Differential interactions of decorin and decorin mutants with type I and type VI collagens Gordon Nareyeck 1 , Daniela G. Seidler 1 , David Troyer 2 ,Ju¨ rgen Rauterberg 2 , Hans Kresse 1 and Elke Scho¨ nherr 1,3 1 Departement of Physiological Chemistry and Pathobiochemistry, University Hospital of Mu ¨ nster, Germany; 2 Institute of Arteriosclerosis Research, University of Mu ¨ nster, Germany; 3 Matrix Biology and Tissue Repair Research Unit, University of Wales College of Medicine, Dental School, Cardiff, UK The small leucine-rich proteoglycan decorin can bind via its core protein to different types of c ollagens such as type I and type VI. To test whether decorin can act as a bridging molecule between these collagens, the binding properties of wild-type decorin, two full-length decorin species with single amino acid substitutions (DCN E180K, DCN E180Q), which previously showed reduced binding to collagen type I fibrils, and a t runcated form of decorin (DCN Q153) to the these collagens were investi- gated. In a solid phase assay dissociation constants for wild-type decorin bound to methylated, therefore mono- meric, triple helical type I collagen were in the order of 10 )10 M , while dissociation constants for fibrillar type I collagen were  10 )9 M . The dissociation constant for type VI was  10 )7 M . Using real-time analysis for a more detailed investigation DCN E180Q and DCN E180K exhibited lower association and higher dissoci- ation constants t o type I collagen, compared to wild-type decorin, deviating by at least one order of magnitude. In contrast, the affinities of these mutants to type VI colla- gen were 10 times higher than the affinity of wild-type decorin ( K D  10 )8 M ). Further investigations verified that complexes of type VI collagen and decorin bound type I collagen and that the affinity of collagen type VI to type I was increased by the presence of decorin. These data show that decorin not only can regulate collagen fibril formation but that it also can a ct as an intermediary between type I and type VI collagen and that these two types of collagen interact via different binding sites. Keywords: collagen t ype I; collagen type VI; decorin; surface plasmon r esonance measurements. Collagens can be divided into several subfamilies according to their quarternary structure a nd their localization in tissue [1,2]. The largest subfamily is represented by the banded fibril forming collagens type I, II and III, which are characterized by long, uninterrupted triple helical domains that assemble laterally to form fibrils. I n contrast, another subfamily, of which type VI collagen is t he only m ember, is characterized by the formation of multimolecular, filamen- tous beaded structures [3]. Although banded fibril forming and filamentous beaded collagens form independent net- works, they intermingle with each other in vivo,this association providing for mechanical stab ilization o f t issues. Electron microscopic studies indicate that the banded fibril forming collagens are traversed specifically near their ÔdÕ bands, within the gap region of the collagen fibrils, by the filamentous beaded structures of the type VI collagen- containing network [4,5]. Collagen fibrils in tissues a re heteropolymers of several types of collagen and of noncollagenous components. For example, collagen fibrils in skin are composed primarily of type I collagen with minor amounts of type III and type V collagen. Type III collagen is found on the fibrillar surface, while type V collagen is buried mainly within the fibrils [6]. Noncollagenous matrix glycoproteins are additionally associated with the surface of the collagen fibrils. Such glycoproteins may in part substitute for collagen species at the fibrillar surface or perform auxiliary functions [7]. Some of these matrix glycoproteins contain leucine-rich repeat structures and have been shown to modulate c ollagen fibrillogenesis and the spacing between the mature fibrils. The chondroitin/ dermatan sulphate proteoglycan decorin (DCN) is a member of this family of small leucine-rich p roteoglycans (SLRP), which is composed of a core protein and a single covalently linked glycosaminoglycan chain. It b inds to collagen fibrils near the d bands (Ôd ecoratesÕ them) and delays the lateral assembly of collagen fibrils [8,9]. Consequently, targeted disruption of the decorin gene in mice leads to abnormal fusion of collagen bundles and to increased f ragility of skin [ 10]. Recently, mic e twofold Correspondence to E. Scho ¨ nherr, Matrix Biology & Tissue R epair Research Unit, University of Wales C ollege of Medicine, Dental School, Heath Park, Cardiff CF14 4XY, UK. Fax: + 44 29 2074 4509, Tel.: + 44 29 2074 2595, E-mail: schonherreh@cardiff.ac.uk Abbreviations: BGN, biglycan; CS/DS, chondroitin sulphate/derma- tan sulphate; DCN, decorin; GAG, glycosaminoglycan; SLRP, small leucine-rich repeat proteoglycan. Note: G. Nareyeck a nd D. G. Seidler c ontribu ted equally to this w ork. (Received 13 M ay 2004, accepted 30 June 2004) Eur. J. Biochem. 