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(Received for publication, October 18,
1994; and in revised form, November 28, 1994) From the
Tenascin-C binds to cell surface and matrix proteoglycans and to
heparin. Two heparin binding regions have recently been localized per
tenascin-C monomer, one in the C-terminal fibrinogen-like domain and
the other in fibronectin type III repeats 3-5. Here we show that
a single region in each subunit is necessary and sufficient for heparin
binding by whole tenascin-C at physiological ionic strength. First,
native tenascin-C was bound to heparin-agarose and digested with
Pronase. A 29-kDa fragment retained on the heparin column was
recognized by a monoclonal antibody against the fibrinogen-like domain.
In contrast, small fragments labeled by an antibody against fibronectin
type III repeats 2-5 were released. Second, mild tryptic
digestion of tenascin-C yielded two related fragments of 180 and 170
kDa. The latter missed part of the fibrinogen domain and had lost
affinity for heparin, in contrast to the former. Finally, chick
tenascin-C constructs were recombinantly expressed in human cells.
Whereas the complete protein and a mutant lacking fibronectin type III
repeats 1-5 bound to heparin-agarose, recombinant tenascin-C
missing the C-terminal fibrinogen-like globe did not. Thus, whole chick
tenascin-C contains one essential heparin binding region per subunit,
located in the fibrinogen-like domain within 10 kDa from the C
terminus. Tenascin-C (for nomenclature, see Erickson, 1993;
Chiquet-Ehrismann et al., 1994) is an extracellular matrix
protein with highly regulated expression in the embryo (Erickson and
Bourdon, 1989). It acts as both an adhesive and an anti-adhesive
substrate for various cell types (Chiquet-Ehrismann, 1991). Tenascin-C
binds to matrix and cell surface proteoglycans such as CTB-proteoglycan
(Hoffman et al., 1988), neurocan and phosphacan (Grumet et
al., 1994), and syndecan-1 (Salmivirta et al., 1991).
Some of these interactions are mediated by the proteoglycan core
proteins (Hoffman et al., 1988; Grumet et al., 1994).
However, binding of syndecan-1 to tenascin-C depends on the heparan
sulfate side chains of the proteoglycan (Salmivirta et al.,
1991). Syndecan-1 and tenascin-C are coexpressed transiently in organ
primordia at the interface between mesenchyme and epithelium (Thesleff et al., 1987; Vainio et al., 1989). Heparin, a
glycosaminoglycan related to heparan sulfate, inhibits attachment of
peripheral neurites to tenascin-C in culture (Wehrle-Haller and
Chiquet, 1993; Chiquet and Wehrle-Haller, 1994). To understand
tenascin-C function, it is thus important to identify the specific
domains of the protein responsible for binding to glycosaminoglycans. Tenascin-C has an oligomeric structure typically with six arms, each
representing a subunit containing an N-terminal interchain
cross-linking domain, a stretch of EGF(
The generation,
purification, and characterization of specific anti-chick tenascin-C
mAbs TnM1 and Tn68 have been described previously (Chiquet and
Fambrough, 1984; Chiquet-Ehrismann et al., 1988). Monoclonal
antibodies Tn4 and Tn20, which are characterized below, are derived
from the same hybridoma library as mAb Tn68. On immunoblots (Towbin et al., 1979), both react with all chick tenascin-C splice
variants (not shown) and are hence directed against constant domains of
the protein. For epitope mapping by electron microscopy, tenascin-C
(50 µg/ml in 0.2 M ammonium bicarbonate) was incubated
with purified antibody at a molar ratio of about one
antibody/tenascin-C subunit for 1 h at 20 °C. Immunocomplexes were
sprayed onto mica, rotary-shadowed, and viewed in the electron
microscope as reported (Chiquet-Ehrismann et al., 1988). For
mAb Tn20, the distance from its binding site on the tenascin-C arms to
the central globular domain was measured from micrographs as described
previously (Chiquet-Ehrismann et al., 1988). The epitopes of
the various mAbs on the tenascin-C subunit are schematically depicted
in Fig. 9.
Figure 9:
Putative domain structure of the chick
tenascin-C variants and fragments (proteolytic and recombinant)
described. For details on tenascin-C structure, see Erickson (1993);
ChiquetEhrismann et al.(1994). The tenascin-C subunit, i.e. one arm of the oligomeric tenascin-C molecule, is
depicted as a linear array (from N to C terminus) of heptad repeats (zig-zag line) with interchain disulfides (S),
EGF-like repeats (diamonds), fibronectin type III repeats (squares), and a fibrinogen-like domain (circle).
Epitopes of the various mAbs used are indicated by the numbered
triangles. Heparin-binding activity of the various fragments is
indicated on the right.
The
complete coding regions of the three constructs were cut out by XhoI and XbaI and subcloned into the eucaryotic
expression vector pCDNAI/NEO (Invitrogen), resulting in the plasmids
pCDNA/TN 190, pCDNA/TN FB
Large proteolytic fragments
were produced from 0.25 mg of purified 190-kDa chick tenascin-C variant
by mild digestion for 45 min at 37 °C with 5 µg/ml trypsin
(Sigma) in 1 ml of NaCl-Tris containing 2 mM CaCl The putative domain structure
of the different proteolytic fragments of tenascin-C is shown
schematically in Fig. 9.
