interaction of decorin with cnbr peptides from collagens i and ii

Interaction of decorin with CNBr peptides from collagens I and IIEvidence for multiple binding sites and essential lysyl residues in collagen

Ruggero Tenni1,*, Manuela Viola1,*, Franz Welser2, Patrizia Sini1, Camilla Giudici1, Antonio Rossi1

and M. Enrica Tira1

1Dipartimento di Biochimica ‘A. Castellani’, University of Pavia, Italy; 2EMP Genetech, Denzlingen, Germany

Decorin is a small leucine-rich chondroitin/dermatan sulfateproteoglycan reported to interact with fibrillar collagensthrough its protein core and to localize at d and e bands ofthe collagen fibril banding pattern. Using a solid-phaseassay, we have determined the interaction of peptidesderived byCNBr cleavage of type I and type II collagenwithdecorin extracted from bovine tendon and its protein coreand with a recombinant decorin preparation. At least fivepeptides have been found to interact with all three decorinsamples. The interaction of peptideswith tendondecorin hasa dissociation constant in the nanomolar range. The triplehelical conformation of the peptide trimeric species is anecessary requisite for the binding.All positive peptides havea region within the d and e bands of collagen fibrils. Twochemical derivatives of collagens and of positive peptides

were prepared by N-acetylation and N-methylation of theprimary amino group of Lys/Hyl side chains. Chemicalmodifications performed in mild conditions do not signifi-cantly alter the thermal stability of peptide trimeric specieswhereas they affect the interaction with decorin: N-acetyla-tion eliminates both the positive charge and the binding todecorin, whereas N-methylation preserves the cationiccharacter and modulates the binding. We conclude thatdecorin makes contacts with multiple sites in type I collagenand probably also in type II collagen and that some collagenLys/Hyl residues are essential for the binding.

Keywords: collagen; decorin; collagen peptides; proteogly-cans; protein–protein interactions.

Decorin is a member of the family of extracellular matrix(ECM) proteoglycans characterized by a protein corecontaining 10 tandem leucine-rich repeats, each of about24 amino acids, flanked by cysteine clusters. The N-terminaldomain carries one chondroitin/dermatan sulfate glycos-aminoglycan chain and the protein core also has threeconsensus sites for N-linked oligosaccharides [1,2]. Leucine-rich repeats are involved in protein–protein interactions andhave been found in a large number of proteins as well assmall leucine-rich proteoglycans (PGs), such as biglycan,fibromodulin and lumican [1,3,4].

Decorin is considered a key regulator of the assembly andfunction of many ECMs. Decorin interacts with a variety ofECM proteins, e.g. with several collagen types, fibronectinand thrombospondin. Collagens have a characteristic triplehelical conformation, due to the repetition of tripletsGly-X-Y. The triple helix has a high surface to volumeratio and the side chains of all X and Y residues areaccessible by the solvent, Xmore thanY positions [5]. Thesegeometric and molecular aspects determine the ability ofmany collagen types to self-associate, leading to defined

supramolecular structures, and collagen propensity tointeract with many ligands [6].

The specific association of decorin with collagens hasbeen reviewed [1,2]. In particular, decorin plays a role inlateral growth of collagen fibrils, delaying the lateralassembly on the surface of the fibrils [7,8]. This mightcontrol fibril dimensions, uniformity of fibril diameter andthe regular spacing of fibrils. The pathophysiologicalrelevance of decorin–collagen interactions has been shownin decorin null mice: homozygous animals are characterizedby skin with reduced tensile strength, containing collagenfibrils with irregular profiles due to lateral fusion [9]. Recentfindings report the binding of decorin to collagen XIV andto the N-terminal region of collagen VI [10,11].

The interplay between ECMs and cells is mediated byintegrins but recent evidence has shown that there areintegrin-independent effects of decorin and collagen oncellular biological activity and proliferation. These effectsare mediated by interactions with cytokines or cellularreceptors, e.g. interactions between decorin and transform-ing growth factor b or between collagens and interleukin 2,or interactions between decorin and epidermal growthfactor receptors or between fibrillar collagens and discoidindomain receptors [12–16]. Decorin–collagen interactions arethus probably able to modulate the influence of bothmacromolecules on cell activities.

Earlier modeling and recent evidence has shown thatdecorin is an arch-shaped molecule [17–19]. The convexsurface is formed by a helices whereas the b strands liningthe inner concavity contain several charged residues exposedto the solvent. The glycosaminoglycan chain and theN-linked oligosaccharides are on the same side of themolecule.

Correspondence to R. Tenni, Dipartimento di Biochimica

ÔA. CastellaniÕ, University of Pavia, Via Taramelli 3b, 27100 Pavia,

Italy. Fax: + 39 0382423108, Tel.: + 39 0382507228,

E-mail: [email protected]

Abbreviations: ECM, extracellular matrix; LRR, leucine-rich

repeat; PG, proteoglycan; SNHSAc, sulfosuccinimidyl acetate;

Tm, melting temperature.

*Note: these authors contributed equally to this work.

