From the Burnham Institute, La Jolla, California
92037 and the ¶ Department of Pharmacology and Neuroscience,
Johns Hopkins University School of Medicine,
Baltimore, Maryland 21205
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ABSTRACT |
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The lecticans are a group of chondroitin sulfate
proteoglycans characterized by the presence of C-type lectin domains.
Despite the suggestion that their lectin domains interact with
carbohydrate ligands, the identity of such ligands has not been
elucidated. We previously showed that brevican, a nervous
system-specific lectican, binds the surface of B28 glial cells (Yamada,
H., Fredette, B., Shitara, K., Hagihara, K., Miura, R., Ranscht, B.,
Stallcup, W. B., and Yamaguchi, Y. (1997) J. Neurosci. 17, 7784-7795). In this paper, we demonstrate that two
classes of sulfated glycolipids, sulfatides and HNK-1-reactive
sulfoglucuronylglycolipids (SGGLs), act as cell surface receptors for
brevican. The lectin domain of brevican binds sulfatides and SGGLs in a
calcium-dependent manner as expected of a C-type lectin
domain. Intact, full-length brevican also binds both sulfatides and
SGGLs. The lectin domain immobilized as a substrate supports adhesion
of cells expressing SGGLs or sulfatides, which was inhibited by
monoclonal antibodies against these glycolipids or by treatment of the
substrate with SGGLs or sulfatides. Our findings demonstrate that the
interaction between the lectin domains of lecticans and sulfated
glycolipids comprises a novel cell substrate recognition system, and
suggest that lecticans in extracellular matrices serve as substrate for adhesion and migration of cells expressing these glycolipids in vivo.
The lecticans are a family of chondroitin sulfate proteoglycans
(CSPGs)1 characterized by the
presence of a C-type lectin domain in their core proteins (1, 2). The
C-terminal globular domains of lecticans, or "proteoglycan lectin
domain" (PLD), consist of one or two epidermal growth factor
(EGF)-like domains, a C-type lectin domain, and a complement regulatory
protein (CRP) domain. This arrangement of domains is similar to that of
selectins, suggesting that lecticans are also involved in the
recognition of carbohydrate ligands.
Lecticans are the most abundantly expressed family of proteoglycans in
the nervous system. The lectican family includes aggrecan (3), versican
(4), neurocan (5), and brevican (6), all of which are expressed in the
nervous system at certain stages of development (1, 7). Although
aggrecan and versican were initially characterized as connective tissue
proteoglycans, their expression in the nervous system has been
demonstrated in a number of reports (8-12). Brevican and neurocan are
specifically expressed in the nervous system (6, 13-15). Structural
similarities with selectins and the abundant expression in the nervous
system suggest that lecticans play major roles in carbohydrate
recognition in the nervous system.
The identity of the ligand to PLDs has been a focus of our interest. We
previously showed that the PLD of versican binds tenascin-R, an
extracellular matrix (ECM) protein predominantly expressed in the
nervous system (16). More recently, we demonstrated that the PLDs of
all lecticans bind tenascin-R, and that brevican and tenascin-R indeed
form a complex in adult rat brain extracts (17). These results suggest
that the lectican-tenascin-R interactions, especially the
brevican-tenascin-R interaction, are relevant to the assembly of the
adult brain ECM. However, these interactions are not
carbohydrate-protein interactions expected of C-type lectin domains;
they are protein-protein interactions between the PLDs and fibronectin
type III domains (FNIII) 3-5 of tenascin-R (17). Regarding
carbohydrate interactions by PLDs, several studies have demonstrated
that PLDs can bind simple sugars and heparin/heparan sulfate in
vitro (18-20). However, these studies failed to identify the
nature of any physiological carbohydrate ligands for PLDs. Thus the
ability of PLDs to behave as C-type lectins in vivo and the
identity of physiological carbohydrate ligands for PLDs are issues that
have not been addressed.
We have previously shown that purified brevican binds to the surface of
primary astrocytes as well as of several immortalized rat neural cell
lines (21). Binding studies with B28 glial cells demonstrated that the
binding is mediated by the C-terminal 80-kDa fragment of the brevican
core protein which includes the PLD (21). It was initially suspected
that tenascin-R deposited to the surface of these cells may act as the
"receptor" for brevican. However, we have found that these cells do
not have any tenascin-R on their surface nor did they secrete any
tenascin-R into culture supernatants. Furthermore, a number of assays
failed to identify any cell surface protein that specifically binds
brevican PLD. Since it has been reported that the lectin domains of P-
and L-selectins bind sulfated glycolipids, such as
sulfatides (22) and HNK-1-reactive sulfoglucuronylglycolipids (SGGLs)
(23), we examined the possibility that the cell surface brevican
receptor is a glycolipid rather than a glycoprotein. In this paper, we
report that the PLDs of lecticans bind these sulfated glycolipids. We
also show that the interaction between brevican and sulfated
glycolipids supports adhesion of cells expressing these glycolipids on
their surfaces. These observations suggest that the PLD-sulfated
glycolipid interactions are a novel cell substrate recognition system.
Materials--
Mixed bovine brain gangliosides, purified bovine
brain sulfatides, and galactosylceramides were purchased from Sigma.
