Activation of heparin cofactor II by fibroblasts and vascular smooth muscle cells

Inhibition of thrombin by heparin cofactor II (HCII) is accelerated by dermatan sulfate, heparan sulfate, and heparin. Purified HCII or defibrinated plasma was incubated with washed confluent cell monolayers, 125I-thrombin was added, and the rate of formation of covalent 125I-thrombin-inhibitor complexes was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Fibroblasts and porcine aortic smooth muscle cells accelerated inhibition of thrombin by HCII 2.3-7.5-fold but had no effect on other thrombin inhibitors in plasma. Human umbilical vein endothelial cells and mouse macrophage-derived cells did not accelerate the thrombin-HCII reaction. IMR-90 normal human fetal lung fibroblasts treated with heparinase or heparitinase accelerated the thrombin-HCII reaction to the same degree as untreated cells. In contrast, treatment with chondroitinase ABC almost totally abolished the ability of these cells to activate HCII while chondroitinase AC had little or no effect, suggesting that dermatan sulfate was responsible for the activity observed. [35S]Sulfate-labeled proteoglycans were isolated from IMR-90 fibroblast monolayers and conditioned medium and fractionated into two peaks on Sepharose CL-2B. The lower Mr proteoglycans contained 74-76% dermatan sulfate and were 11-25 times more active with HCII than the higher Mr proteoglycans which contained 68-97% heparan sulfate. The activity of the lower Mr proteoglycans decreased 70-90% by degradation of the dermatan sulfate component with chondroitinase ABC. These results confirm that dermatan sulfate proteoglycans are primarily responsible for activation of HCII by IMR-90 fibroblasts. We suggest that HCII may inhibit thrombin when plasma is exposed to vascular smooth muscle cells or fibroblasts.     

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.     

The protease specificity of heparin cofactor II. Inhibition of thrombin generated during coagulation

125I-labeled heparin cofactor II (HCII) was mixed with plasma and coagulation was initiated by addition of CaCl2, phospholipids, and kaolin or tissue factor. In the presence of 67 micrograms/ml of dermatan sulfate, radioactivity was detected in a band which corresponded to the thrombin-HCII complex (Mr = 96,000) upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No other complexes were observed. The thrombin-HCII complex was undetectable when 5 units/ml of heparin was present or when prothrombin-deficient plasma was used. In experiments with purified proteases, HCII did not significantly inhibit coagulation factors VIIa, IXa, Xa, XIa, XIIa, kallikrein, activated protein C, plasmin, urokinase, tissue plasminogen activator, leukocyte elastase, the gamma-subunit of nerve growth factor, and the epidermal growth factor-binding protein. HCII inhibited leukocyte cathepsin G slowly, with a rate constant of 8 X 10(4) M-1 min-1 in the presence of dermatan sulfate. These results indicate that the protease specificity of HCII is more restricted than that of other plasma protease inhibitors and suggest that the anticoagulant effect of dermatan sulfate is due solely to inhibition of thrombin by HCII.     

Heparin cofactor IIOslo. Mutation of Arg-189 to His decreases the affinity for dermatan sulfate

Heparin and dermatan sulfate increase the rate of inhibition of thrombin by heparin cofactor II (HCII) approximately 1000-fold by providing a catalytic template to which both the inhibitor and the proteinase bind. A variant form of HCII that binds heparin but not dermatan sulfate has been described recently in two heterozygous individuals (Andersson, T.R., Larsen, M.L., and Abildgaard, U. (1987) Thromb. Res. 47, 243-248). We have now purified the variant HCII (designated HCIIOslo) from the plasma of ne of these individuals. HCIIOslo or normal HCII (11 nM) was incubated with thrombin (9 nM) for 1 min in the presence of heparin or dermatan sulfate. Fifty percent inhibition of thrombin occurred at 26 micrograms/ml dermatan sulfate with normal HCII and greater than 1600 micrograms/ml dermatan sulfate with HCIIOslo. In contrast, inhibition of thrombin occurred at a similar concentration of heparin (1.0-1.5 micrograms/ml) with both inhibitors. To identify the mutation in HCIIOslo, DNA fragments encoding the N-terminal 220 amino acid residues of HCII were amplified from leukocyte DNA by the Taq DNA polymerase chain reaction and both alleles were cloned. A point mutation (G----A) resulting in substitution of His for Arg-189 was found in one allele. The same mutation was constructed in the cDNA of native HCII by oligonucleotide-directed mutagenesis and expressed in Escherichia coli. The recombinant HCIIHis-189 reacted with thrombin in the presence of heparin but not dermatan sulfate, confirming that this mutation is responsible for the functional abnormality in HCIIOslo.     

