(Received for publication, July 5, 1995; and in revised form, August 23, 1995)
From the
The small splice variant of tenascin-C (TN) has eight fibronectin type III (FN3) domains. The major large splice variant has three (in chicken) or seven (in human) additional FN3 domains inserted between domains five and six. Chiquet-Ehrismann et al. (Chiquet-Ehrismann, R., Matsuoka, Y., Hofer, U., Spring, J., Bernasconi, C., and Chiquet, M. (1991, Cell Regul. 2, 927-938) demonstrated that the small variant bound preferentially to fibronectin in enzyme-linked immunosorbent assay, and only the small variant was incorporated into the matrix by cultures of chicken fibroblasts. Here we have studied human TN, and confirmed that the small variant binds preferentially to purified fibronectin and to fibronectin-containing extracellular matrix. Thus this differential binding appears to be conserved across vertebrate species. Using bacterial expression proteins, we mapped the major binding site to the third FN3 domain of TN. Consistent with this mapping, a monoclonal antibody against an epitope in this domain did not stain TN segments bound to cell culture matrix fibrils. The enhanced binding of the small TN variant suggests the existence of another, weak binding site probably in FN3 domains 6-8, which is only positioned to bind fibronectin in the small splice variant. This binding of domains 6-8 may involve a third molecule present in matrix fibrils, as the enhanced binding of small TN was much more prominent to matrix fibrils than to purified fibronectin.
Tenascin-C (TN) ()is a large extracellular matrix
(ECM) protein localized in many embryonic and some adult tissues
(reviewed in (1) and (2) ). The distribution of TN is
determined primarily by the differential expression of the cells that
secrete it, but probably also involves binding to specific ECM
molecules, including collagens, proteoglycans, and fibronectin. Binding
of TN to collagens and proteoglycans appears to be important in several
functions, but the present study will focus on the interaction of TN
with fibronectin (FN).
Several studies have examined the binding of TN to FN. Chiquet-Ehrismann et al.(3, 4) demonstrated binding of soluble TN, in the range of 5-50 µg/ml, to FN-coated plastic. Faissner et al.(5) and Lightner and Erickson (6) reported no binding in a similar ELISA assay, but their maximum concentrations of soluble TN were less than 2 µg/ml. Because ELISA assays have one component partially denatured on plastic, and the binding is usually limited to a very small fraction of the soluble component, it is important to have independent assays of binding. Lightner and Erickson (6) developed a sedimentation assay that demonstrated binding of TN to FN with both molecules native and soluble. The interaction was obvious at 50 µg/ml FN and stronger at 200 µg/ml, indicating a dissociation constant in the range of 0.2 µM relative to the FN dimer.
Complementing the biochemical demonstration of TN binding to FN, co-distribution of TN with FN fibrils has been demonstrated clearly both in cell cultures and embryos. Chiquet and Fambrough (7) demonstrated substantial overlap of TN with FN immunostaining in primary fibroblast culture. Riou et al.(8) provided convincing evidence that TN was distributed together with FN in the dorsal region of the amphibian embryo. Subsequently, they showed that when TN was injected into the blastocoel cavity of living embryos at the late blastula stage, the injected TN bound to fine FN-containing fibrils assembled at the midblastula stage as well as to the complex FN-rich ECM observed at late gastrula stage(9) . Therefore, it is evident that TN can bind to FN fibrils assembled in cell culture and embryonic tissues.
Vertebrate tenascins have several splice variants, in which a number of FN3 domains, indicated by letters A-D, are inserted between FN3 domains 5 and 6 (Fig. 1). The number of alternative splice domains varies according to the species. In chicken TN the most common splice variants have 0, 1, or 3 added domains, while in human they have 0, 1, or 7(1) . We will refer to the form with 0 added domains as small TN, and the forms with 3 or 7 added domains as large TN. It is reasonable to expect that the alternative splice domains might bind to unique ligands, and indeed we have recently demonstrated that the human large TN binds to a cell-surface receptor, annexin II(10) , through its alternative splice segment, TNfnA-D. More surprising are reports that small TN binds to some ligands more avidly than large TN. Zisch et al.(11) observed that only small TN bound to the cell surface molecule F11/contactin. Chiquet-Ehrismann et al.(4) reported that small TN bound more avidly to FN in ELISA, and that the TN incorporated into FN matrix fibrils in vivo was almost exclusively the small variant. Both of these studies used chicken TN.
Figure 1: The domain structure of human TN subunit is shown above, and the bacterial expression proteins used in this study shown below (see (12) for more details). The large TN splice variant, HxB.L, contains the shaded FN3 domains lettered A-D; HxB.S is missing these seven domains. Bacterial protein TNfnALL contains all 15 FN3 domains of HxB.L, and TNfn1-8 contains only the 8 domains of HxB.S.
