From the Institut de Biologie et Chimie des Protéines, CNRS UMR 5086, Université Claude Bernard, 7 passage du Vercors, 69367 Lyon Cedex 07, France
Received for publication, November 9, 2000, and in revised form, February 6, 2001
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ABSTRACT |
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Tenascin-X is known as a heparin-binding
molecule, but the localization of the heparin-binding site has not been
investigated until now. We show here that, unlike tenascin-C, the
recombinant fibrinogen-like domain of tenascin-X is not involved in
heparin binding. On the other hand, the two contiguous fibronectin type III repeats b10 and b11 have a predicted positive charge at
physiological pH, hence a set of recombinant proteins comprising these
domains was tested for interaction with heparin. Using solid phase
assays and affinity chromatography, we found that interaction with
heparin was conformational and involved both domains 10 and 11. Construction of a three-dimensional model of domains 10 and 11 led us
to predict exposed residues that were then submitted to site-directed
mutagenesis. In this way, we identified the basic residues within each
domain that are crucial for this interaction. Blocking experiments
using antibodies against domain 10 were performed to test the
efficiency of this site within intact tenascin-X. Binding was
significantly reduced, arguing for the activity of a heparin-binding
site involving domains 10 and 11 in the whole molecule. Finally, the
biological significance of this site was tested by cell adhesion
studies. Heparan sulfate cell surface receptors are able to interact
with proteins bearing domains 10 and 11, suggesting that tenascin-X may
activate different signals to regulate cell behavior.
Tenascin-X is an extracellular matrix molecule that belongs to the
tenascin family, comprising five members: TN-C, TN-R, TN-X, and the
more recently characterized TN-Y and TN-W. All are multi-domain proteins consisting of an N-terminal region involved in
oligomerization, a series of epidermal growth factor-like repeats, a
variable number of fibronectin type III
(FNIII)1 modules, and a
C-terminal domain homologous to fibrinogen (Fbg). This complex
structure gives rise to multiple interactions with proteins and
carbohydrates. Alternative splicing events involving FNIII domains
generate a variety of isoforms whose different interaction properties
and functions have been demonstrated for TN-C (1).
Many studies have emphasized the possible role of proteoglycan binding
in the functions of members of the tenascin family. TN-C binding to
glycosaminoglycans, namely heparin, heparan and chondroitin sulfates is
well documented. Two heparin-binding sites have been identified in the
fifth FNIII and the Fbg domain of TN-C by the use of recombinant
domains produced in bacteria (2, 3). The latter domain contains the
active site of the whole molecule (4, 5). Several types of
proteoglycans have been shown to interact with TN-C, e.g.
brain proteoglycans (6), or chondroitin sulfate proteoglycans derived
from smooth muscle cells in culture (7) or from cartilage (8).
Perlecan, an extracellular heparan sulfate proteoglycan, interacts
preferentially with the small splice variant of TN-C; FNIII domains
3-5 are involved in this interaction (9). Moreover, incorporation of
the small variant of TN-C in the fibronectin matrix deposited by cells
in vitro is dependent on the presence of heparan sulfate
proteoglycans (9). Some data suggest that cell surface heparan sulfates
present on fibroblasts (2) or on hematopoietic cells (10) are able to
interact with the Fbg domain. Syndecan from embryonic mesenchyme (11)
as well as glypican (12), both of which are heparan sulfate receptors,
have also been found to interact with TN-C via their glycosaminoglycan
(GAG) chains. Other proteoglycans may interact via N-linked
oligosaccharides (phosphacan/RPTP Heparin binding seems to be a common feature of molecules of the
tenascin family; TN-X and TN-Y are also heparin-binding proteins (16,
17), although the functional significance of this interaction has not
been demonstrated until now. Our study is focussed on the localization
of heparin-binding domains within the TN-X molecule. TN-X has the
typical arrangement of modules characteristic of TNs (16, 18-20). It
has a widespread expression during embryonic and adult life, where it
appears more specifically in striated muscle, tendon and ligament
sheaths, dermis, adventitia of blood vessels, peripheral nerves, and
digestive tract (16, 19, 21-23). Few data are available concerning the
function of TN-X, but in one clinical case, a patient deficient in TN-X
protein, has been reported to present a connective tissue disorder
typical of Ehlers Danlos-like syndrome (24). Symptoms consist of skin
and joint hyperextensibility, vascular fragility, and poor wound
healing. In view of these connective tissue defects, TN-X might be
involved in cell-matrix interactions and in matrix network formation.
