Eur. J. Biochem. 208, 359 - 366 (1 992) 'GI FEBS 1992
Basement-membrane heparan sulfate proteoglycan binds to laminin by its heparan sulfate chains and to nidogen by sites in the protein core Cristina BATTAGLTA, Ulrike MAYER, Monique AUMAILLEY and Rupert TIMPL Max-Planck-Institut fur Biochemie, W-8033 Martinsried, Federal Republic of Gcrmany (Received April 21/June 18, 1992) - EJB 92 0562
A large, low-density form of heparan sulfate proteoglycan was isolated from the EngelbrethHolm-Swarm (EHS) tumor and demonstrated to bind in immobilized-ligand assays to laminin fragment E3, collagen type IV, fibronectin and nidogen. The first three ligands mainly recognize the heparan sulfate chains, as shown by inhibition with heparin and heparan sulfate and by the failure to bind to the proteoglycan protein core. Nidogen, obtained from the EHS tumor or in recombinant form, binds exclusively to the protein core in a heparin-insensitive manner. Studies with other laminin fragments indicate that the fragment E3 possesses a unique binding site of laminin for the proteoglycan. A major binding site of nidogen was localized to its central globular domain G2 by using overlapping fragments. This allows for the formation of ternary complexes between laminin, nidogen and proteoglycan, suggesting a key role for nidogen in basement-membrane assembly. Evidence is provided for a second proteoglycan-binding site in the C-terminal globule G3 of nidogen, but this interaction prevents the formation of such ternary complexes. Therefore, the G3-mediated nidogen binding to laminin and proteoglycan are mutually exclusive.
Basement membranes are large, extracellular protein matrices composed of networks of collagen IV and/or laminin [l] with which several more specific components (nidogen, BM-40) are associated. These complex scaffolds are located close to cells and possess a distinct repertoire of biological activities [2, 31. Ubiquitous to these matrices are also heparan sulfate proteoglycans [2, 31 that provide major polyanionic sites implicated in filtration control 141. An abundant proteoglycan of low buoyant density has been isolated from the mouse Engelbreth-Holm-Swarm (EHS) tumor and other sources and characterized [5 - 81. It is composed of an approximately 500-kDa polypeptide chain with three heparan sulfate chains attached to one end, as seen by electron microscopy. The protein core is elongated and consists of 5-7 globular domains of variable size connected by short rod-like segments [5]. Some smaller heparan sulfate proteoglycans may also exist in basement membranes [6, 7, 9, 101 but their precise relationship to the large major form has not yet been entirely clarified. The complete amino acid sequence of the protein core has been recently deduced from cDNA clones for the large proteoglycan from mouse [ll, 121 and human [13]. The data agree with the electron microscopical analyses [5] and indicate a complex array of five large domains, each of which is composed of several repeating protein motifs. It was suggested that the N-terminal domain I possesses the heparan-sulfateattachment sites. Domains I1 and I11 consist of cysteine-rich repeats similar to epidermal growth factor (EGF) and of Correspondence to R. Timpl, Max-Planck-Institut fur Biochemie, W-8033 Martinsried, Federal Republic of Germany Abbreviations. EGF, Epidermal growth factor; EHS, EngelbrcthHolm-Swarm (tumor).
globular structures. The central domain 1V is composed of a large row of structural elements homologous to the N-CAM motif found in the immunoglobulin gene superfamily. The Cterminal domain V is represented by three laminin G globe structures and two EGF-like repeats. The domain structure of the protein core is similar to laminin A chain [14], except that the N-CAM repeats are replaced by a coiled-coil a helix in laminin. The complex structure of the heparan sulfate proteoglycan suggests that both the polyanionic side chains and some protein core domains may be involved in matrix interactions. This suggestion is supported by the observation that denaturing solvents are required to extract the proteoglycan from the EHS tumor [5, 61. Potential ligands for proteoglycan interactions are laminin and collagen IV, as shown in binding studies with immobilized ligands [15] and by electron microscopy [16]. Yet, the domains involved have so far not been identified. The weak binding of both ligands to heparan sulfate was, however, indicated from studies with a small proteoglycan from the EHS tumor [9]. Nidogen, which has distinct afinities for laminin [17, 181 and collagen IV [19] was recently shown to mediate the formation of ternary complexes [20]. This was interpreted to indicate a key role for nidogen in basement membrane assembly. In the present study, we have shown that the large heparan sulfate proteoglycan binds to all three ligands through either the heparan sulfate side chains or sites in the protein core.
MATERIALS AND METHODS Sources of proteoglycan and glycosaminoglycans The low-density form of heparan sulfate proteoglycan was extracted with 6 M guanidine/HCl from the EHS tumor. It
3 60 was purified by ion-exchange and molecular-sieve chromatography and by CsCl gradient ultracentrifugation following method A as described [5]. It was then subjected to a second chromatographic separation on DEAE cellulose in 7 M urea, 0.05 M TrisiHCl, pH 8.6, with elution by a 00.6 M NaCl gradient (800 ml). The last step reduced laminin contamination to 1 -2.5%, as measured by a radioimmunoassay for laminin fragment P1 [21] which detects epitopes stable against 6 M guanidine/HCl. The protein core was obtained by heparitinasc digestion [5] and purified on DEAE cellulose (see above). The heparan sulfate chains (approximately 36 kDa) were released from the proteoglycan by alkaline degradation [5] and further purified on DEAE cellulose (see above) using a 0-0.4-M NaCl gradient (200 ml). Trypsin digestion was uscd to prepare a fragment consisting of all three heparan sulfate chains connected by a small peptide segment [5]. Other protein ligands and fragments The laminin-nidogen complex was obtained from EDTA extracts of the EHS tumor and its individual constituents were separated after dissociation with 2 M guanidine/HCl by molecular-sieve chromatography [17]. The production of recombinant mouse nidogen in human cell clones and its purification was described previously [20]. A stable complex between recombinant nidogen and laminin fragment P1 was purified on a Superose 6 HR 16/50 column [ZO]. Collagen IV was obtained by reductive extraction from the EHS tumor [22]. Human plasma fibronectin was kindly supplied by Behringwerke AG, and further purified on heparin-Sepharose. Basement-membrane protein BM-40 was prepared from the mouse EHS tumor [23]. Laminin fragments were obtained by digestion with pepsin (Pl) or elastase (ElX, E3, E4,E8 and E10) and purified as described previously [IS, 211. A new fragment, LCX-9, similar to (28-9 [24], was obtained by digesting the laminin-nidogen complex with lysyl endopeptidase (24 h, 37 " C , enzyme/substrate ratio 1: 100) and purified by conventional chromatography [ I X ] . It contained the EX structure but also the entire long arm rod of laminin. The recombinant nidogen fragments Nd-I (domains G1, G2, rod) and Nd-I1 (G3) have been described [20]. Further recombinant products Nd-I a (domains G1, G2) and Nd-Ib (rod) were prepared from the construct Nd-I by established procedures [20] and kindly supplied by D. Reinhardt and R. Nischt, Martinsried. We also prepared the rccornbinant C-terminal nidogen fragment Nd-11, which was previously shown to be poorly soluble [20]. Culture medium (100 ml) containing Nd-I1 (1 - 2 pg/ml) was initially concentrated to 10 ml with an Amicon 1 0 kDa filter and then to 0.6 ml in a Centricon 30 tube. It was then passed over a Superose 12 HR 10/30 column in 0.2 M ammonium acetate, pH 6.9, and yielded Nd-I1 in )70% purity. Nidogen was also cleaved with Staphylococcus aureus V8 protease (2 h, 37"C, enzyme/substrate ratio 1 : 100) to yield an 80-kDa fragment, SV11, which starts with the sequence TGVVF at position 346. This demonstrates that fragment SVll consists of nidogen domains G2 and rod [25]. Further nidogen fragmcnts Th-50 (GI) and Th-100 (G2, G3, rod) were prepared from a thrombin digest of the laminin-nidogen complcx [19]. A schematic illustration of the laminin and nidogen fragments is shown in Figs 1 and 4. Binding assays l h e major binding assay using one ligand immobilized to a plastic surface and the other dissolved in 0.1 M NaC1,
0.05 M Tris/HCl, pH 7.4, followed a standard protocol [19]. Coating efficiency with the heparan sulfate proteoglycan (0.5-1 pg/well) was 2-4% and thus comparable to other ligands used previously. In some tests, coating (4-7% efficiency) and binding was carried out in the presence of 2 mM CaClz or 2 mM EDTA. Binding of the second ligand was detected by antibodies specific for various ligands and their fragments, adjusted to concentrations yielding similar color yields at 492 nm (absorbance 1.2-1.6) in an enzyme-linkedantibody assay. All binding assays were repeated several times. Variations between repeats (see Table 1) are mainly due to different speeds of color development and do not necessarily reflect binding activities of ligands. For the formation of ternary complexes, immobilized ligands were first incubated with a fixed amount of recombinant nidogen (0.1 ml, 70 nM) which was removed prior to the addition of the third ligand [20]. In inhibition assays, constant amounts of soluble ligands were incubated with the inhibitors (2 h, 4°C) prior to adding the mixture to the immobilized ligand. Radioligand assays with both interacting components in soluble form have been previously described [lX]. Analytical methods
Protein concentrations were determined on a Biotronik LC 5001 amino acid analyzer after hydrolyzing samples with 6 M HCI (110°C. 16 h). Amino acid sequences were determined by Edman degradation on an Applied Biosysteins model 473 scquencer, following the manufacturer's instructions. Electrophoresis in SDS/polyacrylamide gels followed established protocols.
