Isolation and characterization of matrix proteoglycans from human nasal cartilage. Compositional and structural comparison between normal and scoliotic tissues

The content, types and the fine structures of proteoglycans (PGs) present in human normal nasal cartilage (HNNC) were investigated and compared with those in human scoliotic nasal cartilage (HSNC). Three PG types were identified in both HNNC and HSNC; the large-sized high buoyant density aggrecan, which is the predominant PG population, and the small-sized low buoyant density biglycan and decorin. HSNC contained a significantly higher amount of keratan sulfate (KS)-rich aggrecan (30%) of smaller hydrodynamic size as compared to HNNC. The average molecular sizes (M(r)s) of aggecan-derived chondroitin sulfate (CS) chains in both HNNC and HSNC were identical (18 kDa), but they significantly differ in disaccharide composition, since CS isolated from HSNC contained higher proportions of 6-sulfated disaccharides as compared to those from HNNC. Scoliotic tissue contained also higher amounts (67%) of the small PGs, biglycan and decorin as compared to HNNC. It is worth noticing that both normal and scoliotic human nasal cartilage contain also non-glycanated forms of decorin and biglycan. Dermatan sulfate (DS) was the predominant glycosaminoglycan (GAG) present on biglycan and decorin in both tissues. The small PGs-derived CS chains in both normal and scoliotic cartilage had the same M(r) (20 kDa), whereas DS chains from scoliotic cartilage were of greater M(r) (32 kDa) than those from normal cartilage (24 kDa). Furthermore, scoliotic tissue-derived DS chains contained higher amounts of iduronate (20%) as compared to those of normal cartilage (12%). Disaccharide analysis of small PGs showed that both HNNC and HSNC were rich in 4-sulfated disaccharides and in each case, the small size PGs contained a considerably higher proportion of 4-sulfated disaccharides than the aggrecan of the same tissue. The higher amounts of matrix PGs identified in scoliotic tissue as well as the differences in fine chemical composition of their GAG chains may reflect the modified architecture and functional failure of scoliotic tissue.     

Characterization of chondroitin sulfate and dermatan sulfate proteoglycans of extracellular matrices of human umbilical cord blood vessels and Wharton's jelly

The chondroitin sulfate/dermatan sulfate proteoglycans (CS/DSPGs) of the human umbilical cord vein, arteries and Wharton's jelly matrices were characterized and localized by immunohistochemical analysis. The CS/DSPGs were found to be decorins and biglycans with 43-48 kDa core proteins and are distributed throughout the umbilical cord. A truncated form of decorin having only the approximately 14 kDa NH(2)-terminal portion of the core protein was found exclusively in the vein. The proteoglycans, regardless of their locations, have two types of CS/DS chains, one with approximately 90% CS and approximately 10% DS and the other with approximately 65% CS and approximately 35% DS. The glycosaminoglycan (GAG) chains of the truncated decorin consist of approximately 53% CS and approximately 47% DS. Both decorin and biglycan including the truncated form of decorin could efficiently bind collagen I and fibronectin. The decorin and biglycan with approximately 10% DS and approximately 90% CS were loosely bound in the extracellular matrices, whereas those with approximately 35% DS bound strongly. Together, these data demonstrate that, the GAG chains with 35-47% DS but not those with 10% DS, interact strongly with the matrix. Our data also show that the GAG chain composition is a significant factor in binding of the decorin and biglycan to matrix proteins. The expression of decorin and biglycan with distinctively different CS/DS proportions implies specific biological functions for these PGs in the umbilical cord. The occurrence of the truncated form of decorin exclusively in the umbilical vein suggests a specific functional role.     