271, 3389–3398 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04273.x deficient in the SLRP decorin, biglycan, fibromodulin and lumican have been generated [11]. Interestingly, double deficiency in decorin and biglycan manifests itself in extremely abnormal architecture of the collagen fibrils. Thus, interactions between the collagens and decorin are of paramount importance in attaining and maintaining tissue integrity. In the present study we investigate the binding properties of wild-type decorin, two decorin mutants and a truncated decorin species with type I and type VI collagen to demonstrate that decorin can act as a bridging molecule. The results indicate that the tertiary structure of decorin is stabilized by the glycosaminoglycan chain. Furthermore, decorin may form a dimer which is capable of interacting concurrently with both type I and type VI collagen molecules. Experimental procedures Expression and preparation of recombinant proteoglycans Wild-type decorin and the decorin mutant DCN E180K were expressed in human kidney 293 cells as previously described [12]. DCN E180K harbours an amino acid exchange at amino acid E180, which is an important site for the interac tion o f decorin with type I collagen fibrils. A plasmid harbouring the cDNA for the decorin mutant DCN E180Q was generated from the respective wild-type plasmid by a one-step site-directed mutagenesis procedure (Stratagene) using the primer pair 5 ¢-GGTGCCCAGTTG TATGACAATC-3¢ and 5¢-GATTGTCTACAACTGGG CACC-3¢.TheaminoacidatE180inDCNE180Qis likewise substituted. A cDNA construct for the truncated decorin species DCN Q153 (M1–Q153), in which six of a total of 10 leucine-rich repeats are lacking [13], was cloned into the EcoRI/XbaI site of pcDNA 3.1 (Invitrogen) and used for transfection with t he Lipofectin (Life Technologies) method. Biglycan (BGN) was expressed in 293 cells as described [14]. All proteoglycan preparations were obtained from conditioned media of transfected 293 cells under condi- tions without denaturing and/or precipitation steps. Media, supplemented with protease inhibitors, were applied directly to a DEAE-Trisacryl M (Serva) and then to a BioGel TSK DEAE-5PW HPLC column (Bio- Rad) as described previously [15]. Proteoglycans were stored at 4 °C in elution buffer (10 m M Tris/HCl pH 7.4, containing  0.6 M NaCl). Immediately prior to use the proteoglycans were dialysed against either 18 m M sodium phosphate pH 7.4, 0.15 M NaCl (NaCl/P i )or10m M Hepes pH 7.4, 0.15 M NaCl, 3.4 m M EDTA, 0.005% (v/v) Tween-20 (HBS) at 4 °C. Glycosaminoglycan-free core protein was generated by exhaustive digestion with chondroitin ABC lyase (Seikagaku Kogyo) as described previously [15]. Glycosaminoglycan chains were liberated by reductive b-elimination with 1 M sodium borohydride in 0.1 M NaOH for 24 h at 37 °C, followed by dialysis and rechromatography on BioGel TSK DEAE-5 PW as described above. [ 35 S]Sulphate-labelled and [ 35 S]methio- nine-labelled decorin from 293 cells and skin fibroblasts were obtained as described previously [12,16]. Preparation of methylated type I collagen and type VI collagen Type I collagen was isolated from calf skin and methylated by treatment with 0.2 M methanolic HCl for 3 d ays at ambient temperature as described [17]. This treatment results in the modification of about 70% of all carboxyl residues. This modification leads to an increase in the pH and a decrease in the hydrophilic properties. Type VI collagen was solubilized from bovine placenta by pepsin treatment and purified by salt fractionation [18]. Surface plasmon resonance analysis All measurements were performed with a BIAcore 1000 analyser (Pharmacia Biosensor). Methylated type I collagen was immobilized via its primary a mino groups to a r esearch grade CM5 sensor chip [19]. During immobilization, a flow rate of 10 lLÆmin )1 of HBS was maintained. The surface of the chip was activated by injecting a mixture of equal volumes of 0.2 M N-ethyl-N¢-(3-dimethylaminopropyl)- carbodiimide and 0.05 M N-hydroxysuccinimide. There- after, 70 lL of a solution of methylated type I collagen (250 lgÆmL )1 )in20m M sodium acetate pH 4.0 was injected followed by 1 M ethanolamine/HCl p H 8.5. Injec- tion times were chosen to achieve about 6000–7000 resonance units (6–7 ng of proteinÆmm )1 [19]. Type VI collagen was immobilized via its free sulfhydryl groups. During immobilization, the flow rate of HBS was main- tained a t 5 lLÆmin )1 . T he surface w as activ ated as described andallowedtoreactwith80 m M 2-(2 pyridinyldithio)ethane amine in 0.1 M sodium borate p H 8.5. At least five coupling pulses of 240 lL type VI c ollagen (25 0 lgÆmL )1 )in0.1 M sodium formiate pH 4.3, were applied until 6000–7000 resonance units were present. The sensor surface was blocked with 50 m ML -cysteine in 0.1 M sodium formiate pH 4.3, 1 M NaCl. BSA was