Mixed neutral glycosphingolipids were obtained from Matreya (Pleasant Gap, PA). The HNK-1 monoclonal antibody was purchased from Becton Dickinson (Bedford, MA). Mouse monoclonal anti-tenascin-R antibody (clone 596) (24) and monoclonal anti-sulfatide antibody were gifts from
Drs. Melitta Schachner (University of Hamburg, Hamburg, Germany) and
Yoshio Hirabayashi (RIKEN, Wako, Japan), respectively. The Fc fusion
protein of L-selectin (L-selectin Ig chimera)
and a HeLa cell line transfected with cDNAs for HNK-1
sulfotransferase, glucuronyltransferase, and N-CAM were obtained from
Dr. Minoru Fukuda (Burnham Institute, La Jolla, CA).
Recombinant Proteins--
A chimeric protein of brevican PLD
fused with Fc region of human IgG (brevican PLD chimera) and
biotinylated recombinant lectin domains (rCLDs) of all four lecticans
were prepared as described previously (17). Brevican PLD chimera
consists of a short segment of the central domain, a EGF domain, a
lectin domain, and a CRP domain of rat brevican. rCLDs consist only of
the lectin domains.
Preparation of Authentic, Full-length Brevican--
For
preparation of recombinant brevican with no fusion partner, CHO cell
were stably transfected with an expression vector pcDNA-rBV, which
has a full-length rat brevican cDNA (3.0-kilobase pair
EcoRI-EcoRI fragment) (25) inserted into a unique
EcoRI site of pcDNAIAmp (Invitrogen, San Diego, CA). For
the preparation of radiolabeled probe for TLC-ligand binding assay,
cultures of a cloned CHO transfectant were metabolically labeled with
100 µCi/ml Tran35S-Label (ICN, Costa Mesa, CA).
35S-Labeled or unlabeled brevican was purified from culture
supernatants by affinity chromatography on the FNIII3-5 fragment of
tenascin-R as follows. The glutathione S-transferase fusion
protein of the FNIII3-5 fragment (17) was bound to glutathione-agarose
and covalently coupled with dimethylpimelimidate according to Gersten and Marchalonis (26). Culture supernatants of CHO transfectants were
incubated overnight at 4 °C with the FNIII3-5 affinity resin preequilibrated with TBS containing 5 mM CaCl2.
After extensive washing, the labeled brevican was eluted from the
affinity resin with 20 mM EDTA.
Flow Cytometry, Immunoblotting, and Ligand Overlay
Assays--
For flow cytometry, cells were dissociated with
trypsin-EDTA (Irvine Scientific, Irvine, CA), suspended in 10% fetal
calf serum in Dulbecco's modified Eagle's medium, and incubated in the medium for 2 h at 37 °C. Cell were then washed three times with PBS containing 0.1% sodium azide, and incubated with PBS containing 1% BSA and 0.1% sodium azide for 20 min on ice. Cells were
then incubated with brevican PLD chimera or primary antibodies for 30 min on ice. After washing three times with PBS containing 0.1% sodium
azide, the cells were incubated with fluorescein-conjugated goat
antibodies to human IgG (Sigma) or to mouse IgG+IgM
(Biosource) for 20 min on ice, washed again, and
resuspended in 0.5-1.0 ml of PBS containing 0.1% sodium azide. The
cells were examined on a FACSort (Becton Dickinson, Oxford, CA). Cell
surface biotinylation was performed with sulfo-NHS-biotin (Pierce)
according to the manufacture's instruction. Biotinylated molecules
were collected by streptavidin-agarose. Ligand overlay and
immunoblotting assays were performed as described previously (17).
Preparation of Glycolipids--
For preparation of total
glycolipid fraction, tissues or cells were extracted by sonication with
chloroform/methanol (2:1) and then with a chloroform/methanol/water
(1:2:0.8). After removal of the remaining precipitates by
centrifugation, the supernatant was dried under N2 stream.
The resulting residue was dissolved in chloroform/methanol (2:1), and
then 1/10 volume of 2 M KOH in methanol was added. The
solution was incubated at 37 °C for 2 h to degrade
phospholipids. The supernatant was collected by centrifugation,
neutralized with 1/20 volume of acetic acid, and dried under
N2 stream. The residue was suspended in water, dialyzed against water, evaporated to dryness, and then dissolved in
chloroform/methanol/water (60:35:8). Acidic glycolipids were purified
as described previously (27). HNK-1-reactive SGGLs were purified from
dog sciatic nerve endoneurium as described previously (28). This sample
contains 41% SO3-GlcU-nLc-4-Cer and 59%
SO3-GlcU-nLc-6-Cer, both of which are recognized by
anti-HNK-1 antibody.
Thin-layer Chromatography (TLC) Ligand Binding Assays--
These
assays were performed according to Taki et al. (29).
Briefly, glycolipid samples were developed twice on high performance thin layer chromatography plates (Baker Inc., Phillipsburg, NJ) in
chloroform/methanol/0.2% aqueous CaCl2 (60:35:8). After
drying, the plates were dipped for 30 s in 0.4%
polyisobutylmethacrylate (2.5% polyisobutylmethacrylate in chloroform
was diluted with hexane to the final concentration of 0.4%), and
air-dried. The dried plates were then incubated with various probes
(antibodies, brevican PLD chimera, rCLDs, or 35S-labeled
native brevican) in TBS containing 1-3% BSA at 4 °C overnight.
