From the Skeletal Research Center, Department of
Biology, Case Western Reserve University, Cleveland, Ohio 44106, the
¶ Department of Cell and Molecular Biology, Section for Connective
Tissue Biology, Lund University, Biomedical Center Floor C12,
SE-221 84 Lund, Sweden, the
Center for Skeletal Development and
Pediatric Orthopedic Research, Shriners Hospital for Children and the
Department of Pharmacology and Therapeutics, University of South
Florida, Tampa, Florida 33612, the ** Department of
Biochemistry, Rush-Presbyterian-St. Luke's Medical Center, Chicago,
Illinois 60612, and the
Department of
Biochemistry, University of Alberta,
Edmonton, Alberta T6G 2H7, Canada
Received for publication, January 6, 2003, and in revised form, February 28, 2003
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ABSTRACT |
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Dramatic changes occur in
skin as a function of age, including changes in morphology, physiology,
and mechanical properties. Changes in extracellular matrix molecules
also occur, and these changes likely contribute to the overall
age-related changes in the physical properties of skin. The major
proteoglycans detected in extracts of human skin are decorin and
versican. In addition, adult human skin contains a truncated form of
decorin, whereas fetal skin contains virtually undetectable levels of
this truncated decorin. Analysis of this molecule, herein referred to
as decorunt, indicates that it is a catabolic fragment of decorin
rather than a splice variant. With antibody probes to the core protein,
decorunt is found to lack the carboxyl-terminal portion of decorin.
Further analysis by matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry shows that the carboxyl terminus of
decorunt is at Phe170 of decorin. This result
indicates that decorunt represents the amino-terminal 43% of the
mature decorin molecule. Such a structure is inconsistent with
alternative splicing of decorin and suggests that decorunt is a
catabolic fragment of decorin. A neoepitope antiserum, anti-VRKVTF, was
generated against the carboxyl terminus of decorunt. This antiserum
does not recognize intact decorin in any skin proteoglycan sample
tested on immunoblots but recognizes every sample of decorunt tested.
The results with anti-VRKVTF confirm the identification of the carboxyl
terminus of decorunt. Analysis of collagen binding by surface plasmon
resonance indicates that the affinity of decorunt for type I collagen
is 100-fold less than that of decorin. This observation correlates with
the structural analysis of decorunt, in that it lacks regions of
decorin previously shown to be important for interaction with type I
collagen. The detection of a catabolic fragment of decorin suggests the existence of a specific catabolic pathway for this proteoglycan. Because of the capacity of decorin to influence collagen
fibrillogenesis, catabolism of decorin may have important functional
implications with respect to the dermal collagen network.
The mechanical properties of the dermis are determined primarily
by the extracellular matrix. These mechanical properties change
dramatically as a function of age (1, 2), perhaps as a direct result of
the known age-related changes in the molecules of the dermal
extracellular matrix. Age-related differences have been shown for
fibrillar collagens (3-7), which are the major extracellular matrix
components of the dermis (8). In addition to collagen, the dermal
extracellular matrix also contains proteoglycans, which show
age-related differences (9-16). Perhaps related to these changes in
proteoglycans are age-related increases in the water content of the
dermis (11) and in the content of mobile water (17).
Although dermal proteoglycans are present in much lower abundance than
collagen, evidence indicates that these molecules are important in the
physiology of skin. For example, the small proteoglycan decorin binds
to type I collagen (18, 19), and targeted disruption of decorin results
in aberrant collagen fibrils and in a reduction in the tensile strength
of skin (20). Similarly, a patient with a variant form of Ehlers-Danlos
syndrome was found to have a substantially reduced amount of dermal
decorin (21). Previous work has shown that decorin and versican are the
major proteoglycans extracted from human skin (16). A truncated form of
decorin is abundant in extracts of postnatal skin, whereas fetal skin
extracts contain little, if any, of this molecule (16). Evidence
presented herein indicates that this molecule is a catabolic fragment
of decorin. This molecule is now referred to as decorunt to reflect its
origin. In addition, data are presented showing that decorunt has a
greatly reduced capacity to bind to type I collagen. The detection of a
catabolic fragment of decorin in adult human skin, but not in fetal
human skin, suggests that there are age-related increases in decorin
turnover in this tissue and/or that decorunt is a nonmetabolized end
product of decorin turnover that accumulates with age in skin.
