Cryptic Self-association Sites in Type III Modules of Fibronectin*

(Received for publication, September 17, 1996, and in revised form, October 28, 1996)

Kenneth C. Ingham Dagger , Shelesa A. Brew , Sheela Huff and Sergei V. Litvinovich

From the American Red Cross Holland Laboratory, Rockville, Maryland 20855

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

The first type III module of fibronectin (Fn) contains a cryptic site that binds Fn and its N-terminal 29 kDa fragment and is thought to be important for fibril formation (Morla, A., Zhang, Z., and Ruoslahti, E. (1994) Nature 367, 193-196; Hocking, D. C., Sottile, J., and McKeown-Longo, P. J. (1994) J. Biol. Chem. 269, 19183-19191). A synthetic 31-mer peptide (NAPQ ... TIPG) derived from the middle of domain III1 was also shown to bind Fn, but the site of its interaction was not determined (Morla, A., and Ruoslahti, E. (1992) J. Cell Biol. 118, 421-429). By affinity chromatography on peptide-agarose, we tested a set of fragments representing the entire light chain of plasma Fn. Only 40-kDa Hep-2 (III12-15) failed to bind. The concentration of urea required for peak elution of Fn and the other fragments decreased in the order Fn > 42-kDa GBF (I6II1-2I7-9) > 19-kDa Fib-2 (I10-12) > 110-kDa CBF(III2-10) > 29-kDa Fib-1 (I1-I5). Neither Fn nor any of the fragments bound immobilized intact III1, confirming the cryptic nature of this activity. In an effort to detect interactions between other Fn domains, all fragments were coupled to Sepharose, and each fragment was tested on each affinity matrix before and after denaturation. The only interaction detected was that of fluid phase III1 with immobilized denatured 110-kDa CBF and 40-kDa Hep-2, both of which contain type III domains. Analysis of subfragments revealed this activity to be dominated by domains III7 and III15. Fn itself did not bind to the denatured fragments. Thus, domain III1 contains two cryptic "self-association sites," one that is buried in the core of the fold but recognizes many Fn fragments when presented as a peptide and another that is concealed in Fn but exposed in the native isolated domain and recognizes cryptic sites in two other type III domains. These interactions between type III domains could play an important role in assembly of Fn multimers in the extracellular matrix.


INTRODUCTION

Fibronectin (Fn)1 circulates in plasma as a 550-kDa 2-chain monomer that can be transformed by cultured fibroblasts into an insoluble fibrillar structure during the cell-driven process of matrix assembly (1). Several regions of Fn have been implicated in this process. N-terminal fragments containing the first five type I modules bind to cell layers and inhibit matrix assembly but are not themselves incorporated into the insoluble matrix unless bivalent, and then most efficiently in the presence of intact Fn (2-8). For a long time, it was thought that N-terminal fragments were interacting with a cell-surface "matrix assembly receptor" that was distinct from integrins that interact with Fn primarily via the Arg-Gly-Asp in the 10th type III domain. At least two unidentified molecules have been proposed as candidates for the matrix assembly receptor by virtue of their interaction with N-terminal fragments of Fn (9, 10). However, none has been further characterized or shown to play a role in matrix assembly. More recently, it is beginning to appear that the matrix assembly receptor, i.e. the molecule responsible for binding N-terminal fragments, might be Fn itself, perhaps conformationally altered by incorporation into the matrix (5, 11, 12).

The fact that Fn matrix assembly could be inhibited with a monoclonal antibody directed to module III1 (13) prompted efforts to examine the interaction of this module with Fn. Morla and Ruoslahti (14) showed that a synthetic 31-mer peptide with N-terminal sequence NAPQ ... , derived from the middle of III1, was able to bind Fn, but the site of its interaction was not determined. The same group later reported that a longer recombinant peptide with the same N terminus, when incubated with whole Fn, was able to induce the formation of polymers that were stable in SDS in the absence of reducing agents and exhibited superior adhesive properties toward fibroblasts (15). At the same time, it was shown by Hocking et al. (11) that module III1, when adsorbed to plastic above its denaturation temperature, was able to bind Fn and its N-terminal 70-kDa fragment; other fragments C-terminal to this were not tested, nor were a sufficient number of denatured type III domains tested to determine the specificity of the observed interaction. More recently, the same group reported that native III1 was able to bind heat-denatured III10 and hypothesized that integrin receptors might induce a conformational change in III10 that would expose a binding site for III1 (16).