Dermatan sulfate remodeling associated with advanced Dupuytren's contracture

Dermatan sulfate (DS) widespread as a component of extracellular matrix proteoglycans, is characterized by great bio-reactivity and remarkable structural heterogeneity due to distinct degrees of sulfation and glucuronosyl epimerization and different polymerization degrees. However, DS metabolism under various biological conditions is poorly known. Dupuytren's contracture is a benign fibromatosis leading to complex remodeling of the palmar fascia structure and properties. However, it remains unclear whether the disease affects the structure of DS, which is the major tissue glycosaminoglycan. Thus the aim of the study was to examine the structure of the total DS in Dupuytren's fascia. DS chains were extracted from 5 samples of normal fascia and 7 specimens of Dupuytren's tissue by papain digestion followed by fractionation with cetylpyridinium chloride. Then, DS structure analysis was performed comprising the evaluation of its molecular masses and sensitivity to hyaluronidase and chondroitinase B. Dupuytren's contracture is associated with significant remodeling of DS chain structure revealed by (1) a distinct profile of chain molecular masses characterized by the appearance of long size components as well as the increase in the content of small size chains; (2) a different glucuronosyl epimerization pattern connected with the enhanced content of glucuronate disaccharide blocks; (3) chain oversulfation. These structural alterations in total DS may modify the GAG interactions especially affecting collagen fibrillogenesis and growth factor availability. Thus, Dupuytren's contracture associated DS remodeling may promote the phenomena typical for advanced disease: apoptosis and reduction in cell number as well as the appearance of dense pseudotendinous collagen matrix.     

Isolation and characterization of matrix proteoglycans from human nasal cartilage: Compositional and structural comparison between normal and scoliotic tissues

The content, types and the fine structures of proteoglycans (PGs) present in human normal nasal cartilage (HNNC) were investigated and compared with those in human scoliotic nasal cartilage (HSNC). Three PG types were identified in both HNNC and HSNC; the large-sized high buoyant density aggrecan, which is the predominant PG population, and the small-sized low buoyant density biglycan and decorin. HSNC contained a significantly higher amount of keratan sulfate (KS)-rich aggrecan (30%) of smaller hydrodynamic size as compared to HNNC. The average molecular sizes (M_rs) of aggecan-derived chondroitin sulfate (CS) chains in both HNNC and HSNC were identical (18 kDa), but they significantly differ in disaccharide composition, since CS isolated from HSNC contained higher proportions of 6-sulfated disaccharides as compared to those from HNNC. Scoliotic tissue contained also higher amounts (67%) of the small PGs, biglycan and decorin as compared to HNNC. It is worth noticing that both normal and scoliotic human nasal cartilage contain also non-glycanated forms of decorin and biglycan. Dermatan sulfate (DS) was the predominant glycosaminoglycan (GAG) present on biglycan and decorin in both tissues. The small PGs-derived CS chains in both normal and scoliotic cartilage had the same M_r (20 kDa), whereas DS chains from scoliotic cartilage were of greater M_r (32 kDa) than those from normal cartilage (24 kDa). Furthermore, scoliotic tissue-derived DS chains contained higher amounts of iduronate (20%) as compared to those of normal cartilage (12%). Disaccharide analysis of small PGs showed that both HNNC and HSNC were rich in 4-sulfated disaccharides and in each case, the small size PGs contained a considerably higher proportion of 4-sulfated disaccharides than the aggrecan of the same tissue. The higher amounts of matrix PGs identified in scoliotic tissue as well as the differences in fine chemical composition of their GAG chains may reflect the modified architecture and functional failure of scoliotic tissue.     

