Showing papers on "Keratan sulfate published in 1993"

The SV2 protein of synaptic vesicles is a keratan sulfate proteoglycan.

Abstract: We have determined that synaptic vesicles contain a vesicle-specific keratan sulfate integral membrane proteoglycan. This is a major proteoglycan in electric organ synaptic vesicles. It exists in two forms on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, i.e., the L form, which migrates like a protein with an M(r) of 100,000, and the H form, with a lower mobility that migrates with an M(r) of approximately 250,000. Both forms contain SV2, an epitope located on the cytoplasmic side of the vesicle membrane. In addition to electric organ, we have analyzed the SV2 proteoglycan in vesicle fractions from two other sources, electric fish brain and rat brain. Both the H and L forms of SV2 are present in these vesicles and all are keratan sulfate proteoglycans. Unlike previously studied synaptic vesicle proteins, this proteoglycan contains a marker specific for a single group of neurons. This marker is an antigenically unique keratan sulfate side chain that is specific for the cells innervating the electric organ; it is not found on the synaptic vesicle keratan sulfate proteoglycan in other neurons of the electric fish brain.

Tissue variation of two large chondroitin sulfate proteoglycans (PG-M/versican and PG-H/aggrecan) in chick embryos

Abstract: PG-M and PG-H, chick large chondroitin sulfate proteoglycans corresponding to versican (fibroblasttype proteoglycan) and aggrecan (cartilage-characteristic proteoglycan), respectively, which are found in mammals, have been characterized in various tissues of chick embryos. Their distribution and the compositions of the core molecules were analyzed by immunofluorescence staining and immunoblotting, respectively, using various monospecific antibodies. Molecules reactive to a monoclonal antibody to the PG-M core protein (designated MY-174) were distributed in various tissues, including aorta, lung, cornea, brain, skeletal muscle and dermis. Immunoblotting with MY-174 of the chondroitinase ABC-digested tissue extracts showed a tissue variation of MY-174-reactive core molecules (550-kD, 500-kD, 450kD, and 350-300-kD). In contrast, PG-H, besides massive occurrence in cartilage, was only found in a few tissues such as aorta and brain. In addition, PG-H in aorta, cornea, and skin was atypical in structure, because it lacked keratan sulfate. The expression of PG-M in developing chick embryos was then examined. PG-M was found in some developmentally active areas, such as the perinotochordal mesenchyme between notochord and neural tube, the basement membranes facing neuroepithelial cells, and condensing mesenchymal cells in limb buds, suggesting some functions distinctive of the developing tissues.

Temporal matrix synthesis and histologic features of a chondrocyte-laden porous collagen cartilage analogue.

Abstract: Cartilage resurfacing by chondrocyte transplantation, using porous collagen matrices as a vehicle to secure the cells in cartilage defects, has been used experimentally in animals. This in vitro study evaluated the temporal morphologic features and proteoglycan synthesis of chondrocyte-laden collagen matrices. Forty-two porous collagen disks were implanted with a minimum of 6 x 10(6) viable chondrocytes, covered by a polymerized collagen gel layer, and 6 disks were harvested after 0, 3, 7, 10, 14, 18, or 22 days of incubation in supplemented Ham's F12 medium at 37 C and 5% CO2. Histologic and histochemical evaluation of formalin-fixed segments of the cultured disks indicated that the chondrocytes proliferated in the implant, producing small groups and linear segments of cells by day 14. The collagen framework remained intact over the course of the study with thick areas attributable to depositions of matrix material after day 10. Alcian blue-stained matrix was evident in the pericellular region of chondrocytes in sections of disks harvested on days 14, 18, and 22. Glycosaminoglycan (GAG) assay by dimethylmethylene blue dye binding after papain digestion of the disk segments revealed negligible amounts of GAG at day 0. Significant (P < or = 0.0001) increase in total GAG content was observed by day 3 (0.329 micrograms/mg of disk) and further increases were observed until a plateau in GAG quantity was seen on day 14. Mean peak GAG content was 0.553 +/- 0.062 micrograms/mg. Secondary treatment of the papain-digested implants with keratanase and chondroitinase ABC yielded similar trends in chondroitin sulfate (CS) and keratan sulfate (KS) concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Keratan sulfate and dermatan sulfate proteoglycans associate with type VI collagen in fetal rabbit cornea.