Denaturation of any protein or independently folded domain will expose hydrophobic groups that are normally buried in the native structure. These sticky residues, at least some of which are also present in the synthetic III1 peptide, could interact with other proteins or fragments in a nonspecific manner, in the same way for example that a number of Fn fragments have been shown to interact with alkyl- and aryl-Sepharose columns (17-19). In the present study, we used analytical affinity chromatography to test a complete set of fragments representing almost the entire light chain of Fn for their ability to bind native or denatured III1. In addition, we tested each of the native fragments against each of the others to identify additional interactions. We found that denatured III1, as represented by the above-mentioned synthetic peptide, bound numerous Fn fragments. The only interaction detected among the native fragments was that of fluid phase III1 with immobilized 40-kDa Hep-2 (III12-15) and 110-kDa cell-binding fragment (III2-10). These interactions were dramatically increased after deliberate denaturation of the immobilized fragments. Additional experiments with subfragments indicate that module III7 in 110 kDa and III15 in 40 kDa contain the cryptic sites for binding of native III1 to the respective fragments.


MATERIALS AND METHODS

Human plasma fibronectin was prepared as described previously (20). The major fragments of Fn were generated with thermolysin (21). The methods of purification and documentation of their purity have been published previously. The heparin-binding fragments (40-kDa III12-15 and 30-kDa III12-14) and 8-kDa III15 were purified according to Ingham et al. (22). The gelatin-binding fragments (42, 30, and 21 kDa) were purified according to Ingham et al. (23). The 56-kDa gelatin-binding fragment was separated from the others by adsorption of the gelatin-binding fraction on heparin-Sepharose; it was homogeneous by SDS-PAGE. Two versions of III1, designated III-1a and III-1b by Litvinovich et al. (24), were used. The first begins with ITETP and was used in all of the experiments in which III1 was chromatographed on affinity columns. It is herein referred to as 9-kDa III1. The other is slightly shorter, beginning with IQWN, and is the one coupled to Sepharose. The 110-kDa III2-10, 54/60-kDa III2-5/6, 39-kDa III7-8, 26-kDa III8-9, and 11-kDa III10 from the cell-binding region were purified as described (25).2 The 29-kDa Fib-1/Hep-1 (referred to herein as simply Fib-1) and 19-kDa Fib-2 fragments were purified as described (26). Recombinant maltose-binding protein fused to Fn module III7 (MBP-III7) was prepared in Escherichia coli by methods similar to those described (27); it was homogeneous by SDS-PAGE. The synthetic 31-mer peptide from the middle of III1 (NAPQ ... TIPG) was prepared with C-terminal amide on a Milligen model 9050 peptide synthesizer using Fmoc chemistry as described (28). The locations of all fragments and the synthetic peptide within Fn are summarized in Fig. 1.


Fig. 1. Schematic illustration of the modular composition of fibronectin and its fragments and the location of the III1 peptide.
[View Larger Version of this Image (20K GIF file)]


Affinity chromatography matrices were prepared by coupling Fn and its fragments to CNBr-activated Sepharose 4B following the directions of the manufacturer (Pharmacia Biotech Inc.). Generally, proteins were coupled in 0.1 M NaHCO3, 0.5 M NaCl, pH 8.0, overnight at 4 °C except for Fn, which was coupled at room temperature. Unreacted sites were blocked with tris(hydroxymethyl)-aminomethane (Tris) buffer. The degree of substitution varied between 3 and 6 mg of protein per ml of packed gel. Matrices that were eluted with urea had been washed with urea previously. Those eluted with guanidinium chloride (GdmCl) were washed only with alternating acid, base, and 1 M NaCl solutions before equilibration with TBS (0.02 M Tris, 0.15 M NaCl, pH 7.4) to prevent possible denaturation of the coupled protein before the first chromatography run. Heat-denatured affinity matrices were prepared by incubating a slurry of the resin in TBS for 10 min at 100 °C. Chromatography was performed at room temperature on a Pharmacia FPLC system. One hundred-µl volumes of protein solutions with A280>= 0.5 were injected onto affinity matrices in TBS at a flow rate of 1.0 ml/min. After a 5-ml wash with TBS, a 10-ml linear gradient from 0 to 6 M urea in TBS or from 0 to 6 M GdmCl in 0.02 M Tris, 1 M NaCl, pH 7.4, was applied. Elution was monitored by absorbance at 280 nm with the Pharmacia detector or by fluorescence at 340 nm with excitation at 280 nm using a Shimadzu RF-535 detector. Elution profiles were corrected for baseline shifts by subtracting the profile obtained when only TBS was injected.

Blocking of sulfhydryl groups was accomplished by incubating immobilized 8-kDa III15 that had been previously heat-denatured with a 100-fold molar excess of HgCl2 for 15 min at room temperature, followed by washing with TBS. Alternatively, 40-kDa Hep-2 was exposed to 20 mM iodoacetamide in the presence of 6 M urea followed by dialysis into 0.1 M NaHCO3, 0.5 M NaCl, pH 8.0, for coupling to Sepharose as above.

SDS-polyacrylamide gel electrophoresis was performed on eluted fractions of a thermolysin digest (1:200 w/w for 4 h at room temperature) of 1.0 mg of Fn that had been applied to a 2.5-ml column of III1 peptide-Sepharose and eluted with a gradient from 0 to 6 M urea. The fractions were concentrated and electrophoresed on 8-25% polyacrylamide gels using a Phastgel system (Pharmacia). Protein bands were stained with Coomassie Brilliant Blue. Urea was from Life Technologies, Inc., and GdmCl was from U. S. Biochemical Corp. All other chemicals were reagent grade or better.


