Interaction of decorin with CNBr peptides from collagens I and II Evidence for multiple binding sites and essential lysyl residues in collagen Ruggero Tenni 1, *, Manuela Viola 1, *, Franz Welser 2 , Patrizia Sini 1 , Camilla Giudici 1 , Antonio Rossi 1 and M. Enrica Tira 1 1 Dipartimento di Biochimica ‘A. Castellani’, University of Pavia, Italy; 2 EMP Genetech, Denzlingen, Germany Decorin is a small leucine-rich chondroitin/dermatan sulfate proteoglycan reported t o interact with fibrillar c ollagens through its protein core and to localize at d and e bands of the c ollagen fibril banding pattern. Using a s olid-phase assay, we have determined the interaction of peptides derived by C NBr c leavage of t ype I and type II c ollagen w ith decorin e xtracted from bovine tendon and its protein core and with a recombinant decorin p reparation. At least five peptides have been found to in teract with all three decorin samples. The interaction of peptides with tendon decorin has a d issociation constant in the nanomolar range. The t riple helical conformation of the peptide trimeric species is a necessary requ isite f or the binding. All positive p eptides have a region within the d and e bands of collagen fibrils. Two chemical derivatives of collagens and of positive peptides were prepared by N-acetylation and N-methylation of the primary amino group of Lys/Hyl side chains. Chemical modifications performed in m ild conditions do not signifi- cantly alter the thermal stability of peptide trimeric species whereas they affect the interaction with decorin: N-acetyla- tion eliminates both the positive charge and the binding to decorin, whereas N-methylation preserves the cationic character and modulates the binding. We conclude that decorin makes contacts with multiple sites in type I collagen and probably also in type II c ollagen and that some collagen Lys/Hyl residues are essential for the binding. Keywords: collagen; decorin; collagen peptides; proteogly- cans; protein–protein interactio ns. Decorin is a member of the family of extracellular matrix (ECM) proteoglycans characterized by a protein core containing 10 tandem l eucine-rich repeats, each of about 24 amino acids, flanked by cysteine clusters. The N-terminal domain carries one chondroitin/dermatan sulfate glycos- aminoglycan chain and the protein core also has three consensus sites for N-linked oligosaccharides [1,2]. Leucine- rich repeats are involved in protein–protein interactions and have been found in a large number of proteins as well as small leucine-rich p roteoglycans (PGs), such as biglycan, fibromodulin and lumican [1,3,4]. Decorin is considered a key regulator of t he assembly and function of many ECMs. Decorin interacts with a variety of ECM proteins, e.g. with several c ollagen types, fibronectin and thrombospondin. Collagens have a characteristic triple helical conformation, due to the repetition of triplets Gly-X-Y. The triple helix has a high surface to volume ratio and the side c hains o f a ll X and Y r esidues a re accessible by the solvent, X more than Y positions [5]. These geometric and molecular aspects determine the ability of many collagen types to self-associate, leading to defined supramolecular structures, and collagen propensity to interact with many ligands [6]. The specific association of decorin with collagens has been reviewed [1,2]. In particular, decorin plays a role in lateral growth of collagen fibrils, delaying the lateral assembly on the surface of the fibrils [7,8]. This might control fibril dimen sions, uniformity o f fibril diameter and the regular spacing of fibrils. The pathophysiological relevance o f decorin–collagen interactions has been shown in decorin null mice: homozygous animals are characterized by skin with reduced tensile strength, containing collagen fibrils with irregular profiles due to lateral fusion [9]. Recent findings report the binding of decorin t o collagen XIV and to the N-terminal region of collagen VI [10,11]. The interplay between ECMs and cells is mediated by integrins but recent evidence has shown that there are integrin-independent effects of decorin and collagen on cellular biological activity and proliferation. These effects are mediated by interactions with cytokines or cellular receptors, e.g. interactions between decorin and transform- ing g rowth factor b or between collagens and interleukin 2, or interactions between decorin and epidermal growth factor receptors or between fibrillar collagens and discoidin domain receptors [12–16]. Decorin–collagen i nteractions are thus probably able to modulate the influence of both macromolecules on cell activities. Earlier modeling and recent evidence has shown that decorin is an arch-shaped molecule [17–19]. The convex surfac e is formed by a helices whereas the b strands lining the inner c oncavity contain s everal charged residues exposed to the solvent. The glycosaminoglycan chain and the N-linked oligosaccharides are on the same side of the molecul e. Correspondence to R. Tenni, Dipartimento di Biochimica ÔA. CastellaniÕ, University of Pavia, Via Taramelli 3b, 27100 Pavia, Italy. Fax: + 3 9 0382423108, Tel.: + 39 0382507228, E-mail: rtenni@unipv.it Abbreviations: ECM, extracellular matrix; LRR, leucine-rich repeat; PG, proteoglycan; SNHSAc, sulfosuccinimidyl acetate; T m , melting temperature. *Note: these authors contributed equally to this work. (Received 3 December 2001, accepted 1 4 January 2002) Eur. J. Biochem. 