LTBP4 belongs to a family of large secreted TGFÎ² binding glycoproteins that are structurally related to fibrillin-1. LTBP4 mutations are linked to an ARCL1C that is characterised by generalised cutis laxa and severely disrupted elastic fibres in several visceral organs including the lung. LTBP4 assists in regulating TGFÎ²1 biology by mediating its folding and secretion and extracellular matrix bioavailability by targeting latent TGFÎ² and mediating its sequestration via interaction with fibrillin-1. LTBP4 is also essential for promoting elastic fibre assembly via interaction with fibulin-4 and -5. Although LTBP4 plays a vital role in elastogenesis, the molecular mechanism by which it regulates elastic fibre assembly is poorly understood, and its structure is not defined yet. To understand the role of LTBP4 in elastogenesis and how ARCL1C-causing mutations impact on LTBP4 leading to defective elastic fibre assembly, it was essential to determine the structure of LTBP4. Using recombinant wildtype LTBP4 constructs and ARCL1C mutants, LTBP4 hydrodynamics and nanostructure have been determined using complementary biophysical methods and compared to analyze any differences. Findings presented in this thesis demonstrate novel structural information on the wildtype LTBP4 and define conformational changes induced by ARCL1C. The monomeric LTBP4 C-terminal region was found to adopt an elongated and flexible conformation. In a comparison of the mutant, C1286S, to the wildtype, minimal conformational change has been induced. While the C1186R mutation caused a more considerable conformational change and resulted in a conformational transition. The monomeric LTBP4 N-terminal region was found to adopt an elongated and inflexible conformation while the C244G mutant resulted in a more compact protein. Previous studies have demonstrated that LTBP4 promotes elastogenesis by interacting with fibulin-4 and fibulin-5 via its N-terminal region and by interacting with fibrillin-1 via its C-terminal region. Matrix deposition of LTBP4 is dependent on fibrillin-1 deposition. While matrix deposition of fibulin-5 is dependent on the proper deposition of LTBP4, moreover, it has been demonstrated that fibronectin and heparin/heparan sulphate (HS) mediate LTBP4 matrix deposition via its N-terminal region. Using binding studies, how ARCL1C mutations may impact on LTBP4 molecular interactions with these proteins, that are involved in LTBP4 deposition and elastogenesis, has been investigated. Data presented here demonstrate that ARCL1C mutants could bind to these matrix partners but with slightly altered affinity depending on the position of the substituted highly conserved cysteine. Findings shown here also identify tropoelastin and LTBP1 as novel matrix partners, suggesting new roles for LTBP4. Previous work by our group demonstrated that LTBP1 can self-assemble and that transglutaminase 2 (TG2) stabilises its assembly and cross-linking to fibrillin-1. Using binding studies and TG2 cross-linking assays, LTBP4 self-assembly and cross-linking with fibrillin-1 was investigated. Data here demonstrate that LTBP4 does not self-interact or cross-link nor cross-link to fibrillin-1 via TG2. Moreover, data here show a novel non-covalent interaction with small latent complex (SLC) and that interaction of LTBP1 with fibrillin-1 is not altered by the formation of the large latent complex (LLC). Collectively, data presented in this thesis contribute to understanding the role of LTBP4 in elastogenesis and highlight the importance of the highly conserved cysteines in LTBP4 structure and function.
|Date of Award||1 Aug 2020|
- The University of Manchester
|Supervisor||Michael Sherratt (Supervisor) & Clair Baldock (Supervisor)|