Differential interactions of decorin and decorin mutants with type I
and type VI collagens
Gordon Nareyeck
1
, Daniela G. Seidler
1
, David Troyer
2
,Ju¨ rgen Rauterberg
2
, Hans Kresse
1
and Elke Scho¨ nherr
1,3
1
Departement of Physiological Chemistry and Pathobiochemistry, University Hospital of Mu
¨
nster, Germany;
2
Institute of
Arteriosclerosis Research, University of Mu
¨
nster, Germany;
3
Matrix Biology and Tissue Repair Research Unit, University of Wales
College of Medicine, Dental School, Cardiff, UK
The small leucine-rich proteoglycan decorin can bind via
its core protein to different types of c ollagens such as type
I and type VI. To test whether decorin can act as a
bridging molecule between these collagens, the binding
properties of wild-type decorin, two full-length decorin

). Further investigations verified
that complexes of type VI collagen and decorin bound
type I collagen and that the affinity of collagen type VI to
type I was increased by the presence of decorin. These
data show that decorin not only can regulate collagen
fibril formation but that it also can a ct as an intermediary
between type I and type VI collagen and that these two
types of collagen interact via different binding sites.
Keywords: collagen t ype I; collagen type VI; decorin; surface
plasmon r esonance measurements.
Collagens can be divided into several subfamilies according
to their quarternary structure a nd their localization in tissue
[1,2]. The largest subfamily is represented by the banded
fibril forming collagens type I, II and III, which are
characterized by long, uninterrupted triple helical domains
that assemble laterally to form fibrils. I n contrast, another
subfamily, of which type VI collagen is t he only m ember, is
characterized by the formation of multimolecular, filamen-
tous beaded structures [3]. Although banded fibril forming
and filamentous beaded collagens form independent net-
works, they intermingle with each other in vivo,this
association providing for mechanical stab ilization o f t issues.
Electron microscopic studies indicate that the banded fibril
forming collagens are traversed specifically near their ÔdÕ
bands, within the gap region of the collagen fibrils, by the
filamentous beaded structures of the type VI collagen-
containing network [4,5].
Collagen fibrils in tissues a re heteropolymers of several
types of collagen and of noncollagenous components.
For example, collagen fibrils in skin are composed

Note: G. Nareyeck a nd D. G. Seidler c ontribu ted equally to this w ork.
(Received 13 M ay 2004, accepted 30 June 2004)
Eur. J. Biochem. 271, 3389–3398 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04273.x
deficient in the SLRP decorin, biglycan, fibromodulin
and lumican have been generated [11]. Interestingly,
double deficiency in decorin and biglycan manifests itself
in extremely abnormal architecture of the collagen fibrils.
Thus, interactions between the collagens and decorin are
of paramount importance in attaining and maintaining
tissue integrity.
In the present study we investigate the binding properties
of wild-type decorin, two decorin mutants and a truncated
decorin species with type I and type VI collagen to
demonstrate that decorin can act as a bridging molecule.
The results indicate that the tertiary structure of decorin is
stabilized by the glycosaminoglycan chain. Furthermore,
decorin may form a dimer which is capable of interacting
concurrently with both type I and type VI collagen
molecules.
Experimental procedures
Expression and preparation of recombinant
proteoglycans
Wild-type decorin and the decorin mutant DCN E180K
were expressed in human kidney 293 cells as previously
described [12]. DCN E180K harbours an amino acid
exchange at amino acid E180, which is an important site
for the interac tion o f decorin with type I collagen fibrils. A
plasmid harbouring the cDNA for the decorin mutant
DCN E180Q was generated from the respective wild-type
plasmid by a one-step site-directed mutagenesis procedure