269, 1428–1437 (2002) Ó FEBS 2002 The main binding site for collagen w ithin the decorin molecule appears to be located in leucine-rich repeats (LRRs) 4–5 with a glutamate (residue 180 of the protein core) playing a critical role and there a re suggestions that decorin has a second binding site for collagen [20–22]. (For the human decorin sequence, we refer to Swiss-Prot, accession number P-07585, which reports the whole t rans- lated product still bearing a 16-residue signal and a 14-residue propeptide sequence.). As far as collagen fibrils are concerned, there is morphological evidence for the presence of chondroitin/dermatan sulfate PGs at the d and e bands in the gap zone of the fibrils formed by the quarter staggered array of type I collagen molecules, and the presence of keratan sulfate PGs at the a and c bands in the overlap z one [23,24]. A study using isolated type I p rocol- lagen molecules and de corin extracted from tissue has shown that t he binding occurs preferentially at two sites around 50 and 100 nm from the N-terminus of the triple helical domain [25]. In a different study, the sequence GAKGDRGET, at position 853–861 of the a1(I) collagen chain, was reported as the binding site for decorin [26]. The KLER and RELH sequences within decorin were suggested as possible complementary sequences of GDRGET, allow- ing m odelling of the position of decorin on the surface of a collagen fibril [18]. A further, theoretical model was postulated [17]: the molecular dimensions of the decorin structure (6.5 · 4.5 · 3 nm) are consistent with a space ab le to accommodate a single type I collagen triple helical molecule inside the concavity; this suggests that about 10 residues per collagen chain are present in the binding site of decorin. In contrast with previous findings, a very recent paper reported that recombinant decorin never subjected to the action of chaotropic agents binds near the C-terminus of the type I collagen a1(I) chain [19]. In this work we have tested the binding of decorin towards CNBr peptides derived from the a chains of type I and type II collagens, by u sing both decorin purified from tendon and its core as well as a recombinant decorin preparation. The results suggest that multiple binding sites for decorin are present in t hese collagens. We have also tested the influence on d ecorin binding of chemical modi- fication of Lys and Hyl side chains of collagens and peptides. Derivatizations that eliminate the positive charge of Lys/Hyl eliminate the binding to decorin, whereas the binding is modulated b y a modification that preserves the charge. MATERIALS AND METHODS Materials Type I collagen from bovine skin and its CNBr peptides were already available and characterized by our laboratory [27–30]. Sulfosuccinimidyl acetate, p-nitrophenyl phosphate, avidin conju gated with alkaline phosphatase, o-phenylene- diamine dihydrochloride and sulfosuccinimidobiotin were obtained from Pierce, avidin conjugated with horseradish peroxidase and a 30-kDa heparin-binding fragment of fibronectin were purchased from Sigma, chondroi- tinase ABC and AC II from Seikagaku Corporation, endoproteinase Arg-C (sequencing grade) from Roche, NaBH 3 CN (sodium cyanoborohydride) from Fluka, DEAE–Sephacel and PD-10 columns from Pharmacia, microtiter plates from Nunc. Fibronectin was a generous gift of L. Visai (Dipartimento di Biochimica ÔA. CastellaniÕ, University of Pavia, Italy). All other reagents were of analytical grade. Preparation and analysis of decorin from tendon Decorin was purified as described p reviously [31,32]. Briefly, proteoglycans were extracted from bovine tendon with 4 M guanidine hydrochloride in 50 m M acetate buffer, 5 m M benzamidine, 0.1 M e-aminocaproic acid, 10 m M EDTA, 1m M phenylmethanesulfonyl fluoride, pH 5.6, and purified by preparative ultracentrifugation (100 000 g)inaCsCl gradient in the presence of buffered 4 M guanidine hydrochloride. The f raction with density 1.5 g ÆmL )1 was adsorbed on DEAE–Sephacel and eluted with a linear 0–0.8 M NaCl gradient in the presence of 4 M urea. Decorin was desalted on PD-10 columns, freeze-dried and stored at )80 °C. The protein content of the decorin preparation was determined with Bradford’s method [33]. Electrophoretic analysis in denaturing conditions was according to Laemmli [34], both before and after chondroitinase ABC digestion [35]. The analysis of disaccharides of the glycosaminoglycan chains was performed after digestion with chondroi- tinase ABC or AC I I with standard methods [36]. Circular dichroism analysis is d escribed below. Decorin from tendon or its core were labeled with biotin as follows. The samples (1 mgÆmL )1 )inNaCl/P i were incubated with a 20-fold molar excess of sulfosuccinimido- biotin for 2 h at room temperature. Concentrated Tris/HCl buffer, pH 7.5, was then added to 50 m M final concentra- tion and the samples were incubated for 1 h, extensively dialyzed against NaCl/P i andstoredat)20 °C. Preparation and analysis of recombinant decorin A f ull-length cDNA encoding the complete human decorin was inserted into a mammalian expression vector design ed for high-level expression of recombinant proteins. This construct was used for transfection of human embryonic kidney cells (American Type Culture Collection) and antibiotic resistant cells were selected. The synthesis of recombinant decorin was checked by electrophoresis and immunoblotting with an antiserum specific for human decorin (a kind gift from H. Kresse, Mu ¨ nster, Germany). For large scale production, decorin producing cells were cultivated in a controlled fermenter system. The culture medium was DMEM/F12 supplemented with 2% fetal bovine serum. The harvested culture supernatant was centrifuged and purified. For purification, the culture medium was adjusted to 250 m M NaClandappliedona column packed with a DEAE Trisacryl matrix (Sigma) equilibrated in 250 m M NaCl, 20 m M Tris, pH 7.4. The column was washed with the same buffer. Elution of bound decorin was carried out in a step from 350 to 580 m M NaCl in 20 m M Tris, pH 7.4. The eluted fractions were passed over a Superdex 200 HR gel filtration column ( Pharmacia) equilibrated a nd eluted with 250 m M NaCl, 2 0 m M Tris, pH 7.4. The fractions containing recombinant decorin were pooled. Identity was confirmed after electophoresis and immunoblotting with the mentioned decorin antiserum. Ó FEBS 2002 Interaction of decorin with collagen peptides (Eur. J. Biochem. 