169 results found
Pomin VH, Mulloy B, 2018, Glycosaminoglycans and Proteoglycans, PHARMACEUTICALS, Vol: 11
Hogwood J, Naggi A, Torri G, et al., 2018, The effect of increasing the sulfation level of chondroitin sulfate on anticoagulant specific activity and activation of the kinin system, PLoS ONE, Vol: 13, ISSN: 1932-6203
Oversulfated chondroitin sulfate (OSCS) was identified as a contaminant in certain heparin preparations as the cause of adverse reactions in patients. OSCS was found to possess both plasma anticoagulant activity and the ability to activate prekallikrein to kallikrein. Differentially sulfated chondroitin sulfates were prepared by synthetic modification of chondroitin sulfate and were compared to the activity of OSCS purified from contaminated heparin. Whilst chondroitin sulfate was found to have minimal anticoagulant activity, increasing sulfation levels produced an anticoagulant response which we directly show for the first time is mediated through heparin cofactor II. However, the tetra-sulfated preparations did not possess any higher anticoagulant activity than several tri-sulfated variants, and also had lower heparin cofactor II mediated activity. Activation of prekallikrein was concentration dependent for all samples, and broadly increased with the degree of sulfation, though the di-sulfated preparation was able to form more kallikrein than some of the tri-sulfated preparations. The ability of the samples to activate the kinin system, as measured by bradykinin, was observed to be through kallikrein generation. These results show that whilst an increase in sulfation of chondroitin sulfate did cause an increase in anticoagulant activity and activation of the kinin system, there may be subtler structural interactions other than sulfation at play given the different responses observed.
Rider CC, Mulloy B, 2017, Heparin, Heparan Sulphate and the TGF-beta Cytokine Superfamily, MOLECULES, Vol: 22
Lever R, Smailbegovic A, Riffo-Vasquez Y, et al., 2016, Biochemical and functional characterization of glycosaminoglycans released from degranulating rat peritoneal mast cells: Insights into the physiological role of endogenous heparin, PULMONARY PHARMACOLOGY & THERAPEUTICS, Vol: 41, Pages: 96-102, ISSN: 1094-5539
Basáñez M-G, Anderson RM, 2016, Preface.
Pomin VH, Mulloy B, 2015, Current structural biology of the heparin interactome, CURRENT OPINION IN STRUCTURAL BIOLOGY, Vol: 34, Pages: 17-25, ISSN: 0959-440X
Tatsinkam AJ, Mulloy B, Rider CC, 2015, Mapping the heparin-binding site of the BMP antagonist gremlin by site-directed mutagenesis based on predictive modelling, BIOCHEMICAL JOURNAL, Vol: 470, Pages: 53-64, ISSN: 0264-6021
Palma AS, Liu Y, Zhang H, et al., 2015, Unravelling Glucan Recognition Systems by Glycome Microarrays Using the Designer Approach and Mass Spectrometry, Molecular & Cellular Proteomics, Vol: 14, Pages: 974-988, ISSN: 1535-9484
Glucans are polymers of D-glucose with differing linkages in linear or branched sequences. They are constituents of microbial and plant cell-walls and involved in important bio-recognition processes, including immunomodulation, anticancer activities, pathogen virulence, and plant cell-wall biodegradation. Translational possibilities for these activities in medicine and biotechnology are considerable. High-throughput micro-methods are needed to screen proteins for recognition of specific glucan sequences as a lead to structure–function studies and their exploitation. We describe construction of a “glucome” microarray, the first sequence-defined glycome-scale microarray, using a “designer” approach from targeted ligand-bearing glucans in conjunction with a novel high-sensitivity mass spectrometric sequencing method, as a screening tool to assign glucan recognition motifs. The glucome microarray comprises 153 oligosaccharide probes with high purity, representing major sequences in glucans. Negative-ion electrospray tandem mass spectrometry with collision-induced dissociation was used for complete linkage analysis of gluco-oligosaccharides in linear “homo” and “hetero” and branched sequences. The system is validated using antibodies and carbohydrate-binding modules known to target α- or β-glucans in different biological contexts, extending knowledge on their specificities, and applied to reveal new information on glucan recognition by two signaling molecules of the immune system against pathogens: Dectin-1 and DC-SIGN. The sequencing of the glucan oligosaccharides by the MS method and their interrogation on the microarrays provides detailed information on linkage, sequence and chain length requirements of glucan-recognizing proteins, and are a sensitive means of revealing unsuspected sequences in the polysaccharides.