Materials--
The sources of reagents for proteoglycan
extraction and isolation and for SDS-PAGE and immunoblots were
as described elsewhere (16). Additional reagents were obtained from the
following sources. Monoclonal antibody 6B6, which recognizes an epitope
in the core protein of decorin, was purchased from Seikagaku America.
Antiserum to the carboxyl terminus of decorin and monoclonal antibodies 5D1, 3B3, and 6D6 against discrete epitopes in the core protein of
decorin have been described previously (22, 23). Anti-VRKVTF antiserum
was raised by Research Genetics (Huntsville, AL) against the synthetic
peptide CGGVRKVTF conjugated to ovalbumin. Serum that had been
clarified by centrifugation was used to probe immunoblots. Immobilon-N
positively charged transfer membrane was obtained from Millipore, and
octyl-Sepharose CL-4B was purchased from Sigma. Samples of fetal and
adult human skin were obtained in accordance with the policies
established by the Institutional Review Board of Case Western Reserve
University as previously described (16). Fetal skin samples were
obtained through the Central Laboratory for Human Embryology,
University of Washington. Adult skin samples were obtained through the
Tissue Procurement Core Facility, Cancer Center, Case Western Reserve
University. Because the human tissues used for this work were
classified as discarded tissues, informed consents of the donors were
not required.
Proteoglycan Extraction and Isolation--
These procedures were
as reported previously (16). Proteoglycans were isolated by anion
exchange chromatography and fractionated into large molecules
(primarily versican) and small molecules (primarily decorin and
decorunt) by Sepharose CL-2B chromatography with 4 M
guanidinium chloride, 0.5%
CHAPS,1 0.05 M
sodium acetate, pH 6.0, as the eluent. Aliquots of each fraction were
analyzed by dot blot with appropriate antibodies. Selected fractions
were pooled and concentrated with Centricon centrifugal concentrators
to volumes less than 100 µl. Proteoglycans were precipitated by
addition of 20 volumes of cold absolute ethanol followed by overnight
incubation at
For separation of decorin from decorunt, the small proteoglycans from
Sepharose CL-2B were fractionated by hydrophobic interaction chromatography on octyl-Sepharose by a modification of a previously published procedure (25). Samples corresponding to 25-300 µg of
glycosaminoglycan were reconstituted in 1 ml of 2 M
guanidinium chloride, 0.1 M sodium acetate, pH 6.3, and
applied to a 2-ml column. The column was rinsed with 10 ml of the same
solution, and the effluent was collected as unbound material. The
column was then eluted with gradients of 2 and 6 M
guanidinium chloride, both in 0.1 M sodium acetate, pH 6.3. The gradient consisted of 23 ml of each solution, and fractions of 0.45 ml were collected. The fractions were assayed by 6B6 dot blot of
aliquots. Decorunt elutes in the unbound fraction (not shown), whereas
decorin and the small amount of biglycan in the human skin proteoglycan
samples are resolved by the gradient (25). Appropriate fractions were pooled, and all pools, including the unbound material, were brought to
0.5% CHAPS by the addition of CHAPS from a 10% solution. The samples
were concentrated, precipitated, and assayed as described above.
SDS-PAGE and Immunoblotting--
The proteoglycan samples were
reconstituted after lyophilization and prepared for SDS-PAGE as
described (16). SDS-PAGE on 5-17.5% gels, electrotransfer, and
immunoblot analysis were performed as done previously (16), except that
Immobilon-N transfer membrane was used. For detection of decorunt on
immunoblots, positively charged transfer membrane is necessary because
of the previously reported poor binding of decorunt to Immobilon-P and
nitrocellulose (16). Hence, all of the immunoblots in this study were
performed with Immobilon-N.