Because GAGs are likely to be involved in these molecular interactions,
we have analyzed the heparin binding properties of TN-X by mapping the
heparin-binding site(s) within the molecule using recombinant proteins.
In comparison with the localization of the heparin binding site in
TN-C, we have produced the recombinant Fbg domain in bacterial or
eukaryotic cells and found no interaction with heparin. Sequence
analysis of TN-X showed that two successive FN III domains are
positively charged at physiological pH (domains 10 and 11). We produced
a set of recombinant proteins containing this region and localized one
heparin binding site to these domains, the residues crucial for this
interaction being identified by site-directed mutagenesis. The
efficiency of this site in the context of the whole TN-X molecule was
analyzed by blocking experiments using antibodies specific for this
region (domain 10). Finally, the physiological significance of this
heparin binding site was tested by cell adhesion studies.
Purification of TN-X from Bovine Embryonic Skin--
Bovine TN-X
was purified using a procedure previously described (21). Briefly, TN-X
was extracted with 0.5 M NaCl and immunopurified on a
column prepared with the 4E7 monoclonal antibody. Proteins nonspecifically bound to the column were desorbed with 1 M
MgCl2, and TN-X was then eluted with 0.15 M
Na2CO3, pH 12.0. After dialysis against
phosphate-buffered saline (PBS), purified TN-X was stored at
Production and Purification of TN-X Recombinant Fragments in
Escherichia coli--
Recombinant proteins encompassing FNIII modules
of bovine TN-X are termed FNXn, where n
corresponds to the repetition(s) used. For this study, we used the
previously described recombinant fragments FNX9, FNX10, and FNX9-10
(25) and generated new recombinant proteins containing the FNIII b11
repeat, i e., FNX11, FNX10-11, and FNX9-10-11. The DNA
coding for the new proteins was amplified by PCR and cloned in the
overproducing plasmid pT7/7-His6 as previously described.
The oligonucleotides used in the PCR reaction were FNIII b9-forward
(25), FNIII b10-forward (25), FNIII b11-forward (5'-TAAGAATTCTAGAGACCCGCAGAGCTGTGG-3') and FNIII
b11-reverse (5'-TATCTGCAGGGTCACGGCGATGGCGGAGAT-3'). Restriction sites needed for cloning are underlined. Production of TN-X fragments in E. coli strain BL21(DE3) was performed
using previously described procedures (25), with the exception that here the IPTG induction was carried out at 37 °C for 3 h
instead of overnight at room temperature. The His-tagged proteins were purified in two chromatographic steps. first using a metal affinity column and second using an ion exchange MonoQ (FNX9 and FNX9-10) or
Mono-S (FNX10, FNX11, FNX10-11, FNX9-10-11) column (Amersham Pharmacia
Biotech). Production and purification of the fibrinogen domain (FbgX)
in bacteria has been previously described (25).
Mutations were generated according to the procedure of Kamman et
al. (26). Internal mutagenic oligonucleotides are listed in Table
I. This procedure consists of two PCR steps. In the first step, one
primer contains the mutation, whereas the second is one of two
oligonucleotides used to generate the wild type construct. The first
PCR fragment, gel purified, is then used as a primer during the second
PCR together with the wild type oligonucleotide opposite to that used
in PCR1.
Production of Recombinant Fbg Domain in Mammalian Cells--
A
DNA fragment encoding the Fbg domain was amplified by PCR using
Goldstar polymerase (Eurogentec, Seraing, Belgium) and FbgX as matrix.
The sequence of the sense primer
(5'-TATGGCCCAGCCGGCCGGTGGGCTGCGGATCCCCTTCC-3') introduced a
SfiI restriction site (underlined) at the 5' end of the PCR
fragment and allowed in-frame cloning with the Ig
Human embryonic kidney HEK 293 cells were transfected with pSec-Fbg
construct permitting the secretion of recombinant protein harboring
c-myc and His6 tags at the C terminus
(Invitrogen). Petri culture dishes (60 mm) with cells at
half-confluency were rinsed twice in serum-free medium and incubated
for 4 h at 37 °C in 5 ml of serum-free medium containing 33 µl of Perfect lipid
To produce the recombinant protein, HEK293 cells were cultured in
serum-free conditions, and the medium was collected every 48 h.
