 mM EDTA, and 0.1 M Tris prior to use in the
RNase refolding assay. Purity of proteins was assessed using a
discontinuous
Tricine1/SDS-polyacrylamide
gel electrophoresis system according to the method of Schagger and von
Jagow (34) and visualized with either 0.025% Serva blue G (Serva,
Paramus, NJ) or silver nitrate (35).
Production and Reactivation of Reduced and Denatured
RNase--
RNase was reduced and denatured essentially as described by
Pigiet and Schuster (22). Briefly, RNase A (30 mg) was incubated overnight in 6 M guanidine HCl containing 0.15 M dithiothreitol and 0.1 M Tris, pH 8.6, at
room temperature. RNase was then purified on a Sephadex G-25 column
equilibrated with 0.1% acetic acid. Peak samples were pooled,
nitrogen-sparged, and then stored at
80 °C until use. Reduced and
denatured RNase at a final concentration of 400 µg/ml (30 µM) was mixed with various proteins in a reaction mixture
containing 0.1 M Tris, pH 7.4, and 1 mM EDTA as
described (22, 23). The extent of RNase reactivation was determined by
removing aliquots of the reaction mixture at various time intervals and
measuring RNase activity as described (23, 36). The absorbance change
at 284 nm was recorded in a final assay mixture that included 1.4 µM RNase and 0.44 mM cytidine 2':3'-cyclic
monophosphate in 0.1 M MOPS, pH 7.0.
No contaminating RNase activity was detected in any of the purified
proteins used in the RNase refolding assay, as assessed by the
inability of purified proteins to hydrolyze cytidine 2':3'-cyclic monophosphate in the absence of added RNase (data not shown). It is
also unlikely that purified proteins contained small amounts of
contaminating redox reagents since the addition of 5 µM
to 2 mM reduced or 4-100 µM oxidized
dithiothreitol did not accelerate the rate of RNase refolding in
comparison with controls (data not shown).
Statistical Analysis--
Statistical analysis was performed
using a one-way analysis of variance. Significant differences between
groups were identified using the Tukey HSD multiple comparison
post hoc test. Statistics were performed using Statistica
software (StatSoft, Tulsa, OK).
Determination of Enzymatic Parameters--
Plots of substrate
concentration versus initial velocity were fit to the
Michaelis-Menten equation using the Enzfitter program (Biosoft,
Ferguson, MO) or DeltaGraph (Deltapoint, Monterey, CA). Lineweaver-Burk
plots thus derived were used to determine Vmax and Km values. To determine
Vmax and Km, the standard
RNase refolding assay (described above) was performed in the presence
of fibronectin (3 µM), protein-disulfide isomerase (1.5 µM), or thioredoxin (30 µM) using RNase
concentrations ranging from 15 to 75 µM. At various time
intervals, aliquots of the reaction mixture were removed, and the
amount of active RNase was determined by measuring the change in
absorbance at 284 nm over time (as described above). A standard curve
was generated in which the change in absorbance at 284 nm over time was
recorded for various concentrations of native RNase. Initial velocities
for the Michaelis-Menten plots (µM native RNase generated
per min) were calculated from the slope of the line derived from the
plot of native RNase concentration versus time.
19- and 40-kDa Fragment Digestion with
-Chymotrypsin--
The
19- or 40-kDa fragment was incubated with
-chymotrypsin at a protein
ratio of 1:200 (w/w) for 1 h at 37 °C. The reaction was
terminated with the addition of 2 mM phenylmethylsulfonyl fluoride (37). Samples of digested proteins were removed and analyzed
by Tricine/SDS-polyacrylamide gel electrophoresis prior to use in the
RNase refolding assay.
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RESULTS |
Fibronectin Contains Protein-disulfide Isomerase
Activity--
Soluble protomeric fibronectin is believed to be
stabilized in the extracellular matrix as a result of disulfide
cross-linking. Fibronectin contains a Cys-X-X-Cys
motif in I12 (21). This motif has been identified as the
active-site sequence in other proteins that exhibit disulfide isomerase
activity (17-20). To determine whether fibronectin has disulfide
isomerase activity, we used an established protein refolding assay.
This assay has been widely used to assess the isomerase activity of
thioredoxin (22, 23, 38), protein-disulfide isomerase (38), and other
proteins (23, 39) and measures the ability of proteins to catalyze the
refolding of reduced and denatured RNase (36). During the process of
RNase refolding, non-native as well as native disulfide bonds form (22,
40-43). Therefore, both disulfide oxidation as well as isomerization
reactions must occur to achieve the native folded state.