Mass Spectrometry--
Coomassie Blue-stained bands on SDS-PAGE
gels were excised and washed extensively with 40% acetonitrile in 25 mM NH4HCO3, pH 7.8. The gel pieces
were dried in a SpeedVac before digestion overnight at 37 °C with 10 µl of sequencing grade trypsin (Promega) at 20 ng/µl in 25 mM NH4HCO3, pH 7.8. The digestion
was terminated by the addition of 10 µl of 2% trifluoroacetic acid,
which also extracted the peptides out of the gel. After a 1-h
extraction at room temperature, the peptides were purified from the
buffer with miniaturized C-18 reversed phase tips
(ZiptipsTM, Millipore). The peptides were eluted directly
onto the sample target. The matrix 2,5-dihydroxybenzoic acid was used
on an AnchorchipTM target (Bruker Daltonik). For
post-source decay experiments, the matrix
Mass spectrometric studies were performed with a Bruker Scout 384 Reflex III MALDI-TOF mass spectrometer. The instrument was used in the
positive ion mode with delayed extraction and an acceleration voltage
of 25 kV. The peptide samples were analyzed with the reflector detector, and 50-100 single-shot spectra were accumulated for improved
signal-to-noise ratio. The spectra were internally calibrated with
autolysis fragments of trypsin. For samples digested with endoproteinases Lys-C and Glu-C, external calibration was used.
Surface Plasmon Resonance Assay--
The BIAcoreTM
2000 system was used to characterize the interaction between
decorin/decorunt and type I collagen (bovine dermal collagen I;
Invitrogen). The carboxymethylated dextran surface on the chip (CM5
sensor chip; BIAcore) was activated with 35 µl of 50 mM
N-hydroxysuccinimide and 35 µl of 200 mM
N-ethyl-N'-(dimethylaminopropyl)carbodiimide at
25 °C at a flow rate of 5 µl/min. Collagen type I (35 µl at 3 µg/ml in 0.15 M NaCl, 10 mM sodium citrate,
pH 5.0) was immobilized at 25 °C at a flow rate of 5 µl/min. One
surface containing no coupled protein was used as a blank. The
remaining activated groups were blocked with 40 µl of 1 M
ethanolamine, pH 8.5. The immobilization of collagen I resulted in
~600 resonance units (1000 resonance units = 1 ng/mm2). The binding assay was performed at 25 °C with
different concentrations of decorin or decorunt (2-fold dilution
series) ranging from 0.31 to 20 µg/ml in 0.15 M NaCl,
0.005% Tween 20, 10 mM HEPES, pH 7.4. The surface
was regenerated with two injections of 0.5 M NaCl, 0.1 M NaHCO3, pH 9.2, and one injection of 2 M NaCl, 10 mM HEPES, pH 8.0, for decorin.
Decorunt was more easily removed, so in this case the buffer at pH 9.2 was exchanged for the same buffer with pH 8.5 to spare proteins at the
chip surface. To assess the influence of the glycosaminoglycan chain
upon binding, experiments were performed without and after digestion of
the proteoglycan samples with chondroitinase ABC (0.1 milliunit/µg of
proteoglycan in 0.3 M NaCl, 10 mM Tris, pH 7.4 for 4 h). Removal of the glycosaminoglycan was verified by
SDS-PAGE.
Immunohistochemistry--
Skin specimens were fixed in 10%
neutral buffered formalin and embedded in paraffin for sectioning. The
sections were blocked with bovine serum albumin and then incubated with
6B6 mouse monoclonal antibody, anti-VRKVTF antiserum, or nonimmune
rabbit serum. The sections were then washed and incubated with
appropriate second antibody conjugated with peroxidase. Peroxidase was
visualized with the VIP substrate kit, which was purchased from Vector
Laboratories. Photographs were taken with Kodak T-MAX 100 film on an
Olympus BH-2 microscope. In all, skin samples from 10 different donors were examined by immunohistochemistry. These samples ranged in age from
fetal to 82 years old.
Immunoblot Analysis--
Previous analysis of proteoglycans
extracted from human skin revealed that decorin is the major
proteoglycan present and that versican is also present at all ages
examined and is found at its highest abundance in fetal skin (16). In
addition, human skin also contains a truncated form of decorin now
referred to as decorunt. The core protein of decorunt is ~17 kDa, as
determined by SDS-PAGE (16). That decorunt is related to decorin is
indicated by the amino-terminal amino acid sequences of these
molecules, which are identical (16). To determine the molecular
characteristics of decorunt, the molecule was analyzed on immunoblots
with probes that recognize different regions of decorin (Fig.
1). Both molecules are recognized by
monoclonal antibody 6B6 in all adult skin proteoglycan samples tested
(Fig. 2). The lack of strong decorunt
reactivity in fetal skin proteoglycans (Fig. 2) is consistent with the
virtual absence of decorunt from fetal skin, as has been described
previously (16). The exact epitope for 6B6 has not been reported.
