From the Department of Biochemistry, University of
Alberta, Edmonton, Alberta T6G 2H7, Canada, the ¶ Molecular
Biophysics Group, Synchrotron Radiation Department, Central Laboratory
of the Research Councils Daresbury Laboratory, Warrington, Cheshire WA4
4AD, United Kingdom, and the
Wellcome Trust Centre for
Cell-Matrix Research, School of Biological Sciences and Research Group
in Eye & Vision Science, School of Medicine, University of Manchester,
Manchester M13 9PL, United Kingdom
Received for publication, November 22, 2002, and in revised form, February 20, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Decorin is a widely distributed member of the
extracellular matrix small leucine-rich repeat
glycoprotein/proteoglycan family. For investigation of its physical
properties, decorin from two sources (young steer skin and a
recombinant adenovirus) was used. The first sample was extracted
into 7 M urea and purified, while the second was
isolated from medium conditioned by 293A cells infected with adenovirus
and purified without chaotropes. The only chemical differences detected
between these materials were a slightly shorter glycosaminoglycan chain
and the retention of the propeptide on the latter. Circular dichroism
spectra of the two samples were virtually identical, showing a high
proportion of The small proteoglycan decorin is an extracellular matrix protein
that has been identified in many connective tissues. Its functions are
believed to include regulation of the formation and/or organization of
collagen fibrils (1), inhibition of the calcification of soft
connective tissues (2), modulation of the activity of growth factors
such as transforming growth factor- There have been a number of studies of the interactions of decorin with
other extracellular proteins, with cytokines such as transforming
growth factor- Previous studies have shown that some samples of decorin extracted from
tissues with chaotropes do not regain native structure and activity
once the denaturant has been removed (15). There have also been reports
that some preparations of tissue-derived decorin have lower activity in
functional assays than recombinant decorin (20). Therefore, here we
used both extracted decorin and recombinant prodecorin purified
entirely without chaotropes. The results clearly establish that both
forms of decorin are dimers in solution, over a wide concentration
range. Furthermore, the solution x-ray scattering profile gives insight
into the shape of this protein dimer.
Isolation and Purification of Intact Decorin and Core
Protein--
Bovine skin decorin was extracted from the hide of a
young steer of between 18 months and 2 years of age into deionized 7 M urea, 0.15 M NaCl, 0.05 M Tris,
adjusted to pH 6.5 with HCl and containing sodium azide (0.02%, w/v)
and proteinase inhibitors (10 mM disodium EDTA, 0.1 M 6-aminohexanoic acid, 5 mM benzamidine-HCl, 0.5 mM N-ethylmaleimide, 1 mM
phenylmethane sulfonic acid, 5 mg/liter pepstatin, 5 mg/liter
leupeptin). It was purified by a combination of anion-exchange
chromatography on DEAE-cellulose, precipitation with 75% ethanol, and
cetylpyridinium chloride and gel-filtration chromatography (21).
Recombinant bovine prodecorin was isolated from the medium of 293A
cells infected with an adenovirus engineered to express full-length
bovine decorin. The Adeno-QuestTM kit (Quantum Biotechnologies Inc.,
Laval, Quebec, Canada), comprising a transfer vector (pQBI-AdBM5),
ClaI-digested DNA from adenovirus type 5 with E1 and E3
deletions, and associated reagents, was used. The cDNA encoding the
full protein sequence of bovine decorin was kindly provided by Dr.
Larry Fisher of the Bone Research Branch, NIDR, National Institutes of
Health, Bethesda, MD, as an EcoR1 insert in the plasmid
PG-28 (10). For large-scale production of recombinant decorin, host
293A cells were grown to confluence in "triple flasks" of
500-cm2 growth area (Nalge Nunc International), 6 × 107 cells per flask, in DMEM supplemented with 10% fetal
bovine serum (FBS). The medium was changed to DMEM, 5% FBS (90 ml/flask) and 4 × 109 PFU of decorin adenovirus were
added to each flask, which was then incubated at 37 °C for 72 h. Cells and medium were harvested by centrifugation at 1000 × g for 10 min at 4 °C. The supernatant collected from 8 triple flasks (about 700 ml) was thawed and applied to a 2.6 × 10-cm column of DEAE-Sephacel (Amersham Biosciences) in 0.2 M NaCl, buffered at pH 7.2 with sodium phosphate (buffer A)
at 4 °C. The column was washed with 5 volumes of buffer A, and bound
material was then eluted with a linear gradient of 150 ml each of
buffer A and buffer B (buffer A with 1.0 M NaCl). Fractions containing decorin, as detected by the dimethylmethylene blue assay for
glycosaminoglycans (22), were pooled, diluted with water to reduce the
concentration of NaCl to below 0.1 M, and reapplied to the
same DEAE-Sephacel column, which had been washed extensively with
buffer A. Elution of bound proteoglycan, which emerged as a single
broad peak at about 0.5 M NaCl, was repeated as above.
