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(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) ( 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 TN
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 TN
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 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 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.
Volume 270,
Number 48,
Issue of December 1, 1995 pp. 29012-29017
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
)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).
Cells and Cell Cultures
A human glioma cell line
U-251MG (clone 3, obtained from Dr. Darell Bigner, Duke University),
and the BHK cells transfected with the small and large splice variants
of TN (12) were grown in Dulbecco's modified
Eagle's medium, high glucose, supplemented with 10% heat
inactivated fetal calf serum.Proteins and Abs
Splice variants of human TN were
produced in transfected BHK cells(12) . Native TN was purified
from culture supernatant of the BHK cells or U-251MG human glioma cells
by gel filtration and Mono Q ion exchange chromatography(13) .
FN was purified from human plasma or horse serum by gelatin-agarose
affinity chromatography(14) . Bacterial expression proteins
were purified as described(12) . A rabbit polyclonal antibody
(Ab), HxB-9172, was produced by injecting recombinant TNfn1-5;
and HxB-9504 by injecting TNfn6-8 plus TNfbg. Rat monoclonal Abs
8C9 (against an epitope in the EGF or central knob region) and
7-13 (against an epitope in TNfn4-5) (
)were
provided by Dr. Moriaki Kusakabe, RIKEN, Japan. Mouse monoclonal Abs
TN-190 and BC-4 are against epitopes in TNfn3 and the EGF
region(15, 16) .Glycerol Gradient Sedimentation Assay
A glycerol
gradient sedimentation assay was done as described by Lightner and
Erickson(6) . 15 to 40% glycerol gradients, 5 ml in 0.2 M ammonium bicarbonate, pH 7.9, or in TBS (Tris-buffered
saline: 20 mM Tris, pH 7.4, 150 mM NaCl) were
overlaid with 150-µl samples containing 100 µg/ml TN plus 50
µg/ml catalase as an internal sedimentation standard. FN and
competing bacterial expression proteins were added at 100 µg/ml to
the sample and to the 15% glycerol prior to making the gradients.
Gradients were centrifuged in an SW 50.1 rotor at 38,000 rpm for 15 h
at 20 °C. 22 fractions were collected and analyzed by
SDS-polyacrylamide gel electrophoresis on 5% polyacrylamide gels.Immunostaining
BHK cells were grown in Labtek
chamber slides (Nunc, Naperville, IL), washed with PBS, and fixed for 5
min with freshly made 3% para-formaldehyde in PBS or with a 1:1
acetone:methanol mixture. After washing cells with PBS, remaining
formaldehyde was neutralized with 50 mM NH
Cl.
Cells were permeabilized with 0.5% Triton X-100 in PBS for 5 min, then
incubated with 1% bovine serum albumin in PBS. Primary rabbit antiserum
against FN was added at a final dilution of 1:100 in PBS and anti-TN
monoclonal Ab 8C9 was added at a concentration of 10 µg/ml. The
slides were incubated for 1 h at 4 °C and then washed three times
with PBS for 15 min and fluorescein-conjugated anti-rat IgG (Sigma) and
rhodamine-conjugated anti-rabbit IgG (TAGO, Inc.) were added at a final
dilution 1:100. After 1 h incubation, slides were washed three times
and mounted for observation and photography.Solid-phase Binding Assay
A solid-phase binding
assay was used to study the interaction between TN and FN. In this
assay, 96-well plates (Falcon 3912) were coated overnight at 4 °C
or 2 h at 37 °C with FN solution (25 µg/ml FN in TBS containing
1 mM CaCl, and 1 mM MgCl) and
washed, followed by blocking for 1-2 h at 37 °C with 5%
non-fat dry milk in PBS. Plates were then incubated with native TN or
recombinant domains of TN in the TBS + magnesium/calcium for 1 h
at 37 °C. After washing the wells, bound proteins were detected by
conventional ELISA technique using polyclonal or monoclonal anti-TN Ab
and horseradish peroxidase-conjugated secondary Ab.