We decided to pursue this interesting finding to determine first if it held for species other than chicken, and to begin mapping the interaction sites. We employed the recombinant human TN proteins HxB.L and HxB.S, which correspond to the large and small splice variants of human TN. These are produced by transfected BHK cells and are fully assembled into native hexabrachions(12) . In addition we used bacterial expression proteins corresponding to defined small segments of FN3 domains to map the binding sites in TN. Binding measured in solution, in ELISA, and in cell culture gave consistent demonstration of the preferential binding of the small splice variant, and mapping of the primary binding site.
For testing the binding of exogenous proteins to preformed ECM, mouse fibroblast NIH 3T3 or BHK cells were grown to confluence in Dulbecco's modified Eagle's medium with high glucose supplemented with 10% fetal calf serum. After five days, the medium was removed and substituted with Dulbecco's modified Eagle's medium with high glucose, 10% fetal calf serum, and 20 µg/ml of purified large or small human TN or recombinant proteins. After 24 h cells were washed three times with PBS and fixed in cold methanol or acetone:methanol (1:1 by volume) for 20 min. Indirect immunofluorescence used monoclonal Abs that recognize both human TN splice variants, but do not react with mouse TN.
Figure 2: Glycerol gradient sedimentation in 200 mM ammonium bicarbonate of HxB.L and HxB.S in the absence or continuous presence of 100 µg/ml FN. Fractions were assayed by a dot-blot with anti-TN polyclonal Ab. Arrowheads indicate the TN peaks.
In
order to map the domain(s) of TN that bind to FN, recombinant TN
segments were added to the gradient along with FN to test for
competition. These experiments were done in TBS buffer, in which the
shift in TN sedimentation with FN is somewhat larger than in ammonium
bicarbonate. As shown in Fig. 3, TNfn6-8 and TNfnA-D
produced a small displacement of the TNFN complex, while
TNfn3-5 and TNfn3 produced a large displacement, shifting the TN
peak almost back to its position in the absence of FN. A strong
disruption of the TN
FN complex by TNfn3, TNfn3-5, and
TNfn1-5 was consistently observed in several experiments, in both
the TBS and ammonium bicarbonate gradients. TNfn6-8, TNfbg, and
HxB.egf did not show significant displacement of the acceleration of TN
in ammonium bicarbonate (not shown). TNfnA-D showed weak displacement
activity in some experiments in both TBS and ammonium bicarbonate, but
no activity in other experiments. Thus, the major binding site for FN
binding appears to be the domain TNfn3.
Figure 3:
Glycerol gradients of TN (100 µg/ml,
from U-251MG conditioned medium, about 90% large TN) sedimented in the
continuous presence of FN (100 µg/ml) and recombinant TN fragments
(100 µg/ml). The lower band present in all fractions is
FN. The upper band is the large splice variant of TN. In this
experiment, TNfn6-8 and TNfnA-D produced a small displacement in
sedimentation of the TNFN complex; TNfn3-5 and TNfn3
produced large displacements.
Figure 4: Differential binding of HxB.L and HxB.S to FN demonstrated by ELISA. FN (2 µg in 100 µl of PBS per well) was coated overnight at 4 °C, and the remaining binding sites were blocked by incubation with 5% non-fat dry milk for 1 h at 37 °C. Various concentrations of HxB.L and HxB.S were incubated with the FN-coated plastic for 2 h at 37 °C, and the amount of bound TN was measured by assays with polyclonal Ab 9172, which recognizes TNfn1-5. The values represent the mean of three determinations. 10 µg/ml corresponds to 60 and 40 nM subunits for HxB.S and HxB.L respectively.
The bacterial expression proteins were again used as competitors to map the binding domain (Fig. 5). All fragments containing TNfn3 gave strong competition, consistent with mapping in the solution-phase assay. TNfn1-5, TNfn1-8, and TNfn3-5 showed the strongest competition at similar molar concentrations. The single domain TNfn3 required a 3-fold higher molar concentration for similar competition. Thus, TNfn3 appears to be the major FN binding site in TN, but additional binding sites in TNfn4-5 may enhance binding.
Figure 5: Mapping the FN-binding site in TN by competition ELISA. Plastic wells were coated with FN as for Fig. 4. After washing, HxB.S was incubated in the absence or presence of various concentrations of recombinant TN fragments at 37 °C for 2 h. After incubation, wells were washed and the amount of bound TN was determined by ELISA with rat monoclonal Ab 8C9. The values represent the mean of three determinations.
Based on the results with native TN, we expected TNfn1-8, which corresponds to the small splice variant, to bind to FN more avidly than TNfnALL. This was confirmed, especially at lower concentrations of TN proteins, where TNfn1-8 showed significantly higher binding (Fig. 6). At higher concentrations both proteins bound equally, probably saturating the FN substrate. The apparent biphasic nature of TNfn1-8 binding seen in Fig. 6, in particular the weak binding at higher concentrations, was not seen consistently; however, the enhanced binding of TNfn1-8 relative to TNfnALL was reproducibly observed at lower protein concentrations.
