(Received for publication, March 3, 1997, and in revised form, April 8, 1997)
From the Department of Pharmacology, New York
University Medical Center, New York, New York 10016, the
§ Friedrich Miescher Institute, P. O. Box 2543, CH-4002
Basel, Switzerland,
Biocenter, University of Basel, CH-4056
Basel, Switzerland, and the ¶ Department of Pharmacology, State
University of New York, Health Science Center, Brooklyn, New
York 11203
Two nervous tissue-specific chondroitin sulfate
proteoglycans, neurocan and phosphacan (the extracellular domain of
protein-tyrosine phosphatase-/
), are high-affinity ligands of
tenascin-C. Using portions of tenascin-C expressed as recombinant
proteins in human fibrosarcoma cells, we have demonstrated both by
direct radioligand binding assays and inhibition studies that
phosphacan binding is retained in all deletion variants except those
lacking the fibrinogen-like globe and that phosphacan binds to this
single domain with nearly the same affinity (Kd
~12 nM) as to native or recombinant tenascin-C. However,
maximum binding of neurocan requires both the fibrinogen globe and some
of the adjacent fibronectin type III repeats. Binding of phosphacan and
neurocan to intact tenascin-C, and of phosphacan to the fibrinogen
globe, is significantly increased in the presence of calcium.
Chondroitinase treatment of the proteoglycans did not affect their
binding to either native tenascin-C or to any of the recombinant
proteins, demonstrating that these interactions are mediated by the
proteoglycan core proteins rather than through the glycosaminoglycan
chains. These results are also consistent with rotary shadowing
electron micrographs that show phosphacan as a rod terminated at one
end by a globular domain that is frequently seen apposed to the
fibrinogen globe in mixtures of phosphacan and tenascin-C. C6 glioma
cells adhere to and spread on deletion variants of tenascin-C
containing only the epidermal growth factor-like domains or the
fibronectin type III repeats and the fibrinogen globe. In both cases
cell adhesion was inhibited by similar concentrations of phosphacan, demonstrating that the fibrinogen globe is not necessary for this effect, which is apparently mediated by a direct action of phosphacan on the cells rather than by its interaction with the proteoglycan binding site on tenascin-C.
We have previously reported that neurocan and
phosphacan/protein-tyrosine phosphatase-/
, two major nervous
tissue-specific chondroitin sulfate proteoglycans, are high affinity
ligands of tenascin-C (apparent Kd ~3
nM) and that phosphacan inhibits the adhesion of C6 glioma
cells to tenascin-C (1). Neurocan is a multidomain proteoglycan with a
136-kDa core protein (2) and together with versican and brevican is a
member of the aggrecan family of hyaluronan-binding proteoglycans (3).
It contains N-terminal immunoglobulin-like and hyaluronan-binding
domains, a C-terminal domain consisting of EGF-, lectin-, and
complement regulatory protein-like sequences, and a nonhomologous
central domain of 593 amino acids, which contains the attachment sites for the three chondroitin sulfate chains and a large number of O-glycosidic oligosaccharides. In contrast, phosphacan (4, 5), which contains a 173-kDa core protein and three chondroitin sulfate
chains, is an mRNA splicing product that represents the entire
extracellular domain of a receptor-type protein-tyrosine phosphatase
that also occurs as a chondroitin sulfate proteoglycan in brain (3).
Phosphacan and protein-tyrosine phosphatase
/
have an N-terminal
carbonic anhydrase-like domain followed by a fibronectin type III
sequence. The phosphatase has, in addition to two cytoplasmic catalytic
domains, an extracellular juxtamembrane sequence of 860 amino acids
that may be deleted by alternative splicing (6) and appears to contain
most of the chondroitin sulfate attachment sites that are actually
utilized (7). The binding of phosphacan/protein-tyrosine phosphatase
/
to tenascin-C is mediated at least in part by
N-linked oligosaccharides on the proteoglycan (8).