)or10m
M
Hepes pH 7.4, 0.15
M
NaCl, 3.4 m
M
EDTA, 0.005%
(v/v) Tween-20 (HBS) at 4 °C. Glycosaminoglycan-free
core protein was generated by exhaustive digestion with
chondroitin ABC lyase (Seikagaku Kogyo) as described
previously [15]. Glycosaminoglycan chains were liberated
by reductive b-elimination with 1
M
sodium borohydride
in 0.1
M
NaOH for 24 h at 37 °C, followed by dialysis
and rechromatography on BioGel TSK DEAE-5 PW as
described above. [
35
S]Sulphate-labelled and [
35
S]methio-
nine-labelled decorin from 293 cells and skin fibroblasts
were obtained as described previously [12,16].
Preparation of methylated type I collagen and
type VI collagen
Type I collagen was isolated from calf skin and methylated
by treatment with 0.2
M

ethanolamine/HCl p H 8.5. Injec-
tion times were chosen to achieve about 6000–7000
resonance units (6–7 ng of proteinÆmm
)1
[19]. Type VI
collagen was immobilized via its free sulfhydryl groups.
During immobilization, the flow rate of HBS was main-
tained a t 5 lLÆmin
)1
. T he surface w as activ ated as described
andallowedtoreactwith80 m
M
2-(2 pyridinyldithio)ethane
amine in 0.1
M
sodium borate p H 8.5. At least five coupling
pulses of 240 lL type VI c ollagen (25 0 lgÆmL
)1
)in0.1
M
sodium formiate pH 4.3, were applied until 6000–7000
resonance units were present. The sensor surface was
blocked with 50 m
ML
-cysteine in 0.1
M
sodium formiate
pH 4.3, 1
M
NaCl. BSA was immobilized and used to

of Roden and Myszka [20]. To correct for changes in
refractive index and nonspecific binding, the responses
obtained with immobilized albumin were subtracted from
3390 G. Nareyeck et al. (Eur. J. Biochem. 271) Ó FEBS 2004
those obtained with bound collagen. The e xperimental data
were then evaluated with the
BIAEVALUATION
3.0 software.
Other binding assays
The binding of [
35
S]sulphate-labelled decorin species and
[
35
S]sulphate-labelled biglycan to reconstituted type I colla-
gen fibrils was performed as described [12]. Solid phase
assays on hydrophilic ELISA strips (Nunc) were used to
investigate interactions with type VI collagen a nd methyla-
ted type I collagen. Type VI collagen (4 lgÆmL
)1
,
50 lLÆwell
)1
) and methylated type I c ollagen (10 lgÆmL
)1
,
100 lLÆwell
)1
)in50m
M

A Jobin-Yvon CD6-Dichrograph spectropolarimeter
(Yvon, France) was used to measure CD spectra at ambient
temperature in NaCl/P
i
in a 0.1-mm path length quartz
cell. Proteoglycan concentrations of  1mgproteinÆmL
)1
were used. Estimations of secondary structure were per-
formed with the
CDNN
2.1 software (ACGT Progenomics
AG, Halle (Saale), Germany).
Electron microscopy
Suspensions of type I collagen in glycerol were s prayed onto
mica sheets w ith a n air brush and rotary shadowed with
platinium-carbon at an angle of about 7°, followed by pure
carbon as described by Cohen et al. [21]. The replicas were
placed on uncoated g rids and analysed w ith a Philips EM
410 electron microscope.
Results
Characterization of purified type I and type VI collagens
Type I collagen fibrils we re generated by neutralization of
acid soluble calf skin collagen as describe d previously [12].
To obtain type I collagen monomers, type I collagen was
methylated which shifts the isoelectric point of the molecule
to a basic pH and increases hydrophobicity. The treated
collagen does not form fibrils under physiological condi-
tions which was confirmed by rotary shadowing (Fig. 1A).
However, the methylated t ype I collagen was still able to
bind to hydrophilic ELISA strips (see below). Bovine type