RESULTS Binding of heparan sulfate proteoglycan to other basement membrane components
Soluble heparan sulfate proteoglycan binds reproducibly to immobilized laminin-nidogen complex, fibronectin and collagen IV in an antibody-linked detection assay. Similar plateau levels and concentrations (2 - 8 nM) were observed for half-maximal reaction with all three ligands. Similar binding was also observed by using proteoglycan as immobilized ligand with laminin-nidogen and fibronectin. However, the apparent binding was less for collagen IV (Table 1). Typical binding profiles are illustrated in Figs 1 and 4. No binding was, however, observed for another basement membrane protein BM-40. As shown previously, complete separation of laminin from nidogen can be only achieved by dissociating the complex with 2 M guanidine/HCl [17]. The analysis of the separated components showed strong binding of the proteoglycan to nidogen, but only little binding to laminin (Table 3 ). The latter observation, as discussed below, is apparently explained by the loss of a native binding site in laminin, caused by the conditions required to dissociate it from nidogen. Proteoglycan binding of nidogen obtained from the complex was also compared to that of recombinant nidogen which can be obtained in more native form without exposure to denaturing conditions [20]. No differences within the variations of the assay (Table 1) were observed for either form of the protein (data not shown). This is in contrast to the binding activities for laminin and collagen IV which are 5 - 30-fold higher for recombinant nidogen than for nidogen exposed to guanidinei HC1 [20]. Since laminin [26] and nidogen [27] are calcium-
361 Table 1. Parameters of ligand binding of soluble or immobilized heparan sulfate proteoglycan (HSPG) to various basement membrane components. Results are expressed as plateau levels achieved in an antibody-linked detection assay (absorbance, A , at 492 nm) and the concentration of soluble ligand to achieve half maximal reaction (BC,,). All values with A > 0.5 arc means SD of 6 - 12 different determinations including 2-6 diffcrcnt preparations of each ligand. The results for nidogen and lamininate after dissociation with 2 M guanidine/HCl from the lamininnidogen complex. Laminin E3 is elastase fragment E3.
Second ligand
Immobilized HSPG
Soluble HSPG plateau
BCw
plateau
BCSO
nM
Laminin-nidogen Nidogen Laminin Laminin E3
Fibronectin Collagen TV BM-40
0.8 k 0.2 1.1 f 0.4 0.3 1.2 f 0.7 1.5 f 0.4 0.9 f 0.4
nM 1.1 & 0.5 0.7 f 0.3 0.1 1.2 0.4 1.2 0.5
5.2 4.0
8.0 7.0 -1
*
2.8 & 1.6 2.2 0.8 6.6 5.0
*
-o-~-o
1b3
A
+-0-0-A-u
- O - - y O - O-0-
10’ 10’ S o h b l e laminin ligand InM)
- A-
01
100
Fig. 1. Binding of laminin-nidogen complex and laminin fragments to immobilized heparan sulfate proteoglycan. The binding data (B) arc based on an antibody-linked detection system for the soluble ligands which were laminin-nidogen complex ( O ) ,fragment E3 (A),rragment LC8-9 ( A ) and fragments ElX, PI, E4, E8 and El0 (0, Zl), the latter being all of similar low activity. (A) shows the approximative localization of the fragments within the cross-shaped laminin [2, 241, identifying the A, B1 and B2 chains of laminin and the localization of the nidogen-binding site (Nd) within the B2 chain [39].
it showed strong binding to immobilized nidogen, some reduced binding to the laminin-nidogen complex and little or no binding to collagen IV, laminin fragment E3 and fibronectin (Fig. 2). Similar binding profiles, except €ora strongly reduced binding for laminin-nidogen complex, were observed by using the protein core as immobilized ligand. Some of the data were confirmed with a tryptic fragment of the proteoglycan consisting of the three heparan sulfate chains connected by a peptide segment that contained only about 3% of the protein core [S]. This fragment, as immobilized ligand, showed distinct binding for fragment E3 but none for nidogen (data not shown) .
362 L.U
I
I
Table 2. inhibitory capacities (ICs0) of various glycosaminoglycans and dextran sulfate for the binding of soluble ligands to immobilized heparan sulfate proteoglycan. The results for laminin and E3 were the average values of three experiments.
ICso for binding to
Inhibitor 1.0
lamininnidogen
laminin E3
fibronectin
0.05
2 100 38 90
0.5 0 lo2
lo1
100
10-1
Heparin Heparan sulfate Dextran sulfate Chondroitin sulfate
0.8 5
1.4
18
0.7
60
6.0
Protein core ligond InMl
Fig.2. Binding activity of proteoglycan protein core used as soluble ligand for various basement membrane components. The ligands used in immobilized form were recombinant nidogen ( O ) ,laminin-nidogen complex (B),fibronectin (m),collagen IV (A)and laminin fragment E3 ( A ) .
A
rod
100 -
2.0
E
0 .c
1.5
(v
0if
W-
g 1.0
20 -
rn
n L
0 1 Ln 3
a
0 10-1
100 10’ Heparin (pg/mll
102
Fig. 3. Sensitivity to heparin inhibition of proteoglycan binding to basement membrane components. The proteoglycan was either used in immobilized form (closed symbols) or as soluble ligand (open symbols). The second ligands were laminin fragment E3 (@, 0),lamininnidogen complex (A,A), fibronectin (H) and EHS tumor nidogcn
(V).
Further studies on the involvement of heparan sulfate chains were persormed by inhibition assays with heparin and other glycosaminoglycans. The binding of soluble fragment E3, laminin-nidogen and fibronectin to immobilized proteoglycan could be very efficiently and rather completely inhibited by heparin. No inhibitory effect was observed for nidogen (Fig. 3). The inhibitory capacities were different, being highest for fragment E3 and 10-20-fold lower for laminin-nidogen and fibronectin. The inhibition profiles changed, however, when using proteoglycan as soluble ligand. The fragment E3 interaction was still completely inhibited but with a 20-fold-lower efficiency. The laminin-nidogen interaction was only partially inhibited (35--40%) and required a more than 3 00-fold increase in heparin concentration to achieve this effect (Fig. 3). This suggests that proteoglycan binding to fragment E3 is completely dependent on heparan sulfate chains. Apparently, it is also responsible for the binding of laminin-nidogen to immobilized proteoglycan. Yet, soluble proteoglycan can bind to immobilized laminin-nidogen in two possible ways; either by heparan sulfate or its protein core.
0.5
0 102
10’
10
10-1
Proteaglycan (nM)
Fig. 4. Binding of proteoglycan to recombinant nidogen and nidogen fragments used as immobilized ligands. (A) Domain model of nidogen [20] and the approximative localization of the fragments. The binding curves (B) were obtained with nidogen ( O ) ,recombinant fragments Nd-I (@), Nd-Ia (W), rod (V)and SV3 1 ( A ) and fragments Th-100 (n)and Th-50 (A),prepared from the laminin-nidogen complex.
The latter possibility is supported by binding studies with the protein core (Fig. 2). A comparison of the inhibiting capacities of heparin and the heparan sulfate chains prepared from the proteoglycan showed a 5 - 30-Sold increase in the concentration causing half-maximal inhibition for heparan sulfate when competing for soluble laminin-nidogen or fragment E3 binding to proteoglycan. Dextran sulfate or chondroitin sulfate were either of equal or lower potency when compared to heparan sulfate (Table 2). Similar, but not identical, inhibition activities were also observed for the fibronectin interaction. Binding activity of nidogen fragments A panel of fragments, prepared from EHS tumor or recombinant nidogen [18-201, was used to map its binding site(s) for the proteoglycan (Fig. 4). These fragments together
363
1.5
1.2
E
E
w N.