After washing with TBS, the plates were incubated with horseradish
peroxidase (HRP)-conjugated secondary antibodies or HRP-conjugated
avidin for 1.5 h, followed by the visualization of reactive bands
with chemiluminescence. In the case of 35S-labeled
brevican, the dried plates were directly exposed to Kodak BioMax film
for 3 days.
Cell Adhesion Assay--
For preparation of substrates,
solutions of brevican PLD chimera or human IgG (100 µg/ml) in
calcium- and magnesium-free Hank's balanced salt solution (CMF/HBSS)
were applied on nitrocellulose-coated plastic (21), and incubated at
37 °C for 2 h. After washing three times with CMF/HBSS,
uncoated surfaces were blocked by incubating with CMF/HBSS containing
2% heat-inactivated BSA (HBSS/BSA) for 2 h at 37 °C. After
washing three times with CMF/HBSS, cells suspended in Opti-MEM (Life
Technologies, Inc.) containing 0.1% heat-inactivated BSA were plated
at a density of 5 × 105 (B28 cells), or 1 × 106 (MDCK cells) per ml, and incubated for 1 h at
37 °C. After gentle washing, attached cells were fixed with 4%
paraformaldehyde in PBS and counted under microscope at 200×
magnification. For perturbation with antibodies, cells were
preincubated with 100 µg/ml HNK-1 or anti-sulfatide monoclonal
antibodies diluted in CMF/HBSS/BSA for 30 min on ice prior to the
plating of the cells. For perturbation by glycolipids, the substrate of
brevican PLD chimera was preincubated with SGGLs, sulfatide, or
galactosylceramide at concentration of 5 µg/ml in CMF/HBSS for 1 h at 37 °C before plating of the cells.
Brevican Binds to Cell Surfaces through Non-tenascin-R Binding
Sites--
We have shown that the PLD-containing C-terminal 80-kDa
fragment of brevican core protein binds to primary astrocytes and B28
cells, which is an immortalized glial cell line. Binding studies with
cell monolayers ruled out hyaluronan, heparan sulfate, and chondroitin
sulfate as cell surface "receptors" for the brevican C-terminal
fragment (21).
To facilitate the identification of the putative brevican receptor, a
fusion protein of brevican PLD and Fc region of human IgG (brevican PLD
chimera) and a biotin-labeled recombinant lectin domain of brevican
(brevican rCLD) were produced. We first examined the binding of
brevican PLD chimera to a series of immortalized neural cell lines
derived from BDIX rats (30) in flow cytometric assay. Among 16 cell
lines tested, brevican PLD chimera bound to B28 cells (Fig.
1A) and four other glial cell
lines, namely B9, B15, B49, and B92. The chimera showed no binding to
other cell lines with fibroblastic or neuronal phenotypes, including B23 (Fig. 1B), B19, B27, B35, B50, B65, B82, B103, B104,
B108, or B111 cells (data not shown). These results suggest that the cell surface binding of the 80-kDa fragment is mediated by the PLD of
the brevican core protein, and that the brevican receptor is expressed
in various neural cell lines.
The lectin domain of brevican binds tenascin-R by a protein-protein
interaction (17). Although tenascin-R is a secreted protein, it is
possible that tenascin-R is present on the surface of B28 cells through
interaction with cell surface tenascin-R binding proteins
(e.g. contactin) or by nonspecific aggregation, thereby
acting as an apparent brevican receptor. However, as described previously (21), no tenascin-R was detected on the surface of B28 cells
either by flow cytometric assay or by immunocytochemistry (data not
shown). Furthermore, the ligand overlay assay with brevican PLD
chimera, which we used to identify tenascin-R as a protein ligand to
lectican PLDs in adult rat brain extracts (17), did not detect
tenascin-R in surface-biotinylated proteins from B28 cells (Fig.
1C).
Having ruled out tenascin-R as the putative receptor for brevican PLD,
we next examined the possibility that cell surface glycoproteins
carrying specific carbohydrates would be the receptor for brevican PLD,
as is the case with selectins. However, the ligand overlay experiment
demonstrated that B28 cells lacks not only tenascin-R but also any cell
surface proteins that specifically interact with brevican PLD chimera
(Fig. 1C), suggesting that there are no glycoprotein ligands
for the brevican PLD in B28 cells. We further searched for cell surface
brevican-binding proteins in B28 cells by immunoprecipitation of
surface-labeled materials and affinity chromatography on a column
bearing the 80-kDa brevican fragment that includes PLD. None of these
experiments could identify glycoproteins that would specifically bind
brevican PLD (data not shown).
The Proteoglycan Lectin Domain Binds Sulfatides and HNK-1-reactive
SGGLs--
It has been reported that sulfated cell surface
glycolipids, sulfatides and HNK-1-reactive SGGLs, bind to the lectin
domains of P- and L-selectin (22, 23). Therefore, we
investigated the possibility that glycolipids would act as cell surface
receptors for brevican PLD. To test this, we prepared glycolipids from
the adult rat cerebellum and probed with brevican PLD chimera in the TLC-ligand overlay assay. As shown in Fig.
