Free radical generation during chemical depolymerization of heparin

Low-molecular weight heparins (LMWHs), as compared with unfractionated heparin (UFH), present superior bioavailability, much longer plasma half-life, and lower incidence of side effects. For these reasons, over the past two decades LMWHs have become the drugs of choice for the treatment of deep venous thrombosis, pulmonary embolism, arterial thrombosis, and unstable angina. Furthermore, their use in acute ischemic stroke is currently under study. LMWHs are obtained by UFH depolymerization, which can be performed using various methods, including nitrous acid depolymerization, cleavage by beta-elimination of benzyl ester, enzymatic depolymerization, and peroxyl radical-dependent depolymerization. This article addresses the chemical depolymerization, obtained by free radical attack (mainly hydroxyl radical), of heparin. The electron spin resonance (ESR) spectroscopy, coupled to the spin trapping technique, was employed to study this reaction. Free radical-mediated heparin depolymerization was performed under different chemical conditions. The final products of the reactions were purified and classified on the basis of their molecular weight and other characteristics. The level of heparin fragmentation was different depending on the type of depolymerization reaction used. Moreover, the level of reproducibility and the resulting radical species were different for every type of reaction performed.     

Ozonolysis of the double bond of the unsaturated uronate residue in low-molecular-weight heparin and K5 heparosan

Ozone is known to add across and cleave carbon–carbon double bonds. Ozonolysis is widely used for the preparation of pharmaceuticals, for bleaching substances and for killing microorganisms in air and water sources. Some polysaccharides and oligosaccharides, such as those prepared using chemical or enzymatic β-elimination, contain a site of unsaturation. We examined ozonolysis of low-molecular-weight heparins (LMWHs), enoxaparin and logiparin, and heparosan oligo- and polysaccharides for the removal of the nonreducing terminal unsaturated uronate residue. 1D ¹H NMR showed that these ozone-treated polysaccharides retained the same structure as the starting polysaccharide, except that the C4–C5 double bond in the nonreducing end unsaturated uronate had been removed. The anticoagulant activity of the resulting product from enoxaparin and logiparin was comparable to that of the starting material. These results demonstrate that ozonolysis is an important tool for the removal of unsaturated uronate residues from LMWHs and heparosan without modification of the core polysaccharide structure or diminution of anticoagulant activity. This reaction also has potential applications in the chemoenzymatic synthesis of bioengineered heparin from Escherichia coli-derived K5 heparosan.     

Controlled Photochemical Depolymerization of K5 Heparosan, a Bioengineered Heparin Precursor

Heparosan is a polysaccharide, which serves as the critical precursor in heparin biosynthesis and chemoenzymatic synthesis of bioengineered heparin. Because the molecular weight of microbial heparosan is considerably larger than heparin, the controlled depolymerization of microbial heparosan is necessary prior to its conversion to bioengineered heparin. We have previously reported that other acidic polysaccharides could be partially depolymerized with maintenance of their internal structure using a titanium dioxide-catalyzed photochemical reaction. This photolytic process is characterized by the generation of reactive oxygen species that oxidize individual saccharide residues within the polysaccharide chain. Using a similar approach, a microbial heparosan from Escherichia coli K5 of molecular weight >15,000 was depolymerized to a heparosan of molecular weight 8,000. The (1)H-NMR spectra obtained showed that the photolyzed heparosan maintained the same structure as the starting heparosan. The polysaccharide chains of the photochemically depolymerized heparosan were also characterized by electrospray ionization-Fourier-transform mass spectrometry. While the chain of K5 heparosan starting material contained primarily an even number of saccharide residues, as a result of coliphage K5 lyase processing, both odd and even chain numbers were detected in the photochemically-depolymerized heparosan. These results suggest that the photochemical depolymerization of heparosan was a random process that can take place at either the glucuronic acid or the N-acetylglucosamine residue within the heparosan polysaccharide.     

