From the Center for Extracellular Matrix Biology,
Institute of Biosciences and Technology, Houston Texas 77030-3303 and
the ¶ Department of Medical Biochemistry & Genetics, Texas A&M
University System Health Science Center, College
Station, Texas 77843-1114
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ABSTRACT |
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Biglycan and decorin have been
overexpressed in eukaryotic cells and two major glycoforms isolated
under native conditions: a proteoglycan substituted with
glycosaminoglycan chains; and a core protein form secreted devoid of
glycosaminoglycans (Hocking, A. M., Strugnell, R. A.,
Ramamurthy, P., and McQuillan, D. J. (1996) J. Biol.
Chem. 271, 19571-19577; Ramamurthy, P., Hocking, A. M., and
McQuillan, D. J. (1996) J. Biol. Chem. 271, 19578-19584). Far-UV CD spectroscopy of decorin and biglycan
proteoglycans indicates that, although they are predominantly
Decorin and biglycan are small proteoglycans comprising chemically
similar core proteins substituted at the N-terminal end with one or two
chondroitin/dermatan sulfate chains, respectively. Despite the presumed
structural similarity between biglycan and decorin (1), they have
distinct patterns of temporal and spatial expression suggesting
different functions. They are members of a family of glycoproteins
grouped together on the basis of their presence in the extracellular
matrix, and by virtue of a leucine-rich motif that dominates the core
protein (for review, see Ref. 2). Most of the members of this family
exist in tissues as proteoglycans and have been labeled the small
leucine-rich proteoglycans
(SLRPs)1 (3).
The protein core of decorin (Fig. 1a) and biglycan (Fig.
1b) can be divided into
distinct domains, based on amino acid sequence and specific
post-translational modifications: a signal sequence that
targets the nascent polypeptide to the secretory route; a short
propeptide of highly charged amino acids that undergoes differential tissue- and cell- specific cleavage; an N-terminal glycosaminoglycan attachment region containing one (decorin)
or two (biglycan) Ser-Gly dipeptide consensus sequences; a leucine-rich repeat (LRR) domain that represents more than two thirds of the core
protein and that is flanked by highly conserved disulfide-bonded cysteine clusters; and a short C-terminal domain. The core protein of
decorin has three consensus sites for N-linked
oligosaccharides; two of these sites are conserved in biglycan.
-sheet, biglycan has a significantly higher content of
-helical
structure. Decorin proteoglycan and core protein are very similar,
whereas the biglycan core protein exhibits closer similarity to the
decorin glycoforms than to the biglycan proteoglycan form. However,
enzymatic removal of the chondroitin sulfate chains from biglycan
proteoglycan does not induce a shift to the core protein structure,
suggesting that the final form is influenced by polysaccharide addition
only during biosynthesis. Fluorescence emission spectroscopy
demonstrated that the single tryptophan residue, which is at a
conserved position at the C-terminal domain of both biglycan and
decorin, is found in similar microenvironments. This indicates that in
this specific domain the different glycoforms do exhibit apparent
conservation of structure. Exposure of decorin and biglycan to 10 M urea resulted in an increase in fluorescent intensity,
which indicates that the emission from tryptophan in the native state
is quenched. Comparison of urea-induced protein unfolding curves
provide further evidence that decorin and biglycan assume different
structures in solution. Decorin proteoglycan and core protein unfold in
a manner similar to a classic two-state model, in which there is a
steep transition to an unfolded state between 1 and 2 M
urea. The biglycan core protein also shows a similar steep transition. However, biglycan proteoglycan shows a broad unfolding transition between 1 and 6 M urea, probably indicating the presence of
stable unfolding intermediates.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Schematic representation of decorin
(a) and biglycan (b), indicating
major domains, including: signal sequence (sig),
propeptide (pro), glycosaminoglycan attachment
(GAG), putative disulfide bonds (boxed
S), 10 leucine-rich repeats (numbered
boxes), and N-linked oligosaccharide
attachment sites.