N
4
%
0.8
1.0
w-
c m n L o v) n
m
n L v) 0
n
a
2
0.4
Q
\ \-
0.5
I02
Fig. 5. Binding of recombinant nidogen, fragment Nd-I and a nidogenPI complex to immobilized proteoglycan (open symbols) or laminin (closed symbols). The soluble ligands used were nidogen ( 0 , O ) , nidogen fragment Nd-I ( , n) and a preformed complex between recombinant nidogen and laminin fragment PI (A, A). Detection was, in all cases. with an antibody against nidogen, recognizingmainly its rod domain.
cover the three globular domains of nidogen, G I , G 2 and G3, and the rod-like domain in different associations, All the fragments that shared the central globular domain G 2 showed comparable activity with nidogen in binding to the soluble proteoglycan ligand. Fragments comprising the N-terminal globular domain G I or the rod-like segment were inactive (Fig. 4). Binding analyses with immobilized proteoglycan showed, however, different reactivities for nidogen and its fragments. While nidogen showed distinct binding, although with lower plateau levels (compare Figs 4 and 5, see also Table l), little or no activity was observed with fragment Nd-I (Fig. 5 ) that lacks the C-terminal G3 domain of nidogen [20]. Further tests with other nidogen fragments which were active as immobilized ligands (Fig. 4) also failed to show binding. This suggested that nidogen domain G3 or a segment joining G 3 to the rod-like segment are responsible for the binding of nidogen to immobilized proteoglycan. Since G 3 has been identified as thc only nidogen domain which binds laminin [18, 201 a further comparison was performed with immobilized laminin (Fig. 5). There was, as expected, a strong reaction of nidogen with laminin, exceeding that with proteoglycan, but no binding for fragment Nd-I. Furthermore, blocking of the laminin-binding site of nidogen by forming a stoichiomctric complex with laminin fragment P1 [20] inactivated the binding to both laminin and proteoglycan ligands (Fig. 5). The nidogen domain G3 was previously obtained as recombinant fragment Nd-I1 that was poorly soluble and therefore could not be used as a soluble ligand [20].We have now introduced a new purification procedure which allowed us to use Nd-I1 at least as an immobilized ligand (Fig. 6). It showed distinct binding of laminin with half-maximal binding with approximately 2 nM soluble ligand. Distinct binding was also observed with the soluble proteoglycan but at 15-fold-higher concentrations. This observation suggested that the small laminin contamination (1 - 2.5%) in the proteoglycan might have been responsible for binding. This possibility was, however: excluded by showing that only antibodies specific for
10’ 100 lo-’ Soluble Ligand (nM)
Fig. 6. Binding of soluble proteoglycan and laminin to immobilized recombinant domain G3 of nidogen (fragment Nd-11). The binding of proteoglycan (0)and laminin (0 j was detected by antibodies specific for each soluble ligand. Negative control experiments were performed by switching the detecting antibodies using anti-laminin in the proteoglycan ( 0j and anti-proteoglycan in the laminin (B) binding assay.
Table 3. Dependence on antibody specificity of the detection of binding nidogen fragment Th-100 to immobilized ligands. Results are expressed as plateau levels (antibody to rod) or maximal levels (antibody to G3) observed in an antibody-linked detection assay ( A at 492 nm) and the concentration of Th-100 to achieve half plateau levels of reaction (BC,,). Immobilized ligand Parameters for antiserum to G3 domain
rod domain
Pro teoglycan Laminin P1 Collagen IV
1.0 1.6 1.4
nM 25 14 14
0.3 0.3 1.4
nM >400 >400 45
proteoglycan, but not those specific for laminin, were bound to the proteoglycan-Nd-IT complex (Fig. 6). The suggestion that the G3 domain of nidogen binds both laminin and proteoglycan was examined in a further experiment, Antibodies to the G3 domain were used for detecting binding to the nidogen fragment Th-100 that consists of domains G2, G 3 and the rod (see Fig. 4) to immobilized proteoglycan. There was no significant binding. In contrast, there was binding of antibodies to the rod domain that represents a non-binding structure (Table 3). The same result was previously observed for nidogen binding to laminin fragment P1 and interpreted to indicate that the ligand binding to domain G 3 interferes with epitope recognition [20]. Much less interference was observed by comparing both antibodies in a binding assay for immobilized collagen IV (Table 3). The results supported evidence that nidogen domain G2 is involved in this binding ([20, 271; D. Reinhardt et al., unpublished results). Together, the data indicate that there are two binding sites of nidogen for the proteoglycan protein core, one in G2 and the other in G3. The latter binding is, however, distinctly weaker than G3-mediated binding to laminin and its fragment PI.
364 2.0
E
1.5
c N m
.\
s,
2
t
1.0
D
.a
L
0
a ln
a
0.5 0
1o2
10'
1OD
10-1
Proteoglycon ligond ( n M )
Fig. 7. Nidogen-mediated formation of ternary complexes between proteoglycan and laminin or collagen IV used in immobilized form. The imniobilized ligands were laminin fragment P1 (0, O ) , laminin (m, 7)and collagen IV ( A , A) which were used prior to (open symbols) or after (closed symbols) treatment with a fixed amount of recombinant nidogen (70 nM).
Evidence for the formation of ternary complexes Since nidogen mediates the binding of laminin to collagen IV [20], it was of interest to examine whether it has a similar bridging activity for the proteoglycan. The formation of a ternary complex was already shown by the binding of lamininnidogen complex to proteoglycan (Table 1, Fig. I), but the role of nidogen in this interaction was not obvious. We therefore resorted to the use of 2 M guanidine/HCl-treated laminin and laminin fragment P1 as immobilized ligands that both possess binding activity for nidogen [18] but not for the proteoglycan (Table 1, Fig. 1). Addition of recombinant nidogen to these ligands clearly enhanced their binding capacity for soluble proteoglycan up to the usual binding levels (Fig. 7). Similar binding profiles were observed with the complex between fragment PI and nidogen obtained at a preparative scale as immobilized ligand and soluble proteoglycan (data not shown). In addition, nidogen enhanced proteoglycan binding to immobilized collagen IV (Fig. 7), indicating the possibility of the formation of a second ternary complex. When we changed to protocols using immobilized proteoglycan, no nidogen-mediated binding of laminin and fragment P1 (not shown) or the direct binding of the P1-nidogen complex (Fig. 5 ) could be demonstrated. Parallel experiments replacing immobilized proteoglycan by collagen IV demonstrated, as shown before [20], the formation of ternary complexes. This observation indicates that proteoglycan, when used as a coat for plastic surfaces, has a decreased ability to provide a binding site for the formation of ternary complexes. The most likely explanation is that immobilized proteoglycan has a reduced affinity for nidogen domain G2 and that its persistent binding to domain G3 interferes with G3-mediated recognition of laminin.
