(Received for publication, November 18, 1996, and in revised form, February 10, 1997)
From the Burnham Institute, La Jolla Cancer Research
Center, La Jolla, California 92037, § Institut de
Biologie et Chimie des Proteines, 69367 Lyon Cedex 07 France, and
¶ Department of Neurosciences, The Cleveland Clinic
Foundation, Cleveland, Ohio 44195
NG2 is a membrane-spanning proteoglycan with a primary structure unique among cell surface or extracellular matrix proteins. To characterize the interaction between NG2 and extracellular matrix proteins, we have used a eukaryotic expression system to produce and purify several recombinant fragments covering not only the entire ectodomain of NG2 but also distinct subdomains of the molecule. Using a solid phase binding assay with various extracellular matrix proteins, we have identified two main ligands for NG2, namely, collagens V and VI. Consistent with previous models of glycosaminoglycan attachment, roughly 50% of the recombinant NG2 fragments containing the central domain have chondroitin sulfate chains attached to the protein core. These glycosaminoglycan chains are not directly involved in collagen binding, since chondroitinase-treated fragments exhibit an unimpaired ability to bind to both collagens. Using more restricted recombinant fragments of NG2, we mapped the binding site for both collagens to the central domain of NG2. Electron microscopy after rotary shadowing of native NG2 molecules indicates that this extended nonglobular domain provides a flexible connection joining the two N- and C-terminal globular regions of NG2. Rotary shadowing of mixtures of NG2 and collagen V or VI confirms a direct interaction between the molecules and indicates that the collagens align with the central region of NG2, giving the appearance of a rod between the N- and C-terminal globules.
The ability of cells to recognize and interact with the extracellular matrix is fundamentally important for normal development and for continued maintenance of tissue architecture and function throughout adulthood. The pathogenesis of a variety of diseases can be traced to defects in cellular recognition of the matrix. Several types of molecules on cell surfaces are able to recognize extracellular matrix components, the most prominent class of receptors being the integrin family of heterodimeric proteins (1). Another group of cellular receptors for extracellular matrix components is constituted by cell surface proteoglycans. An increasing number of structurally different proteoglycan core polypeptides of this type have been identified by cDNA cloning (2, 3). For example, syndecans, the best characterized of the transmembrane proteoglycans, can interact with a variety of matrix molecules, such as collagen I (4, 5), fibronectin (6), thrombospondin (7), and tenascin (8). Our laboratory has described another proteoglycan, NG2, with the potential to serve as a cell surface receptor for matrix components. This chondroitin sulfate-containing, membrane-spanning proteoglycan has a unique primary structure, which shares few obvious protein motifs with any other cell surface or matrix proteins (9). NG2 is expressed on immature progenitor cells in several types of developing tissues and is down-regulated when these precursors undergo terminal differentiation (10-13). However, high levels of expression are once again observed in some types of malignant cells, such as melanomas, glioblastomas, and chondrosarcomas (14, 15).
Integrin-mediated recognition of the extracellular matrix has been
shown to initiate an intracellular cascade of second
messenger-dependent signaling events that modulate cellular
function such as cell adhesion, spreading, migration, and
differentiation (16). In contrast, little information is available
concerning intracellular events triggered by engagement of
transmembrane proteoglycans. On the one hand, both NG2 and syndecan-1
have been shown to interact with the cytoskeleton (17-19). On the
other hand, it has recently been shown that the engagement of
syndecan-1 by its specific ligands is able to mediate cell spreading
without the involvement of the syndecan cytoplasmic domain (20). This
casts doubt on the idea that the proteoglycan itself can directly
mediate signal transduction that leads to cell spreading. An
alternative suggestion is that cell surface proteoglycans may function
as co-receptors, acting in concert with other types of matrix
recognition molecules. This has been observed in the case of syndecans
and the 5
1-integrin (21, 22). The co-receptor mechanism is
thought to operate either by changing the conformation of the matrix
ligand or by making it more available in some way. In addition, the
human melanoma proteoglycan, sharing high homology with NG2 (23), has
been shown to participate, along with the
4
1-integrin, in
promoting spreading of melanoma cells on a fibronectin fragment (24,
25).
