Assays for determining heparan sulfate and heparin O-sulfotransferase activity and specificity

Sterner, Eric; Li, Lingyun; Paul, Priscilla; Beaudet, Julie M.; Liu, Jian; Linhardt, Robert J.; Dordick, Jonathan S.

doi:10.1007/s00216-013-7470-4

Cited by 21 publications

(17 citation statements)

References 42 publications

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“…Use of ten-fold higher AST IV (Reaction 15 ) showed an elevated TriS disaccharide content compared to two-fold higher AST IV (Reaction 14 ) and Reaction 1 . These results are in agreement with the observed rapid kinetics of AST IV and suggest that cofactor recycling is not the rate limiting step in sulfotransferase coupled in vitro biocatalytic systems (Sterner et al, 2014). …”

Section: Resultssupporting

confidence: 89%

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Combinatorial one-pot chemoenzymatic synthesis of heparin

Bhaskar

et al. 2015

Carbohydrate Polymers

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Contamination in heparin batches during early 2008 has resulted in a significant effort to develop a safer bioengineered heparin using bacterial capsular polysaccharide heparosan and recombinant enzymes derived from the heparin/heparan sulfate biosynthetic pathway. This requires controlled chemical N-deacetylation/ N-sulfonation of heparosan followed by epimerization of most of its glucuronic acid residues to iduronic acid and O-sulfation of the C2 position of iduronic acid and the C3 and C6 positions of the glucosamine residues. A combinatorial study of multi-enzyme, one-pot, in vitro biocatalytic synthesis, carried out in tandem with sensitive analytical techniques, reveals controlled structural changes leading to heparin products similar to animal- derived heparin active pharmaceutical ingredients. Liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy analysis confirms an abundance of heparin’s characteristic trisulfated disaccharide, as well as 3-O-sulfo containing residues critical for heparin binding to antithrombin III and its anticoagulant activity. The bioengineered heparins prepared using this simplified one-pot chemoenzymatic synthesis also show in vitro anticoagulant activity.

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Section: Resultssupporting

confidence: 89%

“…The three different isoforms possess similar substrate specificity and can introduce 6- O -sulfo groups into polysaccharide chains with various levels of N -sulfo and 2- O -sulfo group substitution, however, the 6-OST-1 prefers the absence of 2- O -sulfo group (Bhaskar et al, 2012; Habuchi et al, 2003; Sterner et al, 2014). …”

Section: Resultsmentioning

confidence: 99%

Combinatorial one-pot chemoenzymatic synthesis of heparin

Bhaskar

et al. 2015

Carbohydrate Polymers

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“…Coupling this reaction to OST reactions not only overcomes strong product inhibition of these sulfotransferases by PAP [103], but also provides a reaction monitoring system. PNP produced by the reaction absorbs light at 400 nm wavelengths, which forms the basis of our current colorimetric sulfotransferase assay [104,105]. …”

Section: Bioengineering Ulmwh Lmwh and Heparinmentioning

confidence: 99%

Bioengineered heparins and heparan sulfates

Suflita

Linhardt

2016

Advanced Drug Delivery Reviews

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111

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Heparin and heparan sulfates are closely related linear anionic polysaccharides, called glycosaminoglycans, which exhibit a number of important biological and pharmacological activities. These polysaccharides, having complex structures and polydispersity, are biosynthesized in the Golgi of animal cells. While heparan sulfate is a widely distributed membrane and extracellular glycosaminoglycan, heparin is found primarily intracellularly in the granules of mast cells. While heparin has historically received most of the scientific attention for its anticoagulant activity, interest has steadily grown in the multi-faceted role heparan sulfate plays in normal and pathophysiology. The chemical synthesis of these glycosaminoglycans is largely precluded by their structural complexity. Today, we depend on livestock animal tissues for the isolation and the annual commercial production of hundred ton quantities of heparin used in the manufacture of anticoagulant drugs and medical device coatings. The variability of animal-sourced heparin and heparan sulfates, their inherent impurities, the limited availability of source tissues, the poor control of these source materials and their manufacturing processes, suggest a need for new approaches for their production. Over the past decade there have been major efforts in the biotechnological production of these glycosaminoglycans, driven by both therapeutic applications and as probes to study their natural functions. This review focuses on the complex biology of these glycosaminoglycans in human health and disease, and the use of recombinant technology in the chemoenzymatic synthesis and metabolic engineering of heparin and heparan sulfates.

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“…This reverse reaction can be used as a cofactor regeneration system when coupled to HS or CS sulfotransferase reactions and overcomes strong product inhibition of these sulfotransferases by PAP (Burkart et al 2000). This cofactor regeneration also produces p -nitrophenol, a yellow colored product which can be easily monitored at a 400 nm wavelength, forming the basis of a commonly used sulfotransferase assay (Burkart and Wong 1999; Sterner et al, 2014). Collectively, this cofactor regeneration system and colorimetric assay represents a valuable enzymatic toolbox for GAG synthesis.…”

Section: Bioengineering Approachesmentioning

confidence: 99%

Heparin and related polysaccharides: synthesis using recombinant enzymes and metabolic engineering

Suflita

et al. 2015

Appl Microbiol Biotechnol

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Glycosaminoglycans are linear anionic polysaccharides that exhibit a number of important biological and pharmacological activities. The two most prominent members of this class of polysaccharides are heparin/heparan sulfate and the chondroitin sulfates (including dermatan sulfate). These polysaccharides, having complex structures and polydispersity, are biosynthesized in the Golgi of most animal cells. The chemical synthesis of these glycosaminoglycans is precluded by their structural complexity. Today, we depend on food animal tissues for their isolation and commercial production. Ton quantities of these glycosaminoglycans are used annually as pharmaceuticals and nutraceuticals. The variability of animal-sourced glycosaminoglycans, their inherent impurities, the limited availability of source tissues, the poor control of these source materials, and their manufacturing processes, suggest a need for new approaches for their production. Over the past decade there have been major efforts in the biotechnological production of these glycosaminoglycans. This mini-review focuses on the use of recombinant enzymes and metabolic engineering for the production of heparin and chondroitin sulfates.

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Assays for determining heparan sulfate and heparin O-sulfotransferase activity and specificity

Cited by 21 publications

References 42 publications

Combinatorial one-pot chemoenzymatic synthesis of heparin

Combinatorial one-pot chemoenzymatic synthesis of heparin

Bioengineered heparins and heparan sulfates

Heparin and related polysaccharides: synthesis using recombinant enzymes and metabolic engineering

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