In addition to their high-affinity binding to tenascin-C, neurocan and phosphacan also interact with several immunoglobulin superfamily neural cell adhesion molecules including Ng-CAM/L1, N-CAM, TAG-1/axonin-1, Nr-CAM, and contactin (9-12). Most of these interactions have apparent dissociation constants in the subnanomolar range, and they are affected in different ways by the removal of N-linked oligosaccharides or chondroitin sulfate chains (8, 11). Immunocytochemical studies of embryonic and early postnatal nervous tissue showed an overlapping localization of the proteoglycans with all of their identified ligands, further supporting the biological significance of their ability to interact in vitro. In addition to their demonstrated high-affinity interactions with neural cell adhesion and extracellular matrix proteins, neurocan and phosphacan are also potent inhibitors of cell adhesion and inhibit or stimulate neurite outgrowth depending on the cell type and other factors (1, 9, 10, 12, 13). Our data therefore suggest that these two chondroitin sulfate proteoglycans are components of a multidimensional mechanism for the regulation of cell-cell and cell-matrix interactions at different sites and periods during nervous tissue histogenesis and that the multiplicity of ligands with differing affinities and properties could provide a means for the fine tuning of various regulatory processes.
To better understand the molecular basis for these multiple but
differentially regulated interactions, and perhaps to eventually design
agents that will affect the binding process, we have begun studies
aimed at identifying the functionally active regions of these large
multidomain proteins. In the present report we demonstrate the critical
role of the fibrinogen globe at the C terminus of tenascin-C for its
interactions with both neurocan and phosphacan/protein-tyrosine phosphatase-/
. We also show that some of the adjacent fibronectin type III repeats are involved in interactions of tenascin-C with neurocan and that the inhibitory effect of phosphacan on the adhesion of rat C6 glioma cells is attributable to a direct action of phosphacan on the cells rather than by blocking adhesion sites on tenascin-C.
Native tenascin-C from
chick embryo fibroblasts (CEF TN)1 was
isolated as described (14). The recombinant chick tenascin-C variants
were expressed in stably transfected HT1080 cells (American Type
Culture Collection). The construction and purification of the smallest
naturally occurring tenascin-C splicing variant TN 190, the mutants
missing either the fibrinogen globe in TN FB (15), the
EGF-like repeats in TN EGF
, the fibronectin type III
repeats in TN FN
, the fibronectin type III repeats as
well as the fibrinogen globe in TN FF
, or the EGF-like
repeats and the fibronectin type III repeats in TN EFN
(16), respectively, have been described previously. TN 260, containing
all of the 14 known fibronectin type III repeats of chick tenascin-C,
was produced by inserting the sequence encoding the extra repeats AD2,
AD1, and C (nucleotides 1-800 of GenBankTM/EBI data base entry X73833[GenBank])
into the tenascin-C construct containing the extra repeats A, B, and D
(corresponds to GenBankTM/EBI data base entry M23121[GenBank]) at nucleotide
position 3907 using the method of "splicing by overlap extension"
(17). The mutant lacking the EGF-like repeats and fibronectin type III
repeats 1-5, designated TN EFN1-5
, was created by the
same method. In this case nucleotides 796-4179 of the entry M23121[GenBank]
were deleted. All polymerase chain reaction modified regions were
analyzed to confirm the correct sequence. Constructs were subcloned in
the eukaryotic expression vector pCDNAI/NEO (Invitrogen, San Diego,
CA), and the encoded proteins were purified from the conditioned medium
of stably transfected HT1080 cells as described (15).
Neurocan and phosphacan (previously designated the 1D1 and 3F8 proteoglycans) were isolated from rat brain by ion exchange chromatography, gel filtration, and immunoaffinity chromatography (18) and were labeled to a specific activity of ~3 × 1018 cpm/mol with 125I by the lactoperoxidase/glucose oxidase method using Enzymobeads (Bio-Rad). Typically, 10-25 µg of protein were labeled per reaction, and free iodine was removed using a PD-10 column (Pharmacia Biotech Inc.).