The post-translational modifications of decorin and biglycan are complex and variable, wherein differentially glycosylated forms of these molecules have been isolated from tissues and cells. Decorin core protein devoid of a glycosaminoglycan chain has been isolated (4, 5), although it is yet to be demonstrated whether this is due to post-secretory cleavage of the glycosaminoglycan attachment domain, or synthesis and secretion of core protein that bypasses the glycosaminoglycan synthetic machinery. There is evidence that members of the SLRP family may be "part time" proteoglycans. Overexpression of decorin and biglycan yields significant amounts of secreted core protein devoid of glycosaminoglycan chains (6, 7), the proportion of which appears to be cell type-dependent and related to the endogenous activity of xylosyltransferase2 (the enzyme that catalyzes the first sugar transfer reaction initiating chondroitin sulfate polymerization on a core protein substrate). The decorin core protein is differentially substituted with N-linked oligosaccharides (6, 8, 9) with two and three sites utilized.
The LRR is a structural motif first identified by Patthy (10) and
subsequently refined by Kobe and Deisenhofer (11), which is usually
present in tandem array and has been described in an increasing number
of proteins, giving rise to a LRR superfamily. It is likely that the
conserved residues of each LRR motif define the secondary structure,
while the intervening residues determine specificity of interaction
with ligands. In 1993, Kobe and Deisenhofer solved the crystal
structure of ribonuclease inhibitor (12), a LRR protein that consists
of 15 repeats. This remains the only report of the detailed structure
of a LRR protein and may provide a prototype for all LRR proteins. Each
LRR consists of a -strand parallel to an
-helix forming a hairpin
structure, which is aligned parallel to a common axis resulting in a
non-globular horseshoe-shaped protein. Binding of the ligand to the
concave face (i.e.
-strands) results in a conformational
change of the entire structure and increases the available surface area
for binding.
The LRR domain of the SLRP family members is unique within the superfamily in that it is flanked by cysteine clusters; at the N-terminal end of the LRR domain there are four similarly spaced cysteine residues in a 20-amino acid stretch that are involved in disulfide bonds; and at the C-terminal end there are two cysteine residues also believed to form an intrachain disulfide bond. The 24-amino acid LRR consensus for members of the SLRP family is X-X-(I/V/L)-X-X-X-X-(F/P/L)-X-X-(L/P)-X-X-L-X-X-(L/I)-X-L-X-X-N-X-(I/L), where X is any amino acid, and in the case where more than one amino acid is noted, the first occurs most often (2).
Attempts have been made to predict the structure of decorin (and
related molecules) based on the crystal structure of ribonuclease inhibitor. Computer modeling, constrained by parameters established by
the structure of ribonuclease inhibitor, have suggested that the
decorin core protein forms an arch-shaped protein with the glycosaminoglycan chain and N-linked oligosaccharides
situated on the same side of the molecule (1). High magnification
rotary shadowing electron micrographs of scleral decorin reveal a
similarly "horseshoe-shaped" molecule (13) consistent with the
computer modeling prediction. However, the ribonuclease inhibitor is
composed entirely of LRRs, whereas the N and C termini of SLRPs have
extended non-LRR-containing domains. The inhibitor lacks cysteine
clusters flanking the LRR domain, which, through intramolecular
disulfide bond formation (14), might provide points of stabilization at either end of the LRR domain. The length of the decorin repeat motif is
24 residues, which is shorter than for ribonuclease inhibitor and may
result in a restricted -sheet. However, Kajava has recently modeled
the LRR superfamily (15), and has predicted that structures with
horseshoe curvature are feasible for proteins with shorter leucine-rich
repeats, although the different subfamilies may differ significantly in
tertiary structure. It is also possible that the extensive
glycosylation of SLRPs may influence the folding of the LRR domain.
In the current study, our data indicate that recombinant human decorin
and biglycan have different secondary structures in solution and marked
differences in conformational stability, as assessed by circular
dichroism and fluorescent spectroscopy. Furthermore, we provide
evidence that both the conformation and stability of these molecules is
variably influenced by whether they are synthesized with or without a
glycosaminoglycan chain, whereas removal of polysaccharides after
secretion has no appreciable influence on conformation or stability.