Intact fibronectin at a concentration of 4 µM enhanced
the refolding of RNase at a rate similar to that of 1 µM
protein-disulfide isomerase (Fig. 1) and
30 µM thioredoxin (data not shown). A control protein,
ovalbumin, which contains disulfide bonds as well as free sulfhydryl
residues, but lacks the Cys-X-X-Cys motif, showed little activity in this assay (Fig. 1). The ability of fibronectin to
catalyze RNase refolding was concentration-dependent. Intact fibronectin at a concentration of 3 µM had ~84% of the
activity of 4 µM fibronectin; 2 µM
fibronectin had ~74% of the activity (data not shown). To directly
compare the ability of fibronectin, protein-disulfide isomerase, and
thioredoxin to catalyze the refolding of reduced and denatured RNase,
kcat and Km values were determined. The ability of fibronectin to catalyze RNase refolding followed typical Michaelis-Menten kinetics (Fig.
2B, inset). The reactions catalyzed by protein-disulfide isomerase (Fig. 2A,
inset) and thioredoxin (data not shown) also followed
typical Michaelis-Menten kinetics. Lineweaver-Burk plots (Fig. 2 and
data not shown) were used to determine the Vmax,
Km, and
kcat/Km values for each of
these proteins (Table I). As shown in
Table I, fibronectin is ~9-fold more active than thioredoxin and
9-fold less active than protein-disulfide isomerase in catalyzing RNase refolding.

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Fig. 1.
Fibronectin contains protein-disulfide
isomerase activity. Shown are the kinetics of refolding of reduced
and denatured RNase in the presence of 4 µM fibronectin
(FN; ), 1 µM protein-disulfide isomerase
(PDI; ), or 4 µM ovalbumin ( ) or in an
uncatalyzed reaction ( ). Refolding of reduced and denatured RNase
was performed as described under "Experimental Procedures." RNase
activity is expressed as percent of total RNase activity. The
absorbance change at 284 nm generated by adding 1.4 µM
native RNase to the final RNase assay mixture is designated as 100%
RNase activity. The data shown represent 1 of 10 similar
experiments.
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Fig. 2.
Kinetic analysis of RNase refolding by
fibronectin and protein-disulfide isomerase. For the
Lineweaver-Burk plots, the RNase refolding assay was performed using
various concentrations of reduced and denatured RNase in the presence
of 1.5 µM protein-disulfide isomerase (A) or 3 µM fibronectin (B). The data are plotted as
1/v versus 1/[S], and a linear fit of the data
was calculated using DeltaGraph. The initial velocity was calculated as
described under "Experimental Procedures."
Vmax was calculated from the reciprocal of the
y intercept, and Km from the reciprocal
of the x intercept. Insets,Michaelis-Menten
plots. The data generated in the assay described above were fitted to
the Michaelis-Menten equation as described under "Experimental
Procedures." rdRNase, reduced and denatured RNase.
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Table I
Enzymatic parameters of fibronectin, thioredoxin, and protein-disulfide
isomerase
The standard RNase refolding assay was performed using 15-75
µM reduced and denatured RNase in the presence of 3 µM fibronectin, 30 µM thioredoxin, or 1.5 µM protein-disulfide isomerase as described under
"Experimental Procedures." Lineweaver-Burk plots were used to
determine Km, Vmax, and
kcat (Vmax/enzyme concentration).
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To localize the disulfide isomerase activity within fibronectin,
proteolytic fragments of fibronectin encompassing the entire fibronectin molecule (Fig. 3,
inset) were assayed for activity. Fragments of fibronectin
that demonstrated the most activity in this assay were the 160/180- and
19-kDa fragments, which contain the carboxyl-terminal type I modules
I10-I12 (Fig. 3). The 40- and 70-kDa
fragments, which contain disulfides, but do not contain the
Cys-X-X-Cys motif, had activity only slightly
higher than the control protein, ovalbumin (Fig. 3). The central
110-kDa cell-binding fragment of fibronectin, which contains one
free sulfhydryl residue, but no disulfide bonds (21), had little
activity in this assay (Fig. 3). These data indicate that the
protein-disulfide isomerase activity of fibronectin is localized to the
19-kDa fragment that contains I10-I12.

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Fig. 3.