Binding assays were performed as described previously (10). Briefly, proteins at a concentration of 1-12 nM were coated in removable Immulon-4 wells, and binding of 125I-labeled neurocan and phosphacan was measured in 20 mM Tris, pH 7.4, containing 150 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, 0.02% NaN3, and 1 mg/ml heat-treated BSA, following incubation with gentle shaking (45 rpm) for 9-12 h at room temperature. Scatchard plots were generated, and dissociation constants were determined using the Macintosh version of the Ligand program (19).
Electron MicroscopyProtein samples for rotary shadowing electron microscopy were dialyzed against 0.2 M NH4HCO3 and processed as described (20). To study the interaction between tenascin-C and phosphacan, CEF TN (30 µg/ml) and phosphacan (30 µg/ml) were incubated overnight at room temperature in 0.2 M NH4HCO3 containing 2 mM CaCl2 and 2 mM MgCl2 before processing for electron microscopy.
Cell Adhesion AssaysTenascin-C substrates for cell adhesion assays were prepared by incubating 2 µl of protein (10 µg/ml in PBS containing 0.001% Tween) placed in a circular array of six spots near the center of 35-mm bacteriological polystyrene Petri dishes (Falcon No. 1008) in a moist chamber. After 30 min the spots were individually washed three times with PBS, removing the solution by gentle suction, and blocked for 1 h with 1% BSA. For double coats with phosphacan (1-20 µg/ml in PBS), blocking solution was applied only once, following the second coating.
Rat C6 glioma cells were grown in Ham's F-10 containing 15% horse serum and 2.5% fetal calf serum. For adhesion assays, cells were removed from tissue culture dishes by treating briefly with 0.05% trypsin, 0.1 mM EDTA and washed with the serum-containing medium described above. The cells were then washed by centrifugation through 3.5% BSA/PBS and resuspended in Ham's F-10 containing 1% ITS+. 250 µl of Ham's/ITS+ containing 5 × 104 cells were deposited in the central region of Petri dishes that had been coated with proteins. After 90 min, unbound cells were removed by a gentle wash using PBS. Bound cells were fixed with 1% glutaraldehyde, stained with cresyl violet, and counted in five fields of 2.2 mm2 corresponding to 15% of the protein-coated area.
CEF TN and the recombinant chick
tenascin-C variants were isolated as described under "Materials and
Methods." Models of all tenascin-C proteins used in this study are
shown in Fig. 1A. Since CEF TN consists of a
mixture of three major splicing variants (21), their alternative
structures are indicated. TN 260 is the largest possible tenascin
variant containing all existing extra repeats (22), whereas TN 190 is
the smallest one consisting of constant repeats only. TN
EGF is missing the EGF-like repeat, TN FN
all fibronectin type III repeats, and TN FB
the
fibrinogen globe. Even larger deletions were made in TN
FF
, which is missing the fibronectin type III repeats as
well as the fibrinogen globe, TN EFN
where the EGF-like
repeats and the fibronectin type III repeats have been removed, and in
TN EFN1-5- that lacks both the EGF-like repeats and
fibronectin type III repeats 1-5. The proteins were analyzed by gel
electrophoresis after reduction and found to migrate as single bands
(except for CEF TN, which consists of three bands corresponding to the
three splicing variants) at the expected positions according to their subunit molecular weight (Fig. 1B). All proteins assemble
into hexameric molecules as the authentic tenascin-C isolated from chick embryo fibroblasts. Examples are shown on the electron
micrographs in Fig. 1C. Clearly the TN 260 has much longer
arms than CEF TN, whereas TN EFN
consists of hexamers
with very short arms composed of three fibronectin type III repeats and
terminating in the fibrinogen globe. The hexameric structures of the
other tenascin-C variants have been shown previously (15, 16).
