Heparin-protein interactions

Heparin, a sulfated polysaccharide belonging to the family of glycosaminoglycans, has numerous important biological activities, associated with its interaction with diverse proteins. Heparin is widely used as an anticoagulant drug based on its ability to accelerate the rate at which antithrombin inhibits serine proteases in the blood coagulation cascade. Heparin and the structurally related heparan sulfate are complex linear polymers comprised of a mixture of chains of different length, having variable sequences. Heparan sulfate is ubiquitously distributed on the surfaces of animal cells and in the extracellular matrix. It also mediates various physiologic and pathophysiologic processes. Difficulties in evaluating the role of heparin and heparan sulfate in vivo may be partly ascribed to ignorance of the detailed structure and sequence of these polysaccharides. In addition, the understanding of carbohydrate-protein interactions has lagged behind that of the more thoroughly studied protein-protein and protein-nucleic acid interactions. The recent extensive studies on the structural, kinetic, and thermodynamic aspects of the protein binding of heparin and heparan sulfate have led to an improved understanding of heparin-protein interactions. A high degree of specificity could be identified in many of these interactions. An understanding of these interactions at the molecular level is of fundamental importance in the design of new highly specific therapeutic agents. This review focuses on aspects of heparin structure and conformation, which are important for its interactions with proteins. It also describes the interaction of heparin and heparan sulfate with selected families of heparin-binding proteins.     

Ethylenically unsaturated polycarbosiloxanes for novel silicone hydrogels: synthesis,end‐group analysis,contact lens formulations,and structure–property correlations

A series of novel mono‐ethylenically unsaturated polycarbosiloxanes macromonomers as compatibilizer materials for soft silicon hydrogels were prepared from the anionic ring‐opening polymerization (AROP) of 2,2,5,5‐tetramethyl‐2,5‐disila‐1‐oxacyclopentane followed by methacrylation. The characterization was performed by end‐group analysis and included the determination of molecular weight, molecular weight distributions, end‐group functionality, and impurity profiles using gas chromatography‐mass spectrometry, gel permeation chromatography, nuclear magnetic resonance, and matrix‐assisted laser desorption/ionization time of flight mass spectrometry. The synthetic procedure was optimized to minimize the formation of any dimer that would have the potential to act as a low molecular weight cross linker. In addition, the novel di‐ethylenically unsaturated polycarbosiloxanes were synthesized by cationic polymerization. Use of these silicone derivatives was explored in the formulation of contact lenses, and the structure–property relationship was examined. When copoymerized with hydrophilic monomers, these were able to give transparent and wettable films with desired properties, particularly a low moduli for contact lenses. Contact lens with high modulus is often shown to impart a higher degree discomfort when worn upon the eye. It was clear from the structure–property relationship that the modulus and the tensile strength of the formulated material depend on the nature and length of the polycarbosiloxane used and may be tuned for the purpose as needed. Copyright © 2013 John Wiley & Sons, Ltd.     

3-O-Sulfation of Heparan Sulfate Enhances Tau Interaction and Cellular Uptake

Prion-like transcellular spreading of tau in Alzheimer's Disease (AD) is mediated by tau binding to cell surface heparan sulfate (HS). However, the structural determinants for tau–HS interaction are not well understood. Microarray and SPR assays of structurally defined HS oligosaccharides show that a rare 3-O-sulfation (3-O-S) of HS significantly enhances tau binding. In Hs3st1^−/− (HS 3-O-sulfotransferase-1 knockout) cells, reduced 3-O-S levels of HS diminished both cell surface binding and internalization of tau. In a cell culture, the addition of a 3-O-S HS 12-mer reduced both tau cell surface binding and cellular uptake. NMR titrations mapped 3-O-S binding sites to the microtubule binding repeat 2 (R2) and proline-rich region 2 (PRR2) of tau. Tau is only the seventh protein currently known to recognize HS 3-O-sulfation. Our work demonstrates that this rare 3-O-sulfation enhances tau–HS binding and likely the transcellular spread of tau, providing a novel target for disease-modifying treatment of AD and other tauopathies.     

High-Field Asymmetric-Waveform Ion Mobility Spectrometry and Electron Detachment Dissociation of Isobaric Mixtures of Glycosaminoglycans

High-field asymmetric waveform ion mobility spectrometry (FAIMS) is shown to be capable of resolving isomeric and isobaric glycosaminoglycan negative ions and to have great utility for the analysis of this class of molecules when combined with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) and tandem mass spectrometry. Electron detachment dissociation (EDD) and other ion activation methods for tandem mass spectrometry can be used to determine the sites of labile sulfate modifications and for assigning the stereochemistry of hexuronic acid residues of glycosaminoglycans (GAGs). However, mixtures with overlapping mass-to-charge values present a challenge, as their precursor species cannot be resolved by a mass analyzer prior to ion activation. FAIMS is shown to resolve two types of mass-to-charge overlaps. A mixture of chondroitin sulfate A (CSA) oligomers with 4–10 saccharides units produces ions of a single mass-to-charge by electrospray ionization, as the charge state increases in direct proportion to the degree of polymerization for these sulfated carbohydrates. FAIMS is shown to resolve the overlapping charge. A more challenging type of mass-to-charge overlap occurs for mixtures of diastereomers. FAIMS is shown to separate two sets of epimeric GAG tetramers. For the epimer pairs, the complexity of the separation is reduced when the reducing end is alkylated, suggesting that anomers are also resolved by FAIMS. The resolved components were activated by EDD and the fragment ions were analyzed by FTICR-MS. The resulting tandem mass spectra were able to distinguish the two epimers from each other.

