(Received for publication, November 16, 1995; and in revised form, January 22, 1996)
From the
Fibronectin exists in a soluble form in body fluids and as a
fibrillar component of the extracellular matrix. Matrix fibronectin
associates as large complexes in SDS unless a reducing reagent is also
present. This observation suggests that complex formation is due to
interprotomeric disulfides that form by thiol-disulfide exchange. To
localize the presumptive new disulfides, we labeled protomeric
fibronectin by the chloramine-T method or with I-Bolton-Hunter reagent, incorporated
I-fibronectin into the matrix of cultured fibroblasts,
and subjected matrix fibronectin to acid or cyanogen bromide digestion.
When cyanogen bromide digests of matrix
I-fibronectin and
protomeric
I-fibronectin labeled with Bolton-Hunter
reagent were analyzed by two-dimensional polyacrylamide gel
electrophoresis in SDS, with the first dimension being nonreducing and
the second reducing, we were not able to identify any fragments of
matrix fibronectin that migrated as high molecular weight complexes in
the first dimension. Limited acid digestion of matrix
I-fibronectin also dissociated the majority of the high
molecular weight complexes. Since we could account for all of the parts
of fibronectin that contain cysteine or cystine, we conclude that
matrix fibronectin is not stabilized by interprotomeric disulfides. We
propose, instead, that stabilization is mediated by noncovalent
protein-protein interactions that are sensitive to reduction, cyanogen
bromide digestion, or limited acid digestion.
Fibronectin exists in two forms, soluble and insoluble. Soluble protomeric fibronectin is found at substantial concentrations in plasma and other body fluids and in the conditioned media of cultured cells. Insoluble fibronectin is a fibrillar component of the extracellular matrix(1, 2, 3) . The insoluble form of fibronectin mediates cell attachment, provides a substrate for cell migration during embryogenesis and wound healing, and thus is considered to be the primary functional form of the protein(2, 4, 5, 6, 7) . Absence of fibronectin is lethal to mouse embryos(8) . In vitro and in vivo studies have shown that matrix fibronectin consists of both endogenously synthesized cellular fibronectin and circulating plasma fibronectin(3, 9, 10) .
Unlike many other matrix molecules with conspicuous self-assembly characteristics, purified plasma fibronectin forms insoluble precipitates in vitro only under special conditions of uncertain physiological significance(11, 12, 13, 14, 15, 16, 17) . In fibroblast cultures, fibronectin fibrils are found on the surface of or between cells(1) . Deposited matrix fibronectin is in the form of multimers that resist dissociation with SDS unless sulfhydryl-containing reagents are also present(18, 19, 20, 21, 22, 23) . This observation suggests that the multimers are stabilized by interprotomeric disulfides. Because blockage of the free sulfhydryls of fibronectin does not impede subsequent formation of SDS-stable multimers, it was further hypothesized that the interprotomeric disulfides form by thiol-disulfide exchange rather than by oxidation of free sulfhydryls(4, 23) . Transglutaminase-mediated cross-linking by activated blood coagulation factor XIII also promotes insolubilization(24, 25) .
Identification of the
new disulfides should be as simple as comparing polyacrylamide gel
electrophoresis (PAGE) ()patterns in SDS of fibronectin
fragments analyzed without or with reducing reagent. Fragments of
I-labeled protomer and multimer should be the same after
reduction, whereas without reduction, two fragments should migrate as a
complex in the digest of multimer but not in the digest of protomer.
Using such reasoning, our laboratory has argued that multimerization of
I-fibronectin involves disulfides in the 70-kDa cathepsin
D NH
-terminal fragment (23) . We found it to be
impossible to advance beyond our 1984 study (23) using
proteases and began to question the existence of interprotomeric
disulfides. In the present paper, we report the negative results of a
more rigorous search for interprotomeric disulfides using acid and
cyanogen bromide digestion. Because we could identify all of the parts
of fibronectin that contain disulfides, these experiments indicate that
interprotomeric disulfides do not form during stabilization of
fibronectin and are compatible with the formation of strong noncovalent
protein-protein interactions that resist disruption with SDS.
Fig. 1shows the location of Met-Xaa and Asp-Pro peptide bonds in the A and B subunits of human plasma fibronectin that should be sensitive to cyanogen bromide and acid cleavage, respectively (27, 28, 31) . Fig. 2tabulates the predicted sizes of cyanogen bromide and acid digestion fragments under nonreducing and reducing conditions and identifies fragments that contain carbohydrates and therefore that have larger sizes than predicted from the amino acid sequence. Plasma fibronectin is assumed to be composed of one subunit with a V region and one subunit with no V region(2, 31, 32) .
