Interaction between collagens and glycosaminoglycans investigated using a surface plasmon resonance biosensor

Hidekazu Munakata, Keiichi Takagaki, Mitsuo Majima1 and Masahiko Endo2

Department of Biochemistry, Hirosaki University School of Medicine,5 Zaifu-cho, Hirosaki 036-8562, Japan and 1Kushiro Junior College,1-10-42 Midorigaoka, Kushiro 085-0814, Japan

Received on January 7, 1999; revised on March 17, 1999; accepted on March 17, 1999

The interactions of glycosaminoglycans with collagens and other glycoproteins in extracellular matrix play important roles in cell adhesion and extracellular matrix assembly. In order to clarify the chemical bases for these interactions, glycosaminoglycan solutions were injected onto sensor surfaces on which collagens, fibronectin, laminin, and vitronectin were immobilized. Heparin bound to type V collagen, type IX collagen, fibronectin, laminin, and vitronectin; and chondroitin sulfate E bound to type II, type V, and type VII collagen. Heparin showed a higher affinity for type IX collagen than for type V collagen. On the other hand, chondroitin sulfate E showed the highest affinity for type V collagen. The binding of chondroitin sulfate E to type V collagen showed higher affinity than that of heparin to type V collagen. These data suggest that a novel characteristic sequence included in chondroitin sulfate E is involved in binding to type V collagen.

Key words: proteoglycan/glycosaminoglycan/surface plasmon resonance/extracellular matrix/interaction

Introduction

The extracellular matrix (ECM) is composed mainly of proteoglycans (PG), hyaluronic acid, collagens and other glycoproteins, which all form aggregates with each other. Proteoglycans consist of a protein core, which has one or more glycosaminoglycan (GAG) side chains bound covalently to it (Kjellén and Lindahl, 1991). Proteoglycan interactions with fibrillar collagens have been implicated in the regulation of ECM assembly (Scott, 1988). Decorin has been shown to interact with type I collagen (Vogel et al., 1984; Schönherr et al., 1995a), type II collagen (Vogel et al., 1984), and type VI collagen (Bidanset et al., 1992) and to inhibit fibrillogenesis (Vogel et al., 1984; Schönherr et al., 1995a). Biglycan and NG2 PG are known to bind to type I collagen (Schönherr et al., 1995b) and type VI collagen (Burg et al., 1996), respectively. Binding of the PG to collagens is abolished by pretreatment of the proteoglycans with alkali (Vogel et al., 1984), but not by digestion with chondroitinase ABC (Vogel et al., 1984; Burg et al., 1996). The recombinant core proteins also bind to collagens (Bidanset et al., 1992; Schönherr et al., 1995a,b). These results are consistent with a view that the interactions between PG and collagens are mainly dependent on the core protein. However, GAG interactions with collagens and other glycoprotein in ECM are also thought to play important roles in both cell adhesion and the formation of ECM structures. In fact, some workers have reported that heparin binds to type V collagen (LeBaron et al., 1989; Yaoi et al., 1990; San Antonio et al., 1994), fibronectin (Hayashi and Yamada, 1982), laminin (Sakashita et al., 1980), and vitronectin (Lane et al., 1987). However, in comparison with the core protein, the binding of GAGs to collagens and glycoproteins is not fully understood.

In order to clarify the chemical bases for the interaction of GAGs with collagens and glycoproteins in ECM, we have examined these interactions using a surface plasmon resonance (SPR) biosensor. This type of biosensor technique allows direct monitoring of the interaction, no labeling of the molecules is required, and only a small amount of sample (typically less than 1 µg/injection) is necessary (Chaiken et al., 1992). In addition, using this biosensor, we have measured the equilibrium dissociation constants (Kd) for the interaction of GAG with collagens.

Results

Binding of GAGs to immobilized ECM proteins

GAG solutions were injected onto the sensor surfaces bearing immobilized collagens, fibronectin, laminin, and vitronectin, and the binding of GAGs and ECM proteins was determined from the increased responses on the sensorgrams. Nonspecific binding and the change in refractive index were corrected by subtracting the response of the albumin surface from that of the ECM proteins.

Since among the various GAGs heparin has been studied most extensively, heparin was injected onto the ECM protein surfaces (Figure 1). Heparin has been shown to bind type V collagen (LeBaron et al., 1989; Yaoi et al., 1990; San Antonio et al., 1994), fibronectin (Hayashi and Yamada, 1982), laminin (Sakashita et al., 1980), and vitronectin (Lane et al., 1987). In our method, no significant binding of heparin to the sensor surface coated with type I, type II, and type IV collagen was detected (Figure 2A-C). On the other hand, heparin was found to bind to type V collagen (Figure 2D), type IX collagen (Figure 2E), fibronectin (Figure 2F), laminin, and vitronectin (data not shown), indicating that the method was useful for investigating the affinities of GAGs and ECM proteins.

