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• W2042279293 abstract "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 437 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. 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 437 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. 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type I motifs matrix-assisted laser desorption/ionization time-of-flight mass spectrometry matrix metalloproteinase The mechanical properties of the dermis are determined primarily by the extracellular matrix. These mechanical properties change dramatically as a function of age (1Daly C.H. Odland G.F. J. Invest. Dermatol. 1979; 73: 84-87Abstract Full Text PDF PubMed Scopus (209) Google Scholar, 2Takema Y. Yorimoto Y. Kawai M. Imokawa G. Br. J. Dermatol. 1994; 131: 641-648Crossref PubMed Scopus (254) Google Scholar), 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 (3Bentley J.P. J. Invest. Dermatol. 1979; 73: 80-83Abstract Full Text PDF PubMed Scopus (44) Google Scholar, 4Lavker R.M. Zheng P. Dong G. J. Invest. Dermatol. 1987; 88: 44-51Abstract Full Text PDF PubMed Scopus (231) Google Scholar, 5Mays P.K. Bishop J.E. Laurent G.J. Mech. Ageing Dev. 1988; 45: 203-212Crossref PubMed Scopus (138) Google Scholar, 6Yamauchi M. Woodley D.T. Mechanic G.L. Biochem. Biophys. Res. Commun. 1988; 152: 898-903Crossref PubMed Scopus (128) Google Scholar, 7Moragas A. Garcia-Bonafé M. Sans M. Torán N. Huguet P. Martı́n-Plata C. Anal. Quant. Cytol. Histol. 1998; 20: 493-499PubMed Google Scholar), which are the major extracellular matrix components of the dermis (8Pinnell S.R. Murad S. Goldsmith L.A. Biochemistry and Physiology of the Skin. Oxford University Press, Oxford1983: 385-410Google Scholar). In addition to collagen, the dermal extracellular matrix also contains proteoglycans, which show age-related differences (9Breen M. Johnson R.L. Sittig R.A. Weinstein H.G. Veis A. Connect. Tissue Res. 1972; 1: 291-303Crossref Scopus (30) Google Scholar, 10Fleischmajer R. Perlish J.S. Bashey R.I. Biochim. Biophys. Acta. 1972; 279: 265-275Crossref PubMed Scopus (83) Google Scholar, 11Pearce R.H. Grimmer B.J. J. Invest. Dermatol. 1972; 58: 347-361Abstract Full Text PDF PubMed Scopus (74) Google Scholar, 12Habuchi H. Kimata K. Suzuki S. J. Biol. Chem. 1986; 261: 1031-1040Abstract Full Text PDF PubMed Google Scholar, 13Longas M.O. Russell C.S. He X.-Y. Carbohydr. Res. 1987; 159: 127-136Crossref PubMed Scopus (117) Google Scholar, 14Meyer L.J.M. Stern R. J. Invest. Dermatol. 1994; 102: 385-389Abstract Full Text PDF PubMed Google Scholar, 15Ågren U.M. Tammi M. Ryynänen M. Tammi R. J. Invest. Dermatol. 1997; 109: 219-224Abstract Full Text PDF PubMed Scopus (33) Google Scholar, 16Carrino D.A. Sorrell J.M. Caplan A.I. Arch. Biochem. Biophys. 2000; 373: 91-101Crossref PubMed Scopus (106) Google Scholar). Perhaps related to these changes in proteoglycans are age-related increases in the water content of the dermis (11Pearce R.H. Grimmer B.J. J. Invest. Dermatol. 1972; 58: 347-361Abstract Full Text PDF PubMed Scopus (74) Google Scholar) and in the content of mobile water (17Richard S. Querleux B. Bittoun J. Jolivet O. Idy-Peretti I. de Lacharriere O. Leveque J.-L. J. Invest. Dermatol. 1993; 100: 705-709Abstract Full Text PDF PubMed Google Scholar). 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 (18Brown D.C. Vogel K.G. Matrix. 