The potential for circular dichroism as an additional facile and sensitive method of monitoring low-molecular-weight heparins and heparinoids

The ultraviolet circular dichroism (CD) spectra of commercial low-molecular-weight heparins, heparinoids and other anticoagulant preparations have been recorded between 180 and 260 nm. Principal component analysis of the spectra allowed their differentiation into a number of groups related to the means of their production reflecting the structural changes introduced by each process. The findings suggest that CD provides a complementary technique for the rapid analysis of heparin preparations

Heparan sulfate regulates amyloid precursor protein processing by BACE1, the Alzheimer's β-secretase

Cleavage of amyloid precursor protein (APP) by the Alzheimer's β-secretase (BACE1) is a key step in generating amyloid β-peptide, the main component of amyloid plaques. Here we report evidence that heparan sulfate (HS) interacts with β-site APP-cleaving enzyme (BACE) 1 and regulates its cleavage of APP. We show that HS and heparin interact directly with BACE1 and inhibit in vitro processing of peptide and APP substrates. Inhibitory activity is dependent on saccharide size and specific structural characteristics, and the mechanism of action involves blocking access of substrate to the active site. In cellular assays, HS specifically inhibits BACE1 cleavage of APP but not alternative cleavage by α-secretase. Endogenous HS immunoprecipitates with BACE1 and colocalizes with BACE1 in the Golgi complex and at the cell surface, two of its putative sites of action. Furthermore, inhibition of cellular HS synthesis results in enhanced BACE1 activity. Our findings identify HS as a natural regulator of BACE1 and suggest a novel mechanism for control of APP processing

Heparin binding preference and structures in the fibroblast growth factor family parallel their evolutionary diversification

The interaction of a large number of extracellular proteins with heparan sulfate (HS) regulates their transport and effector functions, but the degree of molecular specificity underlying protein–polysaccharide binding is still debated. The 15 paracrine fibroblast growth factors (FGFs) are one of the paradigms for this interaction. Here, we measure the binding preferences of six FGFs (FGF3, FGF4, FGF6, FGF10, FGF17, FGF20) for a library of modified heparins, representing structures in HS, and model glycosaminoglycans, using differential scanning fluorimetry. This is complemented by the identification of the lysine residues in the primary and secondary binding sites of the FGFs by a selective labelling approach. Pooling these data with previous sets provides good coverage of the FGF phylogenetic tree, deduced from amino acid sequence alignment. This demonstrates that the selectivity of the FGFs for binding structures in sulfated polysaccharides and the pattern of secondary binding sites on the surface of FGFs follow the phylogenetic relationship of the FGFs, and so are likely to be the result of the natural selection pressures that led to the expansion of the FGF family in the course of the evolution of more complex animal body plans

Introduction to the Molecules Special Edition Entitled 'Heparan Sulfate and Heparin: Challenges and Controversies': Some Outstanding Questions in Heparan Sulfate and Heparin Research

The scope of this article is to provide a brief general introduction to heparan sulfate (HS) and heparin, and attempt to identify some of the central challenges regarding research into the chemistry and biology of glycosaminoglycans (GAGs), some of which are the subject of contributions to the special issue of Molecules (published in volume 23, 2018) entitled ‘Heparan Sulfate and Heparin: Challenges and Controversies’ [...

Chemical modification of glycosaminoglycan polysaccharides

The linear anionic class of polysaccharides, glycosaminoglycans (GAGs), are critical throughout the animal kingdom for developmental processes and the maintenance of healthy tis-sues. They are also of interest as a means of influencing biochemical processes. One member of the GAG family, heparin, is exploited globally as a major anticoagulant pharmaceutical and there is a growing interest in the potential of other GAGs for diverse applications ranging from skin care to the treatment of neurodegenerative conditions, and from the treatment and prevention of microbial infection to biotechnology. To realize the potential of GAGs, however, it is necessary to develop effective tools that are able to exploit the chemical manipulations to which GAGs are susceptible. Here, the current knowledge concerning the chemical modification of GAGs, one of the principal approaches for the study of the structure-function relationships in these molecules, is reviewed. Some additional methods that were applied successfully to the analysis and/or processing of other carbohydrates, but which could be suitable in GAG chemistry, are also discussed