A better understanding of the role played by cell surface proteoglycans in cellular function initially requires precise characterization of the interactions of these molecules with specific extracellular matrix ligands. We began to study matrix ligands for NG2 in a solid phase binding assay using NG2 purified from the B49 cell line (26). However, a major limitation in this work was the difficulty in purifying large amounts of the proteoglycan under native conditions. To overcome this difficulty and to achieve better in vitro characterization of the detailed mechanisms governing NG2-matrix interactions, we have produced recombinant fragments of NG2 covering not only the entire extracellular domain of NG2 but also distinct subdomains of the extracellular portion of the molecule. These fragments have been biochemically characterized and used in a solid phase binding assay along with a variety of potential extracellular matrix ligands. Our results identify two main ligands for NG2, namely, collagens V and VI. We show that both collagens bind to the central portion of NG2, which consists of a flexible rodlike domain separating two globular regions located at the N- and C-terminal ends of the NG2 ectodomain.
The full-length NG2 cDNA was excised from the pBluescript vector by digestion with XbaI and partial digestion with HindIII (27) and then subcloned into the EcoRV site of the eukaryotic expression vector pcDNA I/Amp (Invitrogen, La Jolla, CA). This construct (pcDNA Amp NG2) was used to generate several expression vectors coding for different domains of the extracellular part of NG2. cDNA fragments were generated by polymerase chain reaction (PCR)1 with the Pfu DNA polymerase (Stratagene) according to the supplier's instructions.
The pcDNA NG2EC construct coding for the entire extracellular part
of NG2 (amino acid residues 1-2223) was generated by introducing a
stop codon after nucleotide 6738. A fragment containing a stop codon
and an XbaI site was obtained by PCR using 5-primer 1 (nucleotide positions 5605-5622 of NG2 cDNA) and 3
-primer 2 (positions 6724-6739). This product was digested with XhoI
and XbaI and ligated into the expression vector pcDNA
Amp NG2 digested by the same enzymes, thus replacing the C-terminal end
of the coding region with the PCR product and removing nucleotides
6739-7173 of the rat NG2 cDNA.
A construct (pcDNA EC3) coding for domain 1 of NG2 and the major
part of domain 2 (residues 1-1465) was made similarly by ligating a
PCR fragment (5
-primer 3 (nucleotides 3494-3510) and 3
-primer 4 (nucleotides 4450-4464)) digested with SplI-XbaI
into the pcDNA Amp NG2 vector digested with the same enzymes, thus introducing a stop codon after nucleotide 4464.
For expression of internal domains of NG2, pcDNA Amp NG2 was used
as a template for PCR to generate fragments for expression vectors.
5-Primer 5 (nucleotides 1963-1979) and 3
-primer 6 (nucleotides 4402-4419) were used to construct the vector encoding the central domain of rat NG2 (PCEP4/D2) (amino acid residues 632-1450). 5
-Primer 7 (nucleotides 4828-4846) and 3
-primer 8 (nucleotides 6702-6723) were used to generate the expression vector PCEP4/D3 coding for the
third domain of the extracellular part of NG2 (amino acid residues
1587-2218). Both fragments contain a unique 5
-NheI
restriction site and introduce at the 3
-end a sequence coding for 6 histidine residues, followed by a stop codon and an XbaI
site for cloning (28). The expression vector used in these two cases
was the construct PCEP4/
2 III 4 (29) based on the vector PCEP4
(Invitrogen), kindly provided by Dr. Ernst Pöschl (Max-Planck
Connective Tissue Clinical Research Group for Rheumatology, Erlangen,
Germany). The NG2 cDNA fragments were fused via their
NheI site with the sequence coding for the signal peptide of
human BM40 contained in this vector (30). Expression and processing
result in secretion of proteins with four N-terminal amino acid
residues (APLA) preceding the authentic NG2 domains. The correct
in-frame insertion of the inserts in all constructs was verified by
restriction mapping and DNA sequencing.