The binding of various GAGs to collagens, fibronectin, laminin, and vitronectin immobilized on the sensor chip was evaluated using the same method, and the results are summarized in Table I. When reverse situation was investigated, i.e., when collagens, fibronectin, laminin, and vitronectin were injected onto sensor surfaces bearing immobilized GAGs, similar results were obtained (data not shown). None of the GAGs except for heparin bound fibronectin, laminin, and vitronectin. Chondroitin sulfate E and dermatan sulfate bound type V collagen. Moreover, chondroitin sulfate E bound type I, type II, type III, type VII, and type IX collagen (Figure 3). The largest responses were obtained from the interaction of chondroitin sulfate E and type V collagen.

Table I. . Interaction of glycosaminoglycans with extracellular matrix proteins
ECM proteins Glycosaminoglycans
HA Ch Ch4S Ch6S ChS-D ChS-E DS HS Hep
Fiber-forming collagens
   Type I collagen - - - - - ± - - -
   Type II collagen - - - - - + - - -
   Type III collagen - - - - - ± - - -
   Type V collagen - - - - - + ± - +
FACIT collagen
   Type IX collagen - - - - - ± - - +
Sheet collagens
   Type IV collagen - - - - - - - - -
   Type VIII collagen - - - - - - - - -
   Type X collagen - - - - - - - - -
Other collagens
   Type VI collagen - - - - - - - - -
   Type VII collagen - - - - - + - - -
Other ECM proteins
   Fibronectin - - - - - - - - +
   Laminin - - - - - - - - +
   Vitronectin - - - - - - - - +
+, Change in the response of more than 20 RU; ±, change in the response from 10 RU to 20 RU; -, change in the response of less than 10 RU. HA, Hyaluronic acid; Ch, chondroitin; Ch4S, chondroitin 4-sulfate; Ch6S, chondroitin 6-sulfate; ChS-D, chondroitin sulfate D; ChS-E, chondroitin sulfate E; DS, dermatan sulfate; HS, heparan sulfate; Hep, heparin.

Equilibrium dissociation constant for the interaction of collagen with glycosaminoglycan


Figure 1. Sensorgrams of binding of heparin to immobilized type V collagen and albumin. (A) Heparin was injected onto type I collagen (a) and albumin (b) immobilized on the sensor surface, respectively. (B) Sensorgram data of albumin surface as the blank sensor surface was subtraced from that of type V collagen surface. 1, start of heparin injection; 2, end of heparin injection.


Figure 2. Sensorgrams of binding of heparin to immobilized various collagens and fibronectin. Heparin was injected onto type I collagen (A), type II collagen (B), type IV collagen (C), type V collagen (D), type IX collagen (E), and fibronectin (F) immobilized on the sensor surface, respectively.

The differences in the affinity of chondroitin sulfate E and various collagens were compared using the Kd. Different concentrations of chondroitin sulfate E were injected over the sensor surface with type V collagen (Figure 3). Response values were reached at steady states (450-500 s), representing the equilibrium binding of chondroitin sulfate E to immobilized type V collagen. The plots of the response values at steady states and the concentration of chondroitin sulfate E are presented in Figure 4. The Kd values were estimated from the reciprocal of the slope (Figure 5). The Kd values for the interaction of type I, type II, and type IX collagen with chondroitin sulfate E and type V collagen with dermatan sulfate were not evaluated using this method. In terms of Kd, the affinities of the collagens for both chondroitin sulfate E and heparin ranged from 5 to 40 nM (Table II). Chondroitin sulfate E showed a higher affinity for type V collagen than for type II or type VII collagen. Heparin showed a higher affinity for type IX collagen than for type V collagen. The binding of chondroitin sulfate E to type V collagen was higher than that of heparin to type V collagen.


Figure 3. Sensorgrams of binding of chondroitin sulfate E to various immobilized collagens. Chondroitin sulfate E was injected onto type I collagen (A), type II collagen (B), type III collagen (C), type V collagen (D), type VII collagen (E), and type IX collagen (F) immobilized on the sensor surface, respectively.