CDNN
2.1 software. The analysis showed that wild-
type decorin and DCN E180K an d DCN E180Q have 2 1%
a-helical motifs and 29.1% b-sheets (Table 1). These results
show that the point mutations have only minor effects on
the g eneral structure of the decorin core protein. For DCN
Q153, which lacks most of the leucine-rich repeats, 36%
a-helical motifs and only 2 4.1% b-sheets were observed. As
shown in Fig. 4, decorin expressed in 293 cells contained no
free core protein, and the length of the glycosaminoglycan
chain w as similar to that of decorin f rom d ermal fibroblasts .
Fig. 2. Electrophoretic comparison of the composition of pepsin digested
type I collagen, acid treated type I collagen, methylated type I c ollagen
and type V I collagen used in the experiments. Sam ples of the different
types of collagen were applied und er reducing (+DTE) and non-
reducing (–DTE) conditions to a 4–12.5% polyacrylamide gradient
gel. Protein was visualized by staining with Coomassie blue.
Fig. 3. CD spectra of the recombinant decorin e xpressed in 293 cells and
purified under nondenaturing conditions. The spectra were obtained
under physiological conditions in 0.15
M
NaCl. Wild-type d ecorin,
solid line; truncated decorin DCN Q153, dotted line. Spectra for the
decorin mutants DCN E180K and DCN E180Q were indistinguish-
able from that of wild-typ e decorin (not shown).
Table 1. Tentative structural motifs of recombinant decorin. Theoretical
calculation using the program CDNN and the data from by CD
spectra measured between 195 nm to 260 nm. Decorin and the decorin
mutants DCN E 180Q a nd DC N E180K an d t runc ated de corin D CN
Q153 we re purified under nondenaturing co nditio ns from the medium

binding properties of the different ligands involved.
[
35
S]Sulphate-labelled decorin was incubated with reconsti-
tuted type I collagen fibrils and its binding was compared to
the different mutants. Wild-type decorin i nteracted strongly
with collagen fibrils, DCN E180K reacted weakly and
DCN E180Q moderately (Fig. 5) which agreed with earlier
results [12] and confirmed t he suitability of the mutants.
To test whether methylated type I collagen monomers
which were planed to be used as ligands for a decorin/
collagen type VI complex showed the expected properties
solid phase assays with methylated collagen type I and
decorin or biglycan as ligands were performed. ELISA
plates were coated with th e collagen monomers and
[
35
S]sulphate-labelled proteoglycans were added. These solid
phase assays showed dissociation constants of 2.3 ·
10
)10
M
for decorin and 1.4 · 10
)9
M
for biglycan (data
not shown) indicating that the methylated type I collagen
monomers were suitable binding partners and could be used
for further studies.
The further analysis was performed by surface plasmon

obtained from decorin by b-elimination showed on ly weak
interaction with type I collagen monomers. The analysis
of the i nteraction of DCN E180K and DCN E180Q with
type I collagen yielded K
D
¼ 4.1 · 10
–9
M
and K
D
¼
1 · 10
–9
M
, respectively. The truncated form of decorin,
DCN Q153 also showed weak interaction with type I
collagen. For c omparative purposes we analysed the
binding of biglycan, another proteoglycan of the SLRP
family, using surface p lasmon resonance. A b inding affinity
of biglycan fo r type I collagen monomers of K
D
¼
2.7 · 10
)9
M
was obtained. To test the reliability of these
data v
2
-values were compared between the different experi-
ments. Because the v

type I collagen (K
D
¼ 3 · 10
–7
M
). Compared to wild-type
decorin, DCN E180Q exhibited about 10-fold lower affinity
for type VI collagen. Unlike wild-type decorin and DCN
180Q, DCN E180K formed multimers as inferred from the
nonlinear curve for the binding of radioactively labelled
DCN E180K to type VI collagen (Fig. 7). However,
multimers of DCN E180K were still capable of binding to
type VI collagen.
To study the binding of decorin an d the decorin mutants
to type VI collagen i n a real-time experiment, we again u sed
surface plasmon resonan ce measurements. The data for the
binding affinities to type VI collagen are summarized in
Table 3. For wild-type decorin a K
D
of 3.6 · 10
)9
M
was
determined. Because the glycosaminoglycan chain influ-
enced the binding affinity of decorin to type I collagen, we
also studied t he binding properties of glycosaminoglycan-
free core protein to type VI collagen. A K
D
of 3.9 · 10
)8

