From the Biochemistry Department, Jerome H. Holland Laboratory for the Biomedical Sciences, American Red Cross, Rockville, Maryland 20855
Received for publication, December 9, 2002, and in revised form, January 14, 2003
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ABSTRACT |
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The gelatin-binding sites of fibronectin are
confined to a 42-kDa region having four type I and two type II modules
in the following order:
I6-II1-II2-I7-I8-I9.
To determine the relative importance of each module for recognition of
gelatin, recombinant green fluorescent fusion proteins were prepared in
which individual modules or groups of modules were deleted, and the
resulting proteins were tested for binding to gelatin by analytical
affinity chromatography. Deletion of both type II modules did not
eliminate binding, confirming that at least some of the type I modules
in this region are able to bind gelatin. It was found that deletion of
type I module 6 tends to increase the affinity, whereas deletion of any
other module decreases it. Deletion of module I9 had
a large effect but only if module II2 was also
present, suggesting an interaction between these two noncontiguous
modules. Analysis of more than 20 recombinant fusion products led to
the conclusion that all modules contribute to the interaction either
directly by contacting the ligand or indirectly through module-module interactions.
Fibronectin is a large modular glycoprotein found in the
extracellular matrix and body fluids of higher organisms. It interacts with a variety of other macromolecules including integrin receptors, heparan sulfate proteoglycans, fibrin, tenascin, and several types of
denatured collagens (gelatins). Here we focus on the interaction with
gelatin. The gelatin-binding sites of fibronectin are confined to a
region having four type I and two type II modules in the following
order:
I6-II1-II2-I7-I8-I9
(Fig. 1). This region can be isolated as a 42-kDa fragment (42-kDa
GBF)1 that binds to gelatin
with affinity only slightly lower than that of the parent protein (1).
Fragments outside this region including an amino-terminal 29-kDa
fragment (type I modules 1 through 5) and a carboxyl-terminal 19-kDa
fragment (type I modules 10 through 12) do not bind gelatin. The 42-kDa
fragment contains the only type II modules in fibronectin, modules that
are thought to play a role in binding to gelatin not only in
fibronectin but in other gelatin-binding proteins (especially some
matrix metalloproteinases where type II modules are also found) (2).
However, the 42-kDa GBF can be further cleaved into two non-overlapping
subfragments, I6-II1-II2-I7 and
I8-I9, both of which also bind gelatin, albeit with ~10-fold lower affinity than the parent fragment (3). This
suggests that type II modules are not essential for binding of fibronectin to gelatin, although they may contribute, and that some
type I modules can bind. It was recently reported that a recombinant
two-module fragment, II1-II2, bound weakly to
gelatin but that the affinity was about 5-fold higher in a
three-module fragment, I6-II1-II2
(4). The latter fragment was shown in the same study to have a
hairpin-like structure in which module I6 interacts
strongly with II2 despite the intervening II1.
This highlights the fact that affinity for gelatin may be affected not
only by the presence of a given module but by the interaction of that
module with neighboring modules.
In the present study we have prepared a large number of
recombinant fragments in which one or more modules have been deleted from 42-kDa GBF to further understand the importance of all the modules
for binding to gelatin. We have shown that modules
II2 and I9 are most critical for binding but
that all six modules contribute directly or indirectly to the binding site.
Materials--
Polyclonal anti-green fluorescent protein (GFP)
antibody was purchased from Clontech.
Spodoptera frugiperda (Sf9) cells adapted to grow in
serum-free medium Sf900-II were obtained from Invitrogen. Swine
skin gelatin (type I) was from Sigma. The 42-, 30-, and 21-kDa
gelatin-binding fragments were purified from a thermolysin digest of
human plasma fibronectin as described
previously (5).
Plasmid Constructs--
A vector (pENTR11-MHG) encoding a
secretion signal, His6 tag, and GFP tag at the amino
terminus was described previously (6). cDNAs encoding the
gelatin-binding domain of rat fibronectin and its variants were
amplified by Pfu DNA polymerase (Stratagene) followed by
BamHI-HindIII digestion and then subcloned into
BglII-HindIII sites of pENTR11-MHG. The primers
used for amplification are given in Table
I where amino acids are numbered from the
amino terminus (pyroglutamic acid) of the processed fibronectin. The
inserts were transferred to pDEST8 (Invitrogen) using the Gateway
cloning system (Invitrogen).