Localization of fibronectin's disulfide
isomerase activity. Fibronectin (FN; 4 µM), various proteolytic fragments of fibronectin (4 µM), protein-disulfide isomerase (PDI; 1 µM), thioredoxin (30 µM), and ovalbumin (4 µM) were tested for their ability to catalyze the
refolding of reduced and denatured RNase. Reduced and denatured RNase
was incubated with the designated proteins for ~100 h. RNase activity
is expressed as -fold increase over uncatalyzed refolding of RNase,
which has been normalized to 1. Similar results were seen when the 70-h
time point was analyzed. The data shown are the means of three or more
experiments. Error bars represent S.E. Values that are
statistically different from ovalbumin (p < 0.05 when
analyzed using the Tukey HSD test) are shown by an asterisk.
Inset, fibronectin and fibronectin fragments. Modules of
fibronectin are represented as rectangles (type I modules),
ovals (type II modules), and squares (type III
modules). The proteolytic fragments of fibronectin used in this paper
and their apparent molecular masses are indicated, as are the location
of fibronectin's free sulfhydryl residues (SH).
Disulfide-containing subunits (types I and II) are
stippled.
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I12 Demonstrates Disulfide Isomerase Activity in the
RNase Refolding Assay--
I12 is located within the
19-kDa fragment and contains the Cys-X-X-Cys
motif. Therefore, to determine whether the disulfide isomerase activity
of the 19-kDa fragment could be further localized to I12,
recombinant I12 was generated using a baculovirus
expression system and tested in the RNase refolding assay. As shown in
Fig. 4, I12 at a
concentration of 4 µM was more active than 4 µM intact fibronectin and as active as 1 µM
protein-disulfide isomerase. These data suggest that most or all of
fibronectin's protein-disulfide isomerase activity is localized to
I12.

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Fig. 4.
Protein-disulfide isomerase activity of
I12. Shown are the kinetics of refolding of reduced
and denatured RNase in the presence of 4 µM
I12 ( ), 4 µM fibronectin (FN;
), 1 µM protein-disulfide isomerase (PDI;
), or 4 µM ovalbumin ( ) or in an uncatalyzed
reaction ( ). Refolding of reduced and denatured RNase was performed
as described under "Experimental Procedures." RNase activity is
expressed as percent of total RNase activity. The data represent one of
three similar experiments.
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Regulation of the Protein-disulfide Isomerase Activity of
Fibronectin--
Deposition of fibronectin into the extracellular
matrix is a highly regulated process (44-48). If fibronectin's
protein-disulfide isomerase activity is involved in cross-linking of
fibronectin in the extracellular matrix, then it would be expected that
its protein-disulfide isomerase activity would also be tightly
regulated. One mechanism by which this activity may be regulated is
through conformation-induced activation. In such a model, isomerase
activity would be partially masked in the native soluble fibronectin
molecule and enhanced by conformational changes induced upon binding of fibronectin to cell surfaces. If fibronectin's protein-disulfide isomerase activity is partially cryptic, limited proteolysis might enhance its protein-disulfide isomerase activity by generating smaller
fragments whose conformations are distinct from those in the intact
molecule. Others have shown that proteolytic fragments of proteins can
have enhanced or novel activities when compared with native proteins
(49, 50). For example, the 40-kDa gelatin-binding fragment and the
amino- and carboxyl-terminal fibrin-binding fragments of fibronectin
have enhanced chemotactic activity when compared with the activity of
intact fibronectin (49, 50). In addition, proteolytic fragments of
plasminogen (51) and collagen XVIII (52) have anti-angiogenic
properties not associated with the native molecules.
To determine whether fibronectin's protein-disulfide isomerase
activity could be increased by limited proteolysis, the 19-kDa fragment
of fibronectin (containing I10-12) was digested with
chymotrypsin and assayed for its ability to catalyze RNase refolding.
Analysis of the digested and undigested 19-kDa fragment by
Tricine/SDS-polyacrylamide gel electrophoresis indicated that digestion
of the 19-kDa fragment by chymotrypsin resulted in generation of
fragments with apparent molecular masses of ~14, 12, and 10 kDa (data
not shown). The chymotrypsin-generated fragments of the 19-kDa
fragment at a concentration of 1 µM had activity similar to 4 µM intact 19-kDa fragment as measured by the RNase
refolding assay (Fig. 5A).
