From the
Interactions between the core protein of the small dermatan
sulfate proteoglycan decorin and type I collagen have been considered
to influence the kinetics of collagen fibrillogenesis and the diameter
of and the distance between the fibrils. A variety of recombinant core
protein fragments were expressed in Escherichia coli,
extracted from inclusion bodies, and renatured in the presence of
bovine serum albumin, which was essential for obtaining functional
activity. A recombinant protein lacking the first 14 amino acids of the
mature core protein (P15-329) interacted with reconstituted type
I collagen fibrils and inhibited collagen fibrillogenesis almost as
efficiently as intact decorin purified from fibroblast secretions under
non-denaturing conditions. Peptides comprising amino acids 15-183
(P15-183) and 185-329 (P185-329) were able to compete
for the binding of wild-type decorin, with P15-183 being more
active than P185-329. Several other peptides were much less
effective. Binding studies using radioactively labeled peptides
P15-183 and P185-329 gave direct evidence for the
independent binding of both peptides. Peptides 15-183 and
15-125 had the capability of inhibiting collagen fibrillogenesis,
whereas peptide 185-329 was inactive. These data suggest (i) that
there are at least two separate binding domains for the interaction
between decorin core protein and type I collagen and (ii) that binding
is not necessarily correlated with an alteration of collagen
fibrillogenesis.
Decorin, a small dermatan sulfate proteoglycan, is a ubiquitous
component of extracellular matrices
(1, 2) where it is
preferentially found in association with collagen fibrils
(3, 4, 5, 6, 7) . Evidence has
been provided that triple helical type I collagen possesses a specific
decorin core protein-binding site at the d-band in each D period
(5, 6, 8) . Some additional interactions may be
mediated by the dermatan sulfate side chain
(9) . As decorin
binds to the surface of collagen fibrils, the lateral assembly of
individual triple helical collagen molecules is delayed
(10, 11) , and the diameter of the fibrils is decreased
(12) . However, only moderately low dissociation constants on
the order of 10
In addition to collagen binding,
the core protein of decorin interacts with other components of the
extracellular matrix
(16, 17, 18) , with
transforming growth factor-
The three pRSET vectors contain a
T7 RNA polymerase promoter, a hexahistidine metal-binding domain, and
an enterokinase cleavage site at the 5` end of the polycloning site.
The plasmids were used to express recombinant proteins in E. coli strain INV
When indicated, much smaller quantities of BSA were
added to the renaturing solution. After dialysis, urea was added to a
final concentration of 8
M, and the purification and
renaturation protocol was repeated, but this time in the presence of
the standard concentration of BSA.
Radioactively labeled proteins
were obtained after incubation with [
A quantitative
comparison between wild-type decorin and P15-329 for the
inhibition of binding of [
The specific competition
between P15-329 and decorin for collagen binding could also be
shown by Western blotting and immune staining instead of using a
radioactive ligand. It is shown in Fig. 5 that P15-329 does not
precipitate during incubation in the absence of collagen fibrils and
that there is indeed competition between the two ligands for binding.
In a further set of experiments the influence of the recombinant
peptide and of decorin on the kinetics of fibril formation was studied.
During fibrillogenesis, decorin has to interact reversibly with the
growing fibrils, and there should be no steady state in the quantity of
bound decorin. It is seen in Fig. 6 A that P15-329 is
equally as efficient as decorin in reducing the turbidity of the fibril
suspension, which suggests a similar effect of decorin and
P15-329 on the diameter reduction of the collagen fibrils. A
prolonged lag phase was also consistently observed in the presence of
P15-329.
The interference
with decorin binding to collagen by two non-overlapping recombinant
peptides prompted us to prepare P15-183 and P185-329 in a
radioactively labeled form for use in competition assays. Fig. 8 shows
very clearly that the binding of
The situation is less clear for the
inhibition of binding of
The
radioactively labeled recombinant peptides were used for the
determination of dissociation constants. The K
In this study, recombinant decorin core protein fragments
were analyzed for their interaction with type I collagen. All the
peptides had to be extracted from inclusion bodies by a strong
denaturing agent and had to be renatured. This was possible only when
BSA was present as a chaperone protein during the renaturation process.