RESULTS

III1 Peptide-Sepharose Binds Many but not All Fibronectin Fragments

The synthetic III1 peptide (14) was coupled to Sepharose, and a thermolysin digest of Fn was applied to the column in TBS. Bound proteins were eluted with a gradient of urea. Pass-through and bound fractions were collected and analyzed by SDS-PAGE (Fig. 2). The nonbinding fraction contained 30- and 40-kDa Hep-2 fragments and two low molecular weight fragments, 9-kDa III1 and 8-kDa III15. Bound fragments included 19-kDa Fib-2, 29-kDa Fib-1, 42-kDa GBF, and 110-kDa CBF. Fn itself bound quantitatively and was eluted at 4 M urea (data not shown).


Fig. 2. III1 peptide-Sepharose binds numerous fragments of fibronectin. Fn was digested for 4 h at room temperature with thermolysin (1:200 w/w) in TBS. The reaction was terminated with EDTA, and the mixture was applied to a 2.5-ml column of III1 peptide-Sepharose, washed with TBS, and eluted with a gradient of 0 to 6 M urea. Nonbound (NB) and bound fractions eluting at successively higher urea concentration (B1, B2, B3, and B4) were collected and analyzed by SDS-PAGE to determine which Fn fragments display affinity for the synthetic peptide from III1. Arrows indicate the migration positions (data not shown) of standard proteins of molecular masses (from top) of 66, 45, 31, 21, and 14 kDa.
[View Larger Version of this Image (65K GIF file)]


Fn and a variety of its purified thermolytic fragments and subfragments were also applied individually to III1 peptide-Sepharose in TBS and eluted with a urea gradient (Fig. 3A). All of the major fragments except 40-kDa Hep-2 and 9-kDa III1 (ITETP... . ) bound to the column and were eluted with urea. The pattern is identical to that observed when the thermolytic digest was applied (Fig. 2), indicating that the fragments that bound in that experiment were interacting directly with the immobilized peptide and not indirectly via other bound fragments. None of the major fragments bound as tightly as Fn itself, as judged by the concentration of urea required for their elution. In additional experiments not shown, the 30- and 21-kDa subfragments of 42-kDa GBF also bound but eluted earlier than the parent fragment, whereas the 56-kDa GBF, which contains type III1, eluted at a similar position as 42-kDa GBF, which does not. Several subfragments of the 110-kDa CBF (25), including 11-kDa III10, 39-kDa III7-8, and 54/60-kDa III2-5/6 either failed to bind or were merely retarded (data not shown).


Fig. 3. The III1 peptide is not active in the native intact domain. Fn and its purified fragments were chromatographed on III1 peptide-Sepharose (left) and on III1-Sepharose (right) in TBS. A gradient to 6 M urea in TBS was applied beginning with the arrows. Elution was monitored by absorbance at 280 nm with a full-scale range of 0.05. The designations on the left refer to both panels. GBF and Hep-2 refer to the 42-kDa and 40-kDa fragments, respectively.
[View Larger Version of this Image (26K GIF file)]


The synthetic III1 peptide is extremely basic with a net positive charge between five and six. The Fn fragments that fail to bind peptide-Sepharose are also basic (30- and 40-kDa Hep-2 and 9-kDa III1), whereas most of those that do bind are acidic. This suggested that electrostatic forces might be responsible for the the observed interactions. To test this possibility, an attempt was made to elute Fn from the peptide column by increasing the ionic strength. No elution was detected with a NaCl gradient up to 2 M. Therefore, the binding must involve other types of forces that are insensitive to ionic strength.

Fibronectin Fragments Do Not Bind Native III1-Sepharose

Fragment 8-kDa III1 (fragment III-1b of Litvinovich et al. (24) with N-terminal sequence IQW ... ) was coupled to Sepharose. Fn and each of its fragments were applied separately in TBS, eluting with a urea gradient (Fig. 3B). No binding was detected with any of the applied proteins. Thus, the binding sites that are present in the synthetic III1 peptide are truly cryptic and not accessible in the native domain from which its sequence was derived. Attempts to render it accessible by exposure of the immobilized domain with 6 M GdmCl (during elution) or by heat (100 °C for 10 min) were unsuccessful, perhaps because of the highly efficient refolding of this domain (24).