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EXPERIMENTAL PROCEDURES |
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Materials-- Ultrapure urea was obtained from Ambion Inc. (Austin, TX). Ultrafree-15 centrifugal filtration devices were from Millipore Corp. (Bedford, MA). All other materials were obtained as described previously (7).
Protein Purification-- Recombinant decorin and biglycan glycoforms were expressed and purified using the vaccinia virus/T7 bacteriophage expression system, as described previously (6, 7). Briefly, recombinant proteins were purified on a column of iminodiacetic acid immobilized on Sepharose 6B. Proteoglycan and core protein forms were resolved and eluted by a linear gradient of imidazole in column buffer (0.5 M NaCl, 20 mM Tris-HCl, pH 8.0). Pooled fractions were dialyzed against phosphate-buffered saline (PBS), pH 7.4, and concentrated on Ultrafree-15 centrifugal filter devices. Protein concentrations were determined by the molar extinction coefficient (16).
Stock Solutions-- Urea stock solutions (10 M) in a buffer of 50 mM NaH2PO4/K2HPO4, pH 7.0 (17), were prepared daily for each experiment and filtered (0.22-µm pore) prior to use. The urea concentration of each stock solution was calculated by weight and by refractive index (17). The buffer solution without urea is referred to as "phosphate buffer."
Circular Dichroism Spectroscopy--
CD spectra of protein
samples were recorded on a Jasco 720 spectropolarimeter using a 2-mm
path-length quartz cell at room temperature at a concentration of 10 µM in phosphate buffer. The recorded spectra (190-250
nm) were the average of 10 scans and were corrected to background
(phosphate buffer alone). Sample cuvettes were sealed with a Teflon
stopper so that no evaporation occurred. The CD signal (mdeg) was
converted to molar ellipticity () by the following
equation.
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(Eq. 1) |
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Fluorescence Emission Spectroscopy-- Fluorescence emission was measured using a FluoroMax2 spectrofluorometer; the pathlength of the cuvette was 1 mm and the signal averaged for 50 s. The excitation wavelength for tryptophan emission was 296 nm in all experiments. Emission scans were collected from 300 to 500 nm.
Equilibrium Unfolding Curves--
Equilibrium unfolding
experiments were done as described previously (17). Briefly, stock
solutions were prepared in phosphate buffer to be 10 times the desired
final protein concentration. Phosphate buffer, urea from the 10 M stock solution, and 100 µl of stock protein solution to
give a final volume of 1 ml were mixed; this yielded final urea
concentrations of 0-8 M and a final protein concentration
of 10 µM. Protein samples were gently mixed and
equilibrated for 18 h at 25 °C. Unfolding was monitored using circular dichroism spectroscopy and fluorescence emission spectroscopy. The CD signal was determined at 220 nm for the proteoglycan and core
protein forms of biglycan and decorin.
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RESULTS AND DISCUSSION |
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Recombinant biglycan and decorin were expressed in the vaccinia/T7 bacteriophage system (6, 7). Both biglycan and decorin were synthesized as two glycoforms: a proteoglycan form, and a core protein form that was secreted devoid of glycosaminoglycan chains.
Circular Dichroism Spectroscopy--
Far-UV CD spectra were
measured for native recombinant biglycan and decorin glycoforms (Fig.
2). Biglycan proteoglycan had spectra
with minima at 220 nm (Fig. 2a, solid
circles); the CD spectra of decorin proteoglycan was
different, with a minima at 218 nm and a broader curve (Fig.
2c, solid circles). Several
deconvolution computer programs were used to facilitate an objective
comparative analysis of the CD spectra, including SELCON (18), VARSELEC (19, 20), CCA (21), and Contin (22) using file conversion software
(SOFTSECTM: File Conversion for Windows, obtained from
Softwood Co.). The estimated contribution of different structural
motifs (i.e. -sheet,
-turn,
-helix, random coil)
varied between different deconvolution programs (data not shown), but
it was nevertheless consistently determined that biglycan and decorin
assumed different structures in solution. For instance, the method of
Sreerama and Woody (18) predicted that the secondary structure of
biglycan to be 30%
-helix, 14%
-sheet, 15%
-turn, and 46%
random coil, whereas decorin comprised 8%
-helix, 44%
-sheet,
14%
-turn, and 33% random coil.
