Structural studies on the oligosaccharides isolated from bovine kidney heparan sulphate and characterization of bacterial heparitinases used as substrates

We prepared a series of oligosaccharides from commercial bovinekidney heparan sulphate after limited digestion with heparitinaseI from Flavobacterium heparinum, and determined the structuresof eight tetrasaccharides and a hexasaccharide by enzymaticanalysis, fast atom bombardment mass spectrometry and 500 MHz¹H NMR spectroscopy. The tetrasaccharides share the common corestructure     

Mycobacterial lipoarabinomannan: an extraordinary lipoheteroglycan with profound physiological effects

Detailed structural and functional studies over the last decade have led to current recognition of the mycobacterial lipoarabinomannan (LAM) as a phosphatidylinositol anchored lipoglycan with diverse biological activities. Fatty acylation has been demonstrated to be essential for LAM to maintain its functional integrity although the focus has largely been on the arabinan motifs and the terminal capping function. It has recently been shown that the mannose caps may be involved not only in attenuating host immune response, but also in mediating the binding of mycobacteria to and subsequent entry into macrophages. This may further be linked to an intracellular trafficking pathway through which LAM is thought to be presented by CD1 to subsets of T-cells. The implication of LAM as major histocompatibility complex (MHC)-independent T-cell epitope and the ensuing immune response is an area of intensive studies. Another recent focus of research is the biosynthesis of arabinan which has been shown to be inhibitable by the anti- tuberculosis drug, ethambutol. The phenomenon of truncated LAM as synthesized by ethambutol resistant strains provides an invaluable handle for dissecting the array of arabinosyltransferases involved, as well as generating much needed structural variants for further structural and functional studies. It is hoped that with more systematic investigations based on clinical isolates and human cell lines, the true significance of LAM in the immunopathogenesis of tuberculosis and leprosy can eventually be explained.      

Purification and properties of two forms of glucoamylase fromAspergillus niger

A. niger produced α-glucosidase, α-amylase and two forms of glucoamylase when grown in a liquid medium containing raw tapioca starch as the carbon source. The glucoamylases, which formed the dominant components of amylolytic activity manifested by the organism, were purified to homogeneity by ammonium sulfate precipitation, ion-exchange and two cycles of gel filtration chromatography. The purified enzymes, designated GA1 and GA2, a raw starch digesting glucoamylase, were found to have molar masses of 74 and 96 kDa and isoelectric points of 3.8 and 3.95, respectively. The enzymes were found to have pH optimum of 4.2 and 4.5 for GA1 and GA2, respectively, and were both stable in a pH range of 3.5–9.0. Both enzymes were thermophilic in nature with temperature optimum of 60 and 65°C, respectively, and were stable for 1 h at temperatures of up to 60°C. The kinetic parametersK _m andV showed that with both enzymes the branched substrates, starch and amylopectin, were more efficiently hydrolyzed compared to amylose. GA2, the more active of the two glucoamylases produced, was approximately six to thirteen times more active towards raw starches compared to GA1.     

Structural and immunological characterization of O-acetylated GD2. Evidence that GD2 is an acceptor for ganglioside O-acetyltransferase in human melanoma cells.

We have shown previously that Golgi-enriched vesicles from the human melanoma cell line Melur can transfer [3H]acetate from [acetyl-3H]acetyl-CoA to endogenous GD3 to form [acetyl-3H]O-acetyl-GD3 (Manzi, A. E., Sjoberg, E. R., Diaz, S., and Varki, A. (1990) J. Biol. Chem. 265, 13091-13103). Applying the same approach in the human melanoma cell line M21, label was found in [acetyl-3H]O-acetyl-GD3 and also in a species co-migrating with unsubstituted GD3 on TLC. Both were sialidase-sensitive and alkali-labile, indicating incorporation as [3H]O-acetyl esters on sialic acids. Immunological reactivity, sialidase sensitivity, chromatographic behavior, and the known ganglioside pattern of M21 cells suggested that the slower migrating species might be [acetyl-3H]O-acetyl-GD2. Sialic acids released from this labeled molecule by sialidase showed esterification with [3H]acetate at both C7 and C9 hydroxyls. Lipid extracts from cells metabolically labeled with [3H]galactose showed a corresponding ganglioside, which upon alkali treatment yielded a species migrating with GD2. Analysis of purified ganglioside by high performance thin layer chromatography immuno-overlays, fast atom bombardment-mass spectrometry in positive and negative ion modes, periodate oxidation resistance, linkage analysis by permethylation and gas chromatography-mass spectrometry, and 500 MHz 1H NMR was consistent with the following structure: 9-O Ac-Neu5Ac alpha 2-8Neu5Ac alpha 2-3(GalNAc beta 1-4) Gal beta 1-4Gluc beta 1-1' ceramide Total gangliosides from M21 were analyzed by high performance thin layer chromatography immuno-overlay with monoclonal antibodies D1.1, JONES, 27A, and 8A2, all known to, or suspected of reacting with 9-O-acetylated gangliosides. The first three bound well to 9-O-acetyl-GD3 and a slower migrating 9-O-acetylated ganglioside, which was distinct from 9-O-acetyl-GD2. Antibody 8A2 reacted weakly with purified 9-O-acetyl-GD2 and strongly with two other 9-O-acetylated gangliosides migrating slower than 9-O-acetyl-GD2. Thus, the family of O-acetylated gangliosides in melanoma cells is much more complex than previously appreciated.     