Purified recombinant
tenascin-190 and tenascin-FB
Figure 1:
Reactivity of heparin-binding and
-nonbinding tenascin-C fragments with various anti-tenascin-C mAbs.
Chick tenascin-C (a) was bound to heparin-agarose in 150
mM NaCl and digested on the matrix with Pronase as described
under ``Experimental Procedures.'' Fragments released by the
protease (W: b, d, and f) and bound
peptides eluted with 1 M NaCl (E: c, e, and g) were run on SDS-PAGE and immunoblotted with
mAbs Tn20 (a-c), Tn68 (d and e), and Tn4 (f and g), respectively. Note that mAb Tn4 recognizes
the smallest heparin-binding fragments (arrowheads).
Antibody Tn68 is
known to bind to an epitope on the second last fibronectin type III
repeat of chick tenascin-C. Using mAb Tn68, we previously isolated a
proteolytic 60-kDa tenascin-C fragment consisting of the three last
fibronectin type III repeats and the fibrinogen-like domain (Chiquet et al., 1991). In the Pronase digestion assay of this study,
this antibody recognized a series of heparin-binding fragments from 60
to 130 kDa (Fig. 1e). The fact that Tn68 recognizes no
heparin-binding fragments smaller than 60 kDa, but labels
non-heparin-binding fragments between 40 and 70 kDa (Fig. 1d), places the epitope of Tn68 at some distance
from a major heparin-binding site within the C-terminal tenascin-C
domain. A novel mAb, Tn4, was selected because it labeled a set of
Pronase-generated, heparin-binding tenascin-C peptides down to a size
of only 29 kDa (Fig. 1g), in addition to the 60-kDa and
larger bound fragments also recognized by Tn68 (cf.Fig. 1, e and g). This indicated that the
epitope of mAb Tn4 lies 30-60 kDa away from the epitope of mAb
Tn68 on the tenascin-C subunit and that it is much closer to a
heparin-binding site. Mapping of mAb Tn4 on rotary-shadowed tenascin-C
molecules in the electron microscope showed that this mAb binds to the
globular domain at the end of tenascin-C arms (Fig. 2a). Furthermore, on immunoblots mAb Tn4
recognized complete chick tenascin-C 190-kDa variant expressed in human
fibroblasts (Fig. 3e) but not a mutant recombinant
protein missing the fibrinogen-like domain at the C terminus (Fig. 3f). These experiments showed that the epitope of
mAb Tn4 is close to a heparin binding region within the fibrinogen-like
domain. Since the 29-kDa fragment recognized by mAb Tn4 was generated
from tenascin-C immobilized on an affinity column, the corresponding
heparin binding region must be functional in the intact protein.
Figure 2:
Electron microscopy of rotary-shadowed
immunocomplexes between tenascin-C and mAb Tn4 (a) or mAb Tn20 (b), respectively. Arrowheads point to antibody
molecules. While mAb Tn4 is attached to the distal end of the arms of
tenascin-C particles, mAb Tn20 binds to the middle of the arms. Both
antibodies can cross-link two neighboring tenascin-C arms. Bar, 50 nm.
Figure 3:
Reactivity of recombinant, complete, or
mutated chick tenascin-C with anti-tenascin-C mAbs. Purified
recombinant tenascin-C 190-kDa variant (a, c, and e) or a deletion mutant protein lacking the fibrinogen-like
domain (b, d, and f) were run on SDS-PAGE
and stained with Coomassie Blue (a and b) or
immunoblotted with mAb Tn20 (c and d) or mAb Tn4 (e and f), respectively. The TN FB
Another novel mAb, Tn20, attaches to the first half of the thicker
portion of tenascin-C arms when viewed in the electron microscope (Fig. 2b), 37 ± 6 nm (n = 77)
away from the central globular domain. Tn20 labels all three major
splice variants of chick tenascin-C (Fig. 1a) and must
therefore recognize a constant region. On immunoblots, mAb Tn20 does
not react with recombinant tenascin-C missing FNIII repeats 1-5
(not shown). Assuming a length of 30 nm for the combined heptad and EGF
domains (Spring et al., 1989) and of 3.2 nm/FNIII repeat
(Leahy et al., 1992), the epitope of mAb Tn20 lies close to
the third constant FNIII repeat of tenascin-C subunits. The smallest
heparin-binding tenascin-C fragment recognized by mAb Tn20 was 100 kDa
in size (Fig. 1c) and was also labeled by mAbs Tn4 and
Tn68 (cf.Fig. 1, c, e, and g). On the other hand, a 70-kDa fragment not retained by
heparin-agarose contained the epitopes of mAbs Tn20 and Tn68 but not of
Tn4 (cf.Fig. 1, b, d, and f). This pattern can only be explained by mAb Tn20 binding N
terminally of Tn68, confirming the ultrastructural data. It is
noteworthy that all smaller tenascin-C fragments recognized by mAb Tn20
(35-70 kDa) appeared in the flow-through of the heparin column (Fig. 1b). Thus, when intact tenascin-C is immobilized
on heparin-agarose before being fragmented, the region around the third
FNIII repeat apparently does not bind (cf.Fig. 9C for a schematic representation of results presented in this
section).
