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ABSTRACT: Heparin has been the most commonly used anticoagulant drug for nearly a century. The drug heparin is generally categorized into three forms according to its molecular weight (MW), unfractionated (UF, average MW 13,000), low molecular weight (LMW, average MW 5,000), and ultra-low molecular weight heparin (ULMWH, average MW 2,000). Overdose of anticoagulant heparin can lead to very dangerous bleeding in patients. Protamine sulfate can be administered as an antidote to reverse heparin's anticoagulant effect. There is not an effective antidote for ULMWH. In the current study, we examine human N-acetylglucosamine 6-sulfatase (NG6S), expressed in Chinese hamster ovary cells as a reversal agent for ULMWH. NG6S removes a single 6-O-sulfo group at the non-reducing end of the ULMWH Arixtra® (fondaparinux) effectively removing its ability to bind to antithrombin and preventing its inhibition of coagulation factor Xa. These results pave the way to develop human NG6S as an antidote for neutralizing the anticoagulant activity of ULMWHs. © 2013 The Authors Journal compilation © 2013 FEBS.
FEBS Journal 02/2013; · 3.79 Impact Factor
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ABSTRACT: Heparan sulfate (HS) belongs to a major class of glycans that perform central physiological functions. Heparin is a specialized form of HS and is a clinically used anticoagulant drug. Heparin is a natural product isolated from pig intestine. There is a strong demand to replace natural heparin with a synthetic counterpart. Although a chemoenzymatic approach has been employed to prepare synthetic heparin, the scale of the synthesis is limited by the availability of sulfotransferases and the cofactor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Here, we present a novel method to produce secreted forms of sulfotransferases in the yeast cells, Kluyveromyces lactis. Five sulfotransferases including N-sulfotransferase, 2-O-sulfotransferase, 3-O-sulfotransferase 1 and 6-O-sulfotransferases 1 and 3 were expressed using this method. Unlike bacterial-expressed sulfotransferases, the yeast proteins can be directly used to modify polysaccharides without laborious purification. The yeast-expressed sulfotransferases also tend to have higher specific activity and thermostability. Furthermore, we demonstrated the possibility for the gram-scale synthesis of PAPS from adenosine 5'-triphosphate at only 1/5000th of the price purchased from a commercial source. Our results pave the way to conduct the enzymatic synthesis of heparin in large quantities.
Glycobiology 01/2011; 21(6):771-80. · 3.58 Impact Factor
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ABSTRACT: Heparan sulfate is a sulfated glycan that exhibits essential physiological functions. Interrogation of the specificity of heparan sulfate-mediated activities demands a library of structurally defined oligosaccharides. Chemical synthesis of large heparan sulfate oligosaccharides remains challenging. We report the synthesis of oligosaccharides with different sulfation patterns and sizes from a disaccharide building block using glycosyltransferases, heparan sulfate C(5)-epimerase, and sulfotransferases. This method offers a generic approach to prepare heparan sulfate oligosaccharides possessing predictable structures.
Journal of Biological Chemistry 10/2010; 285(44):34240-9. · 4.77 Impact Factor
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ABSTRACT: In quorum sensing (QS) process, bacteria regulate gene expression by utilizing small signaling molecules called autoinducers in response to a variety of environmental cues. Autoinducer 2 (AI-2), a QS signaling molecule proposed to be involved in interspecies communication, is produced by many species of gram-negative and gram-positive bacteria. In Escherichia coli and Salmonella typhimurium, the extracellular AI-2 is imported into the cell by a transporter encoded by the lsr operon. Upstream of the lsr operon, there is a divergently transcribed gene encoding LsrR, which was reported previously to repress the transcription of the lsr operon and itself. Here, we have demonstrated for the first time that LsrR represses the transcription of the lsr operon and itself by directly binding to their promoters using gel shift and DNase I footprinting assays. The beta-galactosidase reporter assays further suggest that two motifs in both the lsrR and lsrA promoter regions are crucial for the LsrR binding. Furthermore, in agreement with the conclusion that phosphorylated AI-2 can relieve the repression of LsrR in previous studies, our data show that phospho-AI-2 renders LsrR unable to bind to its own promoter in vitro.
Cell Research 08/2009; 19(11):1258-68. · 8.19 Impact Factor
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ABSTRACT: Quorum sensing (QS) is a bacterial cell-cell communication process by which bacteria communicate using extracellular signals called autoinducers. Two QS systems have been identified in Escherichia coli K-12, including an intact QS system 2 that is stimulated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex and a partial QS system 1 that consists of SdiA (suppressor of cell division inhibitor) responding to signals generated by other microbial species. The relationship between QS system 1 and system 2 in E. coli, however, remains obscure. Here, we show that an EAL domain protein, encoded by ydiV, and cAMP are involved in the interaction between the two QS systems in E. coli. Expression of sdiA and ydiV is inhibited by glucose. SdiA binds to the ydiV promoter region in a dose-dependent, but nonspecific, manner; extracellular autoinducer 1 from other species stimulates ydiV expression in an sdiA-dependent manner. Furthermore, we discovered that the double sdiA-ydiV mutation, but not the single mutation, causes a 2-fold decrease in intracellular cAMP concentration that leads to the inhibition of QS system 2. These results indicate that signaling pathways that respond to important environmental cues, such as autoinducers and glucose, are linked together for their control in E. coli.
Cell Research 07/2008; 18(9):937-48. · 8.19 Impact Factor