The renatured protein P15-329 exhibited a similar, albeit
quantitatively not exactly the same, collagen binding activity as
wild-type decorin. A strong argument for the general success of the
renaturing procedure is the observation that a protein that did not
bind to collagen could be converted to a species capable of interacting
with collagen, even after additional denaturation with urea, provided
that the appropriate conditions for renaturation were employed. The
presence of large quantities of BSA, however, precluded the use of
physicochemical methods like measurements of CD spectra for an
evaluation of the renaturation process. CD spectra of
N-glycan-free decorin showed a broad minimum around 212 nm in
contrast to the sharper minimum seen for intact decorin
(34) .
These data were explained as an indication of aggregate formation, but
it was also demonstrated that oligosaccharide-free decorin retained its
ability to bind to collagen fibrils and to retard collagen
fibrillogenesis in vitro.
An inherent difficulty of the use
of protein fragments is the fact that negative results of an assay
could be due either to the absence of the active structure within the
partial sequence or to improper renaturation. In addition to focusing
on the full-length peptide P15-329, we focused our studies on the
peptides P15-125, P15-183, P185-329, and
P125-230. P125-230 was almost completely inactive with
regard to collagen binding. However, other than the full-length
peptide, it was the most efficient inhibitor of decorin
endocytosis.
A surprising diversity of the interacting
domains of decorin core protein is the main result of our studies.
Separate high affinity binding sites to collagen were observed for
P15-183 and P185-329. P15-125 was especially active
in the inhibition of collagen fibrillogenesis, whereas higher doses
were required to compete with decorin binding to preformed collagen
fibrils when compared with P15-183. This could suggest that
P15-125 contains a lower affinity binding site. The existence of
a low affinity binding site has been proposed previously
(13) .
A recent preliminary report also arrives at the conclusion that decorin
core protein contains at least two functional domains that are involved
in the interaction with the collagen-like molecule C1q
(35) .
The existence of two high affinity binding sites may be difficult to
understand when one considers the globular structure, determined by
rotary shadowing, of the relatively small core protein
(36) .
However, the crystal structure of ribonuclease inhibitor, which, like
decorin, is a protein with leucine-rich repeats, showed that this
protein has the non-globular shape of a horseshoe and represents a new
class of
Inhibition of binding of
[
We are indebted to M. Bahl for skillful technical
assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
M have been reported for
the complex between type I collagen and decorin
(13, 14) , and in vitro studies suggested a
preferential binding of decorin to type VI collagen
(15) .
Nevertheless, mediating molecules like type VI collagen are not
required to interpret the data on the interaction between decorin and
fibrillar collagens
(9) .
(19) , and with a receptor
required for its endocytosis
(20) . Decorin belongs to a
proteoglycan family that is characterized by core proteins of about 40
kDa that contain 10-12 homologous, leucine-rich repeats of about
25 amino acids in their central sequence and that have disulfide loops
located at conserved positions near the N and C termini
(21, 22) . The single glycosaminoglycan chain of decorin
is linked with serine 4 of the mature core protein
(23) .
Previously, it had been attempted to characterize the collagen-binding
domain of decorin in detail. Chemical and enzymatic degradation studies
indicated that the 17-amino acid N-terminal peptide including the
glycosaminoglycan chain was not required for the interaction of the
proteoglycan with a collagen matrix
(24) , but the inhibition of
fibrillogenesis was abolished by a disulfide bond reduction
(25) . Further studies came to the conclusion that neither the
N-terminal half nor the central leucine-rich repeats of the core
protein can, by itself, interact fully with fibrillar collagen
(26) . In the present study we will describe that at least two
domains of decorin core protein are able to bind to fibrillar collagen
and that binding does not necessarily correlate with an inhibition of
fibrillogenesis.