, which is about
one order o f magnitude higher than that found with type I
collagen.
Considering R
max
(where 1000 resonance units ¼
1ngÆmm
)2
) s toichiometric analysis of the surface plasmon
resonance measurements revealed that a single collagen
molecule binds about 0.186 decorin molecules in the
presence as well as the absence of its glycosaminoglycan
chain (Fig. 6A,B). The number of decorin molecules
binding to type VI collagen increased from 1 : 0.042 to
1 : 0.061 by the presence of the glycosaminoglycan chain
(Fig. 8 A,B), indicating that the glycosaminoglycan chain
does not only stabilize wild-type decorin, but can also
interfere with the function of decorin. The glycosamino-
glycan chain alone did not show binding properties to the
collagen coated chip (data not shown). Furthermore, the
amino acid exchange at position E180 resulted in a change
in the bindin g capacity of decorin to both type I an d to type
VI collagen.
Formation of complexes of decorin, type I collagen
and type VI collagen
In a further investigation we analysed whether the same
site or similar sites on wild-type decorin, DCN E180Q
and DCN E180K bind to type I and to type VI collagen.
Table 2. Binding of decorin and decorin mutants to type I collagen. Type I
collagen monomers were immobilized on CM5 chips. Surface plasmon

DCN E180Q 9.8 · 10
8
1 · 10
)9
349 0.02
DCN E180K 2.4 · 10
8
4.1 · 10
)9
171 0.34
DCN Q153 2.7 · 10
7
3.9 · 10
)8
282 0.24
BGN WT 3.7 · 10
8
2.7 · 10
)9
358 0.12
CS/DS chain 5 · 10
3
2 · 10
)4
– 0.94
Fig. 7. Interaction of [
35
S]sulphate-labelled wild-type decorin and the
decorin mutants DCN E180Q and DCN E180K with pepsin d igested
type VI collagen in t he solid phase binding assay. Wild-type decorin (d),

91 0.044
DCN E180Q 2.9 · 10
9
3.4 · 10
)10
154 0.11
DCN E180K 3.3 · 10
8
2.9 · 10
)9
147 0.01
DCN Q153 7.6 · 10
7
1.3 · 10
)8
143 0.09
BGN WT 4.7 · 10
7
2.1 · 10
)8
116 0.034
CS/DS chain 5 · 10
2
2 · 10
)3
– 0.08
3394 G. Nareyeck et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Type VI collagen w as first immobilized on a CM5 chip and
reacted with decorin prior to adding methylated type I
collagen. The results showed that the initially formed type

measurements.
Measuring t he interaction of decorin with different types
of collagen by surface plasmon r esonance analysis w e f ound
a high affinity of decorin for triple helical type I collagen
compared to previously published values of 10
)8
)10
)9
for
intact and chondroitin ABC lyase-treated decorin to
reconstituted type I collagen fibrils [26,27]. In both of these
studies the proteoglycans were treated with chaotropic
agents. Our studies using decorin isolated f rom fibroblast
culture medium under nondenaturing conditions revealed
two unique high affinity binding sites ( K
D
¼ 7 · 10
)10
M
and K
D
¼ 3 · 10
)9
M
) and 0.043 decorin molecules per
collagen monomer [28]. The present study using methylated
type I collagen revealed only one binding site with K
D
¼
5.8 · 10

phosphate [32]. However, we found no changes in the
affinity of decorin to collagen type I or VI for these
components (Figs 6B and 8B). This does not rule out that
Zn
2+
is interacting with the N terminus of decorin and may
cause dimerization [ 31], but it did not affect the interaction
with the two types o f collagen.
It is known that the amino acid E180 in decorin is
involved in type I collagen binding [12]. Therefore, the
Fig. 8. Surface plasmon reson ance measurement of immobilized pepsin
digested type VI collagen. (A) Interaction with wild -typ e decorin core
protein (ob tained by chondroitin A BC lyase digestion) in HBS bu ffer.
Concentrations of wild-type decorin core protein used were as indi-
cated. (B) Interaction with wild-type decorininHBSbuffer(solidlines)
andinHBSbuffercontaining15l
M
ZnCl
2
(dotted lines). Concen-
trations of wild-type decorin used were a s indicated.
Table 4. Type V I collagen was dige sted with pepsin and imm obilized on
CM5 chips followed by c omplex formation of wild-type decorin and
decorin mutants. Surface plasmon resonance measurements of the
collagen/proteoglycans complexes were performed with monomers
of methylated type I collagen. The proteoglycans were purified
under nondenaturing condition from the medium of 293 cells. WT,
Wild-type.
Type I collagen binding K
A