Figure 4:
Heparin binding by large tryptic fragments
of chick fibroblast tenascin-C. Purified tenascin-C 190 kDa variant (a) was mildly digested with trypsin as described under
``Experimental Procedures.'' Resulting fragments were loaded
onto heparin-agarose in 150 mM NaCl (L, b).
Unbound material washed from the column (W, c and e) and protein retained and eluted with 1 M NaCl (E, d and f) were analyzed by SDS-PAGE after
Coomassie staining (a-d, reducing gel; e and f, nonreducing gel). A 180-kDa fragment binds to
heparin-agarose (d) while one of 170 kDa does not (c).
When the mixture of tryptic
fragments was passed over heparin-agarose in 150 mM NaCl, the
180-kDa fragment was quantitatively retained on the column (Fig. 4d), whereas all of the 170-kDa fragment appeared
in the flow-through (Fig. 4c). It is remarkable that a
very large tenascin-C fragment missing only small regions at both ends,
but containing all constant FNIII repeats, does not bind to heparin
under physiological salt concentrations. This result indicates that
there is one physiologically relevant heparin-binding site/tenascin-C
subunit and localizes it to the C-terminal half of the fibrinogen
globe, within 10 kDa from the distal end of the protein.
Figure 5:
Electron microscopy of recombinant,
complete and mutated chick tenascin-C. Purified recombinant tenascin-C
190-kDa variant (TN 190, a) or the deletion mutant
protein lacking the fibrinogen-like domain (TN
FB
As expected, when isolated recombinant
190-kDa tenascin-C was applied to heparin-agarose in 150 mM NaCl, it was quantitatively retained on the column (Fig. 6, b and c). In contrast, recombinant protein lacking
the fibrinogen-like domain did not bind at all under these conditions (Fig. 6, e and f), thus confirming the results
obtained with large tryptic fragments of chick fibroblast tenascin-C.
However, one could still argue that purification of tenascin-C by high
pH elution from an antibody affinity column might affect a second
heparin-binding site elsewhere in the molecule. To exclude this
possibility, culture supernatants from transfected cells containing
either full-length or truncated tenascin-C were directly passed over
heparin-agarose, and bound material was eluted with high salt.
Tenascin-C in bound and unbound fractions was determined by ELISA. As
seen in Fig. 7, under these conditions all full-length
recombinant tenascin-C bound to the affinity column, while most
tenascin-C lacking the fibrinogen-like domain appeared in the
flow-through. The small fraction which was retained might have bound
indirectly, via another molecule like fibronectin (Chiquet-Ehrismann et al., 1991) present in conditioned medium. We also tested
recombinant tenascin-C which had the fibrinogen domain but missed FNIII
repeats 1-5, and, as expected, was recognized by mAb Tn4 but not
by mAb Tn20 (not shown). Under the same conditions, this mutant protein
was retained on heparin-agarose and eluted at practically the same
ionic strength as wild type protein. This experiment confirms that
under physiological pH and salt concentrations, tenascin-C secreted by
cells binds quantitatively to heparin only if it carries an intact
fibrinogen-like domain at its C terminus. It appears that this domain
is necessary and sufficient for the heparin binding activity of the
whole protein.
Figure 6:
Heparin binding by isolated recombinant,
intact, or mutant chick tenascin-C. Recombinant tenascin-C 190-kDa
variant (TN 190: a-c) or the deletion mutant protein
missing the fibrinogen-like domain (TN FB
Figure 7:
Binding of recombinant chick tenascin-C
from conditioned media to heparin-agarose. Conditioned media from
transfected HT1080 cell lines secreting recombinant chick tenascin-C
190-kDa variant (TN 190) or the deletion mutant proteins
lacking the fibrinogen-like domain (TN FB
Figure 8:
Heparin binding of the peptide
PSSFRNLEGRRKRA from the fibrinogen-like domain of tenascin-C. Bovine
serum albumin coupled with the peptide (a and b) or
serum albumin alone (c and d) was applied to
heparin-agarose, and unbound (W, a and c)
and bound (E, b and d) material were
analyzed by SDS-PAGE after Coomassie staining. In e and f, chick fibroblast 190-kDa tenascin-C (e) and its
180-kDa tryptic fragment (f) were immunoblotted with
polyclonal antibody against tenascin-C peptide-bovine serum albumin
conjugate.
Tenascin-C has complicated effects on cells. Depending on the
cell type and on the assay, it can act both as an anti-adhesive or an
adhesive extracellular matrix substrate (for review, see
Chiquet-Ehrismann, 1991). Glioma cells (Lotz et al., 1989),
endothelial cells (Sriramarao et al., 1993; Joshi et
al., 1993), embryonic Schwann precursor cells (Wehrle-Haller and
Chiquet, 1993), and neural crest cells (Halfter et al., 1989)
are all capable of attaching on tenascin-C substrates, but they do not
spread and are inhibited in their migration. On the other hand, the
cell bodies of embryonic neurons attach poorly to tenascin-C, but under
proper conditions nevertheless establish large, rapidly moving growth
cones and long, well attached neurites (Wehrle and Chiquet, 1990).