Preparation of Proteoglycans
Reference
(wild-type) decorin was purified to about 95% purity from the
secretions of cultured human skin fibroblasts as described
(27) . Briefly, the preparation included sequential anion
exchange chromatographies in the presence of protease inhibitors on a
DEAE-Trisacryl M column (Serva, Heidelberg, Germany) and on a Bio-Gel
TSK DEAE-5 PW high pressure liquid chromatography column (Bio-Rad). Use
of urea and of other denaturing agents was avoided.
[S]Sulfate-labeled decorin was prepared
analogously after incubating the cells for 72 h in the presence of 1.48
MBq/ml [
S]sulfate (carrier-free)
(Amersham-Buchler, Braunschweig, Germany) and 4% (v/v) fetal calf serum
that had been dialyzed against 0.15
M NaCl.
Expression and Purification of Recombinant Decorin Core
Protein Fragments
Clone D6, which contains the coding region of
human decorin core protein, has been described previously
(28) .
In the context of the present investigation, it is noteworthy that this
clone contains a HpaI site in the untranslated downstream
region (position 1383) that had not been described in the original
publication of decorin cDNA
(21) but has been found more
recently
(29) . The clone was used to prepare the following
recombinant peptides (see and Fig. 1). P15-329 was
cloned into the BamHI/ EcoRI restriction site of the
expression vector pRSET A (Invitrogen, San Diego, CA). P15-125
was obtained as follows. An EcoRI/ AlwNI fragment of
the cDNA was made blunt ended at the AlwNI site and cloned
into the EcoRI/ BamHI site of pGEM-4Z (Promega,
Heidelberg), which was propagated in Escherichia coli strain
DH5. This procedure generated a stop codon 3` of leucine 125. The
plasmid was digested with BstUI and HindIII and
subcloned into the BamHI/ HindIII site of pRSET A. To
obtain the P15-183 construct, the cDNA was treated with
EcoRI and BsmI, and additionally with HpaI,
to distinguish between the two EcoRI sites 5` and 3` of the
BsmI site. The EcoRI/ BsmI fragment was
cloned into the EcoRI/ KpnI site of pGEM-4Z, thus
generating the codons for Arg-Tyr-Pro-Gly-Ile-Leu-Stop 3` of threonine
183, and subcloned into pRSET A as described for P15-125. A cDNA
for P33-160 was obtained by digesting the cDNA with
EcoRI/ DdeI. Further subcloning was as described for
P15-125 except that BstUI was replaced by TaqI
for digestion of the pGEM-4Z cloning intermediate. P125-230 was
obtained as follows. A HincII digest of the cDNA was cloned
into the BamHI site of pGEM-4Z. The orientation of the insert
was determined with HinfI before the vector was digested with
AlwNI/ HindIII. This fragment was then ligated into
the BamHI/ HindIII site of pRSET C (Invitrogen),
thereby creating a leucine and a stop codon 3` of the valine 230 codon.
P154-329 was obtained analogously like P15-329, except that
the cDNA was treated with BanI/ EcoRI and subcloned
into pRSET B (Invitrogen). For the generation of P185-329 a
BsmI/ EcoRI fragment of the cDNA was treated with
AlwNI for removal of the 5` sequence and subcloned into pRSET
A as in the case of P15-329.