After extensive dialysis against water, the partially purified decorin
(yield 20 mg) was freeze-dried. Portions (10 mg) of this material were
dissolved in 10 ml of 20 mM Tris/HCl, 0.5 M
NaCl, pH 7.2 (buffer C) and applied to a 1 × 5-cm column of
concanavalinA-Sepharose (Sigma Chemical Co.), equilibrated in buffer C
at 4 °C. After washing to remove any unbound material, as detected
by monitoring the absorbance at 230 nm, bound proteoglycan was eluted
as a single peak with a linear gradient of 15 ml each of buffer C and
the same buffer containing 0.5 M
Decorin core protein was prepared by digesting intact proteoglycan with
chondroitin ABC lyase (EC 4.2.2.4, Seikagaku) at a ratio of 1 unit of
enzyme per 6 mg of proteoglycan in 0.1 M Tris adjusted to
pH 7.3 with acetic acid and containing 0.02% (w/v) sodium azide. The
digest was then applied to a 0.8 × 5-cm column of concanavalin
A-Sepharose equilibrated in 0.05 M sodium phosphate, 0.5 M NaCl, pH 7.0 at a flow rate of 15 ml/h. After washing
with about 8 column volumes of this buffer, bound proteoglycan core was
eluted as a single peak with the same buffer containing 0.3 M Characterization of Decorin Preparations--
Protein contents
were determined by the BCA assay (Pierce) using bovine serum albumin as
a standard (23). Glycosaminoglycan contents were determined by the
dimethylmethylene blue dye-binding assay using pig skin dermatan
sulfate (Seikagaku Corporation, Tokyo, Japan) as a standard (22). The
purified proteoglycans and the protein cores produced from them by
digestion with chondroitin ABC lyase or by chondroitin ABC lyase
followed by N-glycosidase F (EC 3.2.2.18, Roche Molecular
Biochemicals) as described (12) were examined by gel electrophoresis in
10% polyacrylamide gels containing 0.1 M Tris, 0.1 M sodium borate, 0.1% SDS, pH 8.6 (24). The reactivity of
the recombinant decorin with four monoclonal antibodies (7B1, 3B3, 5D1,
and 6D6) prepared against natural bovine decorin was tested by Western
blotting, as described (25). The sequence of the first 27 amino acid
residues of the recombinant decorin was determined in a Biolynx amino
acid sequencer on a sample of the core protein produced by digestion
with chondroitin ABC lyase, purified by SDS-PAGE, and blotted onto a
polyvinylidene difluoride membrane (26).
Circular dichroism spectra were recorded using a Jasco J-810
spectropolarimeter on samples of proteoglycan dissolved in 25 mM sodium phosphate, pH 7.0 at protein concentrations of
about 0.1 mg/ml (determined by the BCA protein assay), in thermostatted cells of 1-mm pathlength.
Gel Chromatography and Light Scattering--
Samples of intact
natural and recombinant decorins were dissolved at various
concentrations (determined by the BCA protein assay, using bovine serum
albumin as standard), in TBS (0.15 M NaCl, 20 mM Tris-HCl, 0.02% (w/v) sodium azide, pH 7.0). After centrifuging at 12,000 × g for 10 min to remove
particulate matter, samples of 0.25 or 0.5 ml were chromatographed on
SuperoseTM 6 HR 10/30 and SuperoseTM 12 HR
10/30 columns (Amersham Biosciences) in TBS with or without 2.5 M GdnHCl. The effluent from these columns was led through a
DAWN-DSP laser light scattering photometer and Optilab 903 interferometer refractometer (Wyatt Technologies, Santa Barbara, CA).
Light scattering data were processed by the Zimm method, using
AstraTM for Windows software, version 4.10 (Wyatt
Technologies). Values of dn/dc of 0.150 and 0.170, as determined
previously (18), were used for the intact skin decorin and the protein
cores produced by digestion with chondroitin ABC lyase. A value of
0.160 was assumed for the recombinant prodecorin based on its lower
glycosaminoglycan content compared with that of skin decorin (see
below). Satisfactory performance of the refractometer and light
scattering detectors was checked by chromatography of samples of bovine
serum albumin.