The medium was loaded on to a Talon metal affinity resin equilibrated
in Tris-buffered saline, and elution was performed in the same buffer
containing 50 mM imidazole, pH 8.0. After dialysis against
50 mM Tris, pH 8.0, the eluate was further purified on a
HiTrap Q-Sepharose column (Amersham Pharmacia Biotech). Separation was
achieved by elution with a linear NaCl gradient from 0 to 1 M. Fractions containing the Fbg domain were pooled and
dialyzed against PBS. Protein purity was checked by SDS-polyacrylamide gel electrophoresis where it was estimated to be greater than 90%.
Protein concentration was determined by absorbance at 280 nm.
Solid Phase Assay of Heparin Binding Activity--
96-well
microtiter plates (Maxisorp, Nunc) were coated overnight at +4 °C
with purified bovine TN-X or recombinant proteins diluted in PBS. Wells
were saturated with T-PBS-BSA (PBS, 0.05% Tween 20, 1% bovine serum
albumin) for 2 h at room temperature and then incubated with
heparin-albumin-biotin (Sigma) in T-PBS-BSA for 2 h at room
temperature. Wells were rinsed with PBS and incubated for 30 min with
peroxidase-conjugated streptavidin (Extravidin, Sigma) diluted 1:2000.
After the last set of rinses, bound peroxidase was detected with
H2O2 and
2,2-azino-bis(3-ethylbenthiazoline-6-sulfonic acid), and the absorbance
was read at 405 nm. Experiments were repeated three to five times. Each
data point represents the mean of duplicate or triplicate determination.
For inhibition experiments, incubation with antibodies was performed
for 1 h before adding the heparin-albumin-biotin complex. Detection of bound heparin was performed as described above.
Analytical Affinity Chromatography--
30 µg of recombinant
FNX10-11 fragments dissolved in buffer A (50 mM phosphate
buffer, pH 7.2, 100 mM NaCl) were loaded onto a Hi-Trap
heparin-Sepharose column (Amersham Pharmacia Biotech) at 0.8 ml/min
using a high performance liquid chromatography system (Waters).
The column was extensively washed with buffer A. Retained proteins were
then eluted with a linear NaCl gradient (0.1-1 M NaCl in
buffer A) with constant monitoring of absorbance at 280 nm.
Cell Adhesion Assay--
Parental CHO-K1 and mutant CHO-677 and
CHO-745 cell lines, developed by Esko et al. (28), were
purchased from the American Type Cell Culture Collection (Manassas,
VA). The cells were cultured in F12K medium supplemented with 10%
fetal bovine serum and 50 µg/ml gentamicin. Cell adhesion to protein
adsorbed to microtiter plates was tested according to a previously
described procedure (19), except that cells were suspended using a
nonenzymatic dissociation solution (Sigma). Inhibition experiments with
GAGs were performed by incubating the FNX10-11 substrate for 15 min with heparin or chondroitin sulfate that was diluted in serum-free medium before adding cells to the plates. The data points are expressed
as means of triplicates, and each experiment was repeated a minimum of
three times.
Molecular Modeling--
A working model of each of the
FNIII-like domains b10 and b11 was constructed based on sequence
comparison with known three-dimensional structures of FNIII repeats
available from the protein data base at RCSB. The program CLUSTAL W
(29) was used to align the FNIII repeats as independent domains with
the following Protein Data Bank codes: 1fnf, 1ttg, 1fna, 1mfn, 1ten,
1cfb, and laz7. The alignment was reformatted, and 10 slightly varying three-dimensional models from each domain were calculated with the
modeling program MODELLER4 (30) on an ORIGIN2000 work station (Silicon
Graphics Inc.) applying molecular dynamics with standard parameters.
The 10 models of each domain were visually inspected with the program O
(31) and then averaged and energy minimized with the program X-PLOR
(32) to obtain a single model of each FNIII domain. Molecular
properties of the three working models, such as potential surface or
side chain interactions, were visualized with the freely available
program "WebLab viewer Light" (Molecular Simulations Inc.) on a
standard PC.
Production and Purification of Recombinant Proteins--
After two
successive purifications by metal affinity resin and ion exchange
chromatography, purified proteins were analyzed by SDS-polyacrylamide
gel electrophoresis, confirming the excellent level of purity for each
protein (Fig. 1). The apparent molecular masses of the recombinant FbgX domain produced in E. coli
and in 293 cells were slightly different. The higher mass of the
protein expressed in mammalian cells was due to (i) the additional
c-myc tag introduced by the pSecTag2/Hygro expression vector
and (ii) the glycosylation that occurred in eukaryotic cells. Indeed,
the analysis of the primary sequence of bovine TN-X allowed us to predict a N-glycosylation site in the Fbg domain, namely the
NIS sequence located at residues 76-78 from this domain (19). The presence of these additional sugars was confirmed by staining gels with
Schiff's reagent (data not shown).
