Domain 36 of Tropoelastin in Elastic Fiber Formation

Abstract

Elastic fiber assembly is a complex stepwise process involving multiple different proteins and enzymes. Domain 36, encoded by the last exon of the elastin gene, is recognized to be an important domain for deposition onto microfibrils, an essential step in elastic fiber assembly. However, the role of domain 36 in elastic fiber assembly has not been clarified. Here, we utilized our established in vitro assembly model to identify the importance of domain 36 for the assembly process. Our results showed that the lack of domain 36 in bovine tropoelastin results in deficient elastic fiber assembly. A similar result was obtained with the point mutation of two cysteine residues and the deletion of the Lysine-Arginine-Lysine-Arginine (RKRK) sequence in domain 36. Double immunofluorescence of tropoelastin and fibrillin-1, a main component of microfibrils, demonstrated reduced localization of these mutant tropoelastin molecules on fibrillin-1 fibers. Moreover, the binding affinity of these mutants to fibrillin-1 and microfibril-associated glycoprotein (MAGP) was significantly decreased. These data indicate that domain 36 of tropoelastin facilitates elastic fiber assembly by interacting with microfibrils via two cysteine residues and the RKRK sequence.

Molecular model of human tropoelastin and implications of associated mutations

“…Domain 36, corresponding to the last 14 residues in the sequence, appeared to be the most mobile of all tropoelastin domains based on rms fluctuation (Figs. 2L, 3J, and 4H), consistent with its role in interacting with cells (37,38) and other elastic fiber proteins (39).…”

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

“…Specific domains and residues that contribute to elastic fiber assembly are identified from across the length of the monomer. Particular attention has been focused on the unique sequence features of C‐terminal domain 36, which includes the only two cysteine residues in the monomer, and unique concentration of charged residues (tetrabasic RKRK motif), responsible for cell‐ and matrix‐binding properties . Interestingly however, tropoelastin expressed from the frog eln2 gene displays a highly unusual evolutionary truncation of the C‐terminal RKRK motif, yet incorporates well into the matrix, albeit with different tissue localization than the product of frog eln1 , which contains the RKRK motif .…”

“…Particular attention has been focused on the unique sequence features of Cterminal domain 36, which includes the only two cysteine residues in the monomer, and unique concentration of charged residues (tetrabasic RKRK motif), responsible for cell-and matrix-binding properties. [3][4][5]14,15 Interestingly however, tropoelastin expressed from the frog eln2 gene displays a highly unusual evolutionary truncation of the C-terminal RKRK motif, yet incorporates well into the matrix, albeit with different tissue localization than the product of frog eln1, which contains the RKRK motif. 16 Other studies reveal the importance of charged residues aspartic acid in domain 6 to fiber assembly, 7 one of only three negatively charged residues in human tropoelastin, and of arginine at the exposed and flexible border of domains 25 and 26.…”

“…Despite the repetitive and modular sequence of tropoelastin, it is clear that fiber formation in tissue culture, and likely in vivo, requires the contribution of specific residues and domains. Abnormal and/or reduced fiber formation has been observed upon mutations to the C‐terminal cell‐interactive domain 36 of mammalian tropoelastins (including deletion of an RKRK motif, or point mutation of the only two cysteine residues in the protein), upon deletion of domains 29 to 36, or by using an antibody to the C‐terminal domain, as well as by mutation of charged residues such as aspartic acid from domain 6, and arginine at the junction of domains 25/26 . Mutations may disrupt the interaction of tropoelastin with other tropoelastin monomers, the cell surface, and/or matrix proteins, affecting globule stability, deposition and/or incorporation into fibers.…”

Tropoelastin and Elastin Assembly

“…In addition to the RKRK sequence, domain 36 contains tropoelastin's sole two cysteines and only disulfide bond. Perturbation of either of these components greatly reduces tropoelastin's ability to self-assemble in vitro and interact with the microfibril scaffold of elastic fibers (Nonaka et al, 2014), indicating that an intact domain 36 is required for correct assembly. Tropoelastin is a low complexity protein on both primary and secondary sequence levels.…”