However, with CNBr-treated human skin decorin, 6B6 is found to
recognize a large fragment containing the glycosaminoglycan (data not
shown). This places the 6B6 epitope somewhere between the amino
terminus of the mature core protein (amino acid 31) and the first
methionine residue of the mature core protein (amino acid 148) (Fig.
1).
Immunoblot analysis was also performed with an antiserum that was
raised against a peptide corresponding to the carboxyl terminus of
decorin (amino acids 348-359) (22). For both of the samples of adult
skin proteoglycans that were tested, this antiserum recognizes decorin,
as expected but does not recognize decorunt (Fig.
3). This result is obtained both with
intact proteoglycans and with core proteins generated by treatment of
the samples with chondroitinase ABC prior to electrophoresis (Fig. 3).
Further analysis of decorunt was performed with three monoclonal
antibodies whose epitopes have been mapped to different regions of the
core protein of decorin (23). All three of these antibodies recognize
human skin decorin in the samples tested (not shown). Antibody 6D6, the
epitope of which is residues 270-273, does not recognize decorunt (not
shown). Antibody 3B3 (residues 173-180) also does not recognize
decorunt, whereas antibody 5D1 (residues 150-157) is able to recognize
decorunt (data not shown). The locations of the epitopes for 3B3 and
5D1 suggest that the carboxyl terminus of decorunt lies between
residues 157 and 173 of decorin (Fig. 1). This corresponds to a
molecular mass of ~15-17 kDa, which correlates well with the
value from SDS-PAGE (16).
Mass Spectrometry--
The first approach to characterize decorunt
by mass spectrometry was to take the chondroitinase-treated sample and
measure its mass by MALDI-TOF MS. However, the sample was found to
contain multiple components, and decorunt showed a broad heterogeneous peak that could not be accurately assigned (~17 kDa). To obtain a
more detailed picture of the cleavage site, decorunt was mapped at the
peptide level after enzymatic digestion with combinations of various
endoproteinases. Trypsin and Lys-C were first used, but the resulting
peptide maps did not contain any unknown peptide masses in the measured
mass range of 750-3500 Da. The amino-terminal part of decorin was
covered by more than 20 peptides (Fig. 4) with the most carboxyl-terminal peptide being AHENEITKVR (residues 157-166) with one missed cleavage. The next peptide to be detected is
VTFNGLNQMIVIELGTNPLK (residues 168-187) with a monoisotopic mass of
2201.20 Da. This peptide was easily detected in intact decorin but was
absent from the decorunt. Thus, the cleavage site appears to be located
within this sequence. The absence of unknown peptides might be a result
of the cleavage site being positioned at the beginning of this
sequence, which would thereby produce a peptide too small for
detection. To provide further information, aliquots were digested with
a different endoproteinase having a different specificity,
endoproteinase Glu-C. The peptide map of this digest contains a strong
signal from a mismatched peptide with a mass of 1091.74 Da. This
peptide mass fits perfectly (expected mass, 1091.69) with the peptide
ITKVRKVTF (residues 162-170). To confirm the identity of the peptide,
a tandem mass spectrometry experiment was performed with
post-source decay to fragment the peptide. The corresponding
post-source decay spectrum is shown in Fig.
5, where the b and y ion series are
annotated and cover 6 of 9 amino acids. The same cleavage site was
identified in decorunt isolated from four different samples of skin
from individuals of 20, 34, 48, and 68 years of age. The cleavage site
would not be detected by trypsin or Lys-C digestion, because the
peptide VTF (365.2 Da) is too small to be accurately detected by
MALDI-TOF MS because of high matrix signals in this part of the
spectrum.
Interaction Studies--
The binding properties of decorunt and
decorin for collagen I were studied with surface plasmon resonance. The
binding and dissociation curves obtained at various decorin/decorunt
concentrations are shown in Fig. 6. The
equilibrium dissociation constants (KD values) for
decorin were experimentally estimated to be 1.2 and 2.9 nM
for intact and chondroitinase-treated decorin, respectively, whereas
the corresponding values for decorunt were 268 and 228 nM.