(Received 3 December 2001, accepted 14 January 2002)

Eur. J. Biochem. 269, 1428–1437 (2002) Ó FEBS 2002

The main binding site for collagen within the decorinmolecule appears to be located in leucine-rich repeats(LRRs) 4–5 with a glutamate (residue 180 of the proteincore) playing a critical role and there are suggestions thatdecorin has a second binding site for collagen [20–22]. (Forthe human decorin sequence, we refer to Swiss-Prot,accession number P-07585, which reports the whole trans-lated product still bearing a 16-residue signal and a14-residue propeptide sequence.). As far as collagen fibrilsare concerned, there is morphological evidence for thepresence of chondroitin/dermatan sulfate PGs at the d and ebands in the gap zone of the fibrils formed by the quarterstaggered array of type I collagen molecules, and thepresence of keratan sulfate PGs at the a and c bands in theoverlap zone [23,24]. A study using isolated type I procol-lagen molecules and decorin extracted from tissue hasshown that the binding occurs preferentially at two sitesaround 50 and 100 nm from the N-terminus of the triplehelical domain [25]. In a different study, the sequenceGAKGDRGET, at position 853–861 of the a1(I) collagenchain, was reported as the binding site for decorin [26]. TheKLER andRELH sequences within decorin were suggestedas possible complementary sequences of GDRGET, allow-ing modelling of the position of decorin on the surface of acollagen fibril [18]. A further, theoretical model waspostulated [17]: the molecular dimensions of the decorinstructure (6.5 · 4.5 · 3 nm) are consistent with a space ableto accommodate a single type I collagen triple helicalmolecule inside the concavity; this suggests that about 10residues per collagen chain are present in the binding site ofdecorin. In contrast with previous findings, a very recentpaper reported that recombinant decorin never subjected tothe action of chaotropic agents binds near the C-terminus ofthe type I collagen a1(I) chain [19].

In this work we have tested the binding of decorintowards CNBr peptides derived from the a chains of type Iand type II collagens, by using both decorin purified fromtendon and its core as well as a recombinant decorinpreparation. The results suggest that multiple binding sitesfor decorin are present in these collagens. We have alsotested the influence on decorin binding of chemical modi-fication of Lys and Hyl side chains of collagens andpeptides. Derivatizations that eliminate the positive chargeof Lys/Hyl eliminate the binding to decorin, whereas thebinding is modulated by a modification that preserves thecharge.

M A T E R I A L S A N D M E T H O D S

Materials

Type I collagen from bovine skin and its CNBr peptideswere already available and characterized by our laboratory[27–30].

Sulfosuccinimidyl acetate, p-nitrophenyl phosphate,avidin conjugated with alkaline phosphatase, o-phenylene-diamine dihydrochloride and sulfosuccinimidobiotin wereobtained from Pierce, avidin conjugated with horseradishperoxidase and a 30-kDa heparin-binding fragment offibronectin were purchased from Sigma, chondroi-tinase ABC and AC II from Seikagaku Corporation,endoproteinase Arg-C (sequencing grade) from Roche,NaBH3CN (sodium cyanoborohydride) from Fluka,

DEAE–Sephacel and PD-10 columns from Pharmacia,microtiter plates from Nunc. Fibronectin was a generousgift of L. Visai (Dipartimento di Biochimica ÔA. CastellaniÕ,University of Pavia, Italy). All other reagents were ofanalytical grade.

Preparation and analysis of decorin from tendon

Decorin was purified as described previously [31,32]. Briefly,proteoglycans were extracted from bovine tendon with 4 M

guanidine hydrochloride in 50 mM acetate buffer, 5 mM

benzamidine, 0.1 M e-aminocaproic acid, 10 mM EDTA,1 mM phenylmethanesulfonyl fluoride, pH 5.6, and purifiedby preparative ultracentrifugation (100 000 g) in a CsClgradient in the presence of buffered 4 M guanidinehydrochloride. The fraction with density 1.5 gÆmL)1 wasadsorbed on DEAE–Sephacel and eluted with a linear0–0.8 M NaCl gradient in the presence of 4 M urea. Decorinwas desalted on PD-10 columns, freeze-dried and storedat )80 °C.

The protein content of the decorin preparation wasdetermined with Bradford’s method [33]. Electrophoreticanalysis in denaturing conditions was according to Laemmli[34], both before and after chondroitinase ABC digestion[35]. The analysis of disaccharides of the glycosaminoglycanchains was performed after digestion with chondroi-tinase ABC or AC II with standard methods [36]. Circulardichroism analysis is described below.

Decorin from tendon or its core were labeled with biotinas follows. The samples (1 mgÆmL)1) in NaCl/Pi wereincubated with a 20-fold molar excess of sulfosuccinimido-biotin for 2 h at room temperature. Concentrated Tris/HClbuffer, pH 7.5, was then added to 50 mM final concentra-tion and the samples were incubated for 1 h, extensivelydialyzed against NaCl/Pi and stored at )20 °C.