2A, brevican PLD chimera reacted with a single band (lane 2) among a
number of glycolipid species extracted from the cerebellum
(lane 1). The reactive band migrated at the same
position as purified sulfatides (compare panel A,
lane 2 with panel B,
lane 1), suggesting that the band represents
sulfatides. To further identify this reactive band, standard
glycolipids were examined by TLC-ligand overlay assay (Fig. 2,
B-F) The brevican PLD chimera bound to purified sulfatides (C, lane 1), but not to any of the
neutral glycosphingolipids (lane 2) or
gangliosides (lane 3). Binding to sulfatides was
abolished in the presence of EDTA (D, lane
1), as expected of a C-type lectin interaction. The brevican
PLD chimera did not bind galactosylceramide, an unsulfated precursor of
sulfatides (indicated by asterisk in B,
lane 2), suggesting that a sulfate group is
necessary for binding. L-selectin Ig chimera also bound to
sulfatides, but not to other glycolipids (E), consistent
with previous reports (23). Human IgG did not bind any glycolipids
(F).
The brevican PLD chimera contains not only the C-type lectin domain but
also EGF and CRP domains flanking the lectin domain (17). To examine
the location of the sulfatide binding site, the binding of biotinylated
recombinant protein consisting of only the lectin domain (brevican
rCLD) was examined. Like the PLD chimera, brevican rCLD specifically
bound to sulfatides (Fig. 3E,
lane 1), but not to neutral glycolipids,
including galactosylceramide, or to gangliosides (Fig. 3E,
lane 2). Moreover, rCLDs of aggrecan, neurocan,
and versican also showed specific binding to sulfatide (Fig. 3,
B-D). All of these interactions were completely suppressed in the presence of EDTA (data not shown). These results show that the
C-type lectin domain of all four lecticans bind sulfatides in a
divalent cation-dependent manner.
Considering that L- and P-selectins bind another class of
sulfated glycolipid, HNK-1-reactive SGGLs (23), we tested the binding
of PLDs to the HNK-1-reactive SGGLs. A sample of dog sciatic nerve-derived SGGLs consisting of roughly equal amounts of
SO3-GlcU-nLc-4-Cer and SO3-GlcU-nLc-6-Cer was
tested for binding to brevican PLD chimera (Fig.
4). The brevican PLD chimera bound to
both SO3-GlcU-nLc-4-Cer and SO3-GlcU-nLc-6-Cer
(lane 3). The binding was EDTA-sensitive (lane 4). Consistent with previous reports,
L-selectin Ig chimera bound SGGLs (lane
5), but human IgG did not (lane 6).
These results demonstrate that PLD binds HNK-1-reactive SGGLs.
HNK-1-reactive carbohydrates are present on several glycoproteins as
well as on glycolipids (31, 32). Thus, there remained a possibility
that glycoproteins carrying HNK-1-reactive glycans could bind PLDs and
act as PLD receptors. To address this question, we performed two
experiments. First, we prepared extracts containing large amounts of
HNK-1-positive glycoproteins from E19 mouse brain. These brain extracts
indeed contained several protein bands intensely reactive with the
HNK-1 antibody in immunoblotting (Fig.
5A, lane 1). We probed this sample with brevican PLD chimera in
ligand overlay assay. Brevican PLD chimera, while it efficiently bound to tenascin-R in adult brain extracts (see Fig. 1C,
lane 4), did not bind any of these HNK-1-positive
glycoproteins (Fig. 5A, lane 2).
Second, we examined by flow cytometry the binding of brevican PLD
chimera to a HeLa-derived cell line that expresses HNK-1 carbohydrates only on glycoproteins, not on glycolipids. This cell line has been
transfected with cDNAs for N-CAM, a glucuronyltransferase (33), and
the HNK-1 sulfotransferase (34). The glucuronyltransferase acts only on
glycoprotein glycans and not on glycolipids (33). Because the parental
HeLa cells express no HNK-1-reactive SGGLs, all of the HNK-1
carbohydrates expressed by these cells are contained on glycoproteins,
mainly on N-CAM.2 Flow
cytometric analysis confirmed that these cells express high levels of
cell surface HNK-1 carbohydrates (Fig. 5B, a).
Despite this, these cells do not bind brevican PLD chimera at all (Fig. 5B, b). In contrast, B28 cells, which express
HNK-1-reactive SGGL (see below), show strong binding of brevican PLD
chimera (see Fig. 1). Although these results do not entirely rule out
the possibility that HNK-1 carbohydrates carried by glycoproteins could
be recognized by PLD under some conditions, they demonstrate that HNK-1
carbohydrates attached to glycoproteins are not recognized by PLDs as
efficiently as HNK-1-reactive SGGLs.
Intact Brevican Also Binds Sulfated Glycolipids--
We next
examined whether intact, full-length brevican, not just the recombinant
PLD fragment, binds these sulfated glycolipids. To test this, we
isolated 35S-labeled brevican from culture supernatants of
CHO cells transfected with full-length rat brevican cDNA, and used
it as a probe in the TLC-ligand overlay assay. As shown in Fig.
