Showing papers on "Keratan sulfate published in 1997"

The Natural History of the Anterior Cruciate Ligament-Deficient Knee Changes in Synovial Fluid Cytokine and Keratan Sulfate Concentrations

Abstract: Restoring knee stability through reconstruction, while providing symptomatic relief, has not been shown to decrease the incidence of degenerative changes after rupture of the anterior cruciate ligament. This suggests that posttraumatic osteoarthritis may not be purely biomechanical in origin, but also biochemical. To test this, we measured the levels of seven cytokine modulators of cartilage metabolism in knee joint synovial fluid after anterior cruciate ligament rupture. We also measured keratan sulfate, a product of articular cartilage catabolism. The sample population consisted of patients with uninjured knee joints (N = 10), and patients with acute (N = 60), subacute (N = 18), and chronic (N = 8) anterior cruciate ligament-deficient knees. Synovial fluid samples were analyzed by enzyme-linked immunosorbent assays. Normal synovial fluids contained high levels of the interleukin-1 receptor antagonist but low concentrations of other cytokines. Immediately after ligament rupture there were large increases in interleukins 6 and 8, tumor necrosis factor alpha, and keratan sulfate. Interleukin-1 levels remained low throughout the course. As the injury became subacute and then chronic, interleukin-6, tumor necrosis factor-alpha, and keratan sulfate levels fell but remained considerably elevated 3 months after injury. Concentrations of interleukin-1Ra fell dramatically. Granulocyte-macrophage colony-stimulating factor concentrations were normal acutely and subacutely but by 3 months after injury were elevated 10-fold. Our data reveal a persistent and evolving disturbance in cytokine and keratan sulfate profiles within the anterior cruciate ligament-deficient knee, suggesting an important biochemical dimension to the development of osteoarthritis there.

CD44 in inflammation and metastasis

Abstract: CD44 is a major cell surface receptor for the glycosaminoglycan, hyaluronan (HA). CD44 binds HA specifically, although certain chondroitin-sulfate containing proteoglycans may also be recognized. CD44 binding of HA is regulated by the cells in which it is expressed. Thus, CD44 expression alone does not correlate with HA binding activity. CD44 is subject to a wide array of post-translational carbohydrate modifications, including N-linked, O-linked and glycosaminoglycan side chain additions. These modifications, which differ in different cell types and cell activation states, can have profound effects on HA binding function and are the main mechanism of regulating CD44 function that has been described to date. Some glycosaminoglycan modifications also affect ligand binding specificity, allowing CD44 to interact with proteins of the extracellular matrix, such as fibronectin and collagen, and to sequester heparin binding growth factors. It is not yet established whether the HA binding function of CD44 is responsible for its proposed involvement in inflammation. It has been shown, however, that CD44/HA interactions can mediate leukocyte rolling on endothelial and tissue substrates and that CD44-mediated recognition of HA can contribute to leukocyte activation. Changes in CD44 expression (mainly up-regulation, occasionally down-regulation, and frequently alteration in the pattern of isoforms expressed) are associated with a wide variety of cancers and the degree to which they spread; however, in other cancers, the CD44 pattern remains unchanged. Increased expression of CD44 is associated with increased binding to HA and increased metastatic potential in some experimental tumor systems; however, in other systems increased HA binding and metastatic potential are not correlated. CD44 may contribute to malignancy through changes in the regulation of HA recognition, the recognition of new ligands and/or other new biological functions of CD44 that remain to be discovered. Abbreviations:aa, amino acid(s); CS, chondroitin sulfate; CSPG, chondroitin sulfate containing proteoglycan; CD44H, ‘hematopoietic’, also called ‘standard’, isoform of CD44 which contains none of the alternatively spliced variant exons; CD44-Rg, CD44 receptor globulin, a secreted chimaeric protein composed of the external domain of the adhesion receptor CD44 and the hinge, CH2 and CH3 regions of human immunoglobulin-G heavy chain; ECM, extracellular matrix; GAG, glycosaminoglycan; HA, hyaluronan; HS, heparan sulfate; KS, keratan sulfate; PB, peripheral blood; PBL, peripheral blood lymphocytes

Insulin-like growth factor binding protein-2 binds to cell surface proteoglycans in the rat brain olfactory bulb.