Low-molecular-weight heparins in the treatment of venous thromboembolism

Venous thromboembolism is a common disease that is associated with considerable morbidity if left untreated. Recently, low-molecular-weight heparins (LMWHs) have been evaluated for use in acute treatment of deep venous thrombosis and pulmonary embolism. Randomized studies have shown that LMWHs are as effective as unfractionated heparin in the prevention of recurrent venous thromboembolism, and are as safe with respect to the occurrence of major bleeding. A pooled analysis did not show substantial differences among different LMWH compounds used, but no direct comparison of the different LMWHs is currently available. Finally, in patients with pulmonary embolism, there is a relative lack of large studies of daily practice. It could be argued that large prospective studies, in patients who were treated with LMWHs from the moment of diagnosis, are needed.     

Role of reducing terminals in unfractionated and low-molecular-mass heparins in causing free radical generation and loss of structure and activity of trypsin.

The role of both the length of saccharide chain and reducing terminals in the heparin molecule in causing oxidative effects on proteins was investigated by employing unfractionated and low-molecular-mass heparins (LMMH), with both intact and reduced reducing terminals on bovine trypsin. The effects of heparin were found to be dependent on both the concentration and time of incubation. Heparins with intact reducing terminals caused significantly higher structural and functional alterations of trypsin compared with heparins with reduced reducing terminals. LMMH was slightly more effective than unfractionated heparin (UNFH) in reducing structural integrity and inhibiting the amidolytic activity of trypsin when used at the same mass, but not molar concentrations. Neither the length of saccharide chains nor the number of intact reducing terminals on the heparin molecule appeared to influence the characteristics of the initial binding of heparin to trypsin, but both these variables crucially affected linkages which in time mediate the inhibition of catalytic activity and the formation of free radicals, ultimately responsible for peptide bond cleavage in trypsin. The results suggest that both a critical number of saccharide units, preferentially lying on shorter chains, and intact reducing terminals in the heparin molecule are involved in setting up the binding which generates radicals and leads to loss of structure and function of the proteinase.     

DNA cleavage induced by antitumor antibiotic leinamycin and its biological consequences

The natural product leinamycin has been found to produce abasic sites in duplex DNA through the hydrolysis of the glycosidic bond of guanine residues modified by this drug. In the present study, using a synthetic oligonucleotide duplex, we demonstrate spontaneous DNA strand cleavage at leinamycin-induced abasic sites through a β-elimination reaction. However, methoxyamine modification of leinamycin-induced abasic sites was found to be refractory to the spontaneous β-elimination reaction. Furthermore, this complex was even resistant to the δ-elimination reaction with hot piperidine treatment. Bleomycin and methyl methanesulfonate also induced strand cleavage in a synthetic oligonucleotide duplex even without thermal treatment. However, methoxyamine has a negligible effect on DNA strand cleavage induced by both drugs, suggesting that the mechanism of DNA cleavage induced by leinamycin might be different from those induced by bleomycin or methyl methanesulfonate. In this study, we also assessed the cytotoxicity of leinamycin against a collection of mammalian cell lines defective in various repair pathways. The mammalian cell line defective in the nucleotide excision repair (NER) or base excision repair (BER) pathways was about 3 to 5 times more sensitive to leinamycin as compared to the parental cell line. In contrast, the radiosensitive mutant xrs-5 cell line deficient in V(D)J recombination showed similar sensitivity towards leinamycin compared to the parental cell line. Collectively, our findings suggest that both NER and BER pathways play an important role in the repair of DNA damage caused by leinamycin.     

Preparation of fluorescein-labelled and biotinylated derivatives of polysaccharides for lectin-saccharide binding studies

Fluorescein- and biotin-labelled polysaccharides were prepared using ethylene diamine coupled to a polysaccharide either by carbodiimide reaction to carboxyl group or after periodate oxidation of saccharide residue and the derivative was used for labelling. Labelled hyaluronic acid, chondroitin sulfate and dextran sulfate were prepared.     

Opposing Effects of Low Molecular Weight Heparins on the Release of Inflammatory Cytokines from Peripheral Blood Mononuclear Cells of Asthmatics

Background

T-cell-mediated inflammatory cytokines, such as interleukin (IL)-4, IL-5, IL-13 and tumor necrosis factor-alpha (TNF-α), play an important role in the initiation and progression of inflammatory airways diseases. Low-molecular-weight heparins (LMWHs), widely used anticoagulants, possess anti-inflammatory properties making them potential treatment options for inflammatory diseases, including asthma. In the current study, we investigated the modulating effects of two LMWHs (enoxaparin and dalteparin) on the release of cytokines from stimulated peripheral blood mononuclear cells (PBMCs) of asthmatic subjects to identify the specific components responsible for the effects.