immobilized and used to determine the proportion of n onspecific binding. The senso r surfaces were regenerated with 1 M NaCl in running buffer for type I collagen-coated chips and with 0.3 M NaCl in the case of immobilized type VI collagen. To form a complex consisting of decorin, type I and type VI collagen, type VI collagen was first immobilized. After binding of decorin the sensor chip surface was not regenerated in order to maintain a high level of bound proteoglycan. Methylated type I collagen in NaCl/P i was then added to the chip and allowed to interact with the proteoglycan. All e xperiments were carried out at 25 °C a t a flow rate of 10 lLÆmin )1 . The response to the running buffer was defined as the baseline level, and all responses were expressed relative to this baseline. Experimental procedures and c onditions leading to precipitation of protein complexes in the flow system and the pump of the BIAcore 1000 instrument had to be strictly avoided to protect the system from damage. For this reason only experiments without the addition of complexes were performed. For the analysis of interactions between proteoglycans and collagens, the sensograms were corrected by a modification of the method of Roden and Myszka [20]. To correct for changes in refractive index and nonspecific binding, the responses obtained with immobilized albumin were subtracted from 3390 G. Nareyeck et al. (Eur. J. Biochem. 271) Ó FEBS 2004 those obtained with bound collagen. The e xperimental data were then evaluated with the BIAEVALUATION 3.0 software. Other binding assays The binding of [ 35 S]sulphate-labelled decorin species and [ 35 S]sulphate-labelled biglycan to reconstituted type I colla- gen fibrils was performed as described [12]. Solid phase assays on hydrophilic ELISA strips (Nunc) were used to investigate interactions with type VI collagen a nd methyla- ted type I collagen. Type VI collagen (4 lgÆmL )1 , 50 lLÆwell )1 ) and methylated type I c ollagen (10 lgÆmL )1 , 100 lLÆwell )1 )in50m M NaHCO 3 pH 9.6, were incubated for 16 h at 4 °C. After blocking w ith 3% BSA in NaCl/P i , 0.05% Tween-20 for 4 h at 37 °C, the wells were washed twice with ice-cold NaCl/P i . Labelled proteoglycans in NaCl/P i (18 m M sodium phosphate pH 7.4, 0 .15 M NaCl) were applied for 6 h or 3 h at 37 °C. After extensive washing with b locking solution, bound proteoglycans were solubilized with 0.1 M NaOH and neutralized with 0.1 M HCl prior to scintillation counting. K D values were deter- mined using PRISM 3.0 (GraphPad Software). CD spectroscopy A Jobin-Yvon CD6-Dichrograph spectropolarimeter (Yvon, France) was used to measure CD spectra at ambient temperature in NaCl/P i in a 0.1-mm path length quartz cell. Proteoglycan concentrations of  1mgproteinÆmL )1 were used. Estimations of secondary structure were per- formed with the CDNN 2.1 software (ACGT Progenomics AG, Halle (Saale), Germany). Electron microscopy Suspensions of type I collagen in glycerol were s prayed onto mica sheets w ith a n air brush and rotary shadowed with platinium-carbon at an angle of about 7°, followed by pure carbon as described by Cohen et al. [21]. The replicas were placed on uncoated g rids and analysed w ith a Philips EM 410 electron microscope. Results Characterization of purified type I and type VI collagens Type I collagen fibrils we re generated by neutralization of acid soluble calf skin collagen as describe d previously [12]. To obtain type I collagen monomers, type I collagen was methylated which shifts the isoelectric point of the molecule to a basic pH and increases hydrophobicity. The treated collagen does not form fibrils under physiological condi- tions which was confirmed by rotary shadowing (Fig. 1A). However, the methylated t ype I collagen was still able to bind to hydrophilic ELISA strips (see below). Bovine type VI collagen containing three polypeptide chains, a1(VI), a2(VI) and a3(VI) covalently linked via disulphide bonds was produced by treatme nt with pepsin t o remove most o f the C- and N-terminal globular domains (Fig. 1B). Fig. 2 shows the composition of the collagen used in the experi- ments. Quantitative analysis of the SDS gel electrophoreses indicated that 63% of the type VI collagen contained the long chain and 37% the s hort fragment o f the a3(VI) chain [22]. Characterization of purified decorin and its mutants Wild-type decorin, DCN E180K, DCN E180Q and DCN Q153 were purified under nondenaturing conditions from conditioned medium of 293 cells transfected with the respective cDNA. No freeze-drying or precipitation steps were performed to avoid artificial complex formation of proteoglycans [23]. CD spectroscopy was u sed to determine whether there are major differences in secondary structure between the wild-type decorin and the mutants. The CD spectra of wild-type decorin and DCN E180Q and D CN E180K appeared similar, whereas that of DCN Q153 differed from tha t of wild -type dec orin (Fig. 3). The CD Fig. 1. Rotary shadowing of isolated type I and type VI collagen mole- cules. (A) Methylated type I collagen was visualized as monomers, whereas (B) pepsin digested type VI collagen appeared as short frag- ments of beaded filaments. Ó FEBS 2004 Decorin interacting with type I and VI collagen (Eur. J. Biochem. 