F` (Invitrogen) as described
(30) . Bacteria
were then collected and treated with 10 ml of 6
M guanidine
hydrochloride, 0.5
M NaCl, and 20 m
M sodium
phosphate, pH 7.8/100 ml of original suspension. For purification, 2 ml
of a nickel-nitriloacetic acid agarose gel (Qiagen, Hilden, Germany)
were put into a spin column and equilibrated with 8
M urea and
0.5
M NaCl in 20 m
M sodium phosphate, pH 7.8 (buffer
A). Solubilized proteins were loaded onto the column by gravity in two
5-ml portions. The column was washed by centrifugation (1000
g
) using 7-ml portions of buffer A until the
A
was below 0.01. Nine washing steps were
usually required. Further washings were by gravity using 8
M urea, 0.5
M NaCl, 0.05% Tween 80 (Sigma) in 20 m
M sodium phosphate, pH 6.5 (buffer B) until the A
was below 0.01 (about 50 ml). Elution was achieved by applying 6
1 ml of 8
M urea, 0.5
M NaCl, and 0.05% Tween
80 in 20 m
M sodium phosphate, pH 4.0. The four fractions with
the highest A
were pooled, diluted with buffer B
to an A
of about 0.03, made 0.05% with
BSA
(
)
, and dialyzed for 48 h at 4 °C against
20 times the sample volume of 1
M urea, 2 m
M glutathione (reduced), 0.02 m
M glutathione (oxidized),
0.005% Tween 80 in 50 m
M Tris-HCl, pH 8.0. After dialysis
overnight against 0.2
PBS, insoluble proteins were removed by
centrifugation, and 1.5-ml aliquots of the supernatant were lyophilized
and reconstituted with 300 µl of H
O by brief
sonication.
S]sulfate
exactly as described
(30) .
Binding to Reconstituted Type I Collagen
Fibrils
Acid-soluble type I collagen from calf skin (Sigma,
Deisenhofen, Germany) was dissolved in 17 m
M acetic acid (4.4
mg/ml), neutralized by adding 2 volumes of 18 m
M sodium
phosphate, pH 7.4, containing 0.14
M NaCl (PBS), and incubated
for 30 min at 37 °C. The fibrils formed were collected by
centrifugation, suspended in 300 µl of 5% (w/v) BSA in PBS/mg of
collagen, and dispersed by ultrasonication. In most experiments,
recombinant peptides were tested in a competition assay. Wild-type
[S]sulfate-labeled decorin (about 8,000 cpm) was
mixed with 0.17 mg of reconstituted collagen fibrils and the respective
peptides in PBS to give a final volume of 600 µl and a final
concentration of 0.875% BSA. Controls were incubated without collagen.
After 5 h at 37 °C under constant mixing, the pellet was collected
by centrifugation (10,000
g for 10 min) and washed 3
times with PBS. Bound decorin was quantitated after solubilizing the
fibrils with 50 µl of 1
M NaOH. All assays were done in
duplicate or triplicate.
S-Labeled recombinant peptides
were assayed analogously. Their quantity was estimated from the protein
content of larger non-radioactive samples prepared in parallel.
Fibrillogenesis Assay
Collagen was prepared from
bovine tendon, and collagen fibrillogenesis was monitored
spectrophotometrically exactly as described
(10) . Appropriate
mixtures of collagen (100 µg) and either decorin or decorin
peptides in a final volume of 1 ml were made on ice and warmed up to 37
°C. The turbidity at 400 nm was measured in 5-min intervals.
Other Methods
SDS-polyacrylamide gel
electrophoresis followed by Western blotting was done as described
earlier
(27) . The polyclonal rabbit antiserum against
deglycosylated human decorin
(31) was found to react with all
recombinant peptides used in this study, but the number of reactive
epitopes of the individual peptides was not determined. For the
quantification of recombinant proteins, standard concentrations of BSA
(1-30 µg) and samples were separated on a 15%
SDS-polyacrylamide gel and transferred to nitrocellulose membranes.
They were stained with 0.1% Amido Black 10B in methanol/acetic
acid/HO (9/2/9, v/v/v) and destained in methanol/acetic
acid/H
O (45/1/4, v/v/v). Stained bands were cut out, eluted
with 300 µl of 25 m
M NaOH and 0.04 m
M EDTA in 50%
aqueous ethanol, and quantitated at 630 nm.
