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

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

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

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

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

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

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

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

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

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-

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).

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

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

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

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

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,

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

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

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