Figure 6: Differential binding of TNfnALL and TNfn1-8 to FN examined by ELISA. TNfnALL (recombinant fragment of TN comprising of all FN3 domains) and TNfn1-8 (missing the 7 alternatively spliced domains) were incubated in plastic wells previously coated with FN. The amount of bound TN was determined with polyclonal Ab 9172, as in Fig. 4.
Figure 7: Distribution of TN variants in BHK cell cultures. Normal BHK cells and three transfected cell lines (12) were grown to confluence in tissue culture slide chambers, and the distribution of TN variants was determined by immunofluorescence staining, using monoclonal Ab 8C9. Bar = 10 µm.
The co-localization of TN and FN was not universal in the ECM, as reported previously by others(7) . Fine FN fibrils without any staining of TN were often observed, and HxB.S was found in some patches that did not stain for FN (data not shown). Thus, HxB.S may bind to other ECM molecules such as collagen or proteoglycans, in addition to FN.
Figure 8: Binding of purified HxB.S (top panel) and HxB.L (bottom panel) to preformed ECM matrix fibrils. 3T3 cells were grown to confluence and the two TN isoforms were added to the medium. After a 24-h incubation, the cultures were washed and the bound human TN was visualized using the monoclonal Ab BC-4 specific for human TN.
Figure 9: Differential incorporation of TNfnALL and TNfn1-8 to ECM of BHK cells. BHK cells were grown to confluence. TNfnALL or TNfn1-8 (30 µg/ml) were added to cultures and incubated for 24 h. Matrix-binding proteins were detected by immunofluorescence staining with HxB-9504 rabbit polyclonal Ab or 190 mouse monoclonal Ab. Bar = 10 µm.
The failure of the two monoclonal Abs to detect the bound TNfn1-8 suggests that their epitopes are buried in the bound TN. The TN-190 epitope is in TNfn3(15) , which our in vitro assays mapped as the primary binding site in TN ( Fig. 3and Fig. 5); the 7-13 epitope has been mapped to TNfn4-5 (data not shown), the adjacent domains that appear to contribute to the binding (Fig. 5).
In the present study, we confirmed the binding of TN to FN
both in solution and in ELISA. The solution-phase binding was observed
in a sedimentation assay with both TN and FN at 100 µg/ml,
essentially the same as in our previous study(6) . We observed
binding of TN to FN-coated plastic at TN concentrations of 2-25
µg/ml, in excellent agreement with the study of Chiquet-Ehrismann et al.(4) . This also explains our previous failure to
observe binding, in ELISAs limited to TN concentrations less than 1
µg/ml(6) . The affinity of the TN-FN binding is sufficient
to saturate the ELISA at 25 µg/ml TN, suggesting that this binding
is saturated in many tissues where TN concentrations reach 200-2000
µg/ml(17) .
Binding of TN to native FN fibrils in cell cultures showed a very strong preference for the small TN splice variant. The preferential binding of small TN was also observed in ELISA and in the sedimentation assay, but the difference was much smaller. Overall, these results confirm and extend the observations of Chiquet-Ehrismann et al.(4) . The much greater differential binding to native FN matrix fibrils suggest that there may be a third molecule that enhances binding of small TN.
An important new finding in the present study is that bacterial expression protein TNfn1-8 binds more avidly than TNfnALL, both to FN matrix fibrils and to purified FN in ELISA. This suggests that the preferential binding is due to the arrangement of FN3 domains, and does not require the other domains nor the hexabrachion structure. However, binding of expression proteins containing only FN3 domains was always much weaker than that binding of native TN, suggesting that the other domains and/or the hexabrachion structure contribute significantly to overall binding.
How can the absence of the splice segment enhance the binding for FN? The simplest model would postulate two binding sites, one in TNfn1-5 and another in TNfn6-8(4) , which can bind simultaneously to FN only when they are brought together in HxB.S. We have now mapped the primary binding site to TNfn3, which is 10 nm distant from TNfn6, but this does not pose a serious problem, because FN itself is elongated and could have two complementary binding sites separated by 10-15 nm.
If there are two sites, one in
TNfn3 and another somewhere in TNfn6-8, we have to address the
question of why the binding of HxB.S to FN could be completely blocked
by 1 µM concentration of segments containing TNfn3, while
TNfn6-8 had no effect. The most likely explanation lies in the
nature of cooperative binding(18) . If the site in TNfn3
produces binding at a K near 1 µM, a
second site in TNfn6-8 could enhance the binding by several
orders of magnitude, even it were far too weak to produce an observable
binding of TNfn6-8 by itself. The very modest 3-10-fold
enhancement we observe in ELISA, or even a 100-fold enhancement that
seems to occur in tissue culture, is fully consistent with a modest
affinity binding site in TNfn3, enhanced by a very weak binding of a
site in TNfn6-8.
We have now confirmed that the preferential binding of HxB.S is conserved in both chickens and humans. The conservation of this activity over the 300 million years that separate chickens and humans suggests that it is biologically important for the functioning of TN in tissues.