Effects of a semi-synthetic N-,O-sulfated glycosaminoglycan K5 polysaccharide derivative in a rat model of cerebral ischaemia/ reperfusion injury

SummaryHeparin and low molecular weight heparins may reduce brain damage evoked by ischaemia/reperfusion (I/R) injury, although their use is hampered by the risk of haemorrhage. Chemical and enzymatic modifications of K5 polysaccharide have shown the possibility to produce heparin-like compounds with low anticoagulant activity and strong anti-inflammatory effects. Using a rat model of transient cerebral I/R, we investigated the effects of an epimerised N-,O-sulfated K5 polysaccharide derivative, K5-N,OSepi, on the infarct size, motor activity and injury caused by ischaemia (30 min) and reperfusion. Reperfusion was allowed for 60 min or 1–5 days. Rats reperfused for 5 days showed an infarct volume of 30.7 ±3.1% and K5-N,OSepi (0.1–1 mg/kg) caused dose-dependent reduction in infarct size (maximum at 1 mg/kg: 13.1 ±2.1% infarct volume). This effect was associated with a significant improvement in motor performance. In the rat hippocampus, one of the brain areas most sensitive to I/R injury, I/R induced a robust increase in myeloperoxidase (MPO) activity, a marker of neutrophil infiltration, that was halved by K5-N,OSepi administration (66.38 ±7.75 µU MPO/tissue g, 30.78 ±5.67 µU MPO/tissue g, respectively). K5-N,OSepi drastically reduced the expression of cyclooxygenase-2,inducible-nitric-oxide-synthase and intercellular-adhesion-molecule-1. I/R-induced activation of nuclear factor-kB was attenuated by drug treatment. Furthermore, K5-N,OSepi administration was associated with a significant modulation of apoptosis markers, such as Bid and Bcl-2. In conclusion, the results demonstrated that the sulfated semi-synthetic K5 derivative K5-N,OSepi protects the brain against I/R injury by disrupting multiple levels of the apoptotic and inflammatory cascade, including inhibition of NF-κB activation.

The Escherichia coli K5 Capsule Is Not Synthesized in a Protected Compartment within the Cytoplasm

ABSTRACT The intracellular expression of the K5 lyase enzyme, which degrades the K5 polysaccharide, decreased cell surface expression of the Escherichia coli K5 capsule. This indicates that biosynthesis of K5 polysaccharide in the cytoplasm is accessible to the action of K5 lyase and is not synthesized within a protected cytoplasmic compartment.

Inhibition of Herpes Simplex Virus Types 1 and 2 In Vitro Infection by Sulfated Derivatives of Escherichia coli K5 Polysaccharide

ABSTRACT Herpes simplex virus type 1 (HSV-1) and HSV-2 are neurotropic viruses and common human pathogens causing major public health problems such as genital herpes, a sexually transmitted disease also correlated with increased transmission and replication of human immunodeficiency virus type 1 (HIV-1). Therefore, compounds capable of blocking HIV-1, HSV-1, and HSV-2 transmission represent candidate microbicides with a potential added value over that of molecules acting selectively against either infection. We report here that sulfated derivatives of the Escherichia coli K5 polysaccharide, structurally highly similar to heparin and previously shown to inhibit HIV-1 entry and replication in vitro, also exert suppressive activities against both HSV-1 and HSV-2 infections. In particular, the N,O-sulfated [K5-N,OS(H)] and O-sulfated epimerized [Epi-K5-OS(H)] forms inhibited the infection of Vero cells by HSV-1 and -2, with 50% inhibitory concentrations (IC50) between 3 ± 0.05 and 48 ± 27 nM, and were not toxic to the cells at concentrations as high as 5 μM. These compounds impaired the early steps of HSV-1 and HSV-2 virion attachment and entry into host cells and reduced the cell-to-cell spread of HSV-2. Since K5-N,OS(H) and Epi-K5-OS(H) also inhibit HIV-1 infection, they may represent valid candidates for development as topical microbicides preventing sexual transmission of HIV-1, HSV-1, and HSV-2.

Mutations in the waaR Gene of Escherichia coli Which Disrupt Lipopolysaccharide Outer Core Biosynthesis Affect Cell Surface Retention of Group 2 Capsular Polysaccharides

ABSTRACT On the basis of increased resistance to K5 capsule-specific bacteriophage, a waaR transposon mutant defective in the biosynthesis of lipopolysaccharide outer core was isolated. In a K1-expressing strain the mutation equally affected sensitivity to K1 capsule-specific bacteriophage, indicating a general effect on group 2 capsules. The waaR mutation affected retention on the cell surface of the K5 polysaccharide, with increased polysaccharide accumulating in the culture supernatant. This indicates that interactions between the outer core of lipopolysaccharide and group 2 capsular polysaccharides are important for the stabilization of group 2 capsular polysaccharides on the cell surface.

Distinct Substrate Specificities of Bacterial Heparinases againstN-Unsubstituted Glucosamine Residues in Heparan Sulfate

The rareN-unsubstituted glucosamine (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{GlcNH}_{3}^{+}\) \end{document}) residues in heparan sulfate have important biological and pathophysiological roles. In this study, four\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{GlcNH}_{3}^{+}\) \end{document}-containing disaccharides were obtained from partially de-N-sulfated forms of heparin and theN-sulfated K5 polysaccharide by digestion with combined heparinases I, II, and III. These were identified as\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \({\Delta}\mathrm{HexA}\mathrm{-}\mathrm{GlcNH}_{3}^{+},{\Delta}\mathrm{HexA}\mathrm{-}\mathrm{GlcNH}_{3}^{+}(6S),{\Delta}\mathrm{HexA}(2S)\mathrm{-}\mathrm{GlcNH}_{3}^{+}\) \end{document}, and\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \({\Delta}\mathrm{HexA}(2S)\mathrm{-}\mathrm{GlcNH}_{3}^{+}(6S)\) \end{document}. Digestions with individual enzymes revealed that heparinase I did not cleave at\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{GlcNH}_{3}^{+}\) \end{document}residues; however, heparinases II and III showed selective and distinct activities. Heparinase II generated\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \({\Delta}\mathrm{HexA}\mathrm{-}\mathrm{GlcNH}_{3}^{+}(6S),{\Delta}\mathrm{HexA}(2S)\mathrm{-}\mathrm{GlcNH}_{3}^{+}\) \end{document}, and\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \({\Delta}\mathrm{HexA}(2S)\mathrm{-}\mathrm{GlcNH}_{3}^{+}(6S)\) \end{document}disaccharides, whereas heparinase III yielded only the\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \({\Delta}\mathrm{HexA}\mathrm{-}\mathrm{GlcNH}_{3}^{+}\) \end{document}unit. Thus, the action of heparinase II requiresO-sulfation, whereas heparinase III acts only on the corresponding non-sulfated unit. These striking distinctions in substrate specificities of heparinases could be used to isolate oligosaccharides with novel sequences of\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{GlcNH}_{3}^{+}\) \end{document}residues. Finally, heparinases were used to identify and quantify\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{GlcNH}_{3}^{+}\) \end{document}-containing disaccharides in native bovine kidney and porcine intestinal mucosal heparan sulfates. The relatively high content ofO-sulfated\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{GlcNH}_{3}^{+}\) \end{document}-disaccharides in kidney HS raises questions about how these sequences are generated.