Functional domains on elastin and microfibril-associated glycoprotein involved in elastic fibre assembly.

Abstract

Studies in vitro suggest that the C-terminus of tropoelastin mediates elastin polymerization through an interaction with microfibril-associated proteins. In this study we have used cultured auricular chondrocytes as a model system to examine whether this interaction is critical for elastic fibre formation in vivo. Auricular chondrocytes, which deposit 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 (ATe) of tropoelastin. Immunofluorescent staining of the extracellular matrix deposited by the cells showed that the CTe antibody inhibited the deposition of elastin without affecting microfibril structure. Cells grown under identical conditions in the presence of ATe, however, formed fibres that stained normally for both elastin and microfibril proteins. Chondrocytes cultured in the presence of microfibril-associated glycoprotein (MAGP):21-35, an antibody directed against a domain near the N-terminus of MAGP, did not organize tropoelastin into fibres. However, immunostaining for MAGP and fibrillin revealed normal microfibrils. In agreement with the immunofluorescence 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, however, contained more soluble elastin, consistent with an antibody-induced alteration of elastin assembly but not its synthesis. Northern analysis of antibody-treated and control cultures substantiated equivalent levels of tropoelastin mRNA. These results confirm that the C-terminus of tropoelastin interacts with microfibrils during the assembly of elastic fibres. Further, the results suggest that the interaction between tropoelastin and microfibrils might be mediated by a domain involving the N-terminal half of MAGP.