An accumulation of proteoglycans in scarred fascia

A little is known about proteoglycan (PG) changes, occuring in the course of scarring of tissues another than skin. The aim of present study was biochemical characterization of glycosaminoglycans (GAGs) and proteoglycans (PGs) of normal and scarred fascia. Samples of normal fascia lata were taken at autopsy from 23 individuals and samples of scarred fascia lata were removed from 23 patients at reoperations for femoral fracture. The obtained tissues were divided into two samples: first of them was submitted to GAG isolation and the second one to PG isolation.GAGs were extracted by extensive papain digestion followed by the fractionation using cetylpyridinium chloride. In order to qualitative and quantitative characterization GAGs were submitted to electrophoresis on cellulose acetate before and after treatment with enzymes, specifically depolymerizing some kinds of GAGs. PGs were extracted using 4 M guanidine HCl followed by purification by forming complexes with Alcian blue. PGs were submitted to gel permeation chromatography on Sepharose 4B. In order to obtain core proteins PGs were depolymerized with chondroitinase ABC. The purified PGs and their core proteins were separated with sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/PAGE). It was found that total GAGs content was significantly elevated in scarred fascia. Both types of fascia contained chondroitin-, dermatan- and heparan sulphates and hyaluronic acid. Dermatan sulphates (DS) were the predominant GAGs of normal and scarred fascia. The contents of all GAG types were increased in scarred fascia. Both types of fascia contained two kinds of dermatan sulphate proteoglycans (DSPGs); first being similar to biglycan and the second one similar to decorin, as it was judged by molecular weight of their native molecules and core proteins as well as type of GAG components. Densitometric analysis showed that decorin is a predominant DSPG in both fascia types, but in scarred tissue the ratio of biglycan to decorin is considerably higher. Moreover, in scarred fascia a large chondroitin sulphate proteoglycan (CSPG) was also observed. The obtained results have shown that the scar formation is accompanied by quantitative and qualitative alterations in GAGs/PGs resembling those observed in hypertrophic skin scars. The biochemical modification of the scarred fascia lata may partly explain the clinically manifested damage to biomechanical properties of this tissue.     

Proteoglycan metabolism during repair of the ruptured medial collateral ligament in skeletally mature rabbits

The metabolism of the chondroitin/dermatan sulfate (CS/DS) proteoglycans (PGs) decorin and biglycan is markedly altered during short-term (3-6 weeks) and long-term (40 weeks-2 years) repair of surgically ruptured medial collateral ligaments from mature rabbits. A PG-rich extracellular matrix accumulates in injury gaps by 3 weeks postsurgery and extends into tissue regions containing the original ligaments, and elevated PG levels remain apparent up to 2 years postinjury. CS/DS PGs were prepared from such ligaments and identified after SDS-polyacrylamide gel electrophoresis by Alcian blue staining or immunoblotting. In normal ligaments, decorin is the most abundant proteoglycan (accounting for approximately 80% of the total); the remainder is biglycan and a large PG, possibly versican. In repairing ligaments, decorin is barely detected, but instead a large proteoglycan and abundant amounts of biglycan accumulate. Biglycan is present in two forms in repairing ligaments, and they can be separated on SDS-PAGE into 200- and 140-kDa forms. The slower migrating species is absent in normal ligaments and may represent a different glycoform (containing either a single or two short chondroitin/dermatan sulfate chains) of biglycan. Alteration in PG expression and posttranslational processing during medial collateral ligament repair are similar to those reported for repair and scar formation of other connective tissues. The accumulation of biglycan observed here may interfere with proper collagen network remodeling and may lead to persistent inflammatory and matrix turnover processes, thus preventing restoration of a long-term functional ligament tissue.     

Human colon adenocarcinoma is associated with specific post-translational modifications of versican and decorin