The decorin constants were obtained by conventional (Langmuir 1:1)
curve fitting with drifting base line, whereas no good fit was obtained
with any model for decorunt. There are several potential explanations
for this. For example, perhaps there are two conformations of decorunt
that act differently in binding to collagen, or decorunt might change
conformation after binding. However, at steady state affinity
(especially in the case of chondroitinase-treated decorunt), the
plateau level just at the end of the injection phase can be used to
provide a rough estimate of the KD value. Because the response from decorunt was significantly lower, the dilution series
starts at twice the concentration to obtain significant signals for
kinetic evaluations. The difference in binding affinity is ~100-fold
between decorin and decorunt. The removal of the glycosaminoglycans had
only marginal effects on the KD values. For decorin,
this value increased from 1.2 to 2.9 nM, whereas for
decorunt it decreased from 268 to 228 nM. Additionally, a
reduced response was observed in the case of decorunt.
Neoepitope Antiserum--
A neoepitope antiserum was generated
against the VRKVTF carboxyl terminus of decorunt. This polyclonal
rabbit antiserum recognizes decorunt in every adult skin proteoglycan
sample tested (Fig. 7). Importantly,
anti-VRKVTF does not recognize intact decorin, which is present in all
of the skin proteoglycan samples tested, as indicated by a companion
blot probed with 6B6 as a control (Fig. 7). Anti-VRKVTF also does not
detect decorunt in the sole fetal skin proteoglycan sample tested (Fig.
7), which is consistent with the absence of decorunt from fetal skin
(16). With adult skin proteoglycan samples treated with chondroitinase
ABC prior to electrophoresis, anti-VRKVTF is observed to recognize the
core protein of decorunt but not the core proteins of decorin (not shown). Incubation of anti-VRKVTF antiserum with 10 µM
CCGVRKVTF, the synthetic peptide used for generation of anti-VRKVTF,
effectively abrogates binding of the antiserum to decorunt on an
immunoblot (not shown). Taken together, these results indicate that
anti-VRKVTF specifically recognizes the VRKVTF carboxyl terminus of
decorunt.
Immunohistochemical staining of normal adult human skin indicates the
presence of decorin and decorunt in the extracellular matrix of the
dermis (Fig. 8). There does not appear to
be any specific staining of either decorin or decorunt in the
epidermis. Although immunostaining for decorin is strong throughout the
entire dermis, immunostaining for decorunt is weak in the outer dermis and strong elsewhere in the dermis (Fig. 8). For both decorin and
decorunt, there is a fibrillar pattern of immunostaining, wherein the
molecules seem to be co-localized with the collagen fibrils of the
dermis.
In our previous analysis of the proteoglycans of human skin, a
truncated form of decorin was detected in adult skin but not in fetal
skin (16). This observation is based on analysis of eight samples of
fetal skin ranging in age from 80 to 120 days estimated gestational age
and of 24 samples of adult skin ranging in age from 20 to 82 years and
from both sun-exposed anatomic sites (face, arm, and thigh) and
sun-protected anatomic sites (breast and abdomen). Although most of the
adult skin samples are from females, the three samples of skin from
males show no obvious differences with respect to the presence of
decorunt. Indeed, the 68-year-old sample in Fig. 2 and the 52-year-old
sample in Fig. 7 are from males, and these samples show similar results to the other skin samples, which are from females. In addition, the
68-year-old sample used for MALDI-TOF MS analysis is the same 68-year-old sample as that in Fig. 2, and this sample gave results similar to those obtained for the three female samples analyzed by
MALDI-TOF MS. Thus, among all of the adult skin samples, there do not
appear to be decorunt-related differences that are due to anatomic site
or gender. The only clear difference with respect to decorunt is the
consistent increase in the observed ratio of decorunt to decorin for
skin samples near the age of 30 years, as indicated by immunoblots
probed with 6B6 (Figs. 2 and 7), gels of intact proteoglycans stained
with toluidine blue (Ref. 16 and data not shown), and gels of core
proteins stained with silver (16) or Coomassie Blue (not shown).
Perhaps this age represents the beginning of the changes that are
manifested as aging. Interestingly, the age range at which decorunt is
most abundant correlates with the age at which products of nonenzymatic
glycation have been shown to begin to accumulate in sun-protected skin
(26). In addition, the evidence obtained by immunohistochemistry
suggests that there are quantitative changes in glycosaminoglycans in
aging skin, although the changes reported were found at older ages than the age at which decorunt is detected at maximal abundance (27).
Truncated forms of decorin have been reported previously in human
post-burn hypertrophic scar (28, 29), calf skin (30), postnatal rat
submandibular glad (31), and bovine cornea (32). However, the exact
nature of these truncated forms of decorin was not ascertained.