Preparation and analysis of recombinant decorin

A full-length cDNA encoding the complete human decorinwas inserted into a mammalian expression vector designedfor high-level expression of recombinant proteins. Thisconstruct was used for transfection of human embryonickidney cells (American Type Culture Collection) andantibiotic resistant cells were selected. The synthesis ofrecombinant decorin was checked by electrophoresis andimmunoblotting with an antiserum specific for humandecorin (a kind gift from H. Kresse, Munster, Germany).For large scale production, decorin producing cells werecultivated in a controlled fermenter system. The culturemedium was DMEM/F12 supplemented with 2% fetalbovine serum. The harvested culture supernatant wascentrifuged and purified. For purification, the culturemedium was adjusted to 250 mM NaCl and applied on acolumn packed with a DEAE Trisacryl matrix (Sigma)equilibrated in 250 mM NaCl, 20 mM Tris, pH 7.4. Thecolumn was washed with the same buffer. Elution of bounddecorin was carried out in a step from 350 to 580 mM NaClin 20 mM Tris, pH 7.4. The eluted fractions were passedover a Superdex 200 HR gel filtration column (Pharmacia)equilibrated and eluted with 250 mM NaCl, 20 mM Tris,pH 7.4. The fractions containing recombinant decorin werepooled. Identity was confirmed after electophoresis andimmunoblotting 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 asdescribed for tendon decorin.

Preparation of type II collagen and its CNBr peptides

Type II collagenwas purified frombovine nasal septum [37].Briefly, the tissue was extracted at 4 °C for 24 h with 4 M

guanidine hydrochloride in Tris/HCl, pH 7.4, in the pres-ence of protease inhibitors. The residue was washed withwater and resuspended at 4 °C for 48 h in 0.5 M acetic acidcontaining 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 removedby centrifugation, dialyzed against 0.1 M acetic acid andfreeze-dried.

Type II collagen CNBr peptides were purified essentiallyfollowing the procedures used for peptides from type Icollagen, by means of a combination of gel filtrationchromatography followed by ion-exchange chromatogra-phy or by reverse-phase chromatography for the twosmaller peptides [27,30].

All collagens and peptides were analyzed for purity bymeans of a quantitative Hyp assay [38], electrophoresis indenaturing conditions [34], N-terminal sequencing for somepeptides and for conformation by means of CD spectro-scopy.

Chemical modification of collagens and CNBr peptides

Chemical modifications have been performed with threedifferent methods, all involving the primary amino group oflysine and hydroxylysine side chains. After the derivatiza-tion, the samples were exhaustively dialyzed against 0.1 M

acetic acid, clarified by centrifugation, freeze-dried andstored at )80 °C. All derivatized samples have beenanalyzed for purity and conformation by the same methodsas the underivatized ones.

N-Methylation. The derivatization was performed withformaldehyde in the presence of NaBH3CN, essentially asdescribed previously [39]. The incubation with HCHO/NaBH3CN was performed for 2 h at room temperaturefollowed by 12–18 h in the cold room. The derivatizedsamples have been dialyzed against 0.1 M NaCl, and thenagainst 0.1 M acetic acid.

N-Acetylation with acetic anhydride. The derivatizationwas performed essentially as described previously [40] at0 °C. Because acetic anhydride quickly hydrolyzes to aceticacid, the pH was maintained constant by additions ofaliquots of 5 M NaOH. These additions, however, intro-duce local strong basic conditions whose consequence is thebreakdown of some peptide bonds and the formation ofnew bonds leading to the presence of molecules bothsmaller and larger than a single monomeric peptide (seeResults).

N-Acetylation with sulfosuccinimidyl acetate (SNHSAc).This procedure is much more mild than the previous one.All operations have been performed at 4 °C. Collagenoussamples (5–15 mg) were suspended overnight in 10 mL of0.5 M borate buffer, pH 8.5. Solid SNHSAc was quicklydissolved at 10.4 mgÆmL)1 (40 mM) in 10 mM acetate

buffer, pH 5.4–5.6, immediately before use. SNHSAcsolution was added under vigorous stirring to the collagensamples in order to have a 10 : 1 molar ratio betweenSNHSAc and primary amino groups. The derivatizationwas allowed to proceed overnight.

The degree of Lys/Hyl modification was determined by acolorimetric method with sodium trinitrobenzenesulfonate,essentially as described [41], using Na-acetyl-L-lysine as thestandard. The extent of derivatization was found to behigher than 80% for most samples. A lower percentage wasfound for type I and II collagens when derivatized withSNHSAc (70 and 76%, respectively) and for two peptidesfrom type II collagen when treated with acetic anhydride(56% for CB6 and 65% for CB8).

Binding assays

Collagenous samples were dissolved in 0.1 M acetic acid at1–1.5 mgÆmL)1 and maintained at 4 °C for ‡ 7 days, withoccasional vortexing. The actual concentration was deter-mined by means of a Hyp assay [38]. After clarification bycentrifugation, working solutions were prepared by dilutionwith NaCl/Pi, at 25 lgÆmL)1 for collagens I and II orequimolecular amounts of their CNBr peptides. Controldilutions determined the amount of sodium hydroxideneeded to neutralize the decrease of pH.

96-Well microtiter plates were coated overnight at 4 °Cwith the solutions of collagenous samples in NaCl/Pi(200 lL per well). Control wells were coated with 200 lLcontaining 5 lg of BSA in NaCl/Pi. All analyses were doneat least in triplicate. After rinsing with 0.15 M NaCl, 0.05%(v/v) Tween-20, the wells were incubated with 200 lL of 1%(w/v) BSA in NaCl/Pi, for 1 h at room temperature. Afterrinsing as above, the coated wells were incubated for 2 h atroom temperature with 20 pmol of biotinylated decorindissolved in 200 lL of NaCl/Pi, 0.05% (v/v) Tween-20. ForScatchard analysis, constant concentrations of collagen orpeptides were used for coating and incubated with increas-ing concentrations of biotinylated decorin. For every solid-phase experiment, control for dose-dependent, nonspecificbinding to coated BSAwells was performed, under identicalconditions.

