Tài liệu Báo cáo Y học: Interaction of decorin with CNBr peptides from collagens I and II Evidence for multiple binding sites and essential lysyl residues in collagen pot

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:
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. Our findings on multiple
binding sites in collagen and on the relevance of Lys/Hyl
residues set some limitations, as do the fact that decorin
might have a second binding site for c ollagen. Because
decorin physiologically interacts with collagens when they
are in their specific aggregation states, multiple contacts are
probably essential for the strength and the specificity of the
interaction.
ACKNOWLEDGEMENTS
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
Italian MURST (grant MM05148132-3) a nd University of Pavia (FAR
and Progetto Giovani Ricercatori 2000/2001).
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