Duru C, Swann C, Dunleavy U, et al., 2015, The importance of formulation in the successful lyophilization of influenza reference materials., Biologicals, Vol: 43, Pages: 110-116, ISSN: 1095-8320
Lyophilized Influenza antigen reference reagents are a critical resource in the quality control of influenza vaccines. A standard formulation has been used successfully at NIBSC for many years however, following the unexpected occurrence of a collapsed appearance in a particular batch a study was carried out to establish the impact of the sugar concentration in the formulation using modulated differential scanning calorimetry (mDSC) and nuclear magnetic resonance spectroscopy (NMR). There was a correlation between the presence and size of the mDSC eutectic temperature events and the freeze dried appearance of the cakes, which became progressively worse with increasing amounts of sugar. NMR spectroscopy could be used to positively identify and quantify the sugars in the formulations. MDSC can rapidly predict if the freeze dried appearance will be acceptable so as to assure the successful lyophilization of influenza reference preparations.
Mulloy B, Rider CC, 2015, The Bone Morphogenetic Proteins and Their Antagonists, BONE MORPHOGENIC PROTEIN, Vol: 99, Pages: 63-90, ISSN: 0083-6729
Mulloy B, Hogwood J, 2015, Chromatographic molecular weight measurements for heparin, its fragments and fractions, and other glycosaminoglycans., Methods Mol Biol, Vol: 1229, Pages: 105-118
Glycosaminoglycan samples are usually polydisperse, consisting of molecules with differing length and differing sequence. Methods for measuring the molecular weight of heparin have been developed to assure the quality and consistency of heparin products for medicinal use, and these methods can be applied in other laboratory contexts. In the method described here, high-performance gel permeation chromatography is calibrated using appropriate heparin molecular weight markers or a single broad standard calibrant, and used to characterize the molecular weight distribution of polydisperse samples or the peak molecular weight of monodisperse, or approximately monodisperse, heparin fractions. The same technology can be adapted for use with other glycosaminoglycans.
Panagos CG, Thomson DS, Moss C, et al., 2014, Fucosylated Chondroitin Sulfates from the Body Wall of the Sea Cucumber Holothuria forskali CONFORMATION, SELECTIN BINDING, AND BIOLOGICAL ACTIVITY, Journal of Biological Chemistry, Vol: 289, Pages: 28284-28298, ISSN: 0021-9258
Fucosylated chondroitin sulfate (fCS) extracted from the sea cucumber Holothuria forskali is composed of the following repeating trisaccharide unit: →3)GalNAcβ4,6S(1→4) [FucαX(1→3)]GlcAβ(1→, where X stands for different sulfation patterns of fucose (X = 3,4S (46%), 2,4S (39%), and 4S (15%)). As revealed by NMR and molecular dynamics simulations, the fCS repeating unit adopts a conformation similar to that of the Lex blood group determinant, bringing several sulfate groups into close proximity and creating large negative patches distributed along the helical skeleton of the CS backbone. This may explain the high affinity of fCS oligosaccharides for L- and P-selectins as determined by microarray binding of fCS oligosaccharides prepared by Cu2+-catalyzed Fenton-type and photochemical depolymerization. No binding to E-selectin was observed. fCS poly- and oligosaccharides display low cytotoxicity in vitro, inhibit human neutrophil elastase activity, and inhibit the migration of neutrophils through an endothelial cell layer in vitro. Although the polysaccharide showed some anti-coagulant activity, small oligosaccharide fCS fragments had much reduced anticoagulant properties, with activity mainly via heparin cofactor II. The fCS polysaccharides showed prekallikrein activation comparable with dextran sulfate, whereas the fCS oligosaccharides caused almost no effect. The H. forskali fCS oligosaccharides were also tested in a mouse peritoneal inflammation model, where they caused a reduction in neutrophil infiltration. Overall, the data presented support the action of fCS as an inhibitor of selectin interactions, which play vital roles in inflammation and metastasis progression. Future studies of fCS-selectin interaction using fCS fragments or their mimetics may open new avenues for therapeutic intervention.
