The collagen fibrillar network: Sequential degradation of its constituents by tissue metalloproteinases

Sammanfattning: The cartilage matrix is made up of a fibrillar meshwork comprising type II collagen and other associated proteins. One main function of this network is to retain the large aggregating proteoglycan, aggrecan, a vital component in the regulation of fluid flux in cartilage. In disease such as osteoarthritis, the breakdown of the collagen network appears to be the final step in destruction of cartilage. The aim of the work described in this thesis was to develop specific tools for the detection of extracellular matrix degradation products as a means to monitor cartilage disease. In the first instance we elaborated an ELISA assay specific for denatured type II collagen. Application of this assay to the detection of collagen denaturation in osteoarthritic versus normal cartilage showed that nearly four times greater percentage unwound collagen could be detected in diseased tissue (denatured/total). The focus was then placed on the fate of some of the fibril associated proteins. Indeed we demonstrated that fibromodulin, a member of the leucine rich repeat proteins, was degraded prior to significant loss of type II collagen in a degradative cartilage explant culture system. Only one major product of degraded fibromodulin could be isolated and this was characterised in order to determine its neo N-terminus. This revealed that this product was lacking the entire tyrosine sulphate rich N-terminus and had an intact C-terminus. Cleavage at the same site could also be performed in vitro with purified MMP-13 (not with MMPs 2, 8 or 9). An antibody directed to the neo-N-terminus of this fragment reacted specifically with degraded fibromodulin in Western blotting, but showed poor reactivity with the natural cleavage product in ELISA assay. Prior to further attempts at raising functional antibodies to the cleavage product we opted to characterise the domains surrounding the site of processing. This work showed that the N-terminal cystine double loop has a cruciform structure. This may cause shielding of the epitope by steric hindrance. We also determined experimentally the presence of sulphation on nearly all of the tyrosine residues found in the N-terminus of fibromodulin including those neighbouring the cleavage site. An error in the sequence was also noted and corrected. The information gained from these characterisation studies will be vital in the choice and composition of immunising peptides used to generate neo-epitope antibodies.