Truncated decorin could arise by alternative splicing, and splice
variants of decorin have been described at the mRNA level, although
not at the protein (proteoglycan) level (33). Our analysis of decorunt
suggests that this molecule arises by catabolism of decorin, because
the VRKVTF carboxyl terminus of decorunt is not consistent with
alternative splicing (34, 35). Decorunt, which represents the
amino-terminal 43% of decorin, contains the amino-terminal domain of
decorin as well as the first four leucine-rich repeats and three amino
acids of the fifth leucine-rich repeat. As such, decorunt lacks almost
half of a region of decorin previously shown to be important for
binding to collagen, namely the fourth and fifth leucine-rich repeats
(36), as well as the glutamate residue reported to be critical for
collagen binding, residue 180 (37). Consequently, decorunt is expected
to have impaired capacity to bind to collagen, and this was confirmed experimentally. In addition, decorunt isolated from hypertrophic scar
has been found to be inactive in affecting the formation of collagen
fibrils in a collagen fibrillogenesis
assay.2 In light of the much
lower affinity of decorunt for type I collagen, retention of decorunt
in the dermis should be less efficient. Nevertheless, decorunt is
present in every adult skin sample thus far examined. It is not clear
whether the carboxyl-terminal fragment is retained in the tissue.
Attempts to raise antibodies that recognize the expected amino-terminal
neoepitope, NGLNQM, have failed.
Decorunt also lacks the carboxyl-terminal 90 residues of a 106-residue
decorin peptide previously shown to bind to transforming growth
factor- The putative cleavage site in decorin that gives rise to decorunt,
VRKVTF-NGLNQM, has not, to our knowledge, been ascribed to any matrix
protease (43-56), although MMP-1, MMP-9, and MMP-12 have been shown to
cleave The occurrence of only a single carboxyl terminus in decorunt, based on
MALDI-TOF MS analysis of decorunt from four different skin samples,
suggests a specific cleavage for the generation of decorunt. This is
further supported by the presence of only one core protein band
recognized by anti-VRKVTF (not shown). Interestingly, for those species
for which decorin core protein amino acid sequences have been
determined (human, bovine, ovine, canine, equine, porcine, rabbit, rat,
murine, chick, and quail), the putative decorunt cleavage site occurs
with that exact sequence only in human, although only rabbit decorin
has an altered sequence at the Phe-Asn site (Phe-Ser) (SWISS-PROT;
www.ebi.ac.uk/swissprot/access.html). Of these sequences, the most
closely related is VRKS(A)VF-NGLNQM in bovine, ovine, canine, equine,
and porcine decorin.
Small leucine-rich repeat proteoglycans, including decorin, have been
reported to be resistant to proteolytic degradation in explant cultures
of bovine nasal cartilage treated with interleukin-1, whereas
proteolysis of aggrecan in these cultures is clearly detected (57). In
another report, decorin was shown to be cleaved by MMP-2, MMP-3, and
MMP-7, although, based on amino acid sequence data of the proteolytic
fragments, the cleavage fragments generated in this study are different
from decorunt (58). In addition, concanavalin A-treated human gingival
fibroblasts have been shown to produce proteases that degrade decorin
(59). In these cultures, the major decorin fragment and its core
protein are slightly larger in size than decorunt and its core protein,
but, like decorunt, the decorin fragment generated by the gingival
fibroblast proteolytic activity is not recognized by monoclonal
antibody 6D6 (59). Because of the larger size of this fragment relative
to decorunt, this fragment is most likely equivalent to the fragments
generated by MMP-2 and MMP-3, which result from cleavage at
Ser240-Leu241 (58). Thus, if decorunt is
generated enzymatically, it appears to be produced by a protease that
has not yet been described or by a known protease whose cleavage
specificity is not yet known.