Abstract: A family of six insulin-like growth factor binding proteins (IGFBPs) bind IGF-I and modulate its biological activity. IGFBPs may bind to macromolecules on the cell surface or pericellular extracellular matrix, and this interaction may modulate their effect on IGF activity. To date, little is known about the specificity of IGFBPs in the regulation of IGF action in the brain. We therefore explored whether IGFBPs were associated with cell membrane or extracellular matrix components in the rat brain. IGF-I binding sites with the characteristics of an IGFBP were found in the olfactory bulb mitral cell layer. This IGFBP was identified as IGFBP-2 by immunoprecipitation of both solubilized membrane preparations and cross-linked 125I-IGF: IGFBP complexes. While binding of IGFBP-2 to cell membranes was unaffected by RGD-containing peptide, it was inhibited by high salt concentration, suggesting interaction with proteoglycans. IGFBP-2 bound in vitro to the glycosaminoglycans chondroitin-4 and -6-sulfate, keratan sulfate, and heparin. IGFBP-2 also bound the proteoglycan aggrecan, an effect reduced by digestion of its glycosaminoglycans. Binding of IGFBP-2 to chondroitin-6-sulfate decreased the binding affinity of IGFBP-2 for IGF-I approximately 3-fold. Finally, an IGFBP-2 antibody coimmunoprecipitated IGFBP-2 and an approximately 200 kDa proteoglycan containing chondroitin-sulfate side chains from the rat olfactory bulb, providing definitive evidence for IGFBP-2 binding to olfactory bulb proteoglycans. These findings indicate that IGFBP-2 binds to proteoglycans in cell membranes of the rat olfactory bulb. Because we have previously shown that IGFs are highly expressed in the rat olfactory bulb, cell associated IGFBP-2 may have an important role in directing IGFs to specific sites in this brain region.

Proteoglycans in the human sclera. Evidence for the presence of aggrecan.

Abstract: PURPOSE: The proteoglycans synthesized and accumulated within the adult human sclera (aged 50 to 80 years) were identified by their size, glycosaminoglycan side chains, and core proteins in an effort to characterize the proteoglycan content of the human sclera. METHODS: Sclerae, unlabeled, or radiolabeled in organ culture with 35SO4 or 3H-proline, were extracted in 4M guanidine-HCl and separated by Sepharose CL-2B and Superose 6 forced-pressure liquid chromatography. Peak fractions, identified by glycosaminoglycan content or radioactivity, were pooled and subjected to G-50 chromatography or sodium dodecyl sulfate-polyacrylamide gel electrophoresis before and after digestion with specific glycosidases. Scleral proteoglycan core proteins were identified in Western blot analysis using specific antisera to decorin, biglycan, and aggrecan. Reverse transcription-polymerase chain reaction analyses were carried out on human scleral fibroblast RNA to confirm the transcription of one scleral proteoglycan. Proteoglycans were localized on sections of scleral tissue using specific antisera. RESULTS: After chromatography on CL-2B, scleral proteoglycans could be resolved into three major peaks, PG-1, PG-2, and PG-3. The largest scleral proteoglycan, PG-1, contained chondroitin sulfate and keratan sulfate glycosaminoglycan side chains. Results of Western blot analyses indicated that the core protein of PG-1 is the aggrecan core protein, migrating at approximately 350 kDa. Reverse transcription-polymerase chain reaction analyses confirmed that human scleral fibroblasts transcribe aggrecan in vitro and in vivo. PG-2 and PG-3 were identified as biglycan and decorin in Western blot analyses using antibiglycan and antidecorin antibodies, respectively. Immunostaining results indicated that aggrecan, biglycan, and decorin are distributed throughout the thickness of the human sclera. CONCLUSIONS: The adult human sclera contains three major proteoglycans; aggrecan, biglycan, and decorin. It is likely that these proteoglycans contribute to the structural properties of the sclera and that the ratios of these proteoglycans will change with age, specific region, and condition of the sclera.