Moreover, chymotrypsin proteolysis of the 19-kDa fragment (4 µM) resulted in a dramatic increase in the rate of RNase
refolding (Fig. 5A and Table
II). Table II compares the time required
for RNase to reach 50% of maximal refolding in the presence of various
proteins or fibronectin fragments. Whereas the intact 19-kDa fragment
required 66 h to refold 50% of the RNase, proteolytic 19-kDa
fragments required only 18 h. In comparison, RNase treated with 4 µM protein-disulfide isomerase reached 50% of its native
refolded state in 28 h, whereas RNase treated with 30 µM thioredoxin required 63 h. In the uncatalyzed
reaction, >100 h was required. The increase in protein-disulfide
isomerase activity after limited proteolysis was specific to the 19-kDa fragment since the ability of the 40-kDa gelatin-binding fragment of
fibronectin to refold RNase did not change substantially upon limited
digestion with chymotrypsin (Fig. 5B and Table II).
Chymotrypsin digestion of the 40-kDa fragment generated fragments of
~31, 28, 27, 16, and 11 kDa (data not shown). These data indicate
that limited proteolysis of the 19-kDa fragment increases its
protein-disulfide isomerase activity, most likely due to generation of
a fragment whose conformation favorably exposes its protein-disulfide
isomerase activity.

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Fig. 5.
Chymotrypsin treatment of the 19-kDa fragment
increases its isomerase activity. The 19- and 40-kDa fragments
were digested with 0.1 mg/ml -chymotrypsin as described under
"Experimental Procedures." A, refolding of reduced and
denatured RNase was performed in the presence of 4 µM
( ) or 1 µM ( ) chymotrypsin-digested 19-kDa
fragment, 4 µM undigested 19-kDa fragment ( ), 4 µM fibronectin (FN; ), 1 µM
protein-disulfide isomerase (PDI; ), or 4 µM ovalbumin ( ) or in an uncatalyzed reaction ( ).
B, refolding of reduced and denatured RNase was performed in
the presence of 4 µM digested ( ) or undigested ( )
40-kDa fragment, 1 µM protein-disulfide isomerase ( ),
or 4 µM ovalbumin ( ) or in an uncatalyzed reaction
( ). The addition of phenylmethylsulfonyl fluoride and
-chymotrypsin to the uncatalyzed reaction did not alter the RNase
refolding rate (data not shown). RNase activity is expressed as percent
of total RNase activity. The data shown represent one of five similar
experiments.
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Table II
Chymotrypsin treatment of the 19-kDa fragment decreases the time
required to reach 50% native refolding
Shown are the half-times required for RNase reactivation by various
proteins and protein fragments. The absorbance change at 284 nm
generated by adding 1.4 µM native RNase to the final
RNase assay mixture is designated as 100% RNase activity. The time
required for various samples to refold reduced and denatured RNase to
50% of maximal activity is indicated with the S.E. of three or more
experiments.
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DISCUSSION |
In this report, we have demonstrated that the extracellular matrix
protein fibronectin contains an intrinsic protein-disulfide isomerase
activity. This is the first demonstration of protein-disulfide isomerase activity in fibronectin and suggests that fibronectin may
catalyze disulfide bond rearrangement during its incorporation into the
extracellular matrix. Other covalent cross-linking events may also lead
to fibronectin multimerization under certain circumstances (53, 54).
For example, fibronectin can be covalently cross-linked in the
extracellular matrix via
-(
-glutamyl)lysyl bonds by activated factor XIII (53, 54). It has also been suggested that fibronectin multimers may be stabilized by noncovalent interactions since attempts
to identify cross-linked regions of fibronectin have not been
successful (16). However, the ability of multimeric extracellular
matrix fibronectin to be converted to monomeric fibronectin by
treatment with disulfide-reducing agents (13, 14) suggests that
extracellular matrix fibronectin is stabilized predominantly by inter-
or intramolecular disulfide exchange.
Our data support a model whereby incorporation of soluble fibronectin
into the extracellular matrix involves a disulfide exchange mechanism
catalyzed by an isomerase activity located within the fibronectin
molecule. Disulfide-stabilized, multimeric fibronectin has been
reported to be a functionally distinct form of fibronectin that has
enhanced adhesive properties, is active in suppressing cell migration
and tumor formation (55, 56), and mediates enhanced binding of bacteria
to host tissue (57). Recent evidence also suggests that the
extracellular matrix multimeric form of fibronectin has
growth-promoting properties not possessed by protomeric fibronectin
(58) and that the effects of matrix fibronectin on cell growth are
dependent on its exact molecular configuration (59). It has also been
shown that treatment of cells with a fragment derived from the first
type III module of fibronectin (III1-C) can lead to
inhibition of fibronectin deposition or disruption of a preexisting
fibronectin matrix and also results in inhibition of cell growth (60,
61). Thus, the identification of protein-disulfide isomerase activity
within the fibronectin molecule is important not only for elucidating
the biochemical mechanisms that regulate fibronectin multimerization,
but also in defining the functional consequence of this multimerization.
