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Originally published In Press as doi:10.1074/jbc.M403031200 on June 28, 2004

J. Biol. Chem., Vol. 279, Issue 36, 37368-37376, September 3, 2004
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Chondroitin Sulfate Chains on Syndecan-1 and Syndecan-4 from Normal Murine Mammary Gland Epithelial Cells Are Structurally and Functionally Distinct and Cooperate with Heparan Sulfate Chains to Bind Growth Factors

A NOVEL FUNCTION TO CONTROL BINDING OF MIDKINE, PLEIOTROPHIN, AND BASIC FIBROBLAST GROWTH FACTOR*

Sarama Sathyaseelan Deepa{ddagger}, Shuhei Yamada{ddagger}, Masahiro Zako§, Olga Goldberger¶, and Kazuyuki Sugahara{ddagger}||

From the {ddagger}Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658-8558, Japan, the §Department of Ophthalmology, Aichi Medical University, Nagakute-Cho, Aichi 480-1195, Japan, and the Department of Medicine, Division of Newborn Medicine, Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115

Received for publication, March 18, 2004 , and in revised form, June 21, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A comparative analysis was carried out of heparan sulfate (HS) and chondroitin sulfate (CS) chains of the ectodomains of hybrid type transmembrane proteoglycans, syndecan-1 and -4, synthesized simultaneously by normal murine mammary gland epithelial cells. Although the HS chains were structurally indistinguishable, intriguingly the CS chains were structurally and functionally distinct, probably reflecting the differential regulation of sulfotransferases involved in the synthesis of HS and CS. The CS chains of the two syndecans comprised nonsulfated, 4-O-, 6-O-, and 4,6-O-disulfated N-acetylgalactosamine-containing disaccharide units and were significantly different, with a higher degree of sulfation for syndecan-4. Functional analysis using a BIAcore system showed that basic fibroblast growth factor (bFGF) specifically bound only to the HS chains of both syndecans, whereas midkine (MK) and pleiotrophin (PTN) bound not only to the HS but also to the CS chains. Stronger binding of MK and PTN to the CS chains of syndecan-4 than those of syndecan-1 was revealed, supporting the structural and functional differences. Intriguingly, removal of the CS chains decreased the association and dissociation rate constants of MK, PTN, and bFGF for both syndecans, suggesting the simultaneous binding of these growth factors to both types of chains, producing a ternary complex that transfers the growth factors to the corresponding cell surface receptors more efficiently compared with the HS chains alone. The involvement of the core protein was also shown in the binding of MK and PTN to syndecan-1, suggesting the possibility of cooperation with the HS and/or CS chains in the binding of these growth factors and their delivery to the cell surface receptors.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Proteoglycans (PG)1 bear glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS). The repeating disaccharide unit in the HS and CS backbones is GlcUA/IdceA-GlcNAc and GlcUA-GalNAc, respectively, onto which are superimposed specific modification patterns, most notably the addition of sulfate groups by a variety of sulfotransferases (1). PGs are distributed ubiquitously in extracellular matrices and at cell surfaces (2). Syndecans are the major cell surface PGs expressed by virtually all epithelial cells. Four kinds of syndecans form a gene family, the transmembrane and cytoplasmic domain being conserved among all of the members (3–7). These syndecans are expressed with different cell-, tissue-, and developmental stage-specific patterns (8, 9), suggesting distinct functions for each family member (10), although some shared activities have been observed, for example, for syndecan-1 and -4 (3, 5, 11, 12).

The majority of GAG chains added to the core proteins of syndecans are of the HS type, although syndecan-1 (13) and syndecan-4 (14) are modified by CS chains as well. The HS chains bind collagen types I, III, and V (15), fibronectin (16), tenascin (17), thrombospondin (18) and basic fibroblast growth factor (bFGF) (19, 20), and other components of the cellular microenvironment, implying roles for syndecan-1 in cell matrix adhesion and cell growth (4). Syndecan-1 core protein contains five potential GAG attachment sites with Ser-Gly motifs, three for HS and two for CS attachment (21, 22). Several syndecan-4 cDNAs have been characterized for rat (ryudocan) (23), human (ryudocan and amphiglycan) (24, 25), and chicken (26). Although rat endothelial cell ryudocan possesses three functional GAG attachment sites capable of bearing either CS or HS chains, chicken syndecan-4 core protein has three potential sites for GAG attachment, modified with HS side chains, and ryudocan isolated from human endothelium-like EAhy926 cells has only HS chains (24).

Syndecan-1 and -4 exhibit similar as well as dissimilar biological activities; both promote intracellular adhesion following their introduction into human B lymphoid cells (27) and are expressed during wound repair (28). Proteolytic cleavage results in the shedding of their ectodomains in acute human dermal wound fluids (29), where they modify the protease/antiprotease balance (30). Unlike syndecan-1, however, syndecan-4 has a distinctive role in the generation and maintenance of focal adhesion complexes (31, 32) and in signal transmission during the formation of dendritic processes (33).