Modulation of sulfated glycosaminoglycan and small proteoglycan synthesis by the extracellular matrix

Cell culture in collagen lattice is known to be a more physiological model than monolayer for studying the regulation of extracellular matrix protein deposition. The synthesis of sulfated glycosaminoglycans (GAG) and dermatan sulfate (DS) proteoglycans by 3 cell strains were studied in confluent monolayers grown on plastic surface, in comparison to fully retracted collagen lattices. Cells were labelled with³⁵S-sulfate, followed by GAG and proteoglycan analysis by cellulose acetate and SDS-polyacrylamide gel electrophoresis, respectively. The 3 cell strains contracted the lattice in a similar way. In monolayer cultures, the major part of GAG was secreted into culture medium whereas in lattice cultures of dermal fibroblasts and osteosarcoma MG-63 cells but not fibrosarcoma HT-1080 cells, a higher proportion of GAGs, including dermatan sulfate, was retained within the lattices. Small DS proteoglycans, decorin and biglycan, were detected in fibroblasts and MG-63 cultures. They were preferentially trapped within the collagen gel. In retracted lattices, decorin had a higher M_r than in monolayer. Biglycan was detected in monolayer and lattice cultures of MG-63 cells but in lattice cultures only in the case of fibroblasts. In this last case, an up regulation of biglycan mRNA steady state level and down regulation of decorin mRNA was observed, in comparison to monolayers, indicating that collagen can modulate the phenotypical expression of small proteoglycan genes.Supported by a fellowship from the Centre National de la Recherche Scientifique     

Site-directed mutagenesis of arginine 103 and lysine 185 in the proposed glycosaminoglycan-binding site of heparin cofactor II

Inhibition of thrombin by heparin cofactor (HCII) is accelerated approximately 1000-fold by heparin or dermatan sulfate. We found recently that the mutation Arg189----His decreases the affinity of HCII for dermatan sulfate but not for heparin (Blinder, M. A., Andersson, T. R., Abildgaard, U., and Tollefsen, D. M. (1989) J. Biol. Chem. 264, 5128-5133). Other investigators have implicated Arg47 and Lys125 of anti-thrombin (homologous to Arg103 and Lys185 of HCII) in heparin binding. To investigate the corresponding residues in HCII, we have constructed amino acid substitutions (Arg103----Leu, Gln, or Trp; Lys185----Met, Asn, or Thr) by oligonucleotide-directed mutagenesis of the cDNA and expressed the products in Escherichia coli. The recombinant HCII variants were assayed for binding to heparin-Sepharose and for inhibition of thrombin in the presence of various concentrations of heparin or dermatan sulfate. All of the Arg103 variants bound to heparin with normal affinity. Furthermore, inhibition of thrombin by the Arg103----Leu variant occurred at a normal rate in the absence of a glycosaminoglycan and was accelerated by normal concentrations of heparin and dermatan sulfate. These results indicate that HCII, unlike anti-thrombin, does not require a positive charge at this position for the interaction with heparin or dermatan sulfate. The Arg103----Gln and Arg103----Trp variants inhibited thrombin at about one-third of the normal rate in the absence of a glycosaminoglycan, suggesting that these mutations exert an effect on the reactive site (Leu444-Ser445) of HCII. All of the Lys185 variants bound to heparin with decreased affinity but inhibited thrombin at approximately the normal rate in the absence of a glycosaminoglycan. These variants required greater than 10-fold higher concentrations of heparin to accelerate inhibition of thrombin and were not stimulated significantly by dermatan sulfate, suggesting that heparin and dermatan sulfate interact with Lys185 of HCII. These results provide evidence that the glycosaminoglycan-binding site in HCII includes Lys185 but not Arg103, both of which were predicted to be involved by homology to anti-thrombin.     