In the case of the pcDNA NG2EC and pcDNA EC3 constructs,
human embryonic kidney 293 cells (American Type Culture Collection) were co-transfected with plasmid psv2pac, which confers puromycin resistance. Stably transfected clones were selected by puromycin resistance following previously described procedures (31). Vectors PCEP4/D2 and PCEP4/D3 were used to transfect the 293 EBNA cell line
(Invitrogen), which constitutively expresses the EBNA-1 protein from
the Epstein-Barr virus, allowing episomal replication of the vector.
Transfected cells were selected by resistance to hygromycin B (300 µg/ml) (29).
293 cell clones or 293 EBNA-resistant cells were tested for expression of the desired NG2 fragments by SDS-PAGE of serum-free culture supernatants followed by Coomassie Blue staining. NG2 products were verified by Western blotting using a polyclonal antiserum against rat NG2 (32). Large amounts of serum-free medium from positive confluent cells were then obtained for protein purification.
Protein Purification0.5-1 liters of serum-free medium
from 293 cells expressing NG2EC or EC3 were applied to a
DEAE-Sepharose column equilibrated in 50 mM Tris/HCl, pH
8.6, containing 0.2 M NaCl, and eluted by a linear gradient
from 0.2 to 0.8 M NaCl in the same buffer. Two peaks were
obtained. The first peak elutes around 0.4 M NaCl and contains NG2 fragments free of chondroitin sulfate chains. The second
peak (0.55 M NaCl) contains the proteoglycan form of the NG2 fragments. Both peaks were individually pooled and will be referred
to as P1 (0.4 M NaCl peak) and P2 (0.55 M NaCl
peak).
Serum-free media from 293 EBNA cells expressing D2 or D3 were purified
on nickel-agarose columns (Qiagen) equilibrated in 20 mM
Tris/HCl, pH 8.0, containing 0.5 M NaCl (buffer A). The column was washed with buffer A and then with the same buffer containing increasing concentrations of imidazole (5-20
mM). More tightly bound proteins were then eluted with 100 mM and then 200 mM imidazole in buffer A. The different eluates were analyzed by SDS-PAGE, and fractions
containing recombinant NG2 fragments were pooled. All recombinant
fragments were dialyzed against 0.2 M ammonium bicarbonate
and concentrated by ultrafiltration before storage at 20 °C.
Protein concentrations were estimated using a BCA protein assay
(Pierce) following the instructions of the supplier. Amino acid
sequence analysis was performed by automated Edman degradation using an
Applied Biosystems 473A protein sequencer.
Several additional proteins were used for binding studies. Native rat NG2 was purified from B49 cells according to the method of Burg et al. (26). Pepsin-solubilized collagen VI purified from human placenta (33) was a gift of Dr R. Timpl (Max-Planck Institut für Biochimie, Martinsried, Germany). Pepsinized collagen V was purified from human amniotic membranes as described (34). Human pepsin-extracted collagen IV, collagen I, and bovine collagen II were purchased from Sigma. Mouse laminin-1 was purchased from Life Technologies, Inc. Recombinant decorin was obtained from Telios (La Jolla, CA).
Enzymatic TreatmentsPurified NG2 fragments were digested
for 1 h at 37 °C with chondroitinase ABC (Seikagaku) (0.01 units/µg of protein) in 0.2 M ammonium bicarbonate. To
remove the free glycosaminoglycan fragments after digestion, proteins
were in some cases dialyzed against 50 mM Tris/HCl, pH 7.4, and 0.15 M NaCl (TBS) prior to use in microtiter plate
binding assays. Complete digestion was verified by SDS-PAGE. Prior to
N-glycanase treatments NG2 fragments were denatured by
boiling for 5 min in sodium phosphate buffer, pH 6, containing 0.4%
SDS and 2% -mercaptoethanol. Nonidet P-40 was then added to a final
concentration of 2.5%. Denatured proteins were digested with
N-glycanase (Genzyme) (0.1 units/µg of protein) for 2 h at 37 °C and analyzed by SDS-PAGE.