Role of phosphoprotein phosphatases in reversible deactivation of chicken adipose tissue hormone-sensitive lipase.

The reversible deactivation of chicken adipose tissue hormone-sensitive lipase alpha(previously activated with Mg2+ ATP and adenosine 3':5'-monophosphate) required Mg2+ and was inhibited by phosphate. These results are consistent with the assumption that deactivation of the protein kinase-activated enzyme is catalyzed by a lipase phosphatase. Cholesterol ester is catalyzed by a lipase phosphatase. Cholesterol ester hydrolase similarly was activated and reversibly deactivated. The activity of endogenous lipase phosphatase in pH 5.2 precipitate fractions was reduced, and in some cases eliminated, by incubation at 50 degrees for 20 min in buffer containing 20% glycerol. Heating at 50 degrees greatly increased the apparent percentage activation of triglyceride and cholesterol ester hydrolases but this was due to a selective decrease in basal (nonactivated) hydrolase activities. Essentially all endogenous lipase phosphatase could be removed by treatment of the pH 5.2 precipitate fraction with ATP-Sepharose affinity gel. The addition of a partially purified preparation of rat liver phosphorylase phosphatase deactivated triglyceride and cholesterol ester hydrolases. The deactivation process was concentration, 5 mM) and was inhibited by 5 mM phosphate and by phosphorylase alpha. Reversible deactivation of hormone-sensitive lipase alpha was also observed with crude prepa- and by phosphorylase alpha. Reversible deactivation of hormone-sensitive lipas alpha was also observed with crude preparations of phosphoprotein phosphatases from rat and turkey hearts, and from rat epididymal fat pads. Thus, hormone-sensitive lipase is deactivated by a variety of phosphoprotein phosphatases from different tissues and different species, implying a low degree of specificity for the deactivating system.     

Cardioprotection mediated by exosomes is impaired in the setting of type II diabetes but can be rescued by the use of non‐diabetic exosomes in vitro

Many patients with ischaemic heart disease also have diabetes. As myocardial infarction is a major cause of mortality and morbidity in these patients, treatments that increase cell survival in response to ischaemia and reperfusion are needed. Exosomes—nano‐sized, lipid vesicles released from cells—can protect the hearts of non‐diabetic rats. We previously showed that exosomal HSP70 activates a cardioprotective signalling pathway in cardiomyocytes culminating in ERK1/2 and HSP27 phosphorylation. Here, we investigated whether the exosomal cardioprotective pathway remains intact in the setting of type II diabetes. Exosomes were isolated by differential centrifugation from non‐diabetic and type II diabetic patients, from non‐diabetic and Goto Kakizaki type II diabetic rats, and from normoglycaemic and hyperglycaemic endothelial cells. Exosome size and number were not significantly altered by diabetes. CD81 and HSP70 exosome markers were increased in diabetic rat exosomes. However, exosomes from diabetic rats no longer activated the ERK1/2 and HSP27 cardioprotective pathway and were no longer protective in a primary rat cardiomyocytes model of hypoxia and reoxygenation injury. Hyperglycaemic culture conditions were sufficient to impair protection by endothelial exosomes. Importantly, however, exosomes from non‐diabetic rats retained the ability to protect cardiomyocytes from diabetic rats. Exosomes from diabetic plasma have lost the ability to protect cardiomyocytes, but protection can be restored with exosomes from non‐diabetic plasma. These results support the concept that exosomes may be used to protect cardiomyocytes against ischaemia and reperfusion injury, even in the setting of type II diabetes.     

