(Received for publication, October 20, 1994; and in revised form, December 22, 1994)
From the
Two sites on tenascin mediate interactions with glycosaminoglycan chains of proteoglycans. One is situated on the fibrinogen-like domain, whereas the other lies within the fibronectin type III homology region (Aukhil, I., Joshi, P., Yan, Y. Z., and Erickson, H. P.(1993) J. Biol. Chem. 268, 2542-2553.). We now characterize the latter binding site more closely by means of recombinant protein fragments derived from the type III homology region of tenascin. Using a heparin-Sepharose column, we localize the second heparin binding site to the fifth fibronectin type III domain. This is confirmed in solid phase assays by incubation of fusion proteins with biotin-labeled heparin. In addition, we demonstrate the binding of heparan sulfate and dermatan sulfate to domain five. Molecular modelling of this domain reveals a conserved heparin-binding motif that we propose as the putative binding site. The fact, that different glycosaminoglycans may bind to this domain, implies that different classes of proteoglycans may in vivo compete for the same site.
Tenascin-C is a large extracellular matrix glycoprotein
implicated in pattern formation during development and in malignant
progression. It commonly occurs as a homohexamer, each polypeptide
chain disulfide linked at its amino terminus to give the characteristic
hexabrachions observed in electron
microgaphs(1, 2, 3) . Each tenascin
polypeptide is composed of colinear domains, similar to epidermal
growth factor, fibronectin type III domains (TNfn) ()or
fibrinogen (TNfbg)(4, 5) . A prominent structural
feature is the variability of a section in the TNfn region arising from
alternative splicing. In chick, for example, three major tenascin
variants exist with either one (TN200) or three (TN220) additional TNfn
domains inserted between TNfn5 and TNfn6 of the TN190 variant (Fig. 1).
Figure 1: Schematic localization of fusion proteins on the tenascin-C isoforms. The three isoforms of chick tenascin-C differ in their number of TNfn domains. The TNfn domains of the TN190 isoform are numbered from 1 to 8. The alternatively spliced domains are designated with letters, based on the sequence of the human protein with seven additional type III domains (14) . The fusion proteins TNfn56, TNfn5D, TNfnD6, and TNfn5ABD6 were constructed as outlined under ``Experimental Procedures.'' They contain at their amino-terminal end 6 histidines and a factor Xa cleavage site (Ile-Glu-Gly-Arg). The bordering domains of the fusion proteins do not start or end with their theoretical first or last amino acid but extend 4 or 5 amino acids into the adjacent TNfn domain. For more details consult sequences listed in Table 1. EGF, epidermal growth factor.
The alternatively spliced region has been linked to ligand binding of tenascin. Contactin/F11, a neuronal cell receptor anchored in the membrane by glycosyl phosphatidylinositol, is a ligand for tenascin-C and binds specifically to the TN190 isoform(6) . This contrasts with the recent isolation and identification of a 35-kDa cell surface tenascin-C receptor as annexin II, which binds specifically to the alternatively spliced region itself(7) . As annexin II binds within the alternatively spliced region and contactin/F11 is specific for the TN190 variant, missing these domains, alternative signaling may be mediated by different tenascin-C isoforms, underlining the functional importance of this region. Consistent with a functional role, the expression of these variants is closely regulated during development. For example, embryonic cartilage contains only the TN190 isoform(2, 8) , whereas the TN220 isoform expression is timed to pave the migration of neural crest cells in the cornea(3) .
Another prominent group of ligands are
proteoglycans, some of which interact with tenascin-C through their
glycosaminoglycan moieties. This includes the chondroitin sulfate
proteoglycans cytotactin-binding proteoglycan(9) , and receptor
tyrosine phosphatase (10) as well as the heparan sulfate
proteoglycans syndecan (11) and glypican(12) . For the
latter two, binding occurs in part through the heparan sulfate chains.
In contrast to contactin/F11 and annexin II, the binding sites of these
proteoglycans have yet to be established. As a first step in this
direction, the binding of tenascin to heparin has been investigated.
Tenascin-C can be isolated over heparin-Sepharose(13) , an
interaction that may be due to one of two putative heparin-binding
regions(14) . Given that one of these sites lies within the
TNfn region and may be adjacent to the functionally important
alternatively spliced domains, we prepared recombinant protein
fragments covering this region of chick tenascin-C. Here we demonstrate
that this heparin-binding site is located on TNfn5. With molecular
modelling, we show that the Tnfn5 domain contains a cluster of basic
amino acids that forms a conserved heparin binding motif.
Recombinant proteins containing a stretch of 6
histidines amino-terminal of the factor Xa cleavage site were prepared
from M15 (pREP4), transformed with the constructs pDS9/56, pDS9/5D,
pDS9/D6, or pDS9/5ABD6, respectively. Transformed bacteria were grown
at 37 °C overnight in a rotary shaker (250 rpm) in 2 YT
medium containing ampicillin and kanamycin. The production of the
fusion proteins was initiated with
isopropyl-1-thio-
-D-galactopyranoside as
described(15) .