Molecular interactions of biglycan and decorin with elastic fiber components: biglycan forms a ternary complex with tropoelastin and microfibril-associated glycoprotein 1.

The interactions of the dermatan sulfate proteoglycans biglycan and decorin have been investigated with the elastic fiber components, tropoelastin, fibrillin-containing microfibrils, and microfibril-associated glycoproteins (MAGP) 1 and 2. Both proteoglycans were found to bind tropoelastin and fibrillin-containing microfibrils but not MAGPs 1 and 2 in solid phase binding assays. The specificity of the binding of biglycan and decorin to tropoelastin was confirmed by co-immunoprecipitation experiments and by the blocking of the interactions with elastin-derived peptides. Isolated core proteins from biglycan and decorin bound to tropoelastin more strongly than the intact proteoglycans, and there were no differences in the tropoelastin binding characteristics of distinct glucuronate-rich and iduronate-rich glycoforms of biglycan. These findings indicated that the binding sites were contained in the protein cores of the proteoglycans rather than the glycosaminoglycan side chains. Scatchard analysis showed that biglycan bound more avidly than decorin to tropoelastin with K(d) values estimated as 1.95 x 10(-7) m and 5.3 x 10(-7) m, respectively. In blocking experiments each proteoglycan showed extensive inhibition of binding of the other to tropoelastin but was most effective at blocking its own binding. This result suggested that biglycan and decorin had closely spaced but distinct binding sites on tropoelastin. Addition of the elastin-binding protein MAGP-1 to the assays enhanced the binding of biglycan to tropoelastin but had no effect on the decorin-tropoelastin interaction. Co-immunoprecipitation experiments showed that MAGP-1 interacted with biglycan but not decorin in the solution phase. The results indicated that biglycan specifically formed a ternary complex with tropoelastin and MAGP-1. Overall the study supports the concept that biglycan may have a specific role in the elastinogenic phase of elastic fiber formation.     

Increase in decorin and biglycan in Duchenne Muscular Dystrophy: role of fibroblasts as cell source of these proteoglycans in the disease

Fibrosis is a common pathological feature observed in muscles of patients with Duchenne muscular dystrophy (DMD). Biglycan and decorin are small chondroitin/dermatan sulfate proteoglycans in the muscle extracellular matrix (ECM) that belong to the family of structurally related proteoglycans called small leucine-rich repeat proteins. Decorin is considered an anti-fibrotic agent, preventing the process by blocking TGF-beta activity. There is no information about their expression in DMD patients. We found an increased amount of both proteoglycans in the ECM of skeletal muscle biopsies obtained from DMD patients. Both biglycan and decorin were augmented in the perimysium of muscle tissue, but only decorin increased in the endomysium as seen by immunohistochemical analyses. Fibroblasts were isolated from explants obtained from muscle of DMD patients and the incorporation of radioactive sulfate showed an increased synthesis of both decorin and biglycan in cultured fibroblasts compared to controls. The size of decorin and biglycan synthesized by DMD and control fibroblasts seems to be similar in size and anion charge. These findings show that decorin and biglycan are increased in DMD skeletal muscle and suggest that fibroblasts would be, at least, one source for these proteoglycans likely playing a role in the muscle response to dystrophic cell damage.     