The characterization of decorunt suggests the occurrence of a specific
catabolic pathway for decorin, which has not been previously reported,
and the generation of the neoepitope antiserum, anti-VRKVTF, provides a
reagent to facilitate investigation of this catabolic pathway. The
detection of decorunt in adult skin, but not in fetal skin, suggests
that there are age-related differences in proteoglycan catabolism in
human skin. The functional consequences of the loss of a portion of the
decorin core protein are not clear. The removal of the portion
containing the transforming growth factor-
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C. Precipitated material was collected by
centrifugation at 4 °C (12,000 rpm for 5 min in a
microcentrifuge). Each pellet was rinsed once with cold 20:1 ethanol:water by brief vortexing and centrifuged again. The final pellet was reconstituted in distilled water (0.1-0.5 ml depending on
its size) and lyophilized to dryness. The samples were reconstituted in
distilled water as before, and small aliquots were withdrawn for
determination of total glycosaminoglycan by the Safranin O procedure
(24). The samples were then split into aliquots based on
glycosaminoglycan content, lyophilized, and stored at
20 °C.
-cyano-hydroxycinnamic
acid was used.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Locations of binding of decorin probes on the
core protein. In the diagrammatic representation of the decorin
core protein, N and C at each end of the core
protein indicate the amino and carboxyl termini, respectively. The
locations of the three consensus sequences for attachment of
asparagine-linked oligosaccharides are indicated by N along
the length of the core protein, whereas the consensus sequence for
attachment of the glycosaminoglycan is indicated by GAG. The
hatched area denotes the leucine-rich repeats. The minimal
known region wherein each probe binds is indicated by an
arrow or a bracket. Other details about the
locations of probe binding are in the text. Reactivity and lack of
reactivity of the various probes with decorunt are indicated by + and
, respectively. Also indicated are the amino-terminal amino acid
sequence, which is the same for decorin and decorunt (16), and the
location of the putative cleavage site, which leads to the formation of
decorunt.
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Fig. 2.
Reactivity of decorin and decorunt with
monoclonal antibody 6B6. Proteoglycans were isolated from adult
human skin of the indicated ages and anatomic sites and from fetal
human skin (combined trunk and scalp skin) of 120 days estimated
gestational age. The proteoglycan samples were subjected to SDS-PAGE
and then electrotransferred to Immobilon-N. The blot was probed with
6B6. brs, breast; abd, abdomen; fac,
face.
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Fig. 3.
Reactivity of decorin and decorunt with
antiserum to the carboxyl terminus of decorin. Proteoglycans from
human skin of the indicated ages were isolated, subjected to SDS-PAGE,
and electrotransferred to Immobilon-N. The anatomic sites of these
samples are breast for the 34-year-old sample and face for the
47-year-old sample. Aliquots of each sample were electrophoresed
without or after prior treatment with chondroitinase ABC
(CSase). Separate blots were probed with 6B6 or anti-decorin
carboxyl terminus (anti-C-terminus) as indicated.
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Fig. 4.
Sequence coverage peptide map of decorunt
digested with trypsin. Peptide masses were obtained with MALDI-TOF
MS, and 22 peptides were matched against the decorin core protein. Some
of these peptides contain oxidized methionine; these are indicated by
black lines, whereas unmodified peptides are shown in
white. The data base matching was made with the program
ProFound, available on the internet
(65.219.84.5/service/prowl/profound.html). The bottom panel
shows experimental masses (Exp) compared with the expected
masses (Theor) along with the peptide sequences and residue
numbers; all of the mass errors are below 50 ppm. MC, number
of missed cleavages.
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Fig. 5.
Post-source decay MALDI-TOF mass spectrum
annotated with b and y ions matching the neoepitope sequence ITKVRKVTF
obtained after digestion with chymotrypsin. The b ion series
covers a sequence tag of six amino acids, TKVRKV, the y ion series
covers VKRV, and the c8 ion represents the loss of Phe from the intact
peptide. The average mass error, calculated by linear mass calibration,
was 0.16 Da. The presence of immonium ions from Arg, Val, Ile, and Lys
residues further supports the inferred sequence. Abs.
Int., absolute intensity.
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Fig. 6.
BIAcore sensorgrams showing interaction of
decorin and decorunt with collagen I. The plots show the
association phase (0-120 s) and the dissociation phase (120-400 s).
