Role of tropoelastin fragmentation in elastogenesis in rat smooth muscle cells

Neonatal rat aortic smooth muscle cell cultures produce two major soluble elastin molecules termed protropoelastin (77 kDa) and tropoelastin (71 kDa). Cell layer extracts are protroproelastin-enriched, while protropoelastin, tropoelastin, and significant amounts of discrete elastin fragments (Mr of 66,000, 61,000, 56,000, and 45,000) are present in preparations from the medium of these cultures. To determine the role of the various elastin molecules in the metabolism of elastin in neonatal rat aortic smooth muscle cell cultures, the amino termini of these proteins were sequenced. All soluble elastin components present in the medium were purified as a single peak by high performance liquid chromatography; further separation of the components was achieved by polyacrylamide gel electrophoresis and electroblotting. The bands were excised and sequenced. The amino-terminal sequences of protropoelastin, tropoelastin, and the 66-kDa, 61-kDa, and 56-kDa fragments were identical: Gly-Gly-Val-Pro-Gly-Ala-Val-Pro-Gly-Gly. This sequence is identical with published amino-terminal sequences of tropoelastins from several other species. As expected, when the cell cultures were pulsed with [3H]valine, all the soluble elastin molecules were radioactive, while only protropoelastin appeared radioactive after [35S] cysteine pulsing. Since cysteine is present only in the carboxyl-terminal end of the molecule, all the data indicate that the cleavage of the elastin fragments identified in the culture are occurring at the carboxyl end of protropoelastin. These results are consistent with the original hypothesis that a precursor-product relationship exists between the 77-kDa and 71-kDa soluble elastin molecules. Based on known tropoelastin sequences and the molecular weights of the discrete fragments, additional fragmentation of protropoelastin and/or tropoelastin most likely occurs at the lysine/alanine-enriched domains presumably involved in cross-link formation.