271) 3391 spectra of the glycosaminoglycan chain alone, obtained by reductive b-elimination, yielded only baseline data (not shown). Evaluation of secondary structure was performed with the CDNN 2.1 software. The analysis showed that wild- type decorin and DCN E180K an d DCN E180Q have 2 1% a-helical motifs and 29.1% b-sheets (Table 1). These results show that the point mutations have only minor effects on the g eneral structure of the decorin core protein. For DCN Q153, which lacks most of the leucine-rich repeats, 36% a-helical motifs and only 2 4.1% b-sheets were observed. As shown in Fig. 4, decorin expressed in 293 cells contained no free core protein, and the length of the glycosaminoglycan chain w as similar to that of decorin f rom d ermal fibroblasts . Fig. 2. Electrophoretic comparison of the composition of pepsin digested type I collagen, acid treated type I collagen, methylated type I c ollagen and type V I collagen used in the experiments. Sam ples of the different types of collagen were applied und er reducing (+DTE) and non- reducing (–DTE) conditions to a 4–12.5% polyacrylamide gradient gel. Protein was visualized by staining with Coomassie blue. Fig. 3. CD spectra of the recombinant decorin e xpressed in 293 cells and purified under nondenaturing conditions. The spectra were obtained under physiological conditions in 0.15 M NaCl. Wild-type d ecorin, solid line; truncated decorin DCN Q153, dotted line. Spectra for the decorin mutants DCN E180K and DCN E180Q were indistinguish- able from that of wild-typ e decorin (not shown). Table 1. Tentative structural motifs of recombinant decorin. Theoretical calculation using the program CDNN and the data from by CD spectra measured between 195 nm to 260 nm. Decorin and the decorin mutants DCN E 180Q a nd DC N E180K an d t runc ated de corin D CN Q153 we re purified under nondenaturing co nditio ns from the medium of 293 cells. Secondary structural motif Decorin mutants (%) DCN Q153 (%) a-Helical 21.7 35.9 b-Sheet 29.1 24.1 b-Turn 15.4 13.8 Coil 31.1 23.5 Fig. 4. Comparison of decorin synthesized in 293 cells and human skin fibroblasts. 293 cells transfected with human wild-type decorin cDNA and human skin fibroblasts were metabolically labelled w ith [ 35 S]methionine. After immunoprecipitation with a monospecific antibody, decorin proteoglycan and c ore p rotein (o btained b y diges- tion with cho ndroitin ABC lyase) were sep arated b y S DS/PAGE on 12.5% polyacrylamide gel. Labelled proteins were visualized by autoradiography. Decorin transfected 293 cells do not synthesize free core protein. The molecular masses of the proteoglycan and the core protein are similar to those of the respective molecules from fibroblasts. 3392 G. Nareyeck et al. (Eur. J. Biochem. 271) Ó FEBS 2004 Interaction of different forms of decorin with type I collagen To test the hypothesis that decorin can act as a bridging molecule between type I and type VI collagen w e first performed solid phase binding assays to determine that the binding properties of the different ligands involved. [ 35 S]Sulphate-labelled decorin was incubated with reconsti- tuted type I collagen fibrils and its binding was compared to the different mutants. Wild-type decorin i nteracted strongly with collagen fibrils, DCN E180K reacted weakly and DCN E180Q moderately (Fig. 5) which agreed with earlier results [12] and confirmed t he suitability of the mutants. To test whether methylated type I collagen monomers which were planed to be used as ligands for a decorin/ collagen type VI complex showed the expected properties solid phase assays with methylated collagen type I and decorin or biglycan as ligands were performed. ELISA plates were coated with th e collagen monomers and [ 35 S]sulphate-labelled proteoglycans were added. These solid phase assays showed dissociation constants of 2.3 · 10 )10 M for decorin and 1.4 · 10 )9 M for biglycan (data not shown) indicating that the methylated type I collagen monomers were suitable binding partners and could be used for further studies. The further analysis was performed by surface plasmon resonance spectroscopy. Collagen monomers of methylated type I collagen were covalently immobilized to a CM5 sensor chip, and the affinities of wild-type decorin, its core protein (Fig. 6A) and of the decorin mutants for the immobilized collagen were measured. Surface plasmon resonance measurements with decorin and t ype I collagen performed in the presence or absence of 15 n M ZnCl 2 showed little influence of the Zn 2+ ions on the binding properties of decorin (Fig. 6B). A single binding site between wild-type decorin and type I collagen