Figure 4. Overlays of sensorgrams showing binding of chondroitin sulfate E at different concentration to immobilized type V collagen. a, 0.5 nM; b, 2.5 nM; c, 5 nM; d, 10 nM; e, 15 nM; f, 25 nM.; g, 35 nM; h, 50 nM

Discussion

In this study, the affinity of GAGs for collagens and ECM glycoproteins was analyzed using a surface plasmon resonance (SPR) biosensor. Compared with known methods, for example affinity chromatography (Yaoi et al., 1990), solid-phase binding assay (LeBaron et al., 1989), and affinity co-electrophoresis (San Antonio et al., 1994), this method is advantageous in that no labeling of samples is required, and the experimental time is short (about 10 min/cycle). Moreover, the intensity of the affinity can be compared using the Kd.

Binding of GAGs to proteins is usually electrostatic in nature (Öbrink, 1973; Kjellén and Lindahl, 1991). At physiological pH and the salt concentration of the solution used in the present experiments, only chondroitin sulfate E and heparin bound to collagens and ECM glycoproteins. The affinities of heparin for collagens, fibronectin, laminin, and vitronectin were reconfirmed by this method. With respect to the interaction between other GAGs and collagens, it was found that chondroitin sulfate E bound especially strongly to type V collagen. The interaction between GAGs and collagens was not affected by the presence of Ca2+ and Mg2+. Thus, it is suggested that the interaction between GAGs and collagens involves not only electrostatic binding, but also other types of specific interaction.

Chondroitin sulfate E from squid cartilage consists mainly of GlcA[beta]1-3GalNAc(4S, 6S) (61.5%) and includes GlcA[beta]1-3GalNAc(4S) (22.9%), GlcA[beta]1-3GalNAc(6S) (9.6%), and GlcA[beta]1-3GalNAc (5.9%) as minor components (Kawai et al., 1966; Suzuki et al., 1968; Yoshida et al., 1989), where 4S and 6S represent 4-O- and 6-O-sulfate, respectively. A few [beta]-D-glucosyl branches are also included in squid cartilage chondroitin sulfate E as an unusual component (Habuchi et al., 1977). Recently, Kinoshita et al., (1997) roughly estimated the glucuronic acid 3-sulfate content of a chondroitin sulfate E preparation to be as high as 10%. Thus, since chondroitin sulfate E has a variety of structures, it is likely that some specific feature of its structure is necessary for binding to type V collagen.

Table II. Equilibrium dissociation constants (Kd) for glycosaminoglycan-extracellular matrix protein interactions
GAGs Collagens Equilibrium dissociation constant (nM)
ChS-E Type I n.d.
ChS-E Type II 39
ChS-E Type III n.d.
ChS-E Type V 5
ChS-E Type VII 38
ChS-E Type IX n.d.
DS Type V n.d.
Hep Type V 26
Hep Type IX 7
n.d., Not determined.


Figure 5. Scatchard plots for the determination of Kd using equilibrium sensor responses. The data (450 s) in Figure 4 were used.

It is known that the structure of chondroitin sulfate E is presented in the GAG of proteoglycan localized to the secretory granules of mast cells (Razin et al., 1982; Stevens et al., 1983) and also at the nonreducing terminal of GAG of aggrecan (Otsu et al., 1985; Midura et al., 1995; Plaas et al., 1997). Moreover, McGee et al. (1995) have demonstrated that procoagulant activity in plasma is inhibited by chondroitin sulfate, and recently Plaas et al., (1997) reported that the N-acetylgalactosamine 4,6-disulfate content of the nonreducing terminal region changes in relation to age. Therefore, it is suggested that a specific domain containing the chondroitin sulfate E structure is involved in ECM formation.

We are now studying the domain structure of chondroitin sulfate E that binds to type V collagen.

Materials and methods

Chemicals

Hyaluronic acid (HA, from human umbilical cord, average molecular weight 900 kDa), chondroitin 4-sulfate (Ch4S, from whale cartilage, average molecular weight 34 kDa), chondroitin 6-sulfate (Ch6S, from shark cartilage, average molecular weight 64 kDa), chondroitin sulfate D (ChS-D, from shark cartilage, average molecular weight 54 kDa), chondroitin sulfate E (ChS-E, from squid cartilage, average molecular weight 70 kDa), dermatan sulfate (DS, from pig skin, average molecular weight 32 kDa), and heparan sulfate (HS, from bovine kidney, average molecular weight 28 kDa) were purchased from Seikagaku Kogyo Co. (Tokyo, Japan). Heparin (Hep, from porcine intestinal mucosa, average molecular weight 19 kDa) was obtained from Sigma Chemical Co. (St. Louis, MO). Chondroitin (average molecular weight 10 kDa) was prepared from chondroitin 6-sulfate by a modification (Nakamura et al., 1990) of the method of Kantor and Schubert, (1957).