Denaturation of Type III-containing Fragments Exposes Binding Sites for Native III1

Fn and all fragments and subfragments were tested on affinity columns containing each of the major fragments, including 29-kDa Fib-1, 42-kDa GBF, 8-kDa III1, 110-kDa CBF, 40-kDa Hep-2, and 19-kDa Fib-2, coupled to Sepharose (Fig. 4). Elution was accomplished with a gradient of 0 to 6 M GdmCl. The only evidence for an interaction was in the case of 9-kDa III1 binding to immobilized 110-kDa CBF and 40-kDa Hep-2. However, the percentage of the applied III1 which bound was low, less than 15%, although the columns contained at least a 5-fold molar excess of the immobilized ligand. All of the columns had been washed with GdmCl prior to application of III1, raising the possibility that the observed binding might have been due to partial denaturation of the immobilized proteins. Therefore, fresh columns of 110-kDa CBF and 40-kDa Hep-2 were prepared, and III1 was applied to determine if it would bind to the native fragments. As shown in Fig. 5, A and B, the amount of protein that was eluted with GdmCl was trivial in comparison to the nonbound fraction.


Fig. 4. Systematic test of interactions between fibronectin fragments by affinity chromatography. Fn and six of its thermolytic fragments were applied to separate columns containing each of the six immobilized fragments, equilibrated, and washed in TBS, then eluted with a gradient from 0 to to 6 M GdmCl plus 1 M NaCl, the start of which is indicated by the arrows. Elution was monitored by intrinsic fluorescence at 340 nm with 280 nm excitation.
[View Larger Version of this Image (49K GIF file)]



Fig. 5. The 110-kDa cell binding and 40-kDa heparin-binding fragments of fibronectin contain cryptic sites for interaction with native 9-kDa III1. Purified 9-kDa III1 was chromatographed on 110-kDa CBF-Sepharose (left panels) and on 40-kDa Hep2-Sepharose (right panels) in TBS before any exposure to GdmCl (A and B), after exposure to GdmCl (C and D), and after heating the affinity matrices at 100 °C for 10 min (E and F). A 10-ml gradient to 6 M GdmCl with 1 M NaCl was used for elution, the start of which is indicated by the arrows. Intrinsic fluorescence at 340 nm with 280 nm excitation was monitored.
[View Larger Version of this Image (27K GIF file)]


After washing the columns with TBS, a second batch of III1 was applied to determine if exposure to 6 M GdmCl during the previous elution would increase the amount of binding. As shown in Fig. 5, C and D, that was indeed the case; approximately 40% of the applied material bound to each column. In the case of the 110-kDa CBF column, the elution occurred in two peaks. Because there was no evidence for heterogeneity of the III1 preparation, this biphasic elution pattern was attributed to heterogeneity of binding sites on the partially denatured immobilized CBF. The percentage of material bound in these experiments is probably underestimated by the fluorescence detector because the native unbound protein has a higher fluorescence efficiency than the denatured bound one, which would be unfolded by the GdmCl used for elution (24).

The affinity resins were then removed from the column, incubated at 100 °C for 10 min, washed with 6 M GdmCl, and again tested in the column. After this treatment, the binding was essentially quantitative in the case of 110-kDa CBF and nearly so with 40-kDa Hep-2 (Fig. 5, E and F). Although the overall recovery of fluorescent material was diminished because of the quenching effect of the GdmCl, the peak representing the unbound unquenched native III1 passing through the column was much lower than in Fig. 5, A and B. The conclusion that both 110-kDa CBF and 40-kDa Hep-2 contain cryptic binding sites for native III1 is inescapable.

Specificity of Binding of Native III1 to Heat-denatured 110-kDa CBF

Fn and its fragments and subfragments were applied to heat-denatured immobilized 110-kDa CBF in TBS followed by elution with a gradient of GdmCl as before. As shown in Fig. 6, the only binding detected was that of native 9-kDa III1 and, to a lesser extent, 56-kDa GBF, which contains III1. A similar pattern was seen with heat-denatured 40-kDa Hep-2 (data not shown). Thus, the binding is quite specific in terms of the number of sites in Fn that recognize heat-denatured type III domains. This is in contrast to the situation with the III1 peptide, assumed to represent denatured III1. Note that the III1 peptide did not bind significantly to the 110-kDa column, whether native or denatured, although the 110-kDa fragment did bind to the immobilized peptide. Note also that Fn itself did not bind to denatured 110-kDa, although it contains III1, indicating that the active site in native III1 is cryptic in whole Fn.


Fig. 6. III1 is the only thermolytic fragment of fibronectin that recognizes cryptic sites in other denatured type III domains. Fn and its fragments were chromatographed on 110-kDa CBF-Sepharose that had been heated at 100 °C for 10 min. A 10-ml gradient to M GdmCl plus 1 M NaCl was used for elution, the start of which is indicated by the arrow. Intrinsic fluorescence at 340 nm with excitation at 280 nm was monitored.
[View Larger Version of this Image (24K GIF file)]


Localization of Binding Sites for Native III1 within Denatured Subfragments of 110-kDa CBF and 40-kDa Hep-2