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After equilibration of recombinant biglycan and decorin in 10 M urea for 16 h, samples were analyzed by CD spectroscopy (Fig. 2, open circles). The CD spectra for both biglycan (Fig. 2a, open circles) and decorin (Fig. 2c, open circles) in 10 M urea reflected a loss of CD signal typical of an absence of secondary structure.
CD spectroscopy was also used to examine the secondary structure of the
core protein glycoforms of biglycan and decorin (Fig. 2). The CD
spectra for the biglycan core protein in phosphate buffer had a minima
at 215 nm (Fig. 2b, solid circles),
with a significantly broader curve than seen for the proteoglycan form (Fig. 2a). Computer deconvolution analysis predicted that
the secondary structure of biglycan core protein was significantly different to the proteoglycan form (13% -helix, 36%
-sheet, 19%
-turn, and 37% random coil; Ref. 18). Thus, the biglycan core
protein, synthesized devoid of glycosaminoglycan chains, had a
secondary structure that is different to the proteoglycan form. The
effect of chondroitin sulfate chain addition on the secondary structure
of biglycan and decorin core proteins was assessed by digestion with
chondroitinase ABC (to remove the glycosaminoglycan chains). There was
no detectable difference between the spectra generated from undigested
and digested proteoglycans. Glycosaminoglycan chains, at 10-fold higher
concentration than in samples analyzed in Fig. 2, did not
contribute to the CD spectra (data not shown). Therefore, removal of
the glycosaminoglycan chains from secreted proteoglycan does not
significantly influence secondary structure.
Comparison of the CD spectra of decorin core protein (Fig.
2d, solid circles) with the spectra of
decorin proteoglycan (Fig. 2b, solid
circles) showed essentially identical curves with the minima
of both glycoforms at 218 nm. The CD scans clearly show that decorin
and biglycan have distinct secondary structures. Furthermore, biglycan
synthesized and secreted devoid of chondroitin sulfate chains assumes a
different structure to biglycan substituted with chondroitin sulfate.
However, removal of the bulk of the chondroitin sulfate mass from
biglycan after purification had no measurable influence on the
structure. Biglycan appears to have more -helical content in its
secondary structure relative to biglycan core and decorin glycoforms,
which are primarily
-sheet in structure. Decorin, on the other hand,
appears to form the same structure in solution irrespective of
substitution with chondroitin sulfate.
Fluorescence Spectroscopy--
The mature core protein of
biglycan and decorin both have a single tryptophan residue situated
between the two conserved cysteine residues at the C-terminal end of
the core protein (Fig. 3a). Peptide sequencing of bovine biglycan has shown that these cysteines form an intramolecular disulfide bond (14). Comparison of the amino
acid sequence in this region reveals that biglycan and decorin share
65% amino acid identity. Fluorescence spectroscopy was used to analyze
the environment of this tryptophan in native and denatured biglycan and
decorin. The fluorescent intensity for all four glycoforms increased in
the presence of 10 M urea (Fig. 3, b-e,
closed circles) relative to the intensity in
phosphate buffer (Fig. 3, b-e, solid circles). These data indicate that the emission from the
tryptophan in the native glycoforms is quenched. Biglycan proteoglycan
in PBS had a maximum emission wavelength of 342 nm (Fig. 2b,
open circles), and after denaturation the peak
emission wavelength was shifted to 352 nm (Fig. 2b,
closed circles). The maximum emission wavelength
of decorin proteoglycan in PBS was 345 nm (Fig. 2d, solid circles); in 10 M urea, the
emission wavelength shifted to 354 nm (Fig. 2d,
open circles). These results suggest that the
tryptophan in native biglycan and decorin is partially buried; in
denatured biglycan and decorin, the tryptophan is exposed to a polar
environment.