269) 1429 Recombinant decorin was analyzed and biotinylated as described for tendon decorin. Preparation of type II collagen and its CNBr peptides Type II collagen was purified from bovine n asal septum [37]. Briefly, the tissue was extracted at 4 °Cfor24hwith4 M guanidine hydrochloride in Tris/HCl, pH 7.4, in the pres- ence of protease inhibitors. The residue was washed with water and resuspended at 4 °Cfor48hin0.5 M acetic acid containing 1 mgÆmL )1 pepsin and 0 .2 M NaCl. The solubi- lized material was dialyzed against 0.9 M NaCl in 0.5 M acetic acid and the precipitate of type II collagen r emoved by centrifugation, dialyzed against 0.1 M acetic acid and freeze-dried. Type II collagen CNBr peptides were pu rified essentially following the procedures used for p eptides from type I collagen, by means of a combination of gel filtration chromatography followed by ion-exchange chromatogra- phy or by reverse-phase chromatography for the two smallerpeptides[27,30]. All collagens and peptides were analyzed for purity by means of a quantitative Hyp assay [38], electrophoresis in denaturing conditions [34], N-terminal sequencing for some peptides and for conformation by means of CD spectro- scopy. Chemical modification of collagens and CNBr peptides Chemical modifications have been performed with three different methods, all involving the primary amino group of lysine and hydroxylysine side chains. After the derivatiza- tion, the s amples were exhaustively dialyzed against 0.1 M acetic acid, clarified by centrifugation, freeze-dried and stored at )80 °C. All derivatized samples have been analyzed for purity and conformation by the same methods as the underivatized ones. N-Methylation. The derivatization was performed w ith formaldehyde in the presence of NaBH 3 CN, e ssentially as described previously [39]. The incubation with HCHO/ NaBH 3 CN was performed for 2 h at room temperature followed by 12–18 h in the cold room. The derivatized samples have been dialyzed against 0.1 M NaCl, and then against 0.1 M acetic acid. N-Acetylation with acetic anhydride. The derivatization was performed essentially as described previously [40] at 0 °C. Because acetic anhydride quickly hydrolyzes to acetic acid, the pH was maintained constant by additions of aliquots of 5 M NaOH. These additions, however, intro- duce local strong basic conditions whose consequence is the breakdown of some peptide bonds and the formation o f new bonds leading to the presence of molecules both smaller and larger than a single monomeric peptide (see Results). N-Acetylation with sulfosuccinimidyl acetate (SNHSAc). This procedure is much more mild than the previous one. All operations have been performed at 4 °C. Collagenous samples (5–15 mg) were s uspended overnight in 10 mL of 0.5 M borate buffer, pH 8.5. Solid SNHSAc was quickly dissolved at 10.4 mgÆmL )1 (40 m M )in10m M acetate buffer, pH 5.4–5.6, immediately before use. SNHSAc solution was a dded under vigorous stirring to the collagen samples in order to have a 10 : 1 molar ratio between SNHSAc and p rimary amino groups. The derivatization was allowed to proceed overnight. The degree of Lys/Hyl modification was determined by a colorimetric method with sodium trinitrobenzenesulfonate, essentially as described [41], using Na-acetyl- L -lysine as the standard. The extent of derivatization w as found to be higher than 80% for most samples. A lower percentage was found for type I and II collagens when derivatized with SNHSAc (70 and 76%, respectively) a nd for two peptides from type II collagen when treated with acetic anhydride (56% for CB6 and 65% for C B8). Binding assays Collagenous samples were dissolved in 0.1 M acetic acid at 1–1.5 m gÆmL )1 and m aintained at 4 °Cfor‡ 7 days, with occasional vortexing. The actual concentration was deter- mined by means of a Hyp assay [38]. After clarification by centrifugation, working solutions were prepared by dilution with NaCl/P i ,at25lgÆmL )1 for collagens I and II or equimolecular amounts of their CNBr peptides. Control dilutions determined the amount of sodium hydroxide needed to neutralize the decrease of pH. 96-Well microtite r plates were coated overnight a t 4 °C with the solutions of collagenous samples in NaCl/P i (200 lL p er well). Control w ells were coated w ith 200 lL containing 5 lgofBSAinNaCl/P i . All analyses were done at least in triplicate. After rinsing with 0.15 M NaCl, 0.05% (v/v) Tween-20, the we lls were incubated with 2 00 lLof1% (w/v) BSA in NaCl/P i , for 1 h at room temperature. After rinsing a s above, the coated wells were incubated for 2 h at room temperature w ith 20 p mol of biotinylated decorin dissolved in 200 lLofNaCl/P i , 0.05% (v/v) Tween-20. For Scatchard analysis, constant concentrations of collagen or peptides were used for coating and incubated with increas- ing concentrations of biotinylated decorin. For every solid- phase experiment, control for dose-dependent, nonspecific binding to coated BSA wells was performed, under identical conditions. Bound decorin from tendon or the recombinant prepar- ation were detected by using avidin conjugated with alkaline phosphatase diluted 1 : 1000 in 1% BSA in NaCl/P i , 0.05% (v/v) Twee n-20 ( 200 lL p er well), f ollowed by a rinse and by 200 lL o f the substr ate solution (p-n itrophenyl phosphate at 1 mgÆmL )1 in 0.9 M diethanolamine/HCl buffer, 0.5 m M MgCl 2 ,3m M NaN 3 , pH 9.5). The absorbance was meas- ured at 405 nm before and after color development. The binding of decorin core was detected as described above but by using avidin conjugated with horseradish peroxidase: all the steps were performed in a final volume of 100 lLper well; horseradish peroxidase was diluted 1 : 1000 in 2mgÆmL )1 BSA solution, followed by a rinse and by the substrate solution (0.04% o-phenylenediamine dihydroch lo- ride and 0.04% (v/v) hydrogen peroxide in a buffer containing 514 m M disodium hydrogen phosphate, 24.3 m M citric acid, pH 5). Color development was stopped by adding 100 lLof3 M hydrochloric acid and the absorbance measured at 490–655 nm. In order to determine the amount of collagen or peptides adsorbed to microtiter wells, 5 lg of each collagen t ype or 1430 R. Tenni et al. (Eur. J. Biochem. 