Seidler1, David Troyer2, Ju¨rgen Rauterberg2, Hans Kresse1 and Elke Scho¨nherr1,3 1 Departement of Physiological Chemistry and Pathobiochemistry, University Hospital of Mu¨nster, Germany
Trang 1Differential interactions of decorin and decorin mutants with type I and type VI collagens
Gordon Nareyeck1, Daniela G Seidler1, David Troyer2, Ju¨rgen Rauterberg2, Hans Kresse1
and Elke Scho¨nherr1,3
1
Departement of Physiological Chemistry and Pathobiochemistry, University Hospital of Mu¨nster, Germany;2Institute of
Arteriosclerosis Research, University of Mu¨nster, Germany;3Matrix 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 collagens 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
species with single amino acid substitutions (DCN
E180K, DCN E180Q), which previously showed reduced
binding to collagen type I fibrils, and a truncated form of
decorin (DCN Q153) to the these collagens were
investi-gated In a solid phase assay dissociation constants for
wild-type decorin bound to methylated, therefore
mono-meric, triple helical type I collagen were in the order of
10)10M, while dissociation constants for fibrillar type I
collagen were 10)9M The dissociation constant for
type VI was 10)7M Using real-time analysis for a
more detailed investigation DCN E180Q and DCN
E180K exhibited lower association and higher dissoci-ation constants to type I collagen, compared to wild-type decorin, deviating by at least one order of magnitude In contrast, the affinities of these mutants to type VI colla-gen were 10 times higher than the affinity of wild-type decorin (KD 10)8M) 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 act as an intermediary between type I and type VI collagen and that these two types of collagen interact via different binding sites Keywords: collagen type I; collagen type VI; decorin; surface plasmon resonance measurements
Collagens can be divided into several subfamilies according
to their quarternary structure and 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 In contrast, another
subfamily, of which type VI collagen is the only member, 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 stabilization of tissues
Electron microscopic studies indicate that the banded fibril
forming collagens are traversed specifically near their d
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 are heteropolymers of several types of collagen and of noncollagenous components For example, collagen fibrils in skin are composed primarily of type I collagen with minor amounts of type III and type V collagen Type III collagen is found on the fibrillar surface, while type V collagen is buried mainly within the fibrils [6] Noncollagenous matrix glycoproteins are additionally associated with the surface
of the collagen fibrils Such glycoproteins may in part substitute for collagen species at the fibrillar surface or perform auxiliary functions [7] Some of these matrix glycoproteins contain leucine-rich repeat structures and have been shown to modulate collagen fibrillogenesis and the spacing between the mature fibrils The chondroitin/ dermatan sulphate proteoglycan decorin (DCN) is a member of this family of small leucine-rich proteoglycans (SLRP), which is composed of a core protein and a single covalently linked glycosaminoglycan chain It binds
to collagen fibrils near the d bands (decorates them) and delays the lateral assembly of collagen fibrils [8,9] Consequently, targeted disruption of the decorin gene
in mice leads to abnormal fusion of collagen bundles and
to increased fragility of skin [10] Recently, mice twofold
Correspondence to E Scho¨nherr, Matrix Biology & Tissue Repair
Research Unit, University of Wales College of Medicine, Dental
School, Heath Park, Cardiff CF14 4XY, UK.
Fax: + 44 29 2074 4509, Tel.: + 44 29 2074 2595,
E-mail: schonherreh@cardiff.ac.uk
Abbreviations: BGN, biglycan; CS/DS, chondroitin
sulphate/derma-tan sulphate; DCN, decorin; GAG, glycosaminoglycan; SLRP, small
leucine-rich repeat proteoglycan.
Note: G Nareyeck and D G Seidler contributed equally to this work.
