Contribution of domain 30 of tropoelastin to elastic fiber formation and material elasticity

Abstract

Elastin is a fibrous structural protein of the extracellular matrix that provides reversible elastic recoil to vertebrate tissues such as arterial vessels, lung, and skin. The elastin monomer, tropoelastin, contains a large proportion of intrinsically disordered and flexible hydrophobic sequences that collectively are responsible for the initial phase separation of monomers during assembly, and are essential for driving elastic recoil. While structural disorder of hydrophobic sequences is controlled by a high proline and glycine residue composition, hydrophobic domain 30 of human tropoelastin is atypically proline-poor, and forms β-sheet amyloid-like fibrils as an individual peptide. We explored the contribution of confined regions of secondary structure at the location of domain 30 in human tropoelastin to fiber assembly and mechanical properties using a set of mutations designed to inhibit or enhance the propensity of β-sheet formation at this location. Our data support a dual role for confined β-sheet secondary structure in domain 30 of tropoelastin in guiding the formation of fibers, and as a determinant of stiffness and viscoelastic properties of cross-linked materials. Together, these results suggest a mechanism for specificity in fiber assembly, and elucidate structure-function relationships for the rational design of elastomeric biomaterials with defined mechanical properties.

Single nucleotide polymorphisms and domain/splice variants modulate assembly and elastomeric properties of human elastin. Implications for tissue specificity and durability of elastic tissue

“…[30,45] The effect of deletion of domain 30 (hTE D30) on self-assembly and material properties has already been published. [17] Variant hTE 122 contains domain 22 inserted between domains 21 and 23. While present in the genomic sequence, exon 22 is essentially always spliced out of hTE.…”

“…Associated with susceptibility to COPD. [8,17,20,21,46,56] Seventy-two hours after transfection, protein products in cell extract and medium compartments were analyzed by western blotting using an antibody to EGFP (Figure 2, upper panel). "|" indicates exon/exon border.…”

“…[13] In general, hydrophobic domains are responsible for phase separation, self-assembly, and elastomeric properties, while crosslinking domains contain the lysine residues destined for covalent cross-linking. [14,[16][17][18][19][20][21][22] Although there is a single gene for human tropoelastin (hTE), several splice variants [23][24][25] and at least 13 RefSeq isoforms have been identified for the protein. [4][5][6][7][8][9][10][11][12][13][14][15][16][17] Such recombinant forms of tropoelastin have been used extensively in vitro to demonstrate that relatively modest changes in sequence and domain arrangements can have significant effects on assembly and material properties.…”

“…[14,[16][17][18][19][20][21][22] Although there is a single gene for human tropoelastin (hTE), several splice variants [23][24][25] and at least 13 RefSeq isoforms have been identified for the protein. However, the necessity for long-term structural/mechanical integrity of elastin, the likelihood that such integrity is related to the architecture of the polymeric elastic matrix, and the increasing awareness of the effects of sequence modification on both assembly and mechanical properties of elastin, [14,[16][17][18][19][20][21][22] have together suggested that variations in the sequence of elastin could also have more subtle effects on the architecture and properties of the final elastic matrix. In the same way, polymorphisms resulting in amino acid substitutions or affecting splice sites could also alter elastin assembly and polymeric properties.…”

Design of an elastin-layered dermal regeneration template

“…We and others [20], [29], [30], [31] have used in vitro cell culture models with the addition of recombinant tropoelastin to investigate elastogenesis by cells. In our model system, human dermal fibroblasts are cultured for 10–12 days prior to the addition of purified recombinant human tropoelastin and then cultured for up to a further 7 days.…”