Figure 1: Schematic domain structure of fibronectin protomer showing locations of cyanogen bromide and acid cleavage sites. Arrows point to locations of Met-Xaa bonds that are the cleavage sites of cyanogen bromide. Arrows with * point to Asp-Pro bonds that are acid cleavage sites. Intramolecular (in the type I and type II modules) and intermolecular disulfide bonds are indicated. SH, free sulfhydryl group. Bars highlight the I-4/I-5 and I-9/III-1 module pairs that are discussed. The open arrowhead denotes the site of insertion of the variably spliced V region that is found in the A subunit of the fibronectin heterodimer.
Figure 2:
Tabulation of cyanogen bromide (A) and acid (B) fragments of human plasma
fibronectin. Met-Xaa (M/X) and Asp-Pro (D/P) peptide
bonds are described by residue numbers and module location. Residues
are numbered beginning with the NH-terminal pyroglutamate
of human fibronectin(31) . The type III modules invariantly
present in the two fibronectin subunits are numbered 1-15 (2) . Fragments generated are described by number of residues (N) and size (kDa, with a (+) to indicate the
need of correction for carbohydrate modification). Lysine content (K) in the reduced form of the fragment is also shown. The
disulfide-bonded covalent structures of fragments without reduction (DSFs) are given. Fragments that are generated due to the
acidic condition of the cyanogen bromide digestion are marked with *.
The fibronectin subunit that contains the V region is denoted A chain. The subunit that lacks the V region is denoted B chain.
In preliminary
experiments, we reproduced the patterns that Gold et al.(33) obtained when nonreduced and reduced cyanogen bromide
digests of unlabeled fibronectin were analyzed by one-dimensional
SDS-PAGE. I-Fibronectin labeled by our standard
chloramine-T technique (26) fragmented less completely than
unlabeled fibronectin did, presumably because of partial oxidation of
methionines. Fibronectin labeled with Bolton-Hunter reagent, in
contrast, fragmented as completely as unlabeled fibronectin did. In
addition, the ratio of radioactivity to protein was fairly constant
among fragments when the protein staining and autoradiographic patterns
were compared (not shown); such a result is predicted by the
distribution of lysines in fibronectin (Fig. 2).
Fibronectin
labeled with Bolton-Hunter reagent bound to cells and became
insolubilized with the same kinetics as fibronectin labeled by the
chloramine-T technique (data not shown). After the 48-h incubation with
cell layers and the 4-h chase, >70% I-fibronectin was
present in SDS-stable multimers when analyzed by SDS-PAGE without
reduction. In order to destroy cellular acid proteases completely
before subjecting cellular extracts to cyanogen bromide digestion in
formic acid, a protocol was developed in which the cells were rapidly
lysed with 1% deoxycholate in the presence of protease inhibitors, and
deoxycholate-insoluble material was treated with warm 1% SDS before
digestion. NEM was also added to block the two free sulfhydryls (Fig. 1) and prevent artifactual formation of disulfide-linked
multimers(11, 23) .
Three disulfide-containing fragments (DSF-1, DSF-2, and DSF-3) account for all of the disulfide-containing parts of fibronectin ( Fig. 2and 3). If disulfide exchange occurs, one of these fragments should be involved. DSF-1 extends from the beginning of module I-1 to the end of module II-2, DSF-2 extends from the beginning of module I-7 to the middle of module III-4, and DSF-3 extends from III-12 through the interchain disulfides to the middle of the V region of the other subunit ( Fig. 1and Fig. 2). Acid cleavage in addition to cyanogen bromide digestion would result in smaller versions of DSF-2 and DSF-3. DSF-1 under reducing conditions should give rise to fragments of 10, 13.2, and somewhat greater than 13.4 kDa and smaller fragments; reduced DSF-2 should contain a major fragment of somewhat greater than 46 kDa; and reduced DSF-3 should contain a fragment of about 39 kDa from the subunit lacking the V region (B subunit), a fragment of somewhat greater than 17 kDa from the subunit with the V region (A subunit), and smaller peptides.
By analysis of two-dimensional gels, we could identify all three DSFs. The small reduced fragments of DSF-1 fell off the diagonal after reduction as expected. A 60-kDa fragment of reduced DSF-2 was recognized by monoclonal antibody 9D2 to the III-1 module (Fig. 3, actual blot not shown). A 39-kDa fragment of reduced DSF-3 reacted with monoclonal antibodies to modules III-12-III-14 constituting the COOH-terminal heparin-binding region (IST-2) (30) and to the III-15 sequence (IST-7)(30) , whereas a 17-kDa fragment reacted only with IST-7 antibody to the III-15 sequence (Fig. 3, actual blot not shown). The two-dimensional protein staining and immunoblotting patterns were more complicated than shown in Fig. 3because of incomplete cleavage giving rise to larger DSFs and acid cleavage at Asp-Pro bonds giving rise to smaller DSFs of DSF-2 and DSF-3.