The Fbg Domain of TN-X Is Not Responsible for Interaction with
Heparin--
Prior to the mapping of the heparin binding site, we
tested the ability of TN-X, purified from embryonic bovine skin, to
interact with heparin. As shown in the solid phase assay (Fig.
2), the heparin biotin-complex was
retained in the wells coated with TN-X in a dose-dependent
manner, with a saturation limit. We produced the individual C-terminal
fibrinogen domain in a bacterial system (FbgX) and tested its
interaction with heparin under the same conditions as intact TN-X. In
this case, no interaction could be observed. Because this recombinant
domain was recovered from the bacterial pellet after urea extraction
and renaturation by dialysis, it is possible that this domain was not
correctly folded and that we missed a conformational binding site
present in this region. To circumvent this problem, we produced the
same domain in mammalian cell cultures. As before, results in the solid
phase heparin-binding test were negative, confirming that the
fibrinogen domain was not responsible for the interaction of TN-X with
heparin.
Localization of a Heparin-binding Site in FNX10-11--
To
identify a heparin-binding site within TN-X, we tested the binding of
recombinant domains spanning FNIII domains 9-11. This region was
chosen because sequence analysis of both domains 10 and 11 predicted a
positive charge at physiological pH. We tested the recombinant proteins
in the solid phase assay, where it clearly appeared that the two
domains 10 and 11 were necessary for interaction with heparin (Fig.
3). Only the recombinant proteins FNX10-11 and FNX9-10-11 exhibited heparin binding activity, whereas the
individual modules in FNX10, FNX11, or FNX9-10 did not.
Interaction with heparin was also tested using soluble recombinant
proteins loaded on to a heparin-Sepharose column (Fig. 4). As expected, FNX10, FNX11, and
FNX9-10 were found in the unbound fraction, and FNX10-11 and FNX9-10-11
were retained in the column. By elution with a linear NaCl gradient,
both proteins were eluted as a single peak whose position corresponded
to a concentration of 0.33 M NaCl. The same apparent
affinity was observed when whole TN-X, purified from skin, was loaded
on the heparin-affinity column (data not shown). When all recombinant
proteins were loaded together in the column, the same result was
obtained, suggesting that domains 10 and 11 must be contiguous in the
same protein to generate a fully active heparin-binding site. A more
careful examination of chromatograms in the unbound fractions suggested
that domain 11 had an higher affinity for heparin than domain 10. Indeed, FNX11 protein was found in the late fractions during column
washing (Fig. 4A).
Identification of Basic Residues Involved in Heparin
Binding--
To further map the heparin binding activity, we decided
to produce recombinant FNX10-11 proteins bearing single or double mutations replacing basic residues Arg and Lys by Gln and Ser, respectively. First, we examined the primary structure of the two
modules 10 and 11. One BBXB sequence, which had been
determined to be a potential heparin binding site, was present in these
two modules, but this sequence was found at a similar position in 13 of
the 30 FNIII-like domains characterized in bovine TN-X (19). Thus, this
sequence was unlikely to be a major binding site in our system. In
agreement with this hypothesis, our data clearly demonstrated that the
site was conformational and involved both domains 10 and 11.
Mutations were dictated by the predicted accessibility of positively
charged residues according to our three-dimensional models of the
isolated FNIII domains. The contributions of Arg3,
Arg4, Lys12, Arg13,
Lys16, and Arg18 located in the region
connecting both domains 10 and 11 were tested. As shown in Fig.
5, a cluster of basic residues was
predicted in domain 11 that comprised Lys12,
Arg13, and Arg90. An additional residue
Arg18 was mutated as a control. In domain 10, the adjacent
residues Arg84, Lys85, and Arg86
that projected outwards in the three-dimensional model, located in a
loop between the F and G
All mutants were tested by heparin-Sepharose chromatography, and their
elution positions were compared with that of the wild type FNX10-11
protein. As shown in Table II, the most
pronounced effect of a single mutation was observed for
Lys16 of domain 11; this mutant was found in the unbound
fraction of the heparin column. All other single mutations realized
within this domain had negligible effects. Double mutants 12-13, 12-90, and 13-90 showed a very moderate decrease (from 63 to 77%) in heparin
affinity, confirming that these residues were not crucial for
interaction with heparin. In domain 10, mutation of Arg86
resulted in strong inhibition of heparin binding because the protein
was eluted at only 30% retention time compared with the wild type.