Subfragments of 110-kDa CBF, including 11-kDa III10, 26-kDa III8-9, 39-kDa III7-8, MBP-III7, and a mixture of 54-kDa III2-5 and 60-kDa III2-6, were coupled to Sepharose and tested for binding of 9-kDa III1 (Fig. 7, A-E, respectively). The only significant binding was seen with 39-kDa III7-8 and MBP-III7, suggesting that III7 is the active module. The amount of binding to these immobilized fragments increased significantly after they had been purposely denatured. The increase was from about 15 to >90% with 39-kDa III7-8 and from about 25 to 50% with MBP-III7. It is clear, however, that some binding occurred, even to the native III7-8 and MBP-III7 columns, suggesting that the active site in III7 is partially exposed in these fragments simply by separating them from other domains. In the case of MBP-III7, it is not certain that all of the recombinant domain was properly folded, even before deliberate denaturation. The other subfragments of 110 kDa as well as MBP alone failed to bind detectable amounts of III1, even after exposure to GdmCl and heat. The failure of 26-kDa III8-9 to show significant binding reinforces the conclusion that III7 is the active module in 39-kDa III7-8. Similar experiments were conducted with 8-kDa III15 and 30-kDa Hep-2 (III12-14), both of which are subfragments of 40-kDa Hep-2 (III12-15). As shown in the lower part of Fig. 7, binding occurred only to 8-kDa III15 (Fig. 7F), and then only after it was denatured. Thus, binding of III1 to denatured 40-kDa Hep-2 appears to occur via module III15.


Fig. 7. Cryptic III1 binding sites are localized to modules III7 and III15. Affinity chromatography of 9-kDa III1 on immobilized subfragments of 110-kDa CBF (A-E) and 40-kDa Hep-2 (F and G): 11-kDa III10 (A), 26-kDa III8-9 (B), 39-kDa III7-8 (C), MBP-III7 (D), 54k/60-kDa III2-5/6 (E), 8-kDa III-15 (F), and 30-kDa III12-14 (G). Fluorescence at 340 nm with excitation at 280 nm was monitored as III1 was applied and eluted with a gradient to 6 M GdmCl/1 M NaCl. ----, before any exposure to GdmCl or heat; --------, after heating the matrices to 100 °C for 10 min. The arrow indicates the start of the 10-ml gradient.
[View Larger Version of this Image (22K GIF file)]


Modules III7 and III15 are unique among type III modules in that each has a free buried sulfhydryl that becomes exposed in the presence of denaturants (29). To determine whether free sulfhydryls might be involved in binding of III1, denatured 8-kDa III15-Sepharose was exposed to 44 mM HgCl2 in TBS for 15 min, a treatment that was shown in separate experiments to block the incorporation of sulfhydryl-specific probes into the protein (data not shown). Such treatment had no effect on the binding of 9-kDa III1. A second experiment in which the sulfhydryl in 40-kDa Hep-2 was blocked with iodoacetic acid in 6 M urea prior to immobilization led to the same conclusion.

Because III7 and III15 are acidic and III1 is basic, we tested whether III1 could be eluted from immobilized heat-denatured 39-kDa III7-8 and 8-kDa III15 with salt alone. No elution was detected up to a NaCl concentration of 1 M.


DISCUSSION

Based on experience with other polymerizing systems such as fibrinogen, it should be possible to localize sites of interaction of Fn by identifying fragments that either self-associate, interact with other fragments, or compete with protomers for binding to complementary polymerization sites and thereby inhibit matrix assembly. Although N-terminal fragments have long been known to inhibit matrix assembly, the sites of their interaction have not been established. The fact that they bind reversibly to cultured cell layers prompted a search for a putative "matrix assembly receptor" (3, 9, 10). Failure to identify a convincing candidate has strengthened the idea that the long sought receptor may be Fn itself, perhaps conformationally altered by its interaction with cells (5, 11, 12). Indeed, experiments in which 125I-labeled 29-kDa Fib-1 was covalently cross-linked to the matrix identified Fn as one of the targets (9, 30).

In the present study, we systematically checked for self-interactions between native fragments of Fn by testing all thermolysin fragments in pairwise fashion by affinity chromatography. Finding no significant interactions between the native fragments, we then followed the lead of Hocking et al. (11, 16) and began to explore the possibility that some of the sites might be cryptic, becoming exposed upon denaturation. The only interaction thus detected was that of native 9-kDa III1 (or 56-kDa GBF, which contains III1) with immobilized denatured type III-containing fragments 110-kDa CBF and 40-kDa Hep-2. These sites were indeed cryptic because: (a) they were much better expressed after denaturation; and (b) immobilized native III1 failed to bind native 110-kDa or 30/40-kDa. The observed binding was rather specific in that III1 was the only native thermolysin fragment that was able to recognize cryptic sites in other heat-denatured type III domains. There was also specificity in terms of the number of type III domains that were recognized by native III1. Testing of a range of subfragments indicated that the ones being recognized upon denaturation were III7 and III15 and that this recognition was independent of the free sulfhydryls contained therein. Subfragment 39-kDa III7-8 and MBP-III7 appeared to bind significant amounts of III1, even before denaturation, as if this site might be at least partially exposed in the smaller subfragment without the need to unfold.