Solution X-ray Scattering--
X-ray scattering data were
collected at station 2.1 (27) of the Synchrotron Radiation Source
(SRS), Daresbury Laboratory. Solutions for the scattering experiments
were prepared in TBS, pH 7.0, chromatographed on Superose-12 or
Superose-6 columns to remove any aggregated material (see below), and
then concentrated by ultrafiltration, as described above. Both
full-length decorin and decorin core were measured at 4 °C, whereas
the latter was also measured at 20 °C. The studies were performed in
a standard cell at sample concentrations of 1 or 5 mg/ml, according to
established procedures (28). This included the examination of the
scattering data, which had been collected in individual time frames of
60 s, to ensure that no observable radiation damage, protein
aggregation, or deposition on the cell windows, had occurred. After
checking and averaging several runs, it was concluded that the samples were unaffected by the synchrotron X-rays and that the profiles obtained over a total data collection time of 90 min (120 min) for
intact decorin (decorin core) were suitable for subsequent data
analysis. A sample-to-detector distance of 1.25 m was used for
both samples to cover the momentum transfer interval 0.03 Å Fig. 1 shows a comparison of the
purified natural and recombinant decorin samples by SDS-PAGE. The only
differences that are apparent are the slightly higher electrophoretic
mobility and more diffuse character of the band obtained with intact
recombinant decorin (lane 4), compared with the natural
material (lane 1). No differences were seen between the core
proteins produced by digestion of the intact proteoglycans with
chondroitin ABC lyase (lanes 2 and 5). Therefore
the higher mobility of the recombinant decorin is a result of its
having a shorter dermatan sulfate chain. This was confirmed by the
lower content of dermatan sulfate measured colorimetrically (27%
compared with 35%). The more diffuse appearance of this band indicates
that the glycosaminoglycan chain on the recombinant decorin is also
more variable in length. The indistinguishable mobilities of the bands
seen after further digestion of the chondroitin ABC lyase core proteins
with N-glycosidase F (lanes 3 and 6), demonstrate that both materials are substituted with
N-linked oligosaccharides to the same extent. The
recombinant and natural decorins are both recognized on Western blots
by four monoclonal antibodies (7B1, 3B3, 5D1, and 6D6) raised against
bovine skin decorin (25) (data not shown).
-sheet and
-turn and little
-helix. The protein
cores were completely denatured in 2.25 M guanidine HCl
(GdnHCl) but recovered their secondary structure on removal of
chaotrope. Light scattering of material eluted from gel-filtration
columns in Tris-buffered saline, pH 7.0, gave molecular mass values of
165 ± 1 kDa and 84.6 ± 4 kDa for intact decorin and the
glycoprotein core produced by digestion with chondroitin ABC lyase,
respectively. Intact recombinant prodecorin had a mass of
148 ± 18 kDa. These values, which are double those estimated from
SDS gel electrophoresis or from the known sequences and compositions,
were halved in 2.5 M GdnHCl. Data from solution
x-ray scattering of intact decorin and its core in Tris-buffered saline
are consistent with a dimeric particle whose protein component has a
radius of gyration of 31.6 ± 0.4 Å, a maximum diameter of
98 ± 5 Å, and approximates two intertwined C shapes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(3), and other effects on cell
proliferation and behavior (4, 5). Indeed, recent evidence suggests
that decorin plays an important role in suppressing tumor growth by two
independent mechanisms (6, 7). Decorin consists of a protein core of 329 (8, 9) or 330 (10) amino acids, to which are attached a single
glycosaminoglycan chain of dermatan or chondroitin sulfate (at Ser-4,
Ref. 11) and three N-linked oligosaccharides (12). In
chicken there is a form of decorin that carries two glycosaminoglycan chains (13). The amino acid sequence of decorin includes a series of at
least 10 leucine-rich repeats
(LRRs),1 averaging 24 residues in length (14). Decorin is a member of the family of
extracellular matrix small leucine-rich repeat
glycoproteins/proteoglycans (15) but the LRR motif is more widely
distributed and has been found in many intracellular, cell surface, and
extracellular proteins (16).
(3) and with cell-surface receptors (4, 6).
Nevertheless, the physical properties of this small proteoglycan, which
are important for the proper interpretation and understanding of these
interactions, have not been well characterized. Early light scattering
studies on decorin (then called proteodermatan sulfate) extracted from
pig skin suggested a very high particle mass in solution: 3.4 × 103 kDa (17). A later study of material extracted from cow
skin, as described here, gave molecular masses for the intact
proteoglycan and the glycoprotein core produced by digestion with
chondroitin ABC lyase in 0.15 M NaCl of 610 and 650 kDa,
respectively (18). In 4 M GdnHCl the molecular masses
reduced to 62 and 39 kDa; values much closer to those estimated from
the mobility in SDS-PAGE or calculated from the amino acid composition
(36,333 Da) plus the single dermatan sulfate chain (~24 kDa, Ref. 18)
and the three N-linked oligosaccharides (~6
kDa).2 The anomalously high
molecular weights measured in solution were attributed to
self-association that has also been reported to be a property of
dermatan sulfate chains in NaCl (19). However, the exponential
dependence of light scattering intensity on particle size can lead to
erroneous results for samples containing small proportions of
aggregated material incompletely removed during sample preparation or
formed in situ. The investigation described here avoided
this pitfall by using continuous monitoring of the refractive index and
light scattering intensity of the effluent from gel chromatography
columns. The contribution of any aggregate present in the solution is
then readily detected and can be corrected for, or, if necessary, it
can be eliminated by re-chromatography.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-methylmannoside. Fractions containing glycosaminoglycan were pooled, dialyzed
exhaustively against water, and freeze-dried. The yield of purified
recombinant decorin was about 14 mg from each batch of 700 ml of
conditioned medium. This material was stored dessicated at
80 °C.