Methods

PBMCs from asthmatic subjects (consist of ~75% of T-cells) were isolated from blood taken from ten asthmatic subjects. The PBMCs were pre-treated in the presence or absence of different concentrations of LMWHs, and were then stimulated by phytohaemagglutinin for the release of IL-4, IL-5, IL-13 and TNF-α. LMWHs were completely or selectively desulfated and their anticoagulant effect, as well as the ability to modulate cytokine release, was determined. LMWHs were chromatographically fractionated and each fraction was tested for molecular weight determination along with an assessment of anticoagulant potency and effect on cytokine release.

Results

Enoxaparin inhibited cytokine release by more than 48%, whereas dalteparin increased their release by more than 25%. The observed anti-inflammatory effects of enoxaparin were independent of their anticoagulant activities. Smaller fractions, in particular dp4 (four saccharide units), were responsible for the inhibitory effect of enoxaparin. Whereas, the larger fractions, in particular dp22 (twenty two saccharide units), were associated with the stimulatory effect of dalteparin.

Conclusion

Enoxaparin and dalteparin demonstrated opposing effects on inflammatory markers. These observed effects could be due to the presence of structurally different components in the two LMWHs arising from different methods of depolymerisation. This study provides a platform for further studies investigating the usefulness of enoxaparin in various inflammatory diseases.     

Structure of a PL17 Family Alginate Lyase Demonstrates Functional Similarities among Exotype Depolymerases

Brown macroalgae represent an ideal source for complex polysaccharides that can be utilized as precursors for cellulosic biofuels. The lack of recalcitrant lignin components in macroalgae polysaccharide reserves provides a facile route for depolymerization of constituent polysaccharides into simple monosaccharides. The most abundant sugars in macroalgae are alginate, mannitol, and glucan, and although several classes of enzymes that can catabolize the latter two have been characterized, studies of alginate-depolymerizing enzymes have lagged. Here, we present several crystal structures of Alg17c from marine bacterium Saccharophagus degradans along with structure-function characterization of active site residues that are suggested to be involved in the exolytic mechanism of alginate depolymerization. This represents the first structural and biochemical characterization of a family 17 polysaccharide lyase enzyme. Despite the lack of appreciable sequence conservation, the structure and β-elimination mechanism for glycolytic bond cleavage by Alg17c are similar to those observed for family 15 polysaccharide lyases and other lyases. This work illuminates the evolutionary relationships among enzymes within this unexplored class of polysaccharide lyases and reinforces the notion of a structure-based hierarchy in the classification of these enzymes.     

Liquid chromatography–diode array detection–mass spectrometry for compositional analysis of low molecular weight heparins

Low molecular weight heparins (LMWHs) are important artificial preparations from heparin polysaccharide and are widely used as anticoagulant drugs. To analyze the structure and composition of LMWHs, identification and quantitation of their natural and modified building blocks are indispensable. We have established a novel reversed-phase high-performance liquid chromatography–diode array detection–electrospray ionization–mass spectrometry approach for compositional analysis of LMWHs. After being exhaustively digested and labeled with 2-aminoacridone, the structural motifs constructing LMWHs, including 17 components from dalteparin and 15 components from enoxaparin, were well separated, identified, and quantified. Besides the eight natural heparin disaccharides, many characteristic structures from dalteparin and enoxaparin, such as modified structures from the reducing end and nonreducing end, 3-O-sulfated tetrasaccharides, and trisaccharides, have been unambiguously identified based on their retention time and mass spectra. Compared with the traditional heparin compositional analysis methods, the approach described here is not only robust but also comprehensive because it is capable of identifying and quantifying nearly all components from lyase digests of LMWHs.     