DISCUSSION The data presented here confirm previous observations [15, 161 that the large heparan sulfate proteoglycan found in basement membranes [2, 31 binds two other basement membrane proteins, laminin and collagen IV. Novel findings were its binding to nidogen and its lack of binding to BM-40. It
was further demonstrated by inhibition studies with heparin or heparan sulfate and binding assays with the protein core, that the heparan sulfate chains provide the major binding sites for laminin, collagen IV and fibronectin. Nidogen binding, however, appears to be mediated entirely by the protein core. Our data on fibronectin are in conflict with other observations for a similar proteoglycan obtained from lung fibroblasts [S] where the protein core seems to be responsible for binding [30]. Yet, variations in the sulfation of heparan sulfate chains of this proteoglycan can modulate antithrombin binding activity [31] and there is evidence for alternative splicing of the protein core [I 31. Such structural differences not fully explored so far may therefore account for the differences in the results. Mapping of a major proteoglycan-binding site of laminin was achieved by a set of non-overlapping fragments which covered more than 95% of the protein structure (see Fig. 1). The results showed a high and exclusive binding activity for fragment E3 that corresponds to the two most C-terminal globular G domains of laminin A chain [14, 321. This binding is to the heparan sulfate chains, as shown by the sensitivity to glycosaminoglycan inhibition and binding of E3 to a small proteoglycan fragment possessing the heparan sulfate chains. The result is in agreement with the strong affinity of E3 for heparin affinity columns [21]. A functional role for this interaction could be modulation of laminin polymerization. Heparin increases this self-assembly process at low concentrations but interferes with it at high concentrations [33, 341. We can, however, not exclude the possibility that laminin may possess more binding sites for the proteoglycan that may have been lost during proteolysis. In addition, potential binding sites could have been destroyed in the protein corc by its exposure to strong denaturing solvents [5, 61. Another binding site of the protein core responsible for proteoglycan oligomerization 1351 apparently resists such treatment. Another laminin fragment, EX, shows distinct binding to heparin affinity columns but no binding for the proteoglycan. This could correlate with the low content of 0-sulfation in the heparan sulfate chains of the EHS tumor [31, 361 compared to heparin. For example, antithrombin binds with high affinity to heparin but only weakly to EHS tumor heparan sulfate [31]. Heparan sulfates obtained from other basement membranes differ in this respect [31, 371 and such proteoglycans could have a more versatile interaction repertoire for laminin. The results support our previous observations [18, 211 that the laminin B1 chain fragments E4 and E l 0 do not bind to heparin or as shown here to the proteoglycan. These structures were implicated in one [34], but not another study [33], to be heparin-binding structures based on the electron microscopical observation of complexes after rotary shadowing. This makes the subsequent assignment of binding sites, using randomly selected synthetic peptides corresponding to laminin fragment E4 and El0 sequences [34, 381 a questionable approach. In order to understand their biological significance, the peptides should at least have been shown to bind to basement-membrane forms of heparan sulfate proteoglycan. A major binding site for the protein core was assigned to the globular domain G2 of nidogen by assays with overlapping fragments. The same nidogen domain or an adjacent structure apparently contains a major binding site for the triple helix of collagen IV [20, 271. Based on the lack of any structural similarity between the collagen-IV triple helix and the proteoglycan sequence [12, 131, it is unlikely that both binding sites on domain G2 are identical. This would also explain the enhancing effect of nidogen for proteoglycan association to collagen IV (Fig. 7) and suggests that direct collagen binding,
365 mediated by heparan sulfdte, could be increased by a second alternative reaction mediated by two non-interfering bindillg sites on the G2 d o m a i n of nidogen. A second binding site on nidogen f o r the proteoglycan protein core was assigned t o its globular d o m a i n G3, but this w a s only possibly d u e t o the fact t h a t the binding activity associated with G2 is strongly reduced in immobilized proteoglycan (Fig. 5). Nidogen domain G 3 has been previously identified a s possessing t h e only binding site for laminin [18, 201 which interacts with a few EGF-like motifs present in t h e short-arm structure of laminin B2 chain [39]. This is of interest since several similar EGF-like repeats a r e f o u n d i n t h e proteoglycan sequence [I 1 - 131. AS discussed belou7, s o m e indications suggest t h a t the binding of nidogen G3 d o m a i n to either laminin or proteoglycan is mutually exclusive. By the use of recombinant nidogen, we were, for the first time, able to show t h a t this protein mediates binding between laminin and collagen TV [20, 271. This suggests a similar function in situ and a key role f o r nidogen in connecting thc networks of collagen TV and laminin in basement membranes [l - 3, 281. Here we show that the addition of heparan sulfate proteoglycan t o these supramolecular assemblies m a y n o t only occur by electrostatic interactions via the h e p a r a n sulfate chains, b u t also involve protein-protein interactions mediated by nidogen. This was clearly demonstrated by using immobilized laminin ligands, nidogen as mediator and t h e proteoglycan as third ligand (Fig. 7). It implicates, i n this constellation, recognition of laminin by the G 3 d o m a i n and of proteoglycan by t h e G2 domain of nidogen. Yet, no ternary complexes were formed using an immobilized proteoglycan ligand a n d examining nidogen-mediated laminin binding or t h e direct binding of preformed laminin P1-nidogen complexes. The immobilized proteoglycan still has, however, a distinct activity f o r binding soluble nidogen. We explain these apparently contradictory d a t a by assuming t h a t the immobilized proteoglycan has lost most of its activity for binding t o the nidogen d o m a i n G2, which is replaced by binding via its G3 domain. This binding, however, does not allow any more interaction with laminin. While the loss of binding activity of immobilized ligands is known in other cases, i.e. for BM-40 [23], our interpretation for the proteoglycan is nevertheless speculative and will need independ e n t confirmation. If correct, it would indicate that alternative choices of nidogen for binding sites m a y exist i n basement membranes a n d contribute t o its variation in architecture [40]. D u e t o the ambiguity of some o f the ternary binding d a t a , we like to emphasize again t h a t the proteoglycan used here was exposed t o strongly denaturing agents for solubilization a n d purification in intact f o r m [ 5 , 6 ] .Circular-dichroism spectroscopy, i n fact, indicates partial denaturation of the protein core by these procedures [5]. To overcome such inherent difficulties i n purifying extracellular matrix proteins, their preparation by recombinant methods i n mammalian cells is an appropriate alternative choice. As shown with recombinant nidogen [20], it may reveal new insights into d o m a i n structure and binding activities. This approach is now feasible for the protein core based on its complete cDNA cloning [12, 131.
Wc thank Mrs Vera van Delden for expert tcchnical assistance, Dr. Karlheinz Mann for amino acid sequencing and Dr. Roswitha Nischt and Mr. Dicter Reinhardt for help with some nidogen fragments. The study wa5 supported by a grant from the Deutache I;hrJchun~spemeinschaft.The fellowshio sunoorl to CR bv the Mario Negri Institute and the Max-Planck-Gescllschaft is appreciated. I
.I