Bound decorin from tendon or the recombinant prepar-ation were detected by using avidin conjugated with alkalinephosphatase diluted 1 : 1000 in 1%BSA in NaCl/Pi, 0.05%(v/v) Tween-20 (200 lLper well), followed by a rinse and by200 lL of the substrate solution (p-nitrophenyl phosphateat 1 mgÆmL)1 in 0.9 M diethanolamine/HCl buffer, 0.5 mM

MgCl2, 3 mM NaN3, pH 9.5). The absorbance was meas-ured at 405 nm before and after color development. Thebinding of decorin core was detected as described above butby using avidin conjugated with horseradish peroxidase: allthe steps were performed in a final volume of 100 lL perwell; horseradish peroxidase was diluted 1 : 1000 in2 mgÆmL)1 BSA solution, followed by a rinse and by thesubstrate solution (0.04% o-phenylenediamine dihydrochlo-ride and 0.04% (v/v) hydrogen peroxide in a buffercontaining 514 mM disodium hydrogen phosphate,24.3 mM citric acid, pH 5). Color development was stoppedby adding 100 lL of 3 M hydrochloric acid and theabsorbance measured at 490–655 nm.

In order to determine the amount of collagen or peptidesadsorbed to microtiter wells, 5 lg of each collagen type or

1430 R. Tenni et al. (Eur. J. Biochem. 269) Ó FEBS 2002

equimolecular amounts of peptides were allowed to adsorbovernight, followed by a brief rinse as above. Then, proteinwas extracted from the wells with two rinses of 200 lL of6 M HCl and subjected to hydrolysis and Hyp quantitation[38]. The percentage of protein adsorbed to the wells wasfound to be 15.1% ± 3.0 for CNBr peptides, 9.4% ± 0.6for type I and II collagen.

Circular dichroism spectroscopy

Solutions of collagens and peptides were preparedby dissolving dry samples in 0.1 M acetic acid at1–1.5 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, theconcentrationwas determined bymeans of aHyp assay [38].Aliquots of the acidic solution were freeze-dried and thendissolved at a concentration of 80 lgÆmL)1 in 0.1 M aceticacid or in NaCl/Pi containing 1 mM EDTA and 1.5 mM

NaN3 [30]. These solutions were equilibrated for ‡ 7 daysat 4–5 °C, with occasional vortexing. Solutions of decorinor its core were prepared in NaCl/Pi at a concentration of4 nmolÆmL)1. All solutions were clarified by centrifugationimmediately before CD analysis. CD spectra were recordedwith a cell of 1 mm path length thermostatted atthe appropriate temperature. Scans were performed at20 nmÆmin)1, collecting data points every 0.05 nm andaveraging the data at least over three scans.

R E S U L T S

Analysis on decorin

Two different decorin preparations have been used: decorinextracted from tendon and a recombinant decorin, asdescribed underMaterials andmethods. The electrophoreticanalysis in denaturing conditions, both before and afterchondroitinase ABC digestion, is present in Fig. 1A. Onsequencing, tendon decorin showed a unique and correctsequence, DEAxGIGPEE, where x is the dermatan/chon-droitin sulfate-bearing serine residue, unrecognized by thesequencer; the recombinant preparation showed a mixtureof decorin with and without the propeptide in an about 1 : 1ratio. CD spectra at 20 °C showed that tendon andrecombinant decorin are very similar to each other, differingbelow 210 nm (Fig. 1B). These spectra are similar toreported spectra of a recombinant decorin purified in theabsence of chaotropic agents, with the exception of thewavelength of the minimum (215–216 instead of 218 nm)and very different to the spectrum of the same preparationpurified in the presence of guanidine hydrochloride [42]. Foreach decorin preparation, the spectra at 4–30 °C aresuperimposable and thermal denaturation occurs at>40 °C with a small difference between tendon andrecombinant decorin (Fig. 1C,D). The protein core oftendon decorin behaved like the whole proteoglycan (datanot shown). Due to the small difference found in theliterature for the wavelength of the minimum between arecombinant decorin (bearing a polyhistidine tag) in thenative state and after denaturation in 10 M urea/renatura-tion in 1 M urea [43], our CD spectra are empirical findingsthat do not necessarily demonstrate a native conformationfor our decorin preparations.

The determination of the disaccharide composition of theglycosaminoglycan chain after chondroitinase ABC diges-tion of tendon decorin showed a high percentage of mono-sulfated species, the 6-sulfated one prevailing: 8% ofunsulfated disaccharide, 56 and 31% of 6- and 4-sulfateddisaccharides, respectively, 5% of disulfated species. Afterchondroitinase AC II digestion the composition was foundto be 11, 71, 15 and 2%, respectively. By applying theformula of Shirk et al. [44], the percentage of iduronic acidcontent was found to be 31%.