Recently, Zako et al. (34) demonstrated that syndecan-1 and -4 synthesized simultaneously by normal mouse mammary gland epithelial cells bore HS chains that were structurally indistinguishable, even though they exhibited spatially different distributions. The expression of both CS and HS chains on the same PG is intriguing but has not been investigated rigorously so far. In this study, we addressed whether the CS chains present in syndecan-1 and -4 are structurally similar, like their HS counterparts, and what functions the CS chains of these hybrid PGs play.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Chondroitinase ABC (EC 4.2.2.4 [EC] ) from Proteus vulgaris, chondroitinase AC-II (EC 4.2.2.5 [EC] ) from Arthrobacter aurescens, heparinase (EC 4.2.2.7 [EC] ) and heparitinase (EC 4.2.2.8 [EC] ) from Flavobacterium heparinum and unsaturated disaccharides derived from CS and HS were purchased from Seikagaku Corp. (Tokyo, Japan). 2-Aminobenzamide was purchased from Nacalai Tesque (Kyoto, Japan). Recombinant human (rh)-midkine (MK) expressed in Escherichia coli was purchased from Peprotech EC LTD (London, UK). rh-Pleiotrophin (PTN) expressed in E. coli was from RELIA TechGmbH (Braunschweig, Germany) and rh-FGF2 (bFGF) expressed in E. coli was from Genzyme Techne (Minneapolis). Cell surface syndecan-1 and -4 ectodomains were prepared from NMuMG cells, as described previously (34), and the same preparations of syndecan-1 and -4 ectodomains as those used in the previous study for a structural analysis of HS were used for the present study. A glutathione S-transferase (GST)-fusion protein of the syndecan-1 ectodomain expressed in a bacterial system and the GST protein were kindly provided by Dr. Alan Rapraeger (University of Wisconsin-Madison). All other chemicals and reagents were of the highest quality available.

Disaccharide Composition Analysis of HS and CS Chains from Syndecan-4 Ectodomain—Enzymatic treatments for analysis of the disaccharide composition of GAG chains were carried out as follows. For HS, the purified syndecan-4 ectodomains (7 ng of core protein) were treated with a mixture of heparinase and heparitinase (0.5 mIU each) in a total volume of 20 µl of 20 mM acetate-NaOH buffer, pH 7.0, containing 10 mM Ca(OAc)2 at 37 °C for 1 h (35). For CS, the purified syndecan-4 ectodomains (48 ng of core protein) were treated with chondroitinase ABC (5 mIU) in a total volume of 20 µl of 0.05 M Tris-HCl buffer, pH 8.0, containing 0.06 M sodium acetate at 37 °C for 30 min (36). The disaccharides released by the above digestions were labeled fluorescently with 2-aminobenzamide at their reducing termini and analyzed by HPLC on an amine-bound silica column as described previously (37).

Biotinylation of Syndecan-1 and -4 Ectodomains—Biotinylation of the primary amines in the core protein of syndecan-1 (3 µg) and syndecan-4 (0.6 µg as core protein) was carried out using EZ-link sulfo-NHS-LC-biotin, as described previously (38). For the preparation of biotinylated syndecan-1 or -4 ectodomains with HS chains (HS/syndecan-1 or -4), one-third of the biotinylated syndecan-1 or -4 ecodomains was treated with a protease-free preparation of CSase ABC (2 mIU) as described above. For the preparation of biotinylated syndecan-1 or -4 ectodomains with CS chains (CS/syndecan-1 or -4), one-third of the biotinylated syndecan-1 or -4 ecodomains was treated with a mixture of heparinase and heparitinase (1 mIU each) as above. Biotinylated syndecan-1 core protein was generated by digesting intact syndecan-1 with a mixture of heparinase and heparitinase followed by digestion with a protease-free preparation of chondroitinase ABC.

Growth Factor Binding Assays Using a BIAcore System—Real time analysis of the interactions of growth factors and biotinylated syndecan-1/-4 ectodomains was performed with a BIAcore J biosensor (BIAcore AB, Uppsala, Sweden). Streptavidin-coated sensor chip SA was used to immobilize the biotinylated samples. Comparable amounts of sample were used for immobilization. The injection of biotinylated samples onto the sensor's surface was controlled to obtain a response of 250–540 resonance units (RU), corresponding to 0.3–0.6 ng of the immobilized protein. The conditions for kinetic analysis were the same as those reported previously (39), except that the association took 180 s and dissociation 120 s, the flow rate being 30 µl/min. The kinetic parameters, viz. the association rate constant (ka), the dissociation rate constant (kd), and the equilibrium dissociation constant (Kd), were calculated with BIAevaluation 3.1 software (BIAcore) using a 1:1 binding model with mass transfer. For most of the analyses, the Kd values were calculated with the "fit kinetics simultaneous ka/kd program," where the ka and kd values were determined simultaneously for the calculation of the Kd value. When this program failed to give a fit because of the unusual pattern of the sensorgram, the Kd values were calculated with the "fit kinetics separate ka/kd program" with 1:1 Langmuir association/dissociation, where the ka and kd values were determined separately for the calculation of Kd.

