Unique heparan sulfate from shrimp heads exhibits a strong inhibitory effect on infections by dengue virus and Japanese encephalitis virus

The structure and biological activities of a highly sulfated heparan sulfate (HS) extracted from shrimp (Penaeus brasiliensis) heads were characterized. Structurally the shrimp HS was more heterogenous than heparin, although it is still highly sulfated. The molecular mass of the shrimp HS preparation was determined to be 32.3 kDa by gel filtration HPLC. Analysis by surface plasmon resonance demonstrated that various growth/differentiation factors specifically bound to the shrimp HS with comparable affinity. Notably, the shrimp HS had a greater inhibitory effect against infections by dengue virus type 2 as well as Japanese encephalitis virus than heparin. Experiments on anticoagulant activity indicated that the shrimp HS exhibited significant anti-thrombin activity, but less than the commercial heparin. Hence, the HS preparation from shrimp heads, an industrial waste, is a prospective agent for a variety of clinical applications.     

ATP-NGF-complex, but not NGF, is the neuroprotective ligand

We have shown previously that nerve growth factor (NGF) requires only low nanomolar ATP concentrations in the cell culture medium to protect cortical rat neurons (CRN) from cellular damage induced by staurosporine (STS). We have also demonstrated before that NGF and other growth factors form stable non-covalent complexes with ATP. Here we demonstrated that 8N₁ATP–NGF, but not NGF, protected CRN against damage. The photo-reactive ATP derivative 8N₃ATP was incubated with NGF and was trapped in its position by UV irradiation forming a covalent bond. The cross-link with a molar ratio of 1:1 (8N₁ATP:NGF) was confirmed by mass spectrometry. Circular dichroism experiments revealed that 8N₁ATP altered the secondary structure of NGF in the same way as ATP did. Covalently bound 8N₁ATP–NGF was shown to be stable in the presence of the ATP-hydrolyzing enzyme alkaline phosphatase while the non-covalent ATP–NGF-complex dissociated with the removal of free ATP from the solution. 8N₁ATP–NGF protected CRN against damage by STS independently of free ATP in the culture medium. These results suggest that the ATP–NGF-complex, but not NGF, is the active ligand.     

Enzymatic Improvement of Food Flavor I. Purification and Characterization of Bovine Liver Mitochondrial Aldehyde Dehydrogenase

Bovine liver mitochondrial aldehyde dehydrogenase (aldehyde: NAD⁺ oxidoreductase, EC 1.2.1.3) has been purified to homogeneity by conventional purification procedures. The enzyme was found to have a molecular weight of 215,000 based on gel filtration. The protein is composed of polypeptides having the same molecular weight, 54,000 and thus it appears to consist of four subunits of equal size. The enzyme exhibited a broad aldehyde specificity, oxidizing irreversibly a wide variety of aliphatic and aromatic aldehydes to corresponding carboxylic acids. Km values for straight-chain saturated aldehydes were below 0.1 µm, and relatively constant independent of the carbon chain lengths of the aldehydes. The maximum velocities for saturated aldehydes also did not vary appreciably with their carbon chain lengths. Maximum activity was observed at pH 9.3 and 50°C. The enzyme activity was affected by some divalent cations. Ca²⁺ enhanced the activity, while Mg²⁺ inhibited it. The enzyme was quite stable at neutral pH, but was unstable above pH 9 or below pH 6. Bovine liver has three isozymes of aldehyde dehydrogenase which are located in the mitochondrial, cytosolic, and microsomal fractions. Comparison of enzymic properties among these isozymes and yeast enzyme indicates that the mitochondrial enzyme is very suitable for improving the objectionable flavor due to aldehydes in foods.     

Extracellular superoxide dismutase in tissues from obese (ob/ob) mice

We have examined the protein content and gene expression of three superoxide dismutase (SOD) isoenzymes in eight tissues from obese ob/ob mice, particularly placing the focus on extracellular-SOD (EC-SOD) in the white adipose tissue (WAT). Obesity significantly increased EC-SOD level in liver, kidney, testis, gastrocnemius muscle, WAT, brown adipose tissue (BAT), and plasma, but significantly decreased the isoenzyme level in lung. Tumor necrosis factor-α and interleukin-1β contents in WAT were significantly higher in obese mice than in lean control mice. Immunohistochemically, both WAT and BAT from obese mice could be stained deeply with anti-mouse EC-SOD antibody compared with those from lean mice. Each primary culture per se almost time-dependently enhanced EC-SOD production, and overtly expressed its mRNA. The loss of heparin-binding affinity of EC-SOD type C with high affinity for heparin occurred in kidney of obese mice. These results suggest that the physiological importance of this SOD isoenzyme in WAT may be a compensatory adaptation to oxidative stress.     

