Paediatrics (RCH) - Theses
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ItemThe role of keratan sulphate in the modulation of aggrecanase activity( 2005)Arthritis is a debilitating disease of the joints caused by the accelerated breakdown of cartilage, resulting in painful, swollen joints. Cartilage protects the joint by absorbing the shock that would otherwise be transferred directly to the underlying bone. One crucial component of cartilage is a specialised molecule known as aggrecan. Aggrecan consists of a core protein with three globular domains (G1, G2 and G3) and is modified with over one hundred highly sulphated glycosaminoglycan chains. Two types of glycosaminoglycans are substituted along the length of the protein, chondroitin sulphate and keratan sulphate. The glycosaminoglycans impart a highly negative charge to the tissue, giving it the ability to retain water and resist compressive forces. Aggrecan is lost from cartilage following cleavage by aggrecanases. Too little aggrecan in cartilage destabilises the structural integrity of the tissue and is associated with arthritis. Of the five known aggrecanase cleavage sites, it is cleavage within the interglobular domain (IGD) between the G1 and G2 domains at NITEGE373 - 374ARGSVI that directly contributes to loss of aggrecan function. The chondroitin sulphate and keratan sulphate located between the G2 and G3 domains is responsible for maintaining the biomechanical properties of aggrecan. The role of keratan sulphate within the G1-G2 domain is unknown, but it is not thought to be essential for aggrecan function. However the literature suggests a possible role of keratan sulphate in facilitating aggrecanase cleavage of NITEGE373 - 374ARGSVI in the IGD. The aim of my project was to examine the role of keratan sulphate in aggrecanase-mediated cleavage of aggrecan in the IGD. Three major goals have been accomplished in this thesis: 1) Identification of a cell type capable of sustained keratan sulphate synthesis. 2) Expression of a recombinant G1-G2 protein substituted with keratan sulphate (rG1-G2). 3) Demonstration that endogenous N-linked keratan sulphate is sufficient to potentiate aggrecanase cleavage of rG1-G2 in the IGD. Cultured cells do not synthesise keratan sulphate. Therefore identifying a cell type, and culture conditions to maximise keratan sulphate synthesis, was a major undertaking. Conditions were identified which allowed for maximal keratan sulphate synthesis, albeit on a small scale, in primary bovine keratocytes. Using a Vaccinia virus expression system, recombinant G1-G2 was expressed in primary bovine keratocytes. Analysis of the rG1-G2 revealed that it was substituted with 5 kDa of keratan sulphate. One important aspect of the study was that the keratan sulphate was all N-linked to the core protein. Subsequent aggrecanase digests, comparing substrates before and after removal of keratan sulphate, showed that aggrecanase cleavage was markedly more efficient when keratan sulphate was present. The results contained in this thesis add significantly to the established literature by providing a greater understanding of the mechanisms involved in aggrecanase-mediated cleavage of aggrecan and cartilage destruction. The results suggest that aggrecan substitution with N-linked keratan sulphate potentiates aggrecanase activity. The results from this study identify N-linked keratan sulphate as a possible target for the development of new drugs for the management of arthritis.