Macrophage receptors for lumican. A corneal keratan sulfate proteoglycan.

Abstract: Purpose Keratan sulfate proteoglycans (KSPGs) of the cornea exhibit a characteristic change in glycosylation resulting from stromal inflammation and scarring. To examine potential roles for these molecules in the pathobiology of the cornea, the authors investigated interaction of inflammatory macrophages with KSPGs in vitro. Methods Attachment and spreading of mouse peritoneal macrophages were examined on surfaces coated with corneal proteoglycans, intact or with modified glycosylation. Solution-phase interactions were demonstrated using soluble proteoglycans labeled with 125I-Iodine or with fluorescein. The affinity and specificity of these interactions were determined by competitive inhibition with unlabeled proteoglycans. Results Macrophages did not adhere to intact corneal KSPGs but did attach and spread rapidly on the lumican core protein after the removal of keratan sulfate chains. Arterial lumican, a nonsulfated form of this proteoglycan, also stimulated macrophage attachment. Labeled arterial lumican specifically bound to macrophages with high affinity. Flow cytometry demonstrated a high proportion of macrophages binding lumican. Lumican binding was inhibited by divalent cation-chelators and by polyanions. Inhibition and kinetics of lumican binding were distinct from interaction of macrophages with maleated bovine serum albumin, collagen, laminin, and fibronectin. Conclusions The highly sulfated KSPGs of cornea do not promote macrophage adhesion; however, the low-sulfate lumican present in pathologic corneas may act to localize macrophages in regions of inflammation. The lumican receptor differs from macrophage scavenger receptors and from receptors for several other extracellular matrix molecules.

Cartilage and bone morphology in osteochondritis dissecans

Abstract: Osteochondritis dissecans (OD) is a syndrome that can be characterized as a non-infectious disturbance of enchondral ossification or as a post-traumatic event. OD occurs in the joint cartilage and physis of long bones, as well as in the talus or metacarpal head especially of young athletes. The medial femoral condyle is the most commonly affected site. The causes of osteochondritis dissecans are poorly understood. Thirty human osteochondral cylinders from patients (aged 16-44 years) with osteochondritis dissecans of the medial femoral condyle (grades IV and V according to Rodegerdts and Gleissner) were harvested intraoperatively from osteochondritic areas as part of a cartilage-bone transplantation. Light microscopy, electron microscopy, and immunohistochemistry using poly- and monoclonal antibodies against collagens and glycosaminoglycans revealed differences between osteochondrotic and normal cartilage. Staining with toluidine blue at pH 1 shows a decrease in acidic glycosaminoglycans in OD. Modified pentachrome staining showed a thinned subchondral growth plate compared to normal osteochondral samples. Borders to the healthy tissue are clearly visible micro- and macroscopically. Scanning electron microscopy revealed structural differences in the subchondral area. Immunohistochemistry found a general decrease in glycosaminoglycan content and a change in composition. Only faint staining for chondroitin and keratan sulfates was observed in osteochondritic cartilage, whereas increased staining was shown for keratan sulfate in bone.

Sulfation of sialyl lactosamine oligosaccharides by chondroitin 6-sulfotransferase

