Showing papers on "Keratan sulfate published in 1984"
TL;DR: The time dependence of HS degradation into particular Mr fragments indicated that HS was cleaved at specific intrachain sites, and 3H-reduced terminal monosaccharides from HS fragments were overwhelmingly L-gulonic acid, the HS-degrading enzyme responsible is an endoglucuronidase (heparanase).
265 citations
Journal Article•
TL;DR: The results of these studies indicate that SH is more effective than TH in decreasing aqueous outflow resistance and that hyaluronic acid is an important GAG contributor to aqueously out flow resistance in the normal rabbit eye.
Abstract: The infusion of two enzymes that degrade hyaluronic acid--testicular hyaluronidase and Streptomyces hyaluronidase--was evaluated by quantitative aqueous perfusion of rabbit eyes and by analyses of glycosaminoglycans (GAGs) isolated from the enzyme-treated eyes. The infusion of 1 and 10 units of Streptomyces hyaluronidase (SH) was considerably more effective than the infusion of 10 and 100 units of testicular hyaluronidase (TH) in reducing aqueous outflow resistance. As a result of the infusion of heat-inactivated enzymes, only a moderate decrease of hyaluronic acid in the aqueous outflow pathway was observed. There was no significant "wash-out" of other GAG material, ie, keratan sulfate, heparan sulfate, and hybrid dermatan sulfate-chondroitin sulfate. The SH enzyme, tested by infusion and isolation of GAGs or by in vitro analyses of the rate and extent of degradation of GAGs, completely removed all hyaluronic acid and did not alter the other GAGs. In contrast, the TH enzyme was only partially effective in degrading susceptible GAGs. The results of these studies indicate that SH is more effective than TH in decreasing aqueous outflow resistance and that hyaluronic acid is an important GAG contributor to aqueous outflow resistance in the normal rabbit eye.
100 citations
TL;DR: Observations indicate that macular corneal dystrophy is caused by an error in the synthesis of keratan sulfate, possibly involving the specific sulfotransferases involved in sulfation of the lactosaminoglycan backbone of the chains.
98 citations
TL;DR: Proteoglycan synthesis can be described as occurring in two stages, translation and N‐glycosylation of a coreprotein precursor which has a long half‐life in the rough endoplasmic reticulum, followed by extensive rapid modification in the Golgi complex in which the majority of glycosaminoglycan and oligosaccharide chains are added to the core protein precursor with subsequent rapid secretion into the extracellular matrix.
Abstract: Biosynthesis of cartilage proteoglycan was examined in a model system of cultured chondrocytes from a transplantable rat chondrosarcoma. Extensive modification with the addition of chondroitin sulfate glycosaminoglycan, N-linkcd oligosac-charide, and O-linked oliogosaccharide is required to convert a newly synthesized core protein precursor into a proteoglycan. Kinetic analyses revealed the presence of a large pool of core protein precursor (t1/2 ∼ 90 min) awaiting completion into proteoglycan. The large t1/2 of this pool allowed kinetic labeling experiments with a variety of radioactive precursors to distinguish between early biosynthetic events associated primarily with the rough endoplasmic reticulum from late events associated primarily with the Golgi apparatus. The results of a series of experiments indicated that the addition of N-linked oligosaccharide chains occurs early in the biosynthetic process in association with the rough endoplasmic reticulum, whereas the initiation and completion of O-linked oligosaccharides occurs much later, at about the same time as chondroitin sulfate synthesis. This also indicated that keratan sulfate chains, when present in the completed molecule, are added in the Golgi apparatus, as they are probably built on oligosaccharide primers closely related to the O-oligosaccharide chains. Furthermore, when 3H-glucose was used as the precursor, the entry of label into xylose, the linkage sugar between the core protein and the chondroitin sulfate chain, was found to occur within 5 min of the entry of label into galactose and galactosamine in the remainder of the chondroitin sulfate chain. This indicated that the initiation and completion of the chondroitin sulfate chain occurs late in the pathway probably entirely in the Golgi apparatus. Thus, proteoglycan synthesis can be described as occurring in two stages in this system, translation and N-glycosylation of a core protein precursor which has a long half-life in the rough endoplasmic reticulum, followed by extensive rapid modification in the Golgi complex in which the majority of glycosaminoglycan and oligosaccharide chains are added to the core protein precursor with subsequent rapid secretion into the extracellular matrix.