Biglycan organizes collagen VI into hexagonal-like networks resembling tissue structures

The ability of the leucine-rich repeat (LRR) proteins biglycan, decorin, and chondroadherin to interact with collagen VI and influence its assembly to supramolecular structures was studied by electron microscopy and surface plasmon resonance measurements in the BIAcore 2000 system. Biglycan showed a unique ability to organize collagen VI into extensive hexagonal-like networks over a time period of only a few minutes. Only the intact molecule, substituted with two dermatan sulfate chains, had this capacity. Intact decorin, with one dermatan sulfate chain only, was considerably less efficient, and aggregates of organized collagen VI were found only after several hours. Chondroadherin without glycosaminoglycan substitutions did not induce any ordered collagen VI organization. However, all three related LRR proteins were shown to interact with collagen VI using electron microscopy and surface plasmon resonance. Biglycan and decorin were exclusively found close to the N-terminal parts of the collagen VI tetramers, whereas chondroadherin was shown to bind close to both the N- and C-terminal parts of collagen VI. In the formed hexagonal networks, biglycan was localized to the intra-network junctions of the collagen VI filaments. This was demonstrated by electron microscopy after negative staining of gold-labeled biglycan in aggregation experiments with collagen VI.     

Characterization of deoxyuridine 5'-triphosphate nucleotidohydrolase from Trypanosoma cruzi

Inhibition of thrombin by heparin cofactor II (HCII) is accelerated 1000-fold by heparin or dermatan sulfate. To investigate the contribution of basic residues of the A helix of HCII to this activation, we constructed amino acid substitutions (K101Q, R103L, and R106L) by site-directed mutagenesis. K101Q greatly reduced heparin cofactor activity and required a more than 10-fold higher concentration of dermatan sulfate to accelerate thrombin inhibition compared with wild-type recombinant HCII. Thrombin inhibition by R106L was not significantly stimulated by dermatan sulfate. These results provide evidence that basic residues of the A helix of HCII (Lys(101) and Arg(106)) are necessary for heparin- or dermatan sulfate-accelerated thrombin inhibition.     

Endocytosis of different members of the small chondroitin/dermatan sulfate proteoglycan family.

The family of small interstitial chondroitin/dermatan sulfate proteoglycans consists of at least three different molecular species: biglycan (proteoglycan I), decorin (proteoglycan II), and proteoglycan-100, which has a glycosylated core protein of about 100 kDa. The core protein of decorin has been shown to be responsible for receptor-mediated endocytosis of this proteoglycan species by a variety of mesenchymal cells. It is now demonstrated that skin fibroblasts and articular chondrocytes endocytose biglycan with an efficiency similar to that of decorin. Uptake of biglycan is also mediated by its core protein and can be inhibited by decorin in a partially competitive manner. In human fibroblasts, endosomal proteins of 51 and 26 kDa, which are known to bind decorin core protein, also interact with biglycan. This interaction can be inhibited by decorin. Bovine articular chondrocytes contained binding proteins of 48 and 25 kDa. Proteoglycan-100 can be distinguished from biglycan and decorin by its low clearance rate, which however, exceeds the rate of fluid phase endocytosis.     

Inhibition of thrombin by sulfated polysaccharides isolated from green algae

Eight different sulfated polysaccharides were isolated from Chlorophyta. All exhibited thrombin inhibition through a heparin cofactor II (HCII)-dependent pathway, and their effects on the inhibition of thrombin were more potent than those of heparin or dermatan sulfate. In particular, remarkably potent thrombin inhibition was found for the sulfated polysaccharides isolated from the Codiales. In the presence of these sulfated polysaccharides, both the recombinant HCII (rHCII) variants Lys(173)-->Leu and Arg(189)-->His, which are defective in interactions with heparin and dermatan sulfate, respectively, inhibited thrombin in a manner similar to native rHCII. This result indicates that the binding site of HCII for each of these eight sulfated polysaccharides is different from the heparin- or dermatan sulfate-binding site. All the sulfated polysaccharides but RS-2 significantly stimulated the inhibition of thrombin by an N-terminal deletion mutant of HCII (rHCII-Delta74). Furthermore, hirudin(54-65) decreased only 2-5-fold the rate of thrombin inhibition by HCII stimulated by the sulfated polysaccharides, while HD22, a single-stranded DNA aptamer that binds exosite II of thrombin, produced an approximately 10-fold reduction in this rate. These results suggest that, unlike heparin and dermatan sulfate, the sulfated polysaccharides isolated from Chlorophyta activate HCII primarily by an allosteric mechanism different from displacement and template mechanisms.     