Abstract: Keratan sulfate proteoglycan (KSPG) and dermatan sulfate proteoglycan (DSPG) are associated with collagen fibrils in adult rabbit cornea. Because certain cytochemical data suggested that proteoglycans are associated with type VI collagen in the fetal rabbit cornea, we developed polyclonal antibodies specific to the core proteins of rabbit corneal KSPG (lumican and/or fibromodulin) and DSPG (decorin and/or biglycan) and used the antibodies as immunocytochemical probes to determine proteoglycan ultrastructural location. Immunogold particles were associated with Type VI collagen filaments but not with collagen fibrils in fetal and neonate rabbit cornea. Association of corneal KSPG and DSPG with Type VI collagen was immunocytochemically confirmed with monoclonal antibodies to low-sulfated keratan sulfate glycosaminoglycan (GAG) and chondroitin-4-sulfate GAG of DSPG. The monospecificity of the polyclonal and monoclonal antibodies and exclusive binding of antibodies to Type VI collagen filaments, together with ...

Heparin as an inducer of hepatocyte growth factor

Abstract: Hepatocyte growth factor (HGF) has mitogenic and unique morphoregulatory functions, and is considered to act as a hepatotrophic and a renotrophic factor for regeneration of the liver and kidney subjected to various insults. We have now obtained evidence that heparin is a potent inducer of HGF production. The addition of heparin to a culture of MRC-5 human embryonic lung fibroblasts increased the HGF concentration in the conditioned medium, in a dose-dependent manner. The maximal stimulation was obtained at 1 microgram/ml heparin and stimulation was 4-fold compared to control cultures. The rate of HGF synthesis in MRC-5 cells, as measured by pulse-labeling with [35S]methionine, and subsequent immunoprecipitation of HGF from both conditioned medium and a cell lysate, was 3-4-fold stimulated by 1 microgram/ml heparin, whereas heparin apparently had no significant effect on the HGF mRNA level. The stimulatory effect of heparin on HGF production was evident in various types of cells, such as MRC-9, IMR-90, and WI-38 human embryonic lung fibroblasts, human skin fibroblasts, HL-60 human promyelocytic leukemic cells and human umbilical vein endothelial cells. In addition to heparin, heparan sulfate also stimulated HGF production, albeit to a lesser extent than heparin; 1.7-fold stimulation with 2 micrograms/ml heparan sulfate. However, other glycosaminoglycans, such as hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, and keratan polysulfate, had no stimulatory effect on HGF production.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonal antibodies to keratan sulfate immunolocalize ramified microglia in paraffin and cryostat sections of rat brain.

Abstract: We used six monoclonal antibodies (MAb) recognizing epitopes within keratan sulfate (KS) chains for an immunocytochemical study of adult rat brain. One of the MAb selectively stained microglia and their ramified processes. KS-positive cells were found throughout the CNS in both paraffin-embedded and cryostat sections; the greatest number were present in hippocampus and brainstem. In the cortex the positive processes of some cells surrounded neuronal somata. In the white matter the processes were both parallel and perpendicular to the axon bundles. Double staining showed that KS-positive cells did not express astrocytic or oligodendroglial markers. By immunoelectron microscopy, the positivity was localized around the perikarya and cell processes of small cells with peripheral chromatin clumps and dark cytoplasm, which often contained secondary lysosomes. The KS-positive cells did not contribute to myelin sheaths and were not surrounded by a basal membrane. In addition to the cellular staining, three other ...

