Abstract
The glycosaminoglycan, heparan sulphate (HS), orchestrates many developmental processes. Yet its biological role has not yet fully been elucidated. Small molecule chemical inhibitors can be used to perturb HS function and these compounds provide cheap alternatives to genetic manipulation methods. However, existing chemical inhibition methods for HS also interfere with chondroitin sulphate (CS), complicating data interpretation of HS function. Herein, a simple method for the selective inhibition of HS biosynthesis is described. Using endogenous metabolic sugar pathways, Ac4GalNAz produces UDP-GlcNAz, which can target HS synthesis. Cell treatment with Ac4GalNAz resulted in defective chain elongation of the polymer and decreased HS expression. Conversely, no adverse effect on CS production was observed. The inhibition was transient and dose-dependent, affording rescue of HS expression after removal of the unnatural azido sugar. The utility of inhibition is demonstrated in cell culture and in whole organisms, demonstrating that this small molecule can be used as a tool for HS inhibition in biological systems.
Highlights
Heparan sulphate (HS) is a prevalent glycosaminoglycan (GAG) attached to protein cores on the cell surface of almost every cell type
HS proteoglycans form an integral part of the extracellular matrix with important roles in development [1], homeostasis [2,3] and disease [4,5]
HS biosynthesis consists of a repeating disaccharide unit structure of glucuronic acid–N-acetylglucosamine (GlcA-GlcNAc) polymerised by the exostoses enzyme complex (EXT1/2) from UDP-GlcA and UDP-GlcNAc active nucleotide donor sugars [6,7,8]
Summary
Heparan sulphate (HS) is a prevalent glycosaminoglycan (GAG) attached to protein cores (proteoglycans) on the cell surface of almost every cell type. HS biosynthesis consists of a repeating disaccharide unit structure of glucuronic acid–N-acetylglucosamine (GlcA-GlcNAc) polymerised by the exostoses enzyme complex (EXT1/2) from UDP-GlcA and UDP-GlcNAc active nucleotide donor sugars [6,7,8] During this process the N-deacetylase/N-sulphotransferase (NDST) enzymes work in tandem to begin modification of the nascent chain. The NDST enzymes can replace the acetyl group on GlcNAc with a sulphate [9], often providing the gateway step for further modifications of the chain. Together, these enzymes contribute to HS functionality by influchain at the 2-, 6- and 3-O position or epimerisation of GlcA to iduronic acid (IdoA) by C5encing the fine patterning of the chain [12]. Ac GalNAz treatment as a potential novel, small chemical inhibitor of HS synthesis
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