Binding assays were performed in microtiter plates (Greiner, Nurtingen, Germany) following previously described protocols (35) with some modifications. Wells coated with the first ligand (5 µg/ml, 100 µl) were blocked with 1% bovine serum albumin in TBS. All further incubation steps were carried out in TBS containing 0.05% Tween 20. Soluble ligands were used over a concentration range of 0.4-50 µg/ml. In inhibition experiments, a fixed concentration (5 µg/ml) of soluble ligand was incubated 3 h at 4 °C with various concentrations of inhibitors (chondroitin sulfate (Sigma) or recombinant NG2 fragments) prior to addition to the coated wells. For detection of bound ligands, different antibodies were used. Antibodies against rat NG2 were affinity-purified from a polyclonal antiserum on a Sepharose column coupled to the recombinant NG2 fragment NG2EC. A polyclonal antiserum against human pepsin-extracted collagen VI was provided by R. Timpl. Antibodies (purified IgG fraction of a polyclonal antiserum) to human pepsinized collagen V were obtained from Dr. D. J. Hartmann (Institut Pasteur, Lyon, France).
Rotary ShadowingNG2 samples were diluted to 10 µg/ml with 0.2 M ammonium bicarbonate. For preparation of NG2 complexes, NG2 molecules were incubated overnight at 4 °C with 1:1 molar ratios of collagen VI, collagen V, or monoclonal antibodies diluted in 0.2 M ammonium bicarbonate. Samples were sprayed onto freshly cleaved mica sheets and immediately placed on the holder of a MED 010 evaporator (Balzers). Rotary shadowing was carried out as described previously (34). Observations of replicas were performed with a Philips CM120 microscope at the Center de Microscopie Electronique Appliquée à la Biologie et à la Géologie, Université Claude Bernard Lyon I.
Expression and Purification of Recombinant NG2 Fragments
Several stable human 293 cell clones were obtained, which
expressed different fragments of the NG2 proteoglycan. These fragments are summarized in Fig. 1. The construct NG2EC codes for
the entire extracellular part of NG2. This region can be roughly
divided into three domains (9): domain 1, the N-terminal region
containing 8 cysteine residues; the central domain 2 lacking cysteine
residues but containing the putative attachment sites for chondroitin
sulfate chains; and domain 3, the membrane proximal region containing two cysteine clusters. A construct coding for domain 1 and the main
part of domain 2 is designated EC3. Two smaller constructs correspond to the internal domains of NG2, D2 and D3. For production of
these four polypeptides, serum-free medium from 293 cell clones or 293 EBNA-transfected cells was screened by SDS-PAGE for the presence of
proteins of the expected sizes, which were absent in medium from
nontransfected 293 cells (36). Expression of the correct protein was
confirmed by Western blotting with a polyclonal antiserum against NG2
(data not shown). Positive cells showed significant production and
secretion of recombinant NG2 fragments, ranging from 0.3 µg/ml for D2
to 8 µg/ml for NG2EC. For both fragments NG2EC and EC
3, ~50% of
the core proteins were substituted by chondroitin sulfate chains and
migrated on SDS-PAGE as broad bands of high molecular mass (~350-450
kDa). The other portion of these two recombinant proteins is
represented by the protein core free of glycosaminoglycan (GAG) chains
(Fig. 2). NG2EC and EC
3 could be purified from the
cell culture medium by DEAE chromatography. Recombinant proteins were
easily separated from media contaminants, since they bind strongly to
the anion exchange resin. Moreover, the DEAE column allows separation
of the protein core from the proteoglycan form of the same fragment. A
first peak, eluting at 0.4 M NaCl, contains the protein
core free of GAG chains (P1). The proteoglycan form (P2) requires 0.55 M NaCl for elution. In the case of both NG2EC and EC
3,
digestion of P2 with chondroitinase ABC gives a pattern similar to that
seen with proteins from P1 (Fig. 2). The EC
3 protein core fragment
appears by electrophoresis as a single band of 180 kDa, but SDS-PAGE of
the NG2EC fragment reveals the presence of at least four bands ranging
from 250 to 290 kDa, all of which are reactive with a polyclonal
antiserum against intact NG2. However, using a polyclonal antiserum
against a synthetic peptide located at the C-terminal end of the
ectodomain of NG2 (residues 2161-2179; Ref. 37), only the high
molecular mass 290-kDa species could be detected by Western blotting.