Specific glycosaminoglycans support the inhibition of thrombin by plasminogen activator inhibitor 1

Abstract: In the absence of accessory components, plasminogen activator inhibitor 1 (PAI-1) rapidly forms equimolar, inactive complexes both with tissue-type (t-PA) and with urokinase-type (u-PA) plamsinogen activator. In the presence of either the glycoprotein vitronectin or the glycosaminoglycan heparin, PAI-1 is endowed with additional, efficient thrombin-inhibitory properties (Ehrlich et al., 1990, 1991a). Here, we have investigated the interaction between PAI-1, thrombin, and glycosaminoglycans in more detail. Inhibition of thrombin by PAI-1 was quantitatively analyzed in the presence of a wide range of concentrations of heparin, heparan sulfate, dermatan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate, keratan sulfate, and hyaluronic acid by measuring residual amidolytic activity. In addition, a qualitative analysis was performed by determining the formation of SDS-stable, equimolar complexes between thrombin and PAI-1 in the presence of various glycosaminoglycans. Heparin, at concentrations between 0.1 and 1 microgram/mL, significantly promoted thrombin inhibition by PAI-1 as well as SDS-stable complex formation. Suboptimal inhibition was observed with dermatan sulfate, chondroitin 4-sulfate, and heparan sulfate at concentrations that are at least 1 order of magnitude higher than that required for optimal inhibition in the presence of heparin. Virtually no inhibition of thrombin and SDS-stable complex formation was detected with any of the other glycosaminoglycans at concentrations between 0.1 and 1 microgram/mL.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning and analysis of the protein modules of aggrecans.

Abstract: The large aggregating chondroitin sulfate proteoglycan of cartilage, aggrecan, has served as a prototype of proteoglycan structure. Molecular cloning has elucidated its primary structure and revealed both known and unknown domains. To date the complete structures of chicken, rat and human aggrecans have been deduced, while partial sequences have been reported for bovine aggrecan. A related proteoglycan, human versican, has also been cloned and sequenced. Both aggrecan and versican have two lectin domains, one at the amino-terminus which binds hyaluronic acid and one at the carboxyl-terminus whose physiological ligand is unknown. Both lectins have homologous counterparts in other types of proteins. Within the aggrecans the keratan sulfate domain may be variably present and also has a prominent repeat in some species. The chondroitin sulfate domain has three distinct regions which vary in their prominence in different species. The complex molecular structure of aggrecans is consistent with the concept of exon shuffling and aggrecans serve as suitable prototypes for comprehending the evolution of multi-domain proteins.

Histamine H2 receptor mediates keratan sulfate secretion in rabbit chondrocytes : role of cAMP

Abstract: We obtained evidence for the presence of a single class of histamine H2 receptor on rabbit chondrocytes. Stimulation of these receptors with specific H2 agonists led to an inhibition of keratan sulfate secretion and rapid (15 min) accumulation of intracellular adenosine 3',5'-cyclic monophosphate (cAMP). Factors such as prostaglandin E2 and parathyroid hormone, which stimulate short-term increases in cAMP, also caused a reduction in keratan sulfate secretion. Conversely, cholera toxin and forskolin, which enhance cAMP accumulation over 48 and 4 h, respectively, as well as a continuous exposure to dibutyryl cAMP, stimulated keratan sulfate secretion. These data suggest that intracellular cAMP must be kept above a certain level for a prolonged period to stimulate keratan sulfate secretion. We conclude that inhibition of keratan sulfate secretion is coupled with activation of the H2 histamine receptor.

Synovial fluid analyses detect and differentiate proteoglycan metabolism in canine experimental models of osteoarthritis and disuse atrophy.

Abstract: Canine experimental models of osteoarthritis (OA) and disuse atrophy were used to study cartilage metabolism. The synovial fluids from the OA joints showed elevated levels of keratan sulfate (KS) epitope and link protein, indicating increased catabolism. Analysis of fluids from joints with disuse atrophy showed high levels of KS epitope, but no increase in link protein. Quantitation of a novel chondroitin sulfate (3B3) epitope showed it to be present only in the synovial fluids and articular cartilage of the OA joints. The results indicate that these may be important indicators, or markers, of degenerative joint disease.

Immunoglobulin G and serum albumin isolated from the articular cartilage of patients with rheumatoid arthritis or osteoarthritis contain covalent heteropolymers with proteoglycans.