Expression of Recombinant Decorin Peptides
A
variety of recombinant decorin peptides (Table I) were expressed as
fusion proteins in E. coli strain INVF`. These constructs
allowed an easy preparation to more than 95% purity by a single
chromatographic step as judged by SDS-polyacrylamide gel
electrophoresis of the peptides prepared in the absence of BSA.
Previous attempts to purify peptides directly expressed in bacteria had
failed. Proteins solubilized with 8
M urea could not be
purified in a single step by chromatography on either DEAE- or
CM-Trisacryl. Chromatography of proteins solubilized with 4
M guanidine hydrochloride on octyl-Sepharose
(32) was
hampered by low recovery. Purification of P15-329 from 100 ml of
bacteria typically yielded 5 mg of peptide. After renaturation in the
presence of BSA, about 800 µg of peptide (with a range of 500-1400
µg in seven separate purifications) were obtained in soluble form.
Their quantity was determined after SDS-polyacrylamide gel
electrophoresis (Fig. 2), electroblotting, and staining of the
respective bands with Amido Black. Because the strong denaturing agent
4
M guanidine hydrochloride had to be used to extract the
peptide from inclusion bodies, the following criteria for successful
renaturation were used. P15-329 becomes insoluble after reduction
and alkylation, indicating that solubility depends on the formation of
disulfide bonds. As functional tests, the inhibition of binding of
wild-type decorin to type I collagen fibrils and to the endocytosis
receptor (not shown) was employed. Successful renaturation required the
presence of sufficient quantities of BSA (Fig. 3). When only 10
µg/ml BSA were included in the renaturing buffer, the resulting
product failed to compete with wild-type decorin for binding to
collagen fibrils. However, refolding to an active state occurred even
after further treatment with 8
M urea provided that sufficient
quantities of BSA were present during renaturation. BSA was therefore
considered to act as a chaperone and to enable the proper folding of
P15-329. The experiment also demonstrates that solubility is not
a sufficient criterion for the functionality of P15-329. It had
recently been reported that decorin core protein, functionally active
with respect to transforming growth factor-
binding, could be
obtained as a fusion protein with E. coli maltose-binding
protein
(33) . Use of this strategy, however, yields a fusion
protein twice the size of the wild-type core protein.
S]sulfate-labeled
decorin to collagen indicated that the doses required for half-maximal
inhibition (IC
) were of the same order of magnitude,
although the doses required for the recombinant peptide were always
somewhat higher (Fig. 4). In seven different preparations the IC
of P15-329 was minimally 1.2-fold and maximally 3.5-fold
higher than that of wild-type decorin.
Existence of Several Distinct Collagen-binding
Sites
The procedure described above was used to renature a
variety of recombinant decorin peptides that were designed to cover
several N- and C-terminal regions as well as a central region
(Fig. 1). One of the peptides, P33-160, was anticipated to
be biologically inactive and to be employable as a negative control
because it contained a single cysteine residue only. With the exception
of P125-230, which covered the central cysteine-free region, all
other peptides contained a paired number of cysteine residues and were
anticipated to be capable of forming disulfide bridges. In support of
this expectation, these peptides formed insoluble complexes upon
reduction and alkylation.
Figure 1:
Schematic structure of mature human
decorin core protein and restriction sites of decorin cDNA. In
A, the consensus sequence
Leu-Xaa-Xaa-Leu-Xaa-Leu-Xaa-Xaa-Asn-Xaa-(Leu/Ile)-(Ser/Thr)-Xaa-(Val/Ile)
is boxed. Numerals above the boxes give the
degree of conservation of the 7 specified residues without ( first numeral) or with ( second numeral)
taking into account conservative exchanges. Small numerals indicate the number of amino acids between the boxed units and the position of the first amino acid of these units,
respectively. The glycosaminoglycan attachment site is indicated by a
large arrow, and cysteine residues are shown by smaller
arrows. In B, the cDNA is related to the exon boundaries.
The restriction sites are indicated by dashed
lines.