Heparin and the phenotype of adult human vascular smooth muscle cells

Summary To study mechanisms controlling growth and phenotype in human vascular smooth muscle cells, we established culture conditions under which these cells proliferate rapidly and achieve life-spans of 50–60 population doublings. In medium containing heparin and heparin-binding growth factors, growth rate and life-span of human vascular smooth muscle cells increased more than 50% relative to cultures with neither supplement, and more than 20% compared to cultures supplemented only with heparin-binding growth factors. In contrast to observations made in rat vascular smooth muscle cells, smooth muscle-specific α-actin in the human cells was expressed only in the presence of heparin and colocalized with β/γ nonmuscle actins in stress fibers, not in adhesion plaques. Heparin, in the presence of heparin-binding growth factors, also caused more than 170% stimulation of tracer glucosamine incorporation into hyaluronic acid and a 7.5-fold increase in hyaluronic acid accumulation. In comparison, total sulfate incorporation into sulfated glycosaminoglycans increased by less than 40%. In light of our previous findings that heparin suppresses collagen gene expression, we conclude that heparin induces human vascular smooth muscle cells exposed to heparin-binding growth factors to remodel their extracellular matrix by altering the relative rates of hyaluronic acid (HA) and collagen synthesis. The resulting hyaluronic-acid-rich, collagen-poor matrix may enhance infiltration of CD44/hyaluronate-receptor-bearing T-lymphocytes and monocytes into the vascular wall, an early event in atherogenesis.     

Purification and characterization of fatty acid synthetase from Cryptococcus neoformans

Fatty acid synthetase has been purified from Cryptococcus neoformans 450 fold to a specific activity of 3.6 units per mg protein with an overall yield of 23%. The purified enzyme contained two non-identical subunits, Mr approximately 2.1×10⁵ and 1.8×10⁵. Under optimum conditions, 100 mM KCl and pH 7.5, apparent Km values for the substrates were: Acetyl CoA, 19 M; Malonyl CoA, 5 M; and NADPH, 6 M. Product inhibition patterns were determined to be: CoA, competitive versus acetyl CoA and malonyl CoA, uncompetitive versus NADPH; NADP, competitive versus NADPH, uncompetitive versus acetyl CoA and malonyl CoA; Palmitoyl CoA, competitive versus malonyl CoA, noncompetitive versus acetyl CoA and NADPH; Bicarbonate, uncompetitive versus malonyl CoA. These product inhibition patterns are consistent with the multisite ping-pong mechanism previously proposed for the avian fatty acid synthetase complex. The cryptococcal fatty acid synthetase was inhibited by the polyanionic polymers, heparin and dextran sulfate, an effect never before demonstrated for a fatty acid synthetase. This inhibition exhibited a marked dependence on the length of the polymer chain, with dextran sulfate fractions with Mr of 6×10⁵ and above having K _i values below 100 nanomolar. A model is presented that involves initial binding of the anionic polymer to the enzyme complex at a region of high positive charge density, followed by interaction of the end of the tethered polymer with the catalytic site. This study represents the first purification of fatty acid synthetase from a basidiomycete.     

Solution dynamics of the 1,2,3,4,6-penta-O-acetyl-α-D-idopyranose ring

The anticoagulant properties of heparin are thought to derive from the inhibition of thrombin and other coagulation-related proteases by the binding of heparin to cofactors such as antithrombin III and heparin cofactor II. The apparent minimum native heparin sequence which can bind to antithrombin III is a highly sulfated pentasaccharide which contains a 2-O-sulfo-α-L-idopyranosyluronic acid residue. The idopyranosyl residue has the unusual property of existing in the solution state as a mixture of ring conformers. Whereas most hexopyranose sugars exist as a single chair conformer (eg D-glucose exists overwhelmingly as a 4C1 chair), the idopyranosyl ring is known to rapidly exchange between at least two and often more distinct conformations, depending on type and number of substituents (hydroxyl, carboxyl, sulfate, etc.) and solvent conditions (solvent pH, salt concentration, temperature). It is believed that this flexibility of the idopyranosyl residue in heparin is related to its binding specificity. In the past, coupling constants and molecular dynamics have been used to estimate the relative populations of conformers in iduronate and related compounds. Here we report extensive NMR measurements, including line shape analysis, T1ρ measurements, T1 and NOE measurements and spectral density mapping, which have been used to study the dynamics of conformer interconversion in model compounds related to idose and glucose. The findings presented here indicate that 1,2,3,4,6-peneta-O-acetyl-α-D-idopyranose can be reasonably well described as existing in a two-state equilibrium consisting of the 4C1 and 0S2 conformers. 13C NMR line shape analysis yields a ΔH+ of 40 kJ mol-1 and a ΔS‡ of 31 J mol-1 K-1 for the 4C1→0S2 interconversion and a ΔH‡ of 31 kJ mol-1 and a ΔS‡ of 13 J mol-1K-1 for the 0S2→4C1 interconversion. This corresponds to exchange rates of 22 and 128 MHz, respectively, at room temperature. This revised version was published online in November 2006 with corrections to the Cover Date.     