81 citations
TL;DR: Heterogeneity of keratan sulfate synthesis at the level of individual chondrocytes, even for cells apparently containing equivalent amounts of intracellular chondroitin sulfate proteoglycan, is suggested.
Abstract: Several monoclonal antibodies which recognize different antigenic determinants of chondroitin sulfate proteoglycan were used to study chondroitin sulfate proteoglycan biosynthesis in chicken chondrocyte cultures. The intracellular sites of synthesis and processing and extracellular deposition in matrix were localized by double immunofluorescence reactions. One rat monoclonal antibody, S103L, which recognizes an antigenic determinant of the core protein of the chicken cartilage chondroitin sulfate proteoglycan monomer, was used to identify both extracellular chondroitin sulfate proteoglycan and intracellular compartments containing chondroitin sulfate proteoglycan precursors. Intracellular staining with S103L was localized to perinuclear regions, and, in some chondrocytes, to a few other cytoplasmic vesicles as well. When chondrocytes were not fed for several days, intracellular chondroitin sulfate proteoglycan precursors were accumulated in larger compartments distributed throughout the cytoplasm. Polyclonal chondroitin sulfate proteoglycan antibodies displayed similar staining characteristics. In contrast, several of the monoclonal antibodies, including the rat monoclonals S11D and P100D, and the mouse monoclonals 1-B-5, 3-B-3 and 9-A-2, did not recognize native chondroitin sulfate proteoglycan, but reacted only with chondroitinase ABC-digested (and/or hyaluronidase-digested) chondroitin sulfate proteoglycan. These antibodies were particularly useful in the demonstration of the extracellular codistribution of chondroitin sulfate proteoglycan with either type II collagen or fibronectin. In other experiments, the monoclonal antibodies to chondroitin sulfate proteoglycan served to demonstrate that the perinuclear subset of intracellular compartments is uniquely involved in the addition of chondroitin sulfate oligosaccharides to the chondroitin sulfate proteoglycan core protein. Lastly, using the mouse monoclonal 5-D-4, which recognizes keratan sulfate determinants, the perinuclear region was identified as the site for keratan sulfate addition. Results suggest heterogeneity of keratan sulfate synthesis at the level of individual chondrocytes, even for cells apparently containing equivalent amounts of intracellular chondroitin sulfate proteoglycan.
28 citations
TL;DR: From the specific radioactivities of the oligosaccharides produced by digestion with endo-beta-galactosidase, it was concluded that both enzyme species reacted best with keratan sulfate segments exhibiting a relatively high degree of sulfation.
26 citations
TL;DR: Culture age and cell density appears primarily to modulate the synthesis of glycosaminoglycan types and chain length and indicates that rapidly dividing chondrocytes were capable of synthesizing proteoglycans which form aggregates with hyaluronic acid.