Hocking et al. (16) reported that recombinant III1 (presumably native) as well as whole Fn were able to bind recombinant III10 but only when the latter was adsorbed to plastic wells at 80 °C, just below the midpoint for thermal denaturation of natural 11-kDa III10 (25). However, a slightly shorter version of recombinant III10, truncated by several kilodaltons from the C terminus, was active even in the fluid phase, as evidenced by its ability to partially inhibit the binding of Fn to heated full-length, plastic-adsorbed recombinant III10 (16). This suggested that a binding site for native III1 was exposed in III10 by proteolytic truncation (or by unfolding, if truncation destabilized the compact structure). In our hands, native 9-kDa III1 failed to bind Sepharose-conjugated 11-kDa III10, even after exposure of the latter to harsh denaturing conditions. Unfolding of 11-kDa III10 is highly reversible in solution (25).3 Although it seems unlikely, we cannot exclude the possibility that refolding of immobilized III10 after boiling or exposure to 6 M GdmCl was sufficiently complete to preclude detectable binding of III1 (Fig. 7, top panel). Note also that domain III11 is not represented by any of the fragments studied and thus cannot be excluded as a possible target of binding by III1 or other fragments.

A role for module III1 in self-interactions of Fn was evident from earlier reports that Fn matrix assembly could be inhibited by a monoclonal antibody that mapped to this region (13). Measurements of specific melting enthalpy suggest that III1 differs from other type III domains in having a smaller compact core flanked by unstructured regions that in other type III domains are part of the overall fold (24). Thus, in a sense, module III1, at least when isolated from neighboring domains, is already partly unfolded (Scheme I), and its unstructured regions and/or its exposed core could be available for interaction with complementary regions on other type III domains through what might be termed "subdomain swapping" (31). The fact that chymotrypsin cleaves Fn in III1 after Trp599 (see Fig. 1) (14), which is highly conserved and deeply buried in other type III domains (32-36), indicates that this region of III1 is accessible in whole Fn. Yet, whole Fn fails to bind denatured immobilized 110-kDa CBF (Fig. 6, top) or 40-kDa Hep-2 (data not shown), suggesting that the binding site(s) in III1 are not accessible in the whole protein. The mechanism by which these and other cryptic sites in type III domains would become exposed in native Fn remains unknown but may depend on mechanical forces exerted by cytoskeletal filaments that align with Fn fibrils across the membrane (1, 5, 12, 37, 38). Cytochalasin and other agents that disrupt actin-containing stress fibers also block or reverse Fn matrix assembly and abolish binding sites for N-terminal fragments (5, 39), consistent with the idea that tensile forces expose cryptic sites. Erickson (40) estimated that forces of readily achievable by contracting cells would be sufficient to completely unfold a type III domain with a Delta G of unfolding of ~10 kcal/mol (24). Partial unfolding would require even less energy.


Scheme I. Folding of module III.
[View Larger Version of this Image (19K GIF file)]


The site or sites in native III1 that recognize denatured III7 and III15 appear to be different from that represented by the 31-residue synthetic peptide of Morla and Ruoslahti (14). This peptide, derived from the core of III1, did not bind to denatured 110k-Sepharose under conditions where intact 9-kDa III1 did. However, we did confirm their finding that the 31-residue peptide, when coupled to Sepharose, is able to bind Fn. This is not surprising in view of the fact that the same peptide also binds most thermolysin fragments, including 29-kDa Fib-1, 42- and 56-kDa GBF, 110-kDa CBF, and 19-kDa Fib-2. The site represented by the peptide is truly cryptic because immobilized intact III1 failed to bind Fn or any of its fragments. It is possible that the structure of III1 in whole Fn is altered by interactions that play a role in maintaining the molecule in a soluble conformation. Additional work is required to test this possibility and to determine which of the numerous interactions of III1 are important for matrix assembly, a process which based on electron microscopic studies (41, 42) can be assumed to be orderly and to involve specific recognition sites between molecules in the fibrils.

Protomeric Fn in solution has long been known to undergo reversible changes in its hydrodynamic properties in response to changes in pH and ionic strength (43). This is thought to arise from long range intramolecular electrostatic interactions between oppositely charged "domains" (44-46). One imagines that an interaction within or between chains of protomeric Fn could involve some of the same recognition elements that govern fibril formation and that the role of cells is to somehow open the molecule and facilitate a switch from intra- to intermolecular self-association. The work presented here as well as additional unpublished work in our laboratory reveals a lack of self- or hetero-interactions between native purified fragments, which collectively cover almost the entire chain. It is possible that multiple interactions involving modules from separate thermolytic fragments, perhaps on separate chains of the covalent dimer, are required to stabilize the solution conformation (47). Additional studies are required to elucidate the structure of Fn in solution, that of its fibrils in the extracellular matrix, and the mechanism by which the former is transformed to the latter. The present work provides additional support for the role of cryptic sites in the self-interactions of this complex protein. Some of these sites may be simply masked by domain-domain interactions within the solution conformation, but others appear to be hidden within the folded domains themselves.