Using proteolytic (Friedlander et al., 1988; Chiquet et
al., 1991) and recombinant (Spring et al., 1989; Prieto et al., 1992; Aukhil et al., 1993) tenascin-C
fragments as well as inhibition by domain-specific mAbs (Lochter et
al., 1991; Husmann et al., 1992), various regions of the
molecule have been implicated in anti-adhesive and adhesive properties.
The neurite-promoting activity of tenascin-C is partially inhibited by
mAbs against the C-terminal FNIII repeats (Lochter et al.,
1991; Chiquet and Wehrle-Haller, 1994). Antibodies to the A first step in testing these
hypotheses is to localize heparin-binding site(s) on the tenascin-C
subunit. Intact tenascin-C (Marton et al., 1989), as well as a
60-kDa C-terminal fragment but not a 80-kDa N-terminal fragment
(Chiquet et al. 1991), bind to immobilized heparin. Using
recombinant fragments produced in bacteria, Aukhil et
al.(1993) further dissected the N-terminal domain of tenascin-C
and showed that the fibrinogen-like globe alone binds heparin while the
adjacent last three FNIII repeats (6, 7, 8) do not. Interestingly, the
recombinant fibrinogen-like domain mediated attachment of fibroblasts
which was blocked by adding heparin and by pretreating the cells with
heparinase or chlorate (an inhibitor of glycosaminoglycan synthesis;
Humphries et al., 1989). These results pointed to a
physiological role in cell adhesion of the heparin-binding site within
the fibrinogen-like domain of tenascin-C. In addition to this domain,
Aukhil et al.(1993) found that recombinant tenascin-C
fragments consisting of FNIII repeats 1-5 and 3-5 were
retained by heparin-Sepharose. Because the isolated FNIII repeat number
3 did not bind, a second, independent heparin-binding site on FNIII
repeats 4-5 was postulated. This domain, however, had no cell
attachment activity (Aukhil et al., 1993). Experiments with
small recombinant fragments cannot solve the question of whether a
certain domain is actually required for heparin binding by the complete
tenascin-C molecule. An active site might be exposed in a small
fragment but masked in the context of adjacent domains (cf. Yurchenco et al., 1993). We therefore took a
complementary approach which was to delete part or all of the
C-terminal fibrinogen-like domain from entire tenascin-C. The results
presented here strongly suggest that an intact fibrinogen-like domain
is necessary and sufficient for heparin binding activity of the whole
tenascin-C subunit. Tenascin-C truncated at the C terminus, either by
proteolysis or by mutation, failed to bind to heparin-agarose when
applied under physiological salt concentrations. In contrast,
tenascin-C lacking FNIII repeats 1-5 still bound to heparin. The
results presented in this paper are summarized schematically in Fig. 9. Our experiments do not exclude completely that a
second putative heparin binding region in tenascin-C might be active
under certain conditions. Aukhil et al.(1993) applied their
recombinant tenascin-C fragments to heparin-Sepharose at low ionic
strength, i.e. in 20 mM Tris-HCl without NaCl, and
found that both the fibrinogen-like globe and FNIII repeats 3-5
eluted at 0.19 M NaCl. Here we used buffer containing 0.15 M NaCl for initial binding of intact tenascin-C to
heparin-agarose and cleaved it proteolytically on the column. Under
these conditions, small tenascin-C fragments recognized by mAb Tn20, i.e. derived from FNIII repeats 2-5, were released from
the heparin matrix (cf.Fig. 9C). Therefore,
the second possible binding region in FNIII repeats 4-5 might be
hidden in the intact protein. Which protein sequence or sequences
are responsible for heparin binding? Cardin and Weintraub(1989)
compiled the known structures of heparin-binding peptides found in
various proteins. They derived two consensus sequence motifs, XBBXBX and XBBBXXBX, where B is a basic
residue and X a nonbasic, most often hydrophobic amino acid.
