|Institution:||University of Manchester|
|Full text PDF:||http://www.manchester.ac.uk/escholar/uk-ac-man-scw:308632|
ADAMTS-Ls (A Disintegrin and Metalloprotease withThrombospondin type 1 motifs-like proteins) have been identified ashaving important roles in the extracellular matrix (ECM) and infibrillinopathies. Mutations in ADAMTSL2 and ADAMTSL4 have beenassociated with geleophysic dysplasia (GD) and ectopia lentis (EL)respectively. Despite their involvement in GD and EL, very littleis known about the structure, function and interactions ofADAMTS-L2 and ADAMTS-L4. Characterisation of these molecules willtherefore enable greater understanding of these molecules and howthey may function in the ECM. The generation of recombinantADAMTS-L2 and ADAMTS-L4 in mammalian cell lines was establishedusing lentiviral and episomal expression systems. Of these twosystems, the lentiviral expression system exhibited long-lastingstable expression of ADAMTS-L2 and ADAMTS-L4 as well as providinggreater yields of protein. Biophysical characterisation ofADAMTS-L2 using OPTIM analysis defined optimal buffer conditionsrequired for protein stability. Biochemical analysis confirmed thatboth ADAMTS-L2 and ADAMTS-L4 have N-linked glycosylation and thatunder native conditions ADAMTS-L2 exists in monomeric form.Negative stain TEM allowed for structural modelling of ADAMTS-L2and ADAMTS-L4. 3D modelling generated a 43.4 asymmetric lobularstructure of ADAMTS-L2. Domain arrangement of ADAMTS-L2 suggeststhat the C-terminal thrombospondin type 1 repeats (TSRs) may beflexible. Structural analysis of ADAMTS-L4 revealed that it adoptedseveral conformations owing to the highly flexible nature of theC-terminal TSRs. Due to this flexibility, 3D reconstruction ofADAMTS-L4 was not possible, however 2D analysis determined theaverage length was 40.6 nm. Interactions of ADAMTS-L2 withfibrillin-1 and latent transforming growth factor -1 (LTBP-1), andco-localisation of ADAMTS-L4 to fibrillin-1 microfibrils havealready been reported. Here, with the use of surface plasmonresonance (SPR), ADAMTS-L2 interactions were confirmed withfibrillin-1 fragment PF17 and a novel interaction with fibronectinfragment FN7-14 was found. Immunofluorescence microscopy ofADAMTS-L2 with fibrillin-1, LTBP-1 and fibronectin in culturedhuman dermal fibroblasts (HDFs) corroborated the interactionsobserved in this study with SPR. Complete co-localisation ofADAMTS-L4 with fibrillin-1 and partial co-localisation with LTBP-1and fibronectin was observed. This investigation reports novelinformation regarding the structure and biomolecular interactionsof ADAMTS-L2 and ADAMTS-L4, contributing towards thecharacterisation of both molecules and towards a betterunderstanding of their function.Advisors/Committee Members: Baldock, Clair.