Inhibition of Binding of MK and PTN to Syndecan-1-immobilized Sensor Chip with Intact Syndecan-1, HS/Syndecan-1, CS/Syndecan-1, Syndecan-1 Core Protein, or GST-Syndecan-1 Ectodomain—HS/syndecan-1, CS/syndecan-1, and syndecan core protein were generated from intact syndecan-1 as described above, except that the preparations were exchanged with phosphate-buffered saline using a filter having a molecular mass cutoff of 10,000 Da (Microcon YM-10, Millipore Corporation, Bedford, MA). For inhibition studies, MK or PTN was mixed with intact syndecan-1, HS/syndecan-1, CS/syndecan-1, syndecan-1 core protein, or bacterially expressed recombinant GST-syndecan-1 ectodomain and then applied to the syndecan-1-immobilized sensor chip, and the interaction was studied using a BIAcore as described previously.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Analysis of the Disaccharide Compositions of HS and CS Chains from the Syndecan-4 Ectodomain—The purified syndecan-4 ectodomain was digested exhaustively with a mixture of heparinase and heparitinase or chondroitinase ABC, separately, and the released disaccharides were labeled fluorescently with 2-aminobenzamide and analyzed by HPLC on an amine-bound silica column. The identity of each peak was confirmed by comparing elution positions with standard 2-aminobenzamide disaccharides. The results are summarized in Table I. The HS chains gave rise to {Delta}DiHS-0S ({Delta}HexUA{alpha}1–4GlcNAc) and {Delta}DiHS-NS ({Delta}HexUA{alpha}1–4GlcN(2-N-sulfate)) as major disaccharides, accounting for 49 and 24%, respectively, of all disaccharide units, where {Delta}HexUA represents 4-deoxy-L-threohex-4-enepyranosyluronic acid. {Delta}DiHS-diS2 ({Delta}HexUA(2-O-sulfate){alpha}1–4GlcN(2-N-sulfate)) was the major disulfated disaccharide unit, accounting for 11% of the total, whereas {Delta}DiHS-6S ({Delta}HexUA{alpha}1–4GlcNAc(6-O-sulfate)), {Delta}DiHS-diS1 ({Delta}HexUA{alpha}1–4GlcN(2-N, 6-O-disulfate)), and {Delta}DiHS-triS ({Delta}HexUA(2-O-sulfate){alpha}1–4GlcN(2-N, 6-O-disulfate)) were minor disaccharide units. The HS disaccharide composition of syndecan-1 ectodomains from NMuMG cells, obtained from our previous study (40), is also listed in Table I for comparison, and the results indicate that the HS disaccharide compositions of the syndecan-1 and -4 ectodomains from NMuMG cells are very similar, if not identical. These results are consistent with those obtained by Zako et al. (34), who reported that the HS chains of syndecan-1 and syndecan-4 derived from NMuMG cells are structurally indistinguishable, even though {Delta}DiHS-0S was not detected in their study because they used 35S-radiolabeled HS chains from syndecan-1 and -4 ectodomains. It should be noted, however, that our study showed that as much as 58 and 49% of the HS disaccharide units were not sulfated in syndecan-1 and -4 ectodomains of NMuMG cells, revealing undersulfation of the HS chains consistent with the degree of sulfation represented by the sulfate: disaccharide molar ratio of 0.58 and 0.77, respectively.


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  		TABLE I
Disaccharide composition of HS-GAG chains from syndecan-1 and syndecan-4

Syndecan-4 ectodomains were digested exhaustively with a mixture of heparinase and heparitinase, and the liberated unsaturated disaccharides were identified and quantified by HPLC on a silica PA-03 column.

 
Chondroitinase ABC digestion of the purified syndecan-4 ectodomain yielded {Delta}Di-0S ({Delta}HexUA{alpha}1–3GalNAc), {Delta}Di-6S ({Delta}HexUA{alpha}1–3GalNAc(6-O-sulfate)), {Delta}Di-4S ({Delta}HexUA{alpha}1–3GalNAc(4-O-sulfate)), and {Delta}Di-diSE ({Delta}HexUA{alpha}1–3Gal-NAc(4,6-O-disulfate)) in a molar ratio of 6:17:68:9 (Table II). The disaccharide composition of the CS chains from syndecan-1 (40) is also listed in Table II for comparison, and {Delta}Di-0S, {Delta}Di-6S, {Delta}Di-4S, and {Delta}Di-diSE were obtained at a molar ratio of 29:5:59:7 upon digestion with chondroitinase ABC. The disaccharide analysis of the CS chains from syndecan-4 and -1 demonstrates a clear difference in composition. A notable difference was observed in the proportions of {Delta}Di-0S and {Delta}Di-6S; {Delta}Di-0S content was 5-fold lower, whereas {Delta}Di-6S content was 3-fold higher, in syndecan-4 compared with syndecan-1. No obvious difference was observed in the proportion of the major component {Delta}Di-4S or oversulfated {Delta}Di-diSE between the two syndecans. The degree of sulfation (1.03) exhibited by the CS chains of syndecan-4 was higher than that (0.78) of the CS chains of syndecan-1 (Table II). Hence, it was concluded that the CS chains on syndecan-1 and -4 from NMuMG cells are structurally different, unlike the HS chains. From 21 pmol of core protein of syndecan-4, ~5 nmol of HS and 0.5 nmol of CS disaccharides were obtained, giving an HS:CS disccharide ratio of 91:9.


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  		TABLE II
Disaccharide composition of CS-GAG chains from syndecan-1 and syndecan-4

Syndecan-4 ectodomains were digested exhaustively with chondroitinase ABC, and the liberated unsaturated disaccharides were identified and quantified by HPLC on a silica PA-03 column.

 
Syndecan-1 and -4 Ectodomains Bound MK, PTN, and bFGF with Comparable Affinities—It was then investigated whether the difference in the structure of the CS chains has any influence on the function of the syndecan-1 and -4 ectodomains (denoted as intact syndecan-1 and -4, respectively). We examined the molecular interaction of syndecan-1 and -4 with three different growth factors, viz. MK and PTN from the MK/PTN family and bFGF from the FGF family. Our previous experiments using MK, PTN, and bFGF with squid cartilage CS-E and heparin indicated that MK and PTN showed comparable or higher affinity for CS-E than for heparin, whereas bFGF had higher affinity for heparin than CS-E (41). Hence, syndecans with both CS and HS chains would be ideal for studying interactions.