-methyl-D-mannoside (Sigma). Fractions
containing protein, as monitored by absorption at 280 nm, were pooled
and desalted by three successive rounds of 10-fold concentration and
dilution with water in a centrifugal concentrator (Millipore
UltrafreeR, nominal molecular mass cutoff 10 kDa) at
1900 × g. This material was stored at 4 °C.
1
q
0.63 Å
1. In view of the
large size of intact decorin, additional measurements at a distance of
4.25 m were performed, permitting q-values between 0.006 Å
1 and 0.18 Å
1. The latter provided a
sufficiently large scattering range for merging profiles collected at
short and long detector distances. The modulus of the momentum transfer
is defined as q = 4
sin
/
, where 2
is the
scattering angle and
is the wavelength (1.54 Å). The q-range was
calibrated using silver behenate powder and wet rat tail collagen
(based on diffraction spacings of 58.38 and 670 Å, respectively). The
radius of gyration, the forward scattering intensity, and the
intraparticle distance distribution function p(r) were
calculated from the experimental scattering data using the indirect
Fourier transform method as implemented in the program GNOM (29).
Particle shapes were restored from the experimental scattering profiles
using the ab initio procedure based on the simulated
annealing algorithm to a set of dummy spheres representing the amino
acid chain of the protein (30). Since both intact decorin and decorin
core exist as dimers in solution (see below), shape restorations were
performed using a 2-fold symmetry axis. The program CRYSOL (31) was
used for the simulation of scattering curves from models based on the
crystal structures of YopM, the leucine-rich effector protein from
Yersinia pestis (PDB codes 1G9U and 1JL5).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (57K):
[in a new window]
Fig. 1.
Comparison by SDS-PAGE of purified natural
decorin and recombinant prodecorin. Lane 1, intact
natural decorin. Lane 4, intact recombinant prodecorin.
Lanes 2 and 5, the glycoprotein cores produced
from the material in lanes 1 and 4 produced by
digestion with chondroitin ABC lyase. Lanes 3 and
6, protein cores of material in lanes 1 and
4 produced by sequential digestion with chondroitin ABC
lyase and N-glycosidase F (lanes 3 and
6). Each lane carries 10 µg of sample. Gels were stained
with Coomassie Blue R250. See text for other details.
Decorin from cow skin has been sequenced in its entirety in our laboratory by Edman degradation (9). The sequence of the first 27 amino acids of the recombinant decorin was determined in the present study by Edman degradation on a sample of the protein core produced by digestion with chondroitin ABC lyase. The major sequence (accounting for about 75% of the yield of phenylthiohydantoin-derivatized amino acid at early cycles) was GPFQQKGLFDFMLEDEA-GIGPEEHFP. This is the sequence of the pro-form of bovine decorin. A second, minor, sequence (DEA-GI-PE), corresponding to the N-terminal sequence of mature bovine decorin, was apparent at cycles 1 to 9. We therefore conclude that proteolytic processing of the recombinant protein by the host 293A cells was inefficient and that this product is predominantly in the pro-form. The gap in the sequence at cycle 18 (or 4) corresponds to the serine residue that is substituted with the single dermatan sulfate chain in bovine decorin (11).
Fig. 2 shows circular dichroism spectra
recorded for intact natural decorin and recombinant prodecorin samples
in 0.025 M sodium phosphate buffer at pH 7.0 and 25 °C.
Three features (Cotton effects) are apparent: a negative at 217 nm, a
weak positive at 204 nm, and a second (weaker) negative at 196 nm. The
spectra for natural and recombinant decorin are very similar and are
characteristic of secondary structures dominated by -sheet and
-turn, with little if any
-helix. We have previously published an
interpretation of an almost identical spectrum that had been obtained
for the protein core of a different sample of natural decorin prepared as described here, namely 2%
-helix, 43%
-sheet, and 14%
-turn (32). Circular dichroism spectra recorded in the presence of 2.25 M GdnHCl (Fig. 2) show essentially complete abolition
of the negative Cotton effect near 217 nm. Removal of GdnHCl by
dialysis resulted in complete recovery of the original secondary
structure, as previously reported by us for urea (32).
