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Abstract Complex carbohydrates (glycans) are major players in all organisms due to their structural, energy, and communication roles. This last essential role involves interacting and/or signaling through a plethora of glycan-binding proteins. The design and synthesis of glycans as potential drug candidates that selectively alter or perturb metabolic processes is challenging. Here we describe the first reported sulfur-linked polysaccharides with potentially altered conformational state(s) that are recalcitrant to digestion by heparanase, an enzyme important in human health and disease. An artificial sugar donor with a sulfhydryl functionality is synthesized and enzymatically incorporated into polysaccharide chains utilizing heparosan synthase. Used alone, this donor adds a single thio-sugar onto the termini of nascent chains. Surprisingly, in chain co-polymerization reactions with a second donor, this thiol-terminated heparosan also serves as an acceptor to form an unnatural thio-glycosidic bond (‘ S -link’) between sugar residues in place of a natural ‘ O -linked’ bond. S -linked heparan sulfate analogs are not cleaved by human heparanase. Furthermore, the analogs act as competitive inhibitors with > ~200-fold higher potency than expected; as a rationale, molecular dynamic simulations suggest that the S -link polymer conformations mimic aspects of the transition state. Our analogs form the basis for future cancer therapeutics and modulators of protein/sugar interactions.more » « less
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Abstract Introduction The endothelial glycocalyx regulates vascular permeability, inflammation, and coagulation, and acts as a mechanosensor. The loss of glycocalyx can cause endothelial injury and contribute to several microvascular complications and, therefore, may promote diabetic retinopathy. Studies have shown a partial loss of retinal glycocalyx in diabetes, but with few molecular details of the changes in glycosaminoglycan (GAG) composition. Therefore, the purpose of our study was to investigate the effect of hyperglycemia on GAGs of the retinal endothelial glycocalyx.
Methods GAGs were isolated from rat retinal microvascular endothelial cells (RRMECs), media, and retinas, followed by liquid chromatography-mass spectrometry assays. Quantitative real-time polymerase chain reaction was used to study mRNA transcripts of the enzymes involved in GAG biosynthesis.
Results and Conclusions Hyperglycemia significantly increased the shedding of heparan sulfate (HS), chondroitin sulfate (CS), and hyaluronic acid (HA). There were no changes to the levels of HS in RRMEC monolayers grown in high-glucose media, but the levels of CS and HA decreased dramatically. Similarly, while HA decreased in the retinas of diabetic rats, the total GAG and CS levels increased. Hyperglycemia in RRMECs caused a significant increase in the mRNA levels of the enzymes involved in GAG biosynthesis (including EXTL-1,2,3, EXT-1,2, ChSY-1,3, and HAS-2,3), with these increases potentially being compensatory responses to overall glycocalyx loss. Both RRMECs and retinas of diabetic rats exhibited glucose-induced alterations in the disaccharide compositions and sulfation of HS and CS, with the changes in sulfation including N,6-O-sulfation on HS and 4-O-sulfation on CS.