Abstract: The effect of cell culture age and concomitant changes in cell density on the biosynthesis of sulfated-proteoglycan by rabbit articular chondrocytes in secondary monolayer culture was studied. Low density (LD, 2 d), middle density (MD, 5-7 d), and high density (HD, 12-15 d) cultures demonstrated changes in cellular morphology and rates of DNA synthesis. DNA synthesis was highest at LD to MD densities, but HD cultures continued to incorporate [3H]-thymidine. LD cultures incorporated 35SO4 into sulfated-proteoglycans at a higher rate than MD or LD cultures. The qualitative nature of the sulfated-proteoglycans synthesized at the different culture ages were analyzed by assessing the distribution of incorporated 35SO4 in associative and dissociative CsCI density gradients and by elution profiles on Sepharose CL-2B. Chondrocytes deposited into the extracellular matrix (cell-associated fraction) 35SO4-labeled proteoglycan aggregate. More aggregated proteoglycan was found in the MD and HD cultures than at LD. A 35SO4-labeled aggregated proteoglycan of smaller hydrodynamic size than that found in the cell-associated fraction was secreted into the culture medium at each culture age. The proteoglycan monomer (A1D1) of young and older cultures had similar hydrodynamic sizes at all cell culture ages and cell densities. The glycosaminoglycan chains of A1D1 were hydrodynamically larger in the younger LD cultures than in the older HD cultures and consisted of only chondroitin 6 and 4 sulfate chains. A small amount of chondroitin 4,6 sulfate was detected, but no keratan sulfate was measured. The A1D2 fractions of young LD cultures contained measurable amounts of dermatan sulfate; no dermatan sulfate was found in older MD or HD cultures. These studies indicated that chondrocytes at LD synthesized a proteoglycan monomer with many of the characteristics of young immature articular cartilage of rabbits. These results also indicated that rapidly dividing chondrocytes were capable of synthesizing proteoglycans which form aggregates with hyaluronic acid. Culture age and cell density appears primarily to modulate the synthesis of glycosaminoglycan types and chain length. Whether or not these glycosaminoglycans are found on the same or different core proteins remains to be determined.
21 citations
Patent•
27 Mar 1984TL;DR: In this article, it was found that abnormal levels of keratan sulfate in the peripheral blood are indicative of abnormalities of cartilage or cartilage-like tissues, and that either substantially complete absence of or very elevated levels of karatansulfate in peripheral blood is indicative of muscular dystrophy and related disorders.
Abstract: It is found that abnormal levels of keratan sulfate in the peripheral blood are indicative of abnormalities of cartilage or cartilage-like tissues. Specifically, elevated levels of keratan sulfate in the peripheral blood plasma or serum are indicative of osteoarthritis and either substantially complete absence of or very elevated levels of keratan sulfate in the peripheral blood are indicative of muscular dystrophy and related disorders. The level of keratan sulfate in the peripheral blood is determined by an immunoassay using a monoclonal antibody. Preferably the immunoassay employs a colorimetric reporter system.
18 citations
TL;DR: The hyalin material in massive cutaneous hyalinosis, a disease characterized by extensive tumorous periodic acid-Schiff-(PAS) positive extracellular cutaneous deposits, has been elucidated by biochemical and immunologic methods.
Abstract: The hyalin material in massive cutaneous hyalinosis, a disease characterized by extensive tumorous periodic acid-Schiff-(PAS) positive extracellular cutaneous deposits, has been elucidated by biochemical and immunologic methods. Three major components were found: kappa light chains, a mannose-rich glycoprotein, and type I collagen. Trace amounts of fibrinogen, fibronectin, laminin, IgG, pregnancy-specific glycoprotein, albumin, and keratan sulfate, but not keratin, were also present. The kappa light chains were monoclonal, cryoprecipiting, and more basic than the kappa chains from two myeloma patients. The glycoprotein, which could not be identified as any known glycoprotein, had an apparent molecular weight of 90,000 D. Amino acid analysis showed that glutamic acid, aspartic acid, leucine, and threonine were abundant, whereas hydroxyproline, hydroxylysine, and sulfhydryl amino acids were absent. The carbohydrate content of the protein was approximately 20%. The major monosaccharides were mannose and N-acetylglu-cosamine. Galactose, N-acetylneuraminic acid and fucose also were present. The third major component of the hyalin material was identified as type I collagen. A humoral immune response to the storage material was found: the patient’s serum contained IgM and IgG class antibodies against the mannosylglycoprotein (90 kD glycoprotein) and against type I collagen.
18 citations
TL;DR: Cartilage proteoglycans decreased in hydrodynamic size between 12 and 22 weeks, corresponding to the period of maximal change in content of keratan and chondroitin sulfate.