Preparation of Recombinant Fragments--
Recombinant fragments
were expressed in Sf9 insect cells using the Bac-to-Bac
expression system (Invitrogen). A bacterial strain (DH10Bac) was
transformed with recombinant plasmids, and colonies containing
recombinant bacmids were grown for isolation of the resulting viral DNA
according to the manufacturer's instructions. Supernatants of
transfected Sf9 cells were used to infect more Sf9 cells
through two or three stages of amplification. Sf9 cells (2 × 108) were then infected with recombinant viruses, and
supernatants were harvested 48 or 72 h after infection.
Recombinant GFP-fibronectin fragments were purified on Talon
metal affinity resin (Clontech), eluting with 150 mM imidazole, and then dialyzed against phosphate-buffered saline (PBS, pH 7.4). The identity of each fragment was verified by
detection of the appropriate viral DNA in the infected cells with
PCR and by the apparent molecular weight of the purified protein
on SDS-PAGE. Yields varied between 0.5 and 1.2 mg/100 ml of culture supernatant.
Gelatin Affinity Chromatography--
Analytical affinity
chromatography was performed as described previously using an FPLC
system (Amersham Biosciences) (7). Briefly, recombinant protein
was injected onto a gelatin-Sepharose column equilibrated with PBS and
pumped at a flow rate of 1 ml/min. After washing, a linear gradient of
urea (0-6 M) was applied between 10 and 30 min, and the
bound GFP fusion proteins were detected with a fluorescence monitor
(Shimadzu Model RF535). In some experiments, unbound and bound
fractions were collected and subjected to immunoblot analysis using
anti-GFP antibody (8). The variable amount of unbound green
fluorescence in the analytical runs is due to varying degrees of
proteolytic cleavage at the flexible linker between GFP and the GBFs
during handling and/or storage of the samples.
Solid-phase Binding Assay--
Ninety-six-well microtiter plates
were coated with 10 µg/ml gelatin in PBS for 16 h at 4 °C.
After washing and blocking with 1% bovine serum albumin in PBS, the
plates were incubated with various concentrations of recombinant
proteins for 2 h at room temperature. Plates were washed three
times with 0.2% bovine serum albumin in PBS and then incubated with
polyclonal anti-GFP antibody for 1 h at room temperature followed
by incubation with horseradish peroxidase-conjugated donkey anti-rabbit
IgG antibody for 40 min at room temperature. After three washes with
PBS, plates were developed with peroxidase substrate. Absorbance
was measured with a Dynatec plate reader, and
C50% values were obtained by fitting the data
to a simple binding isotherm using Sigmaplot.
Since each of the six modules in the 42-kDa gelatin-binding
fragment of fibronectin has a unique number, 6, 1, 2, 7, 8, and 9, those numbers are used as a shorthand notation to
specify the modular composition of the various mutants, and dashes (-)
are used to designate deletions. Various combinations of these modules were expressed as fusion proteins with enhanced GFP using a baculovirus expression system. Initially we used gelatin-Sepharose in conjunction with SDS-PAGE and immunoblotting to assess binding. Typical results are
shown in Fig. 2 where it can be seen that
the pair of type II modules alone does not bind to gelatin-Sepharose,
whereas a fragment containing type I modules 7, 8, and 9 does bind.
These data suggest that type II modules alone are neither sufficient nor essential for binding to gelatin and that at least some type I
modules are able to bind without the help of type II modules.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic illustration of the modular
composition of human plasma fibronectin and its gelatin-binding
region. Type I modules are shown as ovals, type II
modules are shown as circles, and type III modules are shown
as squares; the small diamonds designate
glycosylation sites. The gelatin-binding domain has modular composition
I6-II1-II2-I7-I8-I9,
referred to herein as 612789. Also shown is a rendition of the
structure of subfragment I6-II1-II2
illustrating the folding back of module II2 onto
I6 (4).