6, native brevican bound to both
sulfatides and SGGLs (lane 1). No binding to
neutral glycosphingolipids or gangliosides was found (lane
2). The glycolipid binding by brevican was inhibited with
EDTA (lane 3), as was the case with the PLD
chimera and the rCLDs. These results demonstrate that not only
recombinant PLD fragment but also intact brevican binds sulfated
glycolipids.
Binding of Brevican PLD Chimera to B28 Cells Is Mediated by
HNK-1-reactive Glycolipids--
To determine if the cell surface
binding of brevican observed with the B28 cells (see Fig.
1A) (21) is indeed mediated by sulfated glycolipids, we
examined whether these cells contain sulfatides and/or SGGLs. Analysis
of an acidic glycolipid fraction from B28 cells demonstrated that B28
cells express HNK-1-reactive SGGLs but not sulfatides (Fig.
7A, lanes
2 and 3). Brevican PLD bound to the B28 cell
SGGLs (lane 4).
To confirm the role of HNK-1-reactive SGGLs in the cell surface binding
of brevican, we examined whether the HNK-1 antibody would inhibit
brevican PLD binding to the B28 cells. As shown in Fig. 7B,
binding of the brevican PLD chimera to B28 cells was inhibited by
preincubation of the cells with the HNK-1 antibody by flow cytometric
assay. Taken together, these results indicate that the binding of
brevican to B28 cells is mediated by an interaction between
HNK-1-reactive SGGLs and the brevican PLD.
Brevican PLD Supports Cell Adhesion through Sulfated Cell Surface
Glycolipids--
Our finding that sulfated cell surface glycolipids
bind lecticans, which are ECM components, suggests that these
proteoglycans may act as adhesive substrates for cells expressing
sulfated glycolipids. To test this possibility, we studied the
attachment of B28 cells, which express HNK-1-reactive SGGLs (see
above), and MDCK cells, which have been shown to express sulfatides
(35), on substrates of brevican PLD. The expression of sulfatides by
MDCK cells was confirmed by flow cytometric assay and
TLC-immunoblotting with anti-sulfatide monoclonal antibody (data not
shown). The brevican PLD chimera was adsorbed to nitrocellulose-coated
dishes, and cell adhesion to the substrate was quantitatively analyzed.
Both the B28 and MDCK cells adhered to the brevican PLD substrate (Fig. 8, panels A1 and
B1) but not to a human IgG substrate (panels A5 and B5). Preincubation of cells with the HNK-1
antibody (panel A2) or anti-sulfatide antibody
(panel B2) significantly reduced the number of
cells that adhered to the brevican PLD substrate. This adhesion was
also inhibited by pretreatment of the brevican PLD substrate with
purified SGGLs (panel A3) or sulfatides
(panel B3) before plating of cells. Pretreatment
of the substrates with galactosylceramides did not have any effect on
the adhesion of either cell type (panels A4 and
B4). Quantitative analysis of these results is shown in Fig.
8 (C and D). These results demonstrate that the
brevican PLD-glycolipid interactions can support cell adhesion, and
suggest that brevican present in the brain ECM may serve as an adhesive
substrate for neural cells expressing HNK-1-reactive SGGLs or
sulfatides.
In this paper, we demonstrate that the PLDs of the lectican family
proteoglycans bind two types of sulfated cell surface glycolipids, sulfatides and HNK-1-reactive SGGLs, in a divalent
cation-dependent manner. Not only the Ig chimera of the
brevican PLD but also the intact brevican core protein possesses the
same binding activity. These interactions support the adhesion of
sulfatide- or SGGL-expressing cells.
Previous reports on the carbohydrate interaction of PLDs have
demonstrated that PLDs are capable of binding simple carbohydrates, including fucose, galactose, and N-acetylglucosamine (16,
18, 19), and heparin/heparan sulfate (20). However, these studies failed to provide data indicating biological significance of the carbohydrate interactions or to identify physiological ligands carrying
these carbohydrate determinants. The present study for the first time
identifies sulfatides and SGGLs as physiologically relevant
carbohydrate ligands for PLDs.
The PLDs of lecticans share several binding properties with selectins.
Like PLDs, P- and L-selectins have been shown to bind sulfatides and HNK-1-reactive SGGLs (22, 23, 36). Both the PLD-sulfated
glycolipid and the selectin-sulfated glycolipid interactions are
divalent cation-dependent. The presence of sulfate residues on terminal sugars is required for binding (22, 23, 36). On the other
hand, selectins and PLDs bind differently to gangliosides, another
class of acidic glycolipids containing sialic acids. P- and
L-selectins have been shown to bind not only sulfated
glycolipids but also gangliosides (37, 38), whereas we showed that
brevican PLD does not bind gangliosides. This suggests that the mere
presence of negative charges is not sufficient for the binding of
brevican PLD to sulfated glycolipids.