269) Ó FEBS 2002 equimolecular a mounts of peptides were allowed to adsorb overnight, followed by a brief rinse as above. Then, protein was e xtracted from the wells with two rinses of 200 lLof 6 M HCl and subjected to hydrolysis and Hyp quantitation [38]. The percentage of protein adsorbed to the wells was found to be 15.1% ± 3.0 for CNBr peptides, 9.4% ± 0.6 for type I and II collagen. Circular dichroism spectroscopy Solutions of collagens and p eptides were prepared by dis solving dry s amples in 0.1 M acetic acid at 1–1.5 mgÆmL )1 . All operations were performed at 4–5 °C. The solutions were equilibrated for ‡ 7 days, with occa- sional vortexing. After clarification by centrifugation, the concentration was determined by means of a Hyp assay [38]. Aliquots of the acidic solution were freeze-dried and then dissolved at a concentration o f 80 lgÆmL )1 in 0.1 M acetic acid or in NaCl/P i containing 1 m M EDTA and 1.5 m M NaN 3 [30]. These solutions were equilibrated for ‡ 7 days at 4–5 °C, with occasional vortexing. Solutions of decorin or its core were prepared in NaCl/P i at a c oncentration of 4nmolÆmL )1 . All solutions were clarified by centrifugation immediately before CD analysis. CD spectra were recorded with a cell of 1 mm path length thermostatted at the appropriate temperature. Scans were performed at 20 nm Æmin )1 , collecting data points every 0.05 nm and averaging the data at least over three scans. RESULTS Analysis on decorin Two different decorin preparations have been used: decorin extracted from tendon and a recombinant decorin, as described under Materials and methods. T he electrophoretic analysis in denaturing conditions, both before and after chondroitinase ABC digestion, is present in Fig. 1 A. On sequencing, tendon decorin showed a unique and correct sequence, DEAxGIGPEE, where x is the dermatan/chon- droitin sulfate-bearing serine residue, unrecognized by the sequencer; t he recombinant preparation showed a mixture of decorin with and without the propeptide in an about 1 : 1 ratio. CD spectra at 20 °C showed that tendon and recombinant decorin are very similar to each o ther, differing below 210 nm (Fig. 1 B). These spectra are similar to reported spectra of a recombinant decorin purified in the absence of chaotropic agents, with the exception of the wavelength of the minimum (215–216 instead of 218 nm) and very different to the spectrum of the same preparation purified in the presence of guanidine hydrochloride [42]. For each decorin preparation, the spectra at 4–30 °Care superimposable and thermal denaturation occurs at >40 °C with a small difference between tendon and recombinant decorin (Fig. 1C,D). The protein core of tendon decorin behaved like the whole proteoglycan (data not shown). Due to the small difference found in the literature for the wavelength o f the minimum between a recombinant decorin (bearing a polyhistidine tag) in the native state and after denaturation in 10 M urea/renatura- tion in 1 M urea [43], our CD spectra are empirical findings that do not necessarily demonstrate a native conformation for our decorin preparations. The determination of the disaccharide composition of the glycosaminoglycan chain after chondroitinase ABC diges- tion of tendon decorin showed a high percentage of mono- sulfated species, the 6-sulfated one prevailing: 8% of unsulfated disaccharide, 56 and 31% of 6- and 4-sulfated disaccharides, respectively, 5% of disulfated species. After chondroitinase AC II digestion the composition was found to be 11, 71, 15 and 2%, respectively. By applying the formula of Shirk et al. [44], the percentage of iduronic acid content was found to be 31%. Biotinylated decorins were used in all subsequent binding experiments with collagenous samples. Control experiments showed that competitive b inding to coated type I and II collagens exists between biotinylated decorins and unmodi- fied tendon decorin (Fig. 1E). Fig. 1. Analysis of decorin. (A) S DS/10% PAGE of tendon decorin (lanes 1 a nd 2) and rec ombinant decorin (lanes 3–4) we have used in this work, both b efore (lanes 1,3) and after ( lanes 2,4) chondroi- tinase ABC digestion. About 10 lgand5lg were analyzed for dec- orins and decorin cores, respectively. Left lane: standard protein markers and their molecular masses (in kDa). The core protein is present as two bands with apparent molecular masses of 47 and 42 kDa (arrowheads). (B) CD spectra at 20 °C o f tendon a nd recombinant decorin (continuous and dotted lines, respectively) d is- solved in NaCl/P i at 4 nmolÆmL )1 . (C,D) CD spectra at 30, 40, 45, 50 °C (identifiable from top to bottom at 205 n m) for tendon (C) or recombinant decorin (D). Spectra at 4–25 °C(notshown)aresuper- imposable with the spectrum at 30 °C. (E) Competition experimen ts between b iotinylated decorins (20 pmol) an d increasing amounts of unmodified tendon decorin (data for biotinylated tendon or recom- binantdecorinchallengedwithcollagenIasthecoatedligandare indicted by circles and rectangles, respectively; data for biotinylated tendon decorin with type II collagen are indicated by triangles). Lines are drawn as a visual aid. Ó FEBS 2002 Interaction of decorin with collagen peptides (Eur. J. Biochem. 