In situ chemical cleavage of proteins immobilized to glass-fiber and polyvinylidenedifluoride membranes: cleavage at tryptophan residues with 2-(2'-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine to obtain internal amino acid sequence

Proteins immobilized to glass-fiber supports and polyvinylidenedifluoride membranes are cleaved in situ with a tryptophan residue-specific reagent, 2-(2'-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine. Protein fragments can be eluted from polyvinylidenedifluoride membranes after in situ digestion and electrophoresed and electroblotted to new polyvinylidenedifluoride membranes for subsequent sequence determination of selected bands. Five proteins (bovine serum albumin, ovalbumin, beta-casein, beta-lactoglobulin, and myoglobin) of known sequence containing one to three tryptophan residues each were used as model substrates to evaluate the specificity and extent of cleavage. Under the conditions used, the reaction is rapid and exhibits a high degree of specificity for tryptophan residues since N-terminal sequence analysis of the digested, immobilized samples yields the expected, newly generated N-termini. No detectable cleavage occurred at nontryptophan residues nor at acid labile aspartic acid-proline peptide bonds. Solution cleavage reactions were also performed and the resulting digest was analyzed by gel electrophoresis. We found a positive correlation between the solution and in situ cleavage reactions, in that, proteins which show cleavage in the solution reaction also yield internal sequence data after in situ digestion. Since tryptophan occurs with low frequency in proteins, this cleavage reaction has the potential to generate a small number of fragments and in favorable circumstances allow sequence analysis of the unfractionated reaction mixture. In addition, the sequence obtainable after, but not before, in situ digestion can be used as an indication that the N-terminus of the protein is blocked.     

Polysaccharide hydrolases ofAureobasidium pullulans

The polysaccharide hydrolase activity of a group of selected strains of the genus Aureobasidium pullulans was investigated using a new gel testing assay. A total of 31 strains were tested for alpha-amylase, alpha-glucosidase and glucoamylase, beta-glucosidase, lichenase, cellulase, xylanase and xylosidase, mannanase and mannosidase production during growth of microorganisms on respective meshed polysaccharide gels. Attempts were made to increase the polysaccharide hydrolase activity through selection of some A. pullulans strains by passaging them on the respective modified xylanase- and cellulase-containing gels. The individual saccharide degradation cleavage products were investigated by chromatography.     

Crystal structure of unsaturated glucuronyl hydrolase complexed with substrate: molecular insights into its catalytic reaction mechanism

Unsaturated glucuronyl hydrolase (UGL), which is a member of glycoside hydrolase family GH-88, is a bacterial enzyme that degrades mammalian glycosaminoglycans and bacterial biofilms. The enzyme, which acts on unsaturated oligosaccharides with an alpha-glycoside bond produced by microbial polysaccharide lyases responsible for bacterial invasion of host cells, was believed to release 4-deoxy-l-threo-5-hexosulose-uronate (unsaturated glucuronic acid, or DeltaGlcA) and saccharide with a new nonreducing terminus by hydrolyzing the glycosidic bond. We detail the crystal structures of wild-type inactive mutant UGL of Bacillus sp. GL1 and its complex with a substrate (unsaturated chondroitin disaccharide), identify active site residues, and postulate a reaction mechanism catalyzed by UGL that triggers the hydration of the vinyl ether group in DeltaGlcA, based on the structural analysis of the enzyme-substrate complex and biochemical analysis. The proposed catalytic mechanism of UGL is a novel case among known glycosidases. Under the proposed mechanism, Asp-149 acts as a general acid and base catalyst to protonate the DeltaGlcA C4 atom and to deprotonate the water molecule. The deprotonated water molecule attacks the DeltaGlcA C5 atom to yield unstable hemiketal; this is followed by spontaneous conversion to an aldehyde (4-deoxy-l-threo-5-hexosulose-uronate) and saccharide through hemiacetal formation and cleavage of the glycosidic bond. UGL is the first clarified alpha(6)/alpha(6)-barrel enzyme using aspartic acid as the general acid/base catalyst.     