Biotinylated decorins were used in all subsequent bindingexperiments with collagenous samples. Control experimentsshowed that competitive binding to coated type I and IIcollagens exists between biotinylated decorins and unmodi-fied tendon decorin (Fig. 1E).

Fig. 1. Analysis of decorin. (A) SDS/10% PAGE of tendon decorin

(lanes 1 and 2) and recombinant decorin (lanes 3–4) we have used in

this work, both before (lanes 1,3) and after (lanes 2,4) chondroi-

tinase ABC digestion. About 10 lg and 5 lg 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 of tendon and

recombinant decorin (continuous and dotted lines, respectively) dis-

solved in NaCl/Pi at 4 nmolÆmL)1. (C,D) CD spectra at 30, 40, 45,

50 °C (identifiable from top to bottom at 205 nm) for tendon (C) or

recombinant decorin (D). Spectra at 4–25 °C (not shown) are super-

imposable with the spectrum at 30 °C. (E) Competition experiments

between biotinylated decorins (20 pmol) and increasing amounts of

unmodified tendon decorin (data for biotinylated tendon or recom-

binant decorin challenged with collagen I as the coated ligand are

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.


Purification, chemical modification and analysisof collagenous samples

Type I collagen and its CNBr peptides were alreadyavailable to us and well characterized. Pepsin-soluble typeII collagen was prepared from bovine nasal septum and itsCNBr peptides were purified by a combination of twochromatographic steps. CNBr peptides from collagens typeI and type II used in this work are indicated in Fig. 2. Theonly peptide we have not been able to purify is theC-terminal peptide of the a1(II) chain, namely CB9,7,probably because this peptide is involved in cross-linking.

Chemical modification of collagens and several of theirpeptides was performed by derivatizing the primary aminogroup of Lys andHyl side chains: methylation withHCHO/NaBH3CN that preserves the positive charge, and acetyla-tion, either with acetic anhydride or SNHSAc, that elimin-ates the positive charge.

Chemical modification of Lys/Hyl side chains causes aslower electrophoretic migration of the collagenous samples(Fig. 3A). N-Acetylated samples also have a low affinity forCoomassie Brilliant Blue R 250, the standard anionic dyewe used to stain polyacrylamide gels. It should be noted thatN-acetylation with acetic anhydride is to be avoided becauseit is artifactual: some peptide bonds are broken with theformation of interchain covalent bonds leading tomolecularspecies larger than the original sample. This is particularlyevident for peptides (Fig. 3A), and also smaller molecularspecies, as shown by analytical gel filtration chromatogra-phy in denaturing conditions (data not shown). All this isprobably the consequence of the addition of concentratedsodium hydroxide during the derivatization in order tomaintain the pH constant.

Using CD spectroscopy at increasing temperatures, wehave determined that many CNBr peptides are able to formtrimeric species that at room temperature prevail over therandom-coil monomeric species; only some small CNBrpeptide trimers have low melting temperatures (see Fig. 2for the values of melting temperatures).

Chemical modification of Lys/Hyl side chains incollagenous samples do not significantly modify both the

triple helical conformation of the trimeric species (Fig. 3B)and the thermal stability, with the relevant exception ofN-acetylation with acetic anhydride for the reasonsmentioned above. The greatest decrease of Tm onderivatization in mild conditions was found to be lessthan 3 °C. A detailed thermodynamic analysis of themelting transition of modified peptide trimers will bedescribed elsewhere.

Binding of decorin to collagenous samplesand effect of chemical modifications

Equimolecular amounts of collagen type I and type II andtheir 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 therecombinant preparation. At 23 °C, both collagen typesbind decorin, as well as some CNBr peptides (asterisked inFig. 2), namely peptides CB8, 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 patterntowards the CNBr peptides, with only some differences inthe intensity for some of the peptides (Fig. 4).

The triple helical conformation of collagenous samples isa necessary requisite for the interaction with decorin,because heat denaturation eliminates their binding(Fig. 4). No other peptide showed any binding also whenthe assay was performed at 4 °C (see Tm of peptides inFig. 2 with respect to the temperature of the bindingexperiments).

It is worth noting that peptide CB10 from type II collagendoes not bind decorin, regardless of the fact that it ishomologous to and in the homologous region of CB7. Wecannot comment on a1(II) CB9,7, because we did not find itin the chromatographic purifications of our CNBr digest oftype II collagen. Peptide a2(I) CB3,5 has some bindingability but the data should be judged with caution becausethis peptide showed a positive CD signal at 221 nm that istypical of native collagen and trimeric peptides but it ispossible that it does not form trimers with the three a chainsin register [28].

Fig. 2. CNBr peptides from type I and type II collagen alpha chains. The scheme shows the names (in bold), position along the triple helical domain,

size (number of residues) andmelting temperature of the trimeric species of CNBr peptides. The bottom two lines indicate theN fi Cdirection with

a length scale (in residues) and the banding pattern of type I collagen fibrils [51]. Melting temperatures have been measured in NaCl/Pi containing

1 mM EDTA and 1.5 mMNaN3 (in 0.1 M acetic acid for a2(I) CB3,5 because of its low solubility in NaCl/Pi); values for type I collagen peptides are

data reported previously [27, 30]. We have determined the ability to bind decorin for all peptides reported in the scheme (positive ones are marked

with an asterisk) and also for the composite peptide a1(I) CB2,4 (Tm � 28° in 0.1 M acetic acid), whereas we could not use peptide a1(II) CB9,7.