Figure 3: Sketch of nonreducing/reducing two-dimensional SDS-PAGE of cyanogen bromide digestion of protomeric fibronectin. Arrows point to expected migration of DSF-1, -2, and -3 in the first (nonreduced) dimension as in Fig. 4. The major spot detected by 9D2 is stippled. The major spot detected only by IST-7 is single cross-hatched. The major spot detected by IST-2 and IST-7 is double cross-hatched.
Figure 4:
Autoradiography of two-dimensional
SDS-PAGE of protomeric and matrix fibronectin. Two-dimensional SDS-PAGE
was performed as described under ``Materials and Methods.''
5-14% gradient gels were used for both dimensions. A,
autoradiography of cyanogen bromide digest of protomeric Bolton-Hunter
reagent-labeled I-fibronectin. B,
autoradiography of digest of stabilized matrix Bolton-Hunter
reagent-labeled
I-fibronectin.
C-Methylated
molecular size markers (Amersham Corp.) indicated are: myosin,
approximately 200 kDa; phosphorylase b, 97.4 kDa; bovine serum
albumin, 69 kDa; ovalbumin, 46 kDa; carbonic anhydrase, 30 kDa; and
lysozyme, 14.3 kDa. C, location of DSFs. a, fragments
that arise due to acid cleavage in addition to cyanogen bromide
cleavage; i, fragments that arise due to incomplete cyanogen
bromide cleavage; A and B, fragments that are
generated from A or B subunits of a fibronectin
dimer.
Autoradiographic patterns of cyanogen bromide
digestion of protomers and multimers shown in Fig. 4were chosen
because the intensities of exposure were similar and pertinent spots
were well separated. The patterns are representative of seven
independent comparisons of digest patterns of protomeric and matrix
fibronectin from two different preparations of labeled protomeric
fibronectin. We were not able to identify any fragments that migrated
reproducibly as higher molecular weight complexes in the first
nonreducing dimension of matrix I-fibronectin digest.
Some differences were noted among individual matched autoradiograms, e.g. the vertical spikes above the diagonal in Fig. 4B that are not present in Fig. 4A and the intensities of some spots including DSF-2 and DSF-3 that
seem to be weaker in some of the matrix digests. However, these
differences were not reproducible. Since we could account for all of
the parts of fibronectin that contained disulfides as in DSF-1, DSF-2,
and DSF-3, and none of these fragments were lost or linked in the
digest of matrix, we can only conclude that insolubilization of
fibronectin by the cells is not mediated by formation of specific
disulfides between fibronectin protomers. These results, albeit with
less sensitivity, are also evidence against the possibilities that
disulfide exchange occurs between fibronectin and other (unlabeled)
matrix molecules or that disulfide exchange is highly degenerate and
involves many fragments so that no one fragment disappears or no one
disulfide-linked pair of fragments appears strongly enough to be
detected.
The evidence against interprotomeric disulfides from the
two-dimensional gels is best for DSF-1, which appeared as intense spots
in autoradiograms of cyanogen bromide-digested protomeric and matrix
fibronectin. DSF-2 and DSF-3 fragments were less intense because of
lighter labeling and the presence of the acid cleavage sites that
generated additional fragments ( Fig. 1and Fig. 2). In
order to analyze the DSF-2 region better, we digested matrix I-fibronectin with formic acid alone (Fig. 5).
After 24 h, 94-, 63-, and 31-kDa fragments were present, and only a
minor portion migrated as high molecular weight complexes under
nonreducing conditions (Fig. 5). By immunoblotting with 9D2 (to
the III-1 module), the 94- and 31-kDa fragments were positive, whereas
the 94-, 63-, and 31-kDa fragments reacted with polyclonal antibodies
to the NH
-terminal 70-kDa region (I-1-I-9 modules).
Thus, the major fragments released by acid digestion represent cleavage
at the Asp-Pro sequences between I-8 and I-9 (Asp-525) and between
III-2 and III-3 (Asp-785), with incomplete cleavage at Asp-525 yielding
the 94-kDa band. These results are more convincing evidence that the
DSF-2 region is not involved in interprotomeric disulfides in
fibronectin matrix.
Figure 5:
Acid
digestion of matrix I-fibronectin. Phosphorimages of acid
digest of matrix
I-fibronectin labeled by the
chloramine-T method are shown. Isolated matrix
I-fibronectin was incubated in 70% formic acid at 37
°C for designated time lengths. ND, matrix
I-fibronectin that was incubated at 37 °C for 24 h in
the absence of acid. Samples were separated by SDS-PAGE with a 9%
separating gel without reducing reagent.
