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Characterisation of ADAMTS-L2 and ADAMTS-L4.
[Thesis]. Manchester, UK: The University of Manchester; 2017.
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Abstract
ADAMTS-Ls (A Disintegrin and Metalloprotease with Thrombospondin type 1 motifs-like proteins) have been identified as having important roles in the extracellular matrix (ECM) and in fibrillinopathies. Mutations in ADAMTSL2 and ADAMTSL4 have been associated with geleophysic dysplasia (GD) and ectopia lentis (EL) respectively. Despite their involvement in GD and EL, very little is known about the structure, function and interactions of ADAMTS-L2 and ADAMTS-L4. Characterisation of these molecules will therefore enable greater understanding of these molecules and how they may function in the ECM. The generation of recombinant ADAMTS-L2 and ADAMTS-L4 in mammalian cell lines was established using lentiviral and episomal expression systems. Of these two systems, the lentiviral expression system exhibited long-lasting stable expression of ADAMTS-L2 and ADAMTS-L4 as well as providing greater yields of protein. Biophysical characterisation of ADAMTS-L2 using OPTIM analysis defined optimal buffer conditions required for protein stability. Biochemical analysis confirmed that both ADAMTS-L2 and ADAMTS-L4 have N-linked glycosylation and that under native conditions ADAMTS-L2 exists in monomeric form. Negative stain TEM allowed for structural modelling of ADAMTS-L2 and ADAMTS-L4. 3D modelling generated a 43.4 Å asymmetric lobular structure of ADAMTS-L2. Domain arrangement of ADAMTS-L2 suggests that the C-terminal thrombospondin type 1 repeats (TSRs) may be flexible. Structural analysis of ADAMTS-L4 revealed that it adopted several conformations owing to the highly flexible nature of the C-terminal TSRs. Due to this flexibility, 3D reconstruction of ADAMTS-L4 was not possible, however 2D analysis determined the average length was 40.6 nm. Interactions of ADAMTS-L2 with fibrillin-1 and latent transforming growth factor β-1 (LTBP-1), and co-localisation of ADAMTS-L4 to fibrillin-1 microfibrils have already been reported. Here, with the use of surface plasmon resonance (SPR), ADAMTS-L2 interactions were confirmed with fibrillin-1 fragment PF17 and a novel interaction with fibronectin fragment FN7-14 was found. Immunofluorescence microscopy of ADAMTS-L2 with fibrillin-1, LTBP-1 and fibronectin in cultured human dermal fibroblasts (HDFs) corroborated the interactions observed in this study with SPR. Complete co-localisation of ADAMTS-L4 with fibrillin-1 and partial co-localisation with LTBP-1 and fibronectin was observed. This investigation reports novel information regarding the structure and biomolecular interactions of ADAMTS-L2 and ADAMTS-L4, contributing towards the characterisation of both molecules and towards a better understanding of their function.