Binding of Neurocan and Phosphacan to Native and Recombinant Tenascins
From the data summarized in Fig. 2, it
can be seen that phosphacan bound well (11-17% of input counts/min)
to native chick tenascin-C as well as to recombinant TN 260 (containing
the six extra fibronectin type III repeats present in a tenascin-C
splice variant), to deletion variants lacking either all of the EGF or fibronectin type III repeats, and to TN 190 (which serves as a control
for the deletion variants). Approximately the same degree of binding
was observed to the fibrinogen globe alone (TN EFN) and
to a recombinant fragment containing the fibrinogen globe and
fibronectin repeats 6-8 (TN EFN1-5-), whereas there was
<3% binding to deletion variants lacking the fibrinogen globe (TN
FB
) or the fibrinogen globe and the fibronectin repeats
(TN FF
).
In relation to a TN 190 control, neurocan binding was reduced by 20%
in the deletion variant lacking only the fibronectin type III repeats
(TN FN), by 50% in variants lacking the fibrinogen globe
(TN FB
), and by 80% in the deletion variant (TN
FF
) lacking both domains (Fig. 2). The importance of both
the fibrinogen globe and the fibronectin type III repeats for neurocan
interactions with tenascin-C was further supported by experiments using
the complementary proteins, insofar as there was only 5% binding to the fibrinogen globe, but binding increased to 12% if fibronectin repeats 6-8 were also present (Fig. 2). However, binding to TN EFN1-5- was still 30% less than to TN 190, suggesting
that fibronectin repeats 1-5 may also enhance neurocan interactions
with tenascin-C, and TN 260, which contains six extra FN III repeats,
bound neurocan somewhat better than TN 190.
Binding of neurocan and phosphacan to native tenascin-C and to the recombinant tenascins was not affected by chondroitinase treatment of the proteoglycans (Ref. 1 and data not shown). These results demonstrate that although the fibrinogen globe also contains the major heparin binding site of tenascin-C (15), its interactions with neurocan and phosphacan are mediated by the proteoglycan core (glyco)proteins rather than through the glycosaminoglycan chains.
Because binding of neurocan and phosphacan to the deletion variant lacking the EGF repeats was in both cases ~44% greater than to TN 190 (Fig. 2), it would appear that the EGF domain may inhibit the interactions of both proteoglycans. The relative amounts of neurocan or phosphacan bound to the various proteins was generally similar in experiments using an 8-fold range of concentrations (1.6-12.5 nM) for coating the plastic wells, although the percent binding increased as a function of increasing protein concentration (data not shown). These results, together with the direct demonstration of binding to the fibrinogen globe (with or without the three adjacent FN III repeats), indicate that the major differences in binding to the tenascin deletion variants do not merely reflect different substrate coating efficiencies. Binding of both proteoglycans reached equilibrium after 9-12 h and was fully reversible after incubation with an excess of unlabeled ligand (data not shown).
Saturation and Binding Affinity of Neurocan and Phosphacan to Recombinant TenascinsPhosphacan bound to recombinant TN 190 with
the same affinity (Kd ~2 nM) that we
previously found for native tenascin-C (1), whereas binding to the
fibrinogen globe alone (TN EFN) showed a somewhat lower
affinity (Kd ~12 nM; Fig.
3). The <3% residual binding of phosphacan to the
deletion variant lacking only the fibrinogen globe (TN
FB
) was probably nonspecific, since it did not yield
a meaningful saturation curve (data not shown). Neurocan also
demonstrated high affinity binding to both TN EFN1-5
and
TNEF
, with apparent dissociation constants of ~10
nM (Fig. 4).
Inhibition by Recombinant Tenascins of Neurocan and Phosphacan Binding to Tenascin-C
Binding of phosphacan to chick tenascin-C
was inhibited to the extent of 55-65% by tenascin-C, TN 190, TN
FN, TN EFN
, and TN EFN1-5
,
but there was no inhibition by deletion variants that do not contain
the fibrinogen globe (i.e. TN FB
and TN
FF
; Fig. 5). These results are in
excellent agreement with those from direct binding studies of
phosphacan to the various recombinant tenascins described above (Fig.