monomers with an affinity of K D ¼ 5.8 · 10 )10 M was found (Table 2). In addition, a K D of 2.1 · 10 )8 M was observed for g lycosaminoglycan-free core protein. This value was two magnitudes h igher t han t hat for wild-type d ecorin. B inding experiments with isolated glycosaminoglycan chains obtained from decorin by b-elimination showed on ly weak interaction with type I collagen monomers. The analysis of the i nteraction of DCN E180K and DCN E180Q with type I collagen yielded K D ¼ 4.1 · 10 –9 M and K D ¼ 1 · 10 –9 M , respectively. The truncated form of decorin, DCN Q153 also showed weak interaction with type I collagen. For c omparative purposes we analysed the binding of biglycan, another proteoglycan of the SLRP family, using surface p lasmon resonance. A b inding affinity of biglycan fo r type I collagen monomers of K D ¼ 2.7 · 10 )9 M was obtained. To test the reliability of these data v 2 -values were compared between the different experi- ments. Because the v 2 -values ranged between 0 and 2, the data were considered to be reliable ( Table 2). Interaction of decorin and decorin mutants with type VI collagen Decorin is known to interact directly with banded fibril forming collagens, whereas an additional, yet undefined component, is thought to be involved in the binding of Fig. 5. Interaction o f radioactively labelled d ecorin and d ecor in mutants with reconstituted type I collagen fibrils. The proteoglycans were puri- fied under nondenaturing conditions as described above. Wild-type decorin (j), DCN E180Q (h),DCNE180K(m). Fig. 6. Surface plasmon resonance measurements of decorin binding to immobilized methylated type I collagen. (A) Interaction with wild-type decorin core protein (obtained b y chondroitin ABC lyase digestio n) in HBS. Decorin core protein concentrations were as indicated. (B) Interaction with wild-type decorin in H BS buffer (solid lines) a nd in HBS buffer containing 15 l M ZnCl 2 (dotted lines). Wild-type decorin concentrations were as indicated. Ó FEBS 2004 Decorin interacting with type I and VI collagen (Eur. J. Biochem. 271) 3393 decorin to type VI collagen [5]. Recent data showed that a complex of decorin/matrilin-1 can act as a possible linke r between type VI and type II collagen [24]. In initial experiments we studied the interaction of the various decorinformswithtypeVIcollageninasolidphase binding assay. Decorin bound less avidly to type VI than to type I collagen (K D ¼ 3 · 10 –7 M ). Compared to wild-type decorin, DCN E180Q exhibited about 10-fold lower affinity for type VI collagen. Unlike wild-type decorin and DCN 180Q, DCN E180K formed multimers as inferred from the nonlinear curve for the binding of radioactively labelled DCN E180K to type VI collagen (Fig. 7). However, multimers of DCN E180K were still capable of binding to type VI collagen. To study the binding of decorin an d the decorin mutants to type VI collagen i n a real-time experiment, we again u sed surface plasmon resonan ce measurements. The data for the binding affinities to type VI collagen are summarized in Table 3. For wild-type decorin a K D of 3.6 · 10 )9 M was determined. Because the glycosaminoglycan chain influ- enced the binding affinity of decorin to type I collagen, we also studied t he binding properties of glycosaminoglycan- free core protein to type VI collagen. A K D of 3.9 · 10 )8 M was obtained for the core protein alone. Isolated glycos- aminoglycan chains from decorin showed a weak inter- action with type VI collagen m onomers, as reported previously [25]. These binding studies constitute further evidence that the glycosaminoglycan chain stabilizes the decorin core protein. The analysis of the affinity of DCN E180K for type V I collagen yielded a K D of 4.1 · 10 –9 M , which is similar to that obtained for wild-type decorin. Su rprisingly, DCN E180Q, which showed a moderate affinity for type I collagen displayed a high affinity to collage n type V I (K D of 3.4 · 10 )10 M ), which is one magnitude lower than that found for wild-type decorin. DCN Q153 interacted only weakly with type VI collage n. For biglycan, however, the experiments revealed a K D of 2.1 · 10 )8 M , which is about one order o f magnitude higher than that found with type I collagen. Considering R max (where 1000 resonance units ¼ 1ngÆmm )2 ) s toichiometric analysis of the surface plasmon resonance measurements revealed that a single collagen molecule binds about 0.186 decorin molecules in the presence as well as the absence of its glycosaminoglycan chain (Fig. 6A,B). The number of decorin molecules binding to type VI collagen increased from 1 : 0.042 to 1 : 0.061 by the presence of the glycosaminoglycan chain (Fig. 8 A,B), indicating that the glycosaminoglycan chain does not only stabilize wild-type decorin, but can also interfere with the function of decorin. The glycosamino- glycan chain alone did not show binding properties to the collagen coated chip (data not shown). Furthermore, the