FUNCTIONAL DOMAINS ON ELASTIN AND MICROFIBRIL-ASSOCIATED GLYCOPROTEININVOLVED IN ELASTIC FIBER ASSEMBLY

Studies in vitro suggest that the C-terminus of tropoelastin mediates elastin polymerization through an interaction with microfibril-associa ted proteins. In this study we have used cultured auricular chondrocyt es as a model system to examine whether this interaction is critical f or elastic fibre formation in vivo. Auricular chondrocytes, which depo sit an abundant elastic fibre matrix, were cultured in the presence of Fab fragments of antibodies directed against the C-terminus (CTe) or an N-terminal domain (AT(e)) of tropoelastin. Immunofluorescent staini ng of the extracellular matrix deposited by the cells showed that the CTe antibody inhibited the deposition of elastin without affecting mic rofibril structure. Cells grown under identical conditions in the pres ence of AT(e), however, formed fibres that stained normally for both e lastin and microfibril proteins. Chondrocytes cultured in the presence of microfibril-associated glycoprotein (MAGP):21-35, an antibody dire cted against a domain near the N-terminus of MAGP, did not organize tr opoelastin into fibres. However, immunostaining for MAGP and fibrillin revealed normal microfibrils. In agreement with the immunofluorescenc e staining patterns, fewer elastin-specific cross-links, indicative of insoluble elastin, were detected in the extracellular matrix of cells cultured in the presence of CTe. The medium from these cultures, howe ver, contained more soluble elastin, consistent with an antibody-induc ed alteration of elastin assembly but not its synthesis. Northern anal ysis of antibody-treated and control cultures substantiated equivalent levels of tropoelastin mRNA. These results confirm that the C-terminu s of tropoelastin interacts with microfibrils during the assembly of e lastic fibres. Further, the results suggest that the interaction betwe en tropoelastin and microfibrils might be mediated by a domain involvi ng the N-terminal half of MAGP.