Distinct secondary structures of the leucine-rich repeat proteoglycans decorin and biglycan. Glycosylation-dependent conformational stability

Biglycan and decorin have been overexpressed in eukaryotic cells and two major glycoforms isolated under native conditions: a proteoglycan substituted with glycosaminoglycan chains; and a core protein form secreted devoid of glycosaminoglycans (Hocking, A. M., Strugnell, R. A., Ramamurthy, P., and McQuillan, D. J. (1996) J. Biol. Chem. 271, 19571-19577; Ramamurthy, P., Hocking, A. M., and McQuillan, D. J. (1996) J. Biol. Chem. 271, 19578-19584). Far-UV CD spectroscopy of decorin and biglycan proteoglycans indicates that, although they are predominantly beta-sheet, biglycan has a significantly higher content of alpha-helical structure. Decorin proteoglycan and core protein are very similar, whereas the biglycan core protein exhibits closer similarity to the decorin glycoforms than to the biglycan proteoglycan form. However, enzymatic removal of the chondroitin sulfate chains from biglycan proteoglycan does not induce a shift to the core protein structure, suggesting that the final form is influenced by polysaccharide addition only during biosynthesis. Fluorescence emission spectroscopy demonstrated that the single tryptophan residue, which is at a conserved position at the C-terminal domain of both biglycan and decorin, is found in similar microenvironments. This indicates that in this specific domain the different glycoforms do exhibit apparent conservation of structure. Exposure of decorin and biglycan to 10 M urea resulted in an increase in fluorescent intensity, which indicates that the emission from tryptophan in the native state is quenched. Comparison of urea-induced protein unfolding curves provide further evidence that decorin and biglycan assume different structures in solution. Decorin proteoglycan and core protein unfold in a manner similar to a classic two-state model, in which there is a steep transition to an unfolded state between 1 and 2 M urea. The biglycan core protein also shows a similar steep transition. However, biglycan proteoglycan shows a broad unfolding transition between 1 and 6 M urea, probably indicating the presence of stable unfolding intermediates.     

A Monoclonal Antibody which Recognizes a Glycosaminoglycan Epitope in Both Dermatan Sulfate and Chondroitin Sulfate Proteoglycans of Human Skin

Studies have been initiated to identify various cell surface and matrix components of normal human skin through the production and characterization of murine monoclonal antibodies. One such antibody, termed PG-4, identifies both cell surface and matrix antigens in extracts of human foetal and adult skin as the dermatan sulfate proteoglycans, decorin and biglycan, and the chondroitin sulfate proteoglycan versican. Treatment of proteoglycans with chondroitinases completely abolishes immunoreactivity for all of these antigens which suggests that the epitope resides within their glycosaminoglycan chains. Further evidence for the carbohydrate nature of the epitope derives from competition studies where protein-free chondroitin sulfate chains from shark cartilage react strongly; however, chondroitin sulfate chains from bovine tracheal cartilage fail to exhibit a significant reactivity, an indication that the epitope, although present in some chondroitin sulfate chains, does not consist of random chondroitin 4- or 6-sulfate disaccharides. The presence of the epitope on dermatan sulfate chains and on decorin was also demonstrated using competition assays. Thus, PG-4 belongs to a class of antibodies that recognize native epitopes located within glycosaminoglycan chains. It differs from previously described antibodies in this class in that it identifies both chondroitin sulfate and dermatan sulfate proteoglycans. These characteristics make PG-4 a useful monoclonal antibody probe to identify the total population of proteoglycans in human skin.     

Expression and localization of the two small proteoglycans biglycan and decorin in developing human skeletal and non-skeletal tissues

