Modification and functional inactivation of the tropoelastin carboxy-terminal domain in cross-linked elastin

Abstract

The carboxyl terminus of tropoelastin is a highly conserved, atypical region of the molecule with sequences that define both cell and matrix interactions. This domain also plays a critical but unknown role in the assembly and crosslinking of tropoelastin during elastic fiber maturation. Using a competitive ELISA with an antibody to an elastase-resistant epitope in the carboxy-terminus of tropoelastin (domain-36), we quantified levels of the domain-36 sequence in elastase-derived peptides from mature, insoluble elastin. We found that the amount of carboxy-terminal epitope in elastin is ∼0.2% of the expected value, assuming each tropoelastin monomer that is incorporated into the insoluble polymer has an intact carboxy-terminus. The low levels suggest that the majority of domain-36 sequence is either removed at some stage of elastin assembly or that the antigenic epitope is altered by posttranslational modification. Biochemical evidence is presented for a potential lysinederived cross-link in this region, which would alter the extractability and antigenicity of the carboxyterminal epitope. These results show that there is little or no unmodified domain-36 in mature elastin, indicating that the cell and matrix binding activities associated with this region of tropoelastin are lost or modified as elastin matures. A crosslinking function for domain-36 may serve to help register the multiple crosslinking sites in elastin and explains why mutations that alter the domain-36 sequence have detrimental effects on elastic fiber assembly.

Characterization of an unusual tropoelastin with truncated C-terminus in the frog

“…However, the strong sequence conservation in this exon of tropoelastin genes is consistent with several reports of an important role for the C-terminal domain in interactions with other proteins and cells to contribute to the assembly and final architecture of the extracellular matrix in vivo (Broekelmann et al, , 2008Brown-Augsburger et al, 1994, 1996Hinek and Rabinovitch, 1993;Hsiao et al, 1999;Rodgers and Weiss, 2004). This view is also supported by several reports of human tropoelastin mutations affecting this C-terminal domain.…”

“…In the penultimate exon, the conserved final two amino acids are in bold and the crosslinking motif normally positioned near the N-terminal of the exon is underlined. matrix, at least in mammalian tissues (Broekelmann et al, , 2008Brown-Augsburger et al, 1994, 1996Hinek and Rabinovitch, 1993;Hsiao et al, 1999;Rodgers and Weiss, 2004), might initially suggest that this second elastin gene represents an actual or incipient pseudogene which is either not expressed or results in a protein product which cannot be incorporated into elastic tissues.…”

Cell Adhesion to Tropoelastin Is Mediated via the C-terminal GRKRK Motif and Integrin αVβ3

“…Others have shown that the C terminus of tropoelastin may be lost in mature elastic fibers, which would account for the lack of cell interaction to soluble bovine elastin (37).…”

Elastomeric recombinant protein-based biomaterials

“…These soluble forms of elastin have shown properties similar to the native tropoelastin, such as ability to coacervate as well as alteration of cell signaling via elastin receptors. However, animal-derived elastin is a heterogeneous partially crosslinked mixture of peptides with inadequate cell binding sites [40]. Elastin derivatives can also be produced via peptide synthesis to generate ELPs, or biosynthetically to form recombinant proteins.…”