After 5 h, the cells were collected by
centrifugation and then resuspended in one-thirtieth of the original
volume in ice-cold sonification buffer (25 mM Tris/HCl; 150
mM NaCl; 0.2 mM phenylmethylsulfonyl fluoride; 2
mM iodacetamide; pH 8.0) and lysed in aliquots of 15 ml with a
sonifier (cell disrupter, SKAN; setting 4, 6 min, 75%). During the
sonification, the suspension was cooled with ice-water. The extract was
cleared by centrifugation at 12,000 g for 15 min at 4
°C (Sorvall, SS34). The fusion protein containing supernatant was
diluted with 1.5 volumes of loading buffer (67 mM sodium
phosphate; 300 mM NaCl; 0.02% NaN
; pH 8.0) and
loaded onto a Ni-NTA column. Under these conditions, fusion proteins
containing the 6-His tag bound tightly to the column, whereas other
bacterial proteins were eluted with washing buffer (67 mM sodium phosphate; 300 mM NaCl; 0.02% NaN
; pH
6.0). The fusion proteins were subsequently eluted with 100 mM sodium acetate; 300 mM NaCl; 0.02% NaN
; pH
3.8. The pH of the collected fractions was adjusted to 7.5 with 1 M Tris/HCl, pH10, and subjected to analysis on 10-15%
SDS-polyacrylamide gel electrophoresis followed by Coomassie Blue
staining.
The
wells were blocked for 1 h at room temperature with 200 µl of 1%
heat denatured bovine serum albumin in TBS (5 min, 85 °C). The
binding of glycosaminoglycans was performed for 2 h at room
temperature. Bound biotin labeled glycosaminoglycans were detected with
alkaline phosphatase conjugated to streptavidin (1:2500) in TBS and p-nitrophenyl phosphate-Na as substrate. Between
each step, the wells were washed 3 times with 200 µl of TBS.
A second mutation was
observed in domain D (nucleotide 3915: AC) of the construct 5D
but not D6. The nucleotide substitution changes the amino acid 1227
from a Glu to an Asp. This mutation close to the priming site of DL2
was introduced by polymerase chain reaction and could not be avoided by
repeating the polymerase chain 1 reaction with the same or other
primers.
Figure 2: Purity of the isolated fusion proteins. The fusion proteins TNfn56 (lane1), TNfn5D (lane2), TNfnD6 (lane3), and TNfn5ABD6 (lane4) were analyzed on 10-15% SDS-polyacrylamide gel electrophoresis and stained with Coomassie Blue.
Figure 3: Binding of fusion proteins to heparin-Sepharose. Fusion proteins TNfn5ABD6 (left), TNfn56 (middle), and TNfnD6 (right), were each applied to heparin-Sepharose in 20 mM NaCl and eluted with a linear gradient of NaCl. Detection was by enzyme-linked immunosorbent assay using tenascin-specific polyclonal antibodies and phosphatase-coupled secondary antibodies, with absorbance measured at 405 nm.
First, we examined the effect of increasing NaCl concentrations on the binding of heparin to fusion proteins. Protamine sulfate, TNfn56, TNfn5D, TNfnD6, and TNfn5ABD6 were coated in TBS, and, after incubating heparin at 150 mM NaCl with these substrates, washing was performed under increasing NaCl concentrations (Fig. 4). Heparin binds to all fusion proteins carrying the TNfn5 domain (TNfn56, TNfn5D, or TNfn5ABD6) but not to TNfnD6. The binding to TNfn5 decreases with increasing NaCl concentrations and is fully abolished between 450 and 600 mM NaCl, similar to the concentration required to elute TNfn56 from heparin-Sepharose. The binding of heparin to protamine sulfate is not influenced by the NaCl concentrations tested. In a separate set of experiments (not shown), the binding of heparin to TNfn56 was found to be saturable. The binding to TNfn56 was also similar to that between heparin and the fibrinogen-like domain of tenascin, consistent with previously published data(14) .
Figure 4: Heparin binding to tenascin fusion proteins. The substrates were coated to 96-well plates as outlined under ``Experimental Procedures.'' After a 2-h incubation with 1 µM biotin-labeled heparin, the wells were washed with different concentrations of NaCl, and the remaining heparin was detected. The strong binding of heparin to the positive control protamine sulfate was not influenced by the NaCl concentrations used.
Figure 5: Binding of other glycosaminoglycans to TNfn56. Biotin-labeled glycosaminoglycans were incubated at different concentrations with coated TNfn56. Heparin shows the strongest binding. Chondroitin sulfates A and C (CSA and CSC) do not bind to TNfn56. Dermatan sulfate (DS) and heparan sulfate (HS) bind with a similar half-maximal concentration of 50 µg/ml
Here we present evidence for a conserved heparin binding site in tenascin-C on the TNfn5 domain. This conclusion is based on the binding of heparin at physiological NaCl concentrations to fusion proteins containing TNfn5. This is true for the fusion proteins derived from chicken tenascin, shown here, and for fusion proteins derived from human tenascin(14) . Heparin binds most strongly of the glycosaminoglycans tested, likely due to its higher level of modification. Heparin and heparan sulfate differ in their degree of sulfation and extent of epimerization of D-glucuronic to L-iduronic acid(19) . The lower affinity of heparan sulfate and dermatan sulfate may not only be explained by their lower degree of sulfation, but also by their reduced content of L-iduronic acid. The latter may be essential for the binding to TNfn5, as glycosaminoglycans lacking L-iduronic acid (chondroitin sulfates A and C) did not bind to TNfn5. Involvement of L-iduronic acid has also been observed for the binding of heparin or heparan sulfate to basic fibroblast growth factor(20, 21) .