Fibronectin (FN, from human fibroblasts), laminin (LM, from mouse) and vitronectin (VN, from human plasma) were purchased from Fibrogenex Inc. (Chicago, IL), Iwaki Glass Co. (Funabashi, Japan), and Chemicon International Inc. (Temecula, CA), respectively. Type I collagen (from porcine skin), type II collagen (from porcine cartilage), type III collagen (from porcine skin), type IV collagen (from porcine skin), and type V collagen (from porcine placenta) were obtained from Wako Pure Chemical Co. (Osaka, Japan). Type VI collagen (from human placenta), type VII collagen (from rat tail), type VIII collagen (from human placenta), type IX collagen (from human placenta), and type X collagen (from human placenta) were purchased from Sigma Chemical Co.

A research grade sensor chip CM5 and an amine coupling kit containing N-hydroxysuccinimide (NHS), N-ethyl-N[prime]-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), 1 M ethanolamine hydrochloride adjusted to pH 8.5 with NaOH and HBS buffer containing 10 mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA and 0.005% surfactant P20 were purchased from Pharmacia Biosensor AB (Uppsala, Sweden). Other reagents were of analytical grade and obtained from commercial sources.

Surface plasmon resonance analysis

Binding of GAG to ECM proteins was determined using a BIAcore 2000 (Pharmacia Biosensor AB, Uppsala, Sweden). Binding reactions caused a change in the surface plasmon resonance (SPR), which was detected optically and measured in resonance units (RU). One thousand RU corresponds to a 0.1° shift in the surface plasmon resonance angle and a surface concentration change of about 1 ng/mm2 for the average protein (Johnsson et al., 1991).

Immobilization of ECM proteins on the sensor surface

Immobilization of ECM proteins on the sensor surface via primary amine groups was performed in the following manner (Löfås and Johnsson, 1990; Johnsson et al., 1991). During immobilization, the flow rate of running buffer (HBS buffer) was maintained at 5 µl/min. The surface was activated by injection of a mixture of EDC/NHS (0.2 M/0.05 M). Then, a 100 µg/ml solution of ECM protein in 10 mM sodium acetate buffer, pH 4.0, was injected followed by 1 M ethanolamine hydrochloride, pH 8.5. The amounts of ECM proteins immobilized onto the sensor surface were controlled within the range 2500-4500 RU by changing the injection time.

Determination of binding of GAGs and immobilized ECM proteins

All experiments were carried out at flow rate of 10 µl/min at 25°C. GAGs in the running buffer were injected onto the sensor surface. The responses of the running buffer were defined as the baseline level, and all responses were expressed relative to this baseline. The sensor surfaces were regenerated with 1.4 M NaCl in HBS buffer.

For analysis of interactions between GAGs and ECM proteins, the sensorgrams were corrected by a modification of the method of Roden and Myszka, (1996). To correct for refractive index change and nonspecific binding, the responses obtained from the surface of albumin as a blank control were subtracted from the ECM protein surface data (Figure 1). When each GAG solution was injected onto the albumin surface followed by running buffer, the responses returned to the baseline immediately, indicating that each GAG did not bind to the albumin.

Determination of equilibrium dissociation constant

The equilibrium dissociation constant (Kd) was calculated using the equation (Chaiken et al., 1992):

Where [Delta]Req is the response value at steady state, [Delta]Rmax is the maximal capacity of the sensor chip for binding GAGs, and C is the molar concentration of GAGs. The molar concentration of GAGs was calculated from the average molecular weight. Kd was determined by plotting [Delta]Req/C vs. [Delta]Req.

Acknowledgments

This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan (Nos. 08457032, 09358013, and 10134203).

Abbreviations

ECM, extracellular matrix; PG, proteoglycan; GAG, glycosaminoglycan; SPR, surface plasmon resonance; HA, hyaluronic acid; Ch4S, chondroitin 4-sulfate; Ch6S, chondroitin 6-sulfate; ChS-D, chondroitin sulfate D; ChS-E, chondroitin sulfate E; DS, dermatan sulfate; HS, heparan sulfate; Hep, heparin; NHS, N-hydroxysuccinimide; EDC, N-ethyl-N[prime]-(3-dimethyl-aminopropyl)-carbodiimide; Kd, equilibrium dissociation constant.

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