(Received 13 May 2004, accepted 30 June 2004)
Trang 2deficient 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 interaction of 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
(Stratagene) using the primer pair 5¢-GGTGCCCAGTTG
TATGACAATC-3¢ and 5¢-GATTGTCTACAACTGGG
CACC-3¢ The amino acid at E180 in DCN E180Q is
likewise substituted A cDNA construct for the truncated
decorin species DCN Q153 (M1–Q153), in which six of a
total of 10 leucine-rich repeats are lacking [13], was cloned
into the EcoRI/XbaI site of pcDNA 3.1 (Invitrogen) and
used for transfection with the Lipofectin (Life Technologies)
method Biglycan (BGN) was expressed in 293 cells as
described [14]
All proteoglycan preparations were obtained from
conditioned media of transfected 293 cells under
condi-tions without denaturing and/or precipitation steps
Media, supplemented with protease inhibitors, were
applied directly to a DEAE-Trisacryl M (Serva) and
then to a BioGel TSK DEAE-5PW HPLC column
(Bio-Rad) as described previously [15] Proteoglycans were
stored at 4C in elution buffer (10 mMTris/HCl pH 7.4,
containing 0.6M NaCl) Immediately prior to use the
proteoglycans were dialysed against either 18 mM sodium
phosphate pH 7.4, 0.15M NaCl (NaCl/Pi) or 10 mM
Hepes pH 7.4, 0.15M NaCl, 3.4 mM EDTA, 0.005%
(v/v) Tween-20 (HBS) at 4C 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 1M sodium borohydride
in 0.1M NaOH for 24 h at 37C, followed by dialysis
and rechromatography on BioGel TSK DEAE-5 PW as
described above [35S]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 0.2M methanolic HCl for 3 days at ambient temperature as described [17] This treatment results in the modification of about 70% of all carboxyl residues This modification leads to an increase in the pH and a decrease in the hydrophilic properties Type VI collagen was solubilized from bovine placenta by pepsin treatment and purified by salt fractionation [18]
Surface plasmon resonance analysis All measurements were performed with a BIAcore 1000 analyser (Pharmacia Biosensor) Methylated type I collagen was immobilized via its primary amino groups to a research grade CM5 sensor chip [19] During immobilization, a flow rate of 10 lLÆmin)1of HBS was maintained The surface of the chip was activated by injecting a mixture of equal volumes of 0.2M N-ethyl-N¢-(3-dimethylaminopropyl)-carbodiimide and 0.05M N-hydroxysuccinimide There-after, 70 lL of a solution of methylated type I collagen (250 lgÆmL)1) in 20 mM sodium acetate pH 4.0 was injected followed by 1Methanolamine/HCl pH 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 at 5 lLÆmin)1 The surface was activated as described and allowed to react with 80 mM2-(2 pyridinyldithio)ethane amine in 0.1Msodium borate pH 8.5 At least five coupling pulses of 240 lL type VI collagen (250 lgÆmL)1) in 0.1M sodium formiate pH 4.3, were applied until 6000–7000 resonance units were present The sensor surface was blocked with 50 mM L-cysteine in 0.1M sodium formiate
pH 4.3, 1M NaCl BSA was immobilized and used to determine the proportion of nonspecific binding The sensor surfaces were regenerated with 1MNaCl in running buffer for type I collagen-coated chips and with 0.3MNaCl in the case of immobilized type VI collagen
To form a complex consisting of decorin, type I and type
VI collagen, type VI collagen was first immobilized After binding of decorin the sensor chip surface was not regenerated in order to maintain a high level of bound proteoglycan Methylated type I collagen in NaCl/Piwas then added to the chip and allowed to interact with the proteoglycan
All experiments were carried out at 25C at a flow rate of
10 lLÆmin)1 The response to the running buffer was defined as the baseline level, and all responses were expressed relative to this baseline Experimental procedures and conditions leading to precipitation of protein complexes
in the flow system and the pump of the BIAcore 1000 instrument had to be strictly avoided to protect the system from damage For this reason only experiments without the addition of complexes were performed For the analysis
of interactions between proteoglycans and collagens, the sensograms were corrected by a modification of the method
of Roden and Myszka [20] To correct for changes in refractive index and nonspecific binding, the responses obtained with immobilized albumin were subtracted from
Trang 3those obtained with bound collagen The experimental data
were then evaluated with theBIAEVALUATION3.0 software
Other binding assays
The binding of [35S]sulphate-labelled decorin species and
[35S]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 and
methyla-ted type I collagen Type VI collagen (4 lgÆmL)1,
50 lLÆwell)1) and methylated type I collagen (10 lgÆmL)1,
100 lLÆwell)1) in 50 mMNaHCO3pH 9.6, were incubated