FOOTNOTES

*   Supported by U. S. National Institutes of Health Grant HL21791. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Holland Laboratory, 15601 Crabbs Branch Way, Rockville, MD 20855. Tel.: 301-738-0731; Fax: 301-738-0794.
1    The abbreviations used are: Fn, fibronectin; GdmCl, guanidinium chloride; PAGE, polyacrylamide gel electrophoresis; GBF, 42-kDa fragment; Hep-2, 40-kDa fragment.
2    More recent analyses have caused us to revise our earlier assessment of the C termini of the 110-kDa thermolytic fragment and its 39-kDa subfragment (25). Based on identification of all peptides in a CNBr digest of 110-kDa CBF, we conclude that it is lacking domain III11. The 39-kDa subfragment, designated as such because of its mobility on SDS-PAGE, was originally thought to contain type III modules 7-9. It has now been shown by sedimentation equilibrium to have a molecular mass of 25.2 kDa under physiological conditions, consistent with two domains, 7 and 8, plus carbohydrate on domain 7. The presence of carbohydrate was confirmed by direct analysis of sugars. By mass spectroscopic analysis, the fragment has a mass of 22.8 kDa. A recombinant fragment with the same peptide composition but lacking carbohydrate also exhibits abnormal mobility by SDS-PAGE, with an apparent molecular weight of Mr ~36,000, indicating that the abnormal mobility is not dependent on the presence of carbohydrate.
3    K. C. Ingham, and S. V. Litvinovich, unpublished results.

