Heparan sulphate (HS) is critical for embryonic development with involvement in a myriad of biological processes, centrally mediating morphogenic movements and facilitating the specification and differentiation of tissues. Complicated by its structural micro-heterogeneity along with expression on numerous different proteoglycan cores, the plethora of roles for HS in biology and their underlying mechanisms have not yet been fully defined. The discovery and characterisation of new reagents and methods for modification of HS expression and/or structure will aid efforts in elucidating the structure and activity of this glycosaminoglycan. Until now, azido sugars have been utilised as labelling reagents for various types of glycosylation, including N-glycans, O-linked mucin-type glycosylation and O-GlcNAcetylation of proteins. Incorporation of the unnatural azido sugar into the glycan of interest inserts a chemically reactive abiotic azide for subsequent detection via Staudinger ligation or click chemistries. However, to our knowledge, application of these azido sugars has not been explored for glycosaminoglycans. A metabolic labelling approach using Ac4GalNAz yields UDP-GalNAz and UDP-GlcNAz (Boyce et al., 2011), ready to target CS/DS and HS, respectively. We hypothesised that HS synthesis might be altered in the presence of UDP-GlcNAz due to the location of the azide on the acetyl group and the potential for interference with endogenous N-deacetylase-N-sulphotransferase biosynthetic enzyme activity. In mammalian cell culture (Chinese hamster ovary cells), treatment with Ac4GalNAz led to a decrease in total HS abundance accompanied by significant increases in 6-O-sulphation within the chains. Incorporation of a radiolabelled metabolic precursor revealed that average HS chain length was decreased in azido sugar-treated CHO cells. The modifications to HS were dose-dependent and HS inhibition was transient. Following removal of Ac4GalNAz from cell culture, HS expression returned to baseline levels within 24 hours. Previous work from the Bertozzi group has demonstrated the utility of Ac4GalNAz for visualising GalNAc- and O-GlcNAc-modified proteins in vivo. Using Xenopus, we were able to show that treatment of fertilised eggs with Ac4GalNAz decreased the abundance of HS in a similar way to that seen in vitro, with an associated impact on embryonic development. Embryonic axial elongation was impaired, with defective myotomal development and aberrant axonal patterning along the trunk and tail. Posterior somite cell nuclei were disorganised, with loss of distinct chevron patterning and skeletal muscle development was impaired with muscle fibres spanning some of the somite boundaries. Removal of the inhibitor partially rescued tail extension defects, as well as muscle development, but not axonal patterning. Therefore, these experiments illustrate a novel application for Ac4GalNAz as a soluble and reversible inhibitor of HS synthesis for in vitro and in vivo studies. The observed potential for control of inhibition via time- and dose-dependent effects enables targeted and selective inhibition of HS and potentially provides a powerful new inhibitor for the study of HS-mediated events.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:634965 |
| Date | January 2015 |
| Creators | Maciej, Marissa Lucy |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/azido-sugars-for-the-modification-of-glycosaminoglycans-in-biology(cf34d9aa-d20b-4225-b871-93a5983832b6).html |
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