|
Fig. 3A shows the results of
chromatography on Superose-12 of a sample of bovine skin decorin
dissolved in TBS. The refractive index signal shows a major peak,
accounting for more than 90% of the total mass of recovered material,
with a small shoulder on the leading edge accounting for about 7%. The
90° light scattering detector shows three peaks: one at
V0 not corresponding to any detectable refractive index
signal and therefore presumably caused by a small amount of fine
particulate material, which was not removed by centrifugation at
12,000 × g, a second associated with the shoulder on
the leading edge of the main peak, and a third, corresponding to the
main refractive index peak. Results for recombinant prodecorin were
identical (data not shown). Light scattering by the small amount of
material associated with the shoulder, which was always present in
freshly dissolved samples of intact natural decorin or recombinant
prodecorin, is strong and interferes with the estimation of the
molecular weight of the material in the major peak. Therefore fractions
corresponding to the major peak, pooled as indicated, were
re-chromatographed on the same column at least once (Fig.
3B), prior to the calculation of molecular weights.
Duplicate analyses gave molecular masses for natural and recombinant
decorin of 165 ± 1 kDa (average and range) and 148 ± 18 kDa, respectively. The effects of concentration on chromatography and
mass were investigated by re-running material from the main peak on the
same column. No decrease in average mass was seen for intact decorin
samples at peak concentrations of between 2.53 µM and
59.5 nM. Chromatographic behavior, as assessed by
monitoring the refractive index, was unchanged even at 2.7 nM, although values for mass could not be estimated
reliably at such low protein concentrations. The dimer is therefore
stable at very high dilutions and does not have a measurable
dissociation constant under the conditions used here.
|
Fig. 4A shows gel
chromatography profiles and plots of molecular weight against elution
volume for intact decorin and the protein core in the presence and
absence of 2.5 M GdnHCl. Denaturation of intact decorin by
GdnHCl caused a slight increase in elution volume but an ~2-fold
decrease in mass (to 88.3 ± 2.8 kDa). The native core protein
gave only a single peak of mass 84.6 ± 4 kDa when
chromatographed in TBS but in 2.5 M GdnHCl it eluted
earlier, with a 2-fold reduction in mass (to 48.2 ± 3.6 kDa).
This value is in reasonable agreement with the molecular mass of 42.3 kDa that can be calculated for the decorin monomer from its composition (36.3 kDa for the protein and 6 kDa for the N-linked
oligosaccharides). Fig. 4B compares the chromatographic
behavior and mass of samples dissolved in TBS containing 2.5 M GdnHCl and then chromatographed in TBS alone, with those
dissolved directly in TBS. Material under the peaks obtained in the
first case gave mass values of 112.8 kDa (for intact decorin) and 68.9 kDa (for the protein core). These values are intermediate between those
determined above for the denatured monomers (in 2.5 M
GdnHCl) and the native dimers (in TBS), indicating that they are
mixtures of monomer and dimer. Core protein recovered from these
columns gave CD spectra that were indistinguishable from those of the
native protein shown in Fig. 2. Taken together, these results show that
denatured monomeric decorin can reassemble into native dimers in
solution rapidly when separated from chaotrope.
|
Solution x-ray scattering profiles of intact natural decorin and
decorin core and their distance distribution functions are shown in
Fig. 5, A and B,
respectively. The insets to the p(r) plots in
Fig. 5B show the low angle regions in the form of Guinier plots (i.e. ln(I) versus q2), from
which the radius of gyration (Rg) can be
extracted from the slope (Rg2/3) of
the straight line. Here the curves confirm the expected linearity for
q 1/Rg based on the
Rg values derived from the p(r)
analysis for each protein form, emphasizing that p(r)
analysis and Guinier approximation are in good agreement. When
calculating the radius of gyration via the p(r) function,
one has to bear in mind that the smallest measured scattering angle
qmin
/Dmax (see e.g. Ref. 33),
a condition which has been met in our experiments.
|
The profile for intact decorin (Fig. 5A) shows a significant contribution from glycosaminoglycan and oligosaccharides (only ~60% of the molecular weight is due to protein), noticeable from the steep rise in the scattering signal at very low angles, which is independent of protein concentration. This is expected from an extended, random chain-like behavior of the sugar chains. Therefore it can be assumed that the sugar chains point away from the decorin core mixing with the solvent. In contrast to the result for intact decorin, for the decorin core scattering from protein dominates the curve, which therefore shows no significant increase in intensity at very low angles. Thus the results are consistent with a globular, dimeric decorin core particle. Molecular masses of intact decorin and decorin core were checked against the forward scattering from a bovine serum albumin solution (66 kDa) and were consistent with dimers in solution. Radii of gyration (Rg) for intact decorin and decorin core at 4 °C were 92.8 ± 0.7 Å and 31.6 ± 0.4 Å, respectively. The latter was also measured at 20 °C (Rg = 31.8 ± 0.4 Å) with identical scattering features, showing that the conformation of the core structure does not change in this temperature range. The maximum particle dimension as deduced from the p(r) function, which represents the distribution of distances between atoms (i.e. scattering centers) within the intact decorin dimer, is Dmax = 350 ± 15 Å and for the decorin core dimer 98 ± 5 Å.