The messenger RNAs and core proteins of the two small chondroitin/dermatan sulfate proteoglycans, biglycan and decorin, were localized in developing human bone and other tissues by both 35S-labeled RNA probes and antibodies directed against synthetic peptides corresponding to nonhomologous regions of the two core proteins. Biglycan and decorin expression and localization were substantially divergent and sometimes mutually exclusive. In developing bones, spatially restricted patterns of gene expression and/or matrix localization of the two proteoglycans were identified in articular regions, epiphyseal cartilage, vascular canals, subperichondral regions, and periosteum, and indicated the association of each molecule with specific developmental events at specific sites. Study of non-skeletal tissues revealed that decorin was associated with all major type I (and type II) collagen-rich connective tissues. Conversely, biglycan was expressed and localized in a range of specialized cell types, including connective tissue (skeletal myofibers, endothelial cells) and epithelial cells (differentiating keratinocytes, renal tubular epithelia). Biglycan core protein was localized at the cell surface of certain cell types (e.g., keratinocytes). Whereas the distribution of decorin was consistent with matrix-centered functions, possibly related to regulation of growth of collagen fibers, the distribution of biglycan pointed to other function(s), perhaps related to cell regulation.     

Decorin and biglycan expression: its relation with endothelial heterogeneity

Decorin and biglycan proteoglycans play important roles in the organization of the extracellular matrix, and in the regulation of cell adhesion and migration. Given morphological and functional endothelial heterogeneity, information is needed regarding whether endothelial cells (ECs) from different vascular beds possess different profiles of proteoglycan constituents of the basement membranes. Here, we report that endothelia from different murine organs and EC lines derived thereof produce and secrete different patterns of proteoglycans. A faint colocalization between decorin and PECAM/CD31 was found on tissue sections from mouse heart, lung and kidney by immunofluorescence. Three EC lines derived from these organs produced decorin (100-kDa) and its core protein (45-kDa). Extracellular decorin recognition in culture supernatant was only possible after chondroitin lyase digestion suggesting that the core protein of secreted proteoglycan is more encrypted by glycosaminoglycans than the intracellular one. Heart and lung ECs were able to produce and release decorin. Kidney ECs synthesized the proteoglycan and its core protein but no secretion was detected in culture supernatants. Although biglycan production was recorded in all EC lines, secretion was almost undetectable, consistent with immunofluorescence results. In addition, no biglycan secretion was detected after EC growth supplement treatment, indicating that biglycan is synthesized, secreted and quickly degraded extracellularly by metalloproteinase-2. Low molecular-mass dermatan sulfate was the predominant glycosaminoglycan identified bound to the core protein. ECs from different vascular beds, with differences in morphology, physiology and cell biology show differences in the proteoglycan profile, extending their heterogeneity to potential differences in cell migration capacities.     

Comparison of heparin- and dermatan sulfate-mediated catalysis of thrombin inactivation by heparin cofactor II.

Heparin and dermatan sulfate activate heparin cofactor II (HCII) comparably, presumably by liberating the amino terminus of HCII to bind to exosite I of thrombin. To explore this model of activation, we systematically substituted basic residues in the glycosaminoglycan-binding domain of HCII with neutral amino acids and measured the rates of thrombin inactivation by the mutants. Mutant D, with changes at Arg(184), Lys(185), Arg(189), Arg(192), Arg(193), demonstrated a approximately 130-fold increased rate of thrombin inactivation that was unaffected by the presence of glycosaminoglycans. The increased rate reflects displacement of the amino terminus of mutant D because (a) mutant D inactivates gamma-thrombin at a 65-fold slower rate than alpha-thrombin, (b) hirudin-(54-65) decreases the rate of thrombin inactivation, and (c) deletion of the amino terminus of mutant D reduces the rate of thrombin inactivation approximately 100-fold. We also examined the contribution of glycosaminoglycan-mediated bridging of thrombin to HCII to the inhibitory process. Whereas activation of HCII by heparin was chain-length dependent, stimulation by dermatan sulfate was not, suggesting that dermatan sulfate does not utilize a template mechanism to accelerate the inhibitory process. Fluorescence spectroscopy revealed that dermatan sulfate evokes greater conformational changes in HCII than heparin, suggesting that dermatan sulfate stimulates HCII by producing more effective displacement of the amino terminus.     

The glucuronyl C5-epimerase activity is the limiting factor in the dermatan sulfate biosynthesis.