Moreover, the cluster of basic residues 84-86 appeared important for
heparin interaction because the double mutants 85-86 and 84-86 were not
retained in the column.
Efficiency of the FNIII 10-11 Heparin-binding Site in the Whole
TN-X Molecule--
To analyze the accessibility of this site in the
native TN-X molecule, we performed inhibition studies using polyclonal
and monoclonal antibodies. The polyclonal antibody was prepared by immunizing guinea pigs with the FNX9-10 protein. Because these two
domains showed only a moderate sequence identity with other FNIII
domains, one can assume that the polyclonal antibody is specific to the
region of domains 9 and 10 in the intact TN-X. Moreover, when the
reactivity of this antibody was tested on FNX recombinant proteins by
enzyme-linked immunosorbent assay, we found a strong affinity for FNX9,
FNX10, FNX9-10, and FNX9-10-11, with negligible affinity for FNX11
(data not shown), even though sequence identity between domain 11 and
domains 9 and 10 is 62 and 58%, respectively. To confirm the
inhibition data, we also used a monoclonal antibody whose epitope is
located within domain 10 (clone14G5) and control monoclonal antibodies
(clones 15F12 and 19D1) that recognize the region encompassing FNIIII
domains 12-15. Localization of epitopes was determined by reactivity
with clones derived from the cDNA expression library from bovine
embryonic skin (19) and by enzyme-linked immunosorbent assay with the set of recombinant FNX proteins. Clone 14G5 reacted only with proteins
bearing domain 10, whereas 15F12 and 19D1 clones were negative with any
of the proteins derived from domains 9-11 (data not shown).
When polyclonal anti-FNX9-10 or monoclonal 14G5 antibodies were
incubated with the proteins before adding heparin, an inhibition of
interaction was observed for both FNX10-11 and intact TN-X purified
from bovine skin (Fig. 6). A maximum of
93% inhibition was obtained using purified polyclonal antibodies and
FNX10-11 protein, whereas with intact TN-X, inhibition was only 67%.
Monoclonal antibody 14G5 was also able to inhibit this interaction but
to a lesser extent, 61 and 52% for FNX10-11 and TN-X, respectively. Control incubations using buffer, nonimmune guinea pig IgG, 15F12, or
19D1 clones did not give a significant inhibition of heparin binding.
Taken together, these results confirm first that the FNIII domain 10 was involved in interaction with heparin and second that the site
located within domains 10 and 11 that we described above was functional
in the whole TN-X molecule.
Cell Interaction with FNX10-11 Protein--
To analyze the
possible interaction of heparan sulfate receptors in cell adhesion to
TN-X, we performed adhesion studies on the recombinant protein
FNX10-11. CHO cells were chosen firstly because they do not express
Since the early studies, TN-X, has been identified as a
heparin-binding molecule (16), but the localization of this activity has not been further investigated. Considering that in TN-C the Fbg
domain is responsible for this activity and in view of the high level
of sequence identity of Fbg within the family of TN molecules, it has
been postulated that this domain is involved in the heparin binding
properties of TN-X (16). Our first set of experiments was designed to
test this hypothesis using solid phase assays. We found that whole TN-X
is able to bind heparin, whereas the recombinant Fbg domain, produced
either in bacteria or in mammalian cells, is not. In TN-C, the heparin
binding activity, mapped to the Fbg domain (2, 5), has been shown, by
mild tryptic digestion, to be located within 10 kDa from the C terminus of the domain. Two possible regions comprising clusters of basic residues were candidates for this interaction: the sequence AKTRYRLRV (amino acids 1702-1710 from the chicken sequence) and the extreme C-terminal sequence GRRKRA (amino acids 1803-1808) (5). It is quite
remarkable that these basic residues are conserved in TN-C from
different species, whereas these sequences are changed respectively to
DSADEYYRLHL and GRGG in bovine TN-X. These sequential sites are not
responsible for the heparin binding properties of TN-X. Moreover, we
can also exclude the presence of another conformational binding site
within the Fbg domain, because the recombinant domain prepared from
mammalian cell expression was not effective in heparin binding. Thus,
the mechanisms of interaction of TN-X with heparin are different from
those of TN-C.