A noteworthy property of PLDs is that they do not recognize
glycoproteins carrying HNK-1 carbohydrates (see Fig. 5), whereas they
do bind to HNK-1 carbohydrates carried by a glycolipid. There are two
possible explanations for this selective binding. PLDs may recognize
not only the terminal sulfoglucuronyllactosamine, the structure that is
shared by HNK-1-reactive glycolipids and glycoprotein glycans, but also
additional sugar residues of glycolipid chains. In this case, it would
be necessary for the PLD to recognize more than four sugar residues to
distinguish between HNK-1-reactive glycolipids and glycoproteins. This
seems unlikely, because functional groups of carbohydrate determinants
interacting with selectins are restricted to the three terminal sugar
residues (39, 40). It seems more likely that the low density of HNK-1
carbohydrates on neural glycoproteins limits their ability to bind
PLDs. In fact, this situation would be similar to what has been
encountered with selectins. Ordinary glycoproteins, even if they carry
a selectin-binding carbohydrate determinant such as sialyl
Lewisx, are weak selectin binders; the presence of clusters
of the carbohydrate determinant on a polypeptide backbone is required
for efficient selectin binding (41, 42). As a result, essentially all
known glycoprotein ligands for selectins are mucin-type glycoproteins that contain clusters of O-linked glycans (43-46). On the
other hand, neural glycoproteins that carry HNK-1 carbohydrates are not
mucin-type glycoproteins, and therefore presumably are incapable of
sustaining high affinity interactions. Although it is possible for PLDs
to bind to HNK-1-reactive oligosaccharides when presented at a high
density, it is unlikely that neural glycoproteins carrying HNK-1
carbohydrates act as the receptor for the PLDs in vivo.
Several lines of evidence suggest that the PLD-sulfated glycolipid
interactions are physiologically significant. For instance, the tissue
distribution of lecticans overlaps with the distribution of sulfatides
and SGGLs. Sulfatides and SGGLs are enriched in nervous tissues, as are
the lecticans. SGGLs are particularly abundant in embryonic cerebral
cortex and adult cerebellum (31). Neurocan and brevican are highly
expressed in embryonic brain and adult cerebellum, respectively (21,
47). Sulfatides are a major component of myelin (48) and are expressed
in oligodendrocytes and Schwann cells in culture (49, 50). Versican has
been shown to be expressed in cultured oligodendrocytes and Schwann
cells (51, 52) and brevican in oligodendrocytes (53).
Our demonstration that PLD-sulfated glycolipid interactions support
cell adhesion also suggests that these interactions have biological
significance. Roles for sulfatides and SGGLs in neural cell
interactions have been proposed in earlier studies. For example, it has
been suggested that SGGLs and sulfatides are involved in the adhesion
of dissociated cerebellar cells and outgrowth of neurites and
astrocytic processes (54). SGGLs have also been shown to have effects
on Schwann cell adhesion (28, 55). More recently, production of higher
levels of SGGLs in cerebellar granule neurons has been shown to
correlate with enhanced neurite outgrowth from these cells (56).
Despite these observations, the identity of the SGGL/sulfatide-binding
molecule(s) involved in these adhesion events has been elusive.
Although several heparin-binding proteins have been shown to bind
sulfatides and SGGLs, including laminin (57, 58), thrombospondin (59),
von Willebrand factor (60), tenascin-C (61), tenascin-R (62), and SBP-1
(63), no lectin-type molecule expressed in the nervous system has been
identified as a ligand for these sulfated glycolipids. Lecticans, on
the other hand, bind sulfatides and SGGLs in the manner expected of
C-type lectin interactions and their distribution overlaps with that of
SGGLs and sulfatides. Thus, lecticans are strong candidates as
physiological ligands for these sulfated glycolipids in nervous tissues. It is possible that at least some of the cell surface interactions reported by the studies cited above are mediated by lecticans.
The fact that the PLD promotes cell adhesion is, at first glance,
somewhat perplexing, since chondroitin sulfate can be inhibitory to
cell adhesion and neurite outgrowth. In fact, it has been shown that
brevican can inhibit neurite outgrowth from cerebellar granule neurons,
and that the chondroitin sulfate moiety is responsible for this
activity (6). However, it should be noted that lecticans do not always
carry chondroitin sulfates. One of the versican splicing variants
expressed mainly in the brain lacks domains that contain
glycosaminoglycan attachment sites (64). Brevican is a so-called
part-time proteoglycan and roughly 20-50% of brevican molecules
isolated from whole brain are devoid of chondroitin sulfates (6, 7).
Thus, our present findings suggest the possibility that brevican may
act as a bifunctional substrate for cell adhesion, depending on whether
it contains chondroitin sulfate chains. Such a putative dual effect on
cell adhesion suggests that the regulation of chondroitin sulfate chain
synthesis could serve as a biological switch determining whether a CSPG
is inhibitory or promoting to cell adhesion.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Brevican PLD chimera binds B28 cells.
A and B, cell surface binding of brevican PLD
chimera to B28 (A) and B23 (B) cells analyzed by
flow cytometry. Cells were stained with brevican PLD chimera followed
by fluorescence-conjugated anti-human IgG (solid line), or
with fluorescence-conjugated anti-human IgG only (dotted
line). Note that B28 cells bind brevican PLD chimera to their
surface but B23 cells do not. C, ligand overlay assay of
biotinylated cell surface proteins probed with brevican PLD chimera.