amino acid exchange at position E180 resulted in a change in the bindin g capacity of decorin to both type I an d to type VI collagen. Formation of complexes of decorin, type I collagen and type VI collagen In a further investigation we analysed whether the same site or similar sites on wild-type decorin, DCN E180Q and DCN E180K bind to type I and to type VI collagen. Table 2. Binding of decorin and decorin mutants to type I collagen. Type I collagen monomers were immobilized on CM5 chips. Surface plasmon resonance measurement s were performed with decorin, different dec- orin mutants, biglycan and the chondroitin sulphate/dermatan sul- phate (CS/DS) c hain rele ased by b-elimination from decorin. T he samples were purified under non-denaturing conditions from the medium of 293 cells. WT, Wild-type; core, decorin digested with chondroitin ABC lyase; CS/DS, glycosaminoglycan chain from deco- rin released by b-elimination; RU, resonance units. Proteoglycan K A ( M )1 ) K D ( M ) R max (RU) v 2 DCN WT 1.7 · 10 9 5.8 · 10 )10 386 0.92 DCN core 4.7 · 10 7 2.1 · 10 )8 199 1.9 DCN E180Q 9.8 · 10 8 1 · 10 )9 349 0.02 DCN E180K 2.4 · 10 8 4.1 · 10 )9 171 0.34 DCN Q153 2.7 · 10 7 3.9 · 10 )8 282 0.24 BGN WT 3.7 · 10 8 2.7 · 10 )9 358 0.12 CS/DS chain 5 · 10 3 2 · 10 )4 – 0.94 Fig. 7. Interaction of [ 35 S]sulphate-labelled wild-type decorin and the decorin mutants DCN E180Q and DCN E180K with pepsin d igested type VI collagen in t he solid phase binding assay. Wild-type decorin (d), DCN E180Q ( h), DCN E 180K (n). Table 3. Binding of decorin and dec orin mutants to type VI collagen. Type VI collagen was digested wi th pepsin and immobilized on CM5 chips. Surface plasmon resonance measurements w ere performed with decorin, d ifferent decorin mutants, biglyc an a nd th e CS/DS chain released by b-elimination from decorin. The samples were purified under nondenaturing conditions from the medium of 293 cells. For abbreviations see Table 2. Proteoglycan K A ( M )1 ) K D ( M ) R max (RU) v 2 DCN WT 2.9 · 10 8 3.6 · 10 )9 120 0.024 DCN core 2.6 · 10 7 3.9 · 10 )8 91 0.044 DCN E180Q 2.9 · 10 9 3.4 · 10 )10 154 0.11 DCN E180K 3.3 · 10 8 2.9 · 10 )9 147 0.01 DCN Q153 7.6 · 10 7 1.3 · 10 )8 143 0.09 BGN WT 4.7 · 10 7 2.1 · 10 )8 116 0.034 CS/DS chain 5 · 10 2 2 · 10 )3 – 0.08 3394 G. Nareyeck et al. (Eur. J. Biochem. 271) Ó FEBS 2004 Type VI collagen w as first immobilized on a CM5 chip and reacted with decorin prior to adding methylated type I collagen. The results showed that the initially formed type VI collagen–decorin c omplexes subsequently bound methy- lated type I collagen with high affinity. K D values were 7 · 10 )9 M for wild-type decorin and 6 · 10 )8 M for DCN E180Q. For DCN E180K we found a K D an order of magnitude lower than f or DCN E180Q. As the K D value f or a complex of type VI collagen and methylated type I collagen in the absence of decorin was only 1.5 · 10 )7 M ,it is obvious that the presence of decorin significantly increases the binding affinity between the two collagens (Table 4). Discussion In this study we investigated the interaction of wild-type decorin and decorin mutants with type I and type VI collagen a nd, for the first time, w e analysed the formation of triple complexes consisting of type VI collagen, decorin and type I collagen using surface plasmon resonance measurements. Measuring t he interaction of decorin with different types of collagen by surface plasmon r esonance analysis w e f ound a high affinity of decorin for triple helical type I collagen compared to previously published values of 10 )8 )10 )9 for intact and chondroitin ABC lyase-treated decorin to reconstituted type I collagen fibrils [26,27]. In both of these studies the proteoglycans were treated with chaotropic agents. Our studies using decorin isolated f rom fibroblast culture medium under nondenaturing conditions revealed two unique high affinity binding sites ( K D ¼ 7 · 10 )10 M and K D ¼ 3 · 10 )9 M ) and 0.043 decorin molecules per collagen monomer [28]. The present study using methylated type I collagen revealed only one binding site with K D ¼ 5.8 · 10 )10 M , a value corresponding to the measurements for the highest affinity binding site in our earlier study. These r elatively high v alues may be attributable to enhanced accessibility of the binding domain of methylated compared to nonmethylated type I collagen and/or to decorin prepared under nondenaturing conditions. The binding data of decorin to monomeric collagen type I are also in agreement with studies using t ype I procollagen molecules [29]. Interestingly, Tenni and coworkers [30], using a different technique, found a l ower affinity for the interaction of decorin with methylated type I collagen peptide frag- ments generated by CNBr cleavage. The lower affinity could be due to the fact that in t his study the lysyl residues were methylated, and so might interact with t he amino a cid E180 of decorin. Thus, compared to previously