The role of the carboxy terminus of tropoelastin in its assembly into the elastic fiber.

Tropoelastin, the soluble precursor protein of insoluble amorphous elastin, contains repeating segments that are important for the characteristic elasticity and crosslinking sites of mature elastin. In addition, there is a unique carboxy terminal domain that is encoded by exon 36 of the elastin gene, and it has been suggested that this region may play a role in the process of insolubilization. The contribution of exon 36 to the maturation of tropoelastin into insoluble elastin was probed in these studies. Neonatal rat aortic smooth muscle cells were cultured and the fate of [3H] Lys labeled human recombinant tropoelastin (hrTE) molecules added to the cultures was monitored. In comparison to the hrTE containing the region encoded by exon 36, hrTE molecules lacking this domain were less efficiently incorporated into elastin, as evidenced by a decrease in NaOH insoluble radioactivity. Specific residues within the domain encoded by exon 36 were targeted for further study in experiments in which the two Cys residues were reduced and alkylated, and/or the four basic Arg-Lys-Arg-Lys residues at the carboxy terminus were removed. Both of these modifications resulted in decreased incorporation into elastin equivalent to the complete removal of the carboxy terminus. Prior treatment of the cell layer with elastase reduced the efficiency of insolubilization of hrTE containing the domain encoded by exon 36, but had no effect on the processing of molecules lacking this region. These data suggest that exon 36 of the elastin gene contributes to normal efficient incorporation of tropoelastin into the elastin fiber.