269) 1431 Purification, chemical modification and analysis of collagenous samples Type I collagen and its CNBr peptides were already available to u s and well characterized. Pepsin-soluble type II collagen was prepared from bovine nasal septum and its CNBr peptides were purified by a combination of two chromatographic steps. CNBr peptides from collagens type I and type II used in this work are indicated in Fig. 2. The only peptide we have not been able to purify is the C-terminal peptide of the a1(II) chain, namely CB9,7, probably because this p eptide is involved in cross-linking. Chemical modification of collagens and several of their peptides was performed by derivatizing the primary amino group of Lys and Hyl side c hains: methylation with H CHO/ NaBH 3 CN that preserves the positive charge, and a cetyla- tion, either with acetic anhydride or SNHSAc, that elimin- ates the positive charge. Chemical modification of Lys/Hyl side chains causes a slower electrophoretic migration of the collagenous samples (Fig. 3A). N-Acetylated samples also have a low affinity for Coomassie Brilliant Blue R 250, the standard anionic dye we used to stain polyacrylamide gels. It should be noted that N-acetylation with acetic a nhydride is t o be avoided because it is artifactual: some peptide bonds are broken w ith the formation of i nterchain covalent bonds leading to molecular species larger than the original sample. This is particularly evident for peptid es (Fig. 3A), and also smaller m olecular species, as shown by analytical gel filtration chromatogra- phy in den aturing conditions (data not shown). A ll this is probably the consequence of the addition of concentrated sodium hydroxide during the d erivatization in order to maintain the pH constant. Using CD spectroscopy at increasing temperatures, we have determined that many CNBr peptides are able to form trimeric species that at room temperature prevail over the random-coil monomeric species; only some small CNBr peptide trimers have low melting temperatures (see Fig. 2 for the values of melting temperatures). Chemical modification of L ys/Hyl side chains in collagenous samples do not significantly modify both the triple helical conformation of the trimeric species (Fig. 3 B) and the thermal stability, with the relevant exception of N-acetylation with acetic anhydride for the reasons mentioned above. The greatest decrease of T m on derivatization in mild conditions was found to be less than 3 °C. A detailed thermodynamic analysis of the melting transition of modified peptide trimers will be described elsewhere. Binding of decorin to collagenous samples and effect of chemical modifications Equimolecular amounts of collagen type I and type II and their CNBr peptides have been used in a solid-phase assay, challenged with a constant amount of biotinylated decorin, either from tendon (intact or the protein core) or the recombinant preparation. At 23 °C, both collagen types bind decorin, as well as some CNBr peptides (asterisked in Fig. 2), namely peptides C B8, CB7 and CB6 from the a1(I) chain, CB4 from a2(I) and only peptide CB11 from a1(II). The different decorins show the same binding pattern towards t he CNBr peptides, with only some differences in the intensity for some of the peptides (Fig. 4 ). The triple helical conformation of collagenous samples is a necessary requisite for the interaction with decorin, because heat denaturation eliminates their binding (Fig. 4 ). No other p eptide showed any binding als o when the assay was performed at 4 °C(seeT m of peptides in Fig. 2 with respect to the temperature of the binding experiments). It is worth n oting that p eptide CB10 from type II collagen does not bind decorin, regardless of the fact that it is homologous to and in the homologous region of CB7. We cannot comment on a1(II) CB9,7, because we did not find it in the chromatographic purifications of our CNBr digest of type II collagen. Peptide a2(I) CB3,5 has some binding ability but the data should be judged with caution because this peptide showed a positive CD signal at 221 nm that is typical of native collagen and trimeric peptides but it is possible that it does not form trimers with the three a chains in register [28]. Fig. 2. CNBr peptides fr om typ e I and type II collagen alpha chains. Th e s ch eme shows the n ame s (in bold), position along the triple helical domain, size (number o f residues) and melting temperature of t he trimeric species of C NBr peptides. The b ottom two lines in dicate the N fi C direction with a length scale (in residues) and the banding patte rn of type I collagen fibrils [51]. Melting temperatures have be en measured in NaCl/P i containing 1m M EDTA and 1.5 m M NaN 3 (in 0 .1 M acetic acid for a2(I) CB 3,5 because of its low so lubility in NaCl/P i ); values for type I collagen p eptides are data reported previo usly [27, 30]. We h ave de termined th e abilit y to bind d ecorin f or all peptid es reported i n the sch eme (positive one s are m arked with an asterisk) and also for the composite peptide a1(I) CB2,4 (T m  28° in 0.1 M acetic acid), whereas we could not use peptide a1(II) CB9,7. 1432 R. Tenni et al. (Eur. J. Biochem. 269) Ó FEBS 2002 As controls, we h ave tested the interaction of decorin w ith other proteins: BSA, as a negative control, sho wed a much lower response than collagens and positive peptides, whereas fibronectin and a 30-kDa fibronectin fragment having heparin-binding ability showed interaction with tendon decorin (Fig. 4). Fibronectin is known to interact with decorin protein core [45]. Our data suggest that decorin interacts with multiple regions of collagen. In competition experiments, we have found that CNBr peptides in solution are not able to compete with t ype I or type II collagen for decorin. When increasing amounts of peptide a1(I) CB7 or a1(II) CB11 (up to 50-fo ld excess with respect to the collagen amount) were preincubated in solution with decorin (at room temperature for 1 h) we observed no variation in binding of decorin to microwells coated with collagen type I or type II, respec t- ively. The same null result was obtained in a competition experiment between CB11 as t he coated ligand and the same peptide in solution with decorin. The reason for this is probably t he interaction of collagen or peptide in solution with the coated c ollagenous molecules [46]. I t is a lso possible that isolated collagen trimers in solution have no or much lower affinity for decorin and that decorin binding to collagen depends on the aggregation status of collagen itself. The affinity between decorin and collagens and p eptides was determined by using constant equimolecular amounts of the collagenous samples with increasing amounts of tendon decorin (Fig. 5). The graphs in Fig. 5C,D indicate a bimodal behaviour of decorin for collagen I and II, suggesting