1989; 9: 468-478Crossref PubMed Scopus (158) Google Scholar, 19Fleischmajer R. Fisher L.W. MacDonald E.D. Jacobs Jr., L. Perlish J.S. Termine J.D. J. Struct. Biol. 1991; 106: 82-90Crossref PubMed Scopus (151) Google Scholar), and targeted disruption of decorin results in aberrant collagen fibrils and in a reduction in the tensile strength of skin (20Danielson K.G. Baribault H. Holmes D.F. Graham H. Kadler K.E. Iozzo R.V. J. Cell Biol. 1997; 136: 729-743Crossref PubMed Scopus (1182) Google Scholar). Similarly, a patient with a variant form of Ehlers-Danlos syndrome was found to have a substantially reduced amount of dermal decorin (21Fushimi H. Kamayama M. Shinkai H. J. Int. Med. 1989; 226: 409-416Crossref PubMed Scopus (21) Google Scholar). Previous work has shown that decorin and versican are the major proteoglycans extracted from human skin (16Carrino D.A. Sorrell J.M. Caplan A.I. Arch. Biochem. Biophys. 2000; 373: 91-101Crossref PubMed Scopus (106) Google Scholar). A truncated form of decorin is abundant in extracts of postnatal skin, whereas fetal skin extracts contain little, if any, of this molecule (16Carrino D.A. Sorrell J.M. Caplan A.I. Arch. Biochem. Biophys. 2000; 373: 91-101Crossref PubMed Scopus (106) Google Scholar). 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. The sources of reagents for proteoglycan extraction and isolation and for SDS-PAGE and immunoblots were as described elsewhere (16Carrino D.A. Sorrell J.M. Caplan A.I. Arch. Biochem. Biophys. 2000; 373: 91-101Crossref PubMed Scopus (106) Google Scholar). 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 (22Roughley P.J. White R.J. Magny M.-C. Liu J. Pearce R.H. Mort J.S. Biochem. J. 1993; 295: 421-426Crossref PubMed Scopus (120) Google Scholar, 23Scott P.G. Dodd C.M. Pringle G.A. J. Biol. Chem. 1993; 268: 11558-11564Abstract Full Text PDF PubMed Google Scholar). 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 (16Carrino D.A. Sorrell J.M. Caplan A.I. Arch. Biochem. Biophys. 2000; 373: 91-101Crossref PubMed Scopus (106) Google Scholar). 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. These procedures were as reported previously (16Carrino D.A. Sorrell J.M. Caplan A.I. Arch. Biochem. Biophys. 2000; 373: 91-101Crossref PubMed Scopus (106) Google Scholar). 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 mguanidinium chloride, 0.57 CHAPS,1 0.05 msodium 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 ॖl. 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 (24Carrino D.A. Arias J.A. Caplan A.I. Biochem. Int. 1991; 24: 485-495PubMed Google Scholar). 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 (25Choi H.U. Johnson T.L. Pal S. Tang L.-H. Rosenberg L. Neame P.J. J. Biol. Chem. 1989; 264: 2876-2884Abstract Full Text PDF PubMed Google Scholar). Samples corresponding to 25–300 ॖg of glycosaminoglycan were reconstituted in 1 ml of 2 mguanidinium 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 mguanidinium 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 (25Choi H.U. Johnson T.L. Pal S. Tang L.-H. Rosenberg L. Neame P.J. J. Biol. Chem. 1989; 264: 2876-2884Abstract Full Text PDF PubMed Google Scholar). Appropriate fractions were pooled, and all pools, including the unbound material, were brought to 0.57 CHAPS by the addition of CHAPS from a 107 solution. The samples were concentrated, precipitated, and assayed as described above. The proteoglycan samples were reconstituted after lyophilization and prepared for SDS-PAGE as described (16Carrino D.A. Sorrell J.M. Caplan A.I. Arch. Biochem. Biophys. 2000; 373: 91-101Crossref PubMed Scopus (106) Google Scholar). SDS-PAGE on 5–17.57 gels, electrotransfer, and immunoblot analysis were performed as done previously (16Carrino D.A. Sorrell J.M. Caplan A.I. Arch. Biochem. Biophys. 2000; 373: 91-101Crossref PubMed Scopus (106) Google Scholar), 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 (16Carrino D.A. Sorrell J.M. Caplan A.I. Arch. Biochem. Biophys. 