The kinetic parameters for the binding of MK, PTN, and bFGF to the intact syndecan-1 and -4 were analyzed using a surface plasmon resonance biosensor. Biotinylated syndecan-1 and -4 were immobilized separately on streptavidin-coated sensor chips, as described under "Experimental Procedures," varying concentrations of individual growth factors were injected onto the sensor's surface, and the interactions were analyzed. Fig. 1, A, B, and C, represents an overlay of the sensorgrams for the binding of MK, PTN, and bFGF to an intact syndecan-1 immobilized chip; and Fig. 2, A, B, and C, represents their binding to an intact syndecan-4-immobilized chip.



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  		FIG. 1.
Sensorgrams for the binding of MK, PTN, and bFGF to syndecan-1. Various concentrations of MK (A, D, and G), PTN (B, E, and H) and bFGF (C, F, and I) were injected over sensor chips immobilized with the syndecan-1 ectodomain (A–C), syndecan-1 ectodomain with HS chains (D–F), and syndecan-1 ectodomain with CS chains (G–I). The long arrows indicate the beginning of the association phase initiated by the injection of varying concentrations of the growth factors, and short arrows indicate the beginning of the dissociation phase initiated with the running buffer.

 



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  		FIG. 2.
Sensorgrams for the binding of MK, PTN, and bFGF to syndecan-4. Various concentrations of MK (A, D, and G), PTN (B, E, and H), and bFGF (C, F, and I) were injected over sensor chips immobilized with the syndecan-4 ectodomain (A–C), syndecan-4 ectodomain with HS chains (D–F), and syndecan-4 ectodomain with CS chains (G–I). The long arrows indicate the beginning of the association phase initiated by the injection of varying concentrations of the growth factors, and short arrows indicate the beginning of the dissociation phase initiated with the running buffer.

 
The kinetic parameters ka, kd, and Kd for the binding of MK, PTN, and bFGF to syndecan-1 and -4 are presented in Tables III and IV. All three growth factors displayed Kd values in the low nM range, suggesting high affinity and a physiological significance of the interactions. The Kd values observed for the interactions of these growth factors were as follows: 29 and 20 nM (MK); 27 and 16 nM (PTN), and 3.4 and 5.0 nM, respectively (bFGF). Thus, MK, PTN, and bFGF bound to intact syndecan-1 and -4 with comparable affinities. For intact syndecan-1, bFGF exhibited the strongest affinity among the growth factors tested, as indicated by a ~9-fold lower Kd value than that obtained for the binding with MK or PTN. Likewise, bFGF exhibited the strongest affinity for intact syndecan-4 among the growth factors; the binding affinity of MK or PTN for intact syndecan-4 was ~4- and 3-fold lower than that exhibited by bFGF. Thus, the three growth factors exhibited significantly different binding affinities for syndecan-1 and -4, most likely reflecting structural differences in the GAG chains. Because, however, syndecan-1 and -4 possess both HS and CS chains, it was not clear at this point whether the difference in binding to syndecan-1 and -4 was the result of HS only or a combination of HS and CS.


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  		TABLE III
Kinetic parameters for the interaction of MK, PTN, and bFGF with immobilized syndecan-1

The biotinylated form of the intact syndecan-1 ectodomain was digested either with a protease-free preparation of chondroitinase ABC to generate a syndecan-1 ectodomain with HS chains (HS/syndecan-1) or with a mixture of heparinase and heparitinase to generate a syndecan-1 ectodomain with CS chains (CS/syndecan-1). The intact and digested ectodomains were immobilized individually on streptavidin-coated sensor chips. The ka, kd, and Kd values for the interaction of MK, PTN, and bFGF with intact syndecan-1, HS/syndecan-1 and CS/syndecan-1 were determined using the fit kinetics simultaneous ka/kd program. The values are expressed as the mean ± S.D.

 


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  		TABLE IV
Kinetic parameters for the interaction of MK, PTN, and bFGF with immobilized syndecan-4

Intact syndecan-4, HS/syndecan-4, and CS/syndecan-4-immobilized sensor chips were prepared as described in the legend to Table I. The ka, kd, and Kd values for the interaction of MK, PTN, and bFGF with intact syndecan-4, HS/syndecan-4, and CS/syndecan-4 were determined using the fit kinetics simultaneous ka/kd program. The values are expressed as the mean ± S.D.

 
Removal of CS Chains from Syndecan-1 and -4 Ectodomains Alters the Kinetics of Binding to MK, PTN, and bFGF—The syndecan-1 and -4 ectodomains were digested individually with a protease-free preparation of chondroitinase ABC to generate ectodomains devoid of CS chains. Each ectodomain thus generated was immobilized on a streptavidin-coated sensor chip and its interactions with MK, PTN, and bFGF analyzed as described above. Fig. 1, D, E, and F, represents the binding of MK, PTN, and bFGF to the syndecan-1 ectodomain with HS chains (HS/syndecan-1) and Fig. 2, D, E, and F, that to the syndecan-4 ectodomain with HS chains (HS/syndecan-4), respectively. As evident from the sensorgrams, removal of the CS chains affected the responses to the binding of MK and PTN to syndecan-1 (Fig. 1, D and E), but not the responses of the binding of bFGF to syndecan-1 (Fig. 1F). For example, the binding of MK at a concentration of 224 nM to HS/syndecan-1 showed a decreased response from 152 to 97 RU, and the response to the binding of PTN at 130 nM to HS/syndecan-1 decreased from 152 to 97 RU compared with the binding to intact syndecan-1. In contrast, the responses for the binding of bFGF at 14 nM to intact syndecan-1 and HS/syndecan-1 were virtually the same.