C-Methylated
molecular size markers (myosin, approximately 200 kDa; phosphorylase b, 97.4 kDa; bovine serum albumin, 69 kDa; ovalbumin, 46 kDa;
and carbonic anhydrase, 30 kDa) migrated as indicated. Arrows point to the top of the stacking gel and the interface between the
stacking and separating gels.
Fragments from the DSF-3 region also labeled less intensely in the cyanogen bromide digests (Fig. 4) and were not readily identified in the acid digests (Fig. 5). The lack of evidence that these fragments were disulfide-linked other than through the interchain disulfides that hold the dimer together (Fig. 4) is in accord with the finding that deletion of modules I-10-I-12 in the DSF-3 region results in dimeric molecules capable of forming disulfide-stable complexes(34) .
SDS-stable fibronectin
multimers did not dissociate with short treatments with 70% formic acid (Fig. 5) or, in other experiments, with several denaturing
reagents other than SDS including 0.2 M potassium thiocyanate,
8 M urea, and 6 M guanidine (results not shown). We
also tested 1,10-phenanthroline because of the possibility that
fibronectin-matrix complexes might be stabilized by Zn binding to vicinal histidinal residues in I-7 and I-8 (2, 31) and could show no dissociation (result not
shown).
The acid cleavage sites are adjacent to the I-9/III-1 module
pair that our laboratory has implicated in fibronectin-fibronectin
interactions on the basis of antibody and fragment inhibition
studies(29) . I-9 is a stable module that is independently
folded(35) . III-1 is unique among type III modules for having
a stable core structure, even after removal of strands that are
expected to be essential for folding(36) . Module III-1 has
received considerable interest by virtue of its interaction with other
parts of fibronectin(15, 37, 38) . Studies of
the interactions of recombinant III-1 with a deletion set of
recombinant 70-kDa fragment constructs indicate that III-1 binds to the
I-4/I-5 module pair. (
)NMR spectroscopy of the recombinant
I-4/I-5-module pair reveals that a tryptophan unique to I-4 interacts
with an arginine unique to I-5; this interaction is a striking feature
of the hydrophobic interface that docks the two modules together into a
fixed structure(39) . One can speculate that a conformational
change takes place whereby the tryptophan of I-4, instead of anchoring
the cis interaction of I-4 with I-5, interacts in trans with I-9 or III-1. Such a scenario would explain the
susceptibility of fibronectin multimers to dissociation by disulfide
bond reduction, which would destabilize I-4, I-5, and I-9, and by acid
cleavage, which might destabilize I-9 and III-1. This scenario would
also account for the observation that in partial cathepsin D digests of
matrix fibronectin, the NH
-terminal 70-kDa fragment of
fibronectin comprised of I-1-I-9 binds in an SDS-stable manner to
high molecular weight material(23) . Another scenario is that
fibronectin assembly is an example of ``three-dimensional domain
swapping'' in which oligomers form by virtue of formation of an
interprotomeric interface that is identical to an intraprotomeric
interface between domains of the protomer(40) . Against this
scenario, however, is the fact that SDS-stable interactions have not
been identified between parts of protomeric fibronectin.
Can the
strength of noncovalent association of fibronectin multimers in aqueous
solution resist dissociation with SDS and the other protein
denaturants? We have considered two examples of such stable
interactions in other proteins. The binding of biotin to streptavidin
causes the subunits of tetrameric streptavidin to be associated so
tightly as to withstand dissociation with SDS and heat(41) .
The large negative free energy of association is thought to be due to
van der Waals' forces dispersion effects in the nearly ideal
preformed cavity that streptavidin presents to biotin(42) .
Mutation of a critical tryptophan that helps to form this cavity and
that contributes to the hydrophobic subunit interface results in
streptavidin that binds biotin but that dissociates when treated with
SDS and heat(41) . Some ternary complexes of the and
subunits of class II major histocompatibility complex
glycoproteins and antigenic or class II-associated invariable chain
peptides are stable in SDS. This stability varies considerably with the
peptides even though the peptides bind to a common groove in a common
conformation(43, 44) . Binding of the peptide is
stabilized by multiple hydrogen bonds and by contacts with pockets in
the binding groove(44, 45) . Some SDS-stable, peptide
class II glycoprotein complexes dissociate in the presence of reducing
agent(46) , presumably due to disruption of the disulfide
bridge in the class II
domain(47) . Thus, the
notion of protein-protein interactions that are stable in SDS and are
broken in SDS plus reducing agent has a well studied precedent. Both of
these examples of SDS-stable interactions are characterized by proteins
associating to form a site for strong binding of a third moiety. By
analogy, multimerization of fibronectin may occur by a scenario that
involves ternary complexes, e.g. the I-4/I-5 and I-9/III-1
module pairs and a third site in polymerizing fibronectin or an
associated protein.