Bober Barkalow and Schwarzbauer(1991) mutated recombinant fibronectin
by changing the sequence PRRARV in the thirteenth FNIII repeat to
PTMARV. This resulted in an almost complete loss of heparin binding
activity of fibronectin. Hence, this sequence of the consensus XBBXBX is necessary for binding of entire fibronectin to
heparin. However, the presence of a putative consensus sequence per
se is not a sufficient criterion to predict heparin binding by a
protein domain. Two other XBBXBX consensus sequences found in
fibronectin have no heparin binding activity (Bober Barkalow and
Schwarzbauer, 1991). Nevertheless, it is quite clear that specific
structural motifs rather than just nonspecific ionic interactions are
required for protein binding to heparin (Cardin and Weintraub, 1989). No amino acid sequence is found in tenascin-C which perfectly fits
one of the postulated consensus motifs (Cardin and Weintraub, 1989). We
therefore screened the chick tenascin-C sequence (Spring et
al., 1989) for clusters of basic amino acids which are conserved
among chick, mouse (Weller et al., 1991), and human (Siri et al., 1991) tenascin-C. Seven such clusters containing at
least 3 basic residues within a hexapeptide stretch (as in XBBXBX or XBBBXBX) are found. The first within the putative
signal peptide is likely to be cleaved in mature tenascin-C. The entire
N-terminal half of the protein contains no other conserved basic
cluster. The next three are found in the fourth and the fifth FNIII
repeat. The remaining three basic clusters are all contained within the
fibrinogen-like domain of tenascin-C. The sequence EKGRHKSKP (amino
acids 1027-1035) is located at the C terminus of the fifth FNIII
repeat and therefore also at one end of the corresponding tenascin-C
fragments prepared by Aukhil et al. (1993). This might explain
why the isolated domains clearly bind to heparin whereas the same
region is obviously not sufficient to mediate binding of large
tenascin-C fragments. Our results with large tryptic fragments indicate
that the region essential for heparin binding by whole tenascin-C it is
located within 10 kDa from the C terminus of the subunit, i.e. in the second half of the fibrinogen-like domain. Of the two
conserved basic clusters found in this region, the sequence
AKTRYRLRV(1702-1710) of alternating basic and hydrophobic amino
acids resembles a
Volume 270,
Number 7,
Issue of February 17, 1995 pp. 3378-3384
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)-like repeats, a
variable number of fibronectin type III domains, and a C-terminal
fibrinogen homology (Jones et al., 1989; Spring et
al., 1989). Intact tenascin-C binds to heparin at physiological
salt concentrations (Marton et al., 1989; Faissner et
al., 1990; Chiquet et al., 1991). This activity was
attributed to a proteolytic, C-terminal 60-kDa fragment of chick
tenascin-C (Chiquet et al., 1991). Using small recombinant
fragments of human tenascin-C expressed in bacteria, Aukhil et
al.(1993) showed that the fibrinogen-like domain (26 kDa) as well
as fibronectin type III repeats 3-5 (30 kDa) bound to
heparin-Sepharose. The question of whether the two active sites were
equally important for binding of whole tenascin-C to heparin was not
addressed. Here, we tested the heparin binding activity of tenascin-C
truncated at its C-terminal fibrinogen-like domain, either
proteolytically or by deletion from recombinant full-length protein. We
present evidence suggesting that only one of the two putative heparin
binding regions is important in the context of the complete protein
chain. Hence, a single site per tenascin-C subunit is likely to be
necessary for recognition of heparin-related glycosaminoglycans in the
matrix and on cell surfaces.
Chick Fibroblast Tenascin-C and Monoclonal
Antibodies
Endogenous tenascin-C was purified from the
conditioned medium of 11-day-old chick embryo fibroblasts using
immunoaffinity chromatography with monoclonal antibody (mAb) TnM1
(Chiquet and Fambrough, 1984; Chiquet et al., 1991). The
smallest tenascin-C splice variant (190 kDa) was separated from larger
variants by using mAbs against the differentially spliced fibronectin
type III repeats (Chiquet et al., 1991).
Construction of Recombinant Chick Tenascin-190,
Tenascin-FN 1-5
A full-length cDNA for the 230-kDa
variant of chick tenascin-C was constructed by joining two overlapping
partial cDNAs which were cloned into pBluescript KS
, and
Tenascin-FB
(provided by Dr. J. Spring, University of Basel). This construct
is called pCTN 230 and represents nucleotides 1-5956 of the EMBL
data base entry M23121 (Spring et al., 1989). The open reading
frame starts at position 235 and ends at position 5658. The smallest
naturally occurring splicing variant, tenascin-190 (TN 190), was
constructed by precise deletion of the extra fibronectin type III
(FNIII) repeats (nucleotides 3361-4179 of the EMBL data base
entry M23121). This was done using a PCR-based method called
``splicing by overlap extension'' (Horton et al.,
1989) and resulted in the plasmid pCTN 190. The PCR amplified regions
of pCTN 190 were analyzed by sequencing (Sequenase DNA Sequencing Kit;
United States Biochemicals Corp.) to verify the correct primary
structure. To construct a tenascin-C without the fibrinogen-like globe
(TN FB), we deleted the fibrinogen domain
(nucleotides 4999-5658 of the EMBL data base entry M23121) from
pCTN 190 using the splicing by overlap extension method, resulting in
plasmid pCTN FB. In the same way, we deleted FNIII
repeats 1-5 (TN FN1-5; nucleotides
2005-3360 of the EMBL data base entry M23121) from pCTN 190.
