Investigating the structure and function of ADAMTSL2

Abstract

ADAMTSL (A Disintegrin and Metalloprotease with Thrombospondin type 1 motifs-like) proteins have important roles in the extracellular matrix (ECM), highlighted by their link to fibrillinopathies. For example, mutations in ADAMTSL2 cause Geleophysic Dysplasia (GD), also caused by mutations of fibrillin-1. Despite the involvement in GD, little is known about the structure, function, and interactions of ADAMTSL2, therefore this project aimed to define the structure and interactions of ADAMTSL2. We have analysed the structure of ADAMTSL2, a ~150 kDa glycoprotein to 12.8 Å using cryo-EM. SAXS analysis is consistent with the cryo-EM structure, where ADAMTSL2 is a globular, slightly elongated flexible molecule. In order to increase the resolution of the EM structure, various approaches have been tried including complexing with known binding partners and epitope labelling. Of these, using a TwinStrep-tag to bind StrepTactin-XT may help in further structural characterisation of ADAMTSL2. The AlphaFold prediction of the structure of ADAMTSL2 showed folded and disordered regions and the low confidence in domain-domain positioning reflects the observed flexibility and challenges encountered in structure determination. SAXS analysis of the C-terminal domains of ADAMTSL2 reveals this region is also flexible which is supported by a negative stain-TEM structure. Molecular modelling of the C-terminal region of ADAMTSL2 has identified a potential interaction with the Wnt3 ligand though conserved residues. Interestingly, the homologous C-terminal region of ADAMTSL4 forms multimeric, flexible string-like fibres, as observed by negative-stain TEM and SAXS. Furthermore, while ADAMTSL2 CT interacted with several ECM components, ADAMTSL4 CT did not, suggesting they have different functionality in the matrix. Using surface plasmon resonance (SPR) the interactions of ADAMTSL2 with the N-terminus of fibrillin-1 been further mapped and several new high affinity interactions with the heparin-binding domains of fibronectin, heparan sulphate (HS), the N-terminal region of LTBP1 and the C-terminus of LTBP4 have also been discovered. Using quartz crystal microbalance and dissipation (QCM-D) we show that fibrillin-1 and fibronectin compete for ADAMTSL2 binding, and over-expression of ADAMTSL2 increases the deposition of fibronectin and LTBP1. Thus far a mechanism for the function of ADAMTSL2 has not been defined, however, our findings and comparison to published data suggests ADAMTSL2 plays a vital role in the deposition of other matrix components, as well as processes such as proliferation and adhesion. We propose that at the cell surface, ADAMTSL2 is facilitating fibrillin assembly mediated by interactions with fibronectin, HS and LTBP1. Additionally, interactions with these matrix molecules and potential involvement in the TGFβ and Wnt/β-catenin signalling pathways suggests a role for ADAMTSL2 in the maintenance of the ECM. These findings contribute to further understanding the importance of the role of ADAMTSL2 in the ECM and will broaden our understanding of healthy and diseased elastic tissues.

Date of Award	1 Aug 2023
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Paul Bishop (Supervisor), Clair Baldock (Supervisor) & Alan Roseman (Supervisor)