No significant difference was observed in the affinity, in terms of the Kd value, of the binding of MK and bFGF to intact syndecan-1 and HS/syndecan-1, but the Kd value for the binding of PTN to HS/syndecan-1 was 2-fold lower than that for the binding to syndecan-1 (Table III). However, it became evident that the removal of CS chains from intact syndecan-1 altered the ka and kd values for the binding of MK, PTN, and bFGF to HS/syndecan-1 (Table III). The ka for the binding of MK, PTN, and bFGF to HS/syndecan-1 was 1.5-, 1.9-, and 1.9-fold lower than that for the binding to intact syndecan-1. The kd values for the dissociation of MK, PTN, and bFGF from HS/syndecan-1 were also 1.7-, 5.1-, and 2.4-fold lower than those for the dissociation from intact syndecan-1. Thus, the removal of CS chains resulted in slower binding and a slower dissociation of the growth factors from intact syndecan-1. These results indicate that the CS chains are involved in both the association of these growth factors with and their dissociation from intact syndecan-1 and that the presence of CS chains in intact syndecan-1 results in a faster binding as well as a faster release of the growth factors, which would facilitate their transfer to the corresponding receptors.

In marked contrast to the results obtained with intact syndecan-1 and HS/syndecan-1, no significant difference in response was observed for the binding of MK, PTN, and bFGF to intact syndecan-4 and to HS/syndecan-4 (Fig. 2, A and D; B and E; C and F). In addition, the Kd value for the binding of bFGF to HS/syndecan-4 was comparable with that for the binding of bFGF to intact syndecan-4, although a significant increase (3-fold) in the Kd was observed for the binding of MK to HS/syndecan-4 compared with intact syndecan-4, whereas a significant decrease (4-fold) was observed for the binding of PTN to HS/syndecan-4 compared with intact syndecan-4 (Table IV). However, alterations to the kinetics of the interactions of these growth factors with syndecan-4 by removal of the CS chains became evident when ka and kd values were compared before and after removal of the CS chains (Table IV). The association constants (ka values) for the binding of MK and bFGF to HS/syndecan-4 were 4.3- and 3.7-fold lower than those for the binding to intact syndecan-4. In contrast, no significant change in the ka value was observed for the binding of PTN to HS/syndecan-4 compared with the binding of PTN to intact syndecan-4. Removal of the CS chains from intact syndecan-4 lowered the kd value by 1.3-, 3.7-, and 2.5-fold for the dissociation of MK, PTN, and bFGF from HS/syndecan-4, respectively, compared with their dissociation from intact syndecan-4. These results altogether suggest that removal of the CS chains altered the kinetics of the syndecan-4 interactions with these growth factors, resulting in a lowering of the association rates of MK and bFGF and the dissociation rates of all three growth factors and in turn suggest that the CS chains of syndecan-4 are likely involved in the efficient binding and transfer to a high affinity receptor of these growth factors as in the case of the CS chains of syndecan-1.

Syndecan-1 Ectodomain with CS Chain(s) Is Capable of Binding MK and PTN but Not bFGF—Intact syndecan-1 and -4 were digested individually with a mixture of heparinase and heparitinase to generate ectodomains devoid of HS chains. Each ectodomain thus prepared was immobilized on a streptavidin-coated sensor chip, and its interactions with MK, PTN, and bFGF were evaluated as described above. bFGF completely failed to interact with the syndecan-1 ectodomain with CS chains (CS/syndecan-1), confirming the removal of HS chains (Fig. 1I). Interestingly, MK and PTN were still capable of interacting with CS/syndecan-1 (Fig. 1, G and H). For example, the responses for the binding of MK at 224 nM and PTN at 130 nM to CS/syndecan-1 were 54 and 40 RU, respectively, which were, however, significantly lower than the responses for the binding of MK and PTN at the same concentrations to intact syndecan-1 (152 and 170 RU, respectively).

The kinetic parameters ka, kd, and Kd for the interaction of MK and PTN with CS/syndecan-1 are presented in Table III. Because of the unusual pattern of the sensorgrams generated by these interactions (Fig. 1, G and H), especially during the dissociation phase, the Kd values were calculated using the fit kinetics separate ka/kd program (see "Experimental Procedures"). The ka for the binding of MK and PTN to CS/syndecan-1 was 7.9- and 218-fold lower, respectively, than that for the binding to intact syndecan-1. In contrast, the kd values did not change markedly, only a 1.6-fold increase and 1.3-fold decrease, for the dissociation of MK and PTN from CS/syndecan-1, compared with the dissociation from intact syndecan-1. The calculated Kd value for the interaction of MK and PTN with CS/syndecan-1 was as high as 0.4 and 5.2 µM, respectively, indicating a lower affinity for the CS chain of syndecan-1 than for intact syndecan-1. These results indicate that the CS chains of synedcan-1 interact with MK and PTN with low affinity in the absence of HS chains.