Summary of primers used for the various GFP fusion proteins
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
SDS-PAGE and immunoblotting of recombinant
GFP fusion proteins containing the 42-kDa gelatin-binding fragment of
fibronectin, designated 612789, and two of its subfragments, -12--- and
---789. Lanes 1, starting material; lanes 2,
unbound fraction; lanes 3, bound fraction.
Analytical affinity chromatography on gelatin-Sepharose was
used to rank the relative affinities of the various fusion proteins using green fluorescence to monitor elution in a urea gradient. The
results for all products are presented in Fig.
3, where panel A
refers to the wild type fusion protein, designated 612789. Its peak
elution occurs at 3.3 M urea, very close to that observed with natural 42-kDa GBF as reported previously (6). This elution profile is reproduced as a dotted line in all other panels
for easy comparison. Except for those panels where no peak is evident, the profiles have been normalized to give the same amplitude at their
peak elution. The urea concentrations at peak elution are summarized in
Table II.
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The next six panels, B through G, in Fig. 3 show results for all single module deletions from amino to carboxyl terminus, respectively. Although none of these deletions abolishes binding, all of them have a measurable effect on the elution position. Interestingly removal of module 6 actually increases the concentration of urea required for elution to about 3.7 M (panel B). The other deletions shift the elution to lower urea in varying degrees with loss of module 7 having the smallest effect and module 9 having the largest. These results alone lead to the conclusion that all six modules play a role, directly or indirectly, in binding to gelatin.
Panels G, H, and I of Fig. 3 show the effects of successive deletions from the carboxyl-terminal end. Once module 9 has been removed, further deletion of module 8 has very little effect as if its only role is to tether 9 in the appropriate position. Further deletion of module 7 yields a three-module fragment, 612---, which fails to bind to gelatin-Sepharose, although slight retardation is evident. This lack of binding of 612--- was confirmed in a separate experiment using an immunoblotting approach like that in Fig. 2 (data not shown).
Panels J, K, and L of Fig. 3 represent successive deletions of modules from the amino-terminal end. As noted above, deletion of module 6 increases the affinity (panel J is identical to panel B). Further deletion of module 1 seems to reverse the effects of deleting module 6 so that the elution of the four-module fragment --2789 is indistinguishable from that of the full-length fragment. The next deletion produces the three-module fragment ---789, which despite its lack of type II modules is able to bind (as in Fig. 2), albeit with lower affinity than --2789. This proves again that type II modules are not essential for binding of fibronectin to gelatin, although the second one has some effect.
Fig. 3, panel M, shows the results for a four-module fragment lacking both terminal type I modules 6 and 9. It was shown above (panel G) that deletion of module 9 causes the largest decrease in affinity of any single module deletion, while deletion of module 6 (panel B) caused an increase in affinity. Here it is seen that the enhancing effect of deleting module 6 also occurs upon its removal from 61278-, shifting the peak elution from 2.15 to 2.45 M urea and partially compensating for the effect of removing module 9.
Deletion of either terminal module 1 or 8 from -1278- had very little further effect (Fig. 3, panels N and O), but deletion of both to produce the bimodular fragment --27-- (panel P) caused a noticeable weakening with substantial amounts of protein bleeding from the column during the wash phase prior to application of the urea gradient. Module 7 binds slightly better when partnered with module 8 (panel R) than with module 2 (panel P). Panel Q shows that a lone pair of type II modules (-12---) does not bind tightly enough to survive the wash phase, consistent with its absence in the sixth lane of Fig. 2. However, deletion of this type II pair from the full-length fragment to yield 6--789 significantly lowers the affinity (panel S).