REFERENCES 1. Yurchcnco, P. D. & Schittny, J. C. (1990) FASEB J . 4 , 15771590. 2. ’Iimpl, R. (1989) Eur. J . Biochenz. 180,487-502. 3. Paulsson, M. (1992) Crit. Rev. Biochem. Mol. Bid. 27, 93-127. 4. Farquhar, M. G. (1991) in Cell biology of extrclcrllular matrix (Hay. E. D., ed.) pp. 335 -378, Plenum Press, New York. 5. Paulsson, M., Yurchenco, P. D., Ruben, G. C., Engel, J. & Timpl, R. (1987) .I. M o l . Biol. 197, 297-313. 6. Hassell, J. R., Leyshon, W . C., Ledbetter, S. R., Tyrce, B., Suzuki, S . , Kato, M., Kimata, K. & Kleinman, H. K. (1985) J. Biol. Chem. 260,8098-8105. 7. Kato, M., Koike, Y., Ito, Y., Suzuki, S. & Kimata, K. (1987) J . Biol. Chem. 262, 7180-7188. 8. Heremans, A., Van der Schueren, B., dc Cock, B., Paulsson, M., Cassiman, J.-J., van dcn Berghe, H. & David, G. (1989) J . Cell Biol. 109, 3299-3211. 9. Fujiwara, S., Wiedemann. H., Timpl, R., Lustig, A. & Engel, J. (1984) Eur. .I. Biochem. 143, 145-157. 10. Kanwar, Y. S., Veis, A., Kimura, J . H. & Jakubowski, M. L. (1984) Proc. Natl Acad. Sci. USA 81,762-766. 11. Noonan, D. M., Horigan, E., Ledbetter, S . , Vogeli, G., Sasaki, M., Yamada, Y. & Hassell, J . R. (1988) J . B i d . Chem. 263. 16379- 16387. 12. Noonan, D. M., Fuller, A , , Valente, P., Cai, S . , Horigan, E.. Sasaki, M., Yamada, Y. & Hassell, J. R . (1991) J. Biol. Chem. 266,22939-22947. 13. Kallunki, P. &Tryggvason,K. (1992)J. CellBiol. 116,559-571. 14. Sasaki, M., Kleinman, H. K., Hubcr, H., Deutzmann, R. & Yamada, Y. (1988) J. Biol. Chem. 263,16536-16544. 15. Woodley. D. T., Rao, C. N., Hassell, .I R., . Liotta, L. A., Martin, G. R. & Kleinman, H. K. (1983) Biochim. Biophys. Acta 761, 278-283. 16. Laurie, G. W.. Bing, J. T., Kleinman, H. K., Hassell, J . R.. Aumailley, M., Martin, G. R. & Feldmann, J. R. (1986) J . Mol. Biol. 189, 205 - 216. 17. Paulsson, M., Aumailley, M., Deutzmann, R., Timpl, R., Beck, K. & Engel, J. (1987) Eur. J . Biochem. 166, 11 - 1 Y . 18. Mann, K., Deutzmann, R. & Timpl, R. (1988) Eur. J. Biochem. 178, 71 - 80. 19. Aumailley, M., Wiedemann, H., Mann, K. & Timpl, R. (1989) Eur. J . Biochem. 184, 241 -248. 20. Fox, J. W., Mayer, U., Nischt, R., Aumailley, M., Reinhardt, D., Wiedemann, H., Mann, K., Timpl, R., Krieg, T., Engel, J. & Chu, M.-L. (1991) EMBU J . 10, 3137-3146. 21. Ott, U.. Odermatt, E., Engcl, J., Furthmayr, H. & Timpl, R. (1982) Eur. J. Biochem. 123, 63--72. 22. Yurchenco, P. D. & Furthmayr, H. (1984) Biochemistry 23, 1839- 1850. 23. Mayer, U., Aumailley, M., Mann, K., Timpl, R. & Engel, J. (1991) Eur. J. Biochem. 198, 141 -150. 24. Hunter, I., Schulthess, T., Bruch, M., Beck, K. & Engel, J. (1 990) Eur. J . Biochem. 188, 205 -211. 25. Mann, K., Deutzmann, R., Aumaillcy, M., Timpl, R., Raimondi, I,., Yamada, Y., Pan, T., Conway, D. & Chu, M.-L. (1989) EMBO J . 8,65 -72. 26. Paulsson, M. (1988) J . Biol. Chem. 263, 5425-5430. 27. Aumailley, M., Battaglia, C., Mayer, U., Rcinhardt, D., Nischt, R., Timpl, R. & Fox, J. W. (1992) Kid~rej)Int., in the press. 28. Yurchenco, P. D., Tsilibary, E. C., Charonis, A. S. & Furthmayr, H. (1985) J . Biol. Chem. 260,7636-7644. 29. Sakashita, S., Engvall, E. & Ruoslahti, E. (1980) FEBS Lett. 116. 243 246. 30. Heremans, A., De Cock, B., Cassiman, J.-J., Van den Berghe, H. &David, G. (19YO) J . Bid. Chem. 265, 8716-8724. 31. Pejler, G., Backstrom, G., Lindahl. U., Paulsson, M.. Dziadek, M., Fujiwara, S. & Timpl, R. (1987) J . Biol. Chem. 262, 50365043. 32. Deutzmann, R.. Huber. J., Schmctz, K. A,, Oberbaumer, I. & Hartl, L. (1988) Eur. J . Biochem. 177, 35-45. 33. Yurchenco, P. D., Cheng, Y.-S. & Schittny. J. C. (1990) .I. Bid. Chem. 265, 3981 ~-39’11. -
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40. Furthmayr, H., Yurchenco, P. D., Charonis, A. S. & Tsilibary, E.C. (1985) in Basement membranes (Shibata, S., ed.) pp. 169-
180, Elsevier, Amsterdam.
Basement-membrane heparan sulfate proteoglycan binds to laminin by its heparan sulfate chains and to nidogen by sites in the protein core Cristina BATTAGLTA, Ulrike MAYER, Monique AUMAILLEY and Rupert TIMPL Max-Planck-Institut fur Biochemie, W-8033 Martinsried, Federal Republic of Gcrmany (Received April 21/June 18, 1992) - EJB 92 0562
A large, low-density form of heparan sulfate proteoglycan was isolated from the EngelbrethHolm-Swarm (EHS) tumor and demonstrated to bind in immobilized-ligand assays to laminin fragment E3, collagen type IV, fibronectin and nidogen. The first three ligands mainly recognize the heparan sulfate chains, as shown by inhibition with heparin and heparan sulfate and by the failure to bind to the proteoglycan protein core. Nidogen, obtained from the EHS tumor or in recombinant form, binds exclusively to the protein core in a heparin-insensitive manner. Studies with other laminin fragments indicate that the fragment E3 possesses a unique binding site of laminin for the proteoglycan. A major binding site of nidogen was localized to its central globular domain G2 by using overlapping fragments. This allows for the formation of ternary complexes between laminin, nidogen and proteoglycan, suggesting a key role for nidogen in basement-membrane assembly. Evidence is provided for a second proteoglycan-binding site in the C-terminal globule G3 of nidogen, but this interaction prevents the formation of such ternary complexes. Therefore, the G3-mediated nidogen binding to laminin and proteoglycan are mutually exclusive.
Basement membranes are large, extracellular protein matrices composed of networks of collagen IV and/or laminin [l] with which several more specific components (nidogen, BM-40) are associated. These complex scaffolds are located close to cells and possess a distinct repertoire of biological activities [2, 31. Ubiquitous to these matrices are also heparan sulfate proteoglycans [2, 31 that provide major polyanionic sites implicated in filtration control 141. An abundant proteoglycan of low buoyant density has been isolated from the mouse Engelbreth-Holm-Swarm (EHS) tumor and other sources and characterized [5 - 81. It is composed of an approximately 500-kDa polypeptide chain with three heparan sulfate chains attached to one end, as seen by electron microscopy. The protein core is elongated and consists of 5-7 globular domains of variable size connected by short rod-like segments [5]. Some smaller heparan sulfate proteoglycans may also exist in basement membranes [6, 7, 9, 101 but their precise relationship to the large major form has not yet been entirely clarified. The complete amino acid sequence of the protein core has been recently deduced from cDNA clones for the large proteoglycan from mouse [ll, 121 and human [13]. The data agree with the electron microscopical analyses [5] and indicate a complex array of five large domains, each of which is composed of several repeating protein motifs. It was suggested that the N-terminal domain I possesses the heparan-sulfateattachment sites. Domains I1 and I11 consist of cysteine-rich repeats similar to epidermal growth factor (EGF) and of Correspondence to R. Timpl, Max-Planck-Institut fur Biochemie, W-8033 Martinsried, Federal Republic of Germany Abbreviations. EGF, Epidermal growth factor; EHS, EngelbrcthHolm-Swarm (tumor).
globular structures. The central domain 1V is composed of a large row of structural elements homologous to the N-CAM motif found in the immunoglobulin gene superfamily. The Cterminal domain V is represented by three laminin G globe structures and two EGF-like repeats. The domain structure of the protein core is similar to laminin A chain [14], except that the N-CAM repeats are replaced by a coiled-coil a helix in laminin. The complex structure of the heparan sulfate proteoglycan suggests that both the polyanionic side chains and some protein core domains may be involved in matrix interactions. This suggestion is supported by the observation that denaturing solvents are required to extract the proteoglycan from the EHS tumor [5, 61. Potential ligands for proteoglycan interactions are laminin and collagen IV, as shown in binding studies with immobilized ligands [15] and by electron microscopy [16]. Yet, the domains involved have so far not been identified. The weak binding of both ligands to heparan sulfate was, however, indicated from studies with a small proteoglycan from the EHS tumor [9]. Nidogen, which has distinct afinities for laminin [17, 181 and collagen IV [19] was recently shown to mediate the formation of ternary complexes [20]. This was interpreted to indicate a key role for nidogen in basement membrane assembly. In the present study, we have shown that the large heparan sulfate proteoglycan binds to all three ligands through either the heparan sulfate side chains or sites in the protein core.
