EFFECT OF HORMONES ON THE TURNOVER OF POLYSACCHARIDES IN CONNECTIVE TISSUES.
(57/460)
A number of hormones somehow modify the turnover of the polysaccharides in a variety of connective tissues. In hypophysectomized animals the turnover of chondroitin sulfate and hyaluronic acid is decreased; when such animals are given growth hormone the turnover of chondroitin sulfate is enhanced but that of hyaluronic acid is unaltered. The effect of parathyroid extracts may be of a dual nature: in some connective tissues there may be an increase in the rate at which chondroitin sulfate is catabolized, in other tissues its synthesis may be stimulated. Thyroxine effectively restores toward normal the depressed synthesis and breakdown of polysaccharides in hypothyroid animals. Estradiol, in addition to inhibiting the resorption of the metaphyses in rats, inhibits the synthesis of chondroitin sulfate in cartilage and aorta. Cortisone too inhibits the synthesis of chondroitin sulfates and hyaluronic acid; its effect on their catabolism is not as striking. (+info)
ISOLATION, CHARACTERIZATION, AND DISTRIBUTION OF ACID MUCOPOLYSACCHARIDES IN RABBIT LEUCOCYTES.
(58/460)
Acid mucopolysaccharides have been extracted from whole rabbit polymorphonuclear leucocytes and from the cytoplasmic granules of these cells. The leucocyte acid mucopolysaccharides can be separated into two fractions by the solubility of their CPC complexes in solutions of differing salt concentration. One of these fractions appears to be identical with hyaluronic acid; the other appears to be an atypical chondroitin sulfate. On both a dry weight and total protein basis the polymorphonuclear leucocyte granule contains approximately 2.6 times as much acid mucopolysaccharide as does the whole cell. Hyaluronic acid is concentrated in the granules in particular; its function is unknown. These results do not indicate that all lysosomes contain abundant acid mucopolysaccharides, for no detectable carbohydrate of this class could be extracted from lysosome-rich alveolar macrophages. (+info)
Secondary and tertiary structures involving chondroitin and chondroitin sulphates in solution, investigated by rotary shadowing/electron microscopy and computer simulation.
(59/460)
Rotary shadowing/electron microscopy of chondroitin 6-sulphate (CS6) and 4-sulphate (CS4) showed that the former, but not the latter, aggregated to mesh works. Preparations made from salt (ammonium acetate) solutions showed enhanced aggregation. Computer modelling, using molecular mechanics and dynamics, was applied to secondary structures (twofold helices) derived from NMR studies, to determine geometric and energetic constraints on duplex and higher-aggregate formation. The calculations suggested that chondroitin, CS6 and undersulphated CS4 could form duplexes, while CS4 could not, thus bridging the gap between atomic dimensions (NMR) and high polymer scale (electron microscopy). Calculations suggested that water structure helped to stabilise the twofold helix. It is proposed that the twofold helical, flat, tape-like molecules aggregate via hydrophobic bonding between the very extensive hydrophobic patches (9 CH units) repeated on alternating sides of the polymers. The negative charge of the polyanions opposes aggregate formation. Calculations showed that duplexes were formed with decreasing stability as the charge density increased, and as the charge was concentrated towards the centre line of the polymer (i.e. in CS4). The unsulphated polymer chondroitin could form duplexes and higher aggregates as readily as hyaluronan. Hyaluronan was calculated to form stable heteroduplexes with CS6 and CS4. The frequency and positioning of the sulphate-ester group within the polymer thus determines whether the molecule participates in duplex formation. (+info)
Structures of Streptococcus pneumoniae hyaluronate lyase in complex with chondroitin and chondroitin sulfate disaccharides. Insights into specificity and mechanism of action.
(60/460)
Streptococcus pneumoniae hyaluronate lyase is a surface enzyme of this Gram-positive bacterium. The enzyme degrades hyaluronan and chondroitin/chondroitin sulfates by cleaving the beta1,4-glycosidic linkage between the glycan units of these polymeric substrates. This degradation helps spreading of this bacterial organism throughout the host tissues and facilitates the disease process caused by pneumococci. The mechanism of this degradative process is based on beta-elimination, is termed proton acceptance and donation, and involves selected residues of a well defined catalytic site of the enzyme. The degradation of hyaluronan alone is thought to proceed through a processive mode of action. The structures of complexes between the enzyme and chondroitin as well as chondroitin sulfate disaccharides allowed for the first detailed insights into these interactions and the mechanism of action on chondroitins. This degradation of chondroitin/chondroitin sulfates is nonprocessive and is selective for the chondroitin sulfates only with certain sulfation patterns. Chondroitin sulfation at the 4-position on the nonreducing site of the linkage to be cleaved or 2-sulfation prevent degradation due to steric clashes with the enzyme. Evolutionary studies suggest that hyaluronate lyases evolved from chondroitin lyases and still retained chondroitin/chondroitin sulfate degradation abilities while being specialized in the degradation of hyaluronan. The more efficient processive degradation mechanism has come to be preferred for the unsulfated substrate hyaluronan. (+info)
Tandem mass spectrometric strategies for determination of sulfation positions and uronic acid epimerization in chondroitin sulfate oligosaccharides.