To map further the heparin binding activity on TN-X, FNIII domains were
also checked for this property. In other extracellular matrix proteins
such as TN-C (3) or fibronectin (34) some of these domains are involved
in heparin-binding. Although other amino acids may participate in the
binding, stretches of basic residues that are positively charged at
physiological pH, namely Arg and Lys, are good candidates for
electrostatic interactions with negatively charged GAG chains. Sequence
analyses indicate that the two contiguous FNIII domains 10 and 11 of
TN-X are positively charged and that these domains were tested for
heparin binding activity. Results obtained with solid phase assays and
analytical chromatography on heparin-Sepharose both led to the same
conclusion that an heparin-binding site, involving both domains 10 and
11, is present in this region. The data also indicate that the relative orientation of the two domains is important because mixtures of FNX10
and FNX11 did not bind heparin in affinity chromatography (Fig. 4) and
solid phase (data not shown). Thus, our results indicate the presence
of one conformational binding site whose nature might be either a
mutivalent site involving two separate binding regions in each domain
or a unique site located in a pocket between the two domains.
Using site-directed mutagenesis, we identified Lys85 and
Arg86 in domain 10 and Lys16 in domain 11 as
crucial for interaction with heparin. None of these residues were
located in sequences matching those previously determined by Cardin and
Weintraub (33) as consensus sequences for heparin binding such as
XBBXBX or
XBBBXXBX, where B is a basic residue
and X a nonbasic, frequently hydrophobic amino acid. The mutagenesis results thus confirmed the conformational nature of the
site. Accessibility of the residues involved in the interaction was
predicted from our three-dimensional working model of isolated domains
10 and 11 (Fig. 5). Residue Arg86 of domain 10 is located
in a region predicted as a loop between the F and G In TN-C, two heparin-binding regions, i.e. FNIII domain 5 and the Fbg domain, were identified using isolated recombinant domains (2, 3), but only the Fbg domain was found to be functional in the
intact TN-C molecule (5). To examine the efficiency of heparin-binding
of domains 10 and 11 within intact TN-X, we carried out inhibition
studies using antibodies specific for this region. The antibodies were
able to decrease specifically the interaction between TN-X and heparin,
thus indicating that the conformational binding site that we detected
using recombinant proteins is accessible within the intact molecule.
However, because inhibition was weaker for the intact molecule than for
the recombinant proteins, we cannot exclude the possibility that an
additional heparin-binding site might exist in TN-X. In TNs, the FNIII
domain region is subjected to alternative splicing events that may
modulate the interaction properties of these proteins (1). Such a
mechanism might be involved in the interaction of TN-X with heparin or
glycosaminoglycans. From studies of mouse TN-X cDNA, in the region
corresponding to bovine domains 10 and 11, no alternative transcripts
could be detected (20). In contrast to these studies, our analyses of human TN-X transcripts from MG63 cells and human fetal tissues revealed
isoforms lacking the human domain 8, which corresponds to the bovine
domain 10.2 The existence of
these isoforms, probably deficient in heparin binding, may be important
for the spatio-temporal regulation of TN-X functions.
The interaction of TN-X with heparin-related GAGs may be necessary for
interaction with extracellular matrix and/or cell surface proteoglycans. Heparan sulfate receptors are involved in numerous cellular events and function as coreceptors, acting in concert with
integrins or growth factor receptors (37). In previous studies, we have
shown that the interaction of cells with TN-X is partially inhibited by
heparin, suggesting that heparan sulfate receptors may be involved in
this interaction (19). In this study, we have tested the ability of
different CHO cell lines to interact with a recombinant fragment
bearing the heparin-binding site. The results demonstrate that CHO
cells interact with the fragment via GAG chains of cell surface
proteoglycans and that the interaction is mediated by heparan and not
chondroitin sulfate chains. Because we have previously shown that
integrin receptors are also involved in the interaction of TN-X with
cells (25), we suggest that the combined effects of both signals
induced by integrins and by heparan sulfate receptors might regulate
the behavior of cells in contact with a TN-X substratum.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
) or their core proteins
(neurocan) (13-15).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
70 °C.