FABMS/derivatisation strategies for the analysis of heparin-derived oligosaccharides

Derivatisation/FABMS strategies applicable to the structure analysis of low microgramme quantities of heparin-derived oligosaccharides are described. Negative and positive FAB data from permethyl derivatives and positive FAB data from the products of subsequent methanolysis are reported for sulfated tetrasaccharides prepared by nitropus acid degradation of heparin. The preparation and FAB behaviour of acetylated derivatives of sulfated oligosaccharides are described for the first time, and the stability of the sulfate groups to base-catalysed acetylation is demonstrated. The acetylation/FABMS methodology, which yields high quality data, shows promise for the characterisation of a wide range of sulfated glycoconjugates.     

Interactions of calcium and other metal ions with caldolysin, the thermostable proteinase from Thermus aquaticus strain T351.

Caldolysin, the extracellular proteinase from the extreme thermophile Thermus aquaticus strain T351, is stabilized by Ca2+. A variety of metal ions were able to substitute for Ca2+. Most were unable to confer as much stability as Ca2+, with the exception of the lanthanide ions, which increased the half-life at 95 degrees C from 1 h to more than 4 h. Results from a variety of separation methods indicated that caldolysin binds 6 Ca2+ ions/molecule of enzyme. The presence of non-linear Ca2+ titration plots, and the removal of 4 Ca2+ ions/molecule by treatment with a cationic ion-exchange gel suggested that caldolysin possesses at least two different types of Ca2+-binding sites, with different affinities. Average binding constants of the two types of binding sites were 2.8 X 10(4)M-1 (for the low-affinity sites) and 7.5 X 10(5) M-1 (for the high-affinity sites). The total Ca2+-binding free energy for caldolysin was shown to be greater than for either thermolysin or Bacillus subtilis neutral proteinase. It appears that the higher thermostability of caldolysin is due to the presence of 6 Ca2+ ions rather than 4 Ca2+ ions/molecule.     

A precise structural analysis of a fertilization-associated carbohydrate-rich glycopeptide isolated from the fertilized eggs of euryhaline killi fish (Fundulus heteroclitus). Novel penta-antennary N-glycan chains with a bisecting N-acetylglucosaminyl residue

A novel carbohydrate-rich sialoglycopeptide of apparent molecularmass $$$6 kDa was isolated from the fertilized eggs of Fundulusheteroclitus (euryhaline killi fish). This glycopeptide is amember of the L-hyosophorin family, characterized by its highcontent of carbohydrate (80–90% by weight) and formedby depolymerization of the precursor glycopolyprotein (H-hyosophorin)upon fertilization. The structures of the N-glycan chains wereunambiguously established by a combination of compositionalanalysis, methylation analysis, selective chemical degradation(periodate oxidation-Smith degradation and hydrazinolysis-nitrousacid deamination), enzymatic (peptide:N-g]ycosidase F, severalß-galactosidases, (ß-hexosaminidase and     

Chlorotriphenyl(quinolinium-2-carboxylato)tin(IV) monohydrate

The crystal structure of chlorotriphenyl(quinolinium-2-carboxylato)tin(IV) monohydrate is reported. The crystals are monoclinic, space group C2/c with cell parameters a = 20.048(3) Å, b = 11.724(1) Å, c = 23.291(3) Å, ]gb = 113.42(1), Z = 8, refined to R_F = 0.034 on 3331 observed reflections. The tin(IV) atom is five-coordinate, being found to three phenyl groups, the chlorine atom and an oxygen from the quinaldic acid. The geometry around the tin atom is trigonal bipyramidal, with the three phenyl groups occupying the equatorial positions, and the chlorine and quinaldic acid oxygen, the apical ones. The acidic proton of quinaldic acid has shifted position in the complex, and is bound to the heterocyclic nitrogen atom.The acid is thus coordinated in the form of a zwitterion. These trigonal bipyramidal units are linked together as dimers by pairs of water molecules, each of which hydrogen-bonds to the non-coordinated carboxylate oxygen atoms of both quinaldic acid molecules, plus the heterocyclic nitrogen atom of one quinaldic acid molecule. For complex formation with the protonated acid, the heterocyclic nitrogen should be alpha to the carboxylic acid group.