This indicates that proteolytic processing of the recombinant NG2EC occurs at the C-terminal end (data not shown).
The two internal NG2 fragments D2 and D3 have 6 histidine residues added to their C-terminal ends and were affinity purified by nickel-agarose chromatography. D3 is expressed as a 100-kDa polypeptide free of glycosaminoglycan chains (Fig. 2). Purified D2 appeared on electrophoresis as two bands of 110 and 125 kDa. Edman degradation of both bands showed the same N-terminal sequence, APLARGGPAQD, which corresponds to the expected sequence from the construct PCEP4/D2 (starting at position 632 in the NG2 sequence). Since both the 110- and 125-kDa proteins have been affinity-purified by means of the 6 histidine residues at their C-terminal ends, it seems likely that the difference in electrophoretic migration of the two polypeptides results from posttranslational modifications other than proteolytic processing. These modifications did not involve N-glycosylation, as judged by the electrophoretic pattern seen after N-glycanase treatment; the increase in electrophoretic mobility of both species indicates that they are equally N-glycosylated. In addition, a portion of the recombinant D2 molecules contain some GAG chains and migrate in SDS-PAGE as a broad band of 250-300 kDa. However, the proteoglycan form of D2 does not exceed 20% (Fig. 2).
The molecular masses of all NG2 fragments were higher than the calculated masses based on the amino acid sequence. Treatment of the recombinant polypeptides with N-glycanase resulted in a reduction of the apparent molecular masses by 6-15%. The highest extent of glycosylation was observed in the D3 domain. This indicates that most if not all of the predicted N-glycosylation sites are occupied in this domain, whereas only 50% of the sites in the more N-terminal part of the molecule appear to be glycosylated.
Binding Properties of Recombinant NG2 Fragments
Binding to the Large Fragments NG2EC and ECWe have
previously shown that the NG2 proteoglycan interacts with collagen VI
both in vitro in solid phase assays (26) and in
situ, in which it anchors the collagen at the cell surface (27,
32). In addition, NG2 seemed to be able to interact with other collagen
types, particularly the fibrillar collagens II and V. The recombinant
NG2 fragments give us the ability to study in more detail these
interactions between NG2 and collagens. When NG2EC and EC3 fragments
were used as soluble ligands for different immobilized collagens,
strong and saturable binding of both fragments was obtained to
pepsin-solubilized human collagens V and VI (Fig. 3,
A and B). In contrast, no binding of either
fragment to collagens I and II was detected. Using radiolabeled ligand,
apparent dissociation constants of 140 and 70 nM were
derived for binding of EC
3 to type V and VI collagens, respectively
(data not shown).
Additional studies were carried out to compare the binding ability of
the two different pools of the NG2EC and EC3 recombinant proteins
obtained from the DEAE purification, P2 containing GAG chains and P1
representing the protein core. Fig. 3, A and B, shows that only the recombinant fragments possessing chondroitin sulfate chains (P2) are able to bind to immobilized collagens V and VI.
Although these results initially indicated to us that NG2 binding to
type V and VI collagens was mediated by the chondroitin sulfate chains,
additional experiments showed that this was not the case. First, the P2
pools of the fragments NG2 EC and EC
3 were digested by
chondroitinase ABC prior to use in binding assays. Although digestion
appeared complete when analyzed by SDS gel electrophoresis (see Fig.
2), both undigested and digested P2 samples bound more strongly to
collagen V or VI than the corresponding P1 samples (Fig.
3C). Second, we could show that increasing concentrations of
free chondroitin sulfate in the binding assay do not inhibit the
binding of either P2 recombinant fragment to collagen VI (Fig. 4). Finally, when collagens were used as the soluble
ligands, no difference was seen between the ability of immobilized P1
and P2 forms of EC
3 to bind both collagens V and VI (Fig.