that decorin has two distinct binding sites for these collagens, as already indicated by o thers [20–22]. Scatchard-type plots, drawn according to Hedbom & Heinegard [47], allowed the calculation of the dissociation constants reported in Table 1. Because our data for collagens I and II did not allow us to obtain meaningful values for both binding sites, we performed linear interpo- lation on all the data points (Fig. 5C,D) obtaining a single dissociation constant that is only indicative of the range. The values of K d are in t he nanomolar range and similar to the values reported in literature f or decorin f rom cartilage or tendon, using type I collagen as the ligand (30 and 16 n M ) [47,48]. Other experiments (not shown) indicated that ionic interactions play an impor tant role in the bin ding between decorin and collagen. Whereas the presence of 50 m M NaCl in the phosphate buffer improved the interaction with respect to analysis performed in NaCl/P i (150 m M NaCl), a higher concentration of salt (250 m M ) resulted in dramat- ically reduced binding. On the contrary, no influence of detergents was found, as determined b y the addition of 1% Triton to the binding solution. To further characterize the nature of the interaction between decorin and collagen, we have chemically modified collagen samples using agents that either disrupt or maintain the po sitive charge, e.g. acetylation and methyla- tion, respectively. Our results indicate that elimination of the positive charge of the side chains of Lys/Hyl residues disrupts the interaction with decorin (Fig. 6). This does not depend on the derivatizing agent, SNHSAc or acetic anhydride, indicating that the side-effects of the treatment with acetic anhydride described above are not responsible for the loss of binding. Methylation of Lys/Hyl residues by treatment with HCHO/NaBH 3 CN preserved the positive charge and this resulted in a more complex effect on binding to decorin (Fig. 6). Whereas two peptides, a1(I) CB8 and a1(II) CB11, showed an increased binding, methylation of the C-terminal half of the a1(I) resulted in either reduced binding for a1(I) CB7, or a complete l oss of the binding for a1(I) CB6. The variation of the binding ability for N-methylated samples Fig. 3. Analysis of collagen samples. Representative analyses for type I I collagen ( left column ) and two C NB r peptides (cen tral and right columns) are reported. Lane 1 indicates underivatized samples; 2, samples derivatized with HCHO/NaBH 3 CN; 3, with SNHSAc; 4, with acetic anhydride. (A) S DS/ PAGE pattern (6% acrylamide for type II collagen; 15% for peptides ). The standard anionic dye Coomassie Brilliant Blue R250 showed a low affinity for the acetylated samples whose band intensity quickly faded d uring destaning. The figures reported were obtained during the very early destaining steps. (B) CD spectra at 30 °C for type II collagen and at 20 °C for the two peptides. All samples were dissolved at 80 lgÆmL )1 in NaCl/P i containing 1 m M EDTA and 1 .5 m M NaN 3 . The figure s re port on ly th e po rtion o f t he spect rum ce ntered o n th e m aximum o f t he po sitive peak ( 221 nm); this positive signal is present only for collagenous samples with triple helical conformation. Ó FEBS 2002 Interaction of decorin with collagen peptides (Eur. J. Biochem. 269) 1433 with respect to the unmodified ones is not related to the percentage of Lys/Hyl side chains that did not react with the derivatizing agent (the percentage ranged from 3 to 12%). Taken together, these data demonstrate the essential role of the positive charge of collagen Lys/Hyl residues for interaction with decorin. DISCUSSION The binding of decorin with fibrillar collagens has been extensively investigated (reviewed in [1,2]), but in vitro studies have not yet conclusively identified the collagen domains responsible for the specific association with decorin. In this study, we have analyzed the binding between decor in and CNBr peptides from type I and type II collagens, both unmodified and chemically derivatized. We have recently characterized CNBr peptides from collagen type I [27–30]. The present work indicates that CNBr peptides from type II collagen have a very similar behaviour. Our data on the interactions between type I a nd type II collagens, their peptides and decorin reveal the following. (a) Type I and p robably also type II collagen appear to have multiple b inding sites for decorin, because several CNBr peptid es are able to interact with t his s mall proteo- glycan. (b) The side chain of Lys/Hyl residues in collagen is relevant for t he binding, because the elimination of their positive charge eliminates the interaction. On the contrary, the chemical modification preserving the ionic character modulates the binding to decorin. This leads to a differential behaviour for the different peptides. (c) Decorin might have two binding sites for collagen, as suggested by others and by the differential behaviour of collagen peptides. Decorin is able to bind several CNBr pe ptides and type I and II collagens only when they are in triple helical conformation. Among the binding peptides, CB8, CB4 and CB11 are found in a homologous region of the N-terminal half of the respective a chains (residues 124–327 of the triple helical domain). On the contrary, CB7 and CB6 lie in the C-terminal half of the chain . Binding specificity is demonstrated by the following. (a) The absence of interaction with deco rin(s) of some peptides that are in triple helical conformation in our assay conditions [CB2, CB2,4 and CB3 from a1(I), CB12, CB8 and CB10 from a1(II)]. (b) A ll peptides able to bind decorin contain a region corresponding to the d and e bands of collagen fibrils (Fig. 2 ). This is in accordance with morphological findings showing that chondroitin/dermatan sulfate PGs, such as Fig. 4. Binding of biotinylated decorins to collagenous samples. A constant amount of type I or II collagen (5 lg) or equim olecular amounts of their CNBr pe ptides were used to coat polystyrene wells. A