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The magnitude of the peak emission wavelength shift for the native and denatured core protein forms of biglycan (Fig. 3c) was similar to that observed for the proteoglycan form (Fig. 3b). The intrinsic fluorescence spectra for biglycan core protein revealed that the native protein had an emission maxima at 341 nm (Fig. 3c, solid circles), which shifted to 353 nm for the denatured protein (Fig. 3d, open circles). Therefore, it appears that the tryptophan is in a similar environment in both glycoforms of biglycan. The spectra for native decorin core protein had a peak emission wavelength maxima of 350 nm (Fig. 3e, solid circles). The maxima for the fluorescence spectra of decorin core protein in 10 M urea was 355 nm (Fig. 3e, open circles). The peak emission wavelength for decorin is different between core protein (350 nm) and proteoglycan (345 nm), which indicates that the microenvironment of the tryptophan in these two glycoforms may be different. The decorin core protein (Fig. 3e) is more solvent-exposed in the native state relative to the decorin proteoglycan (Fig. 3d). However, taken together, these subtle differences suggest that the C-terminal domain is structured similarly among all four glycoforms.
Urea Denaturation Curves--
To further characterize structural
differences between biglycan and decorin, the conformational stability
was investigated. Urea denaturation curves were determined for each
glycoform of biglycan and decorin. The CD spectra (shown in Fig. 2)
revealed the maximal CD signal difference between native and denatured proteoglycan was at 220 nm, and this wavelength was used to monitor changes in the CD signal as unfolding occurred. The denaturation curves
generated for the proteoglycan forms of biglycan and decorin are
complex (Fig. 4), but highly
reproducible, and were not amenable to curve fitting algorithms. The
denaturation curve for biglycan proteoglycan indicates the protein is
very susceptible to urea denaturation based on the limited
pretransition baseline from 0-0.5 M urea (Fig.
4a). However, the transition from the folded to unfolded
state occurs gradually from 1 to 6 M urea, and it is not
clear where the post-transition baseline begins. The unfolding of
biglycan proteoglycan probably proceeds through stable intermediates, reflecting sequential disruption of domains. Unfolding of decorin proteoglycan follows a simpler, possibly two-state, mechanism (Fig.
4c), with a pretransition baseline from 0 to 1.0 M urea, a sharp transition between 1.0 and 2.0 M urea, and an apparent post-transition baseline with
increasing urea concentration.
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The urea-induced unfolding of the core protein glycoforms of biglycan and decorin was also examined. Biglycan core protein (Fig. 4b) had a pretransition between 0 and 1.5 M urea, a sharp transition between 1.5 and 2.5 M, followed by a slowly increasing post-transition above 3.5 M urea. The denaturation curve for decorin core protein was similar to both the biglycan core protein and the decorin proteoglycan glycoforms, with a pretransition region at 0-1.0 M, a transition from 1.0 to 2.0 M urea, and a similar post-transitional baseline at higher urea concentrations (Fig. 4d).
Reversibility of Unfolding--
Reversibility of unfolding is an
important parameter when defining conformational stability. All four
glycoforms were equilibrated in 10 M urea and then diluted
to a urea concentration of 1.0 M urea, at a final
concentration of 10 µM. This preparation was compared
with glycoforms (10 µM) that had been equilibrated
directly in 1 or 10 M urea. The effect on secondary
structure was examined by far-UV CD spectroscopy (Fig.
5). All of the refolding profiles (Fig.
5, triangles) demonstrate that none of the glycoforms were able to refold to their original conformation after exposure to 10 M. The biglycan proteoglycan in PBS (Fig. 2a,
solid circles) and 1.0 M urea (Fig.
5a, solid circles) had similar CD
profiles, with a minima of 215 nm. After exposure to 10 M
urea and subsequent dilution to 1 M urea, the minima
shifted to 213 nm and the curve was significantly broader (Fig.
5a, triangles). Decorin proteoglycan equilibrated
in 1 M urea overnight had a similar spectra to native proteoglycan (Fig. 5c, closed
circles). When the urea concentration was diluted back to 1 M urea, the secondary structure was reproducibly different
to the CD spectra of the native proteoglycan, exhibiting a minima at
214 nm and a significantly broader curve (Fig. 5c, triangles). The biglycan core protein in PBS and in 1 M urea (Fig. 5b, squares) had a
minima ~218 nm, which was shifted to 209 nm in the refolded spectra
(Fig. 5b, triangles). The spectra for decorin
core protein in 1 M urea (Fig. 5d,
circles) had a minima at 216 nm, and the refolded protein
(Fig. 4d, triangles) had a spectra with a minima
at 214 nm. These spectra clearly demonstrate these refolded glycoforms
do not appear to assume the same secondary structure as in the native
state, under the conditions used. Indeed, it is possible that the
refolded form has a molten globule-like structure that may regain a
secondary structure but does not resemble the native folded protein
(23).