Mulloy B, Heath A, Shriver Z, et al., 2014, USP compendial methods for analysis of heparin: chromatographic determination of molecular weight distributions for heparin sodium, ANALYTICAL AND BIOANALYTICAL CHEMISTRY, Vol: 406, Pages: 4815-4823, ISSN: 1618-2642
Mulloy B, Feizi T, 2014, Tricks of the trade in glycoscience: the preparation and analysis of a blood group A-active mucin glycoprotein., Biochem J, Vol: 2014, Pages: c1-c3
Many aspects of glycosylation are conserved among animals, and it can be advantageous and sometimes critical to identify a readily available and abundant source of carbohydrate material that harbours a hard-to-characterize antigen or ligand of interest. The Biochemical Journal Classic paper by Morgan and King is a well-written account of serviceable methods for the extraction and quantification of a carbohydrate antigen. These methods were highly influential in subsequent studies of the blood group antigens. Some of these tricks of the trade still have a place in modern glycobiology.
Khan S, Fung KW, Rodriguez E, et al., 2013, The Solution Structure of Heparan Sulfate Differs from That of Heparin IMPLICATIONS FOR FUNCTION, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 288, Pages: 27737-27751
Khan S, Rodriguez E, Patel R, et al., 2013, The solution structure of heparan sulfate differs from that of heparin. IMPLICATIONS FOR FUNCTION (vol 286, pg 24842, 2011), JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 288, Pages: 21209-21209
Khan S, Gor J, Mulloy B, et al., 2013, Semi-Rigid Solution Structures of Heparin by Constrained X-ray Scattering Modelling: New Insight into Heparin-Protein Complexes (vol 395, pg 504, 2010), JOURNAL OF MOLECULAR BIOLOGY, Vol: 425, Pages: 1847-1847, ISSN: 0022-2836
Clark SJ, Ridge LA, Herbert AP, et al., 2013, Tissue-Specific Host Recognition by Complement Factor H Is Mediated by Differential Activities of Its Glycosaminoglycan-Binding Regions, JOURNAL OF IMMUNOLOGY, Vol: 190, Pages: 2049-2057, ISSN: 0022-1767
Zhang H, Zhang S, Tao G, et al., 2013, Typing of Blood-Group Antigens on Neutral Oligosaccharides by Negative-Ion Electrospray Ionization Tandem Mass Spectrometry, Analytical Chemistry, Vol: 85, Pages: 5940-5949-5940-5949
Foley SA, Szegezdi E, Mulloy B, et al., 2012, Erratum: An unfractionated fucoidan from Ascophyllum nodosum: Extraction, characterization, and apoptotic effects in vitro (Journal of Natural Products (2011) 749 (1851-1861) DOI: 10.1021/np200124m), Journal of Natural Products, Vol: 75, ISSN: 0163-3864
Foley SA, Szegezdi E, Mulloy B, et al., 2012, An Unfractionated Fucoidan from Ascophyllum nodosum: Extraction, Characterization, and Apoptotic Effects in Vitro (vol 74, pg 1851, 2011), JOURNAL OF NATURAL PRODUCTS, Vol: 75, Pages: 1674-1674, ISSN: 0163-3864
Vipond C, Mulloy B, Rigsby P, et al., 2012, Evaluation of a candidate International Standard for Meningococcal Group C polysaccharide, BIOLOGICALS, Vol: 40, Pages: 353-363, ISSN: 1045-1056
Khan S, Nan R, Gor J, et al., 2012, Bivalent and co-operative binding of complement Factor H to heparan sulfate and heparins, BIOCHEMICAL JOURNAL, Vol: 444, Pages: 417-428, ISSN: 0264-6021
Troeberg L, Mulloy B, Ghosh P, et al., 2012, Pentosan polysulfate increases affinity between ADAMTS-5 and TIMP-3 through formation of an electrostatically driven trimolecular complex, BIOCHEMICAL JOURNAL, Vol: 443, Pages: 307-315, ISSN: 0264-6021