Abstract: The present study was undertaken to identify the cartilage matrix molecules that are bound with intermolecular disulfide bonds to IgG and serum albumin molecules recovered from the articular cartilage of patients with rheumatoid arthritis (RA) or osteoarthritis (OA). The cartilage specimens were extracted sequentially with three changes of neutral buffer, three changes of 6 M guanidine hydrochloride and then partiallydegraded with bacterial collagenase. The extracted IgG and albumin, along with matrix molecules bound to these proteins, were isolated with affinity chromatography using antibodies to IgG or human serum albumin conjugated to agarose beads. The isolated materials were characterized with sodium dodecyl sulfate polyacrylamide gel electrophoresis and transfer blotting, using specific antibodies to IgG, albumin, and proteoglycans. In the isolated materials, heteropolymers with IgG or albumin wer identified. These polymers contained keratan sulfate and less frequently chondroitin-4-sulfate and chondroitin-6-sulfate. These findings identified the keratan sulfate rich proteoglycans, prevalent at the surface of joint cartilage, as the most common cartilage matrix molecules that are covalently bound to IgG or to serum albumin by disulfide bonds in the articular cartilage of patients with RA or OA.

Keratan sulfate inhibits its release in rabbit chondrocyte

Abstract: Keratan sulfate (KS) is one of the major glycosaminoglycans in cartilage matrix proteoglycan. We find that exogenously added KS totally blocks keratanase sensitive glycosaminoglycan release measured by radiolabeled ligand. We also find that KS release is potentially inhibited by its own presence as measured by ELISA. When KS is digested with keratanase before its addition to the medium, its inhibitory effect on KS secretion is partially blocked. Exogenously added KS promotes the accumulation of the KS epitope in the cell layer. These data suggest that KS negatively regulates its own secretion.

Effect of glycosaminoglycans on the growth of cultured tumor cells.

Abstract: The effect of various glycosaminoglycans on the growth of cultured Tawa sarcoma cells (CTS cells) were determined under both fast and slow growth conditions. Hyaluronic acid, chondroitin, chondroitin 4-sulfate, and chondroitin 6-sulfate (all of which have only one type of uronic acid, glucuronic acid) inhibited the growth of CTS cells during fast growth and accelerated it during slow growth. Both keratan sulfate and keratan polysulfate (containing galactose) inhibited the growth of CTS cells during both growth conditions. Only glycosaminoglycans containing iduronic acid (heparin, heparan sulfate, and dermatan sulfate) accelerated the growth of the cells during fast growth. However, heparin inhibited the growth during slow growth while heparan sulfate and dermatan sulfate accelerated it. Growth regulation seems to require complete structural integrity of the glycosaminoglycans. The component subunits alone lack such activity when not linked together.

Sensitivity of monoclonal antibody, 5-D-4, for the detection of aggrecan, aggrecan fragments, and keratan sulfate

Abstract: Bovine nasal septum aggrecan and selected proteinase-digested products of aggrecan were evaluated in an inhibition ELISA using the anti-keratan sulfate (KS) monoclonal antibody 5-D-4 (5D4). Undegraded aggrecan was recognized with an IC50 of 0.27 μg/ml. When aggrecan was treated with human stromelysin (SLN), human leukocyte elastase (HLE), or papain, the degradation fragments had different hydrodynamic sizes. Treatment with SLN produced the largest fragments, HLE generated intermediate fragments, and papain the smallest fragments. Whereas degradation of aggrecan by SLN had little effect on recognition of proteoglycan in the ELISA (IC50-0.5 μg/ml), degradation by both HLE and papain significantly decreased the sensitivity for detection of KS epitope (IC50-170 and 215 μg/ml, respectively). In addition, 5D4 detected single chain costal and corneal KS with much less sensitivity (IC50-21 and 469 μg/ml, respectively) than undegraded aggrecan (IC50-0.27 μg/ml).