5). Similar results were obtained with the NG2EC
fragment (not shown). Taken altogether, these results suggest that the
chondroitin sulfate chains of NG2 do not participate directly in the
binding of NG2 to collagens. The inability of P1 pools of NG2 EC and
EC
3 to bind effectively to immobilized collagens V and VI remains an anomaly. Although this result may represent a quirk of the solid phase
binding assay (involving hidden versus exposed epitopes on
the immobilized molecules), it could also indicate that addition of GAG
chains to the NG2 core polypeptide induces a conformational state that
facilitates binding to the collagens. Adsorption of P1 polypeptides to
plastic might produce a conformation that mimics that of the P2
polypeptide.
Binding to the Restricted Fragments D2 and D3
In an attempt
to localize more precisely the collagen binding site of NG2, binding
assays with the two smaller fragments, D2 and D3, were performed using
collagens V and VI as soluble ligands (Fig. 6). For the
binding to collagen VI, decorin served as a positive control (38).
Negative controls included the basement membrane proteins laminin-1 and
collagen IV. Significant binding could be obtained with the central D2
fragment of NG2 to collagen VI (Fig. 6A) or V (Fig.
6B). Binding was similar to or higher than that obtained
with the larger fragments NG2EC and EC3. Binding of the third domain
of NG2, D3, to collagen VI was as low as that seen with the controls
laminin and collagen IV. Binding of D3 to collagen V occurred at the
background level seen for bovine serum albumin, confirming that this
domain is not involved in the binding of NG2 to collagens. Thus, the
central portion of NG2 appears sufficient to achieve binding of the
proteoglycan to collagens V and VI. To confirm the specificity in
binding of recombinant fragments to collagen VI, inhibition experiments
with soluble fragments were performed. Preincubation of soluble
collagen VI (30 nM) with an excess of the different
fragments NG2EC, EC
3, and D2 (150 nM) inhibits from 65 to 90% of the collagen binding to wells coated with the same fragments
(Fig. 7). These results provide evidence of specific
interaction of the NG2 fragments with collagen VI.
Electron Microscopy of Rotary-shadowed Preparations
Rotary shadowing of native NG2 purified from the B49 cell line
revealed abundant amounts of large globular domains (Fig.
8A). Two globules were often found in
apposition, separated by a distance ranging from 30 to 110 nm
(n = 60). In rare cases, a threadlike connection was
observed between the globules (Fig. 8A, inset 1). Moreover,
this central region could be labeled by a monoclonal antibody, the
epitope of which has been localized to the recombinant D2 domain by
enzyme-linked immunosorbent assay and dot blot experiments (Fig.
8A, inset 2). These observations indicate first that the structure of the central domain cannot be easily resolved by the rotary-shadowing technique due to the absence of extensive folding, and
second, that the central portion of NG2 is flexible. These data are
consistent with the putative folding pattern of NG2 deduced from its
primary structure (9), which indicates the presence of N- and
C-terminal cysteine-rich domains (respectively, D1 and D3) separated by
a 978-amino acid segment free of cysteine residues (D2). Rotary
shadowing of mixtures of native NG2 and collagen V or VI were performed
to visualize directly the interaction between the molecules.
Pepsin-extracted collagen VI consists mainly of a mixture of dimers and
tetramers (39). Interestingly, collagen VI tetramers could often be
seen aligning with the central domain of NG2, giving the appearance of
a rod joining the two N- and C-terminal globules of NG2 (Fig.
8B). In these cases, the distance between the two globules
is no longer variable and corresponds exactly to the length of collagen
VI tetramers (about 100 nm). In the case of type V collagen, the 300-nm
rodlike collagen molecule could often be seen in alignment with the NG2
molecules, giving the appearance of a thickening of the rodlike segment
between the two NG2 globules. In these cases the distance between the two NG2 globules remained variable, ranging from 40 to 100 nm (Fig. 8C).
