Structural characterisation of marine glycosaminoglycans and their interactions with proteins.
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Glycosaminoglycans (GAGs) are a group of structurally related, naturally occurring polysaccharides, found as the carbohydrate moieties of proteoglycans and sometimes as free polysaccharides. GAGs are expressed ubiquitously on animal cell surfaces and within extracellular matrices. All GAGs are sulfated to various degrees, except hyaluronan, which is always non-sulfated. In this project, GAGs and other sulfated carbohydrates were purified from different marine sources and an algae species living in soil, and their structures were characterised, mainly by NMR spectroscopy. Oversulfated dermatan sulfate (DS), fucosylated chondroitin sulfate (fCS) with interesting anti-metastatic properties, a sulfated fucosylated GlcNAc polymer, naturally-occurring cellulose sulfate and a polysaccharide with a repeating disaccharide unit of IdoA-Gal, were some of the more unusual carbohydrates identified. Common GAGs like hyaluronan, chondroitin sulfate A and C, DS and heparin/heparin sulfate-like polysaccharides were also purified from lumpsuckers and ragoworms. Different depolymerisation techniques were also investigated. The effects of Fentontype reaction and photochemical free radical depolymerisation on DS were studied. Elimination of the reducing end IdoA in the case of Fenton-type produced oligosaccharides was established. The oxidisation of reducing end GalNAc to Nacetylgalactosaminic acid, as a result of both depolymerisation techniques, was also identified. In addition, the effect of mild acid hydrolysis on fCS polysaccharides were studied and this technique was deemed unfitting for the depolymerisation of the molecule, as it causes defucosylation and partial desulfation. Finally, human beta-defensin 2 (HBD2) was expressed recombinantly, as a fusion protein with pE-Sumo, in an attempt to design a high yield expression system capable of producing correctly folded protein. The significance of NMR in the identification of the state of the protein and the need for an expression system capable of carrying out correctly the post-translational modifications was demonstrated.