MATERIALS AND METHODS Sources of proteoglycan and glycosaminoglycans The low-density form of heparan sulfate proteoglycan was extracted with 6 M guanidine/HCl from the EHS tumor. It
3 60 was purified by ion-exchange and molecular-sieve chromatography and by CsCl gradient ultracentrifugation following method A as described [5]. It was then subjected to a second chromatographic separation on DEAE cellulose in 7 M urea, 0.05 M TrisiHCl, pH 8.6, with elution by a 00.6 M NaCl gradient (800 ml). The last step reduced laminin contamination to 1 -2.5%, as measured by a radioimmunoassay for laminin fragment P1 [21] which detects epitopes stable against 6 M guanidine/HCl. The protein core was obtained by heparitinasc digestion [5] and purified on DEAE cellulose (see above). The heparan sulfate chains (approximately 36 kDa) were released from the proteoglycan by alkaline degradation [5] and further purified on DEAE cellulose (see above) using a 0-0.4-M NaCl gradient (200 ml). Trypsin digestion was uscd to prepare a fragment consisting of all three heparan sulfate chains connected by a small peptide segment [5]. Other protein ligands and fragments The laminin-nidogen complex was obtained from EDTA extracts of the EHS tumor and its individual constituents were separated after dissociation with 2 M guanidine/HCl by molecular-sieve chromatography [17]. The production of recombinant mouse nidogen in human cell clones and its purification was described previously [20]. A stable complex between recombinant nidogen and laminin fragment P1 was purified on a Superose 6 HR 16/50 column [ZO]. Collagen IV was obtained by reductive extraction from the EHS tumor [22]. Human plasma fibronectin was kindly supplied by Behringwerke AG, and further purified on heparin-Sepharose. Basement-membrane protein BM-40 was prepared from the mouse EHS tumor [23]. Laminin fragments were obtained by digestion with pepsin (Pl) or elastase (ElX, E3, E4,E8 and E10) and purified as described previously [IS, 211. A new fragment, LCX-9, similar to (28-9 [24], was obtained by digesting the laminin-nidogen complex with lysyl endopeptidase (24 h, 37 " C , enzyme/substrate ratio 1: 100) and purified by conventional chromatography [ I X ] . It contained the EX structure but also the entire long arm rod of laminin. The recombinant nidogen fragments Nd-I (domains G1, G2, rod) and Nd-I1 (G3) have been described [20]. Further recombinant products Nd-I a (domains G1, G2) and Nd-Ib (rod) were prepared from the construct Nd-I by established procedures [20] and kindly supplied by D. Reinhardt and R. Nischt, Martinsried. We also prepared the rccornbinant C-terminal nidogen fragment Nd-11, which was previously shown to be poorly soluble [20]. Culture medium (100 ml) containing Nd-I1 (1 - 2 pg/ml) was initially concentrated to 10 ml with an Amicon 1 0 kDa filter and then to 0.6 ml in a Centricon 30 tube. It was then passed over a Superose 12 HR 10/30 column in 0.2 M ammonium acetate, pH 6.9, and yielded Nd-I1 in )70% purity. Nidogen was also cleaved with Staphylococcus aureus V8 protease (2 h, 37"C, enzyme/substrate ratio 1 : 100) to yield an 80-kDa fragment, SV11, which starts with the sequence TGVVF at position 346. This demonstrates that fragment SVll consists of nidogen domains G2 and rod [25]. Further nidogen fragmcnts Th-50 (GI) and Th-100 (G2, G3, rod) were prepared from a thrombin digest of the laminin-nidogen complcx [19]. A schematic illustration of the laminin and nidogen fragments is shown in Figs 1 and 4. Binding assays l h e major binding assay using one ligand immobilized to a plastic surface and the other dissolved in 0.1 M NaC1,
0.05 M Tris/HCl, pH 7.4, followed a standard protocol [19]. Coating efficiency with the heparan sulfate proteoglycan (0.5-1 pg/well) was 2-4% and thus comparable to other ligands used previously. In some tests, coating (4-7% efficiency) and binding was carried out in the presence of 2 mM CaClz or 2 mM EDTA. Binding of the second ligand was detected by antibodies specific for various ligands and their fragments, adjusted to concentrations yielding similar color yields at 492 nm (absorbance 1.2-1.6) in an enzyme-linkedantibody assay. All binding assays were repeated several times. Variations between repeats (see Table 1) are mainly due to different speeds of color development and do not necessarily reflect binding activities of ligands. For the formation of ternary complexes, immobilized ligands were first incubated with a fixed amount of recombinant nidogen (0.1 ml, 70 nM) which was removed prior to the addition of the third ligand [20]. In inhibition assays, constant amounts of soluble ligands were incubated with the inhibitors (2 h, 4°C) prior to adding the mixture to the immobilized ligand. Radioligand assays with both interacting components in soluble form have been previously described [lX]. Analytical methods
Protein concentrations were determined on a Biotronik LC 5001 amino acid analyzer after hydrolyzing samples with 6 M HCI (110°C. 16 h). Amino acid sequences were determined by Edman degradation on an Applied Biosysteins model 473 scquencer, following the manufacturer's instructions. Electrophoresis in SDS/polyacrylamide gels followed established protocols.
RESULTS Binding of heparan sulfate proteoglycan to other basement membrane components
Soluble heparan sulfate proteoglycan binds reproducibly to immobilized laminin-nidogen complex, fibronectin and collagen IV in an antibody-linked detection assay. Similar plateau levels and concentrations (2 - 8 nM) were observed for half-maximal reaction with all three ligands. Similar binding was also observed by using proteoglycan as immobilized ligand with laminin-nidogen and fibronectin. However, the apparent binding was less for collagen IV (Table 1). Typical binding profiles are illustrated in Figs 1 and 4. No binding was, however, observed for another basement membrane protein BM-40. As shown previously, complete separation of laminin from nidogen can be only achieved by dissociating the complex with 2 M guanidine/HCl [17]. The analysis of the separated components showed strong binding of the proteoglycan to nidogen, but only little binding to laminin (Table 3 ). The latter observation, as discussed below, is apparently explained by the loss of a native binding site in laminin, caused by the conditions required to dissociate it from nidogen. Proteoglycan binding of nidogen obtained from the complex was also compared to that of recombinant nidogen which can be obtained in more native form without exposure to denaturing conditions [20]. No differences within the variations of the assay (Table 1) were observed for either form of the protein (data not shown). This is in contrast to the binding activities for laminin and collagen IV which are 5 - 30-fold higher for recombinant nidogen than for nidogen exposed to guanidinei HC1 [20]. Since laminin [26] and nidogen [27] are calcium-
361 Table 1. Parameters of ligand binding of soluble or immobilized heparan sulfate proteoglycan (HSPG) to various basement membrane components. Results are expressed as plateau levels achieved in an antibody-linked detection assay (absorbance, A , at 492 nm) and the concentration of soluble ligand to achieve half maximal reaction (BC,,). All values with A > 0.5 arc means SD of 6 - 12 different determinations including 2-6 diffcrcnt preparations of each ligand. The results for nidogen and lamininate after dissociation with 2 M guanidine/HCl from the lamininnidogen complex. Laminin E3 is elastase fragment E3.
Second ligand
Immobilized HSPG
Soluble HSPG plateau
BCw
plateau
BCSO
nM
Laminin-nidogen Nidogen Laminin Laminin E3
Fibronectin Collagen TV BM-40
0.8 k 0.2 1.1 f 0.4 0.3 1.2 f 0.7 1.5 f 0.4 0.9 f 0.4
nM 1.1 & 0.5 0.7 f 0.3 0.1 1.2 0.4 1.2 0.5
5.2 4.0
8.0 7.0 -1
*
2.8 & 1.6 2.2 0.8 6.6 5.0
*
-o-~-o
1b3
A
+-0-0-A-u
- O - - y O - O-0-
10’ 10’ S o h b l e laminin ligand InM)
- A-
01
100
Fig. 1. Binding of laminin-nidogen complex and laminin fragments to immobilized heparan sulfate proteoglycan. The binding data (B) arc based on an antibody-linked detection system for the soluble ligands which were laminin-nidogen complex ( O ) ,fragment E3 (A),rragment LC8-9 ( A ) and fragments ElX, PI, E4, E8 and El0 (0, Zl), the latter being all of similar low activity. (A) shows the approximative localization of the fragments within the cross-shaped laminin [2, 241, identifying the A, B1 and B2 chains of laminin and the localization of the nidogen-binding site (Nd) within the B2 chain [39].
it showed strong binding to immobilized nidogen, some reduced binding to the laminin-nidogen complex and little or no binding to collagen IV, laminin fragment E3 and fibronectin (Fig. 2). Similar binding profiles, except €ora strongly reduced binding for laminin-nidogen complex, were observed by using the protein core as immobilized ligand. Some of the data were confirmed with a tryptic fragment of the proteoglycan consisting of the three heparan sulfate chains connected by a peptide segment that contained only about 3% of the protein core [S]. This fragment, as immobilized ligand, showed distinct binding for fragment E3 but none for nidogen (data not shown) .
362 L.U
I
I
Table 2. inhibitory capacities (ICs0) of various glycosaminoglycans and dextran sulfate for the binding of soluble ligands to immobilized heparan sulfate proteoglycan. The results for laminin and E3 were the average values of three experiments.
ICso for binding to
Inhibitor 1.0
lamininnidogen
laminin E3
fibronectin
0.05
2 100 38 90
0.5 0 lo2
lo1
100
10-1
Heparin Heparan sulfate Dextran sulfate Chondroitin sulfate
0.8 5
1.4
18
0.7
60
6.0
Protein core ligond InMl
Fig.2. Binding activity of proteoglycan protein core used as soluble ligand for various basement membrane components. The ligands used in immobilized form were recombinant nidogen ( O ) ,laminin-nidogen complex (B),fibronectin (m),collagen IV (A)and laminin fragment E3 ( A ) .