Syndecan-4 Ectodomain with CS Chains Is Capable of Binding MK and PTN but Not bFGF—The sensorgrams for the binding of MK, PTN, and bFGF to CS/syndecan-4 are shown in Fig. 2, G, H, and I. Although bFGF failed to bind CS/syndecan-4, confirming the removal of HS chains, MK and PTN showed significant binding. The binding of MK at 149 nM and that of PTN at 130 nM to CS/synedcan-4 generated a response of 101 and 180 RU, respectively, which was lower than the responses (160 and 240 RU) for the binding to syndecan-4 of MK and PTN, respectively, at the same concentrations. The ka, kd, and Kd for the binding of MK and PTN to CS/syndecan-4 are presented in Table IV. The ka values for the binding of MK and PTN to CS/syndecan-4 were decreased 5- and 21.7-fold compared with the binding to intact syndecan-1, whereas the kd values showed a 2.4-fold increase and 3.9-fold decrease for the dissociation of MK and PTN from CS/syndecan-4 compared with the dissociation from intact syndecan-4. The Kd value for the binding of MK to CS/syndecan-4 was 191 nM, which was 9-fold higher than that for intact syndecan-4, indicating a lower affinity of CS/syndecan-4 for MK. The Kd value for the interaction of PTN with CS/syndecan-4 was 81.3 nM, which was 5-fold higher than that for intact syndecan-4. These results appear to indicate that the CS chains of syndecan-4 can interact directly with MK and PTN with moderate affinity, which is consistent with the notion that removal of the CS chains from syndecan-4 altered the kinetics of the binding to HS/syndecan-4 compared with the kinetics of the binding to intact syndecan-4 (see above).

No direct binding of bFGF to CS/syndecan-4 was observed after removal of the HS chains. In addition, alterations of the kinetics of binding to HS/syndecan-4 were noted compared with the binding to intact syndecan-4. These findings suggest that although bFGF cannot interact with the CS chains in the absence of the HS chains, it interacts with the CS chains in the presence of HS chains. Thus, although bFGF can interact with HS chains alone, it binds to syndecan-4 more efficiently when it binds both HS and CS chains simultaneously, resulting in a faster association and faster dissociation. Hence, the HS chains, which are located away from where the growth factors bind, may share the growth factors with a CS chain that is in close proximity to the cell surface receptor (3), forming a ternary complex or quaternary complex involving the core protein and then transfer the growth factors to the corresponding receptors. These results prompted us to speculate that in intact syndecan-1 and -4, the HS and CS chains cooperate with each other in some way for the binding of MK, PTN, and bFGF. Because the core protein of syndecan-1 may cooperate with the CS chains and/or HS chains for the binding of MK and PTN, possible interactions of MK, PTN, and bFGF with syndecan-1 core protein were also examined.

The Core Protein of Syndecan-1 Cooperates with the CS Chains to Bind MK and PTN—Biotinylated syndecan-1 devoid of both HS and CS chains was immobilized on a streptavidin-coated sensor chip, and the interactions with MK, PTN, and bFGF were investigated as described previously. bFGF did not bind to the core protein of syndecan-1 (data not shown), consistent with the absence of binding of bFGF to CS/syndecan-1 (Fig. 1I). The response generated by the binding of MK at 224 nM to the core protein of syndecan-1 was 20 RU, compared with 54 RU generated by the binding to CS/syndecan-1 at the same MK concentration (data not shown). The binding of PTN at 195 nM to the core protein of syndecan-1 gave a response of 60 RU compared with 80 RU generated by the binding to CS/syndecan-1 at the same PTN concentration. Because the protocol used for the biotinylation of core protein was the same as that for the biotinylation of functional IgG, as provided by the manufacturer, no structural alterations caused by biotinylation are expected. These results suggest that the core protein of syndecan-1 may also be involved in the binding of MK and PTN, cooperating with the CS chains or HS chains or both. The possibility cannot be excluded that MK or PTN forms a quaternary complex with the HS chain, CS chain, and the core protein of syndecan-1, either during the binding of the growth factors or during the transfer of the growth factors from one HS chain to another or from HS and CS chains to cell surface receptors. Because the amount of syndecan-4 was limited, no such experiment for syndecan-4 core protein could be performed.

The difference in the binding affinity of CS chains derived from syndecan-1 and -4 for MK and PTN probably reflects the fundamental difference in the fine structure of the CS chains. In this context, it should be remembered that the CS chains derived from syndecan-1 have a higher proportion of nonsulfated GalNAc residues than the CS chains from syndecan-4 (Table II). For the binding of MK and PTN to the CS chains of syndecan-4, the sequential arrangement of the oversulfated E-unit (GlcUA{beta}1–3GalNAc(4S,6S)) in combination with the C-unit (GlcUA{beta}1–3GalNAc(6S)) or A-unit (GlcUA{beta}1–3GalNAc(4S)) is likely a more important determinant than the amount of nonsulfated GalNAc residues (41). This structural and functional difference in the CS chains derived from syndecan-1 and -4 may suggest that they perform different biological functions in the cell.