In this study, the amounts and the fine structural characteristics of versican and decorin present in human colon adenocarcinomas (HCC) were investigated and compared with those in human normal colon (HNC). HCC is characterized by significant increase in the amounts of versican and decorin (13- and 8-fold in terms of protein, respectively). These two proteoglycans (PGs) were the predominant in HCC (86% of total uronic acid). In HNC, versican and decorin contained both chondroitin sulfate/dermatan sulfate chains (CS/DS), with DS to be the predominant one (90-93%). The molecular sizes (M(r)s) estimated for DS and CS chains were 25-28 and 21-28 kDa, respectively. In CS/DS chains isolated from both versican and decorin, 4-sulfated disaccharides accounted for 79-86% of total disaccharide units, respectively, whereas lower amounts of 6- and non-sulfated units were also recorded. In contrast, the tumor-associated versican and decorin were of smaller hydrodynamic size with lower glycosaminoglycan (GAG) content per PG molecule as compared with those found in HNC. In HCC, both PGs contained mainly CS chains (up to 86%) and the M(r)s of CS and DS chains were also found to be of smaller size (12 and 16 kDa, respectively). The sulfation patterns of CS/DS chains from both PGs were also significantly different. They were composed mainly of 6-sulfated disaccharides (63-70%), whereas 4-sulfated units accounted for 23-31%. A significant increase in the proportion of non-sulfated disaccharides was also recorded. These findings indicate that the colon adenocarcinoma is characterized by a remarkable increase in the concentration of versican and decorin. Furthermore, these PGs are significantly modified at the post-translational level, i.e. the type, length and the sulfation pattern of their GAG chains. These specific structural alterations of versican and decorin may influence the biology of cancer cells in HCC.     

Cell density-dependent regulation of proteoglycan synthesis by transforming growth factor-beta(1) in cultured bovine aortic endothelial cells

The regulation of vascular endothelial cell behavior during angiogenesis and in disease by transforming growth factor-beta(1) (TGF-beta(1)) is complex, but it clearly involves growth factor-induced changes in extracellular matrix synthesis. Proteoglycans (PGs) synthesized by endothelial cells contribute to the formation of the vascular extracellular matrix and also influence cellular proliferation and migration. Since the effects of TGF-beta(1) on vascular smooth muscle cell growth are dependent on cell density, it is possible that TGF-beta(1) also directs different patterns of PG synthesis in endothelial cells at different cell densities. In the present study, dense and sparse cultures of bovine aortic endothelial cells were metabolically labeled with [(3)H]glucosamine, [(35)S]sulfate, or (35)S-labeled amino acids in the presence of TGF-beta(1). The labeled PGs were characterized by DEAE-Sephacel ion exchange chromatography and Sepharose CL-4B molecular sieve chromatography. The glycosaminoglycan M(r) and composition were analyzed by Sepharose CL-6B chromatography, and the core protein M(r) was analyzed by SDS-polyacrylamide gel electrophoresis, before and after digestion with papain, heparitinase, or chondroitin ABC lyase. These experiments indicate that the effect of TGF-beta(1) on vascular endothelial cell PG synthesis is dependent on cell density. Specifically, TGF-beta(1) induced an accumulation of small chondroitin/dermatan sulfate PGs (CS/DSPGs) with core proteins of approximately 50 kDa in the medium of both dense and sparse cultures, but a cell layer-associated heparan sulfate PG with a core protein size of approximately 400 kDa accumulated only in dense cultures. Moreover, only in the dense cell cultures did TGF-beta(1) cause CS/DSPG hydrodynamic size to increase, which was due to the synthesis of CS/DSPGs with longer glycosaminoglycan chains. The heparan sulfate PG and CS/DSPG core proteins were identified as perlecan and biglycan, respectively, by Western blot analysis. The present data suggest that TGF-beta(1) promotes the synthesis of both perlecan and biglycan when endothelial cell density is high, whereas only biglycan synthesis is stimulated when the cell density is low. Furthermore, glycosaminoglycan chains are elongated only in biglycan synthesized by the cells at a high cell density.     

Platelet derived growth factor BB is a ligand for dermatan sulfate chain(s) of small matrix proteoglycans from normal and fibrosis affected fascia

