Domains in Tropoelastin That Mediate Elastin Depositionin Vitro and in Vivo

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 ␤-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.The inherent complexity of the elastic fiber, combined with the unique physical properties of its component proteins, has made understanding elastin structure and assembly one of the most difficult problems in matrix biology. Although technical advances in cell and molecular biology have given us new information about elastin gene expression and elastin synthesis, surprisingly little is known about how the elastic fiber is assembled at the molecular level. The major component of the mature fiber is a covalently cross-linked polymer of the protein elastin. Elastin is secreted from the cell as a soluble monomer called tropoelastin. In the extracellular space, lysine residues within tropoelastin are specifically modified to form covalent cross-links between tropoelastin chains. This cross-linked polymer has a high degree of reversible distensibility, including the ability to deform to large extensions with small forces.The tropoelastin molecule is characterized by a series of tandem repeats, each including a lysine-containing cross-linking region followed by a hydrophobic motif (1). Cross-linking is initiated by the extracellular enzyme(s) lysyl oxidase, which catalyzes the oxidative deamination of lysyl ⑀-amino groups. Under normal conditions, the cross-linking process is extremely efficient, with all but ϳ5 of the ϳ40 lysine residues in tropoelastin participating in covalent linkages that form the functional polymer. How the cross-linking sites within the monomer are aligned prior to cross-linking is unclear. It has long been assumed that microfibrils provide a scaffold or template for elastin assembly by binding and aligning tropoelastin monomers so that lysine-containin...

Charge transport and intrinsic fluorescence in amyloid-like fibrils

“…It may be possible that the aggregation into such well defined, nanoordered assemblies represents a state of an efficient minimal energy arrangement of polypeptide chains (15)(16)(17). Recent results have shown that fibrillar structures similar to amyloid fibrils are formed by sequences like poly(XGlyGlyYGly) (X, Y ϭ Val, Leu, or Ala), which are highly repeated in the hydrophobic domains of elastin (18,19).…”

Supramolecular Amyloid-like Assembly of the Polypeptide Sequence Coded by Exon 30 of Human Tropoelastin

“…In the alternate succession of hydrophobic and cross-linking domains, peculiar of tropoelastin (27), this peptide belongs to the C-terminal hydrophobic domains of human tropoelastin and has been indicated as fundamental for a correct assembly of elastin (10). It is a glycine-rich domain, containing 13 glycine residues up to 25 residues, and contains only hydrophobic residues.…”

Identification and characterization of seven novel mutations of elastin gene in a cohort of patients affected by supravalvular aortic stenosis

“…It has been shown that the disruption of the disulfide bond dramatically reduces the ability of tropoelastin to assemble and be included in developing fibers, as it does the removal of the C-terminal region. 16 Therefore, we can speculate that the mutant tropoelastin, if secreted from the cell, would likely impair the tropoelastin from assembling in a dominant-negative manner, resulting in a deleterious effect on the normal elastogenesis in those patients. A definitive resolution of this issue would require arterial smooth muscle in order to investigate whether these mutations yield abnormal elastin and/or normal elastogenesis, but this kind of tissue is not available.…”