published data, variations in measurements of the affinities between decorin and c ollagen seem to be attributable to differences in the isolation and purification of the collagens an d proteogly- cans. H ow important the purificatio n method is has recently been shown by Goldoni and coworkers [23] who demon- strated that f reeze-drying and p recipitation step s can lead to the formation of nonfunctional complexes of decorin and biglycan. Differences in affinity may also be due to other factors such as complex formation of decorin and biglycan in the presence of physiological concentrations of Zn 2+ [31] or phosphate [32]. However, we found no changes in the affinity of decorin to collagen type I or VI for these components (Figs 6B and 8B). This does not rule out that Zn 2+ is interacting with the N terminus of decorin and may cause dimerization [ 31], but it did not affect the interaction with the two types o f collagen. It is known that the amino acid E180 in decorin is involved in type I collagen binding [12]. Therefore, the Fig. 8. Surface plasmon reson ance measurement of immobilized pepsin digested type VI collagen. (A) Interaction with wild -typ e decorin core protein (ob tained by chondroitin A BC lyase digestion) in HBS bu ffer. Concentrations of wild-type decorin core protein used were as indi- cated. (B) Interaction with wild-type decorininHBSbuffer(solidlines) andinHBSbuffercontaining15l M ZnCl 2 (dotted lines). Concen- trations of wild-type decorin used were a s indicated. Table 4. Type V I collagen was dige sted with pepsin and imm obilized on CM5 chips followed by c omplex formation of wild-type decorin and decorin mutants. Surface plasmon resonance measurements of the collagen/proteoglycans complexes were performed with monomers of methylated type I collagen. The proteoglycans were purified under nondenaturing condition from the medium of 293 cells. WT, Wild-type. Type I collagen binding K A ( M )1 ) K D ( M ) v 2 Type VI collagen 6.9 · 10 8 1.5 · 10 )7 0.319 Type VI collagen/DCN WT 1.4 · 10 8 7.2 · 10 )9 0.149 Type VI collagen/DCN E180K 1.6 · 10 6 6.2 · 10 )7 0.18 Type VI collagen/DCN E180Q 1.7 · 10 7 5.9 · 10 )8 0.141 Ó FEBS 2004 Decorin interacting with type I and VI collagen (Eur. J. Biochem. 271) 3395 moderate K D value for binding of DCN E180Q to reconstituted type I collagen fibrils and an even lower affinity of DCN E180K was expected. In contrast to reports that the glycosaminoglycan c hains have no influence on the binding of decorin to collagen fibrils [26,33], we observed reduced binding affinity for the glycosaminoglycan-free core protein. Evidently t he glycosaminoglycan chain of decorin stabilizes the tertiar y structure of the proteoglycans the reby causing difference in binding affinity. As decorin is not the only SLRP that interacts with type I collagen the homo- logous proteoglycan biglycan was investigated. The affinity of biglycan for methylated type I collagen was lower than the affinity of decorin which corroborated previous data obtained with biglycan from bacteria and f rom osteosarcoma cells using fibrillar type I collagen [28]. To investigate the interaction o f d ecorin m utants E180K and E180Q with type VI collagen a solid phase assay was performed. One mutant, DCN E180K, which has a 10-fold lower affinity to collagen t ype I than wild-type decorin had a similar affinity to type VI collagen as wild-type decorin. DCN E 180Q showed an even stronger binding to type VI collagen than wild-type decorin, while its affinity to type I collagen was reduced. These data suggest that amino acid E180 may b e not only important for the binding of decorin to type I collagen, but may a lso be involved in t he binding to type VI. The interaction of type VI collagen with decorin has to be seen in the context that type VI collagen is responsible for the formation of the beaded microfibrillar network and interacts with a wide range of molecules including membrane components such as integrins [34], matrix molecules, proteoglycans [35] and matrilins [24]. The complexity of the interaction of decorin with type VI collagen suggests a possible role of decorin as a bridging molecule between the c ollagen molecules. Previous studies have shown that the binding of decorin to type VI collagen is less efficient th an the b inding of decorin t o type I collagen [36]. Therefore, we tried to optimize binding of type VI collagen t o the sensor chip by using a free sulfhydryl group and not amino groups as in previous studies [37]. This method was avoided as it has been described that immo- bilization via amino groups may lead to manifold inter- actions of the c ollagen with t he dextran m atrix o f the sen sor chip, possibly masking important binding domains on the collagen and leading to rapid saturation of the chip surface [19]. Previous studies indicated that biglycan interacts with type VI collagen and is involved in the o rganization of type VI collagen networks [37]. Our findings indicate that the binding of biglycan to type VI collagen is weaker (K D ¼ 2 · 10 )8 M ) than the binding of decorin (K D ¼ 3 · 10 )9 M ) to this type of collagen. These findings may indicate that the lower affinity of biglycan is necessary for the fast organiza- tion of the t ype VI network while decorin may have a more stabilizing function [24]. A further difference in the interac- tion of decorin and biglycan with type VI collagen was that decorin without glycosaminoglycan chain had a reduced binding affinity, whereas the interaction of biglycan with type VI collagen was independent of the presence of the glycosaminoglycan chains. Nevertheless, the glycosamino- glycan chain plays a role in guiding type VI collagen into the organized structure both in v itro [37] and in t issue [38]. These findings are of biological importance, because decorin is involved in fibrillogenesis of type I collagen and also in the generation of the microfibrillar n etwork [37]. Analysis of the secondary structure of decorin and its mutants by CD spectroscopy showed that no significant alterations were induced by the amino acid substitutions in the mutations compared to wild-type decorin. However, as CD spectra give only the overall proportion of different secondary structures, small changes in the distribution might not have been registered. DCN Q153, which lacks six of the 1 0 leucine-rich repeats of wild-type decorin, showed significantly changed C D s pectra as expected. Some changes were observed to previous results [39–41] which may be due to different expression and purification procedures. In our expression system transfected 293 cells syn thesize decorin with its normal pre- and propeptide sequences and have expression and secretion rates similar to those in normal skin fibroblasts (Fig. 4). Therefore, this system resembles more the situation in normal fibroblasts than does t hat i n an overexpressing system [39]. Crystal structures of the core proteins of small leucine-rich repeat p roteoglycans have not yet been published, although i n analogy to the structure of the ribonuclease A inhibitor, a horseshoe arch stru cture has been proposed by computer modelling [42] and t his structure is supported by electron m icroscopic observation [43]. In t his model t he a-helical motifs are located on the outer face of the horseshoe, while the parallel b-sheets are located inside [44]. These results do not clarify whether decorin can bind type I collagen and type VI collagen concurrently therefore Fig. 9. Models of the potential interaction of decorin with type I a nd type VI co llagens. Decorin is shown by the grey arrow, the tip of the arrow represents the C-terminus; the dashed line indicates the glycos- aminoglycan chain; type I collagen, black circle; type VI collagen, white c ylinder. (A) Interaction involving d imerization of decorin and binding of both collagens to the i nner surface of decorin. (B) Inter- action of type I collagen with t he inner surface of the decorin core protein and type VI c ollagen b inding with its noncollagenous domain to the outer surface of d ecorin. 3396 G. Nareyeck et al. (Eur. J. Biochem. 271) Ó FEBS 2004 we used a different app roach to study this interaction in greater detail. We formed dimeric complexes consisting of type VI collagen and decorin on th e CM5 sensor chip and applied methylated type I collagen to the complexes. As expected the complexes were still able to bind type I collagen. Surprisingly, the stability of the te rnary c omplex was higher than that of the dimeric complex of decorin with type I or type VI collagen. The existence of an interaction of de corin with type I and type VI collage n has been shown in vivo in skin [45]. More recently a comple x formation between the globular domains of collagen type VI and a decorin/matrilin-1 complex has been described which can act as a bridge between type VI and type II collagen in c artilage, whereas decorin binds to the globular N-terminal domain of type V I collagen [24]. Even though in our study type VI collagen was treated with pepsin, electron micrographs still demonstrate the presence of globular domains, so decorin could act as a bridging molecule alone, by binding to the N terminus of type VI collagen and to type I collagen. Furthermore, the dissoci- ation constants for wild-type decorin and the two decorin mutants showed a similar relation to each other in the tertiary complex compared to the interaction w ith the type I or type VI collagen alone. Therefore, two binding models are possible: (a) decorin forms a dimer and can interact with the same binding site either with type I collagen or type VI collagen (Fig. 9A). This agrees with findings of Scott and coworkers [40], w ho reported that d ecorin a nd glycosamiminoglycan-free core proteins form dimers, how- ever, the purification described in this paper was with freeze-drying. 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