Structural requirements of the short isoform of platelet derived growth factor BB (PDGF-BB) to bind dermatan sulfate (DS)/chondroitin sulfate (CS) are unknown. Meanwhile the interaction may be important for tissue repair and fibrosis which involve both high activity of PDGF-BB and matrix accumulation of DS. We examined by the solid phase assay the growth factor binding to DS chains of small proteoglycans from various fasciae as well as to standard CSs. Before the assay a structural analysis of DSs and CSs was accomplished involving the evaluation of their epimerization and/or sulfation patterns. In addition, in vivo acceptors for PDGF-BB in fibrosis affected fascia were detected. PDGF-BB binding sites on DSs/CSs are located in long chain sections with the same type of hexuronate isomer however without any apparent preference to glucuronate or iduronate residues. Alternatively, the interaction seems to involve two shorter DS chain sections assembling disaccharides with the same type of hexuronate isomer which are separated by disaccharide(s) with another hexuronate one. Moreover, DS/CS affinity to the growth factor most probably depends on an accumulation of di-2,4-O-sulfated disaccharides in binding site while the presence of 6-O-sulfated N-acetyl-galactosamine residues rather attenuates the binding. All examined fascia DSs and standard CSs showed significant PDGF-BB binding capability with the highest affinity found for normal palmar fascia decorin DS. In fibrosis affected palmar fascia DS/CS proteoglycans are able to form with PDGF-BB supramolecular complexes also including other matrix components such as type III collagen and fibronectin which bind the growth factor covalently. Our results suggest that DS chains of fascia matrix small PGs may regulate PDGF-BB availability leading to restriction of fibrosis associated with Dupuytren's disease or to control of normal fascia repair.     

Fluoride-induced changes to proteoglycan structure synthesised within the dentine-pulp complex in vitro

Fluoride is known to influence mineralisation patterns within dentine, where alterations in the post-translational modification of proteoglycans (PG) have been proposed as an implicating factor. In light of recent studies elucidating changing PG profiles in the transition of predentine to mineralised dentine, this study investigates the influence of fluoride on the major PG populations (decorin, biglycan and versican) within the pulp, predentine and dentine. Tooth sections from rat incisors were cultured for 14 days in the presence 0, 1 and 6 mM sodium fluoride and the PG extracted from the pulp, predentine and dentine matrices. PG species and corresponding metabolites were identified by their immuno-reactivity to antibodies against decorin, biglycan and versican. Component glycosaminoglycan chains were characterised with respect to their nature, chain length and disaccharide composition. Levels of PG extracted from pulp and predentine were reduced, particularly for biglycan. Fluoride did not influence levels of decorin or versican within predentine or dentine, although the processing of these macromolecules within pulp and predentine was affected, particularly at higher fluoride concentrations. Levels of dermatan sulfate were reduced within pulp and predentine, although the effect was less pronounced for predentine. Fluoride reduced sulfation of glycosaminoglycan chains within pulp and predentine tissues, with a notable reduction in Deltadi6S evident. In all three tissues, glycosaminoglycan chain length was reduced. Considering the various roles for PG in the dentine-pulp complex, either directly or indirectly in the mineralisation process, changes in the synthesis, structure and processing of the different PG species within the pulp, predentine and dentine matrices provides a further molecular explanation for the altered mineralisation patterns witnessed during fluorosis.     

The role of decorin and biglycan dermatan sulfate chain(s) in fibrosis-affected fascia

Organ fibrosis is associated with excessive deposition of dermatan sulfate (DS) in the extracellular matrix (ECM) of the affected tissue. However, the significance of DS in fibrosis process is poorly known. Thus, we have analyzed both in vitro and in vivo the binding potential toward fibroblast growth factor-2, platelet-derived growth factor BB and fibronectin (FN) of DS representing glycosaminoglycan (GAG) chains of two proteoglycans decorin and biglycan derived from fascia undergoing fibrosis due to Dupuytren's disease. Moreover, to investigate the relation between DS structure and its binding properties to above ligands, we have also studied the interactions of the GAG chains from normal porcine skin decorin and biglycan. The examined interactions, especially those engaging extractable pool of both human and porcine decorin DS, are characterized by very high affinity and low capacity. Moreover, the presence of iduronate residues is not essential for the DS binding to all studied ligands and the interactions more strongly depend on the GAG sulfation pattern. All investigated interactions have biological relevance as judged from the coexistence of decorin (and biglycan) DS, both growth factors and FN in supra-molecular complexes localized in ECM of both fibrous and normal human fascia. Moreover, these complexes also include collagen type III. It seems that fascia fibrosis process when compared with physiological circumstances is associated with the preservation of at least some functions of decorin and biglycan DSs such as the regulation of growth factor bioavailability and most probably influence FN fibrillogenesis as well as coupling of various fibrilar matrix element assembly.     