Constructs were analyzed by sequencing the PCR modified regions. For
further experiments, we selected from the sequenced clones those which
had no nucleotide exchanges resulting in amino acid mutations., and pCDNA/TN
FN1-5. The domain structure of the
corresponding recombinant proteins is represented in Fig. 9A.Transfection and Expression of Recombinant
Tenascin-C
HT1080 human fibrosarcoma cells (American Tissue
Culture Collection) were stably transfected with pCDNA/TN 190, pCDNA/TN
FB, or pCDNA/TN FN1-5 by the
calcium phosphate method using the CellPhect Transfection Kit protocol
(Pharmacia). Dulbecco's medium with 10% fetal calf serum was used
for the transfection as well as for subsequent cell cultures. After 16
h of transfection, the cells were washed one to two times with fresh
medium and incubated for 1 additional day. The cells were harvested
with trypsin-EDTA and plated on 10-cm dishes (Falcon) at various
dilutions in selective medium containing 700 µg/ml G-418
(Calbiochem). After 1 week the medium was changed and after 1
additional week, the first clones were picked. The clones were plated
on multiwell plates (24 wells; Falcon) in medium containing 700
µg/ml G-418 and 10
M dexamethasone
(which down-regulates endogenous production of human tenascin-C by
HT1080 cells; not shown). One week later, the conditioned media were
analyzed for the presence of secreted chick tenascin-C variants by
ELISA (Pearson et al., 1988) using mAb TnM1 (Chiquet and
Fambrough, 1984). Cells from positive wells were subcloned, and the
screening procedure was repeated until all subclones were positive for
tenascin-C secretion.Isolation and Characterization of Recombinant
Tenascin-C
Conditioned media from tenascin-C secreting
HT1080-pCDNA/TN 190 and HT1080-pCDNA/TN FB cells were
collected (850 ml), concentrated by precipitation with 50% saturated
ammonium sulfate, and dissolved in 20 ml of phosphate-buffered saline
(NaCl/P
: 150 mM NaCl, 20 mM NaP, pH 7.4). After the probes had been dialyzed
overnight against NaCl/P, the material was slowly passed
over a gelatine-agarose column (20-ml bed volume; Sigma) to remove
fibronectin. This column was connected to a Sepharose 4B column (10-ml
bed volume; Pharmacia) coupled with a monoclonal antibody against human
tenascin-C, TnC18-13 (Schenk et al., 1995) to eliminate
traces of endogenous human tenascin-C produced by the HT1080 cells.
Subsequently, a Sepharose 4B column coupled with anti-chick tenascin-C
mAb Tn68 was applied (Chiquet-Ehrismann et al., 1988; 20-ml
bed volume). After washing with NaCl/P, the first two
columns were disconnected, and the mAb Tn68 column was washed with
0.05% Triton X-100/1 M NaCl in NaCl/P. After a
second wash with NaCl/P, tenascin-190 or
tenascin-FB, respectively, were eluted with 50 mM diethylamine in H
O, pH 11. Before the fractions were
frozen, they were neutralized with 1 M Tris-HCl, pH 7.4. All
buffers contained 0.01% Tween 20 (Fluka), which we have found to
prevent sticking of tenascin-C to plastic tubes. Samples were analyzed
by SDS-PAGE (6% polyacrylamide gel; Laemmli, 1970) and immunoblotting
(Towbin et al., 1979) using mAbs Tn20 and Tn4 (2 µg/ml)
and peroxidase-labeled goat anti-mouse IgG (1:1000; Cappel). Blots were
developed by chemiluminescence (ECL kit; Amersham). Molecular mass
standards were from Sigma; calibrations (
10
kDa) are indicated on one side of each gel figure. For rotary
shadowing and electron microscopy, recombinant proteins were processed
as described (Chiquet-Ehrismann et al., 1988).Heparin-binding Proteolytic Fragments of Chick Fibroblast
Tenascin-C
To generate small heparin-binding fragments from
chick tenascin-C, 1 mg of the protein was bound to a column of 1.5 ml
of heparin-agarose (Sigma) in Tris-buffered saline (NaCl-Tris: 150
mM NaCl, 20 mM Tris-HCl, pH 7.4). After washing with
NaCl-Tris, bound tenascin-C was digested on the column by applying 1.5
ml of Pronase solution (Sigma; 50 µg/ml in NaCl-Tris containing 1
mM CaCl and 1 mM MgCl) and
incubating at 20 °C for 30 min. Tenascin-C fragments released by
the protease were washed from the column with 2.5 ml of NaCl-Tris, and
4 mM phenylmethanesulfonyl fluoride (Sigma) were added to stop
further digestion. Tenascin-C fragments retained on the heparin-agarose
were eluted with 1 M NaCl, 20 mM Tris-HCl, pH 7.4.
Unbound and bound fragments were analyzed by SDS-PAGE (3-15%
acrylamide gradient gel) and immunoblotting (Chiquet et al.,
1988) using anti-chick tenascin-C mAbs..