HS/syndecan-1, CS/syndecan-1, and Syndecan-1 Core Protein Inhibit the Binding of MK and PTN to Syndecan-1-immobilized Chip to Varying Degrees—The interaction analysis studies have demonstrated that MK and PTN can bind CS/syndecan-1 and core protein with lower affinity compared with their binding to HS/syndecan-1. To clarify these results, inhibition studies were carried out using a BIAcore system. Intact syndecan-1, HS/syndecan-1, CS/syndecan-1, syndecan-1 core protein, and bacterially expressed GST-syndecan-1 ectodomain, which is not modified by sugar chains, were used as inhibitors, and all of the tested inhibitors were capable of inhibiting the binding of MK or PTN to syndecan-1-immobilized chip in a dose-dependent manner (Fig. 3, A and B), whereas control experiments carried out with chondroitinase or a mixture of heparinase and heparitinase but without syndecan-1 did not show any inhibition, indicating that the inhibition observed with HS/syndecan-1, CS/syndecan-1, or syndecan-1 core protein was not the result of the enzymes used. Premixing of MK or PTN (100 ng each) with 2.5 ng of intact syndecan-1 reduced the binding of MK or PTN to immobilized syndecan-1 by 50% compared with the binding of MK (RU = 43) or PTN (RU = 103) alone (Fig. 3, A and B, respectively). Comparable degrees of inhibition for the binding of MK to immobilized syndecan-1 were achieved with 5 ng of HS/syndecan-1, 50 ng of CS/syndecan-1, 100 ng of syndecan-1 core protein, and 300 ng of the GST-syndecan-1 ectodomain, which were 2-, 20-, 40-, and 120-fold higher in concentration than intact syndecan-1. Comparable degrees of inhibition for the binding of PTN to immobilized syndecan-1 were achieved with 10 ng of HS/syndecan-1, 100 ng of CS/syndecan-1, 200 ng of syndecan-1 core protein, and 3,000 ng of GST-syndecan-1 ectodomain, which were 4-, 40-, 80-, and 1,200-fold higher in concentration than intact syndecan-1. The GST protein itself showed no inhibition even at a high dose (6 µg) (data not shown). The difference in the amount of syndecan-1 core protein and GST-syndecan-1 ectodomain required for comparable degrees of inhibition for the binding of MK or PTN may be caused by the presence or absence of glycoprotein-type N-linked and/or O-linked oligosaccharides on the protein core. It remains to be investigated whether such oligosaccharides interact with these growth factors or influence the interaction by causing conformational changes of the core protein. The results are in good agreement with those obtained from direct binding studies using a BIAcore, strengthening the notion that not only the HS chains but the CS chains and the core protein of syndecan-1 are also capable of binding MK and PTN, although to varying degrees of affinity.



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  		FIG. 3.
Inhibition of the binding of MK or PTN to syndecan-1-immobilized sensor chip by intact syndecan-1, HS/syndecan-1, CS/syndecan-1, syndecan-1 core protein, or GST-syndecan-1 ectodomain. A fixed concentration of MK (A) or PTN (B) was mixed with varying concentrations of intact syndecan-1, HS/syndecan-1, CS/syndecan-1, syndecan-1 core protein, or GST-syndecan-1 ectodomain, and the interaction-immobilized syndecan-1 was studied using a BIA-core as described under "Experimental Procedures."

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, the CS chains of syndecan-4 from NMuMG cells were analyzed for the first time for determination of the disaccharide units. The major disaccharide unit (70%) was sulfated at C4 of the GalNAc residue with an appreciable proportion of oversulfated E-unit (9%). Analysis of the disaccharide composition of CS chains of syndecan-1 and -4 from the same cells indicated structural differences, especially in the nonsulfated GalNAc residue content and in the degree of sulfation. The CS chains of syndecan-4, bearing a lower proportion of nonsulfated units and a higher proportion of 6-O-sulfated GalNAc-containing disaccharide units, had a higher degree of total sulfation than those of syndecan-1. The CS chains of syndecan-1 and -4 had a higher degree of sulfation than their HS counterparts. Interestingly, whereas the HS chains on syndecan-1 and -4 are structurally and functionally similar, the CS chains are distinct in both respects and appear to have regulatory roles in modulating growth factor functions, cooperating with the HS chains. Concomitant synthesis of undersulfated HS and highly sulfated CS in the same tissue has been observed previously for a hybrid PG, presumable perlecan, produced by the Engelbreth-Holm-Swarm mouse tumor (42), probably reflecting the differential regulation of sulfotransferases involved in the synthesis of HS and CS.

The presence of significant proportions of the E-unit in the CS chains of syndecan-1 and -4 was also noted. Interestingly, however, the CS chains on syndecan-1, with an E-unit content comparable with that of syndecan-4, bound MK and PTN with much lower affinity, suggesting that the sequence is more important for the binding of these growth factors than the E-unit content. Our previous studies demonstrated that CS chains with an E-unit content of 14% from appican (43), a CS-PG form of amyloid precursor protein, strongly bind bFGF, MK, and PTN, and the E unit is essential for the binding (38). Hence, the lack of binding of bFGF to CS/syndecan-4 with an E-unit content of 9% suggests that the sequential arrangement of an E-unit with either an A- and/or C-unit in the CS chains is essential for binding the growth factor. A simple methodology to sequence such long stretches of CS or HS chains is currently unavailable, although a few sophisticated procedures have been developed (44–46) for HS and CS chains, and the sequences that bind FGF family members (47) and type V collagen (48) have been examined. The MK and PTN binding domains remain to be sequenced.

Experiments using MK, PTN, and bFGF in this study demonstrated that syndecan-1 and -4 bound these growth factors with comparable affinities, supporting a physiological role for these syndecans in the signaling of growth factors. The removal of the HS chains resulted in a complete loss of bFGF binding, but interestingly it lowered the binding affinity of MK and PTN only partially, demonstrating for the first time that not only the HS but also the CS chains of a hybrid PG are capable of binding MK and PTN. It has been suggested that the mammary cell surface PG takes part in the anchorage of the cell to the extracellular matrix through the HS chains, and the CS chains may alter the binding specificity and affinity of the HS-PG (13). The GAGs have also been proposed to aid in diffusion, delivering an effector protein to a particular location in the mammary epithelium or in the stroma (49). Removal of the CS chains from both syndecans in this study revealed that they accelerate the association and dissociation of MK, PTN, and bFGF from their HS counterparts. These results prompted us to speculate that the CS chains form a ternary or quaternary complex with the HS chains and a growth factor, possibly involving the core protein depending on the nature of the growth factor, and then efficiently transfer the growth factor to its corresponding cell surface receptor. The results of the present study support the proposal that alterations to binding parameters, viz. the ka and kd values, can have a substantial biological impact, even if there is no change in the value of Kd (50), by elucidating the binding properties of MK, PTN, and bFGF.