Isolation and characterization of rat skeletal muscle proteoglycan decorin and comparison with the human fibroblast decorin.

1. The extracellular matrix (ECM) of rat skeletal muscle contains several proteoglycans (PGs). The more abundant correspond to a chondroitin/dermatan sulfate PG or decorin. 2. Decorin isolated from rat skeletal muscle ECM has a smaller molecular size than human fibroblast decorin. 3. The difference in size is mainly due to the glycosaminoglycan (GAG) chain length rather than the core protein size. 4. Peptide analysis of trypsin treated decorins shows at least three peptides with the same electrophoretic mobility.     

Temporal regulation of hyaluronan and proteoglycan metabolism by human bone cells in vitro

Osteoblasts elaborate a dynamic extracellular matrix that is constructed and mineralized as bone is formed. This matrix is primarily composed of collagen, along with noncollagenous proteins which include glycoproteins and proteoglycans. After various times in culture, human bone cells were labeled with [35S]sulfate, [3H] leucine/proline, or [3H]glucosamine and the metabolism of hyaluronan and four distinct species of proteoglycans (PGs) was assayed in the medium, cell layer, and intracellular pools. These cells produce hyaluronan (Mr approximately 1,400,000; a chondroitin sulfate PG (CSPG), Mr approximately 600,000; a heparan sulfate PG (HSPG), Mr approximately 400,000; and two dermatan sulfate PGs with Mr approximately 270,000 (biglycan, PG I) and Mr approximately 135,000 (decorin, PG II) that distribute between the medium and cell layer. Two days following subculture, 12 h [35S]sulfate steady-state labeling yielded a composition of 24, 27, 31, and 18% for total CSPG, HSPG, biglycan, and decorin, respectively. While HSPG and decorin levels and distribution between medium and cell layer remained relatively constant during steady-state labeling at different times in culture, CSPG and biglycan levels increased dramatically at late stages of growth, and their distribution changed throughout culture. These results were independent of cell density, media depletion, and labeling pool effects. In contrast, hyaluronan synthesis was uncoupled from PG synthesis and apparently density-dependent. Pulse chase labeling at different stages of culture showed that the CSPG and decorin behaved as secretory PGs. Both HSPG and biglycan underwent catabolism, with HSPG possessing a t1/2 of 8 h and biglycan a t1/2 of 4 h. While the rate of HSPG turnover did not appreciably change between early and late culture, that of biglycan decreased. The mRNA for decorin was constant, while that of biglycan changed during culture. These results suggest that each PG possesses a distinct pattern of cellular and temporal distribution that may reflect specific stages in matrix formation and maturation.     

The Identification of Proteoglycans and Glycosaminoglycans in Archaeological Human Bones and Teeth

Bone tissue is mineralized dense connective tissue consisting mainly of a mineral component (hydroxyapatite) and an organic matrix comprised of collagens, non-collagenous proteins and proteoglycans (PGs). Extracellular matrix proteins and PGs bind tightly to hydroxyapatite which would protect these molecules from the destructive effects of temperature and chemical agents after death. DNA and proteins have been successfully extracted from archaeological skeletons from which valuable information has been obtained; however, to date neither PGs nor glycosaminoglycan (GAG) chains have been studied in archaeological skeletons. PGs and GAGs play a major role in bone morphogenesis, homeostasis and degenerative bone disease. The ability to isolate and characterize PG and GAG content from archaeological skeletons would unveil valuable paleontological information. We therefore optimized methods for the extraction of both PGs and GAGs from archaeological human skeletons. PGs and GAGs were successfully extracted from both archaeological human bones and teeth, and characterized by their electrophoretic mobility in agarose gel, degradation by specific enzymes and HPLC. The GAG populations isolated were chondroitin sulfate (CS) and hyaluronic acid (HA). In addition, a CSPG was detected. The localization of CS, HA, three small leucine rich PGs (biglycan, decorin and fibromodulin) and glypican was analyzed in archaeological human bone slices. Staining patterns were different for juvenile and adult bones, whilst adolescent bones had a similar staining pattern to adult bones. The finding that significant quantities of PGs and GAGs persist in archaeological bones and teeth opens novel venues for the field of Paleontology.     