As controls, we have tested the interaction of decorin withother proteins: BSA, as a negative control, showed a muchlower response than collagens and positive peptides,whereas fibronectin and a 30-kDa fibronectin fragmenthaving heparin-binding ability showed interaction withtendon decorin (Fig. 4). Fibronectin is known to interactwith decorin protein core [45].

Our data suggest that decorin interacts with multipleregions of collagen. In competition experiments, we havefound that CNBr peptides in solution are not able tocompete with type I or type II collagen for decorin. Whenincreasing amounts of peptide a1(I) CB7 or a1(II) CB11 (upto 50-fold excess with respect to the collagen amount) werepreincubated in solution with decorin (at room temperaturefor 1 h) we observed no variation in binding of decorin tomicrowells coated with collagen type I or type II, respect-ively. The same null result was obtained in a competitionexperiment between CB11 as the coated ligand and the samepeptide in solution with decorin. The reason for this isprobably the interaction of collagen or peptide in solutionwith the coated collagenousmolecules [46]. It is also possiblethat isolated collagen trimers in solution have no or muchlower affinity for decorin and that decorin binding tocollagen depends on the aggregation status of collagen itself.

The affinity between decorin and collagens and peptideswas determined by using constant equimolecular amountsof the collagenous samples with increasing amounts oftendon decorin (Fig. 5). The graphs in Fig. 5C,D indicate abimodal behaviour of decorin for collagen I and II,suggesting that decorin has two distinct binding sites forthese collagens, as already indicated by others [20–22].Scatchard-type plots, drawn according to Hedbom &Heinegard [47], allowed the calculation of the dissociationconstants reported in Table 1. Because our data forcollagens 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 singledissociation constant that is only indicative of the range.The values of Kd are in the nanomolar range and similar tothe values reported in literature for decorin from cartilage ortendon, using type I collagen as the ligand (30 and 16 nM)[47,48].

Other experiments (not shown) indicated that ionicinteractions play an important role in the binding betweendecorin and collagen. Whereas the presence of 50 mMNaClin the phosphate buffer improved the interaction withrespect to analysis performed in NaCl/Pi (150 mM NaCl), ahigher concentration of salt (250 mM) resulted in dramat-ically reduced binding. On the contrary, no influence ofdetergents was found, as determined by the addition of 1%Triton to the binding solution.

To further characterize the nature of the interactionbetween decorin and collagen, we have chemically modifiedcollagen samples using agents that either disrupt ormaintain the positive charge, e.g. acetylation and methyla-tion, respectively.

Our results indicate that elimination of the positivecharge of the side chains of Lys/Hyl residues disrupts theinteraction with decorin (Fig. 6). This does not depend onthe derivatizing agent, SNHSAc or acetic anhydride,indicating that the side-effects of the treatment with aceticanhydride described above are not responsible for the loss ofbinding.Methylation of Lys/Hyl residues by treatment withHCHO/NaBH3CN preserved the positive charge and thisresulted 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-terminalhalf of the a1(I) resulted in either reduced binding for a1(I)CB7, or a complete loss of the binding for a1(I) CB6. Thevariation of the binding ability for N-methylated samples

Fig. 3. Analysis of collagen samples. Representative analyses for type II collagen (left column) and two CNBr peptides (central and right columns)

are reported. Lane 1 indicates underivatized samples; 2, samples derivatized with HCHO/NaBH3CN; 3, with SNHSAc; 4, with acetic anhydride.

(A) SDS/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 during 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/Pi containing 1 mM EDTA and 1.5 mM NaN3. The figures report only the portion of the spectrum centered on the maximum of the positive

peak (� 221 nm); this positive signal is present only for collagenous samples with triple helical conformation.


with respect to the unmodified ones is not related to thepercentage of Lys/Hyl side chains that did not react with thederivatizing agent (the percentage ranged from 3 to 12%).

Taken together, these data demonstrate the essential roleof the positive charge of collagen Lys/Hyl residues forinteraction with decorin.

D I S C U S S I O N

The binding of decorin with fibrillar collagens has beenextensively investigated (reviewed in [1,2]), but in vitrostudies have not yet conclusively identified the collagendomains responsible for the specific association withdecorin. In this study, we have analyzed the bindingbetween decorin and CNBr peptides from type I and typeII collagens, both unmodified and chemically derivatized.

We have recently characterized CNBr peptides fromcollagen type I [27–30]. The present work indicates thatCNBr peptides from type II collagen have a very similarbehaviour.

Our data on the interactions between type I and type IIcollagens, their peptides and decorin reveal the following.

(a) Type I and probably also type II collagen appear tohave multiple binding sites for decorin, because severalCNBr peptides are able to interact with this small proteo-glycan.

(b) The side chain of Lys/Hyl residues in collagen isrelevant for the binding, because the elimination of theirpositive charge eliminates the interaction. On the contrary,the chemical modification preserving the ionic charactermodulates the binding to decorin. This leads to a differentialbehaviour for the different peptides.