Mulloy B, Khan S, Perkins SJ, 2012, Molecular architecture of heparin and heparan sulfate: Recent developments in solution structural studies, PURE AND APPLIED CHEMISTRY, Vol: 84, Pages: 65-76, ISSN: 0033-4545
Gray E, Hogwood J, Mulloy B, 2012, The anticoagulant and antithrombotic mechanisms of heparin., Handb Exp Pharmacol, Pages: 43-61, ISSN: 0171-2004
The molecular basis for the anticoagulant action of heparin lies in its ability to bind to and enhance the inhibitory activity of the plasma protein antithrombin against several serine proteases of the coagulation system, most importantly factors IIa (thrombin), Xa and IXa. Two major mechanisms underlie heparin's potentiation of antithrombin. The conformational changes induced by heparin binding cause both expulsion of the reactive loop and exposure of exosites of the surface of antithrombin, which bind directly to the enzyme target; and a template mechanism exists in which both inhibitor and enzyme bind to the same heparin molecule. The relative importance of these two modes of action varies between enzymes. In addition, heparin can act through other serine protease inhibitors such as heparin co-factor II, protein C inhibitor and tissue factor plasminogen inhibitor. The antithrombotic action of heparin in vivo, though dominated by anticoagulant mechanisms, is more complex, and interactions with other plasma proteins and cells play significant roles in the living vasculature.
Mulloy B, 2012, Structure and physicochemical characterisation of heparin., Handb Exp Pharmacol, Pages: 77-98, ISSN: 0171-2004
Heparin is a member of the heparan sulphate family of glycosaminoglycans, a linear polysaccharide with a complex sequence resulting from the action of post-polymerisation enzymes on a regular repeating disaccharide background. Its overall conformation is rod-like in solution as well as in the solid state, but the conformational fluctuations of iduronate residues give rise to considerable internal motion and variation in local three-dimensional structure. Structure/function relationships and their relation to sequence are still the subject of argument, but new methodologies to tackle the subject are emerging. Heparin as a therapeutic agent and as the object of research may be characterised by numerous physico-chemical techniques. These include chromatographic methods for measurement of molecular weight; a variety of spectroscopic techniques; separation methods for whole polysaccharides, as well as for oligo- and monosaccharides; and mass spectrometric methods for mapping and sequence analysis. The impetus provided by the discovery of heparin contamination with oversulphated chondroitin sulphate has been influential in bringing combinations of many old and new techniques into use to ensure that heparin is sufficiently consistent and pure to be used safely. Synthetic and semi-synthetic heparins are in development and may become reality in the relatively near future.
Clark J, Ridge LA, Herbert AP, et al., 2011, The CCP6-8 and CCP19-20 'heparin'-binding regions of complement factor H have different and tissue-specific glycosaminoglycan-binding properties, 13th European Meeting on Complement in Human Disease, Publisher: PERGAMON-ELSEVIER SCIENCE LTD, Pages: 1716-1717, ISSN: 0161-5890
Khan S, Rodriguez E, Patel R, et al., 2011, The Solution Structure of Heparan Sulfate Differs from That of Heparin IMPLICATIONS FOR FUNCTION, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 286, Pages: 24842-24854
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