The reaction was stopped by adding 10 µg/ml soybean trypsin
inhibitor (Sigma). After removing a sample, the digested material was
immediately applied to a 1-ml heparin-agarose column. The flow-through
was collected, and bound fragments were eluted with 1 M NaCl,
20 mM Tris-HCl, pH 7.4. All samples were analyzed by SDS-PAGE
(3-15% acrylamide gradient gel).Heparin Binding of Recombinant Chick
Tenascin-C
Conditioned media samples (200 µl) from HT1080
cells secreting complete or mutated recombinant chick tenascin-C were
applied to a 0.5-ml heparin-agarose column which was equilibrated with
150 mM NaCl, 100 mM Tris-HCl, pH 7.4, containing
0.01% Tween 20 (Fluka) (NaCl-Tris-Tween). Using starting buffer with 20
mM instead of 100 mM Tris-HCl gave the same results
(not shown). During elution, 200-µl fractions were collected. The
column was first washed with 1 ml of the starting buffer. To release
bound material, a gradient from 0.15 to 1 M NaCl was applied
(in increments of 50 mM NaCl in the same buffer; 200
µl/step). Samples were analyzed for the presence of tenascin-C by a
solid-phase ELISA using mAb TnM1 (2 µg/ml) and peroxidase-labeled
goat anti-mouse IgG (1:1000; Cappel) as described previously
(Chiquet-Ehrismann et al., 1988). were dialyzed against
NaCl-Tris-Tween and applied separately onto a 0.5-ml heparin-agarose
column equilibrated in the same buffer. The flow-through was collected,
and bound material was eluted with 1 M NaCl, 100 mM Tris-HCl, pH 7.4. All fractions were analyzed by SDS-PAGE (6%
polyacrylamide gel) and subsequent immunoblotting using mAb Tn20 as
described above.Heparin Binding of a C-terminal Tenascin-C
Peptide
The peptide PSSFRNLEGRRKRA which comprises the last 14
C-terminal amino acids of the chick tenascin-C sequence was synthesized
and purified by Dr. Paul Jenö at the Biocenter
facilities. Four mg of peptide was coupled to 4 mg of keyhole lamprey
hemocyanin (Sigma) or bovine serum albumin (Serva), respectively, with
1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (Sigma) as described
(Jennes and Stumpf, 1983; Arechiga et al., 1986). A rabbit
antiserum against the peptide-hemocyanin conjugate was generated
according to a published protocol (Arechiga et al., 1986). A
small column of tenascin-C coupled to CNBr-activated Sepharose 4B
(Pharmacia) was used to affinity-purify specific IgG from this
antiserum, which was used for immunoblots (see above). The
peptide-bovine serum albumin conjugate (and untreated serum albumin as
a control) was dialyzed against 150 mM NaCl, 20 mM Tris-HCl, pH 7.4, and incubated with a 100-µl aliquot of
heparin-agarose beads. Supernatants and material eluted from the beads
with 1 M NaCl, 20 mM Tris-HCl, pH 7.4, were analyzed
by SDS-PAGE as described above.
Mapping of Anti-tenascin-C mAb Epitopes Relative to
Heparin-binding Sites
In order to identify anti-tenascin-C mAbs
with epitopes close to the heparin-binding site(s) of intact chick
tenascin-C, the protein was allowed to bind to heparin-agarose in
buffer containing 150 mM NaCl, and digested on the column with
Pronase. Tenascin-C fragments not retained by heparin were released by
the protease. Bound fragments were eluted with 1 M NaCl. Bound
and unbound fractions were run on SDS-PAGE and immunoblotted with a
series of anti-tenascin-C mAbs (Fig. 1).
mutant protein does not react with mAb Tn4 (f).
Binding of a Tryptic 180- But Not a 170-kDa Tenascin-C
Fragment to Heparin
In order to assess the importance of the
C-terminal heparin-binding site within the intact tenascin-C molecule,
we sought to generate large proteolytic fragments which only missed
small parts at the extreme ends of the subunits. When chick tenascin-C
is mildly digested with proteases, the 230-kDa splice variant is easily
cleaved within the extra FNIII repeats giving rise to the N-terminal
60-kDa heparin-binding fragment (Chiquet et al., 1991). In
contrast, the 190-kDa variant is more stable, and fragments around 170
kDa are produced (Chiquet et al., 1991). Purified 190-kDa
tenascin-C variant (Fig. 4a) was therefore incubated
with low amounts of trypsin, resulting in only two kinds of fragments,
180 and 170 kDa (Fig. 4b). Analysis on nonreducing
SDS-PAGE revealed that both fragments were partially monomeric and
partially still disulfide-linked to dimers (Fig. 4, e and f). This indicates that both are missing about 10 kDa
at the N-terminal end of the tenascin-C subunit, up to the region of
the interchain disulfide bridges (Chiquet et al., 1991). Thus,
whereas both fragments have a similar N terminus, they must differ at
their C terminus, the 170-kDa fragment lacking about half of the
fibrinogen-like domain (cf.Fig. 9B). Both
fragments were still recognized by mAb Tn4, indicating that the epitope
of this antibody is located within the first half of the
fibrinogen-like domain (not shown).