2), in which it was determined that only the fibrinogen globe is
required.
Neurocan binding was inhibited to a lesser extent (25-35%) and was
seen only with proteins containing fibronectin type III repeats
(i.e. tenascin-C, TN 190, and TN FB), whereas
the smaller recombinant fragments and deletion variants that were
lacking fibronectin type III repeats actually enhanced binding of
neurocan to tenascin-C (data not shown). The explanation for the
enhanced binding is not clear, although neurocan may interact with
small soluble tenascin fragments or deletion variants to form a complex
that is capable of binding to immobilized tenascin-C. However, these
results do support the conclusion from the direct binding studies that
some of the fibronectin type III repeats are necessary for maximal
interaction of neurocan and tenascin-C.
We previously
obtained evidence suggesting that calcium may play a role in the
interaction of phosphacan with human tenascin-C (8). More detailed
studies have now shown that calcium is required for maximal interaction
of phosphacan with native or recombinant tenascin-C and with smaller
tenascin variants containing the fibrinogen globe, as demonstrated by
the ~75% decrease in binding observed in calcium- and magnesium-free
buffers containing EDTA (Fig. 6). Magnesium can
partially substitute for calcium in phosphacan binding to the
full-length tenascins but has relatively little effect on its
interactions with the fibrinogen globe alone, and divalent cations also
had some effect on the small residual binding of phosphacan to the
tenascin-C deletion variant (TN FF) in which the
fibrinogen globe and the fibronectin type III repeats are lacking.
There was a similar calcium requirement for neurocan binding to
full-length native and recombinant tenascin-C, whereas the absence of
calcium and magnesium had only minor and variable effects on neurocan
interactions with the tenascin-C deletion variants tested (data not
shown).
Because a potential calcium-binding site in the fibrinogen globe of
tenascin-C might involve disulfide bonds, we also evaluated the effects
of reducing the recombinant tenascins with dithiothreitol. This
treatment decreased phosphacan binding to full-length tenascin-C by
>70% but had less effect on binding to the fibrinogen globe alone,
and interactions of neurocan were either unaffected (with TN 190) or
actually enhanced in the case of several of the deletion variants (Fig.
7).
Rotary Shadowing Electron Microscopy of Phosphacan Complexes with Tenascin-C
In electron micrographs shown in Fig.
8A phosphacan appeared as a rod with kinks, a
globular domain at one end, and several fine thread-like lateral
projections. The globular region can be assumed to correspond to the
N-terminal carbonic anhydrase-like domain and the fibronectin type III
repeat of phosphacan, which we have shown previously to be involved in
binding to tenascin-C (8), and the long rod to the remainder of the
core protein, which is known to contain numerous
O-glycosidic oligosaccharides and chondroitin sulfate side
chains visible as the fine lateral projections. In mixtures of
phosphacan with tenascin-C the two molecules are often seen in
association with each other (Fig. 8, B and C).
The globular region of phosphacan was frequently seen next to the
fibrinogen globe of tenascin-C, confirming the results from our study
of deletion variants; both types of data indicate that the fibrinogen
globe of tenascin-C is the phosphacan binding domain.
Effects of Phosphacan on Adhesion and Spreading of C6 Glioma Cells on Recombinant Tenascins
We have reported previously that
phosphacan (but not neurocan) inhibits the adhesion of rat C6 glioma
cells to tenascin-C, whereas there is no effect of either proteoglycan
on adhesion to laminin (1). We have now analyzed this effect in more
detail using deletion variants and recombinant fragments of tenascin-C. C6 cells showed good adhesion and spreading only on native chick tenascin-C and on deletion variants lacking the EGF-like repeats (TN
EGF) or both the fibronectin type III repeats and the
fibrinogen globe (TN FF
) when coated on the
bacteriological dishes employed in our previous studies (Fig.