A novel strategy to generate biologically active neo-glycosaminoglycan conjugates

Heparin and heparan sulfate are structurally related polysaccharides with a variety of biological effects/functions. Most of these effects are due to interactions, of varying specificity, between the negatively charged polysaccharide chains and proteins. While such interactions generally involve a single saccharide domain of decasaccharide size or less, ternary complexes of two protein molecules binding to separate domains on a single polysaccharide chain are known to occur. To facilitate studies on domain organization and its importance for biological function a strategy was developed to chemically conjugate defined heparin oligomers in linear and chemoselective fashion. The procedure requires that the oligosaccharide to provide the reducing-terminal domain of the conjugate is generated by lyase degradation of a parent polysaccharide, whereas the nonreducing-terminal domain is obtained through deaminative cleavage with nitrous acid. The applicability of the method was demonstrated by constructing a conjugate composed of two heparin 12-mers, of which the reducing-terminal component contained the antithrombin-binding region, whereas the nonreducing-terminal domain did not. Contrary to any of the unconjugated oligomers, the product was found to efficiently promote the inactivation of thrombin by antithrombin.     

Properties and applications of the (2-nitrofluoren-9-yl)methoxycarbonyl group.

This paper presents a new protecting group, the (2-nitrofluoren-9-yl)methoxycarbonyl group. Investigations on the properties of this new modification of the Fmoc-system, such as the solvent-dependent photochemical cleavage, and enhanced lability towards bases, are described, as well as UV-kinetic measurements of the cleavage reaction. In addition, the incorporation of the (2-nitrofluoren-9-yl)methoxycarbonyl group into two peptides, and a sequence-dependent photochemical cleavage reaction are reported.     

The properties of precipitation reactions between secretion products in the oviduct of pleurodeles: identification of a lectin

The properties of two precipitation reactions occurring between secretory products from the oviduct of Pleurodeles waltl have been studied. It has been demonstrated that a lectin is involved in one of the reactions. This lectin precipitated glycogen and starch and required calcium; the most potent saccharide inhibitors were 2-amino-2-deoxy-D-glucose and D-glucose, respectively. The other reaction was related to glycoproteins (probably sulfated glycoproteins) that contained sulphur. The properties of this reaction were not the same as purely ionic interactions; basic protein-acidic polysaccharide interactions have been compared. A lectin was probably implicated but this could not be demonstrated because no saccharide inhibitor was found. There are several similitudes between this reaction and the lectin-galactoside reaction which occurs in the reaction between cortical granule content and egg jellies in anurans.     

Improved procedures for the selective chemical fragmentation of rhamnogalacturonans

The structural characterization of branched rhamnogalacturonans (RGs) requires the availability of methods that selectively cleave the Rhap-(1→4)-α-GalAp linkage and thereby generate oligosaccharide fragments that are suitable for mass spectrometric and NMR spectroscopic analyses. Enzymic cleavage of this linkage is often ineffective, especially in highly branched RGs. Therefore, we have developed an improved chemical fragmentation method based on β-elimination of esterified 4-linked GalpA residues. At least 85% of the carboxyl groups of the GalA residues in Arabidopsis thaliana seed mucilage RG is esterified using methyl iodide or 3-iodopropanol in Me₂SO containing 8% water and 1% tetrabutylammonium fluoride. However, β-elimination fragmentation at pH 7.3 and 120 °C is far more extensive with hydroxypropyl-esterified RG than with methyl-esterified RG. The non-reducing 4-deoxy-β-l-threo-hex-4-enepyranosyluronic acid residue formed by the β-elimination reaction is completely removed by treatment with aqueous N-bromosuccinimide, thereby simplifying the structural characterization of the chemically generated oligoglycosyl fragments. This newly developed procedure was used to selectively fragment the branched RG from peppergrass seed mucilage. The products were characterized using MALDI-TOF mass spectrometry, glycosyl residue composition analysis, and 1 and 2D NMR spectroscopy. Our data show that the most abundant low-molecular weight fragments contained a backbone rhamnose residue substituted at O-4 with a single sidechain, and suggest that peppergrass seed mucilage RG is composed mainly of the repeating unit 4-O-methyl-α-d-GlcpA-(1→4)-β-d-Galp-(1→4)-[→4)-α-d-GalpA-(1→2)-]-α-l-Rhap-(1→.     