Several putative extracellular matrix ligands have been previously proposed to interact with the NG2 proteoglycan, i.e. collagens II, V, and VI as well as the glycoproteins laminin-1 and tenascin-C (26). However, the difficulty of purifying large amounts of NG2 precluded a complete analysis of the binding interactions between purified molecules. In the present study, we have successfully produced and purified several recombinant fragments of NG2. High yields of these fragments have allowed us to confirm and characterize in more detail the interactions of NG2 with collagens V and VI, which in our assays represent the extracellular matrix ligands with the strongest affinities for the proteoglycan. Electron microscopy of rotary-shadowed molecules confirms not only the collagen binding properties of NG2 but also several aspects of NG2 biochemistry. Rotary-shadowed preparations of native NG2 indicate that this molecule comprises two globular domains joined by a rodlike central domain that is difficult to visualize. This structure is compatible with the model deduced from the cDNA sequencing of NG2 in which domains 1 and 3, with globular conformations stabilized by intrachain disulfide bonds, lie at either end of the extended, cysteine-free second domain (9). The high levels of expression obtained in our recombinant system with the D3 region are consistent with the idea that this domain represents a stable, independent unit, capable of folding efficiently in the absence of the rest of the molecule. The D2 domain was expressed at a lower level and was partially modified posttranslationally, although the nature of the modification remains to be determined. The largest discrepancy in expression was seen with a construct encoding the 650 N-terminal amino acid residues of domain 1. Extremely low levels of a 90-kDa protein were obtained with this construct, even though it contained all 8 cysteine residues that might be involved in disulfide-mediated stabilization of its globular structure (not shown). Preliminary results suggest that additional residues C-terminal to this 650-amino acid segment (i.e. in the initial portion of domain 2) may be necessary for proper folding of the N-terminal globular domain. The case of the larger NG2EC fragment also seems complex, since several distinct proteolytic events appear to occur at the C-terminal end of this polypeptide. It has been shown that recombinant proteins expressed in 293 cells can undergo proteolytic trimming, most frequently after a BXBB site (B for basic amino acid) (28, 36). Such sequences are absent in NG2, but two sites in the ectodomain (Arg1774-Arg and Arg2095-Arg) may represent cleavage sites for dibasic endoproteases (40). Truncated NG2 species of 275 and 290 kDa have been characterized in various NG2-positive cell lines (37), indicating a sensitivity to proteolysis in this part of the molecule. Although this process could represent a pathway for regulation of NG2 as a cell surface receptor, no information is available at present concerning in vivo occurrence of cleaved forms of the proteoglycan.
Biochemical studies with the recombinant NG2 fragments confirm that the
D2 region is the site for attachment of chondroitin sulfate chains to
the NG2 core polypeptide. This raises the question of whether the
chondroitin sulfate chains are involved in the collagen binding
mechanism. We previously suggested that binding of native NG2 to
collagen VI was independent of the presence of the chondroitin sulfate
chains (26, 32). Our current data confirm that chondroitin sulfate is
not directly involved in the binding mechanism and that the protein
core of NG2 derived by chondroitinase digestion can bind effectively to
collagens V and VI. However, we have also demonstrated that soluble NG2
fragments synthesized without GAG chains fail to interact with
immobilized collagens V and VI. One possible explanation for this
result is that addition of the GAG chains may modulate folding of the
protein core to produce a conformation that facilitates recognition of the collagen ligands. Thus the process of chondroitin sulfate chain
attachment could represent a means of regulating the receptor-ligand interaction. In this respect, it is of interest that NG2 is expressed both as a proteoglycan and as a protein core free of GAG chains. Although the GAG content of the NG2 core polypeptide in tissues has not
been characterized in detail, different cell lines exhibit different
ratios of mature proteoglycan and core protein (41), with one or two
extreme cases expressing the core protein almost exclusively.2 This phenomenon seems to be
rather common, since both proteoglycan and core protein forms of other
cell surface molecules such as CD44 and -glycan have also been
described (42, 43). Interestingly, although CD44 also serves as a cell
surface receptor for extracellular matrix effectors such as
fibronectin, in contrast to NG2, its chondroitin sulfate chains are
directly involved in the binding mechanism (44, 45). The NG2
extracellular domain binds collagens V and VI with moderately high
affinities, i.e. apparent dissociation constants of 140 and
70 nM, respectively, as determined from solid phase assays.