moderate K
D
value for binding of DCN E180Q to
reconstituted type I collagen fibrils and an even lower
affinity of DCN E180K was expected. In contrast to reports
that the glycosaminoglycan c hains have no influence on the
binding of decorin to collagen fibrils [26,33], we observed
reduced binding affinity for the glycosaminoglycan-free core
protein. Evidently t he glycosaminoglycan chain of decorin
stabilizes the tertiar y structure of the proteoglycans the reby
causing difference in binding affinity. As decorin is not the
only SLRP that interacts with type I collagen the homo-
logous proteoglycan biglycan was investigated. The affinity
of biglycan for methylated type I collagen was lower than
the affinity of decorin which corroborated previous
data obtained with biglycan from bacteria and f rom
osteosarcoma cells using fibrillar type I collagen [28].
To investigate the interaction o f d ecorin m utants E180K
and E180Q with type VI collagen a solid phase assay was
performed. One mutant, DCN E180K, which has a 10-fold
lower affinity to collagen t ype I than wild-type decorin had a
similar affinity to type VI collagen as wild-type decorin.
DCN E 180Q showed an even stronger binding to type VI
collagen than wild-type decorin, while its affinity to type I
collagen was reduced. These data suggest that amino acid
E180 may b e not only important for the binding of decorin
to type I collagen, but may a lso be involved in t he binding to
type VI. The interaction of type VI collagen with decorin
has to be seen in the context that type VI collagen is
responsible for the formation of the beaded microfibrillar

M
)
to this type of collagen. These findings may indicate that the
lower affinity of biglycan is necessary for the fast organiza-
tion of the t ype VI network while decorin may have a more
stabilizing function [24]. A further difference in the interac-
tion of decorin and biglycan with type VI collagen was that
decorin without glycosaminoglycan chain had a reduced
binding affinity, whereas the interaction of biglycan with
type VI collagen was independent of the presence of the
glycosaminoglycan chains. Nevertheless, the glycosamino-
glycan chain plays a role in guiding type VI collagen into the
organized structure both in v itro [37] and in t issue [38]. These
findings are of biological importance, because decorin is
involved in fibrillogenesis of type I collagen and also in the
generation of the microfibrillar n etwork [37].
Analysis of the secondary structure of decorin and its
mutants by CD spectroscopy showed that no significant
alterations were induced by the amino acid substitutions in
the mutations compared to wild-type decorin. However, as
CD spectra give only the overall proportion of different
secondary structures, small changes in the distribution
might not have been registered. DCN Q153, which lacks six
of the 1 0 leucine-rich repeats of wild-type decorin, showed
significantly changed C D s pectra as expected. Some changes
were observed to previous results [39–41] which may be due
to different expression and purification procedures. In our
expression system transfected 293 cells syn thesize decorin
with its normal pre- and propeptide sequences and have
expression and secretion rates similar to those in normal

with type I or type VI collagen. The existence of an
interaction of de corin with type I and type VI collage n has
been shown in vivo in skin [45]. More recently a comple x
formation between the globular domains of collagen type
VI and a decorin/matrilin-1 complex has been described
which can act as a bridge between type VI and type II
collagen in c artilage, whereas decorin binds to the globular
N-terminal domain of type V I collagen [24]. Even though
in our study type VI collagen was treated with pepsin,
electron micrographs still demonstrate the presence of
globular domains, so decorin could act as a bridging
molecule alone, by binding to the N terminus of type VI
collagen and to type I collagen. Furthermore, the dissoci-
ation constants for wild-type decorin and the two decorin
mutants showed a similar relation to each other in the
tertiary complex compared to the interaction w ith the type
I or type VI collagen alone. Therefore, two binding models
are possible: (a) decorin forms a dimer and can interact
with the same binding site either with type I collagen or
type VI collagen (Fig. 9A). This agrees with findings of
Scott and coworkers [40], w ho reported that d ecorin a nd
glycosamiminoglycan-free core proteins form dimers, how-
ever, the purification described in this paper was with
freeze-drying. The dimer formation described in this paper
cannot result from the purification method, because
purification under nondenaturing conditions without
freeze-drying avoids artificial dimerization [23]; (b) decorin
binds via a binding site on the outer surface of the molecule
to globular domains of collagen t ype VI and via a different
binding site which is affected by E180 to collagen type I

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