In order to visualize conformational properties of the core protein,
shape reconstructions were performed ab initio from the scattering profiles using the dummy spheres model (30), assuming a
2-fold molecular symmetry. As can be seen from Fig.
6, the shape of the decorin core can be
approximated by two "C" shapes intertwining. With the purpose of
providing a simple comparison between the reconstructed shape and a
known protein structure containing leucine-rich repeats, a ribbon model
of the Y. pestis cytotoxin YopM (residues 34-330, only)
(34) has been arranged and displayed in the form of an intertwined
dimer (Fig. 6, right hand side). The choice of YopM for this
purpose was based on the fact that it has no -helices within the LRR
domain, unlike most other known leucine-rich repeat protein structures,
which contain a considerable proportion of
-helix. As noted above,
the CD spectrum of decorin indicates little if any
-helix. The
lengths of the repeats are also quite similar (20 or 22 residues for
YopM and, on average, 24 for decorin), as is the overall length.
|
Finally, a scattering pattern simulation using different dimer
arrangements based on the YopM crystal structure (see Fig. 7), demonstrates that shapes represented
by a "horseshoe" or "doughnut," among others, are not in
harmony with the data presented here. We note that despite the lack of
a contribution from carbohydrate chains, which have not been modeled
due to their intrinsic flexibility, the model shown in Fig. 6 provides
an excellent fit to the experimental scattering data of the decorin
core (yielding a goodness of fit value of = 1.5).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
By carrying out the light scattering analysis of intact decorin and its glycoprotein core on the continuously flowing effluent from gel filtration columns, we demonstrate here that the predominant species in solution at room temperature are, in both cases, dimers. A small amount of aggregated material with strong light scattering elutes as a shoulder ahead of the main peak when freshly dissolved samples of either natural decorin or recombinant prodecorin are chromatographed. This shoulder corresponds to about 7% of the total mass, as measured by refractive index. Once removed by chromatography it does not reform in solution, even after 2 weeks at 4 °C.2 Further characterization of this minor fraction, which appears to be generated when salt-free solutions are freeze-dried, will be described elsewhere. Failure to remove aggregates such as this in earlier light scattering studies may have led to the erroneously high estimates of particle mass.
Repeated re-chromatography on the same column of the material from the major peak, demonstrated that the decorin dimer is stable over a wide range of concentrations: no dissociation could be detected even at peak concentrations as low as 2.7 nM. Exposure to 2.5 M GdnHCl, however, converted both the intact decorin and glycoprotein core dimers to monomers with estimated values for Mw close to those that can be calculated from the compositions. This concentration of GdnHCl is sufficient to destroy all secondary structure (Fig. 2), and the dimerization therefore depends on the existence of a native protein core.
Removal of GdnHCl from solutions of decorin by dialysis or by dilution during gel filtration restores the secondary structure and facilitates the rapid reformation of dimers. Since the sample of natural decorin used here had been isolated from skin using a chaotropic agent (7 M urea), it might be argued that the reversible denaturation and dimerization are artifactual. However, identical behavior was seen for a sample of recombinant prodecorin that had been isolated and purified without the use of chaotropes. The tendency to form a stable dimer in solution would therefore seem to be an intrinsic property of decorin.
Decorin belongs to a family of 12 extracellular matrix proteins that
contain leucine-rich repeats. Biglycan has been shown to form dimers
reversibly (35) but the aggregation states of the other extracellular
matrix LRR proteins have not been characterized. However these are
likely to adopt similar structural folds, resembling those in
ribonuclease inhibitor (36) and other related proteins of known
structure. With one exception (an Azotobacter vinelandii protein, PDB code: 1LRV) these all take the form of a partial "solenoid" with an inner face made up of parallel -strands, a structural feature that has been suggested to provide sites for interaction with other proteins. In the case of decorin we suggest that
this feature facilitates the dimerization.
Electron-microscopic images of decorin preparations have been
interpreted as showing horseshoe- or arch-shaped structures, such as
might be expected for a molecule with the same overall topology as
ribonuclease inhibitor (37, 38). The resolution available in the
electron microscope does not, however, permit the detailed shape to be
deduced, nor does it allow the unambiguous distinction of monomers and
dimers. The appearance of the specimens may also be influenced by the
preparative techniques used. Solution x-ray scattering can give a
higher (but still low) resolution structure of macromolecules directly
in solution. We have applied this technique to both intact decorin and
its glycoprotein core. Satisfactory reconstruction of a shape for the
intact decorin dimer is difficult in view of the significant
contribution to the scattering from the two dermatan sulfate chains.