Nuclear Magnetic Resonance and Molecular Dynamics Simulation of the Interaction between Recognition Protein H7 of the Novel Influenza Virus H7N9 and Glycan Cell Surface Receptors

Avian influenza A viruses, which can also propagate between humans, present serious pandemic threats, particularly in Asia. The specificity (selectivity) of interactions between the recognition protein hemagglutinin (HA) of the virus capsid and the glycoconjugates of host cells also contributes to the efficient spread of the virus by aerosol between humans. Some avian origin viruses, such as H1N1 (South Carolina 1918), have improved their selectivity for human receptors by mutation in the HA receptor binding site, to generate pandemic viruses. Molecular details and dynamics of glycan–HA interactions are of interest, both in predicting the pandemic potential of a new emerging strain and in searching for new antiviral drugs. Two complementary techniques, ¹H saturation transfer difference (¹H STD) nuclear magnetic resonance and molecular dynamics (MD) simulation, were applied to analyze the interaction of the new H7 (A/Anhui/1/13 H7N9) with LSTa [Neu5Ac α(2→3) Gal β(1→3) GlcNAc β(1→3) Gal β(1→4) Glc] and LSTc [Neu5Ac α(2→6) Gal β(1→4) GlcNAc β(1→3) Gal β(1→4) Glc] pentasaccharides, models of avian and human receptor glycans. Their interactions with H7 were analyzed for the first time using ¹H STD and MD, revealing structural and dynamic behavior that could not be obtained from crystal structures, and contributing to glycan–HA specificity. This highlighted aspects that could affect glycan–HA recognition, including the mutation H7 G228S, which increases H2 and H3 specificity for the human receptor. Finally, interactions between LSTc and H7 were compared with those between LSTc and H1 of H1N1 (South Carolina 1918), contributing to our understanding of the recognition ability of HAs

Mobility shift-based electrophoresis coupled with fluorescent detection enables real-time enzyme analysis of carbohydrate sulfatase activity

Sulfated carbohydrate metabolism is a fundamental process, which occurs in all domains of life. Carbohydrate sulfatases are enzymes that remove sulfate groups from carbohydrates and are essential to the depolymerisation of complex polysaccharides. Despite their biological importance, carbohydrate sulfatases are poorly studied and challenges remain in accurately assessing the activity, specificity and kinetic parameters. Most notably, separation of desulfated products from sulfated substrates is currently a time-consuming process. In this paper, we describe the development of rapid capillary electrophoresis coupled to substrate fluorescence detection as a high-throughput and facile means of analysing carbohydrate sulfatase activity. The approach has utility for the determination of both kinetic and inhibition parameters and is based on existing microfluidic technology coupled to a new synthetic fluorescent 6S-GlcNAc carbohydrate substrate. Furthermore, we compare this technique in terms of both time and resources, to high performance anion exchange chromatography and NMR-based methods, which are the two current ‘gold standards’ for enzymatic carbohydrate sulfation analysis. Our study clearly demonstrates the advantages of mobility shift assays for the quantification of near real-time carbohydrate desulfation by purified sulfatases, and could support the search for small molecule inhibitors of these disease-associated enzymes. One sentence summary Sulfatases remove sulfate groups from biomolecules; in this study we report a rapid and robust capillary electrophoresis assay for the quantification of carbohydrate desulfation

Molecular Origins of the Compatibility Between Glycosaminoglycans and Aβ40 Amyloid Fibrils