REFERENCES

  1. Hynes, R. O. (1990) Fibronectins, Springer-Verlag, NY
  2. McKeown-Longo, P., and Mosher, D. (1983) J. Cell Biol. 97, 466-472 [Abstract]
  3. McKeown-Longo, P. J., and Mosher, D. F. (1985) J. Cell Biol. 100, 364-374 [Abstract]
  4. Quade, B. J., and McDonald, J. A. (1988) J. Biol. Chem. 263, 19602-19609 [Abstract/Free Full Text]
  5. Mosher, D. F. (1993) Curr. Opin. Struct. Biol. 3, 214-222
  6. Sottile, J., and Wiley, S. (1994) J. Biol. Chem. 269, 17192-17198 [Abstract/Free Full Text]
  7. Ichihara-Tanaka, K., Titani, K., and Sekiguchi, K. (1995) J. Cell Sci. 108, 907-915 [Abstract/Free Full Text]
  8. Sechler, J. L., Takada, Y., and Schwarzbauer, J. E. (1996) J. Cell Biol. 134, 573-583 [Abstract]
  9. Limper, A. H., Quade, B. J., LaChance, R. M., Birkenmeier, T. M., Rangwala, T. S., and McDonald, J. A. (1991) J. Biol. Chem. 266, 9697-9702 [Abstract/Free Full Text]
  10. Moon, K.-Y., Shin, K. S., Song, W. K., Chung, C. H., Ha, D. B., and Kang, M.-S. (1994) J. Biol. Chem. 269, 7651-7657 [Abstract/Free Full Text]
  11. Hocking, D. C., Sottile, J., and McKeown-Longo, P. J. (1994) J. Biol. Chem. 269, 19183-19191 [Abstract/Free Full Text]
  12. Mosher, D. F. (1995) Thromb. Haemostasis 74, 529-533 [Medline] [Order article via Infotrieve]
  13. Chernousov, M. A., Fogerty, F. J., Koteliansky, V. E., and Mosher, D. F. (1991) J. Biol. Chem. 266, 10851-10858 [Abstract/Free Full Text]
  14. Morla, A., and Ruoslahti, E. (1992) J. Cell Biol. 118, 421-429 [Abstract]
  15. Morla, A., Zhang, Z., and Ruoslahti, E. (1994) Nature 367, 193-196 [CrossRef][Medline] [Order article via Infotrieve]
  16. Hocking, D. C., Smith, R. K., and McKeown-Longo, P. J. (1996) J. Cell Biol. 133, 431-444 [Abstract]
  17. Morgenthaler, J. (1982) FEBS Lett. 150, 81-84 [CrossRef]
  18. Hayashi-Nagai, A., Kitagaki-Ogawa, H., Matsumoto, I., Hayashi, M., and Seno, N. (1991) J. Biochem. (Tokyo) 109, 83-88 [Abstract]
  19. Harumiya, S., Jung, S.-K., Sakano, Y., and Fujimoto, D. (1993) J. Biochem. (Tokyo) 113, 710-714 [Abstract]
  20. Miekka, S. I., Ingham, K. C., and Menache, D. (1982) Thromb. Res. 27, 1-14 [Medline] [Order article via Infotrieve]
  21. Borsi, L., Castellani, P., Balza, E., Siri, A., Pellecchia, C., De Scalzi, F., and Zardi, L. (1986) Anal. Biochem. 155, 335-345 [Medline] [Order article via Infotrieve]
  22. Ingham, K. C., Brew, S. A., Migliorini, M. M., and Busby, T. F. (1993) Biochemistry 32, 12548-12553 [Medline] [Order article via Infotrieve]
  23. Ingham, K. C., Brew, S. A., and Migliorini, M. M. (1989) J. Biol. Chem. 264, 16977-16980 [Abstract/Free Full Text]
  24. Litvinovich, S. V., Novokhatny, V. V., Brew, S. A., and Ingham, K. C. (1992) Biochim. Biophys. Acta 1119, 57-62 [Medline] [Order article via Infotrieve]
  25. Litvinovich, S. V., and Ingham, K. C. (1995) J. Mol. Biol. 248, 611-626 [CrossRef][Medline] [Order article via Infotrieve]
  26. Novokhatny, V. V., and Ingham, K. C. (1994) J. Mol. Biol. 238, 833-844 [CrossRef][Medline] [Order article via Infotrieve]
  27. Busby, T. F., Argraves, W. S., Brew, S. A., Pechik, I., Gilliland, G. L., and Ingham, K. C. (1995) J. Biol. Chem. 270, 18558-18562 [Abstract/Free Full Text]
  28. Ingham, K. C., Brew, S. A., and Migliorini, M. (1994) Arch. Biochem. Biophys. 314, 242-246 [CrossRef][Medline] [Order article via Infotrieve]
  29. Wagner, D. D., and Hynes, R. O. (1979) J. Biol. Chem. 254, 6746-6754 [Abstract]
  30. Barry, E. L. R., and Mosher, D. F. (1988) J. Biol. Chem. 263, 10464-10469 [Abstract/Free Full Text]
  31. Bennett, M. J., Schlunegger, M. P., and Eisenberg, D. (1995) Protein Sci. 4, 2453-2468 [Free Full Text]
  32. Main, A. L., Harvey, T. S., Baron, M., Boyd, J., and Campbell, I. D. (1992) Cell 71, 671-678 [Medline] [Order article via Infotrieve]
  33. Leahy, D. J., Hendrickson, W. A., Aukhil, I., and Erickson, H. P. (1992) Science 258, 987-991 [Medline] [Order article via Infotrieve]
  34. Leahy, D. J., Aukhil, I., and Erickson, H. P. (1996) Cell 84, 155-164 [Medline] [Order article via Infotrieve]
  35. Dickinson, C. D., Veerapandian, B., Dai, X., Hamlin, R. C., Xuong, N., Ruoslahti, E., and Ely, K. R. (1994) J. Mol. Biol. 236, 1079-1092 [Medline] [Order article via Infotrieve]
  36. Huber, A. H., Wang, Y., Bieber, A. J., and Bjorkman, P. J. (1994) Neuron 12, 717-731 [Medline] [Order article via Infotrieve]
  37. Halliday, N. L., and Tomasek, J. J. (1995) Exp. Cell Res. 217, 109-117 [CrossRef][Medline] [Order article via Infotrieve]
  38. Zhang, Q., Checovich, W. J., Peters, D. M., Albrecht, R. M., and Mosher, D. F. (1994) J. Cell Biol. 127, 1447-1459 [Abstract]
  39. Wu, C., Keivens, V. M., O'Toole, T. E., McDonald, J. A., and Ginsberg, M. H. (1995) Cell 83, 715-724 [Medline] [Order article via Infotrieve]
  40. Erickson, H. P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10114-10118 [Abstract/Free Full Text]
  41. Peters, D. M. P., Portz, L. M., Fullenwider, J., and Mosher, D. M. (1990) J. Cell Biol. 111, 249-256 [Abstract]
  42. Dzamba, B. J., and Peters, D. M. P. (1991) J. Cell Sci. 100, 605-612 [Abstract]
  43. Alexander, S. S., Jr., Colonna, G., and Edelhoch, H. (1979) J. Biol. Chem. 254, 1501-1505 [Abstract]
  44. Williams, E. C., Janmey, P. A., Ferry, J. D., and Mosher, D. F. (1982) J. Biol. Chem. 257, 14973-14978 [Abstract/Free Full Text]
  45. Markovic, Z., Lustig, A., Engel, J., Richter, H., and Hormann, H. (1983) Hoppe-Seyler's Z. Physiol. Chem. 364, 1795-1804 [Medline] [Order article via Infotrieve]
  46. Hormann, H., and Richter, H. (1986) Biopolymers 25, 947-958 [Medline] [Order article via Infotrieve]
  47. Lai, C.-S., Wolff, C. E., Novello, D., Griffone, L., Cuniberti, C., Molina, F., and Rocco, M. (1993) J. Mol. Biol. 230, 625-640 [CrossRef][Medline] [Order article via Infotrieve]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.