Lack of Heparin Binding by Recombinant Tenascin-C Missing
the Fibrinogen-like Domain
To exclude artifacts introduced by
cleaving chick fibroblast tenascin-C with proteases, it was important
to repeat a similar experiment as presented in the last section using
recombinant, intact as well as mutated protein. Full-length chick
190-kDa tenascin-C variant was expressed in stably transfected human
HT1080 cells. Recombinant protein of the correct size was secreted into
the medium and could be isolated from there by antibody affinity
chromatography (Fig. 3a). Electron microscopic
examination of the purified material showed exclusively oligomeric
particles; many had six arms with all structural features of intact
tenascin-C molecules (Fig. 5a). A second vector was
constructed with a truncation of the tenascin-C coding sequence exactly
at the C-terminal end of the last FNIII repeat, i.e. lacking
the entire fibrinogen-like domain but otherwise intact. A comparable
amount of the resulting recombinant protein of 165 kDa was produced
upon expression and was purified (Fig. 3b). The mutant
protein reacted with anti-tenascin-C mAbs (Fig. 3d)
with the exception of mAb Tn4 (Fig. 3f). When viewed in
the electron microscope, the truncated recombinant tenascin-C had
somewhat shorter arms, lacking the terminal globule (Fig. 5b).
, b) were sprayed onto mica and
rotary-shadowed. The top half of each panel shows a representative
overview, while the bottom half depicts enlarged selected molecules. Bar, 250 nm (top halves) or 100 nm (bottom halves),
respectively.
, d-f) was purified and applied to heparin-agarose. Loaded
material (L, a and d), unbound protein
washed from the column (W, b and e), and
protein bound and eluted with 1 M NaCl (E, c and f) was analyzed by reducing SDS-PAGE after
immunoblotting with mAb Tn20. Intact (c) but not mutant (f) tenascin-C binds to
heparin-agarose.
)
or FNIII repeats 1-5 (TN FN1-5)
were passed over heparin-agarose. After washing with 150 mM NaCl, protein bound to the column was eluted with a NaCl
concentration gradient. Chick tenascin-C was detected in collected
fractions by solid-phase ELISA using mAb TnM1. Complete recombinant
tenascin-C and the TN FN1-5 mutant protein
elute at about 300 mM NaCl, while most of the TN
FB mutant protein is washed out at 150 mM NaCl.
A Basic Peptide from the Extreme C Terminus of Tenascin-C
Binds to Heparin
At the C terminus of the fibrinogen-like
domain, and hence at extreme end of the coding sequence of tenascin-C,
a conspicuous stretch of basic amino acids is found which is conserved
among species (but which is not found in fibrinogen itself). This
peptide (PSSFRNLEGRRKRA) was synthesized, coupled to keyhole lamprey
hemocyanin, and injected into a rabbit. The resulting antiserum (not
shown) and antibodies affinity-purified from it reacted on immunoblots
with chick fibroblast tenascin-C as well as with its heparin-binding
tryptic 180-kDa fragment, but not with the non-binding 170-kDa fragment
on immunoblots (Fig. 8, e and f). This
indicates that the C-terminal peptide sequence is present at least in a
fraction of mature tenascin-C molecules. The synthetic peptide was also
covalently linked to bovine serum albumin, and the heparin binding
ability of the resulting conjugate was tested. While control serum
albumin appeared in the flow-through when applied to heparin-agarose in
150 mM NaCl (Fig. 8c), the tenascin-C
peptide-albumin conjugate was retained (Fig. 8b).
Hence, this sequence might contribute to the heparin binding of native
tenascin-C (see ``Discussion'').
1
integrin chain also strongly suppress neurite growth on tenascin-C,
indicating that this class of cell surface receptors is involved
(Wehrle-Haller and Chiquet, 1993). In addition, we found that heparin
affects neurite adhesion on a tenascin-C substrate. When present at the
beginning of a primary culture, it inhibits neurite outgrowth
completely (Wehrle-Haller and Chiquet, 1993). Addition of heparin at
later times results in partial detachment and fasciculation of already
formed neurites, whereas growth cones remain attached and continue to
migrate (Chiquet and Wehrle-Haller, 1994). Therefore, as in the case of
fibronectin (Saunders and Bernfield, 1988) and laminin (Yurchenco et al., 1993), heparin-binding site(s) on the tenascin-C
subunit might interact with cell surface proteoglycans, thereby
enhancing neurite adhesion. Indeed, tenascin-C has been reported to
bind to the heparan sulfate side chains of syndecan (Salmivirta et
al., 1991). The same heparin-binding site(s) might help to anchor
tenascin-C within the extracellular matrix, e.g. by mediating
interaction with perlecan (Noonan et al., 1991) or other
heparan sulfate proteoglycans.-pleated sheet structure known to bind heparin
in apoE; it is classified as a XBBXBX motif by Cardin and
Weintraub(1989). The basic cluster at the very C terminus of the
tenascin-C subunit, GRRKRA(1803-1808), can bind heparin in
vitro and is present at least in a fraction of the protein, as
shown here. However, which of the basic clusters in the fibrinogen-like
domain is most important for heparin binding activity remains to be
shown by further mutational analysis. The approach described here to
express recombinant whole tenascin-C with small deletions or mutations
will be essential to elucidate the function of this complicated
multidomain protein in cell and matrix interactions.
)
We thank Jürg Spring for providing
the full-length chick tenascin-C cDNA, Theres Schulthess and Charlotte
Fauser for their invaluable help with the electron microscopy, Paul
Jenö for peptide synthesis, Gerd Kleemann for
advice with computer graphics, and John Daly and Stefan Baumgartner for
critical reading of the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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