Abstract
Elastic fiber assembly is a complicated process involving multiple different proteins and enzyme activities. However, the specific protein-protein interactions that facilitate elastin polymerization have not been defined. To identify domains in the tropoelastin molecule important for the assembly process, we utilized an in vitro assembly model to map sequences within tropoelastin that facilitate its association with fibrillin-containing microfibrils in the extracellular matrix. Our results show that an essential assembly domain is located in the C-terminal region of the molecule, encoded by exons 29-36. Fine mapping studies using an exon deletion strategy and synthetic peptides identified the hydrophobic sequence in exon 30 as a major functional element in this region and suggested that the assembly process is driven by the propensity of this sequence to form beta-sheet structure. Tropoelastin molecules lacking the C-terminal assembly domain expressed as transgenes in mice did not assemble nor did they interfere with assembly of full-length normal mouse elastin. In addition to providing important information about elastin assembly in general, the results of this study suggest how removal or alteration of the C terminus through stop or frameshift mutations might contribute to the elastin-related diseases supravalvular aortic stenosis and cutis laxa.
Highlights
Elastic fiber assembly is a complicated process involving multiple different proteins and enzyme activities
The Presence of the Tropoelastin C Terminus Is Required for Its Deposition into the Extracellular Matrix of pigmented epithelial (PE) Cells—To determine which regions of tropoelastin are necessary for its association with the extracellular matrix, we generated expression constructs consisting of full-length elastin as well as mutant and deletion forms of the molecule
Because these cells produce all of the components necessary to form the scaffolding for elastic matrices, but not tropoelastin itself, they provide a useful system for studying the early stages of elastic fiber assembly
Summary
Elastic fiber assembly is a complicated process involving multiple different proteins and enzyme activities. It has long been assumed that microfibrils provide a scaffold or template for elastin assembly by binding and aligning tropoelastin monomers so that lysine-containing regions are in register for cross-linking This idea evolved from electron microscopic images showing that the appearance of microfibrils is the first ultrastructural indication of the elastic fiber [2,3,4] and that microfibrils are associated with elastin throughout the elastogenic period. Studies with synthetic peptides and tropoelastin deletion constructs suggest that the hydrophobic sequence encoded by exon 30 is a major functional element in this region These findings provide an explanation for how SVAS mutations could lead to haploinsufficiency when the truncated product of the mutant allele is secreted without this critical assembly domain
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