9), whereas there was good adhesion and varying degrees
of spreading on all recombinant tenascins when applied to tissue
culture plastic (data not shown). The adhesion and spreading on the
variant lacking the EGF-like repeats are consistent with other reports
that this domain has antiadhesive properties for various cell types
(23, 24), but the EGF-like domain itself is evidently not antiadhesive
for C6 cells insofar as TN FF
, containing only the
EGF-like domain and the central N-terminal oligomerization region,
supported adhesion and spreading almost as well as TN
EGF
. However, adhesion and spreading on the EGF-like
domains (TN FF
) were significantly more sensitive to
inhibition by phosphacan, as demonstrated by our finding that
concentrations as low as 1 µg/ml have a noticeable effect on the
number of adherent cells and on process length (Fig. 9E),
and very few processes were seen at a phosphacan concentration of 5 µg/ml (Fig. 9F), whereas there was good spreading of C6
cells on substrates coated with TN EGF
and 5 µg/ml of
phosphacan (Fig. 9B). Concentrations of phosphacan approaching 20 µg/ml were required to completely inhibit process extension on TN EGF
(Fig. 9C).
While the TN FB deletion variant did not support adhesion
on bacteriological plates (data not shown), the fibrinogen globe alone
(TN EFN
) or together with fibronectin type III repeats
6-8 (TN EFN1-5-) allowed good adhesion and cell spreading
(Fig. 10, A/B and D/E). Process
extension on both substrates was completely inhibited by phosphacan at
a concentration of 10 µg/ml (Fig. 10, C and F). However, the fibrinogen globe is not an essential adhesion domain for
C6 cells, because deletion of both the fibronectin type III repeats and
the fibrinogen globe (in TN FF
) provided an
excellent substrate, as shown in Fig. 9.
C6 cell adhesion to native chick tenascin-C and to deletion variants
lacking either the EGF-like repeats or the fibronectin type III repeats
and the fibrinogen globe was inhibited by phosphacan in a
concentration-dependent manner, reaching ~90% inhibition at a phosphacan coating concentration of 20 µg/ml (Fig.
11). These results indicate both that the tenascin-C
domains involved in C6 cell adhesion are not confined to the fibrinogen
globe that mediates interactions of tenascin-C with phosphacan and that
the antiadhesive effect of phosphacan is exerted directly on the cells independent of its ability to bind to tenascin-C (and potentially block
cell adhesion domains).
This study has utilized a new approach to identify the regions of tenascin-C that are involved in its interactions with two nervous tissue-specific chondroitin sulfate proteoglycans, neurocan and phosphacan. Rather than expressing and studying only isolated domains, whose properties may not reflect those seen when they are present in the context of the entire molecule, we have employed deletion variants of tenascin-C that lack one or more homology domains and compared their effects in molecular and cell interactions with those obtained using native or full-length recombinant tenascin-C. All tenascin-C variants assembled correctly to hexameric molecules of the expected characteristics as determined by gel electrophoresis, their reactivity with monoclonal antibodies, and their molecular dimensions as revealed by electron microscopy. The expression of these tenascin-C variants by mammalian cells also increases the probability that they will be properly folded and glycosylated. Our studies demonstrated both by direct radioligand binding assays and by inhibition experiments that phosphacan binding is retained in all deletion variants except those lacking the fibrinogen globe and that phosphacan binds to this single domain with nearly the same high affinity as to native or recombinant tenascin-C. However, maximum binding of neurocan and inhibition of its interactions with tenascin-C require both the fibrinogen globe and some of the adjacent fibronectin type III repeats.
The C-terminal globular region of tenascin-C is homologous to the
globular domain of the and
chains of fibrinogen and contains a
sequence that is similar to the calcium-binding site identified in the
fibrinogen
chain (21, 25). This site has been defined as the EF
hand (consisting of an
-helix, a calcium binding loop, and another
-helix) based on a structural analysis of parvalbumin (26). It has
been shown that 45Ca binds to tenascin-C immobilized on
nitrocellulose (27) and that the binding of cytotactin-binding
proteoglycan to tenascin-C in covasphere coaggregation assays is
decreased in the presence of EDTA (28). Based on this information, it
has been proposed that the fibrinogen globe contains an EF hand calcium
binding site and that binding of calcium to this region may determine a
specific conformation that is important for its function (29).