constant a mount of biotinylated decorin was added (20 pmol); the bound decorin was de termined using avidin conjugated with alkaline phosphatase or, for tendon protein core, horseradish peroxidase. The absorbance plotted in t he panels for a ll collagens and peptides we h ave tested was determined by exploiting a c olorimetric reactio n catalyzed by the enzyme. The absorbance is the mean of analyses performed at least in triplicate; the highest standard deviation for samples able to bind decorin w as 17% o f t he m ean. T op: analysis w ith tend on de corin on collagen samples in native an d in denaturin g conditions (wh ite and black columns, r espectively). The righ t panel reports t he binding of tendon decorin to BSA, fibronectin and a 30-kDa heparin-binding fragment of fi bronectin. Bottom: analys is on collagen samples with tendon decorin core (dark gray) and recombinant decorin (light gray). (n.d. not determined.) Fig. 5. Affinity of collagenous samples with decorin. Increasing amounts of b iotinylated t endon d ecorin w ere a dded t o polystyre ne wells coat ed with a constan t a mount of collagen (5 lg) or equi- molecular a mounts of CNBr peptides. The b indin g was determined by using avidin c onjugated with alkaline phosphatase . (A,B) Saturation curves of two collagens and two peptides reported as examples. Each data p o int is the average va lue of a determination performed at least i n triplicate. The highest standard deviation was 1 8% of the m ean. Lines are add ed as a visua l h elp. (C,D) Scatchard-type plots [ 47] on the same samples. Lines interpolating the d ata have been computed with the least square method. For type I a nd II collagens, linear interpolation was performed taking into account all data points (see text). The resulting dissociation constants are reported in Table 1. 1434 R. Tenni et al. (Eur. J. Biochem. 269) Ó FEBS 2002 decorin, localize in these bands, whereas keratan sulfate PG are present at a and c bands [23,24]. However, not all collagen peptides that contain regions of the collagen molecule falling within the d band interact with decorin, e.g. the homologous peptides a1(I) CB3 and a1(II) CB8, or peptide a1(II) CB10. Collagen binding to decorin does not therefore depend on the clusters of charged residues responsible of the banding pattern but on specific sequences that contain ionic residues. (c) The action on platelet adhesion and activation by only two peptides from type II collagen (data not shown) and only by peptide a1(I) CB3 from t ype I collagen, as already known from the literature [49]. Peptide CB10 from type II collagen i s homologous to a1(I) CB7, but does not interact with decorin. One possible explanation o f this d iscrepancy could lie in the fact that type II collagen is more glycosylated than type I collagen. It seems to us probable that glycosylation of hydroxylysine will block the binding. However, the glycosylation pattern of Hyl residues is known for CB7 [50] but not for CB10. Aliquots of both CB10 and CB7 have also been digested at 37 °C for 18 h with endoproteinase Arg-C, according to the manufacturer’s guidelines, with an e nzyme to s ubstrate ratio of 1 : 130. None of the most abundant fragments, separated by reverse-phase HPLC with the same protocol used to separe small CNBr peptides, showed at 4 °C any binding ability to tendon decorin (data not shown). This suggests that also some Arg-containing sequences are relevant in collagen for its interaction with decorin, or that none o f the fragment was p resent in our assay conditions as a trimeric species, or that the minor enzyme activity cleaving Lys peptide bonds had a relevant effect. The affinity o f the b inding peptides for decorin is in the nanomolar range with the same magnitude reported by others for type I collagen [47,48], and the dissociation constants are within one order of magnitude (Table 1). Our determinations showed also that the binding between decorin and collagens or their CNBr peptides is quite sensitive to the ionic strength of the buffer, suggesting an ionic character of the binding. The main decorin r egion implicated in the binding to collagens was h ypothesized to lie inside the concave area of the arch-shaped protein core [17]. Residues in LRR 4 and 5 were considered responsible for the binding [21]. The concave surface, formed by b strands, is lined by many charged residues and several hydrophobic side chains. Charged residues probably make ionic con tacts; in partic- ular, carboxylate ions might bridge two positive residues, and/or Lys ammonium ions or Arg guanidinium ions might bridge two negative groups. One of the relevant residues is glutamate-180 found by Kresse and coworkers to be relevant for the collagen binding [22]. It should however, be noted that the constructs lacking LRR 5 or bearing the substitution Glu180 to Lys [22] bring several positive charges close to each other. This might have a direct influence on the conformation of decorin core, and only an indirect one on the collagen binding. However, this remains a h ypothesis, as, to our knowledge, no conformational analysis was reported on these constructs. The presence in the decorin molecule of a second binding site for collagen was suggested previously [20–22]. The results we have obtained from the Scatchard-type plots for type I and type II collagens (Fig. 5) and the different behavior of the N-terminal collagen peptides with respect to CB7 and CB6 might be a further support to this hypothesis. Chemical modification of collagens and their CNBr peptides demonstrated that acetylation eliminates their binding to decorin. Lys/Hyl side chains are therefore present at, or very near to the binding site(s) and their positive charge is a stringent requisite for the binding. This is not surprising, if i ndeed collagen binds inside the concave surface o f decorin, owing t o the presence of an ele vated Fig. 6. Effect of c hemical modifications. A constant amount o f type I or II collagen (5 lg) or equimolecular amou nts of their C NBr peptides were use