15 citations
Journal Article•
TL;DR: The control of glycosaminoglycan biosynthesis was investigated by studying the kinetic and regulatory properties of some enzymes involved in the formation of UDP-sugar precursors, which appeared to be regulated by the value of the NAD/NADH ratio.
Abstract: The control of glycosaminoglycan biosynthesis was investigated by studying the kinetic and regulatory properties of some enzymes involved in the formation of UDP-sugar precursors: UDP-N-acetylglucosamine 4'-epimerase, catalyzing the interconversion of hexosamine precursors and UDP-glucose dehydrogenase and UDP-glucose 4'-epimerase, utilizing UDP-glucose for the formation of uronic acid and galactose precursors. The study was carried out in tissues with different glycosaminoglycan production: bovine cornea, producing both chondroitin sulfate and keratan sulfate, and newborn-pig epiphysial-plate cartilage, producing mostly chondroitin sulfate. The biosynthesis of hexosamine precursors appeared to be regulated by the value of the NAD/NADH ratio. This control mechanism regulated also the activities of both UDP-glucose dehydrogenase and UDP-glucose 4'-epimerase and, therefore, it could correlate the biosynthesis of glycosaminoglycan precursors with the redox activity of the cell. At the level of UDP-glucose utilization two other control mechanisms were demonstrated: the different affinities of UDP-glucose dehydrogenase and UDP-glucose 4'-epimerase for UDP-glucose in tissues with different glycosaminoglycan production and the cellular concentration of UDP-xylose. This sugar-nucleotide inhibited UDP-glucose dehydrogenase, but did not affect the UDP-glucose 4'-epimerase activity; therefore, and increase of its cellular concentration may result in a decreased chondroitin sulfate synthesis and in an increased keratan sulfate formation.
01 Jan 1984
TL;DR: Proteoglycans constitute a major macro-molecular component in the extracellular matrix of connective tissues and have also been identified as components of basement membranes and cell surface coats.
Abstract: In addition to the fibrillar proteins collagen and elastin, proteoglycans constitute a major macro-molecular component in the extracellular matrix of connective tissues. They have also been identified as components of basement membranes and cell surface coats. Chemically, the proteoglycans consist of a protein core to which glycosaminoglycan chains are covalently attached. The latter are composed of alternating uronic acid and hexosamine residues and are all polyanions with acidic sulfate and/or carboxyl groups (1). A particularly high content of proteoglycans is found in cartilage and much of the information that is available today derives from studies on this tissue. An average cartilage proteoglycan has a molecular weight of about 2.5 af09106 and is composed of a protein core to which about 100 chon-droitin sulfate and 50–60 keratan sulfate chains are bound. Within the cartilaginous matrix, most of these monomers occur in large aggregates, formed by noncovalent interaction with hyaluronic acid and link proteins (2–4). The structure of proteoglycans in other connective tissues, in basement membranes, and in cell surface coats is less well known. The type, number, and size of glycosaminoglycan chains per molecule have been found to vary considerably, but the supramolecular organization (e.g. aggregate formation) is still poorly defined.
TL;DR: Based on these observations, isolation of heparan sulfate from pig lung crude heparin sulfate fractions which contain chondroitin sulfates and dermatan sulfates was attempted, using this chromatographic technique.
TL;DR: Keratan sulfate was isolated from the skin of Pacific mackerel (Scomber japonicus) after exhaustive digestion with pronase followed by ethanol precipitation and fractionation on a cellulose column with 0.3% recovery of dried material.
TL;DR: The findings suggest that the synthesis and/or degradation of the various types of glycosaminoglycan chains (chondroitin sulfate and keratan sulfate) of cartilage proteoglycan can be regulated differentially by serum growth factors.
TL;DR: The results support that the linkage region of cornea proteokeratan sulfate is synthesized via the assembly of a glucosylated dolichyl pyrophosphoryl oligosaccharide, its transfer to protein and subsequent processing by glycosidases.