A
rod
100 -
2.0
E
0 .c
1.5
(v
0if
W-
g 1.0
20 -
rn
n L
0 1 Ln 3
a
0 10-1
100 10’ Heparin (pg/mll
102
Fig. 3. Sensitivity to heparin inhibition of proteoglycan binding to basement membrane components. The proteoglycan was either used in immobilized form (closed symbols) or as soluble ligand (open symbols). The second ligands were laminin fragment E3 (@, 0),lamininnidogen complex (A,A), fibronectin (H) and EHS tumor nidogcn
(V).
Further studies on the involvement of heparan sulfate chains were persormed by inhibition assays with heparin and other glycosaminoglycans. The binding of soluble fragment E3, laminin-nidogen and fibronectin to immobilized proteoglycan could be very efficiently and rather completely inhibited by heparin. No inhibitory effect was observed for nidogen (Fig. 3). The inhibitory capacities were different, being highest for fragment E3 and 10-20-fold lower for laminin-nidogen and fibronectin. The inhibition profiles changed, however, when using proteoglycan as soluble ligand. The fragment E3 interaction was still completely inhibited but with a 20-fold-lower efficiency. The laminin-nidogen interaction was only partially inhibited (35--40%) and required a more than 3 00-fold increase in heparin concentration to achieve this effect (Fig. 3). This suggests that proteoglycan binding to fragment E3 is completely dependent on heparan sulfate chains. Apparently, it is also responsible for the binding of laminin-nidogen to immobilized proteoglycan. Yet, soluble proteoglycan can bind to immobilized laminin-nidogen in two possible ways; either by heparan sulfate or its protein core.
0.5
0 102
10’
10
10-1
Proteaglycan (nM)
Fig. 4. Binding of proteoglycan to recombinant nidogen and nidogen fragments used as immobilized ligands. (A) Domain model of nidogen [20] and the approximative localization of the fragments. The binding curves (B) were obtained with nidogen ( O ) ,recombinant fragments Nd-I (@), Nd-Ia (W), rod (V)and SV3 1 ( A ) and fragments Th-100 (n)and Th-50 (A),prepared from the laminin-nidogen complex.
The latter possibility is supported by binding studies with the protein core (Fig. 2). A comparison of the inhibiting capacities of heparin and the heparan sulfate chains prepared from the proteoglycan showed a 5 - 30-Sold increase in the concentration causing half-maximal inhibition for heparan sulfate when competing for soluble laminin-nidogen or fragment E3 binding to proteoglycan. Dextran sulfate or chondroitin sulfate were either of equal or lower potency when compared to heparan sulfate (Table 2). Similar, but not identical, inhibition activities were also observed for the fibronectin interaction. Binding activity of nidogen fragments A panel of fragments, prepared from EHS tumor or recombinant nidogen [18-201, was used to map its binding site(s) for the proteoglycan (Fig. 4). These fragments together
363
1.5
1.2
E
E
w N.
N
4
%
0.8
1.0
w-
c m n L o v) n
m
n L v) 0
n
a
2
0.4
Q
\ \-
0.5
I02
Fig. 5. Binding of recombinant nidogen, fragment Nd-I and a nidogenPI complex to immobilized proteoglycan (open symbols) or laminin (closed symbols). The soluble ligands used were nidogen ( 0 , O ) , nidogen fragment Nd-I ( , n) and a preformed complex between recombinant nidogen and laminin fragment PI (A, A). Detection was, in all cases. with an antibody against nidogen, recognizingmainly its rod domain.
cover the three globular domains of nidogen, G I , G 2 and G3, and the rod-like domain in different associations, All the fragments that shared the central globular domain G 2 showed comparable activity with nidogen in binding to the soluble proteoglycan ligand. Fragments comprising the N-terminal globular domain G I or the rod-like segment were inactive (Fig. 4). Binding analyses with immobilized proteoglycan showed, however, different reactivities for nidogen and its fragments. While nidogen showed distinct binding, although with lower plateau levels (compare Figs 4 and 5, see also Table l), little or no activity was observed with fragment Nd-I (Fig. 5 ) that lacks the C-terminal G3 domain of nidogen [20]. Further tests with other nidogen fragments which were active as immobilized ligands (Fig. 4) also failed to show binding. This suggested that nidogen domain G3 or a segment joining G 3 to the rod-like segment are responsible for the binding of nidogen to immobilized proteoglycan. Since G 3 has been identified as thc only nidogen domain which binds laminin [18, 201 a further comparison was performed with immobilized laminin (Fig. 5). There was, as expected, a strong reaction of nidogen with laminin, exceeding that with proteoglycan, but no binding for fragment Nd-I. Furthermore, blocking of the laminin-binding site of nidogen by forming a stoichiomctric complex with laminin fragment P1 [20] inactivated the binding to both laminin and proteoglycan ligands (Fig. 5). The nidogen domain G3 was previously obtained as recombinant fragment Nd-I1 that was poorly soluble and therefore could not be used as a soluble ligand [20].We have now introduced a new purification procedure which allowed us to use Nd-I1 at least as an immobilized ligand (Fig. 6). It showed distinct binding of laminin with half-maximal binding with approximately 2 nM soluble ligand. Distinct binding was also observed with the soluble proteoglycan but at 15-fold-higher concentrations. This observation suggested that the small laminin contamination (1 - 2.5%) in the proteoglycan might have been responsible for binding. This possibility was, however: excluded by showing that only antibodies specific for
10’ 100 lo-’ Soluble Ligand (nM)
Fig. 6. Binding of soluble proteoglycan and laminin to immobilized recombinant domain G3 of nidogen (fragment Nd-11). The binding of proteoglycan (0)and laminin (0 j was detected by antibodies specific for each soluble ligand. Negative control experiments were performed by switching the detecting antibodies using anti-laminin in the proteoglycan ( 0j and anti-proteoglycan in the laminin (B) binding assay.
Table 3. Dependence on antibody specificity of the detection of binding nidogen fragment Th-100 to immobilized ligands. Results are expressed as plateau levels (antibody to rod) or maximal levels (antibody to G3) observed in an antibody-linked detection assay ( A at 492 nm) and the concentration of Th-100 to achieve half plateau levels of reaction (BC,,). Immobilized ligand Parameters for antiserum to G3 domain
rod domain
Pro teoglycan Laminin P1 Collagen IV
1.0 1.6 1.4
nM 25 14 14
0.3 0.3 1.4
nM >400 >400 45
proteoglycan, but not those specific for laminin, were bound to the proteoglycan-Nd-IT complex (Fig. 6). The suggestion that the G3 domain of nidogen binds both laminin and proteoglycan was examined in a further experiment, Antibodies to the G3 domain were used for detecting binding to the nidogen fragment Th-100 that consists of domains G2, G 3 and the rod (see Fig. 4) to immobilized proteoglycan. There was no significant binding. In contrast, there was binding of antibodies to the rod domain that represents a non-binding structure (Table 3). The same result was previously observed for nidogen binding to laminin fragment P1 and interpreted to indicate that the ligand binding to domain G 3 interferes with epitope recognition [20]. Much less interference was observed by comparing both antibodies in a binding assay for immobilized collagen IV (Table 3). The results supported evidence that nidogen domain G2 is involved in this binding ([20, 271; D. Reinhardt et al., unpublished results). Together, the data indicate that there are two binding sites of nidogen for the proteoglycan protein core, one in G2 and the other in G3. The latter binding is, however, distinctly weaker than G3-mediated binding to laminin and its fragment PI.
364 2.0
E
1.5
c N m
.\
s,
2
t
1.0
D
.a
L
0
a ln
a
0.5 0
1o2
10'
1OD
10-1
Proteoglycon ligond ( n M )
Fig. 7. Nidogen-mediated formation of ternary complexes between proteoglycan and laminin or collagen IV used in immobilized form. The imniobilized ligands were laminin fragment P1 (0, O ) , laminin (m, 7)and collagen IV ( A , A) which were used prior to (open symbols) or after (closed symbols) treatment with a fixed amount of recombinant nidogen (70 nM).
