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Stewart K.L.,University of Leeds | Hughes E.,Lancaster University | Yates E.A.,University of Liverpool | Akien G.R.,Lancaster University | And 7 more authors.
Journal of the American Chemical Society

The amyloid plaques associated with Alzheimer's disease (AD) comprise fibrillar amyloid-β (Aβ) peptides as well as non-protein factors including glycosaminoglycan (GAG) polysaccharides. GAGs affect the kinetics and pathway of Aβ self-assembly and can impede fibril clearance; thus, they may be accessory molecules in AD. Here we report the first high-resolution details of GAG-Aβ fibril interactions from the perspective of the saccharide. Binding analysis indicated that the GAG proxy heparin has a remarkably high affinity for Aβ fibrils with 3-fold cross-sectional symmetry (3Q). Chemical synthesis of a uniformly 13C-labeled octasaccharide heparin analogue enabled magic-angle spinning solid-state NMR of the GAG bound to 3Q fibrils, and measurements of dynamics revealed a tight complex in which all saccharide residues are restrained without undergoing substantial conformational changes. Intramolecular 13C-15N dipolar dephasing is consistent with close (<5 Å) contact between GAG anomeric position(s) and one or more histidine residues in the fibrils. These data provide a detailed model for the interaction between 3Q-seeded Aβ40 fibrils and a major non-protein component of AD plaques, and they reveal that GAG-amyloid interactions display a range of affinities that critically depend on the precise details of the fibril architecture. © 2016 American Chemical Society. Source

Uniewicz K.A.,University of Liverpool | Uniewicz K.A.,PromoCell GmbH Sickingenstr | Ori A.,Structural and Computational Biology Unit | Rudd T.R.,University of Liverpool | And 5 more authors.
Methods in Molecular Biology

Studies of the structural changes invoked in proteins by the binding of the glycosaminoglycan (GAG) polysaccharide portion of proteoglycans are of increasing importance to research in a wide range of fields, from biochemistry and molecular biology to biotechnology and medicine. One important aspect is the degree of stabilisation or destabilisation induced in a protein by the binding of these anionic materials, and this can affect enzyme activity, the stability of complexes, folding and the formation of aggregates, including those in neurodegenerative processes. A simple method, able to determine the effect of interactions with GAG polysaccharides on protein stability is described, based on the propensity of a fluorescent dye-Sypro™ Orange-to present differentiable fluorescence emission spectra following contact with exposed core amino acid residues. The method requires only commonly available and inexpensive equipment and is suitable for a multi-well format, allowing multiple readings to be made simultaneously. © 2012 Springer Science+Business Media, LLC. Source

Schlesinger M.,University of Bonn | Schmitz P.,University of Bonn | Zeisig R.,Max Delbruck Center for Molecular Medicine | Naggi A.,Ronzoni Institute for Chemical and Biochemical Research | And 3 more authors.
Thrombosis Research

Introduction: The integrin VLA-4-mediated binding is important for the metastatic dissemination of melanoma cells. Recently we found that heparin possesses a binding capacity to VLA-4. This could contribute to the heparin function to attenuate metastasis in a selectin-dependent manner. Aiming to a purposive, anti-adhesive heparin application, structural requirements of heparin for VLA-4 recognition have to be elucidated. Materials and methods: A series of non-anticoagulant heparin derivatives were investigated concerning their inhibitory capacities for VLA-4 mediated binding of human melanoma MV3 cells to VCAM-1 under physiological flow conditions in vitro. A surface acoustic wave biosensor was applied to detect kinetic constants of selected derivatives binding to both, VLA-4 or P- and L-selectin. Results: Experimental metastasis of MV3 cells in mice confirmed the relevance of VLA-4 for metastatic dissemination. LMWHs (enoxaparin, tinzaparin) efficiently blocked VLA-4 cell binding, dominantly via the integrin's α-chain. Desulfation at 2-O-position, N-acetylation or a size smaller than tetradecasaccharide disfavoured VLA-4 inhibition. Glycol-splitting of heparin and thus higher chain flexibility is a tolerable parameter. A derivative with 50% 6-O-desulfation appeared promising and exceeded tinzaparin in VLA-4 inhibition, both compounds displayed binding affinities to VLA-4 in the low micromolar range. Conclusions: These findings provide structure-activity relationships for heparin VLA-4 binding, which partly differ from P- and L-selectin requirements. The data confirm that anti-coagulative and anti-adhesive function of heparin can be distinguished favouring applications of non-anticoagulant heparins in antimetastatic approaches without the risk of bleeding complications. The 50% 6-O-desulfated heparin-derivative appears promising to further evaluate the interference with selectin and VLA-4 binding functions in vivo. © 2012 Elsevier Ltd. All rights reserved. Source