for 16 h at 4C After blocking with 3% BSA in NaCl/Pi,
0.05% Tween-20 for 4 h at 37C, the wells were washed
twice with ice-cold NaCl/Pi Labelled proteoglycans in
NaCl/Pi(18 mMsodium phosphate pH 7.4, 0.15MNaCl)
were applied for 6 h or 3 h at 37C After extensive
washing with blocking solution, bound proteoglycans were
solubilized with 0.1M NaOH and neutralized with 0.1M
HCl prior to scintillation counting KDvalues were
deter-mined usingPRISM3.0 (GraphPad Software)
CD spectroscopy
A Jobin-Yvon CD6-Dichrograph spectropolarimeter
(Yvon, France) was used to measure CD spectra at ambient
temperature in NaCl/Pi in a 0.1-mm path length quartz
cell Proteoglycan concentrations of 1 mg proteinÆmL)1
were used Estimations of secondary structure were
per-formed with theCDNN2.1 software (ACGT Progenomics
AG, Halle (Saale), Germany)
Electron microscopy
Suspensions of type I collagen in glycerol were sprayed onto
mica sheets with an 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 grids and analysed with a Philips EM
410 electron microscope
Results
Characterization of purified type I and type VI collagens
Type I collagen fibrils were generated by neutralization of
acid soluble calf skin collagen as described 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 type I collagen was still able to
bind to hydrophilic ELISA strips (see below) Bovine type
VI collagen containing three polypeptide chains, a1(VI),
a2(VI) and a3(VI) covalently linked via disulphide bonds
was produced by treatment with pepsin to remove most of
the C- and N-terminal globular domains (Fig 1B) Fig 2
shows the composition of the collagen used in the
experi-ments Quantitative analysis of the SDS gel electrophoreses
indicated that 63% of the type VI collagen contained the
long chain and 37% the short fragment of the a3(VI) chain [22]
Characterization of purified decorin and its mutants Wild-type decorin, DCN E180K, DCN E180Q and DCN Q153 were purified under nondenaturing conditions from conditioned medium of 293 cells transfected with the respective cDNA No freeze-drying or precipitation steps were performed to avoid artificial complex formation of proteoglycans [23] CD spectroscopy was used to determine whether there are major differences in secondary structure between the wild-type decorin and the mutants The CD spectra of wild-type decorin and DCN E180Q and DCN E180K appeared similar, whereas that of DCN Q153 differed from that of wild-type decorin (Fig 3) The CD
Fig 1 Rotary shadowing of isolated type I and type VI collagen mole-cules (A) Methylated type I collagen was visualized as monomers, whereas (B) pepsin digested type VI collagen appeared as short frag-ments of beaded filafrag-ments.
Trang 4spectra of the glycosaminoglycan chain alone, obtained by
reductive b-elimination, yielded only baseline data (not
shown) Evaluation of secondary structure was performed
with theCDNN2.1 software The analysis showed that
wild-type decorin and DCN E180K and DCN E180Q have 21%
a-helical motifs and 29.1% b-sheets (Table 1) These results
show that the point mutations have only minor effects on the general structure of the decorin core protein For DCN Q153, which lacks most of the leucine-rich repeats, 36% a-helical motifs and only 24.1% 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 was similar to that of decorin from dermal fibroblasts
Fig 2 Electrophoretic comparison of the composition of pepsin digested
type I collagen, acid treated type I collagen, methylated type I collagen
and type VI collagen used in the experiments Samples of the different
types of collagen were applied under 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 expressed in 293 cells and
purified under nondenaturing conditions The spectra were obtained
under physiological conditions in 0.15 M NaCl Wild-type decorin,
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-type 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 E180Q and DCN E180K and truncated decorin DCN Q153 were purified under nondenaturing conditions from the medium
of 293 cells.
Secondary structural motif
Decorin mutants (%)
DCN Q153 (%)
Fig 4 Comparison of decorin synthesized in 293 cells and human skin fibroblasts 293 cells transfected with human wild-type decorin cDNA and human skin fibroblasts were metabolically labelled with [ 35 S]methionine After immunoprecipitation with a monospecific antibody, decorin proteoglycan and core protein (obtained by diges-tion with chondroitin ABC lyase) were separated by SDS/PAGE on 12.5% polyacrylamide gel Labelled proteins were visualized by autoradiography Decorin transfected 293 cells do not synthesize free core protein The molecular masses of the proteoglycan and the core protein are similar to those of the respective molecules from fibroblasts.