(c) Decorin might have two binding sites for collagen, assuggested by others and by the differential behaviour ofcollagen peptides.

Decorin is able to bind several CNBr peptides and type Iand II collagens only when they are in triple helicalconformation. Among the binding peptides, CB8, CB4and CB11 are found in a homologous region of theN-terminal half of the respective a chains (residues 124–327of the triple helical domain). On the contrary, CB7 and CB6lie in the C-terminal half of the chain. Binding specificity isdemonstrated by the following.

(a) The absence of interaction with decorin(s) of somepeptides that are in triple helical conformation in our assayconditions [CB2, CB2,4 and CB3 from a1(I), CB12, CB8and CB10 from a1(II)].

(b) All peptides able to bind decorin contain a regioncorresponding to the d and e bands of collagen fibrils(Fig. 2). This is in accordance with morphological findingsshowing 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 equimolecular

amounts of their CNBr peptides were used to coat polystyrene wells.

A constant amount of biotinylated decorin was added (20 pmol); the

bound decorin was determined using avidin conjugated with alkaline

phosphatase or, for tendon protein core, horseradish peroxidase. The

absorbance plotted in the panels for all collagens and peptides we have

tested was determined by exploiting a colorimetric reaction 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 was 17% of the mean. Top: analysis with tendon decorin

on collagen samples in native and in denaturing conditions (white and

black columns, respectively). The right panel reports the binding of

tendon decorin to BSA, fibronectin and a 30-kDa heparin-binding

fragment of fibronectin. Bottom: analysis 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 biotinylated tendon decorin were added to polystyrene

wells coated with a constant amount of collagen (5 lg) or equi-

molecular amounts of CNBr peptides. The binding was determined by

using avidin conjugated with alkaline phosphatase. (A,B) Saturation

curves of two collagens and two peptides reported as examples. Each

data point is the average value of a determination performed at least in

triplicate. The highest standard deviation was 18% of the mean. Lines

are added as a visual help. (C,D) Scatchard-type plots [47] on the same

samples. Lines interpolating the data have been computed with the

least square method. For type I and II collagens, linear interpolation

was performed taking into account all data points (see text). The

resulting dissociation constants are reported in Table 1.


decorin, localize in these bands, whereas keratan sulfate PGare present at a and c bands [23,24]. However, not allcollagen peptides that contain regions of the collagenmolecule falling within the d band interact with decorin, e.g.the homologous peptides a1(I) CB3 and a1(II) CB8, orpeptide a1(II) CB10. Collagen binding to decorin does nottherefore depend on the clusters of charged residuesresponsible of the banding pattern but on specific sequencesthat contain ionic residues.

(c) The action on platelet adhesion and activation by onlytwo peptides from type II collagen (data not shown) andonly by peptide a1(I) CB3 from type I collagen, as alreadyknown from the literature [49].

Peptide CB10 from type II collagen is homologous toa1(I) CB7, but does not interact with decorin. One possibleexplanation of this discrepancy could lie in the fact thattype II collagen is more glycosylated than type I collagen. Itseems to us probable that glycosylation of hydroxylysinewill block the binding. However, the glycosylation patternof Hyl residues is known for CB7 [50] but not for CB10.Aliquots of both CB10 and CB7 have also been digested at37 °C for 18 h with endoproteinase Arg-C, according to themanufacturer’s guidelines, with an enzyme to substrate ratioof 1 : 130. None of the most abundant fragments, separatedby reverse-phase HPLC with the same protocol used tosepare small CNBr peptides, showed at 4 °C any bindingability to tendon decorin (data not shown). This suggeststhat also some Arg-containing sequences are relevant incollagen for its interaction with decorin, or that none of thefragment was present in our assay conditions as a trimericspecies, or that the minor enzyme activity cleaving Lyspeptide bonds had a relevant effect.

The affinity of the binding peptides for decorin is in thenanomolar range with the same magnitude reported byothers for type I collagen [47,48], and the dissociationconstants are within one order of magnitude (Table 1). Ourdeterminations showed also that the binding betweendecorin and collagens or their CNBr peptides is quitesensitive to the ionic strength of the buffer, suggesting anionic character of the binding.

The main decorin region implicated in the binding tocollagens was hypothesized to lie inside the concave area ofthe arch-shaped protein core [17]. Residues in LRR 4 and 5were considered responsible for the binding [21]. The

concave surface, formed by b strands, is lined by manycharged residues and several hydrophobic side chains.Charged residues probably make ionic contacts; in partic-ular, carboxylate ions might bridge two positive residues,and/or Lys ammonium ions or Arg guanidinium ions mightbridge two negative groups. One of the relevant residues isglutamate-180 found by Kresse and coworkers to berelevant for the collagen binding [22]. It should however,be noted that the constructs lacking LRR 5 or bearing thesubstitution Glu180 to Lys [22] bring several positivecharges close to each other. This might have a directinfluence on the conformation of decorin core, and only anindirect one on the collagen binding. However, this remainsa hypothesis, as, to our knowledge, no conformationalanalysis was reported on these constructs.

The presence in the decorin molecule of a second bindingsite for collagen was suggested previously [20–22]. Theresults we have obtained from the Scatchard-type plots fortype I and type II collagens (Fig. 5) and the differentbehavior of the N-terminal collagen peptides with respect toCB7 and CB6might be a further support to this hypothesis.