Saccharide anions as inhibitors of the malaria parasite

The asexual erythrocytic stage of Plasmodium falciparum was grown in culture in the presence or absence of glycoconjugate polyanions of varying structure, size and substitutions. Heparin, dextran sulfate, fucoidan and pentosan polysulfate had antimalarial IC50 values between one and 11 μg ml−1. Constituent heparin disaccharides were ineffective against the malaria parasite and desulfation from either the O- or N-substitution sites of heparin or reduction of the uronic acid carboxyl group neutralized the antimalarial response to varying degrees. Immobilization of heparin onto agarose beads still permitted antimalarial activity suggesting that parasite uptake of the glycoconjugate is not required for inhibition. Accordingly, it is concluded that invasion of free parasites into the erythrocytes was inhibited rather than parasite maturation within the red cell. Merozoite surface antigen-1 was apparently prevented from binding to human erythrocytes in the presence of highly sulfated polyanions and, in a dose-dependent fashion, heparin. Abbreviations: MSA-1, merozoite surface antigen-1 This revised version was published online in November 2006 with corrections to the Cover Date.     

Analysis of sulfates on low molecular weight heparin using mass spectrometry: structural characterization of enoxaparin

Introduction: Structural characterization of low molecular weight heparin (LMWH) is critical to meet biosimilarity standards. In this context, the review focuses on structural analysis of labile sulfates attached to the side-groups of LMWH using mass spectrometry. A comprehensive review of this topic will help readers to identify key strategies for tackling the problem related to sulfate loss. At the same time, various mass spectrometry techniques are presented to facilitate compositional analysis of LMWH, mainly enoxaparin.

Areas covered: This review summarizes findings on mass spectrometry application for LMWH, including modulation of sulfates, using enzymology and sample preparation approaches. Furthermore, popular open-source software packages for automated spectral data interpretation are also discussed. Successful use of LC/MS can decipher structural composition for LMWH and help evaluate their sameness or biosimilarity with the innovator molecule. Overall, the literature has been searched using PubMed by typing various search queries such as ‘enoxaparin’, ‘mass spectrometry’, ‘low molecular weight heparin’, ‘structural characterization’, etc.

Expert commentary: This section highlights clinically relevant areas that need improvement to achieve satisfactory commercialization of LMWHs. It also primarily emphasizes the advancements in instrumentation related to mass spectrometry, and discusses building automated software for data interpretation and analysis.     

The Responses of Hyperglycemic Dividing Mesangial Cells to Heparin Are Mediated by the Non-reducing Terminal Trisaccharide

Our previous studies showed: (i) that growth-arrested G₀/G₁ rat mesangial cells stimulated to divide in hyperglycemic medium initiate intracellular hyaluronan synthesis that induces autophagy and the cyclin D3-induced formation of a monocyte-adhesive extracellular hyaluronan matrix after completing cell division; and (ii) that heparin inhibits the intracellular hyaluronan and autophagy responses, but after completing division, induces hyaluronan synthesis at the plasma membrane with the formation of a larger monocyte-adhesive hyaluronan matrix. This study shows: (i) that the non-terminal trisaccharide of heparin is sufficient to initiate the same responses as intact heparin, (ii) that a fully sulfated tetrasaccharide isolated from bacterial heparin lyase 1 digests of heparin that contains a Δ-2S-iduronate on the non-reducing end does not initiate the same responses as intact heparin, and (iii) that removal of the Δ-2S-iduronate to expose the fully sulfated trisaccharide (GlcNS(6S)-IdoUA(2S)-GlcNS(6S)) does initiate the same responses as intact heparin. These results provide evidence that mammalian heparanase digestion of heparin and heparan sulfate exposes a cryptic motif on the non-reducing termini that is recognized by a receptor on dividing cells.