-chain leader
sequence. The antisense primer
(5'-TATGGGCCCGCCTCCCCTGCCCAGGGGCCGGTAG-3') introduced an
ApaI site (underlined) at the 3' end of the DNA and
permitted in-frame cloning with the c-myc and
His6 tags. The PCR fragment was introduced between the
SfiI and ApaI sites of the mammalian expression
vector pSecTag2/hygro (Invitrogen, Groningen, The Netherlands).
8 (Invitrogen) and 11 µg of DNA. After
removing the transfection solution, the cells were incubated for
36 h in Dulbecco's modified Eagle's medium containing 10% fetal
calf serum. Selection was then performed by adding 400 µg/ml
hygromycin B in the culture medium. Clones were passaged in 24-well
tissue culture plates, and the medium was checked for the presence of
the Fbg domain. One milliliter of medium was incubated with 50 µl of
Talon resin. The resin was rinsed in Tris-buffered saline and heated in
Laemmli sample buffer. The supernatant was analyzed by Western blotting using routine procedures (21), using a monoclonal antibody that recognizes the His6 tag. This antibody was obtained
serendipitously by immunization with recombinant proteins corresponding
to SURF modules of sea urchin fibrillar collagen, produced in a
bacterial system using the same vector as for the FNX proteins
(27).
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
SDS-polyacrylamide gel electrophoresis
analysis of purified TN-X recombinant proteins. Samples were
loaded on 15% acrylamide gels under reducing conditions. Proteins were
successively purified using Talon metal affinity and ion exchange
chromatography. Molecular masses of migration standards are indicated
on the left of figure. Lane 10, FNX10; lane
11, FNX11; lane 9-10, FNX9-10; lane 10-11,
FNX10-11; lane 10-11*, FNX10-11 mutated on Lys48
of domain 10; lane 9-10-11, FNX9-10-11; FbgX
Proc., Fbg domain produced in E. coli; FbgX
Euc., Fbg domain produced in mammalian cells.
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Fig. 2.
Solid phase heparin binding assays of TN-X
and recombinant FbgX domain. Wells were coated with proteins (3 µg/ml), saturated with BSA. Various concentrations of
Heparin-Biotin-BSA (HB-BSA) complex were added. The amount
of bound heparin was quantitated by incubation with
streptavidin-peroxidase and development with
ABTS/H2O2. Each data point is the mean of
duplicates.
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Fig. 3.
Binding of FNIII domains from TN-X (FNX) to
heparin. All recombinant proteins were coated at 30 nM
concentration, and incubations were performed as described in Fig. 2.
Results are expressed as the means of duplicate experiments.
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Fig. 4.
Chromatography of FNIII domains on
heparin-Sepharose. Proteins were loaded in 50 mM
phosphate buffer, pH 7.2, 0.15 M NaCl and eluted with a
linear gradient of NaCl (from 0.15 to 1 M). A,
elution profiles of individual proteins (FNXn) or a mixture
of all of these proteins (mix). B,
SDS-polyacrylamide gel electrophoresis of unbound (lanes
1-7) and eluted (lanes E1-E3) fractions recovered
after chromatography of the recombinant protein mixture.
-sheets, were considered as good candidates
for the interaction. These residues, together with Arg45, a
control located on the other side of the module, were mutated. Mutation
sites within domains 10 or 11 of FNX10-11 are summarized in Table
I (see "Materials and Methods").
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Fig. 5.
Calculated three-dimensional molecular work
models of FNIII domains 10 and 11 from TN-X. The three amino acids
Lys16 of domain 11 and Arg85 and
Arg86 of domain 11 are colored magenta. The
hydophobic surface that represents a possible interface between the two
domains is indicated as an ellipse in domain 10 and hidden
by its orientation of domain 10. The two N-terminal sequences
that are on top of both domains were not modeled because of missing
structural information and are represented by linear chain; the length
of the N-terminal sequence of domain 11 might be of sufficient length
to be connected to the C terminus of domain 10.
Nucleotide sequences of primers used for mutagenesis
Heparin binding of mutated recombinant proteins FNX 10-11
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Fig. 6.