The binding of MK and PTN to syndecan-1 suggests that the core protein also participates. It has been demonstrated that the core protein of phosphacan, the extracellular domain of the brain-derived CS-PG protein-tyrosine phosphatase {zeta}, together with the CS chain, constitute the binding sites for PTN (51). Milev et al. (52) have demonstrated that the core protein of phosphacan shows high affinity binding to bFGF and potentiates its mitogenic effect. Our findings in this study indicate that although the core protein of syndecan-1 may participate in the binding of MK and PTN, such an interaction does not seem to be necessary for the binding of bFGF.

MK and PTN constitute a unique family of Hep-binding proteins implicated in the regulation of growth and differentiation (53). In this study, we observed strong binding of MK and PTN to syndecan-1 and -4. To our knowledge, the direct binding of PTN to syndecan-4 is a new finding, although strong binding of MK to syndecan-1 (54), syndecan-3/N-syndecan (55), and syndecan-4/ryudocan (56) has been reported. Recent reports indicate that CS-PGs in the brain indeed bind MK and PTN: the high affinity binding of MK to protein-tyrosine phosphatase {zeta} through the CS chain (57); the binding of MK and PTN to PG-M/versican isolated from E13 mouse embryos (58); and the high affinity binding (Kd = 0.25–3.0 nM) of PTN to phosphacan (59). The CS chain from appican (43) and the CS/dermatan sulfate chain from embryonic pig brain (39) are capable of binding MK and PTN in addition to bFGF, although the latter chains are assumed to be derived from various PGs. A model of MK signaling through protein-tyrosine phosphatase {zeta} has been proposed (60), where the MK receptor is a molecular complex of protein-tyrosine phosphatase {zeta} and a transmembrane protein, viz. low density lipoprotein receptor-related protein, which reportedly functions as a receptor of signals from Wnt and reelin. The involvement of MK in breast cancer has been indicated by the finding that loss of MK expression correlates with tumor progression (61). Expression of PTN by mammary cells in vivo and a putative role for PTN in controlling the proliferation and/or differentiation of different mammary cell types have been proposed (62). Expression of PTN in nontumorigenic, attachment-dependent epithelial cells leads to an attachment-independent, highly tumorigenic phenotype (63). Therefore, it is essential to investigate interactions of MK and PTN with not only HS but also CS chains to clarify the molecular mechanism of their functions.

Various FGFs including FGF-1, -2, -7, and -8 are produced by cells of the mammary gland at different stages of development (49). bFGF stimulates branching morphogenesis during mammary gland development (64) and also acts as a survival factor for mammary epithelial cells (65). The amount of bFGF in the mammary gland increases with puberty and pregnancy but decreases markedly during lactation, and the number of bFGF receptors in epithelial cells changes in parallel (66). In this study, we demonstrated that bFGF bound intact syndecan-1 and -4 with similar affinities (Kd = 3.4 and 5.0 nM, respectively), consistent with the report of identical affinity (Kd = 28 nM) of bFGF for intact syndecan-1 and -4, obtained by affinity coelectrophoresis (34). The differences in the Kd values observed in the present and previous study could be accounted for by the difference in the methods used for the calculation. The strong binding of bFGF to human syndecan-4/ryudocan with a Kd value of 0.5 nM has also been reported (56).

The fine structures of HS chains in syndecan-1 vary in a cell type-specific manner (8, 67). Proteins with the same core but structurally different HS chains differ in their ability to modulate the biological activities of acidic FGF and bFGF (68). On the other hand, two unrelated HS-PGs, viz. syndecan-4 and glypican-1, extracted from fibroblasts, possess HS chains with no major domain or fine structural difference and indistinguishable affinities for the Hep-II domain of the matrix component fibronectin (69), suggesting that the variation in structure and binding affinity of HS on cell surface syndecan enables the cell to respond to specific HS-binding effectors in the cellular microenvironment. The attachment of different numbers of HS and CS chains to syndecan family members may alter interactions with specific proteins and hence modify the biological function of these components.


   FOOTNOTES
 
This article is dedicated to the memory of Prof. Merton Bernfield.

* This work was supported in part by the Scientific Research Promotion Fund of the Japan Private School Promotion Foundation Grant-in aid for Exploratory Research 15659021 (to K. S.), Grant-in-aid for Priority Areas 14082207 (to K. S.), and the National Project on Functional Glycoconjugate Research Aimed at Developing New Industry (to K. S.) from the Ministry of Education, Science, Sports, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed: Dept. of Biochemistry, Kobe Pharmaceutical University, 4-19-1 Motoyama-Kita-Machi, Higashinada-ku, Kobe, Hyogo 658-8558, Japan. Tel.: 81-78-441-7570; Fax: 81-78-441-7569; E-mail: k-sugar{at}kobepharma-u.ac.jp.

1 The abbreviations used are: PG, proteoglycan; bFGF, fibroblast growth factor; CS, chondroitin sulfate; GAG, glycosaminoglycan; Gal-NAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; GlcUA, glucuronic acid; GST, glutathione S-transferase; HPLC, high performance liquid chromatography; HS, heparan sulfate; IdceA, iduronic acid; MK, midkine; NMuMG, normal murine mammary gland; PTN, pleiotrophin; rh, recombinant human; RU, resonance units. Back


   ACKNOWLEDGMENTS
 
We thank Momoyo Ueno and Mika Kanbara, Kobe Pharmaceutical University, for technical assistance.



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