The functional conservation of
heparin binding between chicken and human TNfn5 implies that the
binding site itself must also be conserved. To identify this binding
site within domain five, we modeled chicken TNfn5 (Fig. 6),
based on the known structure of human TNfn3(18) . The
fibronectin type III repeats are formed from two -sheets, giving
rise to a minor face of three
-strands (designated A, B, and E)
and a major face of four
-strands (C, C`, F, and G). The
-strands F and G, shown facing the viewer in Fig. 6,
contain a consensus heparin-binding motif
(BXBXBXXXXB)(22) ,
which is highly conserved in tenascin-C between different species
ranging from chick, mouse, human, and pig(23) . The spacing
between residues Lys-1032 and Lys-1039 (Table 3), which are
separated by 21 Å in the model (Fig. 6), is likely to be
critical here. In both apolipoprotein E and antithrombin III, a
heparin-binding motif is formed from similarly conserved basic amino
acids, which are spaced about 23 Å apart along
-strands (Table 3). This is consistent with that predicted for efficient
binding to a pentasaccharide sequence(22) , thought to be the
minimal functional unit of heparin(24) . The conservation of
the consensus heparin-binding motif stands in contrast to the poor
conservation of domain five as a whole(25) , providing further
evidence for a likely functional importance. However, other basic
residues may also be involved in heparin binding. Inspection of the
TNfn5 model reveals that other pairs of conserved basic amino acids lie
within a radius of 20-23 Å on the major face of TNfn5. In
particular Arg-985 and Arg-987 on the C-strand are possible candidates
in combination with Lys-1034 and Lys-1039, respectively. Which
combination of amino acids are involved in binding to heparin can now
be studied using site-directed mutagenesis or by x-ray diffraction
methods should crystallization of the fusion protein-glycosaminoglycan
complex prove to be possible.
Figure 6:
Chicken TNfn5 modelled on human TNfn3. The blue and red highlighted stretches represent the
basic amino acids Lys and Arg of chicken TNfn5. The basic amino acids,
which are conserved in human, pig, mouse, and chicken are represented
by red, and nonconserved residues are represented by blue. The conserved basic amino acids are concentrated on the
major face of the domain. The heparin binding motif
(BXBXBXXXXB) is located on the -strand
G, with Lys-1032 (K`) and Lys-1039 (K) separated by
21 Å.
Presumably the correct conformation of
the binding region is critical for the specificity of interaction with
glycosaminoglycan chains(22) . An advantage of fusion proteins
compared with peptides is that a correct folding of the domains should
lead to the binding site being presented to the glycosaminoglycan in a
close to native form. With this in mind, the strategy for preparation
of the fusion proteins was chosen to maximize the likelihood of
producing native proteins that are correctly folded and soluble under
nondenaturing conditions. To this end, the constructs begin 4-5
amino acids amino- and carboxyl-terminal outside the theoretical
domains. The length of the extraneous sequence at the amino termini
(polyhistidine tail for purification and factor Xa cleavage site) was
kept to a minimum to reduce the influence on folding of the type III
homology domains. This approach appears to be successful, as the fusion
proteins were released in soluble form from the Escherichia coli merely by sonification in isotonic buffer. A further indication of
the native structure comes from the ready cleavage by factor Xa.
Incorrect folding of fusion proteins would lead to the factor Xa
recognition sequence being buried, which then only becomes exposed to
the proteinase after denaturation/renaturation cycles. Both our fusion
proteins ()and those produced by a similar protocol (14, 18) can be crystallized for structural analysis.
This further strengthens the case for native folding. The proposed
heparin-binding site is likely to be available in the intact tenascin-C
as fusion protein TNfn1-5 from human (14) and TNfn56
(shown here), both encompassing TNfn5, are active.
A number of candidate proteoglycan ligands have been reported for tenascin-C(9, 10, 11, 12) . Among them, evidence for binding via the glycosaminoglycans has been presented for syndecan (11) and glypican (12) . The degree of modification of the heparan sulfate chains of syndecan and glypican is likely to be tissue-dependent. Whether a particular sequence of charged residues is necessary for optimal binding to TNfn5, such as the heparin pentasaccharide specific for antithrombin or the dermatan sulfate heptasaccharide binding to heparin cofactor(19) , is the subject of further investigations. The assignment of glycosaminoglycan binding activity to TNfn5 and the availability of the recombinant fragments should stimulate the search for further in vivo proteoglycan ligands and lead to a better understanding of the role of tenascin-C in development and disease.