Method for measuring hyaluronic acid

Abstract: A method for measuring hyaluronic acid in a biological sample which comprises (a) coating a solid support with hyaluronic acid; (b) incubating the sample with cartilage proteoglycan; (c) exposing the incubated sample to the coated solid support; (d) then exposing the coated solid support to a keratan sulfate-reactive antibody; (e) determining the amount of antibody linked to keratan sulfate; and (f) correlating the amount of antibody linked to the keratan sulfate to the amount of hyaluronic acid in the sample.

Biochemical profile of proteoglycans and glucosaminoglycans of the mammalian tectorial membrane

Abstract: The tectorial membrane is an acellular connective tissue which plays an essential role in cochlear function. While a comparatively large amount of information is available on the collagen network of the tectorial membrane, studies on the biochemical nature of this highly hydrated matrix, which is composed of proteoglycans (PGs) and glycosaminoglycans (GAGs), have been quite limited. Previous reports on the biochemical analysis of the tectorial membrane have failed to detect uronic acid, which is present in large amounts in all mammalian GAGs except keratan sulfate. Applying a colorimetric assay based on the binding of GAGs to cationic dye Safranin-0 in combination with enzymatic techniques, we were able to measure GAGs in the murine tectorial membrane. Approximately 0.3% uronic acid-containing GAGs (mainly in the form of chondroitin/dermatan sulfate) and 0.17% keratan sulfate were detected in the tectorial membrane (both on a wet weight basis). In addition, various types of electrophoresis revealed one large PG with a molecular mass similar to that of the large type cartilage PGs and three small PGs, containing chondroitin sulfate and keratan sulfate side chains, respectively. Judging by coelution of standards, one of the small PGs seemed to correspond to fibromodulin, which has at least one keratan sulfate side chain, and binds to type I and type II collagen to regulate collagen organization in tissues. Our results suggest: (1) Donnan equilibrium is established in the tectorial membrane because sulfated GAGs are highly negatively charged and consequently bring about an influx of large amounts of water and cations into the matrix.(ABSTRACT TRUNCATED AT 250 WORDS)

Glycosidase and sulfatase activities and their possible role in keratan sulfate degradation in metamorphosing bonefish (Albula sp.) leptocephali.

Abstract: During metamorphosis of leptocephalous larvae of the bonefish (Albula sp.), keratan sulfate, the principal glycosaminoglycan of the extracellular gelatinous body matrix, is degraded. Artificial substrates have been utilized to demonstrate the presence of β-N-acetylglucosaminidase, β-galactosidase and sulfatase activities in whole-body homogenates of early metamorphosing leptocephali. The concerted action of these enzymes has been shown to degrade the keratan sulfate polymer in other tissues. This paper describes the extraction, partial purification and some of the physical and kinetic properties of these enzymes. Additionally, starch gel electrophoresis was used to follow glycosidase activities in early, intermediate and advanced metamorphosing larvae. No differences were observed in electrophoretic migration or banding pattern of either β-N-acetylglucosaminidase or β-galactosidase during metamorphosis.

Location of the Keratan Sulfate Attachment Sites in the Hyaluronate Binding Region of the Proteoglycan, Aggrecan

Abstract: Publisher Summary This chapter presents a study examining location of the keratan sulfate (KS) attachment sites in the hyaluronate binding region (HAABR) of the proteoglycan, aggrecan. HABR from pig laryngeal cartilage was isolated and digested, and the peptides were separated. The KS-containing peptide was isolated by virtue of its high molecular weight relative to other glycosylated and nonglycosylated peptides. It was detected by its positive reaction in ELISA assays using monoclonal antibody 5D4. The peptide was coupled to arylamine-PVDF using (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The membrane was washed with 0.1% aqueous TFA to remove uncoupled material. The membrane was cut into small pieces and placed directly into the standard reaction cartridge, without a glass fiber filter. Tryptic digestion of the aggrecan HABR followed by gel permeation chromatography and reversed-phase HPLC indicated that all the highly sulfated KS in the HABR was in a single tryptic peptide from the C-terminal of the domain. The 27-residue peptide had a high molecular weight (8–15 kDa) as a result of the KS GAG chain(s).