3‐O‐Sulfation of Heparan Sulfate Enhances Tau Interaction and Cellular Uptake

Prion‐like transcellular spreading of tau in Alzheimer's Disease (AD) is mediated by tau binding to cell surface heparan sulfate (HS). However, the structural determinants for tau–HS interaction are not well understood. Microarray and SPR assays of structurally defined HS oligosaccharides show that a rare 3‐O‐sulfation (3‐O‐S) of HS significantly enhances tau binding. In Hs3st1^?/? (HS 3‐O‐sulfotransferase‐1 knockout) cells, reduced 3‐O‐S levels of HS diminished both cell surface binding and internalization of tau. In a cell culture, the addition of a 3‐O‐S HS 12‐mer reduced both tau cell surface binding and cellular uptake. NMR titrations mapped 3‐O‐S binding sites to the microtubule binding repeat 2 (R2) and proline‐rich region 2 (PRR2) of tau. Tau is only the seventh protein currently known to recognize HS 3‐O‐sulfation. Our work demonstrates that this rare 3‐O‐sulfation enhances tau–HS binding and likely the transcellular spread of tau, providing a novel target for disease‐modifying treatment of AD and other tauopathies.     

Capillary electrophoresis for the analysis of glycosaminoglycans and glycosaminoglycan-derived oligosaccharides

Glycosaminoglycans are a family of polydisperse, highly sulfated complex mixtures of linear polysaccharides that are involved in many life processes. Defining the structure of glycosaminoglycans is an important factor in elucidating their structure-activity relationship. Capillary electrophoresis has emerged as a highly promising technique consuming an extremely small amount of sample and capable of rapid, high-resolution separation, characterization and quantitation of analytes. Numerous capillary electrophoresis methods for analysis of intact glycosaminoglycans and glycosaminoglycan-derived oligosaccharides have been developed. These methods allow for both qualitative and quantitative analysis with a high level of sensitivity. This review is concerned with separation methods of capillary electrophoresis, detection methods and applications to several aspects of research into glycosaminoglycans and glycosaminoglycan-derived oligosaccharides. The importance of capillary electrophoresis in biological and pharmaceutical samples in glycobiology and carbohydrate biochemistry and its possible applications in disease diagnosis and monitoring chemical synthesis are described.     

Non-covalent stabilization of a beta-hairpin peptide into liposomes

An oligopeptide modified on both the N- and C-termini with hydrophobic moieties was prepared on a solid phase and anchored into a liposome, stabilizing the fold of the peptide into a beta-hairpin, which would otherwise be a random coil.     

Capillary electrophoresis of complex natural polysaccharides

Complex natural polysaccharides, glycosaminoglycans (GAGs), are a class of ubiquitous macromolecules that exhibit a wide range of biological functions and participate and regulate multiple cellular events and (patho)physiological processes. They are generally present either as free chains (hyaluronic acid and bacterial acidic polysaccharides) or as side chains of proteoglycans (PGs; chondroitin/dermatan sulfate, heparin/heparan sulfate, and keratan sulfate) and are most often found in cell membranes and in the extracellular matrix. The recent emergence of modern analytical tools for their study has produced a virtual explosion in the field of glycomics. CE, due to its high resolving power and sensitivity, has been useful in the analysis of intact GAGs and GAG-derived oligosaccharides and disaccharides affording concentration and structural characterization data essential for understanding the biological functions of GAGs. In this review, novel off-line and on-line CE-MS and MS/MS methods for screening of GAG-derived oligosaccharides and disaccharides will be discussed.     

Solution structures of chemoenzymatically synthesized heparin and its precursors

We report the first chemoenzymatic synthesis of the stable isotope-enriched heparin from a uniformly labeled [(13)C,(15)N]N-acetylheparosan (-GlcA(1,4)GlcNAc-) prepared from E. coli K5. Glycosaminoglycan (GAG) precursors and heparin were formed from N-acetylheparosan by the following steps: chemical N-deacetylation and N-sulfonation leading to N-sulfoheparosan (-GlcA(1,4)GlcNS-); enzyme-catalyzed C5-epimerization and 2-O-sulfonation leading to undersulfated heparin (-IdoA2S(1,4)GlcNS-); enzymatic 6-O-sulfonation leading to the heparin backbone (-IdoA2S(1,4)GlcNS6S-); and selective enzymatic 3-O-sulfonation leading to the anticoagulant heparin, containing the GlcNS6S3S residue. Heteronuclear, multidimensional nuclear magnetic resonance spectroscopy was employed to analyze the chemical composition and solution structure of [(13)C,(15)N]N-acetylheparosan, precursors, and heparin. Isotopic enrichment was found to provide well-resolved (13)C spectra with the high sensitivity required for conformational studies of these biomolecules. Stable isotope-labeled heparin was indistinguishable from heparin derived from animal tissues and is a novel reagent for studying the interaction of heparin with proteins.