Binding of TN to FN in Solution
Our previous
study (6) demonstrated accelerated sedimentation of TN when FN
was present throughout the glycerol gradient. In these gradients the FN
was added only to the 15% glycerol, so its concentration was initially
100 µg/ml at the top of the gradient and 50 µg/ml in the
middle. However, FN sediments at about 8 S, compared to 13 S for TN, so
its concentration at the position of TN should be continuously in the
range of 75-100 µg/ml. The accelerated sedimentation of TN
indicates the formation of larger complexes resulting from the
association of TN with FN. Our previous study used human TN purified
from glioma cell culture, which is 90% large TN. Here we tested
individually HxB.L and HxB.S, obtained from our transfected BHK cell
lines. Both TN variants showed accelerated sedimentation of about one
or two fractions in the presence of FN (Fig. 2). HxB.S gave a
somewhat larger shift (two fractions) than HxB.L (one fraction).
FN 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 TNFN 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.
FN complex; TNfn3-5 and TNfn3
produced large displacements.
Binding of TN to FN in Solid-phase Binding
Assay
To characterize further the interaction of TN variants
with FN, an ELISA-type solid-phase binding assay was employed. FN was
coated on plastic at 20 µg/ml and overlaid with different
concentrations of soluble HxB.L or HxB.S proteins. Bound TN was
detected by ELISA using a polyclonal Ab against the TNfn1-5
region of TN, which should bind equally to both splice variants (Fig. 4). HxB.S achieved a higher level of binding and reached
saturation at lower concentrations than HxB.L.
Incorporation of TN into FN Matrix Fibrils in Cell
Culture
Chiquet-Ehrismann et al.(4) used Abs
specific for alternative splice domains to show that cultures of
chicken fibroblasts, which secrete both small and large splice
variants, incorporate only small TN into FN matrix fibers. We used our
transfected BHK cells (12) to test this in separate cultures
secreting HxB.egf, HxB.S, or HxB.L. Cells were grown to be fully
confluent, and FN and TN were localized by immunostaining using anti-FN
polyclonal Ab and rat monoclonal Ab 8C9, which binds to HxB.egf and
therefore recognizes all three forms (Fig. 7). All four BHK cell
lines deposited prominent FN fibrils in the ECM (not shown).
Immunostaining of BHK-HxB.egf showed weak cellular staining, but no TN
staining in the ECM. BHK-HxB.L likewise showed weak staining inside or
on cells but little fibrillar staining. In contrast, BHK-HxB.S showed a
prominent fibrillar pattern of staining. Most HxB.S was co-localized
with FN fibrils and was associated especially with thick, well
developed FN fibrils.
Binding of Purified TN Splice Variants to FN Matrix
Fibrils
Binding of the two TN splice variants to the ECM was
also tested by adding purified HxB.L and HxB.S separately to NIH 3T3
cell cultures. These cells make a prominent FN matrix, but no
detectable TN. After a 24-h incubation with added human TN, the
cultures were washed and the bound human TN was visualized using the
species-specific monoclonal Ab BC-4. Cultures treated with HxB.L did
not show any staining, while cells treated with HxB.S showed the
typical fibrillar pattern of TN (Fig. 8).
Differential Binding of TNfn1-8 and TNfnALL to FN
Matrix Fibers
We finally tested whether bacterial expression
proteins containing only FN3 domains could bind to FN fibrils in cell
culture and whether TNfn1-8, which contains only the FN3 domains
of small TN, bound better than TNfnALL, which contains all the FN3
domains of large TN. BHK cells were grown to be fully confluent, and
TNfn1-8 or TNfnALL (30 µg/ml) were added to the medium. After
24 h the cultures were washed and bound protein was detected by
immunostaining using four different Abs: HxB-9172, a rabbit polyclonal
Ab recognizing TNfn1-5; HxB-9504, a rabbit polyclonal Ab
recognizing TNfn6-8 plus TNfbg; TN-190, a mouse monoclonal Ab
recognizing TNfn3; and 7-13, a rat monoclonal Ab recognizing
TNfn4-5. Cells not treated with recombinant protein did not show
any staining, demonstrating no cross-reaction of Abs with hamster TN,
if any. With all four Abs, TNfnALL showed punctate staining at
pericellular spaces and over cell bodies, but no fibrillar staining (Fig. 9). In contrast, TNfn1-8 showed virtually no
specific staining with either monoclonal Ab, but both polyclonal Abs
demonstrated prominent fibrillar staining. The staining of
TNfn1-8 to ECM was not as prominent as that of native small TN,
but the localization pattern was similar. The enhanced binding of
TNfn1-8 to these ECM fibers appeared to be absolute (TNfnALL did
not bind), and certainly much greater than to purified FN in the
solid-phase ELISA.
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) .
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.
))
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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