Design of molecules that specifically recognize and cleave apurinic sites in DNA

We have prepared a series of a tailor-made molecules that recognize and cleave DNA at apurinic sites in vitro. These molecules incorporate in their structure different units designed for specific function: an intercalator for DNA binding, an nucleic base for abasic site recognition and a linking chain of variable length and nature (including amino and/or amido functions). The cleavage efficiency of the molecules can be modulated by varying successively the nature of the intercalating agent, the nucleic base and the chain. All molecules bind to native calf thymus DNA with binding constants ranging from 10⁴ to 10⁶ M^?1. Their cleavage activity was determined on plasmid DNA (pBR 322) containing 1.8 AP-sites per DNA-molecule. The minimum requirements for cleavage are the presence of the three units, the intercalator, the nucleic base and at least one amino function in the chain. The most efficient molecules cleaved plasmid DNA at nanomolar concentrations. Enzymatic experiments on the termini generated after cleavage of AP-DNA suggest a strand break induced by a β-elimination reaction. In order to get insight into the mode of action (efficiency, selectivity, interaction), we have used synthetic oligonucleotides containing either a true abasic site at a determined position to analyse the cleavage parameters of the synthetic molecules by HPLC or a chemically stable along (tetrahydrofuran) of the abasic site for high field ¹H NMR spectrometry and footprinting experiments. All results are consistent with a β-elimination mechanism in which each constituent of the molecule exerts a specific function as indicated in the scheme: DNA targeting, abasic site nucleases and can be used advantageously as substitutes for the natural enzyme for in vitro cleavage of AP-sites containing DNA.     

Model for general acid-base catalysis by the hammerhead ribozyme: pH-activity relationships of G8 and G12 variants at the putative active site

We have used nucleobase substitution and kinetic analysis to test the hypothesis that hammerhead catalysis occurs by a general acid-base mechanism, in which nucleobases are directly involved in deprotonation of the attacking 2'-hydroxyl group and protonation of the 5'-oxygen that serves as the leaving group in the cleavage reaction. We demonstrate that simultaneous substitution of two important nucleobases, G8 and G12, with 2,6-diaminopurine shifts the pH optimum of the cleavage reaction from greater than 9.5 to approximately 6.8 in two different hammerhead constructs. Controls involving substitution with other nucleobases and combinations of nucleobases at G5, G8, and/or G12 do not show this behavior. The observed changes in the pH-rate behavior are consistent with a mechanism in which N1 protonation-deprotonation events of guanine or 2,6-diaminopurine at positions 8 and 12 are essential for catalysis. Further support for the participation of G8 and G12 comes from photochemical cross-linking experiments, which show that G8 and G12 can stack upon the two substrate nucleobases at the reactive linkage, G(or U)1.1 and C17 (Heckman, J. E., Lambert, D., and Burke, J. M. (2005) Photocrosslinking detects a compact active structure of the hammerhead ribozyme, Biochemistry 44, 4148-4156). Together, these results support a model in which the hammerhead undergoes a transient conformational change into a catalytically active structure, in which stacking of G8 and G12 upon the nucleobases spanning the cleavage site provides an appropriate architecture for general acid-base catalysis. The hammerhead and hairpin ribozymes may share similarities in the organization of their active sites and their catalytic mechanism.     

Structural and Functional Comparison of Polysaccharide-Degrading Enzymes

Sugar molecules as well as enzymes degrading them are ubiquitously present in physiological systems, especially for vertebrates. Polysaccharides have at least two aspects to their function, one due to their mechanical properties and the second one involves multiple regulatory processes or interactions between molecules, cells, or extracellular space. Various bacteria exert exogenous pressures on their host organism to diversity glycans and their structures in order for the host organism to evade the destructive function of such microbes. Many bacterial organism produce glycan-degrading enzymes in order to facilitate their invasion of host tissues. Such polysaccharide degrading enzymes utilize mainly two modes of polysaccharide-degradation, a hydrolysis and a β-elimination process. The three-dimensional structures of several of these enzymes have been elucidated recently using X-ray crystallography. There are many common structural motifs among these enzymes, mainly the presence of an elongated cleft transversing these molecules which functions as a polysaccharide substrate binding site as well as the catalytic site for these enzymes. The detailed structural information obtained about these enzymes allowed formulation of proposed mechanisms of their action. The polysaccharide lyases utilize a proton acceptance and donation mechanism (PAD), whereas polysaccharide hydrolases use a direct double displacement (DD) mechanism to degrade their substrates.