An early step in the biosynthesis of dermatan sulfate is polymerization to chondroitin, which then is modified by the D-glucuronyl C5-epimerase and mainly 4-O-sulfotransferase. The final structure of the dermatan sulfate side chains varies and our aim was to identify, which of the two enzymes that are crucial to generate dermatan sulfate copolymeric structures in tissues. Dermatan sulfate side chains of biglycan and decorin were prepared from fibroblasts and nasal and articular chondrocytes and characterized regarding detailed structure. Microsomes were prepared from these cells and the activities of D-glucuronyl C5-epimerase and 4-O-sulfotransferase were determined. Chondrocytes from nasal cartilage synthesized biglycan and decorin containing 10%, articular chondrocytes 20--30%, and fibroblast 80% of the uronosyl residues in the l-iduronyl configuration. All three tissues contained high amount of 4-O-sulfotransferase activity. The activity of d-glucuronyl C5-epimerase showed different relationships. Fibroblasts contained a high level of the epimerase activity, articular chondrocytes intermediary activity, and in nasal cartilage it was barely detectable. The data indicate that the activity of the d-glucuronyl C5-epimerase is the main factor for formation of dermatan sulfate in tissues.     

Lumican is a major small leucine-rich proteoglycan (SLRP) in Atlantic cod (Gadus morhua L.) skeletal muscle

Knowledge on fish matrix biology is important to ensure optimal fish -quality, -growth and -health in aquaculture. The aquaculture industry face major challenges related to matrix biology, such as inflammations and malformations. Atlantic cod skeletal muscle was investigated for collagen I, decorin, biglycan, and lumican expression and distribution by real-time PCR, immunohistochemical staining and Western blotting. Immunohistochemical staining and Western immunoblotting were also performed using antibodies against glycosaminoglycan side chains of these proteoglycans, in addition to fibromodulin. Real-time PCR showed highest mRNA expression of lumican and collagen I. Collagen I and proteoglycan immunohistochemical staining revealed distinct thread-like structures in the myocommata, with the exception of fibromodulin, which stained in dense structures embedded in the myocommata. Chondroitinase AC-generated epitopes stained more limited than cABC-generated epitopes, indicating a stronger presence of dermatan sulfate than chondroitin sulfate in cod muscle. Lumican and keratan sulfate distribution patterns were strong and ubiquitous in endomysia and myocommata. Western blots revealed similar SLRPs sizes in cod as are known from mammals. Staining of chondroitin/dermatan sulfate epitopes in Western blots were similar in molecular size to those of decorin and biglycan, whereas staining of keratan sulfate epitopes coincided with expected molecular sizes of lumican and fibromodulin. In conclusion, lumican was a major proteoglycan in cod muscle with ubiquitous distribution overlapping with keratan sulfate. Other leucine-rich proteoglycans were also present in cod muscle, and Western blot using antibodies developed for mammalian species showed cross reactivity with fish, demonstrating similar structures and molecular weights as in mammals.     

Molecular size of dermatan sulfate oligosaccharides required to bind and activate heparin cofactor II

Heparin cofactor II (HCII) inhibits thrombin rapidly in human plasma in the presence of heparin or dermatan sulfate. To determine the minimum structure of dermatan sulfate required to activate HCII, the glycosaminoglycan was partially degraded by sequential treatment with periodate, [3H]borohydride, and sulfuric acid. Labeled oligosaccharide fragments were separated by gel filtration chromatography. Purified fragments were then applied to a column of HCII bound to concanavalin A-Sepharose, and bound oligosaccharides were eluted with a gradient of sodium chloride. Di-, tetra-, and hexasaccharide fragments did not bind to HCII, while 15% of the octasaccharides and up to 45% of larger fragments bound. Octasaccharides that bound to the HCII column had a greater negative charge than the run-through material based on anion-exchange chromatography, suggesting that they contained a greater number of sulfate groups per molecule. Fragments of dermatan sulfate containing a minimum of 12-14 sugar residues accelerated inhibition of thrombin by HCII. Fragments of this length that bound to the column of immobilized HCII had molar specific activities greater than those of the fragments that did not bind. These studies suggest that HCII is activated by dermatan sulfate fragments greater than or equal to 12 residues in length that contain a specific octasaccharide sequence required for binding to the inhibitor.