It was therefore of interest to examine the effects of divalent cations on the interactions of phosphacan and neurocan with tenascin-C. Our results indicate that calcium is required for maximal binding of phosphacan to both native and full-length recombinant tenascin-C as well as to the fibrinogen globe alone (~75% decrease in binding in the absence of calcium; Fig. 6), whereas in the case of neurocan interactions this requirement for calcium was seen only with respect to the complete tenascin-C molecule. Local changes in the concentration of extracellular calcium could play a regulatory role in tenascin-proteoglycan interactions, both directly by affecting the conformation of the proteoglycan binding site in the fibrinogen globe of tenascin-C, and because calcium may serve as a major counterion for the carboxyl and sulfate groups on the chondroitin sulfate chains of neurocan and phosphacan. However, it must also be recognized that the calcium-binding site in the fibrinogen globe of tenascin-C is only putative and that other regions of the molecule may be affected, as would appear to be the case for neurocan-tenascin interactions.
We also examined the effects of reducing disulfide bonds in tenascin-C to evaluate their importance for its binding properties. Treatment of tenascin-C with dithiothreitol would be expected to convert the tenascin hexabrachions to trimers and possibly to monomers, since the trimers are thought to be formed by a triple coiled-coil region in the molecule that is stabilized by disulfide bonds, and hexamers are formed by disulfide linkage of two trimers. Reduction should also alter the conformation of the disulfide-bonded EGF-like repeats and the fibrinogen globe (21). Our data indicate that the integrity of disulfide-stabilized structures in tenascin-C contributes significantly to its interaction with phosphacan, because treatment of TN 190 with dithiothreitol reduced binding by >70%, and reduction had a lesser but still significant effect on binding to the fibrinogen globe (Fig. 7). In contrast, interactions with neurocan were not affected by reduction of TN 190, and in the case of some deletion variants binding of neurocan was actually increased. These results support other evidence that the fibronectin type III repeats have an ancillary role in neurocan-tenascin interactions, since they do not contain cysteine residues and would therefore not be expected to be directly affected by reducing agents.
We have demonstrated previously that two tryptic glycopeptides derived from the N-terminal carbonic anhydrase-like and fibronectin type III domains of phosphacan bind to tenascin-C. This interaction is mediated at least in part by sialylated complex-type oligosaccharides occupying the single N-glycosylation site on each glycopeptide, insofar as their binding is abolished following treatment of phosphacan with peptide N-glycosidase (8). It has also recently been reported that a 35-kDa human serum protein with a fibrinogen domain has C-type lectin activity that is thought to be mediated by this domain (30). Rotary shadowing electron micrographs of phosphacan show a rod that is terminated at one end by a globular domain that interacts with the fibrinogen globe of tenascin-C (Fig. 8). The results of our deglycosylation studies indicate that the globular domain represents the N-terminal portion of phosphacan, and this conclusion is supported by the distribution of O-glycosylation sites on the phosphacan core protein. From the concentration and monosaccharide composition of glycoprotein-type oligosaccharides in phosphacan (originally designated the 3F8 proteoglycan; 18), it can be calculated that the number of GalNAc residues in phosphacan from 7-day postnatal brain (64 mol/mol for a 173-kDa core protein) is in excellent agreement with the sum of 33 threonine and 30 serine GalNAc-Ser/Thr O-glycosylation sites indicated by a neural network analysis (31, 32) of the phosphacan amino acid sequence. All of these sites (which account for only 17% of the total serine and threonine residues) are in the C-terminal portion of the protein outside of the carbonic anhydrase and fibronectin type III homology domains (3), and the high concentration of O-glycosidic oligosaccharides would tend to give this region an extended conformation. GalNAc-linked oligosaccharides essentially disappear from phosphacan during the course of postnatal brain development (18), but these are replaced by a significant number of oligosaccharides (and keratan sulfate chains) containing mannosyl-O-serine/threonine linkages (33, 34). Based on the concentration of mannose residues and the proportion of these that are converted to mannitol after alkaline borohydride treatment of the proteoglycan (18), it can likewise be calculated that the number of oligosaccharides containing O-glycosidic mannose linkages increases during the course of postnatal development to 73 mol/mol of protein in adult brain. Both types of O-glycosidic oligosaccharides would confer mucin-like properties on the protein and in the case of the transmembrane phosphatase would also serve to extend the N-terminal ligand-binding domain away from the cell surface.
