Age-related Changes in the Proteoglycans of Human Skin

SPECIFIC CLEAVAGE OF DECORIN TO YIELD A MAJOR CATABOLIC FRAGMENT IN ADULT SKIN*

David A. CarrinoDagger §, Patrik Önnerfjord§, John D. Sandy||, Gabriella Cs-Szabo**, Paul G. ScottDagger Dagger , J. Michael SorrellDagger , Dick Heinegård, and Arnold I. CaplanDagger

From the Dagger  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 Dagger Dagger  Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada

Received for publication, January 6, 2003, and in revised form, February 28, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 -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.

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 alpha -cyano-hydroxycinnamic acid was used.

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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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).


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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.

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).


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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.

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.


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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.

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.


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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.

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.


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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.

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.


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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

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-beta (38). Thus, decorunt may also be unable to bind effectively to transforming growth factor-beta , 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.

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 alpha 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.

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-beta -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.

    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.

    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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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