Trang 5Interaction of different forms of decorin with type I
collagen
To test the hypothesis that decorin can act as a bridging
molecule between type I and type VI collagen we first
performed solid phase binding assays to determine that the
binding properties of the different ligands involved
[35S]Sulphate-labelled decorin was incubated with
reconsti-tuted type I collagen fibrils and its binding was compared to
the different mutants Wild-type decorin interacted strongly
with collagen fibrils, DCN E180K reacted weakly and
DCN E180Q moderately (Fig 5) which agreed with earlier
results [12] and confirmed the 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 the collagen monomers and
[35S]sulphate-labelled proteoglycans were added These solid
phase assays showed dissociation constants of 2.3·
10)10M for decorin and 1.4· 10)9M 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
resonance spectroscopy Collagen monomers of methylated
type I collagen were covalently immobilized to a CM5
sensor chip, and the affinities of wild-type decorin, its core
protein (Fig 6A) and of the decorin mutants for the
immobilized collagen were measured Surface plasmon
resonance measurements with decorin and type I collagen
performed in the presence or absence of 15 nM ZnCl2
showed little influence of the Zn2+ ions on the binding
properties of decorin (Fig 6B) A single binding site
between wild-type decorin and type I collagen monomers
with an affinity of KD¼ 5.8 · 10)10M was found
(Table 2) In addition, a KDof 2.1· 10)8Mwas observed
for glycosaminoglycan-free core protein This value was two
magnitudes higher than that for wild-type decorin Binding
experiments with isolated glycosaminoglycan chains obtained from decorin by b-elimination showed only weak interaction with type I collagen monomers The analysis
of the interaction of DCN E180K and DCN E180Q with type I collagen yielded KD¼ 4.1 · 10–9
M and KD¼
1· 10–9
M, respectively The truncated form of decorin, DCN Q153 also showed weak interaction with type I collagen For comparative purposes we analysed the binding of biglycan, another proteoglycan of the SLRP family, using surface plasmon resonance A binding affinity
of biglycan for type I collagen monomers of KD¼ 2.7· 10)9Mwas obtained To test the reliability of these data v2-values were compared between the different experi-ments Because the v2-values ranged between 0 and 2, the data were considered to be reliable (Table 2)
Interaction of decorin and decorin mutants with type VI collagen
Decorin is known to interact directly with banded fibril forming collagens, whereas an additional, yet undefined component, is thought to be involved in the binding of
Fig 5 Interaction of radioactively labelled decorin and decorin mutants
with reconstituted type I collagen fibrils The proteoglycans were
puri-fied under nondenaturing conditions as described above Wild-type
decorin (j), DCN E180Q (h), DCN E180K (m).
Fig 6 Surface plasmon resonance measurements of decorin binding to immobilized methylated type I collagen (A) Interaction with wild-type decorin core protein (obtained by chondroitin ABC lyase digestion) in HBS Decorin core protein concentrations were as indicated (B) Interaction with wild-type decorin in HBS buffer (solid lines) and in HBS buffer containing 15 l M ZnCl 2 (dotted lines) Wild-type decorin concentrations were as indicated.
Trang 6decorin to type VI collagen [5] Recent data showed that a
complex of decorin/matrilin-1 can act as a possible linker
between type VI and type II collagen [24] In initial
experiments we studied the interaction of the various
decorin forms with type VI collagen in a solid phase
binding assay Decorin bound less avidly to type VI than to
type I collagen (KD¼ 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 and the decorin mutants
to type VI collagen in a real-time experiment, we again used
surface plasmon resonance measurements The data for the
binding affinities to type VI collagen are summarized in
Table 3 For wild-type decorin a KDof 3.6· 10)9M was
determined Because the glycosaminoglycan chain influ-enced the binding affinity of decorin to type I collagen, we also studied the binding properties of glycosaminoglycan-free core protein to type VI collagen A KDof 3.9· 10)8M was obtained for the core protein alone Isolated glycos-aminoglycan chains from decorin showed a weak inter-action with type VI collagen monomers, as reported previously [25] These binding studies constitute further evidence that the glycosaminoglycan chain stabilizes the decorin core protein
The analysis of the affinity of DCN E180K for type VI collagen yielded a KDof 4.1· 10–9
M, which is similar to that obtained for wild-type decorin Surprisingly, DCN E180Q, which showed a moderate affinity for type I collagen displayed a high affinity to collagen type VI (KDof 3.4· 10)10M), which is one magnitude lower than that found for wild-type decorin DCN Q153 interacted only weakly with type VI collagen For biglycan, however, the experiments revealed a KDof 2.1· 10)8M, which is about one order of magnitude higher than that found with type I collagen
Considering Rmax (where 1000 resonance units¼
1 ngÆmm)2) stoichiometric 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 8A,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 binding capacity of decorin to both type I and 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
resonance measurements were performed with decorin, different
dec-orin mutants, biglycan and the chondroitin sulphate/dermatan
sul-phate (CS/DS) chain released by b-elimination from decorin The
samples were purified under non-denaturing conditions from the
medium of 293 cells WT, Wild-type; core, decorin digested with
chondroitin ABC lyase; CS/DS, glycosaminoglycan chain from
deco-rin released by b-elimination; RU, resonance units.
