Functional and Structural Characterisation of Mutant Tropoelastin Constructs

Abstract

The major component of elastic fibres is elastin, formed from its soluble precursor, tropoelastin. The tropoelastin nanostructure is characterised by an N-terminal coil region, hinge region, bridge region and C-terminal foot region. Tropoelastin assembly into elastic fibres consists of distinct stages of tropoelastin synthesis, coacervation and cross-linking into mature fibres. However, the contributions of specific structural regions in tropoelastin to elastic fibre assembly are insufficiently understood. This thesis explores the significance of specific residues/regions in human tropoelastin. Mutant constructs were designed in which the bridge region R515, the hinge region E345/E414, or the N-terminal D72 have been inactivated by alanine substitution. Another mutant that contains domain 22, which is typically spliced out in native human tropoelastin, was also produced. Functional impairment of the R515A isoform has been reported in assays modelling each stage of assembly. A system was optimised to define the elastogenic potential of R515A. When added to human cells, R515A tropoelastin assembled less efficiently into elastic fibres with atypical morphology, which is partially attributed to an altered bridge and C-terminal structure. The E345A, E414A, E345A+E414A, D72A and EX22 constructs were extensively characterised via their ability to coacervate, cross-link, interact with cells, and form elastic fibres. All mutant constructs displayed varying degrees of impaired self-assembly. The shapes of the mutant species were further analysed to correlate their functional attributes to structural effects of the mutation/s. All mutants showed conformational changes consistent with biochemical properties and the expected mutation site/s. These results identify for the first time the role of these regions in maintaining the wild-type structure of human tropoelastin, and the importance of this native structure to normal tropoelastin assembly and function.