Abstract: We have previously shown that chondroitin 6-sulfotransferase (C6ST) catalyzes transfer of sulfate not only to position 6 of GalNAc residue of chondroitin but also to position 6 of Gal residue of keratan sulfate. In this study, we examined the sulfation of sialyl lactosamine oligosaccharides by C6ST. C6ST catalyzed transfer of sulfate to NeuAc alpha 2-3Gal beta 1-4GlcNAc (SLN), NeuAc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc (SL1L1), NeuAc alpha 2-3Gal beta 1-4(6-sulfo)GlcNAc beta 1-3(6-sulfo)Gal beta 1-4(6-sulfo)GlcNAc (SL2L4), and their desialylated derivatives, but not to NeuAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc (SLe(x)). The sulfated product formed from SLN was degraded with neuraminidase and reduced with NaBH4. The resulting sulfated disaccharide alditol showed the same retention time in SAX-HPLC as that of [3H]Gal(6SO4) beta 1-4GlcNAc-ol. The sulfated product formed from SLN was also degraded by a reaction sequence of neuraminidase digestion, hydrazinolysis, deamination, and NaBH4 reduction. The final product was coeluted with [3H]Gal(6SO4) beta 1-4anhydromannitol in SAX-HPLC. These observations show that C6ST could transfer sulfate to position 6 of Gal residue of SLN. Incorporation of sulfate into SL2L4 was much higher than the incorporation into SL1L1, suggesting that sulfate moiety attached to adjacent GlcNAc residue may stimulate the transfer of sulfate to Gal residue. The recombinant C6ST also catalyzed sulfation of the sialyl lactosamine oligosaccharides, indicating that a single protein catalyzes sulfation of chondroitin, keratan sulfate, and sialyl lactosamine oligosaccharides. These results raised a possibility that C6ST may be one of the candidates involved in the biosynthesis of sulfated sialyl Lewis x ligand for L-selectin.

Concentrations of keratan sulfate in plasma and synovial fluid from clinically normal horses and horses with joint disease.

Abstract: OBJECTIVE To determine whether keratan sulfate concentrations in plasma or synovial fluid from clinically normal horses were different from concentrations in horses with joint disease and whether concentrations varied with type of joint disease. DESIGN Case-control study. ANIMALS 67 clinically normal horses, 10 clinically normal foals, and 160 horses with joint disease. PROCEDURE ELISA was used to measure keratan sulfate concentrations. RESULTS Mean plasma keratan sulfate concentration (mean +/- SEM, 580 +/- 124 ng/ml) in foals peaked at 10 weeks of age. Mean plasma keratan sulfate concentration in clinically normal horses was 200 ng/ml (95% confidence interval, 157 to 251 ng/ml). Horses with osteochondral (chip) fractures, other closed intraarticular fractures, inflammatory arthritis (synovitis), infectious arthritis, or osteochondrosis had significantly higher plasma keratan sulfate concentrations than did clinically normal horses, but horses with osteoarthritis did not. Breed, gender, and type of joint disease affected keratan sulfate concentration in synovial fluid. Standard-breds with chip fractures of the metacarpophalangeal/ metatarsophalangeal joints had significantly higher keratan sulfate concentrations in synovial fluid than did Thoroughbreds. Keratan sulfate concentrations in synovial fluid from osteoarthritic carpal joints were lower than concentrations in normal carpal joints and tarsocrural joints with inflammatory joint disease. CLINICAL IMPLICATIONS Keratan sulfate concentration alone was not a specific marker of joint disease but was affected by various joint diseases.

Biochemical, histochemical, and immunohistochemical characterization of distal tibial osteochondrosis in horses.

Abstract: OBJECTIVE To compare the biochemical, histochemical, and immunohistochemical profiles of articular cartilage from horses with naturally acquired distal tibial osteochondrosis (OC) with cartilage from a similar location in clinically normal horses. ANIMALS 9 affected horses (group 1, 16 OC lesions) and 4 control horses (group 2, 8 normal osteochondral specimens). PROCEDURE OC specimens were collected during arthroscopic removal of the fragment, and control specimens were collected by aseptic osteotomy. Uronic acid, total protein, total glycosaminoglycan (GAG), chondroitin sulfate (CS), and keratan sulfate (KS) contents were determined. Histomorphologic, histochemical, and immunohistochemical examinations were performed on specimens after snap freezing at -80 C and cryosectioning. Monoclonal antibodies (MAB) 3B3 and 5D4 were applied for location of epitopes of CS and KS, respectively. RESULTS OC lesions had significantly lower quantity of uronic acid, total GAG, and CS, compared with normal cartilage. OC cartilage had significantly less intense staining with toluidine blue, along with irregular cellularity and tidemark characteristics, compared with normal cartilage. Monoclonal antibodies 3B3 and 5D4 stained OC cartilage, whereas MAB 5D4 did not stain control cartilage. Additionally, MAB 3B3 and 5D4 stained the fibrous tissue that was found firmly attached to the OC lesion located between the parent distal portion of the tibia and OC fragment. CONCLUSION OC cartilage lesions of the distal intermediate ridge of the tibia in horses are biochemically, histochemically, and immunohistochemically distinct from normal cartilage from the same location. Results may reflect the inability of the chondrocyte of the developing joint to alter matrix components that would allow proper maturation and differentiation into bone.