Proteoglycan K A ( M )1 ) K D ( M ) R max (RU) v2
DCN WT 1.7 · 10 9 5.8 · 10)10 386 0.92
DCN core 4.7 · 10 7 2.1 · 10)8 199 1.9
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 [35S]sulphate-labelled wild-type decorin and the
decorin mutants DCN E180Q and DCN E180K with pepsin digested
type VI collagen in the solid phase binding assay Wild-type decorin (d),
DCN E180Q (h), DCN E180K (n).
Table 3 Binding of decorin and decorin mutants to type VI collagen Type VI collagen was digested with pepsin and immobilized on CM5 chips Surface plasmon resonance measurements were performed with decorin, different decorin mutants, biglycan and the CS/DS chain released by b-elimination from decorin The samples were purified under nondenaturing conditions from the medium of 293 cells For abbreviations see Table 2.
Proteoglycan K A ( M )1 ) K D ( M ) R max (RU) v 2
DCN WT 2.9 · 10 8 3.6 · 10)9 120 0.024 DCN core 2.6 · 10 7
3.9 · 10)8 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
Trang 7Type VI collagen was 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
VI collagen–decorin complexes subsequently bound
methy-lated type I collagen with high affinity KD values were
7· 10)9Mfor wild-type decorin and 6· 10)8Mfor DCN
E180Q For DCN E180K we found a KD an order of
magnitude lower than for DCN E180Q As the KDvalue for
a complex of type VI collagen and methylated type I
collagen in the absence of decorin was only 1.5· 10)7M, it
is obvious that the presence of decorin significantly increases
the binding affinity between the two collagens (Table 4)
Discussion
In this study we investigated the interaction of wild-type
decorin and decorin mutants with type I and type VI
collagen and, for the first time, we analysed the formation of
triple complexes consisting of type VI collagen, decorin
and type I collagen using surface plasmon resonance
measurements
Measuring the interaction of decorin with different types
of collagen by surface plasmon resonance analysis we found
a high affinity of decorin for triple helical type I collagen compared to previously published values of 10)8)10)9for 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 from fibroblast culture medium under nondenaturing conditions revealed two unique high affinity binding sites (KD¼ 7 · 10)10M and KD¼ 3 · 10)9M) and 0.043 decorin molecules per collagen monomer [28] The present study using methylated type I collagen revealed only one binding site with KD¼ 5.8· 10)10M, a value corresponding to the measurements for the highest affinity binding site in our earlier study These relatively high values may be attributable to enhanced accessibility of the binding domain of methylated compared
to nonmethylated type I collagen and/or to decorin prepared under nondenaturing conditions The binding data of decorin to monomeric collagen type I are also in agreement with studies using type I procollagen molecules [29] Interestingly, Tenni and coworkers [30], using a different technique, found a lower affinity for the interaction
of decorin with methylated type I collagen peptide frag-ments generated by CNBr cleavage The lower affinity could
be due to the fact that in this study the lysyl residues were methylated, and so might interact with the amino acid E180
of decorin Thus, compared to previously published data, variations in measurements of the affinities between decorin and collagen seem to be attributable to differences in the isolation and purification of the collagens and proteogly-cans How important the purification method is has recently been shown by Goldoni and coworkers [23] who demon-strated that freeze-drying and precipitation steps can lead to the formation of nonfunctional complexes of decorin and biglycan
Differences in affinity may also be due to other factors such as complex formation of decorin and biglycan in the presence of physiological concentrations of Zn2+[31] or 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
Zn2+is interacting with the N terminus of decorin and may cause dimerization [31], but it did not affect the interaction with the two types of 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 resonance measurement of immobilized pepsin
digested type VI collagen (A) Interaction with wild-type decorin core
protein (obtained by chondroitin ABC lyase digestion) in HBS buffer.
Concentrations of wild-type decorin core protein used were as
indi-cated (B) Interaction with wild-type decorin in HBS buffer (solid lines)
and in HBS buffer containing 15 l M ZnCl 2 (dotted lines)
Concen-trations of wild-type decorin used were as indicated.