The three potential chondroitin sulfate attachment sites that are most likely to be utilized are all located in the C-terminal portion of the phosphacan core protein, although additional attachment sites may be present more N-terminally at Ser-595 and Ser-645 (3, 4). In electron micrographs one sees fine thread-like structures (Fig. 8) that extend laterally from the core protein and are consistent in appearance with that of glycosaminoglycan chains visualized in other proteoglycans (35, 36). Although these appear to arise from sites throughout the core protein, some of those seen near the globular region may in fact represent chondroitin sulfate chains with attachment sites in the C-terminal half of the proteoglycan but that follow the core protein before reaching out into the surrounding space (as has been observed previously in the case of aggrecan (35)).
Recent investigations have shown a complex pattern of tenascin-C effects on cell adhesion and the promotion of neurite outgrowth, in which the concerted action of several domains leads to the diverse cellular responses observed (16). The effects of phosphacan on C6 cell adhesion to tenascin-C are similar to the previously observed inhibition of neuronal adhesion to the neural cell adhesion molecule Ng-CAM/L1. It was concluded from these studies that inhibition of neuronal adhesion by both neurocan and phosphacan (which bind to Ng-CAM/L1) is mediated by direct effects on the cells rather than via binding to the substrate, since the proteoglycans also inhibited adhesion to a substrate consisting of anti-Ng-CAM antibodies, to which the proteoglycans do not bind (9, 10). Phosphacan also inhibited the adhesion of C6 cells to Ng-CAM (7) but not to laminin (1). The results obtained using an Ng-CAM substrate are probably also mediated by a direct inhibitory effect of phosphacan on the cells, although this question has not yet been addressed experimentally, whereas the lack of an inhibitory effect on a laminin substrate could be due to the involvement of different cell receptors as well as to stronger cell-matrix interactions that are not susceptible to inhibition by phosphacan (at least at the concentrations tested).
Our studies provide the first direct biochemical and electron
microscopic evidence for specific, high-affinity binding of proteins to
the fibrinogen globe of tenascin-C. The terminal globular domain of
fibrinogen is involved in protein-protein interactions in the process
of fibrin polymerization, binding of fibrinogen to bacteria, and to
receptors on platelets (37). Thrombospondin also binds to distinct
sites on the distal parts of the and
chains of fibrinogen (38),
and by analogy with fibrinogen it has been suggested that the
fibrinogen globe of tenascin-C may play a role in its association with
other proteins (39). The fibrinogen globe of tenascin-C has been
indirectly implicated in binding to
-integrins (40) and has been
shown by electron microscopy to be attached to collagen fibrils in
chicken vitreous humor (41). Because of its location at the C-terminal
tips of the hexabrachion arms, the fibrinogen globe is ideally situated to mediate the interactions of tenascin-C with cells and with other
proteins. Phosphacan and neurocan bound to this domain may serve as a
bridge between tenascin-C and neural cell adhesion molecules to which
these proteoglycans also bind with high affinity (3, 9-11) or in other
ways modulate its biological properties.
We thank Dr. Jürgen Engel for helpful suggestions concerning the electron microscopic studies.