In the RNase refolding assay, 4 µM fibronectin had
disulfide isomerase activity similar to that of 1 µM
protein-disulfide isomerase or 30 µM thioredoxin (Figs. 1
and 2). Analysis of kcat/Km values indicated that fibronectin is ~9-fold more active than thioredoxin and 9-fold less active than protein-disulfide isomerase in
catalyzing RNase refolding. The rates determined in this study for
protein-disulfide isomerase are lower than those previously reported
(19, 62, 63). However, in those previous reports, RNase refolding was
performed in the presence of reduced and oxidized glutathione at pH 8, conditions substantially different from those used in our assays. Thus,
these differences likely account for the differences in rates reported.
The RNase refolding assay used in this and other studies predominantly
measures the ability of proteins to catalyze dithiol oxidation
reactions (17). However, refolding of reduced and denatured RNase to a
native state involves formation (oxidation) and rearrangement
(isomerization) of non-native disulfide bonds until the final folded
state is achieved (22, 40-42), a process that is thought to be driven
by the search for the most stable protein conformation (17). The assay
buffer employed in this study does not require the addition of an
external oxidative agent or the prior "activation" of protein
catalysts by the addition of a reducing agent (64, 65) and thus is
likely to more closely approximate the environment outside the cells where fibronectin deposition into the extracellular matrix occurs.
Our data indicate that fibronectin's protein-disulfide isomerase
activity is localized predominantly to the last type I module, I12 (Fig. 4), which contains the
Cys-X-X-Cys motif (21). I12 was more
active on a molar basis than intact fibronectin, indicating that most
or all of fibronectin's isomerase activity is localized to
I12. Localization of the protein-disulfide isomerase
activity of fibronectin to I12 suggests the possibility
that the Cys-X-X-Cys motif of I12 may
be important in catalyzing disulfide cross-linking of fibronectin in
the extracellular matrix since the Cys-X-X-Cys motif in protein-disulfide isomerase and thioredoxin is the active-site sequence required for catalyzing disulfide bond isomerization (17-20).
The Cys-X-X-Cys motif in von Willebrand factor
has also been shown to be critical for the ability of von Willebrand
factor to form disulfide-stabilized multimers (66).
We have previously shown that fibronectin containing mutations in
I12 or lacking I12 can become incorporated into
the extracellular matrix of cells containing a pre-established matrix
(30). However, if the protein-disulfide isomerase activity of
I12 is critical for the formation of disulfide-stabilized
fibronectin multimers, it is possible that the non-mutant fibronectin
present in the assay provided this activity. In support of this, we and
others have shown that fibronectin deletion mutants lacking the
Arg-Gly-Asp sequence (67, 68) or lacking a large internal portion
(III1-I12) (69) become incorporated into the
extracellular matrix of cells containing a pre-established fibronectin
matrix (67, 69), but are not incorporated into the extracellular matrix
of cells lacking a pre-established fibronectin matrix (68, 69).
The cryptic nature of fibronectin's protein-disulfide isomerase
activity is consistent with the highly regulated nature of fibronectin
matrix assembly (44-48). Our data indicate that the protein-disulfide
isomerase activity of fibronectin can be increased by limited
proteolysis, a treatment that likely results in changes in protein
conformation. Conformational changes have also been detected in
fibronectin following binding of fibronectin to surfaces (50, 70) or
following alterations in pH and ionic strength (71, 72). Conformational
alterations in fibronectin have been shown to lead to exposure of
cryptic binding sites (9, 10). Conformational alteration of
III1 generates a binding site for the 70-kDa amino terminus
of fibronectin (10). Similarly, conformational alteration of
III10 leads to exposure of a cryptic binding site for
III1 (9). Fibronectin that is incubated with a fragment of
III1 (III1-C) (55) or with conformationally
altered III10 (9) forms disulfide-stabilized multimers,
presumably due to conformational changes in fibronectin induced by
interaction with III1-C or with altered III10.
These data suggest a model in which the interaction of fibronectin with
cell surfaces triggers a series of conformational changes leading to
exposure of fibronectin-fibronectin interactive sites (9, 10, 55, 68,
73, 74) and activation of fibronectin's disulfide isomerase activity
that may be important for fibronectin fibril formation. Future studies
will be directed toward defining the interactions that regulate
fibronectin's protein-disulfide isomerase activity and determining
whether this activity mediates fibronectin cross-linking during
fibronectin matrix assembly.