These are presumably very flexible and their large contribution to the
total molecular mass makes model building for scattering profile
simulations complicated and beyond the scope of the present report.
However the x-ray scattering by the decorin core particle can be
modeled satisfactorily and the resulting model fitted with a dimer of YopM monomers arranged as 2 intertwined C shapes. Interestingly, YopM
carries two long -helices at the extremities. It is tempting to
suggest that in decorin there are sugar chains contributing equivalent
mass in this location.
We do not present evidence here relating directly to the aggregation
state of decorin in tissues. However, the finding that it is a highly
stable dimer in solution, and that dimerization probably results from
interactions between the -sheet surfaces, has important implications
for understanding interactions with other proteins both in
vivo and in vitro. It has been proposed that a
horseshoe-shaped monomeric decorin, modeled on the
-helix rich
ribonuclease inhibitor, binds to monomers of collagen through its inner
-sheet face (39). However the x-ray scattering data presented here
are not consistent with a horseshoe shape and further imply that part
or all of the
-sheet face is unavailable for binding. Our data
suggest that in functional studies it should not be assumed that
decorin is a monomer when calculating affinity constants etc.;
moreover, as a dimer it may bind more than one ligand. In the light of
the results described here, re-examination of these earlier assumptions
and models would appear necessary.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Sophie Lehocky and Leigh Ann Giebelhaus for technical assistance. We thank Prof. Martin Humphries (University of Manchester and Wellcome Trust Centre for Cell-Matrix Research) for allowing us to use some of his x-ray scattering beam time at the Daresbury SRS.
![]() |
FOOTNOTES |
---|
* This work was supported by the Canadian Institutes for Health Research and The Wellcome Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Tel.: 780-492-5755; Fax: 780-492-0886; E-mail: Paul.Scott@ualberta.ca.
** Present address: Cystic Fibrosis Center, 4019A Thurston Bowles Bu. CB 7248, University of North Carolina, Chapel Hill, NC 27599-7248.
A Wellcome Trust Senior Research Fellow in Clinical Science.
Published, JBC Papers in Press, February 21, 2003, DOI 10.1074/jbc.M211936200
2 P. G. Scott, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: LRR, leucine-rich repeat; BCA, bicinchoninic acid; CD, circular dichroism; Dmax, maximum dimension; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PFU, plaque-forming unit; Rg, radius of gyration; TBS, Tris-buffered saline; GdnHCl, guanidine hydrochloride.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Danielson, K. G.,
Baribault, H.,
Holmes, D. F.,
Graham, H.,
Kadler, K. E.,
and Iozzo, R. V.
(1997)
J. Cell Biol.
136,
729-743 |
2. | Scott, J. E., and Haigh, M. (1985) Biosci. Rep. 5, 71-81[Medline] [Order article via Infotrieve] |
3. | Yamaguchi, Y., Mann, D. M., and Ruoslahti, E. (1990) Nature 346, 281-284[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Iozzo, R. V.,
Moscatello, D. K.,
McQuillan, D. J.,
and Eichstetter, I.
(1999)
J. Biol. Chem.
274,
4489-4492 |
5. | Hakkinen, L., Strassburger, S., Kahari, V. M., Scott, P. G., Eichstetter, I., Iozzo, R. V., and Larjava, H. (2000) Lab. Invest. 80, 1869-1880[Medline] [Order article via Infotrieve] |
6. |
Santra, M.,
Eichstetter, I.,
and Iozzo, R. V.
(2000)
J. Biol. Chem.
275,
35153-35161 |
7. | Grant, D. S., Yenisey, C., Wesley Rose, R., Tootell, M., Santra, M., and Iozzo, R. V. (2002) Oncogene 21, 4765-4777[CrossRef][Medline] [Order article via Infotrieve] |
8. | Krusius, T., and Ruoslahti, E. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 7683-7687[Abstract] |
9. | Scott, P. G. (1993) in Dermatan Sulphate and Dermatan Sulphate Proteoglycans: Chemistry, Biology, Chemical Pathology (Scott, J. E., ed) , pp. 81-101, Portland Press, London |
10. | Day, A. A., McQuillan, C. I., Termine, J. D., and Young, M. R. (1987) Biochem. J. 248, 801-805[Medline] [Order article via Infotrieve] |
11. | Chopra, R. K., Pearson, C. H., Pringle, G. A., Fackre, D. S., and Scott, P. G. (1985) Biochem. J. 232, 277-279[Medline] [Order article via Infotrieve] |
12. | Scott, P. G., and Dodd, C. M. (1990) Connect. Tissue Res. 24, 225-236[Medline] [Order article via Infotrieve] |
13. |
Blaschke, U. K.,
Hedbom, E.,
and Bruckner, P.