Lanes 1-3, B28 cells were surface-biotinylated and
biotinylated proteins were resolved in an 8-16% gradient gel under a
nonreducing condition. Nitrocellulose filters blotted with the gel were
probed with HRP-conjugated avidin (lane 1),
brevican PLD chimera followed by HRP-conjugated anti-human IgG
(lane 2), or HRP-conjugated anti-human IgG alone
(lane 3). Incubation with brevican PLD or
anti-human IgG was performed in the presence of 5 mM
CaCl2. Lanes 2 and 3 were
exposed for extended periods to show any positive bands. Note that
brevican PLD chimera does not bind any specific proteins. This result
indicates that B28 cells do not contain tenascin-R nor other proteins
that specifically interact with brevican PLD on their surface. The band
at ~200 kDa (indicated by asterisk) is due to nonspecific
binding of HRP-conjugated anti-human IgG (see lane
3). Lane 4, positive control for the reactivity
of the brevican PLD chimera. Adult rat brain extracts were resolved by
SDS-polyacrylamide gel electrophoresis as described above, blotted to
nitrocellulose, and probed with the brevican PLD in the presence of 5 mM CaCl2. Note that the brevican PLD chimera,
although it did not bind to any specific proteins in lanes
1-3, recognized tenascin-R present in adult brain extracts
(17).
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Fig. 2.
Brevican PLD binds sulfatide.
A, total glycolipids isolated from adult rat cerebellum were
analyzed for binding to brevican PLD chimera in TLC-ligand overlay
assay. TLC plates were stained by orcinol to visualize total
glycolipids (lane 1) or probed with brevican PLD
chimera followed by HRP-conjugated anti-human IgG (lane
2). Note that brevican PLD chimera binds to a single
glycolipid species (lane 2). This band comigrates
with purified specimen of sulfatides (lane 1 in
B). B-F, purified glycolipid specimens were
analyzed for binding to brevican PLD chimera in TLC-ligand overlay
assay. TLC plates were stained by orcinol (B), or probed
with brevican PLD chimera in the presence of 5 mM
CaCl2 (C), brevican PLD chimera in the presence
of 20 mM EDTA (D), L-selectin Ig
chimera in the presence of 5 mM CaCl2
(E), or normal human IgG in the presence of 5 mM
CaCl2 (F). Lanes 1, bovine
brain sulfatide (2.5 µg); lanes 2, mixed
neutral glycosphingolipids (10 µg); lanes 3,
mixed gangliosides (10 µg). BrePLD, brevican PLD chimera;
LS-Ig, L-selectin Ig chimera; hIgG,
human IgG. Asterisk in B indicates
galactosylceramides. Note that brevican PLD chimera as well as
L-selectin Ig chimera binds sulfatide in the presence of
Ca2+.
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Fig. 3.
Lectin domains of all four lecticans bind
sulfatide. Glycolipid samples were separated in TLC and probed
with rCLDs of lecticans. TLC plates were stained by orcinol
(A), or probed with aggrecan rCLD (B), neurocan
rCLD (C), versican rCLD (D), or brevican rCLD
(E) in the presence of 5 mM CaCl2.
Lanes 1, mixtures of purified sulfatides (2.5 µg) and galactosylceramides (2.5 µg); lanes
2, mixtures of mixed neutral glycosphingolipids (10 µg)
and gangliosides (10 µg). GalC, galactosylceramides. Note
that rCLD of all lecticans bind to sulfatide, but not to
galactosylceramide.
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Fig. 4.
Brevican PLD binds HNK-1-reactive SGGLs.
Purified SGGLs from dog sciatic nerve containing 41%
SO3-GlcU-nLc-4-Cer and 59% SO3-GlcU-nLc-6-Cer
(5 µg/lane) were separated in TLC, and stained with orcinol
(lane 1), or probed with HNK-1 monoclonal
antibody (lane 2), brevican PLD chimera in the
presence of 5 mM CaCl2 (lane
3), brevican PLD chimera in the presence of 20 mM EDTA (lane 4),
L-selectin Ig chimera in the presence of 5 mM
CaCl2 (lane 5), or human IgG in the
presence of 5 mM CaCl2 (lane
6). HNK-1, HNK-1 monoclonal antibody;
BrePLD, brevican PLD chimera; LS-Ig,
L-selectin Ig chimera; hIgG, human IgG. Note
that brevican PLD chimera binds both species of HNK-1-reactive SGGLs in
the presence of Ca2+.
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Fig. 5.
HNK-1 carbohydrates on glycoproteins are not
efficiently recognized by brevican PLD. A, a microsome
fraction from E19 mouse brain was resolved by SDS-polyacrylamide gel
electrophoresis, transferred to nitrocellulose filters, and probed with
the HNK-1 monoclonal antibody (lane 1) or
brevican PLD chimera (lane 2). Note that brevican
PLD chimera does not bind to any of the HNK-1 carrying glycoproteins
present in the fraction. B, flow cytometric analysis of a
HeLa cell line expressing high levels of glycoprotein-attached HNK-1
carbohydrates driven by HNK-1 sulfotransferase (34). These HeLa cells
were incubated with the HNK-1 antibody (a) or brevican PLD
chimera (b). Bound chimera or antibodies were detected with
fluorescein-conjugated anti-mouse IgG+IgM (a) or anti-human
IgG (b). Dotted lines, cells were stained with
secondary antibodies alone. Note that brevican PLD chimera does not
bind to these cells, although they express high levels of
glycoprotein-attached HNK-1 epitope.