骨质中硫酸软骨素类蛋白多糖的类型和特征

采用骨质蛋白质的三步 (盐酸胍 - EDTA-盐酸胍 )提取法 ,较完全地提取兔长骨和人牙槽骨骨质中各类蛋白多糖 ( PGs) ,并采用凝胶过滤和离子交换柱层析等方法进行纯化 ,再用单克隆抗体 ( MAb2 B6、MAb3B3和 MAb1 B5)检测、分析其中 PGs的类型和性质 .结果表明 ,兔长骨中 PGs的主要类型为 DS类 ( 4 5k D)、C6S类 ( 2 0 0 k D)、C4S类 ( 4 5k D)和 COS类 ( 2 0 0 k D) PG;人牙槽骨中则主要含 DS类 PG( 4 5k D) ,和少量 COS类 PG( 4 5k D和 1 1 0 k D) ,未发现 C4S类 PG.根据此结果可以推测 ,兔长骨以混合方式 (软骨成骨和类骨质成骨 )骨化 ,而人牙槽骨则以类骨质成骨为主 .两者骨质结构和损伤后修复方式可能也有一定的差异 .     

Differential regulation of extracellular matrix proteoglycan (PG) gene expression. Transforming growth factor-beta 1 up-regulates biglycan (PGI), and versican (large fibroblast PG) but down-regulates decorin (PGII) mRNA levels in human fibroblasts in culture

Proteoglycans (PGs) comprise a group of extracellular matrix macromolecules which play an important role in matrix biology. In this study, normal human skin and gingival fibroblast cultures were incubated with transforming growth factor-beta 1 (TGF-beta 1), and the expression of three PGs, viz. biglycan (PGI), decorin (PGII), and versican (a large fibroblast proteoglycan) was examined. The results indicate that TGF-beta 1 (5 ng/ml) markedly increased the expression of biglycan (up to 24-fold) and versican (up to 6-fold) mRNAs and the enhancement of biglycan expression was coordinate with elevated type I procollagen gene expression in the same cultures. In contrast, the expression of decorin mRNA was markedly (up to approximately 70%) inhibited by TGF-beta 1. The response to TGF-beta 1 was similar in both skin and gingival fibroblasts, although the gingival cells were clearly more responsive to stimulation by TGF-beta 1 with respect to biglycan gene expression. Analysis of 35S-labeled proteoglycans in the culture media of skin and gingival fibroblasts also revealed stimulation of biglycan and versican production, and reduction in decorin production. Quantitation of both [35S]sulfate and [3H]leucine-labeled decorin in cell culture media by immunoprecipitation revealed a 50% reduction in decorin production in cell cultures treated with TGF-beta 1. This TGF-beta 1-elicited reduction was accompanied by an apparent increase in the size of the decorin molecules, although the size of the core protein was not altered, as judged by Western immunoblotting following chondroitinase ABC digestion. Analysis of the proteoglycans in the matrix and membrane fractions also revealed increased amounts of versican in cultures treated with TGF-beta 1. These results indicate differential regulation of PG gene expression in fibroblasts by TGF-beta 1, and these observations emphasize the role of PGs in the extracellular matrix biology and pathology.     