Injections of ligand were performed in a series of double dilutions
ranging from 10 to 0.31 µg/ml for intact decorin (A) and
chondroitinase-treated decorin (B) and from 20 to 0.62 µg/ml for intact decorunt (C) and chondroitinase-treated
decorunt (D). For intact decorin (A), the
tracings, from uppermost to lowest, show the
responses at concentrations of 10, 5, 1.25, 0.62, and 0.31 µg/ml; for
chondroitinase-treated decorin (B), the tracings, from
uppermost to lowest, show the responses at
concentrations of 10, 5, 2.5, 1.25, 0.62, and 0.31 µg/ml; for intact
decorunt (C) and chondroitinase-treated decorunt
(D), the tracings, from uppermost to
lowest, show the responses at concentrations of 20, 10, 5, 2.5, 1.25, and 0.62 µg/ml. The curves represent the
response after subtraction of the values obtained from the uncoated
control surface. The dissociation constants (KD
values) were experimentally estimated to be 1.2 and 2.9 nM
(intact and chondroitinase-treated, respectively) for decorin and 268 and 228 nM (intact and chondroitinase-treated,
respectively) for decorunt.
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Fig. 7.
Reactivity of decorin and decorunt with
anti-VRKVTF neoepitope antiserum. Proteoglycans were isolated from
adult human skin of the indicated ages and anatomic sites and from
fetal human skin (scalp skin) of 120 days estimated gestational age.
Duplicate sets of proteoglycan samples were subjected to SDS-PAGE on
separate gels and electrotransferred as described in the legend to Fig.
2. Separate blots were probed with 6B6 or anti-VRKVTF as indicated.
brs, breast; abd, abdomen; fac,
face.
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Fig. 8.
Immunolocalization of decorin and decorunt in
human skin. Sections of facial skin from a donor 65 years of age
were immunostained with monoclonal antibody 6B6, which recognizes both
decorin and decorunt (A); rabbit antiserum anti-VRKVTF,
which recognizes decorunt, but not decorin (B); or nonimmune
rabbit serum (C). Nonspecific staining of the epidermis
(E) is observed with nonimmune rabbit serum (C).
The arrows in B indicate the transition points in
the outer dermis where the intensity of immunostaining for decorunt
abruptly changes. The bars in each panel equal
670 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(38). Thus, decorunt may also be unable to bind effectively
to transforming growth factor-
, although this has not been tested
experimentally. It also remains to be determined whether decorunt has a
reduced capacity with respect to other core protein-mediated processes
that decorin can influence, such as inhibition of cell proliferation
through binding to the epidermal growth factor receptor (39-42). A
recent publication presents data indicating that the sixth leucine-rich
repeat is most important in the interaction of decorin with the
epidermal growth factor receptor (42). Because this region of decorin
is missing from decorunt, the expectation is that decorunt is incapable
of binding to the epidermal growth factor receptor.
1-antitrypsin on the carboxyl side of Phe
(Phe376-Leu377) in the sequence AAGAMF-LEAIPM
(45). Treatment of purified decorin with MMP-3, ADAMTS4, or cathepsin D
results in cleavage of the core protein, but the fragment that is
generated is not recognized by
anti-VRKVTF.3 Rather, MMP-3
and ADAMTS4 generate a cleavage at PKTLQE154, which is 16 residues closer to the amino terminus than the putative decorunt
cleavage site.3 Catabolic fragments of versican have been
found in human skin, and the cleavage patterns of these fragments are
consistent with generation by ADAMTS1 or
ADAMTS4.4 This observation
suggests that ADAMTS1 and/or ADAMTS4 are present in skin. That the
putative decorunt cleavage site differs from the site at which ADAMTS4
cleaves decorin suggests that decorin and versican are located in
different extracellular matrix compartments in skin.
-binding site may
influence sequestering of this growth factor in the matrix. The
consequences of the weaker binding to collagen may be altered stability
of skin because of changes in the collagen network.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Amy Wright and Natalie Sell for technical assistance and to Debra Bush for typing the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by the Swedish Research Council, Österlunds Stiftelse, Kung Gastav V:s 80-year Fund, Kungliga Fysiografiska Sällskapet, and Kocks Stiftelser, by L'Oréal (Paris), and by the National Institutes of Health.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.
Published, JBC Papers in Press, March 5, 2003, DOI 10.1074/jbc.M300124200
2 P. G. Scott, unpublished observations.
3 J. D. Sandy and J. Westling, unpublished observations.
4 D. A. Carrino, A. Calabro, M. T. Dours-Zimmermann, J. D. Sandy, D. R. Zimmermann, J. M. Sorrell, V. C. Hascall, and A. I. Caplan, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are: CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; ADAMTS, a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type I motifs; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; MMP, matrix metalloproteinase.
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REFERENCES |
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