©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Single Heparin Binding Region within the Fibrinogen-like Domain Is Functional in Chick Tenascin-C (*)

(Received for publication, October 18, 1994; and in revised form, November 28, 1994)

Doris Fischer (1) Ruth Chiquet-Ehrismann (1)(§) Carlo Bernasconi (2)(¶) Matthias Chiquet (2)(¶)

From the  (1)Friedrich Miescher Institute, P. O. Box 2543, CH-4002 Basel and the (2)Department of Biophysical Chemistry, Biocenter of the University, CH-4056 Basel, Switzerland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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(^1)-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.


EXPERIMENTAL PROCEDURES

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).

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.



Construction of Recombinant Chick Tenascin-190, Tenascin-FN 1-5, and Tenascin-FB

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 (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.

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, 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 10M 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(i): 150 mM NaCl, 20 mM NaP(i), pH 7.4). After the probes had been dialyzed overnight against NaCl/P(i), 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(i), 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(i). After a second wash with NaCl/P(i), tenascin-190 or tenascin-FB, respectively, were eluted with 50 mM diethylamine in H(2)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 (times 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(2) and 1 mM MgCl(2)) 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.

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(2). 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).

The putative domain structure of the different proteolytic fragments of tenascin-C is shown schematically in Fig. 9.

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).

Purified recombinant tenascin-190 and tenascin-FB 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.


RESULTS

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).


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 mutant protein does not react with mAb Tn4 (f).



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).

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).


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.

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).


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, 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.



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, 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.




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) 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'').


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.




DISCUSSION

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 beta1 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.

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 beta-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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Friedrich Miescher Institut, Postfach 2543, CH-4002 Basel, Switzerland. Tel.: 41-61-6972494; Fax: 41-61-6973976.

Supported by grants from the Swiss National Fund (to M. C.).

(^1)
The abbreviations used are: EGF, epidermal growth factor; ELISA, enzyme-linked immunosorbent assay; FB, fibrinogen-like domain; FNIII, fibronectin tye III repeat; mAb, monoclonal antibody; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; TN, tenascin.


ACKNOWLEDGEMENTS

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.


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