Purification and characterization of iduronic acid-rich and glucuronic acid-rich proteoglycans implicated in human post-burn keloid scar

Small proteoglycans (PGs), extracted from human keloid scar tissue with 4M guanidinium chloride and fractionated by DEAE-cellulose chromatography, were separated by ethanol precipitation into one L-iduronic acid-rich and one D-glucuronic acid-rich fraction. The size of the L-iduronic acid-rich PG was 102 kDa with a 27 kDa glycosaminoglycan chain, that of the D-glucuronic acid-rich PG was 90 kDa with a 26 kDa glycosaminoglycan chain, and the protein core of both PGs was 14.5 kDa. The two PGs carried sulfate groups mostly attached at C-4 of the 2-amino-2-deoxy-D-galactose units. The N-terminal amino acid sequence of both was similar to human bone PGII (decorin), normal and hypertrophic scar, and human dermal tissue PG.     

Proteoglycans in human laryngeal cartilage. Identification of proteoglycan types in successive cartilage extracts with particular reference to aggregating proteoglycans

The content, composition and structure of proteoglycans (PGs) in adult human laryngeal cartilage (HLC) were investigated. PGs were extracted from the tissue by using two different extraction protocols. In the first protocol, PGs were extracted under dissociative conditions, 4 M guanidine HCl (GdnHCl), and in the second protocol, sequentially, with phosphate buffered saline (PBS) and solutions of increasing GdnHCl concentration (0.5, 1, 2 and 4 M). Chemical and immunological analyses of dissociate extracts (first protocol) revealed the presence of four, at least, different types of PGs. Aggrecan was the major PG, versican, decorin and biglycan being in small amounts. Galactosaminoglycan-containing PGs (GalAGPGs) represented the vast majority of total PGs present in extracts of HLC. Differential digestion with chondroitinase ABC and AC II showed that the GalAGPGs from HLC contained a significant proportion of dermatan sulphate (DS). In addition, disaccharide analysis showed that 6-sulphated disaccharides predominated in chondroitin sulphate (CS) chains. The sequential extraction (second protocol) indicated that PBS extract contained very little amount of PGs. The 0.5, 1 and 2 M GdnHCl extracts contained 6.3%, 24.5% and 15.2% of total extracted PGs, respectively. Four molar GdnHCl extracted the larger proportion, about 53%, of total PGs. This extract contained almost only proteoglycan aggregate components i.e., G1 bearing aggrecan, hyaluronan and link protein. The characterization of the aggrecan showed that it constituted a polydisperse population of monomers with an average molecular mass of 720 kDa. The glycosaminoglycans (GAGs) present were chondroitin sulphate with a M(r) of 15 kDa, and keratan sulphate (KS) with a M(r) of 10 kDa, in proportions 84% and 16%, respectively.     

Developmental expression of dermatan sulfate proteoglycans in the elastic bovine nuchal ligament.

The nuchal ligament of bovines is a useful system in which to study elastic fibre formation since it contains up to 83% elastin and undergoes a period of rapid elastinogenesis during the last trimester of fetal development and in the first four post-natal months. To identify proteoglycans (PGs) which may be involved in this process we initially investigated changes in the glycosaminoglycan (GAG) profiles during nuchal ligament development. In contrast to the collagenous Achilles tendon, nuchal ligament exhibited: (a) elevated hyaluronan (HA) levels in the peak period of elastin-associated microfibril (fibrillin) synthesis (130-200 days) which precedes elastinogenesis; and (b) markedly increased synthesis of a glucuronate-rich copolymeric form of dermatan sulfate (DS) in the period corresponding to elastin formation (200-270 days). Analysis of DSPGs isolated from 230-day nuchal ligament showed that this copolymer was predominantly associated with a glycoform of biglycan which was specifically elevated at this stage in development. This finding was consistent with Northern blot analysis which showed that steady-state biglycan mRNA levels increased significantly during the elastinogenic period. In contrast, the mRNA levels for decorin, the only other DSPG detected in this tissue, declined rapidly after 140 days of fetal development. In conclusion, the results suggest that HA may play a role in microfibril assembly and that a specific glycoform of biglycan may be associated with the elastinogenic phase of elastic fibre formation.