Inhibition of the interaction with heparin
using polyclonal and monoclonal antibodies. After coating with
FNX10-11 (1 µg/ml) or TN-X (3 µg/ml) and saturation, wells were
incubated with antibodies (10 µg/ml for purified antibodies or
diluted 10-fold for hybridoma supernatants). HB-BSA complex was added
at 1 µg/ml and revealed as described in the legend to Fig. 2.
control, antibody diluent (Tween-Tris-buffered
saline, and BSA); IgG, purified guinea pig IgG;
anti-FNX10-11, IgG purified from serum of guinea pig
immunized with FNX10-11 protein (REF); RPMI, medium used for
hybridoma production; 14G5, 15F12, and
19D1, anti-TN-X hybridoma supernatants (epitope in domain
FNIII 10 for 14G5 and in the region encompassing FNIII domains 12-15
for 15F12 and 19D1). Each data point is the mean of triplicate
experiments, and error bars are calculated as standard error
of the mean.
V
3, an integrin that might interfere with
the adhesion tests on recombinant FNX proteins; indeed, we have
previously shown that this integrin is a ligand for the RGD site of
domain 10 (25). Secondly, the availability of mutant CHO cell lines
deficient in GAG synthesis allowed us to analyze the GAG class involved
in the interaction. CHO-K1 cells were able to adhere the FNX10-11
protein in a dose-dependent manner (Fig. 7A). Two mutant cell lines
derived from CHO-K1 were tested in adhesion experiments: CHO-677,
deficient in heparan sulfate but synthesizing more chondroitin sulfate,
and CHO-745, which lacks both heparan and chondroitin sulfates. These
two mutant cells exhibited a very low interaction with FNX10-11
protein, corresponding to ~25-30%, compared with the parental
CHO-K1 cells (Fig. 7A). As a confirmation of these results,
inhibition of cell interaction by incubating the FNX10-11 substrate
with GAGs was performed. As shown in Fig. 7B, heparin was a
potent inhibitor of the interaction between FNX10-11 and CHO-K1 cells,
whereas chondroitin sulfate had no significant effect. These data
indicated that cell interaction with FNX10-11 and consequently with
TN-X may be mediated by heparan sulfate receptors.
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Fig. 7.
Adhesion of CHO cells to FNX10-11.
A, dose response of different CHO cell lines adhering to
FNX10-11. CHO-K1 is the control cell line, CHO-677 is deficient in
heparan sulfate and synthesizes more chondroitin sulfate, and CHO-745
lacks both heparan and chondroitin sulfates. B, wells were
coated with 500 nM FNX10-11 and saturated. Inhibition by
chondroitin sulfate (CS) or with heparin (HEP)
was performed by preincubating the wells and diluting the CHO-K1 cell
suspension with the indicated concentration of GAG. Results are
expressed as the means of triplicate experiments and standard error of
the mean.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-sheets of the
FNIII module and Lys16 in domain 11 is located in the
region linking domains 10 and 11. Our model was not able to predict the
relative orientation of domains 10 and 11, a parameter that may be
important in the binding site that we have defined. In the model
proposed by Leahy et al. (35), the adjacent FNIII domains of
TN-C are tightly packed to make an extended filament. The tilted
orientation of domains 10 and 11 in TN-X may bring together the basic
residues involved in the interaction to create a cationic pocket. The
crystallization data obtained by Sharma et al. (36), dealing
with repeated arrays of FNIII domains of fibronectin, led to the
conclusion that the inter-repeat interface varies from 340 to 660 Å2; some interfaces are thus flexible, whereas others are
relatively rigid units. In TN-X, the inter-domain sequences have a
variable size but an unusual length when compared with those of
fibronectin or TN-C; we have suggested that the highly flexible
appearance of TN-X in electron microscopy is due to the length of these
inter-domain regions (19). It should be emphasized that in the region
comprising domains 10 and 11, these extensions are relatively short.
This property may favor a closer orientation between domains 10 and 11 and thus preserve the efficiency of the heparin-binding site.
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FOOTNOTES |
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* This work was supported by grants from Association pour la Recherche and by European Community Biotechnology Grant Bio4-CT96-0662.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.
To whom correspondence should be addressed: IBCP, UMR 5086, 7 passage du Vercors, 69367 Lyon cedex 07, France. E-mail:
c.lethias@ibcp.fr.
Published, JBC Papers in Press, February 7, 2001, DOI 10.1074/jbc.M010210200
2 J.-Y. Exposito, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: FNIII, fibronectin type III; Fbg, fibrinogen; FbgX, fibrinogen-like domain of tenascin-X; FNX, Fibronectin-type III domain of tenascin-X; GAG, glycosaminoglycan; PBS, Phosphate Buffered Saline; TN, tenascin; BSA, bovine serum albumin; PCR, polymerase chain reaction; CHO, Chinese hamster ovary.
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REFERENCES |
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