Chemical modification of collagens and their CNBrpeptides demonstrated that acetylation eliminates theirbinding to decorin. Lys/Hyl side chains are thereforepresent at, or very near to the binding site(s) and theirpositive charge is a stringent requisite for the binding. This isnot surprising, if indeed collagen binds inside the concavesurface of decorin, owing to the presence of an elevated

Fig. 6. Effect of chemical modifications. A constant amount of type I

or II collagen (5 lg) or equimolecular amounts of their CNBr peptides

were used to coat polystyrene wells. A constant amount of biotinylated

tendon decorin was added (20 pmol); the bound decorin was deter-

mined using avidin conjugated 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 of the underivatized sample (white column) and for

derivatives with SNHSAc (black) and HCHO/NaBH3CN (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 between biotinylated

tendon decorin and collagenous samples.

Collagen sample Kd (nM)

Type I collagen 41a

CB6 from a1(I) b

CB7 from a1(I) 13

CB8 from a1(I) 44

CB4 from a2(I) 16

Type II collagen 42a

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.


number of ionic residues. On the contrary, reductivemethylation modulates the binding of all peptides todecorin, the largest decrease being shown by a1(I) CB7and CB6 (Fig. 6), suggesting a different specificity of thesepeptides. All these effects are direct, because all peptides wehave derivatized in mild conditions maintain the ability toform trimeric species that are the major species in ourbinding assays.

A previous study reported that decorin binding occurspreferentially at about 50 and 100 nm from the N-terminusof type I collagen [25]. We found the region � 50 nm fromthe N-terminus falls within peptides CB8, CB4 and CB11and was able to bind decorin. Apart from the presence ofLys, we are not able to compare our data with thesuggestion of a collagen sequence able to bind decorin,namely GAKGDRGET, at position 853–861 of the triplehelical domain of the a1(I) chain, within peptide CB7 [26]. Asimilar sequence is present in the homologous region oftype II and III collagens.Without GAK, the sequence G-D/E-R-G-E-Hyp/T is present also at position 623–628 of thesame chain (in peptide CB7) and of homologous sequencesof other collagen alpha chains. The collagen sequenceDRGE might have KLER and RELH as possible comple-mentary sequences in decorin [18], at position 130–133 and272–275, in the LRR 3 and 9, respectively. The modelproposed on the basis of these complementary sequences inthe two interacting proteins showed a double contactbetween decorin and two collagen molecules. However, thisis discordant with the decorin model [3,4,17] where the ionicresidues of KLER/RELH fall inside the concave surface ofdecorin, with the exception of K-130.

It is not possible to reconcile our findings with mostresults recently reported by Keene et al. [19] which are indisagreement with many previous results, as widely dis-cussed in the paper. On one side, a periodicity was noticedby these authors in aggregates of decorin and type IpC-collagen seen in electron micrographs of rotary shad-owed molecules; this was due to the presence of decorin, aspC-collagen alone did not show a similar pattern. CNBrpeptides of the a1(I) chain that we have found to binddecorin are positioned along the chain in a manner thatperiodicity of binding is the natural outcome, even if ourdata do not allow a determination of the size of the periodand even if peptide CB3, unable to bind decorin, interruptsthe periodicity. On the other side, the relevance of Lys/Hylresidues both in collagens and peptides for interaction withdecorin is in contrast with the findings that the binding sitefor decorin is located in a sequence within the peptide a1(I)CB6 devoid of any Lys/Hyl residue and containing, as ionicamino acids, only oneGlu and oneArg, 13 residues apart. Itis interesting to note that the same region of the a2(I) chaincontains the dipeptideHH.The tripletGHH is unique in thetriple helical domain of all collagen chains, as determined bya search in Swiss-Prot. One can thus hypothesize that thepolyhistidine tag present in the recombinant decorinpreparation used by Keene et al. [19] is able, in the presenceof minute amounts of proper cations, to interact with GHHin a2(I) and direct the binding of decorin to the collagenC-terminus in CB6. However, this cannot be deducedbecause no control experiments are reported with decorinlacking the polyhistidine tag or with decorin purified in thepresence of chaotropic agents to compare with conditionsused in previous determinations.

On this basis, we can conclude the precise location andthe relative orientation of the binding sites in decorin andcollagen are not yet known. Our findings on multiplebinding sites in collagen and on the relevance of Lys/Hylresidues set some limitations, as do the fact that decorinmight have a second binding site for collagen. Becausedecorin physiologically interacts with collagens when theyare in their specific aggregation states, multiple contacts areprobably essential for the strength and the specificity of theinteraction.

A C K N O W L E D G E M E N T S

We thank Antonella Forlino for helpful suggestion and criticism, Elena

Campari and Luigi Corazza for technical assistance, ÔCentro Grandi

StrumentiÕ, University of Pavia, for peptide sequencing and free access

to the spectropolarimeter. This work was supported by grants from

ItalianMURST (grantMM05148132-3) andUniversity of Pavia (FAR

and Progetto Giovani Ricercatori 2000/2001).

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interaction of decorin with cnbr peptides from collagens i and ii

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