2000; 373: 91-101Crossref PubMed Scopus (106) Google Scholar). Hence, all of the immunoblots in this study were performed with Immobilon-N. Coomassie Blue-stained bands on SDS-PAGE gels were excised and washed extensively with 407 acetonitrile in 25 mm NH4HCO3, pH 7.8. The gel pieces were dried in a SpeedVac before digestion overnight at 37 °C with 10 ॖl of sequencing grade trypsin (Promega) at 20 ng/ॖl in 25 mm NH4HCO3, pH 7.8. The digestion was terminated by the addition of 10 ॖl of 27 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 α-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. The BIAcoreTM2000 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 ॖl of 50 mmN-hydroxysuccinimide and 35 ॖl of 200 mmN-ethyl-N′-(dimethylaminopropyl)carbodiimide at 25 °C at a flow rate of 5 ॖl/min. Collagen type I (35 ॖl at 3 ॖg/ml in 0.15 m NaCl, 10 mm sodium citrate, pH 5.0) was immobilized at 25 °C at a flow rate of 5 ॖl/min. One surface containing no coupled protein was used as a blank. The remaining activated groups were blocked with 40 ॖl of 1 methanolamine, 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 ॖg/ml in 0.15 m NaCl, 0.0057 Tween 20, 10 mm HEPES, pH 7.4. The surface was regenerated with two injections of 0.5 m NaCl, 0.1m NaHCO3, pH 9.2, and one injection of 2m 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/ॖg 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. Skin specimens were fixed in 107 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. 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 (16Carrino D.A. Sorrell J.M. Caplan A.I. Arch. Biochem. Biophys. 2000; 373: 91-101Crossref PubMed Scopus (106) Google Scholar). 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 (16Carrino D.A. Sorrell J.M. Caplan A.I. Arch. Biochem. Biophys. 2000; 373: 91-101Crossref PubMed Scopus (106) Google Scholar). That decorunt is related to decorin is indicated by the amino-terminal amino acid sequences of these molecules, which are identical (16Carrino D.A. Sorrell J.M. Caplan A.I. Arch. Biochem. Biophys. 2000; 373: 91-101Crossref PubMed Scopus (106) Google Scholar). 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 (16Carrino D.A. Sorrell J.M. Caplan A.I. Arch. Biochem. Biophys. 2000; 373: 91-101Crossref PubMed Scopus (106) Google Scholar). 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).Figure 2Reactivity 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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) (22Roughley P.J. White R.J. Magny M.-C. Liu J. Pearce R.H. Mort J.S. Biochem. J. 1993; 295: 421-426Crossref PubMed Scopus (120) Google Scholar). 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 (23Scott P.G. Dodd C.M. Pringle G.A. J. Biol. Chem. 1993; 268: 11558-11564Abstract Full Text PDF PubMed Google Scholar). 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 (16Carrino D.A. Sorrell J.M. Caplan A.I. Arch. Biochem. Biophys. 2000; 373: 91-101Crossref PubMed Scopus (106) Google Scholar). 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.Figure 5Post-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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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 nmfor 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. 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 (16Carrino D.A. Sorrell J.M. Caplan A.I. Arch. Biochem. Biophys. 2000; 373: 91-101Crossref PubMed Scopus (106) Google Scholar). 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 ॖmCCGVRKVTF, 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 (16Carrino D.A. Sorrell J.M. Caplan A.I. Arch. Biochem. Biophys. 2000; 373: 91-101Crossref PubMed Scopus (106) Google Scholar). This observation is based on analysis of eight samples of fetal skin ranging in age from 80 to 120 days estimated gestational" @default.
• W2042279293 created "2016-06-24" @default.
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• W2042279293 date "2003-05-01" @default.
• W2042279293 modified "2023-10-12" @default.
• W2042279293 title "Age-related Changes in the Proteoglycans of Human Skin" @default.
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