d to c oat polystyrene w ells. A constant amount o f biotinylated tendon decorin was added ( 20 pmol); the b ound decorin was deter- mined using avidin conjugat ed with alkaline phosphatase. The absorbance is the average value of at least three determinations; the highest standard deviation for samples able to bind decorin was 17% of the mean. For each collagenous sample used in native conditions, the results o f the underivatized sam ple (white column) a nd for derivatives with SNHSAc (black) and HCHO/NaBH 3 CN (gray) are reported. The results obtained with samples treated with acetic anhydride (not shown) overlap those with SNHSAc. Table 1. Dissociation constants of the complexes b etween biotinylated tendon decorin and collagenous samples. Collagen sample K d (n M ) Type I collagen 41 a CB6 from a1(I) b CB7 from a1(I) 13 CB8 from a1(I) 44 CB4 from a2(I) 16 Type II collagen 42 a CB11 from a1(II) 22 a The value reported was obtained from the linear interpolation of all data points (Fig. 5C,D), because it was impossible from our data to calculate meaningful values for two binding sites. b It was impossible to calculate the dissociation constant for this peptide because a saturation level was not clearly identifiable. Ó FEBS 2002 Interaction of decorin with collagen peptides (Eur. J. Biochem. 269) 1435 number of ionic residues. On the contrary, reductive methylation modulates the binding of all peptides to decorin, the largest decrease being shown by a1(I) CB7 and CB6 (Fig. 6), suggesting a different specificity of t hese peptides. All these effects are direct, because all p eptides we have derivatize d in mild conditions maintain the ability t o form trimeric species that are the major species in our binding assays. A previous study reported that decorin binding occurs preferentially at about 50 and 100 nm from the N-terminus of type I collagen [25]. We found the region  50 nm from the N-terminus falls w ithin peptide s CB8, CB4 a nd CB11 and was able to bind decorin. Apart from the presence of Lys, we are not able to compare our data with the suggestion of a collagen sequence able to bind decorin, namely GAKGDRGET, at position 853–861 of the triple helical domain of the a1(I) chain, within peptide CB7 [26]. A similar sequence is present in the homologous region of type II and III collagens. Without GAK, the sequence G-D/ E-R-G-E-Hyp/T is present also at position 623–628 of the same chain (in peptide CB7) a nd of homologous sequences of other collagen alpha chains. The collagen sequence DRGE might have KLER an d RELH as possible comple- mentary sequences in decorin [18], at position 130–133 and 272–275, in the L RR 3 and 9, respectively. The m odel proposed on the basis of these complementary sequences in the two interacting proteins showed a double contact between decorin and two collagen molecules. However, this is discordant with the decorin model [3,4,17] where the ionic residues of KLER/RELH fall inside the concave surface o f decorin, with the excep tion of K-130. It is not possible to reconcile our findings with most results recently reported by Keene et al .[19]whicharein disagreement with many previous results, as widely dis- cussed in the paper. On one side, a periodicity was noticed by these authors in aggregates of decorin and type I pC-collagen seen in electron micrographs of rotary shad- owed molecules; this was due to the presence of decorin, as pC-collagen alone did not show a similar pattern. CNBr peptides of the a1(I) chain that we have found to bind decorin are positioned along the chain in a manner that periodicity of binding is the natural outcome, even if our data do not allow a determination of the size of the period and even if peptide CB3, unable to bind decorin, interrupts the periodicity. On the other side, the relevance of Lys/Hyl residues both in collagens and peptides for interaction with decorin is in contrast with the findings that the binding site for decorin is located in a seq uence within the peptide a1(I) CB6 devoid of any Lys/Hyl residue and containing, as ionic amino acids, only one Glu and one Arg, 13 residues apart. It is interesting to note that the same region of the a2(I) chain contains the d ipeptide HH . The triplet GHH is unique in the triple helical domain of all collagen chains, as determined by a search in Swiss-Prot. One c an thus hypothesize that the polyhistidine tag present in the recombinant decorin preparation used by Keene et al. [19] is able, in the presence of minute amounts of proper cations, to interact with GHH in a2(I) and direct the binding of decorin to the collagen C-terminus in CB6. However, this cannot be deduced because no control experiments are reported with decorin lacking the polyhistidine tag or with decorin purified in the presence of chaotropic agents to c ompare with co nditions used in previous determinations. On this basis, we can conclude the precise location and the relative o rientation of the binding sites in decorin and collagen are not yet known. 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(1990) The a 2 b 1 integrin cell surface collagen receptor binds to the a1(I)-CB3 peptide of collagen. J. Biol. Chem 265, 4778–4781. 50. Ibrahim, J. & Harding, J.J. (1989) Pinpointing the sites of hydroxylysine glycosides in peptide a1-CB7 of bovine c orneal collagen, and t heir possible r ole i n de termining fib ril d iameter a nd thus transparency. Biochim. Biophys. A cta 992, 9–22. 51. Chapman, J.A. & Hulmes, D .J.S. (1984) Electron microscopy of the collagen fibril. In Ultrastructure of the Connective Tissue Matrix. (Ruggeri,A.&Motta,P.M.,eds),pp.1–33.M.Nijhoff Publishers, Boston, MA. Ó FEBS 2002 Interaction of decorin with collagen peptides (Eur. J. Biochem. 269) 1437 . Interaction of decorin with CNBr peptides from collagens I and II Evidence for multiple binding sites and essential lysyl residues in collagen Ruggero. the binding of decorin towards CNBr peptides derived from the a chains of type I and type II collagens, by u sing both decorin purified from tendon and its