(1996)
J. Biol. Chem.
271,
30347-30353 |
14. | Patthy, L. (1987) J. Mol. Biol. 198, 567-577[Medline] [Order article via Infotrieve] |
15. | Hocking, A. M., Shinomura, T., and McQuillan, D. J. (1998) Matrix Biol. 17, 1-19[Medline] [Order article via Infotrieve] |
16. | Kobe, B., and Deisenhofer, J. (1994) Trends Biochem. Sci. 19, 415-421[CrossRef][Medline] [Order article via Infotrieve] |
17. | Coster, L., Fransson, L. A., Sheehan, J., Nieduszynski, I. A., and Phelps, C. F. (1981) J. Biol. Chem. 197, 483-490 |
18. | Zangrando, D. W., Gupta, R., Jamieson, A. M., Blackwell, J., and Scott, P. G. (1989) Biopolymers 28, 1295-1308[Medline] [Order article via Infotrieve] |
19. | Fransson, L. A., Nieduszynski, I. A., Phelps, C. F., and Sheehan, J. K. (1979) Biochim. Biophys. Acta 586, 179-188 |
20. | Hildebrand, A., Romaris, M., Rasmussen, L. M., Heinegard, D., Twardzik, D. R., Border, W. A., and Ruoslahti, E. (1994) Biochem. J. 302, 527-534[Medline] [Order article via Infotrieve] |
21. |
Pearson, C. H.,
Winterbottom, N.,
Fackre, D. S.,
Scott, P. G.,
and Carpenter, M. R.
(1983)
J. Biol. Chem.
258,
15101-15104 |
22. | Farndale, R. W., Sayers, C. A., and Barrett, A. J. (1982) Connect. Tissue Res. 9, 247-248[Medline] [Order article via Infotrieve] |
23. | Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, DC. (1985) Analyt. Biochem. 150, 76-85[Medline] [Order article via Infotrieve] |
24. | Mechanic, G. (1979) in Skeletal Research: An Experimental Approach. (Simmons, D. J. , and Kumin, A. S., eds) , pp. 227-241, Academic Press, New York |
25. |
Scott, P. G.,
Dodd, C. M.,
and Pringle, G. A.
(1993)
J. Biol. Chem.
268,
11558-11564 |
26. | Scott, P. G., Nakano, T., and Dodd, C. M. (1997) Biochim. Biophys. Acta 1336, 254-262[Medline] [Order article via Infotrieve] |
27. | Towns-Andrews, E., Berry, A., Bordas, J., Mant, P. K., Murray, K., Roberts, K., Sumner, I., Worgan, J. S., and Lewis, R. (1989) Rev. Sci. Instrum. 60, 2346-2349[CrossRef] |
28. | Grossmann, J. G., Scharff, A. J., O'Hare, P., and Luisi, B. (2001) Biochemistry 40, 6267-6274[CrossRef][Medline] [Order article via Infotrieve] |
29. | Semenyuk, A. V., and Svergun, D. I. (1991) J. Appl. Crystallogr. 24, 537-540[CrossRef] |
30. |
Svergun, D. I.,
Petoukhov, M. V.,
and Koch, M. H. J.
(2001)
Biophys. J.
80,
2946-2953 |
31. | Svergun, D., Barberato, C., and Koch, M. H. J. (1995) J. Appl. Crystallogr. 28, 768-773[CrossRef] |
32. | Scott, P. G., Winterbottom, N., Dodd, C. M., Edwards, E., and Pearson, C. H. (1986) Biochem. Biophys. Res. Communs. 138, 1348-1354[Medline] [Order article via Infotrieve] |
33. | Feigin, L. A., and Svergun, D. I. (1987) Structure Analysis by Small-Angle X-Ray and Neutron Scattering , Plenum Press, New York. |
34. | Evdokimov, A. G., Anderson, D. E., Routzahn, K. M., and Waugh, D. S. (2001) J. Mol. Biol. 312, 807-821[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Liu, J.,
Laue, T. M.,
Choi, H. U.,
Tang, L. H.,
and Rosenberg, L.
(1994)
J. Biol. Chem.
269,
28366-28373 |
36. | Kobe, B., and Deisenhofer, J. (1993) Nature 366, 751-756[CrossRef][Medline] [Order article via Infotrieve] |
37. | Scott, J. E. (1996) Biochemistry 35, 8795-8799[CrossRef][Medline] [Order article via Infotrieve] |
38. |
Keene, D. R.,
San Antonio, J. D.,
Mayne, R.,
McQuillan, D. J.,
Sarris, G.,
Santoro, S. A.,
and Iozzo, R. V.
(2000)
J. Biol. Chem.
275,
21801-21804 |
39. |
Weber, I. T.,
Harrison, R. W.,
and Iozzo, R. V.
(1996)
J. Biol. Chem.
271,
31767-31770 |