Fragment --2789 is the only four-module fragment that binds with full affinity. Removal of either of its terminal modules substantially lowers that affinity by a similar amount (Fig. 3, panels K, L, and N). However, deletion of the interior modules 7 and 8 from --2789 generates a bimodular fragment containing only 2 and 9, which failed to bind with significant affinity although retardation was evident (panel T). If modules 7 and 8 in either --2789 or in the full-length fragment are replaced by homologous modules 4 and 5 from outside the gelatin-binding region, the binding is essentially abolished (panels U and V). The effect of deleting modules 7 and 8 from the full-length fragment (panel W) is much less severe than replacing them with modules 4 and 5. That modules 7 and 8 serve more than a spacer function is evident from the fact that they can bind independently as a pair (panel R). From the foregoing results it is apparent that all six modules in the gelatin-binding domain of fibronectin play a role in ligand recognition, either directly through contact with gelatin or indirectly by influencing neighboring modules.
ELISA analysis was used as an alternative measure of the affinity of
some of the fusion proteins for gelatin. Plastic microtiter wells were
coated with gelatin and incubated with increasing concentrations of the
fusion protein, and binding was detected immunologically with antibody
to GFP. Representative data are shown in Fig.
4. The concentrations required for
half-saturation (C50%) in this and other
experiments not shown varied between 0.2 and ~5 µM as
summarized in Table II. The range of values of
C50% obtained for the full-length fragment is
in good agreement with Kd values between 0.6 and 1.2 µM determined previously in the fluid phase (Ref. 3 and
references therein). In Fig. 5, the
values of C50% are plotted against the urea
concentration required for peak elution. Despite considerable scatter
in the data, there is a reasonable correlation. Most of the scatter can be attributed to uncertainties in the C50%
values determined by ELISA, which is not the optimum method for weak
interactions. The affinity chromatography results, although only
qualitative, are quite reproducible and thus provide a more reliable
determination of the rank order of affinities.
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The difference in urea concentration required for elution
of any mutant relative to the full-length fragment, calculated from the
data in column 3 of Table II, or for any pair of mutants differing by a
single module is summarized visually in Fig.
6 where results are grouped according to
the module that was deleted. Note how module 6 stands out from the
others in that its deletion consistently enhances binding, whereas most
of the others, especially 1 and 9, have a weakening effect when
deleted.
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DISCUSSION |
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The results presented here indicate that recognition of gelatin is a robust property of fibronectin. Any one of the six modules within the 42-kDa gelatin-binding domain (612789) can be deleted without abrogation of binding. At the same time, all six modules appear to play some role since each of their deletions has a measurable effect on affinity. Thus the situation is more complicated than is often found with domain deletion experiments of this type. For example, the fact that fragment --2789 binds with the same affinity as the full-length fragment would suggest that modules 6 and 1 are not important. But this conclusion is rendered invalid by the observation that removal of 6 alone actually increases the affinity, while removal of 2 alone decreases it by a similar degree. When both are removed the effects appear to compensate.
There are two ways in which a given module might contribute to the interaction with gelatin. The first is direct, i.e. the module engages the ligand, and the other is indirect, i.e. the module interacts with and affects the conformation or disposition of neighboring modules that actually make contact. Some modules could operate at both of these levels. The three-dimensional structure of the trimodular fragment I6II1II2 (Fig. 1) shows that modules need not be contiguous to engage in such interactions; I6 forms an extensive interface with II2 despite the intervening II1 (4). The carboxyl terminus of II2 in that structure is located such that the following module, I7, could also interact with I6 as was inferred from earlier observations that the thermal stability of I6 was greater in fragments where I7 was present (5). Thus, there is reason to suspect that the gelatin-binding domain of fibronectin is folded back upon itself providing numerous opportunities for interactions between noncontiguous modules such that the deletion of any one of them could perturb the overall arrangement of the others.
Additional evidence for long range interaction between modules comes
from examination of the effect of deleting module 9. As shown in Fig.
6, deletion of this module from 612789, -12789, or --2789 consistently
reduces the affinity for gelatin by a similar amount; the
[urea] values vary between
1.1 and
1.25 M (Fig. 6). The fact that this effect is lost in fragment ---789 could suggest
some type of interaction between modules 2 and 9 that is important for
binding. It should also be mentioned that deletion of modules could
allow new interactions between other modules. For example, a weak
interaction between modules 1 and 2 that was observed in the isolated
pair (9) was not present in a larger fragment that also contained
module 6 (4).