Table 4 Type VI collagen was digested with pepsin and immobilized on CM5 chips followed by complex 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 ( M )1 ) K D ( M ) v 2
Type VI collagen 6.9 · 10 8 1.5 · 10)7 0.319 Type VI collagen/DCN WT 1.4 · 10 8
7.2 · 10)9 0.149 Type VI collagen/DCN E180K 1.6 · 10 6 6.2 · 10)7 0.18 Type VI collagen/DCN E180Q 1.7 · 10 7 5.9 · 10)8 0.141
Trang 8moderate KD 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 chains 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 the glycosaminoglycan chain of decorin
stabilizes the tertiary structure of the proteoglycans thereby
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 from
osteosarcoma cells using fibrillar type I collagen [28]
To investigate the interaction of decorin mutants 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 type I than wild-type decorin had a
similar affinity to type VI collagen as wild-type decorin
DCN E180Q 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 be not only important for the binding of decorin
to type I collagen, but may also be involved in the 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
network and interacts with a wide range of molecules
including membrane components such as integrins [34],
matrix molecules, proteoglycans [35] and matrilins [24] The
complexity of the interaction of decorin with type VI
collagen suggests a possible role of decorin as a bridging
molecule between the collagen molecules Previous studies
have shown that the binding of decorin to type VI collagen
is less efficient than the binding of decorin to type I collagen
[36] Therefore, we tried to optimize binding of type VI
collagen to the sensor chip by using a free sulfhydryl group
and not amino groups as in previous studies [37] This
method was avoided as it has been described that
immo-bilization via amino groups may lead to manifold
inter-actions of the collagen with the dextran matrix of the sensor
chip, possibly masking important binding domains on
the collagen and leading to rapid saturation of the chip
surface [19]
Previous studies indicated that biglycan interacts with
type VI collagen and is involved in the organization of type
VI collagen networks [37] Our findings indicate that the
binding of biglycan to type VI collagen is weaker (KD¼
2· 10)8M) than the binding of decorin (KD¼ 3 · 10)9M)
to this type of collagen These findings may indicate that the
lower affinity of biglycan is necessary for the fast
organiza-tion of the type 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 vitro [37] and in tissue [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 network [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 10 leucine-rich repeats of wild-type decorin, showed significantly changed CD spectra 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 synthesize decorin with its normal pre- and propeptide sequences and have expression and secretion rates similar to those in normal skin fibroblasts (Fig 4) Therefore, this system resembles more the situation in normal fibroblasts than does that in an overexpressing system [39] Crystal structures of the core proteins of small leucine-rich repeat proteoglycans have not yet been published, although in analogy to the structure of the ribonuclease A inhibitor, a horseshoe arch structure has been proposed by computer modelling [42] and this structure is supported by electron microscopic observation [43] In this model the a-helical motifs are located on the outer face of the horseshoe, while the parallel b-sheets are located inside [44]
These results do not clarify whether decorin can bind type I collagen and type VI collagen concurrently therefore
Fig 9 Models of the potential interaction of decorin with type I and type VI collagens Decorin is shown by the grey arrow, the tip of the arrow represents the C-terminus; the dashed line indicates the glycos-aminoglycan chain; type I collagen, black circle; type VI collagen, white cylinder (A) Interaction involving dimerization of decorin and binding of both collagens to the inner surface of decorin (B) Inter-action of type I collagen with the inner surface of the decorin core protein and type VI collagen binding with its noncollagenous domain
to the outer surface of decorin.
Trang 9we used a different approach to study this interaction in
greater detail We formed dimeric complexes consisting of
type VI collagen and decorin on the CM5 sensor chip and
applied methylated type I collagen to the complexes As
expected the complexes were still able to bind type I
collagen Surprisingly, the stability of the ternary complex
was higher than that of the dimeric complex of decorin
with type I or type VI collagen The existence of an
interaction of decorin with type I and type VI collagen has
been shown in vivo in skin [45] More recently a complex
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 cartilage, whereas decorin binds to the globular
N-terminal domain of type VI 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 with 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], who reported that decorin and
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 type VI and via a different
binding site which is affected by E180 to collagen type I
(Fig 9B) The exact type of interaction remains to be
established, but our findings show that decorin may be a
bridging molecule between type I and type VI collagen
networks in vitro and may be also in vivo
Acknowledgements
We thank Jo¨rg Ro¨sgen and Hans-Ju¨rgen Hinz for help with CD
spectroscopy and Konrad Beck for a critical reading of the manuscript.
This work was supported by the Deutsche Forschungsgemeinschaft,
Sonderforschungsbereich 492 Extracellular Matrix: Biogenesis,
Assem-bly and Cellular Interaction, Projects A6, A9.
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