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Fig. 6.
Native brevican binds sulfatides and
SGGLs. Glycolipid specimens were separated by TLC, and then probed
with 35S-labeled brevican prepared from CHO transfectants.
Samples were: mixtures of 2.5 µg of sulfatide and 5 µg of SGGLs
(lanes 1 and 3) and mixtures of 10 µg of mixed neutral glycosphingolipids and 10 µg of gangliosides
(lanes 2 and 4). Assay was performed
in the presence of 5 mM CaCl2 (lanes
1 and 2) or 20 mM EDTA
(lanes 3 and 4). Note that native
brevican binds both sulfatides and HNK-1-reactive SGGLs. The binding is
calcium-dependent.
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Fig. 7.
Binding of brevican PLD chimera to B28 cells
is mediated by HNK-1-reactive glycolipids. A, B28 cells
have HNK-1-reactive SGGLs. Acidic glycolipids were isolated from B28
cells and analyzed in TLC-ligand overlay assay. TLC plates in which B28
cell-derived acidic glycolipids had been separated were stained with
orcinol (lane 1), or probed with the HNK-1
antibody (lane 2), anti-sulfatide monoclonal
antibody (lane 3), or brevican PLD chimera
(lane 4). Note that B28 cells contain
HNK-1-reactive SGGLs but not sulfatides (lanes 2 and 3). Brevican PLD chimera binds B28 cell-derived SGGLs
(lane 4). B, binding of brevican PLD
chimera to B28 cells is inhibited by HNK-1 antibody. B28 cells that had
been preincubated with or without the HNK-1 antibody, were incubated
with brevican PLD chimera, followed by staining with
fluorescein-conjugated anti-human IgG and analysis by flow cytometry.
Solid line, binding of brevican PLD chimera to untreated B28
cells; broken line, binding of brevican PLD chimera to B28
cells pretreated with the HNK-1 antibody; dotted line,
staining with fluorescein-conjugated secondary antibody alone. Note
that the HNK-1 monoclonal antibody abolished the binding of brevican
PLD chimera to B28 cells (broken line).
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Fig. 8.
Brevican PLD-glycolipid interactions support
cell adhesion. A, adhesion of B28 cells to the
substrate of brevican PLD chimera. Cells were plated on the substrate
of brevican PLD chimera (panels 1-4) or human
IgG (panel 5). In panel 2,
cells were preincubated with 100 µg/ml HNK-1 monoclonal antibody
before plating. In panels 3 and 4,
substrates were preincubated with 5 µg/ml SGGLs or 5 µg/ml
galactosylceramide, respectively, before plating. Photographs were
taken 1 h after plating. B, adhesion of MDCK cells to
the substrate of brevican PLD chimera. The arrangement of the
experiment was the same as in A, except that cells were
preincubated with anti-sulfatide monoclonal antibody instead of HNK-1
monoclonal antibody in panel 2, and with
sulfatide instead of SGGLs in panel 3. C and
D, quantitation of cell adhesion. Cell adhesion assays were
performed as described above. Number of adhered cells per field was
counted under 200× magnification. Results represent the mean ± 1 S.D. of adhered cells from experiments in quadruplicate. Experimental
arrangement in columns 1-5 corresponds to
those of panels 1-5 in A and B. Bre, brevican PLD chimera; hIgG, human IgG;
HNK-1, HNK-1 monoclonal antibody; Sulf,
anti-sulfatide monoclonal antibody; Sulf, sulfatides;
GalC, galactosylceramide.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Drs. Melitta Schachner, Yoshio Hirabayashi, and Minoru Fukuda for providing reagents and cell lines. We also thank Drs. Fukuda, William Stallcup, and Douglas Ethell for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants NS32717 and HD25938 (to Y. Y.) and CA28896 (to E. R.), and by Cancer Center Support Grant CA30199.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Current address: Dept. of Cell and Molecular Biology, Lund University, P. O. Box 94, S-221 00 Lund, Sweden.
To whom correspondence should be addressed: Burnham Institute,
10901 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-646-3124; Fax:
619-646-3199; E-mail: yyamaguchi{at}burnham-inst.org.
2 M. Fukuda, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: CSPG, chondroitin sulfate proteoglycan; PLD, proteoglycan lectin domain; CRP, complement regulatory protein; ECM, extracellular matrix; SGGL, sulfoglucuronylglycolipid; rCLD, recombinant C-type lectin domain; FNIII, fibronectin type III; EGF, epidermal growth factor; CHO, Chinese hamster ovary; TBS, Tris-buffered saline; MDCK, Madin-Darby canine kidney; HRP, horseradish peroxidase; PBS, phosphate-buffered saline; BSA, bovine serum albumin; CMF, calcium- and magnesium-free; HBSS, Hank's balanced salt solution.
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