In comparison, another cell surface proteoglycan, syndecan, has been
shown to have dissociation constants of 5 and 320 nM,
respectively, for these two collagen species (46).
We have been able to localize the binding site for collagens V and VI
to the central, nonglobular domain of NG2. All three recombinant NG2
fragments (NG2EC, EC3, and D2) containing the central domain are
capable of binding to both collagens. The D2 fragment appears to have a
binding capacity equal to or greater than those of the two larger
fragments, suggesting that this segment is largely responsible for the
collagen binding properties of NG2. In contrast, the isolated D3 domain
exhibits no ability to bind collagens.
Rotary-shadowed mixtures of NG2 and collagen V or VI contained examples of NG2 molecules in which the collagenous triple helical domain spanned the space between the N- and C-terminal globules, apparently in alignment with the central D2 domain. This observation suggests that the collagenous domain may have multiple points of contact with the central nonglobular domain of NG2. Thus the collagen binding domain might not be localized to an extremely restricted segment of NG2 but may comprise an extended portion of the central segment. The binding mechanisms of collagens V and VI present some similarities: 1) they interact with the same domain of the NG2 core protein; and 2) the triple helical domain of the collagen is involved in the interaction. Despite the presence of the Gly-X-Y motif in the collagenous domain of both molecules, these two collagens do not share a high degree of homology in their sequence (47). However, binding of NG2 does not occur to all types of collagenous sequences. We did not obtain binding of our recombinant fragments to collagens I or II, regardless of whether these collagens were in a monomeric or fibrillar state (data not shown). These results differ from those of Burg et al. (26), which showed that native NG2 could bind to collagen II. This discrepancy could indicate that NG2 has a weaker affinity for collagen II and/or a different mechanism for binding to collagen II than to collagens V and VI.
Previous suggestions have been made concerning the biological
significance of the collagen VI-NG2 interaction (26, 27, 32). Collagen
VI is considered to be a key component in extracellular matrix assembly
due to its ability to interact with other extracellular matrix proteins
such as collagen I, collagen XIV, von Willebrand factor, and
proteoglycans, either with the protein core of the molecule or with the
glycosaminoglycan side chains (48). The fibrillar collagen V is widely
distributed as a minor component of the extracellular matrix in various
tissues. It is thought to be a crucial component for connective tissue
architecture, as collagen V generally co-polymerizes with the more
abundant collagen I to form heterotypic fibers (49). Mice producing a structurally abnormal subunit, 2(V), present some major disorders in
the organization of the extracellular matrix in different tissues (50).
Collagen V has been shown to bind to many extracellular matrix
components through its triple helical domain, although it is thought to
be rapidly masked in situ by collagen I molecules. However,
accumulation of collagen V has been observed during tissue remodeling
and in neoplasia. In such cases, collagen V could become transiently
available as a potential ligand that fulfills a very specialized
function (51). Collagens V and VI also promote adhesion and spreading
of numerous tumor cell lines, of smooth muscle cells, and of corneal
fibroblasts in primary culture (48, 51). The integrins
1
1 and
2
1 are generally considered the dominant receptors for cell
adhesion to collagens V and VI (52, 53). However, collagen V has also
been shown to be a substrate for glycosaminoglycan-mediated cell
attachment (54). No data are available at present concerning the
possible involvement of NG2 as a receptor in mediating cell adhesion to
collagen V or VI. However, there is evidence that transmembrane
proteoglycans and other cell surface receptors such as integrins can
exert a coordinate effect during interactions with extracellular matrix
components. In particular, it has been shown that the
melanoma-associated proteoglycan, which shares 81% homology with rat
NG2 (23), participates in concert with the
4
1-integrin in the
spreading of melanoma cells on a fibronectin fragment (24, 25). It is
not known whether a similar mechanism may be involved in cell spreading on collagen V or VI, but this type of interaction could be particularly relevant during normal development or tumor metastasis when levels of
NG2 expression are maximal.
We thank Dr. R. Timpl and Dr. E. Pöschl for providing pepsin-extracted collagen VI and expression vectors. We also thank M. M. Boutillon for protein sequencing.