Abstract
Elastin is an essential vertebrate protein responsible for the elasticity of force-bearing tissues such as those of the lungs, blood vessels, and skin. One of the key features required for the exceptional properties of this durable biopolymer is the extensive covalent cross-linking between domains of its monomer molecule tropoelastin. To date, elastin's exact molecular assembly and mechanical properties are poorly understood. Here, using bovine elastin, we investigated the different types of cross-links in mature elastin to gain insight into its structure. We purified and proteolytically cleaved elastin from a single tissue sample into soluble cross-linked and noncross-linked peptides that we studied by high-resolution MS. This analysis enabled the elucidation of cross-links and other elastin modifications. We found that the lysine residues within the tropoelastin sequence were simultaneously unmodified and involved in various types of cross-links with different other domains. The Lys-Pro domains were almost exclusively linked via lysinonorleucine, whereas Lys-Ala domains were found to be cross-linked via lysinonorleucine, allysine aldol, and desmosine. Unexpectedly, we identified a high number of intramolecular cross-links between lysine residues in close proximity. In summary, we show on the molecular level that elastin formation involves random cross-linking of tropoelastin monomers resulting in an unordered network, an unexpected finding compared with previous assumptions of an overall beaded structure.
Highlights
Elastin is an essential vertebrate protein responsible for the elasticity of force-bearing tissues such as those of the lungs, blood vessels, and skin
After cleaving mature elastin by pancreatic elastase (PE), we identified 310 linear elastin peptides covering 78% of TE’s sequence
Recent studies on TE and in vitro cross-linked TE provided valuable information on structural properties of elastin’s precursor in solution [35,36,37,38,39], very little is known with respect to the organization of mature elastin in the elastic fiber core
Summary
The identity of linear (noncross-linked) peptides can indirectly provide important information about putative crosslinking sites. The majority of linear peptides were released from hydrophobic domains, which are not involved in cross-linking. We found that Lys residues belonging to KP domains 4, 8, 13, and 35 as well as to KA domains 6, 15, 19, 21, 23, 27, 29, and 31 were partly unmodified. This demonstrates that most KA and KP domains were not completely involved in cross-linking. The C-terminal Lys of the pairwise-occurring Lys residues was found in linear peptides, and it was neither deaminated nor cross-linked. The other, N-terminal Lys residue, which typically belongs to the AAKAA motif, was partly unmodified only in KA domains 6, 19, and 23. All nine mutations were present in hydrophobic regions of elastin and did not affect any sites of cross-linking
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