Alekseeva A.,Ronzoni Institute for Chemical and Biochemical Research | Casu B.,Ronzoni Institute for Chemical and Biochemical Research | Torri G.,Ronzoni Institute for Chemical and Biochemical Research | Pierro S.,Ronzoni Institute for Chemical and Biochemical Research | Naggi A.,Ronzoni Institute for Chemical and Biochemical Research
Analytical Biochemistry

Glycol-split (gs) heparins, obtained by periodate oxidation/borohydride reduction of heparin currently used as an anticoagulant and antithrombotic drug, are arousing increasing interest in anticancer and anti-inflammation therapies. These new medical uses are favored by the loss of anticoagulant activity associated with glycol-splitting-induced inactivation of the antithrombin III (AT) binding site. The structure of gs heparins has not been studied yet in detail. In this work, ion pair reversed-phase high-performance liquid chromatography (IPRP-HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS) widely used for unmodified heparin has been adapted to the analysis of oligosaccharides generated by digestion with heparinases of gs heparins usually prepared from porcine mucosal heparin. The method was also found to be very effective in analyzing gs derivatives obtained from heparins of different animal and tissue origins. Besides the major 2-O-sulfated disaccharides, heparinase digests of gs heparins contain mainly tetra- and hexasaccharides incorporating one or two gs residues, with distribution patterns typical for individual gs heparins. A heptasulfated, mono-N-acetylated hexasaccharide with two gs residues was shown to be a marker of the gs-modified AT binding site within heparin chains. © 2012 Elsevier Inc. All rights reserved. Source

Alekseeva A.,Ronzoni Institute for Chemical and Biochemical Research | Casu B.,Ronzoni Institute for Chemical and Biochemical Research | Cassinelli G.,Ronzoni Institute for Chemical and Biochemical Research | Guerrini M.,Ronzoni Institute for Chemical and Biochemical Research | And 2 more authors.
Analytical and Bioanalytical Chemistry

Periodate oxidation followed by borohydride reduction converts the well-known antithrombotics heparin and low-molecular-weight heparins (LMWHs) into their "glycol-split" (gs) derivatives of the "reduced oxyheparin" (RO) type, some ofwhich are currently being developed as potential anti-cancer and anti-inflammatory drugs. Whereas the structure of gs-heparins has been recently studied, details of the more complex and more bioavailable gs-LMWHs have not been yet reported. We obtained RO derivatives of the three most common LMWHs (tinzaparin, enoxaparin, and dalteparin) and studied their structures by two-dimensional nuclear magnetic resonance spectroscopy and ion-pair reversed-phase high-performance liquid chromatography coupled with electrospray ionization mass spectrometry. The liquid chromatography-mass spectrometry (LC-MS) analysis was extended to their heparinase-generated oligosaccharides. The combined NMR/LC-MS analysis of RO-LMWHs provided evidence for glycol-splitting-induced transformations mainly involving internal nonsulfated glucuronic and iduronic acid residues (including partial hydrolysis with formation of "remnants") and for the hydrolysis of the gs uronic acid residues when formed at the non-reducing ends (mainly, in RO-dalteparin). Evidence for minor modifications, such as ring contraction of some dalteparin internal aminosugar residues, was also obtained. Unexpectedly, the N-sulfated 1,6-anhydromannosamine residues at the enoxaparin reducing end were found to be susceptible to the periodate oxidation. In addition, in tinzaparin and enoxaparin, the borohydride reduction converts the hemiacetalic aminosugars at the reducing end to alditols. Typical LC-MS signatures of ROderivatives of individual LMWH both before and after digestion with heparinases included oligosaccharides generated from the original antithrombin-binding and "linkage" regions. © Springer-Verlag Berlin Heidelberg 2013. Source

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