Structural changes in the large proteoglycan, aggrecan, in different zones of the ovine growth plate

Abstract: The large cartilage proteoglycan, aggrecan, was found to vary throughout the ovine physis corresponding to the maturational state of the resident chondrocytes. Two populations of proteoglycan monomer were observed in articular, epiphyseal, and in the resting zone of growth plate cartilage. These proteoglycans contained chondroitin sulfate glycosaminoglycan chains sulfated predominantly in the 4 position along with lesser amounts of chondroitin-6-sulfate and keratan sulfate. In the proliferative zone of the growth plate, chondrocytes synthesize one population of proteoglycan monomer which was significantly larger than monomer populations in articular, epiphyseal, or resting zone and this size increase could be attributed to an increase in its constituent chondroitin sulfate side chains. As these chondrocytes progress through their life cycle they continue to modify the structural characteristics of the aggrecan molecule they synthesize. Thus, in the hypertrophic region of the growth plate, the proteoglycan monomer is larger again than in the proliferative region. Variation in sulfation pattern on aggrecan chondroitin sulfate side chains is also observed in the hypertrophic region with an increasing proportion of unsulfated residues present, which may play a role in the initiation of mineralization. In addition, increasing amounts of the carbohydrate sequence recognized by monoclonal antibody 7-D-4 are observed in the hypertrophic zone.

Identification, Partial Characterization, and Distribution of Versican and Link Protein in Bovine Dental Pulp

Abstract: The dynamics of changes in the cellularity and extracellular matrix composition of dental pulp varies considerably during tooth development and maturation. In this paper, we studied matrix proteoglycans where we hypothesized that they played important roles in structural, spatial, and transport aspects of pulpal development and maintenance. The pulpal tissue was collected from partially erupted bovine incisors, pulverized, and then extracted with 6 M guanidine-HCl. The extract was subjected to anion column chromatography (DEAE-8HR), and the fractions collected were screened by dot-blot immunoassay by means of monoclonal antibodies generated against 4- and 6-sulfated chondroitin sulfate isomers, and keratan sulfate, 2-B-6, 3-B-3, and 5-D-4, respectively. The chondroitin-6-sulfate was the major glycosaminoglycan species and occurred as a large-molecular-weight proteoglycan (> 500 kDa). After further purification, it was subjected to agarose/acrylamide composite gel electrophoresis, and it migrated as a single band stained with Stains-All. The band was immunopositive against antibody 3-B-3 by Western blot analysis. The partial amino acid sequence analyses of the core protein clearly indicated this molecule to be versican. The presence of link protein was also confirmed by Western blot analysis with an anti-link protein monoclonal antibody, 8-A-4. Furthermore, immunohistochemical study indicated that the distributions of versican and link protein coincide in the dental pulp and are enriched in the peripheral area of the tissue just beneath the odontoblast layer. Since the dental pulp contains hyaluronan, versican may bind to hyaluronan via its hyaluronan-binding domain, where this association is stabilized by link protein. This complex, then, could form large hydrated proteoglycan aggregates that fill the extracellular space, support odontoblasts, and/or facilitate the transport function of metabolites and nutrients within the tissue.

Macular corneal dystrophy type II: multiple studies on a cornea with low levels of sulphated keratan sulphate.