(61/460)
Chondroitin sulfate (CS) is a glycosaminoglycan consisting of repeating (HexA-GalNAc sulfate) disaccharides, the functions of which depend on patterns of sulfation and uronic acid epimerization. The correlation of biological activities with structure requires a strategy to determine the sequences of CS oligosaccharides without the need for total isolation. Tandem mass spectrometry has enabled the development of proteomics, based on CID fragmentation of ions produced from complex mixtures of proteolytic peptides, and has the potential for rapid sequencing of CS and other glycosaminoglycan classes. The most challenging aspects of CS sequencing are to distinguish GalNAc residues sulfated at the 4- versus the 6-position and uronic acid epimers. This work describes the utility of (1) reducing terminal derivatives and (2) control of precursor ion charge state for tandem mass spectrometric strategies for determining GalNAc sulfation positional isomers of CS. The capability of tandem MS to differentiate uronic acid epimers is also shown, providing evidence that complete or nearly complete information on CS covalent structure may be obtained using tandem MS. (+info)
Purification of hyaluronidase from human placenta.
(62/460)
Hyaluronidase [EC 3.2.1.35] was isolated from human placenta and purified by ammonium sulfate fractionation, DEAE-cellulose column chromatography and gel filtration on Sephadex G-150. Its isoelectric point was at pH 5.2 and the molecular weight was 7 X 10(4) based on Sephadex G-200 gel filtration data. This enzyme was very stable at temperatures below 30 degree, but was almost completely inactivated at 60degree within 30 min. Its optimum pH was 3.9, a characteristic property of a lysosomal hyaluronidase. The Michaelis constant was 1.18 x 10(-1) mg per ml with purified hyaluronate. This enzyme depolymerized hyaluronate, chondroitin, chondroitin 4-sulfate and 6-sulfate, and the end product formed from hyaluronate was tetrasaccharide. Its biological diffusing activity was statistically significant on intracutaneous injection of 1.86 mU of the hyaluronidase into the back skine of a rabbit. (+info)
Nematode chondroitin polymerizing factor showing cell-/organ-specific expression is indispensable for chondroitin synthesis and embryonic cell division.
(63/460)
Chondroitin polymerization was first demonstrated in vitro when human chondroitin synthase (ChSy) was coexpressed with human chondroitin polymerizing factor (ChPF), which is homologous to ChSy but has little glycosyltransferase activity. To analyze the biological function of chondroitin, the Caenorhabditis elegans ortholog of human ChSy (sqv-5) was recently cloned, and the expression of its product was depleted by RNA-mediated interference (RNAi) and deletion mutagenesis. Blocking of chondroitin synthesis resulted in defects of cytokinesis in early embryogenesis, and eventually, cell division stopped. Here, we cloned the ortholog of human ChPF in C. elegans, PAR2.4. Despite little glycosyltransferase activity of the gene product, chondroitin polymerization was demonstrated as in the case of mammals when PAR2.4 was coexpressed with cChSy in vitro. The worm phenotypes including the reversion of cytokinesis, observed after the depletion of PAR2.4 by RNAi, were very similar to the cChSy (sqv-5)-RNAi phenotypes. Thus, PAR2.4 in addition to cChSy is indispensable for the biosynthesis of chondroitin in C. elegans, and the two cooperate to synthesize chondroitin in vivo. The expression of the PAR2.4 protein was observed in seam cells, which can act as neural stem cells in early embryonic lineages. The expression was also detected in vulva and distal tip cells of the growing gonad arms from L3 through to the young adult stage. These findings are consistent with the notion that chondroitin is involved in the organogenesis of the vulva and maturation of the gonad and also indicative of an involvement in distal tip cell migration and neural development. (+info)
Glycosylation-related gene expression in prion diseases: PrPSc accumulation in scrapie infected GT1 cells depends on beta-1,4-linked GalNAc-4-SO4 hyposulfation.
(64/460)
Several lines of evidence indicate that some glycoconjugates are efficient effectors of the cellular prion protein (PrP(C)) conversion into its pathogenic (PrP(Sc)) isoform. To assess how glycoconjugate glycan moieties participate in the biogenesis of PrP(Sc), an exhaustive comparative analysis of the expression of about 200 glycosylation-related genes was performed on prion-infected or not, hypothalamus-derived GT1 cells by hybridization of DNA microarrays, semiquantitative RT-PCR, and biochemical assays. A significant up- (30-fold) and down- (17-fold) regulation of the expression of the ChGn1 and Chst8 genes, respectively, was observed in prion-infected cells. ChGn1 and Chst8 are involved in the initiation of the synthesis of chondroitin sulfate and in the 4-O-sulfation of non-reducing N-acetylgalactosamine residues, respectively. A possible role for a hyposulfated chondroitin in PrP(Sc) accumulation was evidenced at the protein level and by determination of chondroitin and heparan sulfate amounts. Treatment of Sc-GT1 cells with a heparan mimetic (HM2602) induced an important reduction of the amount of PrP(Sc), associated with a total reversion of the transcription pattern of the N-acetylgalactosamine-4-O-sulfotransferase 8. It suggests a link between the genetic control of 4-O-sulfation and PrP(Sc) accumulation. (+info)