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In summary, comparison of the biophysical properties of biglycan and decorin indicates that these small leucine-rich repeat proteoglycans have different overall secondary structures, as assessed by circular dichroism spectroscopy. However, fluorescence spectroscopy indicates that the conserved tryptophan in the C-terminal disulfide-bonded domain is in a similar environment for both biglycan and decorin. A qualitative analysis of conformational stability revealed the possibility of multiple transitions during urea-induced unfolding of biglycan proteoglycan; in contrast, biglycan core protein and the decorin glycoforms appear to follow a two-state unfolding mechanism. Glycosylation also had differential effects on the structure and stability of biglycan, but not decorin. The core protein form of biglycan is more stable than the proteoglycan, and they appear to assume different structures in solution. This is in contrast to the decorin core protein, which assumes a similar conformation independent of substitution with a glycosaminoglycan chain.
A recent study by Font et al. (24) showed that fibromodulin,
a member of the SLRP family, exhibited a CD spectra consistent with a
predominantly -sheet structure (minima at ~210 nm). Furthermore, brief exposure to 6 M urea and heat (60 °C) did not
irreversibly disrupt the secondary structure. This is in contrast to
the present study, in which extended exposure to 10 M urea
alone has serious consequences on the conformation of the core protein.
Furthermore, thermal denaturation of biglycan or decorin (up to
60 °C) is not reversible (data not shown). This further illustrates
that, despite the similarity of SLRP members at the amino acid level,
and presumed evolutionary relatedness, there appears to be significant
divergence in secondary structure and conformational stability.
Functionally, this is likely to be critical to the differential
biological activities and distribution of SLRP molecules (2).
Glycosaminoglycans are long extended polymers of repeating disaccharide
units. It is not unreasonable to speculate that these large
polysaccharides can influence the structural conformation or stability
of a protein. In this study, the chondroitin sulfate chains of biglycan
may have a critical role in stabilizing the secondary structure of the
protein during biosynthesis. Significant differences in the CD spectra
of the native biglycan proteoglycan and core protein provide evidence
that the presence of glycosaminoglycan chains can alter structure. It
is unclear how the glycosaminoglycan affects the secondary structure,
but it should be noted that this is not a general role in
proteoglycans, as the glycosaminoglycan chain of decorin had no
detectable effect on structure or conformational stability. This
observation once again confirms that biglycan and decorin have
different folded conformations. Furthermore, this study clearly
indicates that there are inherent flaws in applying generalizations
from the structure of one member of the LRR superfamily to other
distantly related proteins. It further emphasizes the need for the
structure of one or more members of the SLRP family to be solved, and
this will impact significantly on the biology of these molecules with
respect to their role in collagen fibrillogenesis (25, 26), modulation
of transforming growth factor- activity (27-31), and interaction
with cell surface receptors (32-34).
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ACKNOWLEDGEMENTS |
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We thank Dr. Steve Labrenz and Dr. Rebecca Rich for many useful discussions and for assistance with operating the CD and fluorescent spectrophotometers.
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FOOTNOTES |
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* This work was supported by grants from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (AR42826), and from NASA/Texas Medical Center (NCC9-36).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.
§ These authors contributed equally to this work.
To whom correspondence should be addressed: IBT, 2121 W. Holcombe Blvd., Houston, TX 77030-3303. Tel.: 713-677-7575; Fax: 713-677-7576; E-mail: dmcquill{at}ibt.tamu.edu.
2 N.-S. Seo, A. M. Hocking, and D. J. McQuillan, manuscript submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: SLRP, small leucine-rich repeat proteoglycans; LRR, leucine-rich repeat; PBS, phosphate-buffered saline; CD, circular dichroism.
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REFERENCES |
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