The Aβ peptide forms extracellular plaques associated with Alzheimer's disease. In addition to protein fibrils, amyloid plaques also contain non-proteinaceous components, including glycosaminoglycans (GAGs). We have shown previously that the GAG low molecular weight heparin (LMWH) binds to Aβ40 fibrils with a three-fold-symmetric (3Q) morphology with higher affinity than Aβ40 fibrils in alternative structures, Aβ42 fibrils, or amyloid fibrils formed from other sequences. Solid-state NMR (SSNMR) analysis of the GAG-3Q fibril complex revealed an interaction site at the corners of the 3Q fibril structure, but the origin of the binding specificity remained obscure. Here, employing a library of short heparin polysaccharides modified at specific sites, we show that the NS or 6-OS glucosamine sulfates, but not the 2-OS iduronate sulfate, of heparin is required for 3Q binding, indicating selectivity in the interactions of the GAG with the fibril that extends beyond general electrostatic complementarity. By creating 3Q fibrils containing point substitutions in the amino acid sequence, we also show that charged residues at the fibril three-fold apices provide the majority of the binding free energy, while charged residues elsewhere are less critical for binding. The results indicate, therefore, that LMWH binding to 3Q fibrils requires a precise molecular complementarity of the sulfate moieties on the GAG and charged residues displayed on the fibril surface. Differences in GAG binding to fibrils with distinct sequence and/or structure may thus contribute to the diverse etiology and progression of amyloid diseases

By-Products of Heparin Production Provide a Diverse Source of Heparin-like and Heparan Sulfate Glycosaminoglycans.

Global production of pharmaceutical heparin (Hp) is increasing, and the production process from raw mucosal material results in large amounts of waste by-products. These contain lower sulfated Hp-like and heparan sulfate (HS), as well as other glycosaminoglycans, which are bioactive entities with pharmaceutical potential. Here we describe the first purification, structural and functional characterisation of Hp-like and HS polysaccharides from the four major by-product fractions of standard heparin production. Analysis of the by-products by disaccharide composition analysis and NMR demonstrated a range of structural characteristics which differentiate them from Hp (particularly reduced sulfation and sulfated disaccharide content), and that they are each distinct. Functional properties of the purified by-products varied, each displaying distinct anticoagulant profiles in different assays, and all exhibiting significantly lower global and specific inhibition of the coagulation pathway than Hp. The by-products retained the ability to promote cell proliferation via fibroblast growth factor receptor signalling, with only minor differences between them. These collective analyses indicate that they represent an untapped and economical source of structurally-diverse Hp-like and HS polysaccharides with the potential for enhancing future structure-activity studies and uncovering new biomedical applications of these important natural products

Antithrombin stabilisation by sulfated carbohydrates correlates with anticoagulant activity

Thermal stabilisation of native antithrombin-III (AT), determined using differential scanning fluorimetry, correlated with the anticoagulant activity of heparin and heparin-related saccharides. Similar conformational changes were induced in native AT by a variety of active and inactive heparin-related sulfated carbohydrates, measured in solution using synchrotron radiation circular dichroism, and their anticoagulant activities. Measurement of native AT stabilisation provides a convenient assay for prospective anticoagulants and represents an additional parameter by which to compare biosimilar heparins.National Science Foundation's National High Magnetic Field Laboratory User Program in the Advanced Magnetic Resonance Imaging and Spectroscopy (AMRIS) Facility (McKnight Brain Institute, University of Florida, USA)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo UNIFESP, Dept Biochem, BR-04044020 São Paulo, BrazilDiamond Light Source Ltd, Beam Line Circular Dichroism 23, Didcot OX11 ODE, Oxon, EnglandUniv Liverpool, Dept Struct & Chem Biol, Liverpool L69 7ZB, Merseyside, EnglandIst Ric Chim & Biochim G Ronzoni, I-20133 Milan, ItalyUniv Fed Rio Grande do Norte, Dept Biochem, BR-59072970 Natal, RN, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Biochem, BR-04044020 São Paulo, BrazilWeb of Scienc