Evidence for the formation of ternary complexes Since nidogen mediates the binding of laminin to collagen IV [20], it was of interest to examine whether it has a similar bridging activity for the proteoglycan. The formation of a ternary complex was already shown by the binding of lamininnidogen complex to proteoglycan (Table 1, Fig. I), but the role of nidogen in this interaction was not obvious. We therefore resorted to the use of 2 M guanidine/HCl-treated laminin and laminin fragment P1 as immobilized ligands that both possess binding activity for nidogen [18] but not for the proteoglycan (Table 1, Fig. 1). Addition of recombinant nidogen to these ligands clearly enhanced their binding capacity for soluble proteoglycan up to the usual binding levels (Fig. 7). Similar binding profiles were observed with the complex between fragment PI and nidogen obtained at a preparative scale as immobilized ligand and soluble proteoglycan (data not shown). In addition, nidogen enhanced proteoglycan binding to immobilized collagen IV (Fig. 7), indicating the possibility of the formation of a second ternary complex. When we changed to protocols using immobilized proteoglycan, no nidogen-mediated binding of laminin and fragment P1 (not shown) or the direct binding of the P1-nidogen complex (Fig. 5 ) could be demonstrated. Parallel experiments replacing immobilized proteoglycan by collagen IV demonstrated, as shown before [20], the formation of ternary complexes. This observation indicates that proteoglycan, when used as a coat for plastic surfaces, has a decreased ability to provide a binding site for the formation of ternary complexes. The most likely explanation is that immobilized proteoglycan has a reduced affinity for nidogen domain G2 and that its persistent binding to domain G3 interferes with G3-mediated recognition of laminin.
DISCUSSION The data presented here confirm previous observations [15, 161 that the large heparan sulfate proteoglycan found in basement membranes [2, 31 binds two other basement membrane proteins, laminin and collagen IV. Novel findings were its binding to nidogen and its lack of binding to BM-40. It
was further demonstrated by inhibition studies with heparin or heparan sulfate and binding assays with the protein core, that the heparan sulfate chains provide the major binding sites for laminin, collagen IV and fibronectin. Nidogen binding, however, appears to be mediated entirely by the protein core. Our data on fibronectin are in conflict with other observations for a similar proteoglycan obtained from lung fibroblasts [S] where the protein core seems to be responsible for binding [30]. Yet, variations in the sulfation of heparan sulfate chains of this proteoglycan can modulate antithrombin binding activity [31] and there is evidence for alternative splicing of the protein core [I 31. Such structural differences not fully explored so far may therefore account for the differences in the results. Mapping of a major proteoglycan-binding site of laminin was achieved by a set of non-overlapping fragments which covered more than 95% of the protein structure (see Fig. 1). The results showed a high and exclusive binding activity for fragment E3 that corresponds to the two most C-terminal globular G domains of laminin A chain [14, 321. This binding is to the heparan sulfate chains, as shown by the sensitivity to glycosaminoglycan inhibition and binding of E3 to a small proteoglycan fragment possessing the heparan sulfate chains. The result is in agreement with the strong affinity of E3 for heparin affinity columns [21]. A functional role for this interaction could be modulation of laminin polymerization. Heparin increases this self-assembly process at low concentrations but interferes with it at high concentrations [33, 341. We can, however, not exclude the possibility that laminin may possess more binding sites for the proteoglycan that may have been lost during proteolysis. In addition, potential binding sites could have been destroyed in the protein corc by its exposure to strong denaturing solvents [5, 61. Another binding site of the protein core responsible for proteoglycan oligomerization 1351 apparently resists such treatment. Another laminin fragment, EX, shows distinct binding to heparin affinity columns but no binding for the proteoglycan. This could correlate with the low content of 0-sulfation in the heparan sulfate chains of the EHS tumor [31, 361 compared to heparin. For example, antithrombin binds with high affinity to heparin but only weakly to EHS tumor heparan sulfate [31]. Heparan sulfates obtained from other basement membranes differ in this respect [31, 371 and such proteoglycans could have a more versatile interaction repertoire for laminin. The results support our previous observations [18, 211 that the laminin B1 chain fragments E4 and E l 0 do not bind to heparin or as shown here to the proteoglycan. These structures were implicated in one [34], but not another study [33], to be heparin-binding structures based on the electron microscopical observation of complexes after rotary shadowing. This makes the subsequent assignment of binding sites, using randomly selected synthetic peptides corresponding to laminin fragment E4 and El0 sequences [34, 381 a questionable approach. In order to understand their biological significance, the peptides should at least have been shown to bind to basement-membrane forms of heparan sulfate proteoglycan. A major binding site for the protein core was assigned to the globular domain G2 of nidogen by assays with overlapping fragments. The same nidogen domain or an adjacent structure apparently contains a major binding site for the triple helix of collagen IV [20, 271. Based on the lack of any structural similarity between the collagen-IV triple helix and the proteoglycan sequence [12, 131, it is unlikely that both binding sites on domain G2 are identical. This would also explain the enhancing effect of nidogen for proteoglycan association to collagen IV (Fig. 7) and suggests that direct collagen binding,
365 mediated by heparan sulfdte, could be increased by a second alternative reaction mediated by two non-interfering bindillg sites on the G2 d o m a i n of nidogen. A second binding site on nidogen f o r the proteoglycan protein core was assigned t o its globular d o m a i n G3, but this w a s only possibly d u e t o the fact t h a t the binding activity associated with G2 is strongly reduced in immobilized proteoglycan (Fig. 5). Nidogen domain G 3 has been previously identified a s possessing t h e only binding site for laminin [18, 201 which interacts with a few EGF-like motifs present in t h e short-arm structure of laminin B2 chain [39]. This is of interest since several similar EGF-like repeats a r e f o u n d i n t h e proteoglycan sequence [I 1 - 131. AS discussed belou7, s o m e indications suggest t h a t the binding of nidogen G3 d o m a i n to either laminin or proteoglycan is mutually exclusive. By the use of recombinant nidogen, we were, for the first time, able to show t h a t this protein mediates binding between laminin and collagen TV [20, 271. This suggests a similar function in situ and a key role f o r nidogen in connecting thc networks of collagen TV and laminin in basement membranes [l - 3, 281. Here we show that the addition of heparan sulfate proteoglycan t o these supramolecular assemblies m a y n o t only occur by electrostatic interactions via the h e p a r a n sulfate chains, b u t also involve protein-protein interactions mediated by nidogen. This was clearly demonstrated by using immobilized laminin ligands, nidogen as mediator and t h e proteoglycan as third ligand (Fig. 7). It implicates, i n this constellation, recognition of laminin by the G 3 d o m a i n and of proteoglycan by t h e G2 domain of nidogen. Yet, no ternary complexes were formed using an immobilized proteoglycan ligand a n d examining nidogen-mediated laminin binding or t h e direct binding of preformed laminin P1-nidogen complexes. The immobilized proteoglycan still has, however, a distinct activity f o r binding soluble nidogen. We explain these apparently contradictory d a t a by assuming t h a t the immobilized proteoglycan has lost most of its activity for binding t o the nidogen d o m a i n G2, which is replaced by binding via its G3 domain. This binding, however, does not allow any more interaction with laminin. While the loss of binding activity of immobilized ligands is known in other cases, i.e. for BM-40 [23], our interpretation for the proteoglycan is nevertheless speculative and will need independ e n t confirmation. If correct, it would indicate that alternative choices of nidogen for binding sites m a y exist i n basement membranes a n d contribute t o its variation in architecture [40]. D u e t o the ambiguity of some o f the ternary binding d a t a , we like to emphasize again t h a t the proteoglycan used here was exposed t o strongly denaturing agents for solubilization a n d purification in intact f o r m [ 5 , 6 ] .Circular-dichroism spectroscopy, i n fact, indicates partial denaturation of the protein core by these procedures [5]. To overcome such inherent difficulties i n purifying extracellular matrix proteins, their preparation by recombinant methods i n mammalian cells is an appropriate alternative choice. As shown with recombinant nidogen [20], it may reveal new insights into d o m a i n structure and binding activities. This approach is now feasible for the protein core based on its complete cDNA cloning [12, 131.
Wc thank Mrs Vera van Delden for expert tcchnical assistance, Dr. Karlheinz Mann for amino acid sequencing and Dr. Roswitha Nischt and Mr. Dicter Reinhardt for help with some nidogen fragments. The study wa5 supported by a grant from the Deutache I;hrJchun~spemeinschaft.The fellowshio sunoorl to CR bv the Mario Negri Institute and the Max-Planck-Gescllschaft is appreciated. I
.I
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