Replacement of type I modules 7 and 8 with type I modules 4 and 5 from the amino-terminal region abolishes binding both in the full-length fragment and in --2789. This is consistent with modules 7 and 8 serving as more than mere spacers as further evidenced by the fact that the pair alone is able to bind even better than the pair of type II modules. Modules 4 and 5 constitute a rigid pair with an extensive interface (10). Whether a similar interaction exists between modules 7 and 8 remains to be determined. There is no such interaction between type I modules 1 and 2 in the fib-1 region of fibronectin (11). If the connection between modules 7 and 8 is flexible, then replacing them with modules 4 and 5 may introduce constraints that prevent other interactions that are important for the overall structure and for binding. This is consistent with the fact that simply deleting modules 7 and 8 had less effect than replacing them with 4 and 5.
Module 6 stands out as the only one whose deletion actually enhances
binding (Fig. 6). Note, however, that the enhancing effect is greatly
diminished in fragments that lack module 2. This suggests that the
above-mentioned interaction between modules 2 and 6, which is well
documented in the known structure of 612 (Fig. 1), imposes a constraint
on the overall structure that slightly impedes the interaction with
gelatin. This is opposite to what was reported by the group who
determined that structure. Pickford et al. (4) reported that
both 612--- and -12--- bound to gelatin-Sepharose and provided evidence
from surface plasmon resonance measurements to suggest that removal of
module 6 from 612--- increased the affinity toward immobilized (1)
chain of human type I collagen ~10-fold. The discrepancy could relate
to the different species of collagen and fibronectin used in the two
studies.2 The
Kd values that were estimated from limited SPR data by Pickford et al. (4) are designated by + symbols in our Fig. 5. Their position on the urea axis would
be consistent with the failure of our corresponding fragments, if they
had similar affinities, to bind to our gelatin-Sepharose.
A recent report from this laboratory highlighted the fact that each of
the chains in type I collagen contains multiple sites with similar
affinity for 42-kDa GBF (12). The nature of those sites remains
unclear. Since it is denatured collagen, i.e. gelatin, that
is recognized by fibronectin, it is assumed that what is being
recognized is some linear sequence as opposed to a well defined
tertiary structure. As of yet the relevant sequence has not been
defined, i.e. there is no synthetic collagen-like peptide that has been shown to bind fibronectin with significant affinity. It
is not even certain whether the various active subfragments of 42-kDa
GBF, such as those described here, are all recognizing the same
sequence in gelatin. It is conceivable that neighboring sequences of
the collagen chains interact with different parts of 42-kDa GBF. This
would provide additional opportunities for module-module interactions
to play a role in defining the binding site. The present analysis of
more than 20 recombinant fusion products clearly illustrates that all
six modules in 42-kDa GBF contribute to its interaction with gelatin
either directly by contacting the ligand or indirectly through
module-module interactions. Determining which is which will probably
require a high resolution structure of a complex between GBF and an
appropriate segment of gelatin.
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FOOTNOTES |
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* 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.
Present address: NHLBI, National Institutes of Health, Bethesda,
MD 20892.
§ To whom correspondence should be addressed: Dept. of Biochemistry, American Red Cross Holland Laboratory, 15601 Crabbs Branch Way, Rockville, MD 20855-2743. Tel.: 301-738-0731; E-mail: ingham@usa.redcross.org.
Published, JBC Papers in Press, January 21, 2003, DOI 10.1074/jbc.M212512200
2
We routinely use swine skin gelatin for affinity
chromatography, and the recombinant fragments used in the current study
were encoded by cDNA from rat fibronectin. The full-length
fragment behaves identically to natural 42-kDa GBF derived from
human plasma fibronectin. The species of gelatin used by Pickford
et al. (4) was not specified. We also find that the affinity
of the 42-kDa GBF for chains and CNBr fragments of bovine
type I collagen (Ref. 12 and Y. Katagiri, S. A. Brew, and K. C. Ingham, unpublished results) is significantly lower than for those
from rat tail collagen (1).
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ABBREVIATIONS |
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The abbreviations used are: GBF, gelatin-binding fragment; GFP, green fluorescent protein; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay.
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