When the different peptides were used in a
competition assay, the lowest IClevels were obtained for
the ``full-length'' peptide, P15-329, and for
P15-183, which contains the first six N-terminally located
leucine-rich repeats in addition to the N-terminal cysteine-rich region
(Table II and Fig. 7). A shortened N-terminal peptide, P15-125,
required at least three times higher doses for half-maximal inhibition
(three independent experiments). Unexpectedly, two C-terminally located
peptides, P154-329 and P185-329, containing 5 and 6
leucine-rich repeats, respectively, were also able to interfere with
collagen binding of wild-type decorin, but the centrally located
peptide P125-230 was not. Nevertheless, this peptide exhibited
functional activity because it was well suited in interfering with the
endocytosis of decorin.
(
)
S-labeled P185-329
to collagen fibrils can be best inhibited by the peptide itself and by
the full-length peptide P15-329. Very minor inhibition only could
be achieved by P15-183.
S-labeled P15-183. Although
the full-length peptide and P15-183 itself were the best
competitors, P185-329 and, to a lesser extent, P125-230
were also able to interfere with binding. This could indicate that the
latter two peptides carry a domain interacting with the P15-183
binding site, albeit with lower affinity. Alternatively, the binding of
P185-329 affects, by steric hindrance, the binding of
P15-183 to an adjacent site, whereas the converse does not occur.
Steric hindrance is also a possible explanation for the observation
that P15-183 inhibited the binding of native decorin to collagen,
whereas the same peptide was not an inhibitor of the interaction
between P185-329 and collagen. In the presence of P15-183,
an interaction of native decorin via its C-terminal collagen-binding
domain should be expected in the absence of steric hindrance.
value of P15-329 was 3
10
mol/liter, an order of magnitude smaller than the
K
values for both P15-183 and
P185-329 (Fig. 9).
Interference with Collagen Fibrillogenesis
Several
of the recombinant decorin peptides were tested for their influence on
collagen fibrillogenesis. It is shown in Fig. 6 B that
P15-183 was approximately as efficient as P15-329 and
wild-type decorin in inhibiting the formation of fibrils. The
collagen-binding peptide P185-329, however, was ineffective,
although all recombinant peptides had an influence on the length of the
lag phase preceding fibrillogenesis. It is also noteworthy that
P15-125 is well suited for interfering with fibril formation,
although its ability to compete with decorin for collagen binding is
only moderate. These general findings were corroborated by the analysis
of at least two different peptide preparations. Considering the
previous finding that the reduction of disulfide bonds of decorin
abolishes its capability to inhibit fibrillogenesis
(25) , it is
tempting to speculate that the N-terminal region of decorin, which
contains 4 cysteine residues and is present in P15-183 and in
P15-125, is of special importance for this function.
Figure 6:
Collagen fibril formation in the presence
of decorin ( DCN) and recombinant
peptides.
Thus, all four of the peptides were
consistently found to be active in at least one assay system. The
segment of the core protein represented by the recombinant peptide was
therefore considered to possess at least a cryptic site for the
respective interaction.
/
protein folds
(37) . Provided that decorin
core proteins exhibit an analogous structure, the data presented in
this study are compatible with the assumption that the surface area
formed by the fifth and/or sixth of the leucine-rich repeats, which are
not contained within P15-125 (see Fig. 1), and a repeat
from the C-terminal half of the molecule are involved in the binding to
collagen fibrils. The flexible N-terminal part of the molecule with its
two disulfide bridges is contained within both P15-125 and
P15-183 and is considered to be involved in fibrillogenesis.
Further studies, however, are needed to elucidate the interacting
structures in greater detail.
Table: Recombinant decorin peptides
Table: Inhibition of decorin-type I
collagen interaction by recombinant decorin peptides
S]sulfate-labeled wild-type decorin to collagen
fibrils by recombinant peptides was measured as described in the legend
of Fig. 7.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.