Abstract: We investigated an individual macular corneal dystrophy (MCD) type II cornea from a 42-year-old woman with markedly reduced antigenic keratan sulphate levels. A characteristic 4.6 A X-ray reflection was evident, and the mid-stroma contained 30% less sulphur than normal. Close packing of collagen was restricted to the superficial stroma. Abnormally large proteoglycan filaments were noted throughout the extracellular matrix and Descemet's membrane's posterior non-banded zone, but not its anterior banded zone. Small, collagen-associated stromal proteoglycans were susceptible to digestion with chondroitinase ABC, but not keratanase I or N-glycanase. On occasion, collagen fibrils ranged in size from 20 nm to 58 nm, with preferential diameters of 34 nm and 42 nm. Corneal guttae were evident, as were numerous endothelial inclusions, most probably due to intracellular fibrillogranular vacuoles similar to those found in the stroma. The endothelium expressed reduced anti-keratan sulphate labelling.

Glycosaminoglycans regulate elastase inhibition by oxidized secretory leukoprotease inhibitor.

Abstract: Secretory leukoprotease inhibitor (SLPI) is one of the major physiological inhibitors protecting respiratory epithelium from attack by excess human leukocyte elastase (HLE), a serine protease released by neutrophils upon activation in response to inflammatory stimuli. Reaction with N-chlorotaurine, a major long-lived oxidant generated by activated neutrophils, oxidized all four methionine residues, but no other amino acids, in SLPI, resulting in substantial diminution of its elastase inhibitory activity. Oxidation of the P 1 ' residue, Met 73 , accounted for most of the diminution in activity since a site-directed mutant of SLPI with leucine at the P 1 ' position retained much higher residual activity after reaction with N-chlorotaurine. The diminished activity of oxidized SLPI could be almost completely restored when an iduronate-containing glycosaminoglycan, such as heparin, heparan sulfate, or dermatan sulfate, was added to the reaction medium. Addition of sulfated glucuronate-containing glycosaminoglycan, chondroitin 4- or 6-sulfate, to the medium resulted in smaller but significant restoration of the lost activity, whereas the effects of hyaluronic acid and keratan sulfate were negligible. Kinetic analysis revealed that glycosaminoglycans greatly accelerated the association of oxidized SLPI and HLE, whereas iduronate-containing glycosaminoglycans also stabilized the enzyme-inhibitor complex formed. Based on these findings, we suggest that oxidized SLPI is a functionally active form of the inhibitor but that expression of its elastase inhibitory activity is regulated by sulfated uronate-containing glycosaminoglycans. Because its methionine residues have already been oxidized, this form of SLPI is resistant to the oxidant species that selectively attacks methionine residues in proteins. These findings indicate that SLPI may play a previously unexpected role in elastase inhibitory function in the lungs when significant inflammation is present.

Serum Sulfotransferase Levels in Patients With Macular Corneal Dystrophy Type I

Abstract: Objective: To measure the levels of sulfotransferase activity for keratan sulfate and chondroitin sulfate in serum of patients with macular corneal dystrophy type I, an inherited disorder that is characterized by the absence of sulfate esters on keratan sulfate in the corneal stroma. Methods: The amount of sulfur-35 transferred from 3′-phosphoadenosine 5′-phosphosulfate to partially sulfated keratan sulfate and partially sulfated chondroitin sulfate by the sulfotransferase present in serum from patients with macular corneal dystrophy and agematched controls was determined under conditions where only the added enzyme was rate limiting. Results: Serum from patients with macular corneal dystrophy type I has the same level of sulfotransferase activity for keratan sulfate and chondroitin sulfate as found in age-matched controls. Conclusions: Patients with macular corneal dystrophy type I have sulfotransferase activity for sulfating at least 1 of the 2 sugars in keratan sulfate. It is proposed that the sulfotransferase for N -acetylglucosamine may be deficient.

Polypeptide of glycosaminoglycan sulfotransferase originating from human and DNA coding for the polypeptide

Abstract: A polypeptide of glycosaminoglycan sulfotransferase originating from human and having the following physical and chemical properties: (i) action: sulfate group is transferred from a sulfate group donor to N-acetylgalactosamine residue or galactose residue of glycosaminoglycan; (ii) substrate specificity: sulfate group is transferred to the hydroxyl group position at C-6 of N-acetylgalactosamine residue of chondroitin; and sulfate group is transferred to the hydroxyl group position at C-6 of galactose residue of keratan sulfate; and (iii) molecular weight: about from 50,000 to 55,000 Da.