TL;DR: The heterogeneities observed in the composition of the KS and CS chains, and their occurrence in relatively distinct regions of the protein core(s) indicate, however, that there is still much to be learned about the structure of these complex macromolecules.
01 Jan 1984
TL;DR: Proteoglycans are thus complex glycoproteins and the information available concerning their biosynthesis suggests that they share the same pathways as other secreted glyCoproteins.
Abstract: Proteoglycans are complex macromolecules in which many long carbohydrate chains are linked to a protein backbone. The main carbohydrate chains form a family of related structures, the glycosaminoglycans. These are relatively long, unbranched polysaccharide chains composed of repeating disaccharide units. Each disaccharide carries sulfate and/or carboxyl residues. Seven basic glycosaminoglycan types (Table 1) occur in mammalian tissues (see Muir and Hardingham, 1975). With the exception of hyaluronate, they have all been reported to occur linked to protein. The linkage to protein involves a neutral trisaccharide (gal-gal-xyl) at the reducing end of the glycosaminoglycan chains, with the xylose forming an O-glycosidic linkage with a serine residue of the protein. Keratan sulfate is linked differently. In skeletal keratan sulfate it is linked via an O-glycosidic linkage of N-acetylgalactosamine to serine or threonine. In corneal keratan sulfate it is linked via an N-glycosylamine linkage from N-acetylgalactosamine to as-paragine. Proteoglycans are thus complex glycoproteins and the information available concerning their biosynthesis suggests that they share the same pathways as other secreted glycoproteins.
TL;DR: Glycosaminoglycans complex with constituents of normal human serum, a finding that was exploited to develop a competitive binding assay for heparin polysaccharides in chondroitinase ABC-treated samples and is sensitive to microgram quantities.
TL;DR: It is concluded that teleost skin keratan sulfate is bound to protein via an N -glycosyl linkage between N- acetyl- D -glucosamine and asparagine and the core region apparently consist of trimannosyl- N and N ′-diacetylchitobiose units.
01 Jan 1984
TL;DR: The cartilage proteoglycan is especially designed for resisting compression and deformation by virtue of its aggregation with hyaluronate to form large, highly expanded supramolecular aggregates.
Abstract: Connective tissue is composed of scattered cells embedded in an extracellular matrix consisting of abundant collagen fibers, elastin, and an amorphous ground substance dominated by proteoglycans. Proteoglycans contain a central protein core that is substituted with glycosaminoglycans. With the possible exception of hyaluronate all of the known glycosaminoglycans (Table 1) occur as proteoglycans. The various proteoglycans of the interfibrillar space are considered to control the architecture of the fibrillar network (Muir and Hardingham, 1975). A general observation is that the type of proteoglycan found in a tissue varies with the biomechanical properties of the tissue. The cartilage proteoglycan is especially designed for resisting compression and deformation by virtue of its aggregation with hyaluronate (carbohydrate—protein interaction) to form large, highly expanded supramolecular aggregates (Muir and Hardingham, 1975).
TL;DR: Cultured chick embryonal retinal pigment epithelial (RPE) cells synthesized two main glycosaminoglycans, chondroitin 4-sulfate, and heparan sulfate, which were liberated mainly in the medium and incorporated into the cell and/or basement membrane.
Abstract: Cultured chick embryonal retinal pigment epithelial (RPE) cells synthesized two main glycosaminoglycans (GAGs), chondroitin 4-sulfate, and heparan sulfate. Chondroitin 4-sulfate was liberated mainly in the medium (apical side) and heparan sulfate was incorporated into the cell and/or basement membrane. Glycosaminoglycans liberated into the medium were composed of chondroitin 4-sulfate (44%), keratan sulfate (22%), heparan sulfate (18%) and hyaluronate (12%). On the other hand, the GAGs in the cell and/or basement membrane were composed of heparan sulfate (52%), hyaluronate (19%), and chondroitin 4-sulfate (13%).